DREB2C Interacts with ABF2, a bZIP Protein Regulating Abscisic Acid-Responsive Gene Expression

DREB2C Interacts with ABF2,a bZIP Protein Regulating Abscisic Acid-Responsive Gene Expression,and Its Overexpression Affects Abscisic Acid Sensitivity1[C][OA]

Sun-ji Lee,Jung-youn Kang,Hee-Jin Park,Myoung Duck Kim,Min Seok Bae,

Hyung-in Choi,and Soo Young Kim*

Department of Molecular Biotechnology and Kumho Life Science Laboratory,College of Agriculture and Life Sciences,Chonnam National University,Gwangju500–757,South Korea

ABF2is a basic leucine zipper protein that regulates abscisic acid(ABA)-dependent stress-responsive gene expression.We carried out yeast two-hybrid screens to isolate genes encoding ABF2-interacting proteins in Arabidopsis(Arabidopsis thaliana). Analysis of the resulting positive clones revealed that two of them encode an AP2domain protein,which is the same as AtERF48/DREB2C.This protein,which will be referred to as DREB2C,could bind C-repeat/dehydration response element in vitro and possesses transcriptional activity.To determine its function,we generated DREB2C overexpression lines and investigated their phenotypes.The transgenic plants were ABA hypersensitive during germination and seedling establishment stages,whereas primary root elongation of seedlings was ABA insensitive,suggesting developmental stage dependence of DREB2C function.The DREB2C overexpression lines also displayed altered stress response;whereas the plants were dehydration sensitive,they were freezing and heat tolerant.We further show that other AP2domain proteins,DREB1A and DREB2A,interact with ABF2and that other ABF family members,ABF3and ABF4,interact with DREB2C.Previously,others demonstrated that ABF and DREB family members cooperate to activate the transcription of an ABA-responsive gene.Our result implies that the cooperation of the two classes of transcription factors may involve physical interaction.

Plants are continually exposed to changing environ-ments in nature and frequently encounter harsh envi-ronmental conditions,such as drought,high salinity, and extreme temperatures.Although they vary widely in their adjustability,plants are nonetheless able to respond adaptively to these and other“abiotic stresses”(Bohnert et al.,1995).This adaptive response is in major part controlled by the phytohormone abscisic acid (ABA;Finkelstein et al.,2002;Xiong et al.,2002). Numerous genes,which are involved in various aspects of adaptive stress response,are up-or down-regulated under stress conditions(Kreps et al.,2002; Ramanjulu and Bartels,2002;Takahashi et al.,2004). Promoter analyses of these ABA-regulated genes re-vealed a number of cis-elements known as“ABA response elements”(ABREs;Busk and Pages,1998; Gomez-Porras et al.,2007).Among the ABREs,those sharing the ACGTGGC core sequence are found to be ubiquitous,and their role in ABA-responsive gene expression has been characterized in detail(Hattori et al.,2002).The ABREs are similar to the G-box, CACGTG,which is found in light-regulated gene promoters(Menkens et al.,1995).Several studies showed that the G-box-type ABRE,which will be referred to as G-ABRE,is necessary but not suf?cient for ABA-induced gene expression.An additional ele-ment is usually required for high level ABA induction. These elements,generally known as“coupling ele-ments,”constitute the ABA response complex together with the G-ABRE(Shen et al.,2004).For example,CE1 (CCACC)functions as a coupling element to the G-ABRE in barley(Hordeum vulgare)HVA22promoter, and the two elements constitute the ABA response complex ABRC1(Shen and Ho,1995).Another cou-pling element,CE3,constitutes,with a G-ABRE,the ABA response complex(ABRC3)present in barley HVA1promoter(Shen et al.,1996).It is also known that G-ABRE itself can function as a coupling element to another copy of G-ABRE(Hobo et al.,1999a).

A subfamily of basic leucine zipper(bZIP)class transcription factors that interact with the G-ABRE has been reported.Referred to as ABFs(ABF1–ABF4;Choi et al.,2000)or AREBs(AREB1–AREB3;Uno et al., 2000),they not only bind G-ABRE in vitro but also regulate ABA/stress response in planta(Fujita et al., 2005;Furihata et al.,2006;Kim,2006).Overexpression

1This work was supported by the Crop Functional Genomics Center of the21C Frontier Program(grant no.CG2112to S.Y.K.) funded by the Ministry of Education,Science,and Technology and by the Agricultural Plant Stress Research Center of Chonnam Na-tional University(grant no.R11–2001–092–04000–0to S.Y.K.)funded by the Korea Science and Engineering Foundation.

*Corresponding author;e-mail sooykim@chonnam.ac.kr.

The author responsible for distribution of materials integral to the ?ndings presented in this article in accordance with the policy described in the Instructions for Authors(https://www.wendangku.net/doc/cb16444214.html,)is: Soo Young Kim(sooykim@chonnam.ac.kr).

[C]Some?gures in this article are displayed in color online but in black and white in the print edition.

[OA]Open Access articles can be viewed online without a sub-scription.

https://www.wendangku.net/doc/cb16444214.html,/cgi/doi/10.1104/pp.110.154617

of ABF3or ABF4in Arabidopsis(Arabidopsis thaliana), for instance,confers ABA hypersensitivity and drought tolerance(Kang et al.,2002;Kim et al.,2004a,2004c), whereas their knockout mutants are partially insensi-tive to ABA and susceptible to drought(Kim et al., 2004c;Finkelstein et al.,2005).Several studies indicate that the in vivo functions of ABFs/AREBs are modu-lated by various kinases(Choi et al.,2005;Furihata et al.,2006;Chae et al.,2007).ABFs/AREBs are highly homologous to ABI5(Finkelstein and Lynch,2000; Lopez-Molina and Chua,2000),sun?ower(Helianthus annuus)and Arabidopsis DPBFs(Kim et al.,1997;Kim and Thomas,1998;Kim et al.,2002),and rice(Oryza sativa)TRAB1(Hobo et al.,1999b).

There is a group of stress-responsive genes whose expression is not regulated by ABA.Promoter analy-ses of these ABA-independent genes have revealed the presence of dehydration-responsive element(DRE; TACCGACAT)and C-repeat(CRT)element(G/ ACCGAC)in drought-responsive and cold-regulated gene promoters,respectively(Baker et al.,1994; Yamaguchi-Shinozaki and Shinozaki,1994).The two elements share the consensus sequence CCGAC,and their cognate transcription factors are known as DREB (for DRE-binding protein;Liu et al.,1998)and CBF(for CRT-binding factor;Stockinger et al.,1997),respec-tively.DREB and CBF belong to the same subfamily of AP2/ERF domain transcription factors,and some members of the DREB family(i.e.DREB1A,DREB1B, and DREB1C)are identical to CBF family members (i.e.CBF3,CBF1,and CBF2,respectively). Pathways leading to stress-responsive gene expres-sion are generally divided into two broad categories, ABA-dependent and ABA-independent pathways, depending on the involvement of ABA(Yamaguchi-Shinozaki and Shinozaki,2005).The pathway(s)lead-ing to ABF/AREB-dependent gene expression via G-ABRE belongs to the former category.Meanwhile, the pathway(s)leading to DREB1/CBF-dependent cold/drought-responsive gene expression belongs to the ABA-independent pathways.Several studies, however,suggest that the two categories of signaling pathways may cross talk to each other;thus,they may be interdependent.For instance,the expression of Arabidopsis CBF family members is ABA inducible (Haake et al.,2002;Knight et al.,2004).ICE1,which is a regulator of CBF expression,is also up-regulated by ABA(Chinnusamy et al.,2003).In the case of maize (Zea mays),DRE-binding AP2/ERF domain factors, DBF1and DBF2,are not only ABA inducible but also regulate ABA response in vivo(Kizis and Pages,2002). These observations suggest that DRE/CRT-regulated expression of some genes may be ABA dependent. Furthermore,Narusaka et al.(2003)have demon-strated that a DRE may function as a coupling element to G-ABRE in the RD29A promoter.They also have shown that ABFs/AREBs and DREBs/CBFs synergis-tically activate the RD29A promoter in a transient assay,although the mechanism of synergistic activa-tion has not been reported yet.

In this report,we show that ABF/AREB family transcription factors physically interact with DREB/ CBF family members.To identify proteins that may modulate the ABF function,we carried out yeast two-hybrid screens employing ABF2as bait.One of the resulting positive clones was AtERF48,which is iden-tical to the DREB family member DREB2C(Sakuma et al.,2002;Nakano et al.,2006).The protein binds to DRE/CRT in vitro,and its overexpression affected both ABA sensitivity and stress tolerance.We further show that ABF2interacts with other DREB family members,DREB1A and DREB2A,and that,conversely, DREB2C interacts with other ABF family members, ABF3and ABF4.Our data suggest that the interaction between ABA-dependent and ABA-independent path-ways may involve physical interaction between ABF and DREB family transcription factors.

RESULTS

Isolation of ABF2-Interacting Proteins

To identify ABF2-interacting proteins,we carried out yeast two-hybrid screens(Kim et al.,2004b).Using a partial fragment of ABF2as bait,we screened approx-imately6.5million yeast transformants and were able to isolate?ve positive clones that speci?cally interact with the bait protein(Fig.1A).Sequence analysis of the clones revealed that two of them encoded a protein with an AP2/ERF domain.The three other clones encoded an ARM repeat protein,which have been reported elsewhere(Kim et al.,2004b).

The AP2domain protein(At2g40340)encoded by the two positive clones was partial,missing the N-terminal52amino acids.Subsequently,a full-length cDNA clone was isolated by PCR utilizing the se-quence information available on the Arabidopsis da-tabase(https://www.wendangku.net/doc/cb16444214.html,).The encoded protein(Fig.1B),which was originally named AAP (for ABF2-interacting AP2domain protein),consisted of341amino acids with a calculated mass of37.8kD. The protein was found to be DREB2C and belongs to the group IV/A-2subfamily of AP2/ERF proteins (Sakuma et al.,2002;Nakano et al.,2006),which includes DREB2family proteins.

The interaction between ABF2and the full-length DREB2C was con?rmed by glutathione S-transferase (GST)pulldown assay.Recombinant ABF2was prepared as a fusion to the GST.Subsequently,its interaction with DREB2C was determined using in vitro-translated DREB2C.Figure1C shows that DREB2C was retained by the GST-ABF2fusion protein but not by GST alone,indicating that DREB2C inter-acted with ABF2in vitro.

Expression Patterns of DREB2C

The effects of exogenous ABA and various stress conditions on DREB2C expression were investigated by reverse transcription(RT)-PCR analysis.As shown

DREB2C Interacts with ABF2

in Figure 2A,ABA had little effect on DREB2C expres-sion.However,a relatively high level of DREB2C RNA was detected after salt treatment.Lower level induction was observed after mannitol and cold treatments.

To investigate the expression pattern of DREB2C in more detail,we generated transgenic plants harboring

a promoter-GUS fusion construct and investigated its promoter activity by histochemical GUS staining.Fig-ure 2B shows that the DREB2C promoter was active in mature embryo and the cotyledons of germinating seedlings.As the seedlings grew,the promoter activity gradually disappeared under normal growth condi-tion.DREB2C promoter activity was induced,how-ever,by treating seedlings with NaCl or mannitol,which is consistent with the results of our RT-PCR analysis (Fig.2A).

We also determined the subcellular localization of DREB2C by Agrobacterium tumefaciens in?ltration of tobacco (Nicotiana benthamiana )leaves.The coding re-gion of DREB2C was fused to EYFP ,and the construct was employed to transform Agrobacterium (C58C1).The Agrobacterium was then coin?ltrated with Agro-bacterium containing p19into tobacco leaves (Voinnet et al.,2003;Witte et al.,2004).Observation of the epidermal cells of the in?ltrated leaves revealed that yellow ?uorescent protein (YFP)signals were local-ized in the nuclei (Fig.2D).

DNA-Binding and Transcriptional Activities of DREB2C

DREB2C belongs to the same subfamily of AP2/ERF proteins as DREB2A,which is one of the DRE-binding factors (Sakuma et al.,2002;Nakano et al.,2006).Therefore,we examined whether DREB2C can bind DRE.Recombinant DREB2C protein was pre-pared as a fusion to the GST,and its DNA-binding activity was determined by electrophoretic mobility shift assay.Figure 3A (lane 2)shows that a shifted band was observed with an oligonucleotide probe that contained a DRE core (https://www.wendangku.net/doc/cb16444214.html,GAC).In contrast,no shifted band was detected when an oligonucleotide with a mutated DRE core was employed as a probe (lane 4).In the same assay,a shifted band was observed with a Cor15a promoter fragment contain-ing DRE sequence (lane 6)but not with the same promoter sequence containing a mutated DRE (lane 8).Thus,DREB2C could speci?cally bind DRE-containing sequences.

The transcriptional activity of DREB2C was deter-mined employing a yeast assay system.Full-length or partial DREB2C fragments were fused to the GAL4DNA-binding domain,and the fusion constructs were introduced into a yeast strain (SFY526)harboring a lacZ reporter gene,which had GAL4-binding sites in its promoter.Transcriptional activity was then deter-mined by measuring the b -galactosidase activity.With the full-length DREB2C,reporter activity much higher than the control activity observed with the GAL4-binding domain alone was detected (Fig.3B).Even higher reporter activity was observed with the C-terminal fragment (DREB2C-C).On the other hand,no reporter activity was observed with the AP2do-main or with the N-terminal fragment (DREB2C-N).Thus,our results showed that DREB2C possesses transcriptional activity and that the activity resides within the C-terminal

portion.

Figure 1.Isolation of an ABF2-interacting protein gene by yeast two-hybrid screening.A,Speci?city of interaction.Interaction between a positive clone (clone 27)isolated from the initial screen and ABF2or nuclear lamin is shown.Reporter yeast containing the bait construct (ABF2or lamin)was transformed with the positive clone,and the transformants were grown on SC-His-Leu-Trp-Ura (SC-HLWU;left)or Gal/raf?nose-His-Leu-Trp-Ura (Gal-HLWU;right)medium.The bottom panels show the results of ?lter lift b -galactosidase assays of the transformants shown in the top panels.B,Deduced amino acid sequence of DREB2C.The ?rst 52amino acids missing in clone 27are indicated by italicized characters.The schematic diagram at the bottom depicts the overall structure of DREB2C.C,In vitro interaction between ABF2and DREB2C was investigated by GST pulldown assay.In vitro-translated DREB2C,labeled with [35S]Met,was incubated with GST alone or with GST-ABF2fusion protein,the bound protein was eluted and electrophoresed on a polyacrylamide gel,and autoradiog-raphy was carried out to visualize the protein.The arrow indicates the position of DREB2C,and the numbers on the left indicate the positions of size markers.[See online article for color version of this ?gure.]

Lee et al.

Overexpression of DREB2C Affects ABA Sensitivity

To investigate the in planta function of DREB2C,we generated DREB2C overexpression (OX)lines.Trans-genic Arabidopsis plants expressing DREB2C under the control of a cauli?ower mosaic virus 35S promoter were prepared,and after preliminary analysis of nine homozygous lines (T3or T4),three representative lines with different DREB2C expression levels (Fig.4A)were analyzed in more detail.

The DREB2C OX lines germinated and grew nor-mally,although they displayed minor growth retarda-tion (data not shown).Because DREB2C interacts with ABF2,a regulator of ABA and stress responses,we asked if DREB2C overexpression would affect ABA sensitivity.When the seeds of the 35S-DREB2C plants with high DREB2C expression levels (AP220and AP227)were germinated in the presence of ABA,their germination rates were lower than those of untrans-formed plants at ABA concentrations greater than 0.5m M (Fig.4B).The ABA effect was more pronounced during postgermination growth.When seeds were germinated and grown in the continuous presence of ABA,seedling development was almost completely inhibited after radicle emergence at 0.5m M ABA (Fig.4C).With the AP218line,in which DREB2C expression level is relatively low,inhibition of shoot growth was observed in approximately 80%of the seedlings.At the same ABA concentration,only 20%of the wild-type seedlings were affected.Thus,35S-DREB2C plants were hypersensitive to ABA during germina-tion and seedling growth.

ABA sensitivity of 35S-ABF2plants is developmental stage dependent (Kim et al.,2004c).We asked,there-fore,whether 35S-DREB2C plants also exhibit develop-ment stage dependence in ABA response.35S-DREB2C transgenic seeds were ?rst germinated in ABA-free medium for 4d.Subsequently,seedlings were trans-ferred to medium containing ABA,and root elongation was measured 5d after the transfer.Figure 4D shows that primary roots of all three DREB2C transgenic lines grew faster than those of wild-type plants in the pres-ence of various concentrations of ABA.Thus,unlike young seedling shoot development,root elongation of 35S-DREB2C plants was partially insensitive to ABA,suggesting that ABA sensitivity of 35S-DREB2C plants is developmental stage dependent.

Overexpression of DREB2C Affects Stress Response

One of the major functions of ABA is the control of stomatal movement under water de?cit conditions.To address if DREB2C is involved in the process,we determined the transpiration rates of 35S-DREB2C plants by measuring the rates of weight loss of de-tached leaves.As shown in Figure 5A,relative fresh weight of the transgenic leaves was consistently lower than that of the wild-type leaves,indicating that their transpiration rates are higher.Consistent with this result,survival rates (i.e.approximately 40%)of soil-grown plants under water de?cit conditions were lower than the wild-type rate (i.e.66%;Fig.5B).

Both ABF and CBF/DREB family transcription fac-tors control the abiotic stress response.Hence,

we

Figure 2.Expression patterns of DREB2C.A,Induction patterns of DREB2C expression.ABA and stress induction patterns of DREB2C were determined by RT-PCR.RNA was isolated from seedlings treated with 100m M ABA (4h),250m M NaCl (4h),or 600m M mannitol (4h)in quarter-strength Murashige and Skoog (MS)solution or with cold (24h at 4°C).Untreated indicates control plants without any treatments,and ?MS indicates treatment with quarter-strength MS solution without any supplements.B,DREB2C promoter activity was determined by histo-chemical GUS staining.a,Immature embryo.b,Mature embryo.c,One-day-old seedling.d,Two-day-old seedling.e,Fifteen-day-old seedling.f,Flower.g,Silique.C,High-osmolarity effects on DREB2C promoter activity.Ten-day-old seedlings were treated with 250m M NaCl or 600m M mannitol for 4h before GUS staining.GUS staining was for 12h in B and C.D,Subcellular localization of DREB2C.Bright-?eld,?uorescence (YFP),and merged images of tobacco leaves in?ltrated with Agrobacterium as described in “Materials and Methods”are shown.

DREB2C Interacts with ABF2

investigated whether DREB2C overexpression af-fected other abiotic stress tolerance.We ?rst examined the freezing tolerance of DREB2C OX lines by deter-mining their survival rates after exposing them to subfreezing temperature.When plants were exposed to 26°C for 24h,16%of the untransformed plants survived.At the same condition,the survival rates of AP218,AP227,and AP220were 36%,56%,and 73%,respectively (Fig.5C).Next,we investigated the heat tolerance of the plants by determining their survival rates under high-temperature conditions.Figure 5D shows that both untransformed plants and DREB2C OX lines survived at 43°C and 44°C.At 45°C,however,DREB2C OX lines exhibited much higher survival rates;whereas 37%of the wild-type plants survived the temperature,the survival rates of AP218,AP227,and AP220were 76%,96%,and 96%,respectively.Thus,DREB2C OX lines were freezing and heat toler-ant.We also investigated cold tolerance by growing plants at low,nonfreezing temperature (i.e.4°C),but we did not observe differences in growth between wild-type and DREB2C OX lines.

Overexpression of DREB2C Affects the Expression of Stress-Responsive Genes

The altered ABA and abiotic stress responses sug-gested that DREB2C is likely to have a regulatory role in vivo.To test this possibility,we determined the expression levels of a number of stress-responsive,DRE/CRT-containing genes (Jaglo-Ottosen et al.,1998)in 35S-DREB2C lines.Figure 6shows that the RNA level of COR15a gene,to whose promoter DREB2C binds (Fig.3B),was elevated in the transgenic lines compared with the wild-type level.Similarly,RD29B (LTI65)gene expression was elevated in the transgenic plants.On the other hand,COR6.6gene expression was reduced slightly in 35S-DREB2C plants,and the expression of COR47,COR78/RD29A ,and RAB18was not affected.Thus,DREB2C overexpression affected the expression of a subset of CBF/DREB-regulated genes.

DREB Family Proteins Interact with ABF Family Proteins

As mentioned earlier,DREB2C belongs to the same subfamily of AP2/ERF proteins as DREB2.Narusaka et al.(2003)previously demonstrated that DRE functions as a coupling element to ABRE to induce ABA-dependent gene expression.Therefore,we wanted to know whether ABF2and other ABFs would interact with DREB family factors,especially with DREB1A and DREB2A,whose functions have been characterized in depth (Liu et al.,1998;Gilmour et al.,2000;Sakuma et al.,2006a,2006b).We ?rst carried out a yeast two-hybrid assay to address the question.Bait constructs containing partial frag-ments of ABF2,ABF3,or ABF4were prepared,and their interactions with DREB2C,DREB2A,and DREB1A were determined.The results showed

that

Figure 3.DNA-binding and transcriptional activities of DREB2C.A,DNA-binding activity of DREB2C was determined by electrophoretic mobility shift assay.Oligonucleotides containing DRE,mutant DRE (mDRE),Cor15a promoter fragment,or mutant Cor15a fragment were employed as probes.The wild-type and mutant DRE/CRT core se-quences in probe DNA are indicated by boldface.2,Probe without recombinant DREB2C;+,probe with recombinant DREB2C.B,Tran-scriptional activity of DREB2C.Transcriptional activity of DREB2C was determined employing a yeast assay system,as described in “Materials and Methods.”The various portions of DREB2C used in the assay are shown schematically at the bottom.GBT9,Empty vector without any insert;DREB2C-Full,full-length DREB2C;DREB2C-N,N-terminal por-tion of DREB2C;DREB2C-AP2,AP2region of DREB2C;DREB2C-C,C-terminal portion of DREB2C.The numbers in parentheses indicate amino acid positions.The values represent b -galactosidase activity in Miller units,and the error bars denote SE (n =5each).Lee et al.

DREB2C interacted with ABF3and ABF4as well as ABF2(Fig.7A,top).On the other hand,DERB2A interacted with ABF2and ABF4but not with ABF3(Fig.7A,middle).DREB1A displayed the same inter-action speci?city as DREB2A (i.e.it interacted with ABF2and ABF4but not with ABF3;Fig.7A,bottom).To con?rm the result of the two-hybrid assay,ABF2interaction with DREB1A and DREB2A was examined by GST pulldown assay.Recombinant GST-ABF2fu-sion protein was prepared and allowed to bind the in vitro-translated DREB proteins labeled with [35S]Met.Bound proteins were then visualized by autoradiog-raphy after PAGE.Figure 7B shows that DREB1A bound the GST-ABF2fusion protein but not GST alone.Likewise,DREB2A was retained by GST-ABF2but it

did not bind GST alone (Fig.7B).Thus,our in vitro binding assay also showed that ABF2interacts with DREB1A and DREB2A.

DISCUSSION

The ABF subfamily of bZIP factors consists of four members,ABF1to ABF4(Choi et al.,2000).Analyses of their overexpression and knockout lines showed that ABF2,ABF3,and ABF4play overlapping but distinct roles in ABA and abiotic stress responses (Kim,2006).In particular,ABF2plays an essential role in seedling growth regulation and Glc response,and its overexpression results in altered

ABA/stress

Figure 4.ABA sensitivity of DREB2C overexpression lines.A,RNA gel-blot analysis of DREB2C expression in transgenic lines.The bottom panel shows an ethidium bromide-stained gel.B,ABA dose response during germination.Germination (full emer-gence of radical)was scored 5d after plating.Experiments were done in quadruplicate (n =30each),and the error bars indicate SE .C,ABA dose response during seedling develop-ment.Seeds were germinated and grown on ABA-containing medium for 2weeks,and seedlings with green cotyledons and/or true leaves were counted.Experiments were carried out in quadruplicate (n =30each),and SE values are indicated by error bars.Representative plants are shown in the right panels.D,ABA dose re-sponse of root elongation.Seeds were germinated on ABA-free medium for 4d,the seedlings were transferred to medium containing various concentra-tions of ABA,and root elongation was measured 5d after the transfer.The data represent relative root elongation rates compared with those on ABA-free medium.Experiments were done in quadruplicate (n =12each),and the error bars indicate SE .Ler,Landsberg erecta .

DREB2C Interacts with ABF2

responses.ABF2is unique in that,unlike ABF3and ABF4,its overexpression phenotypes are developmen-tal stage dependent (Kim et al.,2004c).

Our results indicate that ABF2interacts with the AP2/ERF domain factor DREB2C.DREB2C is a mem-ber of the DREB2subfamily of AP2/ERF domain transcription factors,which consists of eight mem-bers (i.e.DREB2A–DREB2H)and ABI4(Finkelstein et al.,1998).Among the subfamily members,DREB2C exhibits highest sequence identity to DREB2H.The

sequence identity (75%),however,is limited to the AP2domain region,because DREB2H lacks the C-terminal half (i.e.it consists of only the AP2do-main).DREB2C does not show signi?cant homology to other DREB2members outside the AP2domain.Several AP2/ERF family proteins,which do not belong to the DREB2subfamily,have been reported that are involved in ABA response.ABI4(Finkelstein et al.,1998)is a positive regulator of ABA and sugar responses.On the other hand,ABR1(Pandey et

al.,

Figure 5.Transpiration rates and stress tol-erance of DREB2C overexpression lines.A,Transpiration rates of DREB2C overexpres-sion lines.Leaves were detached from 3-week-old plants (?fth to eighth leaves)and weighed at 20-min intervals.Relative leaf weights (%)compared with initial weights are shown.Each data point represents the mean of quadruplicate measurements (n =12each),and the error bars indicate SE .B,Drought tolerance of DREB2C overexpres-sion lines.Nine-day-old seedlings were withheld from water for 10d (top row)or 15d (bottom row)before photographs were taken.Survival rates were determined 3d after rewatering at the end of the test.The data represent means of three indepen-dent experiments (n =100each,P =0.087),and the error bars indicate SE .C,Freezing tolerance of DREB2C overexpression lines.Two-week-old plants were placed at 26°C for 24h without any pretreatment,returned to normal growth temperature,and grown for 5d.The data show mean survival rates of six independent measurements (n =13each).D,Heat tolerance of DREB2C over-expression lines.Two-week-old plants were placed at the indicated temperatures for 1h and grown at normal conditions for 4d before photographs were taken.The sur-vival rates are means of ?ve to 15indepen-dent measurements (n =13each),and the error bars indicate SE .In B to D,represen-tative plants are shown with treatment con-ditions and line numbers.In A and D,error bars are generally smaller than the symbols.Ler,Landsberg erecta .

Lee et al.

2005)and AtERF7(Song et al.,2005)are negative regulators.The maize AP2/ERF domain protein DBF1(Kizis and Pages,2002)is a DRE-binding factor that positively regulates ABA-responsive expression of RAB17.DREB2C is not homologous to any of these AP2/ERF family proteins.

Little is known about the functions of the DREB2family transcription factors except those of DREB2A (Sakuma et al.,2006a,2006b),which is involved in drought and heat responses.Others (Lim et al.,2007;Lee et al.,2009)previously showed that DREB2C OX lines are heat tolerant.They also observed DNA-binding and transcriptional activities of DREB2C and reported a number of DREB2C-regulated genes.Our results suggest that DREB2C is involved not only in heat response but also in other stress responses.Fur-thermore,we showed that DREB2C OX lines exhibit a number of phenotypes that suggest its regulatory role in ABA response.Germination of the DREB2C trans-genic seeds and young seedling growth are ABA hypersensitive (Fig.4,B and C).Postgermination root elongation,on the contrary,is ABA insensitive (Fig.4D).Transpiration rates of the transgenic plants are higher than the wild-type rate,and the plants are more susceptible to water-de?cit conditions (Fig.5,A and B).DREB2C overexpression also affected the expression of several ABA/stress-responsive genes and promoted not only thermotolerance but also freezing tolerance (Figs.5,C and D,and 6).Together,these observations strongly suggest that DREB2C may play a regulatory role in ABA and stress responses,

probably by regulating a subset of ABA/stress-responsive genes via DRE/CRT.

Narusaka et al.(2003)showed that DRE/CRT in the RD29A promoter can function as a coupling element to ABRE;whereas a single copy of ABRE cannot direct ABA-responsive reporter gene activation,

DRE/CRT

Figure 6.Expression of ABA-responsive genes in DREB2C overexpres-sion lines.RNA was isolated from normal (untreated)or ABA-treated (100m M for 4h)plants,and the expression of ABA and/or cold-responsive genes was determined by RNA gel-blot analysis.The bottom panel shows an ethidium bromide-stained gel.WT,Wild

type.

Figure 7.Interactions between ABF and DREB family members.A,Two-hybrid assays were carried out to investigate the interactions between ABF and DREB family members.DREB2C,DREB2A,and DREB1A indicate prey constructs carried on https://www.wendangku.net/doc/cb16444214.html,min,ABF2,ABF3,and ABF4denote bait constructs.Yeast containing each bait construct was transformed with individual prey constructs,transform-ants were grown on Gal/raf?nose-His-Leu-Trp-Ura medium,and col-ony lift b -galactosidase assays were performed.B,In vitro interactions between ABF2and DREBs.ABF2interaction with DREB1A or DREB2A was investigated by GST pulldown assy.In vitro-translated DREB1A or DREB2A,each labeled with [35S]Met,was allowed to bind GST or GST-ABF2fusion protein,and bound protein was visualized by autoradiog-raphy after SDS-PAGE.The numbers indicate protein size in kD.[See online article for color version of this ?gure.]

DREB2C Interacts with ABF2

and ABRE together can.Furthermore,they showed that coexpression of DRE-binding and ABRE-binding factors synergistically activates the RD29A promoter.

A similar observation was also made by Xue and Loveridge(2004),who reported that the barley AP2 domain protein HvDRF1cooperates with HvABI5in the activation of the ABA-inducible HVA1promoter. These results imply that ABFs/AREBs and DREBs may synergistically interact with each other in ABA-responsive gene expression.The nature of the interac-tion,however,was not yet known.

Our results suggest that the interaction between ABFs/AREBs and DREBs may involve physical inter-action.ABF2interacts not only with DREB2C(Fig.7) but also with DREB1A and DREB2A in vitro and in yeast.Moreover,ABF4also interacts with DREB1A and DREB2A.The DREB2subfamily(i.e.group IV/ A-2subfamily)of AP2/ERF proteins consists of nine members including ABI4,and DREB1A belongs to another subfamily consisting of six members(Sakuma et al.,2002;Nakano et al.,2006).ABFs/AREBs,on the other hand,are members of a bZIP subfamily consist-ing of nine members(Kim et al.,2002).At present,we do not know whether other members of these bZIP and AP2/ERF subfamilies also interact with each other.Our limited analysis of interactions presented in this paper,however,suggests that,if they do, speci?city would exist in their physical interactions. It remains to be determined what determines the putative interaction speci?city between the two fam-ilies of transcription factors.In addition,in vivo,their interactions would be further limited by their spatial and temporal expression patterns.

Although our data indicate that ABF2interacts with DREB2C and other DREB family members,we do not know the biological signi?cance of their interactions. In the case of ABF2and DREB2C,their expression patterns are similar in several respects.ABF2is nu-cleus localized and highly inducible by high salt and ABA(Kim et al.,2004c).Similarly,DREB2C is localized in the nucleus and inducible by high salinity and high osmolarity(Fig.2),implying that they may interact in vivo under these abiotic stress conditions.Considering the diverse expression patterns of ABF and DREB family members,it would be possible that different members of the two protein families may interact with each other,depending on tissue type and environ-mental conditions.

Also,the interaction mechanism and the outcome of their interactions need to be determined in the future. Both ABF2and DREB2C possess DNA-binding and transcriptional activities,and other members of the two protein families also possess DNA-binding and/ or transcriptional activities.It may be speculated that ABFs/AREBs and DREBs might cooperate in their binding to respective cognate cis-elements,or their physical association might somehow modulate their transcriptional activities.In the transient assay by Narusaka et al.(2003),coexpression of ABFs/AREBs and DREBs enhanced transcription of the RD29A promoter.In our case,overexpression of DREB2C resulted in phenotypes that are different from those of ABF2:shoot development of DREB2C OX lines was hypersensitive to ABA,whereas root elongation was insensitive to ABA.ABF2/AREB1activity is regulated by ABA-dependent phosphorylation(Furihata et al., 2006).Thus,part of the reason for the difference may be the requirement of posttranslational modi?cation for ABF2activity.Also,it may be speculated that equimolar amounts of ABF2and DREB2C need to be present for cooperative interaction,and excess presence of one of the proteins may have an inhibitory effect.

MATERIALS AND METHODS

Manipulation of DNA,RNA Isolation,and

Expression Analysis

Manipulation of DNA and RNA was according to the standard methods (Sambrook and Russell,2001).RNA isolation,RNA gel-blot analysis,and RT-PCR were carried out as described previously(Kang et al.,2002;Kim et al., 2004b).RNA samples used for RT-PCR analysis were treated with DNase I to remove possible contaminating DNA.The?rst-strand cDNA synthesis was carried out employing SuperScript III(Invitrogen)according to the supplier’s instruction,and ampli?cations were carried out within linear range.The primer set5#-AGGATTGTAGCGATGAATATGTTCTC-3#and5#-CCAGT-CAAACGAAATTTGAAATCTTATG-3#was used for the RT-PCR analysis of DREB2C.Probes for northern-blot analysis were prepared by PCR,using primer sets that span gene-speci?c regions whenever possible,and gel puri?ed.Primer sequences are available upon request.

Two-Hybrid Screen and Two-Hybrid Assay

Bait construction,two-hybrid screening,and the analysis of positive clones have been described in detail elsewhere(Kim et al.,2004b).A full-length DREB2C cDNA,which includes parts of5#and3#untranslated regions,was isolated from a cDNA plasmid library by PCR,using the primers 5#-GCTATCGTCTTTGCTACTACTACTAC-3#and5#-CTTGTTCATATGTCA-TATGTATTCACAAG-3#.The PCR product was cloned into the TOPO-TA cloning vector(Invitrogen),and the nucleotide sequence was con?rmed by sequencing the entire insert fragment.

Bait constructs for the two-hybrid assays shown in Figure7were prepared by cloning various portions of ABF coding regions into pGilda(Clontech).The ABF2fragment containing amino acids65to337was prepared by PCR and cloned into the Eco RI-Xho I sites of pGilda,whereas the PCR product of the ABF3fragment(amino acids274–373)was cloned into the Sma I-Xho I sites of the same vector.The ABF4fragment(amino acids89–352)was cloned into the Eco RI-Xho I sites of pGilda.Prey constructs were prepared using pYESTrp-2 (Invitrogen).For DREB2C,the original positive clone(no.27)was used in the assay.For DREB1A and DREB2A,entire coding regions were ampli?ed and cloned into the Bam HI-Not I and the Eco RI-Not I sites of pYESTrp-2,respec-tively.Primer sequences used in the bait and the prey constructs are available upon request.

GST Pulldown Assay

To prepare the GST-ABF2fusion construct,a partial ABF2coding region containing the N-terminal337amino acids but lacking the C-terminal79 amino acids(i.e.the bZIP region)was prepared by PCR using primers 5#-ATGGATGGTAGTATGAATTTGG-3#and5#-gagagctcgagCTCTA-CAACTTTCTCCACAGTG-3#and,after Xho I digestion,cloned into the Sma I-Xho I sites of pGEX6P-2(GE Healthcare).GST-ABF2fusion protein was prepared using a GST puri?cation kit(GE Healthcare)according to the supplier’s instructions.Brie?y,fresh culture(optical density at600nm=0.6)of BL21(DE3)cells transformed with the GST-ABF2fusion construct was in-duced with1m M isopropyl-b-D-thiogalactoside for2h at37°C to express the recombinant protein,the cells were lysed by sonication,and the cell debris were removed by centrifugation.The cleared lysate was loaded onto the

Lee et al.

glutathione-Sepharose4B column(GE Healthcare).The column was washed with13phosphate-buffered saline(PBS)buffer(140m M NaCl,2.7m M KCl,10 m M Na2HPO4,and1.8m M KH2PO4,pH7.4),and bound proteins were eluted with a glutathione elution buffer(50m M Tris-HCl,pH8.0,and10m M glutathione).

For in vitro translation,the open reading frame of DREB2C,DREB1A,or DREB2A was cloned into pGEM-T Easy vector(Promega).In vitro translation was performed using the TNT Quick coupled transcription/translation sys-tem(Promega)according to the manufacturer’s instructions in the presence of [35S]Met.

For the GST pulldown assay,in vitro-translated DREB2C,DREB1A,or DREB2A product was incubated with10m g of GST and glutathione-Sepharose 4B resin for1h at room temperature in13PBS buffer.After brief centrifugation, the supernatant was incubated with GST or GST-ABF2fusion protein(5m g)in the presence of glutathione-Sepharose4B resin for1h at room temperature with constant rotation.At the end of binding,resins were washed three times with 1mL of13PBS buffer to remove unbound proteins,and bound proteins were eluted with a glutathione elution buffer.Proteins were separated on12%SDS-polyacrylamide gels and visualized by autoradiography. Electrophoretic Mobility Shift Assay

To prepare recombinant DREB2C,the entire DREB2C coding region was ampli?ed using the primers5#-ATGCCGTCGGAGATTGTTGACAG-3#and 5#-ttttccttttgcggccgcAATCTTATGTAGATCCATGAACATC-3#and cloned into pGEX6P-2(GE Healthcare)after Not I digestion.The GST-DREB2C fusion construct was used to transform BL21cells,and GST-DREB2C fusion protein was prepared using a GST puri?cation kit(GE Healthcare)from the trans-formed cells according to the supplier’s instructions.

Electrophoretic mobility shift assay was carried out as described(Choi et al.,2000).Brie?y,2m g of GST-AP2fusion protein was incubated with each DNA probe for30min on ice in a binding buffer(25m M HEPES,pH7.6,10% glycerol,1m M MgCl2,1m M dithiothreitol,1m M EDTA,and50m M KCl) containing1m g of poly(dI-dC).Sequences of the forward strand of probe DNA(Stockinger et al.,1997;Liu et al.,1998),which were prepared by Klenow ?ll-in reaction in the presence of[32P]dATP,are as follows:DRE,aattAGATA-TACTACCGACATGAGTTCC;mDRE,aattAGATATACTACTTTTATGAGTTCC; COR15a,ttAATTTCATGGCCGACCTGCTTTTTT;and mCOR15a,ttAATTT-CATGGAATCACTGCTTTTTT.

Transcriptional Assay

Full-length or partial fragments of DREB2C were prepared by PCR using the following primer sets:5#-CATGCCGTCGGAGATTGTTGACAG-3#and 5#-AATCTTATGTAGATCCATGAA CATC-3#for the full-length construct; 5#-ATGCCGTCGGAGATTGTTGACAG-3#and5#-CAAATCCCGTTTTCAGGT-CCACC-3#for DREB2C-N;5#-TGACTATAGAGGAGTTAGACAGAG-3#and 5#-GAGCGATTTGTGATCTCGGGAAG-3#for DREB2C-AP2;and5#-TTCTT-CGACTGCTGCCACTGCC-3#and5#-AATCTTATGTAGATCCATGAACATC-3# for DREB2C-C.The PCR products were cloned into GBT9(Clontech)digested with Sma I.

The constructs were individually introduced into SFY526yeast strain by transformation,and the resulting transformants were kept on SC-Trp-Ura medium.For transcriptional assay,colonies from each transformant group were grown in a SC-Trp-Ura medium to A600of approximately1.The cultures were diluted?ve times with fresh medium and grown further for4h.A600was measured at the end of the growth period,and1.5-mL aliquots were processed according to the instructions for b-galactosidase assay provided by Clontech. For each construct,?ve colonies were assayed.b-Galactosidase activity was expressed in Miller units.

Plant Material,Preparation of Transgenic Plants,and Phenotype Analysis

To prepare DREB2C OX lines,the coding region of DREB2C was ampli?ed using the primers5#-ATGCCGTCGGAGATTGTTGACAG-3#and5#-AATCT-TATGTAGATCCATGAACATC-3#from the full-length DREB2C cDNA clone. The ampli?ed fragment was then cloned into pBI121(Jefferson et al.,1987), which was prepared by Xba I digestion followed by Klenow?ll-in reaction after removal of the GUS coding region.

To prepare the DREB2C promoter-GUS construct,the1.1-kb promoter fragment was ampli?ed using the primer set5#-acgcgtcgacGTTGTAGAT-TGAAAATGAGATAGCTC-3#and5#-GTAAGAAAGCACTCGAACAAAG-TAGC-3#.The ampli?ed fragment was then cloned into Sma I-Sal I sites of pBI101.2after digestion with Sal I.Arabidopsis(Arabidopsis thaliana)plants were transformed according to the vacuum in?ltration method(Bechtold and Pelletier,1998)using Agrobacterium tumefaciens strain GV3101.Nine homozy-gous overexpression lines were recovered,and,after preliminary analysis, three representative lines were analyzed in detail.Phenotype analysis,ABA response,and various stress tests were carried out as described(Kang et al., 2002;Kim et al.,2004a,2004c)using T4homozygous lines.For promoter analysis,four transgenic lines were generated,which displayed the same GUS staining pattern.GUS staining was conducted as described(Kang et al.,2002) with T2or T3generation transgenic plants.

Subcellular Localization

The coding region of DREB2C was prepared by PCR using the primer set 5#-aaggagctcATGCCGTCGGAGATTGTTGACA-3#and5#-TGTAGATCCAT-GAACATCTTTGTCTC-3#,and after Sac I digestion,the ampli?ed fragment was cloned into the Sac I-Sma I sites of p35S-FAST/EYFP in frame with the EYFP coding region.Tobacco(Nicotiana benthamiana)leaves were coin?ltrated with the Agrobacterium strains(C58C1)containing the DREB2C-EYFP con-struct and p19according to Witte et al.(2004).The images of the tobacco epidermal cells were obtained with a?uorescence microscope(Olympus BX51)30to40h after in?ltration.

ACKNOWLEDGMENT

We are grateful to the Kumho Life Science Laboratory of Chonnam National University for providing equipments and plant growth facilities. Received February9,2010;accepted April11,2010;published April15,2010.

LITERATURE CITED

Baker SS,Wilhelm KS,Thomashow MF(1994)The5#-region of Arabidop-sis thaliana cor15a has cis-acting elements that confer cold-,drought-and ABA-regulated gene expression.Plant Mol Biol24:701–713 Bechtold N,Pelletier G(1998)In planta Agrobacterium-mediated transfor-mation of adult Arabidopsis thaliana plants by vacuum in?ltration.

Methods Mol Biol82:259–266

Bohnert HJ,Nelson DE,Jensen RG(1995)Adaptations to environmental stresses.Plant Cell7:1099–1111

Busk PK,Pages M(1998)Regulation of abscisic acid-induced transcription.

Plant Mol Biol37:425–435

Chae MJ,Lee JS,Nam MH,Cho K,Hong JY,Yi SA,Suh SC,Yoon IS(2007)

A rice dehydration-inducible SNF1-related protein kinase2phosphor-

ylates an abscisic acid responsive element-binding factor and associates with ABA signaling.Plant Mol Biol63:151–169

Chinnusamy V,Ohta M,Kanrar S,Lee BH,Hong X,Agarwal M,Zhu JK (2003)ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes Dev17:1043–1054

Choi H,Hong JH,Kang J,Kim SY(2000)ABFs,a family of ABA-responsive element binding factors.J Biol Chem21:1723–1730

Choi H,Park HJ,Park JH,Kim S,Im MY,Seo HH,Kim YW,Hwang I,Kim SY(2005)Arabidopsis calcium-dependent protein kinase AtCPK32in-teracts with ABF4,a transcriptional regulator of ABA-responsive gene expression,and modulates its activity.Plant Physiol139:1750–1761 Finkelstein R,Gampala SS,Lynch TJ,Thomas TL,Rock CD(2005) Redundant and distinct functions of the ABA response loci ABA-INSENSITIVE(ABI)5and ABRE-BINDING FACTOR(ABF)3.Plant Mol Biol59:253–267

Finkelstein RR,Gampala SS,Rock CD(2002)Abscisic acid signaling in seeds and seedlings.Plant Cell(Suppl)14:S15–S45

Finkelstein RR,Lynch TJ(2000)The Arabidopsis abscisic acid response gene ABI5encodes a basic leucine zipper transcription factor.Plant Cell 12:599–609

Finkelstein RR,Wang ML,Lynch TJ,Rao S,Goodman HM(1998)The Arabidopsis abscisic acid response locus ABI4encodes an APETALA2 domain protein.Plant Cell10:1043–1054

Fujita Y,Fujita M,Satoh R,Maruyama K,Parvez MM,Seki M,Hiratsu K, Ohme-Takagi M,Shinozaki K,Yamaguchi-Shinozaki K(2005)AREB1

DREB2C Interacts with ABF2

is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis.Plant Cell17: 3470–3488

Furihata T,Maruyama K,Fujita Y,Umezawa T,Yoshida R,Shinozaki K, Yamaguchi-Shinozaki K(2006)Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.Proc Natl Acad Sci USA103:1988–1993

Gilmour SJ,Sebolt AM,Salazar MP,Everard JD,Thomashow MF(2000) Overexpression of the Arabidopsis CBF3transcriptional activator mimics multiple biochemical changes associated with cold acclimation.

Plant Physiol124:1854–1865

Gomez-Porras JL,Riano-Pachon DM,Dreyer I,Mayer JE,Mueller-Roeber B(2007)Genome-wide analysis of ABA-responsive elements ABRE and CE3reveals divergent patterns in Arabidopsis and rice.BMC Genomics8:260

Haake V,Cook D,Riechmann JL,Pineda O,Thomashow MF,Zhang JZ (2002)Transcription factor CBF4is a regulator of drought adaptation in Arabidopsis.Plant Physiol130:639–648

Hattori T,Totsuka M,Hobo T,Kagaya Y,Yamamoto-Toyoda A(2002) Experimentally determined sequence requirement of ACGT-containing abscisic acid response element.Plant Cell Physiol43:136–140

Hobo T,Asada M,Kowyama Y,Hattori T(1999a)ACGT-containing abscisic acid response element(ABRE)and coupling element3(CE3) are functionally equivalent.Plant J19:679–689

Hobo T,Kowyama Y,Hattori T(1999b)A bZIP factor,TRAB1,interacts with VP1and mediates abscisic acid-induced transcription.Proc Natl Acad Sci USA96:15348–15353

Jaglo-Ottosen KR,Gilmour SJ,Zarka DG,Schabenberger O,Thomashow MF(1998)Arabidopsis CBF1overexpression induces cor genes and enhances freezing tolerance.Science280:104–106

Jefferson RA,Kavanagh TA,Bevan MW(1987)GUS fusions:b-glucuron-idase as a sensitive and versatile gene fusion marker in higher plants.

EMBO J20:3901–3907

Kang J,Choi H,Im M,Kim SY(2002)Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling.Plant Cell14:343–357

Kim JB,Kang JY,Kim SY(2004a)Over-expression of a transcription factor regulating ABA-responsive gene expression confers multiple stress tolerance.Plant Biotechnol J2:459–466

Kim S,Choi HI,Ryu HJ,Park JH,Kim MD,Kim SY(2004b)ARIA,an Arabidopsis arm repeat protein interacting with a transcriptional reg-ulator of abscisic acid-responsive gene expression,is a novel abscisic acid signaling component.Plant Physiol136:3639–3648

Kim S,Kang JY,Cho DI,Park JH,Kim SY(2004c)ABF2,an ABRE-binding bZIP factor,is an essential component of glucose signaling and its overexpression affects multiple stress tolerance.Plant J40:75–87

Kim SY(2006)The role of ABF family bZIP class transcription factors in stress response.Physiol Plant126:519–527

Kim SY,Chung HJ,Thomas TL(1997)Isolation of a novel class of bZIP transcription factors that interact with ABA-responsive and embryo-speci?cation elements in the Dc3promoter using a modi?ed yeast one-hybrid system.Plant J11:1237–1251

Kim SY,Thomas TL(1998)A family of novel basic leucine zipper proteins bind to seed-speci?cation elements in the carrot Dc3gene promoter.

J Plant Physiol152:607–613

Kim SY,Ma J,Perret P,Li Z,Thomas TL(2002)Arabidopsis ABI5 subfamily members have distinct DNA-binding and transcriptional activities.Plant Physiol130:688–697

Kizis D,Pages M(2002)Maize DRE-binding proteins DBF1and DBF2are involved in rab17regulation through the drought-responsive element in an ABA-dependent pathway.Plant J30:679–689

Knight H,Zarka DG,Okamoto H,Thomashow MF,Knight MR(2004) Abscisic acid induces CBF gene transcription and subsequent induction of cold-regulated genes via the CRT promoter element.Plant Physiol 135:1710–1717

Kreps JA,Wu Y,Chang HS,Zhu T,Wang X,Harper JF(2002)Tran-scriptome changes for Arabidopsis in response to salt,osmotic,and cold stress.Plant Physiol130:2129–2141

Lee K,Han KS,Kwon YS,Lee JH,Kim SH,Chung WS,Kim Y,Chun SS, Kim HK,Bae DW(2009)Identi?cation of potential DREB2C targets in Arabidopsis thaliana plants overexpressing DREB2C using proteomic analysis.Mol Cells28:383–388

Lim CJ,Hwang JE,Chen H,Hong JK,Yang KA,Choi MS,Lee KO,Chung

WS,Lee SY,Lim CO(2007)Over-expression of the Arabidopsis DRE/ CRT-binding transcription factor DREB2C enhances thermotolerance.

Biochem Biophys Res Commun362:431–436

Liu Q,Kasuga M,Sakuma Y,Abe H,Miura S,Yamaguchi-Shinozaki K, Shinozaki K(1998)Two transcription factors,DREB1and DREB2, with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis.Plant Cell10: 1391–1406

Lopez-Molina L,Chua NH(2000)A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana.Plant Cell Physiol41: 541–547

Menkens AE,Schindler U,Cashmore AR(1995)The G-box:a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins.Trends Biochem Sci20:506–512

Nakano T,Suzuki K,Fujimura T,Shinshi H(2006)Genome-wide analysis of the ERF gene family in Arabidopsis and rice.Plant Physiol140: 411–432

Narusaka Y,Nakashima K,Shinwari ZK,Sakuma Y,Furihata T,Abe H, Narusaka M,Shinozaki K,Yamaguchi-Shinozaki K(2003)Interaction between two cis-acting elements,ABRE and DRE,in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses.Plant J34:137–148

Pandey GK,Grant JJ,Cheong YH,Kim BG,Li L,Luan S(2005)ABR1,an APETALA2-domain transcription factor that functions as a repressor of ABA response in Arabidopsis.Plant Physiol139:1185–1193 Ramanjulu S,Bartels D(2002)Drought-and desiccation-induced modu-lation of gene expression in plants.Plant Cell Environ25:141–151 Sakuma Y,Liu Q,Dubouzet JG,Abe H,Shinozaki K,Yamaguchi-Shinozaki K(2002)DNA-binding speci?city of the ERF/AP2domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression.Biochem Biophys Res Commun 290:998–1009

Sakuma Y,Maruyama K,Osakabe Y,Qin F,Seki M,Shinozaki K, Yamaguchi-Shinozaki K(2006a)Functional analysis of an Arabidopsis transcription factor,DREB2A,involved in drought-responsive gene expression.Plant Cell18:1292–1309

Sakuma Y,Maruyama K,Qin F,Osakabe Y,Shinozaki K,Yamaguchi-Shinozaki K(2006b)Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress-responsive gene expression.Proc Natl Acad Sci USA103:18822–18827 Sambrook J,Russell DW(2001)Molecular Cloning:A Laboratory Manual, Ed3.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY Shen Q,Ho TH(1995)Functional dissection of an abscisic acid(ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element.Plant Cell7: 295–307

Shen Q,Zhang P,Ho TH(1996)Modular nature of abscisic acid(ABA) response complexes:composite promoter units that are necessary and suf?cient for ABA induction of gene expression in barley.Plant Cell8: 1107–1119

Shen QJ,Casaretto JA,Zhang P,Ho TH(2004)Functional de?nition of ABA-response complexes:the promoter units necessary and suf?cient for ABA induction of gene expression in barley(Hordeum vulgare L.).

Plant Mol Biol54:111–124

Song CP,Agarwal M,Ohta M,Guo Y,Halfter U,Wang P,Zhu JK(2005) Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses.Plant Cell17:2384–2396 Stockinger EJ,Gilmour SJ,Thomashow MF(1997)Arabidopsis thaliana CBF1encodes an AP2domain-containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water de?cit.Proc Natl Acad Sci USA94:1035–1040

Takahashi S,Seki M,Ishida J,Satou M,Sakurai T,Narusaka M,Kamiya A,Nakajima M,Enju A,Akiyama K,et al(2004)Monitoring the expression pro?les of genes induced by hyperosmotic,high salinity,and oxidative stress and abscisic acid treatment in Arabidopsis cell culture using a full-length cDNA microarray.Plant Mol Biol56:29–55

Uno Y,Furihata T,Abe H,Yoshida R,Shinozaki K,Yamaguchi-Shinozaki K(2000)Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity.Proc Natl Acad Sci USA97: 11632–11637

Lee et al.

Voinnet O,Rivas S,Mestre P,Baulcombe D(2003)An enhanced transient expression system in plants based on suppression of gene silencing by the p19protein of tomato bushy stunt virus.Plant J33:949–956

Witte CP,Noe¨l LD,Gielbert J,Parker JE,Romeis T(2004)Rapid one-step protein puri?cation from plant material using the eight-amino acid StrepII epitope.Plant Mol Biol55:135–147

Xiong L,Schumaker KS,Zhu JK(2002)Cell signaling during cold, drought,and salt stress.Plant Cell(Suppl)14:S165–S183

Xue GP,Loveridge CW(2004)HvDRF1is involved in abscisic acid-

mediated gene regulation in barley and produces two forms of AP2 transcriptional activators,interacting preferably with a CT-rich element.

Plant J37:326–339

Yamaguchi-Shinozaki K,Shinozaki K(1994)A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought,low-temperature,or high-salinity stress.Plant Cell6:251–264 Yamaguchi-Shinozaki K,Shinozaki K(2005)Organization of cis-acting regulatory elements in osmotic-and cold-stress-responsive promoters.

Trends Plant Sci10:88–94

DREB2C Interacts with ABF2

with的复合结构和独立主格结构

1. with+宾语+形容词。比如：. The boy wore a shirt with the neck open, showing his bare chest. 那男孩儿穿着一件衬衫，颈部敞开，露出光光的胸膛。Don’t talk with your mouth full. 嘴里有食物时不要讲话。 2. with+宾语+副词。比如：She followed the guide with her head down. 她低着头，跟在导游之后。 What a lonely world it will be with you away. 你不在，多没劲儿呀！ 3. with+宾语+过去分词。比如：He was listening to the music with his eyes half closed. 他眼睛半闭着听音乐。She sat with her head bent. 她低着头坐着。 4. with+宾语+现在分词。比如：With winter coming, it’s time to buy warm clothes. 冬天到了，该买些保暖的衣服了。 He soon fell asleep with the light still burning. 他很快就睡着了，（可）灯还亮着。 5. with+宾语+介词短语。比如：He was asleep with his head on his arms. 他的头枕在臂膀上睡着了。 The young lady came in, with her two- year-old son in her arms. 那位年轻的女士进来了，怀里抱着两岁的孩子。 6. with+宾语+动词不定式。比如： With nothing to do in the afternoon, I went to see a film. 下午无事可做，我就去看了场电影。Sorry, I can’t go out with all these dishes to wash. 很抱歉，有这么多盘子要洗，我不能出去。 7. with+宾语+名词。比如： He died with his daughter yet a school-girl.他去逝时，女儿还是个小学生。 He lived a luxurious life, with his old father a beggar . 他过着奢侈的生活，而他的老父亲却沿街乞讨。(8)With so much work to do ,I can't go swimming with you. (9)She stood at the door,with her back towards us. (10)He entered the room,with his nose red with cold. with复合结构与分词做状语有啥区别 [ 标签：with, 复合结构, 分词状语] Ciro Ferrara 2009-10-18 16:17 主要是分词形式与主语的关系 满意答案好评率：100%

With的用法全解

With的用法全解 with结构是许多英语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 一、 with结构的构成 它是由介词with或without+复合结构构成，复合结构作介词with或without的复合宾语，复合宾语中第一部分宾语由名词或代词充当，第二部分补足语由形容词、副词、介词短语、动词不定式或分词充当，分词可以是现在分词，也可以是过去分词。With结构构成方式如下： 1. with或without-名词/代词+形容词； 2. with或without-名词/代词+副词； 3. with或without-名词/代词+介词短语； 4. with或without-名词/代词 +动词不定式； 5. with或without-名词/代词 +分词。 下面分别举例： 1、 She came into the room，with her nose red because of cold.（with+名词+形容词，作伴随状语）

2、 With the meal over ， we all went home.（with+名词+副词，作时间状语） 3、The master was walking up and down with the ruler under his arm。（with+名词+介词短语，作伴随状语。） The teacher entered the classroom with a book in his hand. 4、He lay in the dark empty house，with not a man ，woman or child to say he was kind to me.（with+名词+不定式，作伴随状语）He could not finish it without me to help him.（without+代词 +不定式，作条件状语） 5、She fell asleep with the light burning.（with+名词+现在分词，作伴随状语） Without anything left in the with结构是许多英 语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 二、with结构的用法 with是介词，其意义颇多，一时难掌握。为帮助大家理清头绪，以教材中的句子为例，进行分类，并配以简单的解释。在句子中with结构多数充当状语，表示行为方式，伴随情况、时间、原因或条件（详见上述例句）。 1.带着，牵着…… (表动作特征)。如： Run with the kite like this.

转录因子ERF家族

Arabidopsis thaliana ERF Family l ERF Family Introduction l Download Sequences l Multiple Sequences Alignment l Phylogenetic Tree Plant Transcription Factor Database v3.0 Center for Bioinformatics , Peking University , China Previous versions:v1.0v2.0Home | Blast | Search | Download | Prediction | Help | About | Links LFY) Species TF ID Description AT1G01250.1 Integrase-type DNA-binding superfamily protein AT1G03800.1ERF domain protein 10AT1G04370.1 Ethylene-responsive element binding factor 14AT1G06160.1 octadecanoid-responsive Arabidopsis AP2/ERF 59AT1G12610.1 Integrase-type DNA-binding superfamily protein AT1G12630.1 Integrase-type DNA-binding superfamily protein AT1G12890.1 Integrase-type DNA-binding superfamily protein AT1G12980.1 Integrase-type DNA-binding superfamily protein AT1G15360.1 Integrase-type DNA-binding superfamily protein AT1G19210.1 Integrase-type DNA-binding superfamily protein AT1G21910.1 Integrase-type DNA-binding superfamily protein AT1G22190.1 Integrase-type DNA-binding superfamily protein AT1G22810.1 Integrase-type DNA-binding superfamily protein AT1G22985.1 Integrase-type DNA-binding superfamily protein AT1G24590.1 DORNROSCHEN-like AT1G25470.1 AP2 domain-containing transcription factor family protein AT1G25470.2 AP2 domain-containing transcription factor family protein AT1G28160.1 Integrase-type DNA-binding superfamily protein AT1G28360.1 ERF domain protein 12AT1G28370.1 ERF domain protein 11AT1G33760.1 Integrase-type DNA-binding superfamily protein AT1G36060.1 Integrase-type DNA-binding superfamily protein AT1G43160.1 related to AP2 6AT1G44830.1 Integrase-type DNA-binding superfamily protein AT1G46768.1 related to AP2 1AT1G49120.1 Integrase-type DNA-binding superfamily protein AT1G50640.1 ethylene responsive element binding factor 3AT1G53170.1 ethylene response factor 8AT1G53910.1 related to AP2 12AT1G53910.2 related to AP2 12AT1G53910.3 related to AP2 12AT1G63030.1 Integrase-type DNA-binding superfamily protein AT1G63030.2 Integrase-type DNA-binding superfamily protein AT1G64380.1 Integrase-type DNA-binding superfamily protein AT1G68550.1 Integrase-type DNA-binding superfamily protein AT1G68550.2 Integrase-type DNA-binding superfamily protein AT1G71130.1 Integrase-type DNA-binding superfamily protein AT1G71450.1 Integrase-type DNA-binding superfamily protein AT1G71520.1 Integrase-type DNA-binding superfamily protein AT1G72360.1 Integrase-type DNA-binding superfamily protein AT1G72360.2 Integrase-type DNA-binding superfamily protein AT1G72360.3 Integrase-type DNA-binding superfamily protein AT1G74930.1 Integrase-type DNA-binding superfamily protein AT1G75490.1 Integrase-type DNA-binding superfamily protein AT1G77200.1Integrase-type DNA-binding superfamily protein

伴随状语的用法

伴随状语的用法 WITH作伴随状语,或说明造成某一局面的原因.至于是用现在分词,还是过去分词得看与主句主句的关系是被动或主动. Fg: with time passing by随着时间的流逝He sat on the chair with his eyes closed.他坐在椅子上,眼睛闭着. 总结一下： with的复合结构可以分以下几种: 一. w ith + 名词 + 形容词.表处于一种状态. He often sleeps with windows open.他经常开着窗户睡觉. 二.with +名词 + 副词. There is a temple with no table in. 三.with +名词 + 介词短语. The teacher came into the classroom with a book under his arm. 四.with + 名词 + 现在分词.这种结构表动作的主动和进行态. The old woman left the her house with water running all the time. 五.with + 名词 + 过去分词,这种结构表被动和完成. The thief was taken to the police station with his hands tied to his back. 六.with + 名词 + 不定式,这种结构表动作的将来式. The mamager has been busy these days with a lot of work to do . 伴随状语是指状语从句的动作伴随主句发生，它的特点是：它所表达的动作或状态是伴随着句子谓语动词的动作而发生或存在的。 例如： ①He sat in the armchair，reading a newspaper. 他坐在扶手椅里读报。 ②All night long he lay awake，thinking of the problem. 他整夜躺在床上睡不着，思考着那个问题。 伴随状语的判断 The dog entered the room, following his master.一般在分词短语（现在分词表主动过去表被动）前有逗号的是伴随状语。

with的用法大全

with的用法大全----四级专项训练with结构是许多英语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 一、 with结构的构成 它是由介词with或without+复合结构构成，复合结构作介词with或without的复合宾语，复合宾语中第一部分宾语由名词或代词充当，第二部分补足语由形容词、副词、介词短语、动词不定式或分词充当，分词可以是现在分词，也可以是过去分词。With结构构成方式如下： 1. with或without-名词/代词+形容词; 2. with或without-名词/代词+副词; 3. with或without-名词/代词+介词短语; 4. with或without-名词/代词+动词不定式; 5. with或without-名词/代词+分词。 下面分别举例：

1、 She came into the room，with her nose red because of cold.(with+名词+形容词，作伴随状语) 2、 With the meal over ， we all went home.(with+名词+副词，作时间状语) 3、The master was walking up and down with the ruler under his arm。(with+名词+介词短语，作伴随状语。) The teacher entered the classroom with a book in his hand. 4、He lay in the dark empty house，with not a man ，woman or child to say he was kind to me.(with+名词+不定式，作伴随状语) He could not finish it without me to help him.(without+代词 +不定式，作条件状语) 5、She fell asleep with the light burning.(with+名词+现在分词，作伴随状语) 6、Without anything left in the cupboard， she went out to get something to eat.(without+代词+过去分词，作为原因状语) 二、with结构的用法 在句子中with结构多数充当状语，表示行为方式，伴随情况、时间、原因或条件(详见上述例句)。

bHLH转录因子家族研究进展

HEREDITAS (Beijing) 2008年7月, 30(7): 821―830 ISSN 0253-9772 https://www.wendangku.net/doc/cb16444214.html, 综 述 收稿日期: 2007?12?04; 修回日期: 2008?02?15 基金项目:国家自然科学基金项目(编号: 30370773)资助[Supported by the National Natural Science Foundation of China (No. 30370773)] 作者简介:王勇(1965?), 男, 浙江人, 副研究员, 博士研究生, 研究方向: 昆虫生物信息学。E-mail: ywang@https://www.wendangku.net/doc/cb16444214.html, 姚勤(1961?), 女, 安徽人, 研究员, 研究方向: 昆虫病毒分子生物学。E-mail: yaoqin@https://www.wendangku.net/doc/cb16444214.html, 王勇、姚勤同为第一作者。 通讯作者:陈克平(1962?), 男, 安徽人, 博士, 研究员, 博士生导师, 研究方向: 昆虫分子生物学、昆虫生物信息学。E-mail: kpchen@https://www.wendangku.net/doc/cb16444214.html, DOI: 10.3724/SP.J.1005.2008.00821 bHLH 转录因子家族研究进展 王勇1, 陈克平2, 姚勤2 1 江苏大学食品与生物工程学院, 镇江 212013; 2 江苏大学生命科学研究院, 镇江 212013 摘要: bHLH 转录因子在真核生物生长发育调控中具有重要作用, 它们组成了转录因子的一个大家族。已经有20种生物基因组中bHLH 家族的成员得到鉴定, 其中动物17种、植物2种、酵母1种。动物bHLH 因其调控基因表达的功能不同而被分成45个家族; 此外, 根据它们所作用DNA 元件和自身结构特点又被分成6个组。A 组包含22个家族, 主要调控神经细胞生成、肌细胞生成和中胚层形成; B 组包含12个家族, 主要调控细胞增殖与分化、固醇代谢与脂肪细胞形成以及葡萄糖响应基因的表达; C 组包含7个家族, 主要负责调控中线与气管发育和昼夜节律、激活环境毒素响应基因的转录; D 组只有1个家族, 它与A 组bHLH 蛋白形成无活性的异源二聚体; E 组有2个家族, 调控胚胎分节、体节形成与器官发生等; F 组也只有1个家族, 调控头部发育、嗅觉神经元生成等。文章综述了bHLH 转录因子家族分类、起源、功能方面的研究进展情况。 关键词: bHLH; 转录因子; 家族 Progress of studies on bHLH transcription factor families WANG Yong 1, CHEN Ke-Ping 2, YAO Qin 2 1 School of Food and Biological Engineering , Jiangsu University , Zhenjiang 212013, China ; 2 Institute of Life Sciences , Jiangsu University , Zhenjiang 212013, China Abstract: bHLH transcription factors are important players in various developmental processes of eukaryotes. They consti-tute a large family of transcription factors. bHLH family members have been identified in genomes of 20 organisms inclu- ding 17 animals, two plants, and one yeast. Animal bHLHs are classified into 45 families based on their different functions in the regulation of gene expression. In addition, they are divided into 6 groups according to target DNA elements they bind and their own structural characteristics. Group A consists of 22 families. They mainly regulate neurogenesis, myogenesis and mesoderm formation. Group B consists of 12 families. They mainly regulate cell proliferation and differentiation, sterol metabolism and adipocyte formation, and expression of glucose-responsive genes. Group C has seven families. They are responsible for the regulation of midline and tracheal development, circadian rhythms, and for the activation of gene tran-scription in response to environmental toxins. Group D has only one family. It forms inactive heterodimers with group A bHLH proteins. Group E has two families, which regulate embryonic segmentation, somitogenesis and organogenesis etc. Group F also has one family. It regulates head development and formation of olfactory sensory neurons etc. This article presents a brief review on progress achieved in studies related to the classification, origination and functions of bHLH tran-scription factor families.

现在分词作状语的分类(伴随、让步、条件、时间等等)资料讲解

谓语动词: 有提示词, 句子缺谓语(与主语构成主谓结构) I. I _______ (tell) by my classmates about that. 2. My mother often __________ (stop) me from watching TV. 时态语态变化, 及主谓一致 非谓语动词(主动---doing , 被动---done, 目的/结果/将要to do ,) 1. We must also consider the reaction of the person __________ (receive) the gift. 2. My pupils, Tom__________ (include), liked her. 1. He entered, ________ (hold) a book in his hand. 2. He entered the room and _______ (hold) a book in his hand. 3. I politely refused her invitation and _____ (walk) away. 4. I politely refused her invitation, ______ (walk) away. 两个动词是同时发生的时候 主语+ 谓语1 + and / but + 谓语2 主语+ 谓语，+非谓语 1. When he _______ (come) in, I was reading a book. 2. Unless I ________ (invite), I won’t attend he party. 3. When _____ (hear) the news, I was excited. 4. Unless ____ (invite), I won’t attend he party. --- When / if / unless / /After/Before 等连词后没有主语+非谓语（--- ing /---ed ) , 主句---When / if / unless / /After /Before等连词+ 主语+ 谓语，主句 1. A boy ________( call ) Jack came here today 2. A boy who ________( call ) Jack came here today 3. We enjoy the movie _________ (direct) by a world famous artist. 4. We enjoy the movie which_________ (direct) by the world famous artist. 名词后没关系词时+ 非谓语, 非谓语动词修饰前面的名词做定语 名词后有关系词时+谓语，做定语从句中的谓语 1.“You can’t catch me!” Jan et shouted, _______ (run) away. 2.He said thanks and ____ (smile) a row of teeth. 3.When first ___________ (introduce) to the market, these products enjoyed great success. 4.When he_______( arrive ) at the corner , he met his friend. 5.________ ( sleep ) late, he turned off the alarm clock. 6.Don’t use words, expressions, or phrases _______(know) only to people with specific knowledge. [例1] I got on the bus and found a seat near the back, and then I noticed a man 18 (sit) at the front. （2011广东卷） [例2] He spit it out, __37___(say) it was awful. （2010广东卷） [例3]The fact that so many people still smoke in public places _______ that we may need a nationwide campaign to raise awareness of the risks of smoking. A. suggest B. suggests C. suggested D. suggesting turn

with 复合结构的常见形式及句法功能

with 复合结构的常见形式及句法功能 1with 复合结构的常见形式 1、with + 名词(或代词) + 现在分词 此时，现在分词和前面的名词或代词是逻辑上的主谓关系。 With prices going up so fast, we can't afford luxuries. 由于物价上涨很快，我们买不起高档商品。（原因状语） With the crowds cheering, they drove to the palace. 在人群的欢呼声中，他们驱车来到皇宫。（伴随状语） 2、with + 名词(或代词) + 过去分词 此时，过去分词和前面的名词或代词是逻辑上的动宾关系。 I sat in my room for a few minutes with my eyes fixed on the ceiling. 我在房间坐了一会儿，眼睛盯着天花板。（伴随状语） She had to walk home with her bike stolen. 自行车被偷，她只好步行回家。（原因状语） 3、with + 名词(或代词) + 形容词 I like to sleep with the windows open. 我喜欢把窗户开着睡觉。（伴随状语） With the weather so close and stuffy, ten to one it'll rain presently. 大气这样闷，十之八九要下雨。（原因状语） 4、with + 名词(或代词) + 介词短语 With the children at school, we can't take our vacation when we want to. 由于孩子们在上学，所以当我们想度假时而不能去度假。（原因状语）

转录因子

角朊细胞 角朊细胞的增殖和分化是一个受到精细调节的过程，并伴随着一系列形态学和生化改变，最终形成角质细胞，这就必然涉及到许多结构基因的同时活化与灭活，即基因表达的调控，而转录水平的调控尤为重要。现已发现许多转录因子如AP1、AP2、Sp1、POU结构域及C/EBP等可调节角朊细胞基因的表达。 目录

转录水平、翻译水平及翻译后水平，其中最常见的调控方式就是转录调控。现已发现AP1、AP2、NFκB、C/EBP、ets、Sp1及POU结构域等转录因子可作为表皮中的调控蛋白，从而调节编码套膜蛋白（involucrin, iNV）、转谷氨酰胺酶（transglutaminase，TG）、SPRR2A、兜甲蛋白（loricrin）、角蛋白及BPAG1等蛋白的基因的表达。本文就与角朊细胞基因表达有关的转录因子作一简要综述。 编辑本段转录因子的一般特征 转录因子（transcription factor）是能与位于转录起始位点上游50～5000bp的顺式作用元件（cis-acting elements）、沉默子（silencer）或增强子（enhancer）结合并参与调节靶基因转录效率的一组蛋白，并能将来自细胞表面的信息传递至核内基因。转录因子通常有几个功能域，可分为DNA结合域、转录调控域及自身活性调控域，DNA结合域可与特定的DNA序列（一般长8～20bp）相互作用，使转录因子与靶基因结合起来，随之转录调控域就可发挥其激活或抑制作用，通常这些结构域在结构与功能上是独立分开的。不同的转录因子还可结合于紧密相邻的DNA序列而形成一种多聚体结构来调节基因表达，这种组合调控（combinatorial regulation）不论转录因子是否激活及其含量多少均可激活基于靶基因中特定转录因子结合位点的转录。除启动基础转录活性外，转录因子还能整合从细胞表面经信号转导途径传递而来的信号[2]。 编辑本段激活角朊细胞基因表达的转录因子 （一）AP1 AP1转录因子通常以jun(c-jun、junB、junD)与Fos（Fra-1、Fra-2、c-fos、fosB）家族成员组成的同源或异源二聚体表达其活性，即结合于5’-GTGAGCTCAG-3’序列。目前已知AP1位点对于编码角蛋白（K1、K5、 K6及K19）、丝聚合蛋白原（profilaggrin）基因的最适转录活性十分重要[3,7]，编码角质化包膜（cornified envelope）相关蛋白-TG1、兜甲蛋白及INV的基因也含有功能性AP1 位点[8，9]，如hINV基因启动子在其转录起始位点上游2.5kb内有5个AP1共有结合位点（AP1-1～5），其中2个AP1位点AP1-1和AP1-5若同时发生突变时角朊细胞的转录水平就可下降80%；佛波酯（TPA）则可使AP1与hINV启动子处AP1-1及AP1-5位点的结合能力增强10～100倍，后经点突变实验证实AP1-1和AP1-5位点可部分介导佛波酯（TPA）诱导的效应[10]。丝聚合蛋白原、K1、兜甲蛋白及K19基因中的AP1位点可活化转录[3,6,7]，

现在分词短语作伴随状语

其实分词做伴随状语就是分词做方式状语的一种，伴随是一种方式。大多情况下其实方式状语从句都等于伴随状语从句。只有在just) as…so…结构中位于句首，这时as从句带有比喻的含义，意思是"正如…"，"就像"，多用于正式文体 分词短语作伴随状语 伴随状语的特点是：它所表达的动作或状态是伴随着句子谓语动词的动作而发生或存在的 1）现在分词与过去分词作状语的区别。 现在分词做状语与过去分词做状语的最主要区别在于两者与所修饰的主语的主动与被动关系的区别。 1）现在分词作状语时，现在分词的动作就是句子主语的动作，它们之间的关系是主动关系。 ）过去分词作状语时，过去分词表示的动作是句子主语承受的动作，它们之间的关系是被动关系。 比如 The teacher came in the classroom with handing a book in his hand. with handing就是个例子 过去分词，现在分词都可以做伴随状语,即在某件事情发生的时候相伴发生的事情这个句子老师进入了教室，他是拿着本书进入教室的，在他进入教室的时候他的手中有一本书（相伴）知道意思了吧。 做题的时候要注意区分什么时候使用过去分词和什么时候使用现在分词做伴随状语。一般的无非又几种情况： 1，过去分词表示一种完成了的或者是被动意义的动作 分词作状语（关键找逻辑主语） a)放在句首的分词往往看作时间状语1以及原因状语2 1. Looking (when I looked) at the picture, I couldn't help missing my middle school days. 2.Seriously injured, Allen was rushed to the hospital. =As he was seriously injured, Allen was rushed to the hospital. b)放在句中或句末常常看作为伴随状态(并列句) The girl was left alone in the room，weeping（crying ）bitterly. (但注意特殊：Generally/frankly speaking... / taken as a whole(总的来讲）不考虑逻辑主语，看作为独立成分)

with_作伴随状语

As 与with 引导伴随状语 as 与with 都可以构成结构来表示伴随状语，as 在此结构中是一个连词，而with 是一个介词，因此构成不同的结构来表示伴随状语： 区别：结构不同！with 引导的不是句子而是短语 As＋主语＋谓语动词，主句 With ＋名词、代词＋介词、形容词，副词，分词，不定式 随着产量增长20％，....... As the production increased by 20 percent, we have had another good harvest year. With the production up by 20 percent, we have had another good harvest year. He enjoys listening to music with his eyes closed. 他喜欢闭着眼睛听音乐。 He came out of the room with his eyes shining. 他走出房间，眼里闪着亮光。 Don't speak with your mouth full. 嘴里吃东西时不要讲话。 Mother looked at me with tears in her eyes. 母亲含泪看着我。 with独立结构的位置可前可后，如： With a lot of work to do,he felt even busier. 有大量工作要做，他感到更忙了。 With a lot of work done,he felt he would have a good rest. 做完了工作，他感到他要好好休息一下。 练习： 1.with ＋名词（代词）＋介词短语 他手托下巴chin，坐在那儿沉思。 He sat there thinking, with 那位老人背倚着墙站在那里。 2.with ＋名词（代词）＋形容词 He stared at his friend 他张大嘴巴凝视着他的朋友。 The man raised his head 这人抬起头来，眼里充满了好奇 3.with ＋名词（代词）＋现在分词 She stood there chatting with her friend, with她站在那儿跟朋友闲聊，孩子在旁边玩。With , I feel very obliged to you． 无论我什么时候遇到困难你总是帮助我，真是太感激你了。 4.with ＋名词（代词）＋过去分词 The goalkeeper left Japan with….这位守门员离开了日本，而他的缺点却暴露无遗。 5.with ＋名词（代词）＋不定式 With……, you’d better hurry． 还有十分钟，你最好快一点。 With….., our group is sure to succeed． 有你领导我们，我们组肯定能成功。 随着社会的发展 随着经济技术的发展progress 随着时间的流逝pass by /go by/elapse/pass 随着时间的推移go on

转录因子正文

转录因子 摘要：随着众多生物基因组计划的完成及其蛋白质组学研究的不断深入，人类步入了系统生物学时代。基因组计划的完成提供了大量的DNA内在信息，解析出基因组中可能存在的全部基因的阅读框架，因此，接下来研究基因的表达调控特别是转录调控就显得非常迫切。另一方面，蛋白组学研究的突飞猛进给我们描绘出了细胞的蛋白质表达谱和网络谱，接下来研究蛋白质与蛋白质，蛋白质与DNA的相互作用将成为现在及以后相当长一段时间内的研究主题。有生物学家认为，21世纪对人类最具有挑战性的生物学主题就是“基因的全基因组调控”和”细胞的全蛋白质的生理功能”这两大难题。 然而，转录因子是可与基因调控序列结合并调控基因转录的一类核蛋白，研究转录因子就是研究转录调控的分子机制，研究一种或一类特定的蛋白质分子与DNA的结合特性，研究与DNA结合的蛋白质分子是怎样调控基因转录等问题。转录因子的研究实际上已构成上述两大生物学难题的一个交叉点，因此，对转录因子的深入研究已是一件极其迫切而且重要的课题。 DNA转录及转录因子 定义 转录：是指以DNA为模板，在RNA聚合酶的作用下合成mRNA，将遗传信息从DNA分子上转移到mRNA分子上，这一过程成为转录。真核生物DNA的转录在细胞核中进行，原核生物的转录在细胞质的核质区

内进行。 转录单元 转录单元是一段以启动子开始至终止子结束的DNA序列。 转录起始（transcription initiation）：转录因子通过识别基因启动子上的特异顺式元件并募集多种蛋白质因子，形成具有RNA聚合酶活性的转录起始复合体，从转录起始位点启动转录的过程。 转录终止子（transcription terminator）：基因编码区下游使RNA聚合酶终止mRNA合成的密码子，是一种位于poly(A)位点下游，长度在几百碱基以内的结构。 终止子可分为两类。一类不依赖于蛋白质辅因子就能实现终止作用。另一类则依赖蛋白辅因子才能实现终止作用。这种蛋白质辅因子称为释放因子，通常又称ρ因子 转录因子：能够结合在某基因上游特异核苷酸序列上的蛋白质，活化后从胞质转位至胞核，通过识别和结合基因启动子区的顺式作用元件,启动和调控基因表达。 转录因子是转录起始过程中RNA聚合酶所需的辅助因子。真核生物基因在无转录因子时处于不表达状态，RNA聚合酶自身无法启动基因转录，只有当转录因子(蛋白质)结合在其识别的DNA序列上后，基因才开始表达。转录因子是结合在某基因上游特异核苷酸序列上的蛋白质，这些蛋白质能调控该基因的转录。转录因子可以调控核糖核酸聚合酶（RNA聚合酶）与DNA模板的结合。转录因子不单与DNA序列上的启动子结合，也可以和其它转录因子形成-转录因子聚合体，来影

伴随状语定义

定义 定义:伴随状语是指状语从句的动作伴随主句发生，它的特点是：它所表达的动作或状态是伴随着句子谓语动词的动作而发生或存在的。 例如： ①He sat in the armchair，reading a newspaper. 他坐在扶手椅里读报。 ②All night long he lay awake，thinking of the problem. 他整夜躺在床上睡不着，思考着那个问题。 伴随状语出现的条件 是由一个主语发出两个动作或同一个主语处于两种状态，或同一个主语发出一个动作时又伴随有某一种状态。伴随状语的逻辑主语一般情况下必须是全句的主语，伴随状语与谓语动词所表示的动作或状态是同时发生的。 伴随状语几种表示方法 一、使用分词形式 The dog entered the room, following his master(这条狗跟着主人进了屋)。 The master entered the room,followed by his dog(主人进了屋，后面跟着他的狗)。 二、用with复合结构 The little girls were playing with snow with their hands frozen red(小女孩们在玩雪，手都冻红了)。 三、用独立主格结构 The little boy goes to school, the little dog accompanying him every day(这小孩每天去上学，那条小狗陪伴着他)。 四、用形容词 Crusoe went home, full of fear(克鲁索满怀恐惧地回家)。 The match will be broadcast live(这场比赛将作实况转播)。 He left home young and came back old(他少小离家老大回)。 五、用名词 He went away a beggar but returned a millionaire(他讨吃要饭离家，腰缠万贯回归)。。 六、用介词短语 The girl came back to her mother in tears.(这女孩眼泪汪汪地回到母亲身边)。 How can you go to the wedding party in rags(你怎能衣衫褴褛地去参加婚宴)？ I went home out of breath(我上气不接下气地回家)。 伴随状语的判断 The dog entered the room, following his master.一般在分词短语（现在分词表主动过去表被动）前有逗号的是伴随状语。 让步状语从句