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Overexpression of the Transcription Factor AP37 in Rice Improves Grain Yield under Drought Condition

Overexpression of the Transcription Factor AP37in Rice Improves Grain Yield under Drought Conditions 1[W][OA]

Se-Jun Oh 2,3,Youn Shic Kim 2,Chang-Woo Kwon,Hye Kyong Park,Jin Seo Jeong,and Ju-Kon Kim*School of Biotechnology and Environmental Engineering,Myongji University,Yongin 449–728,Korea

Transcription factors with an APETELA2(AP2)domain have been implicated in various cellular processes involved in plant development and stress responses.Of the 139AP2genes predicted in rice (Oryza sativa ),we identi?ed 42genes in our current study that are induced by one or more stress conditions,including drought,high salinity,low temperature,and abscisic acid.Phylogenic analysis of these 42stress-inducible AP2genes revealed the presence of six subgroups (I–VI)with distinct signature motifs.Two genes,AP37and AP59,representing subgroups I and II,respectively,were functionally characterized.

Both genes were found to be induced upon 2h of exposure to drought and high-salinity conditions but to differ in their expression pro?le upon exposure to low temperature and abscisic acid.The overexpression of AP37and AP59in rice under the control of the constitutive promoter OsCc1increased the tolerance to drought and high salinity at the vegetative stage.Increased tolerance to low temperatures was observed only in OsCc1:AP37plants.More importantly,the OsCc1:AP37plants showed signi?cantly enhanced drought tolerance in the ?eld,which increased grain yield by 16%to 57%over controls under severe drought conditions,yet exhibited no signi?cant difference under normal growth conditions.In contrast,grain yield in OsCc1:AP59plants in the ?eld was reduced by 23%to 43%compared with controls under both normal and drought stress conditions.Microarray experiments identi?ed 10and 38genes that are up-regulated by AP37and AP59,respectively,in addition to 37genes that are commonly induced by both factors.Our results suggest that the AP37gene has the potential to improve drought tolerance in rice without causing undesirable growth phenotypes.

Drought stress is among the most serious challenges to crop production worldwide.Upon exposure of plants to drought conditions,many stress-related genes are induced,and their products are thought to function as cellular protectants of stress-induced damage (Thomashow,1999;Shinozaki et al.,2003).The expression of stress-related genes is largely regu-lated by speci?c transcription factors.Members of the APETELA2(AP2),bZIP ,zinc ?nger,and MYB families have been shown to have regulatory roles in stress responses.The rice (Oryza sativa )and Arabidopsis (Arabidopsis thaliana )genomes code for more than 1,300transcriptional regulators,accounting for about

6%of the estimated total number of genes in both cases.About 45%of these transcription factors were reported to be from plant-speci?c families (Riechmann et al.,2000;Kikuchi et al.,2003).One example of such a plant-speci?c family of transcription factors is APETALA2(AP2),whose members share a highly con-served DNA-binding domain known as AP2(Weigel,1995).AP2factors appear to be widespread in plants,with the genomes of rice and Arabidopsis predicted to contain 139and 122AP2genes,respectively (Nakano et al.,2006).Members of the AP2family have been implicated in diverse functions in cellular processes involving ?ower development,spikelet meristem de-terminacy,plant growth,and stress tolerance (Chuck et al.,1998;Liu et al.,1998;Haake et al.,2002;Dubouzet et al.,2003;Gutterson and Reuber,2004).Of these diverse functions,the involvement of the AP2family in stress response has been relatively well characterized.In particular,CBF/DREB genes from Arabidopsis have been shown to play crucial roles in response to low temperature,salt,and drought stresses in transgenic Arabidopsis (Stockinger et al.,1997;Gilmour et al.,1998;Liu et al.,1998;Jaglo et al.,2001).CBF/DREBs are members of the AP2family and identi?able by the presence of CBF/DREB signature motifs (PKK/RPAGRxKFxETRHP and DSAWR)di-rectly ?anking the AP2domain (Jaglo et al.,2001).Overexpression of CBF/DREB s in transgenic Arabi-dopsis increases the transcript levels of stress-related genes and enhances tolerance to drought,high-salinity,and freezing stresses (Jaglo-Ottosen et al.,1998;Kasuga

This work was supported by the Ministry of Education,Science,and Technology of Korea through the Crop Functional Genomics Center (grant no.CG2111to J.-K.K.),by the Biogreen21Program (grant to J.-K.K.),and by the Korea Science and Engineering Foun-dation through the Plant Metabolism Research Center at Kyung-Hee University (grant to J.-K.K.)2

These authors contributed equally to the article.3

Present address:Syngenta Seeds Co.,Ltd.,100Gongpyeong-dong Jongro-gu,Seoul 110–702,Korea.

*Corresponding author;e-mail jukon306@https://www.wendangku.net/doc/789263834.html,.

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/789263834.html,)is:Ju-Kon Kim (jukon306@https://www.wendangku.net/doc/789263834.html,).[W]

The online version of this article contains Web-only data.[OA]

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et al.,1999;Haake et al.,2002).CBF/DREB s are also heterologously effective in canola(Brassica napus;Jaglo et al.,2001),tomato(Solanum lycopersicum;Hsieh et al., 2002),tobacco(Nicotiana tabacum;Kasuga et al.,2004), and rice(Oh et al.,2005b),enhancing stress tolerance in the corresponding transgenic plants.CBF/DREB orthologs have also been identi?ed in canola,tomato, wheat(Triticum aestivum),rye(Secale cereale),barley (Hordeum vulgare),and rice,and all of them are induc-ible by low-temperature treatments(Jaglo et al.,2001; Choi et al.,2002;Xue,2002,2003;Dubouzet et al.,2003; Skinner et al.,2005;Oh et al.,2007).The AP2gene family from other plant species,including DBF1and DBF2(Kizis and Pages,2002)from maize(Zea mays), AhDREB1(Shen et al.,2003)from Atriplex hortensis, OPBP1(Guo et al.,2004)from tobacco,CaPF1(Yi et al., 2004)from pepper(Capsicum annuum),HvRAF(Jung et al.,2006)from barley,and SodERF3(Trujillo et al., 2008)from sugarcane(Saccharum of?cinarum),have been found to be involved in responses to various abiotic stress conditions.

Approximately20%of rice-growing areas world-wide are prone to drought.Although drought condi-tions can alter the growth and development of rice at any time during its life cycle,drought stress during reproductive growth directly results in a loss of grain yield.To evaluate improvements in grain yield under drought conditions,it is important to subject the plants to the stress during the transition to the reproductive phase.To date,a number of studies have suggested that overexpression of stress-related genes could im-prove drought tolerance in rice to some extent(Xu et al.,1996;Garg et al.,2002;Jang et al.,2003;Ito et al., 2006;Hu et al.,2006,2008;Nakashima et al.,2007). Despite such efforts to develop drought-tolerant rice plants,very few of these have been shown to improve grain yields under?eld conditions.Examples of pos-itive effects include transgenic rice plants expressing SNAC1(Hu et al.,2006)and OsLEA3(Xiao et al.,2007), which was shown to improve grain yield under?eld drought conditions.

In this study,we identi?ed42rice genes encoding transcription factors with the AP2domain that were stress inducible.Two closely related yet distinct genes, AP37and AP59,were functionally characterized.The overexpression of these genes in transgenic rice im-proved the plant tolerance to both drought and high salinity at the vegetative stage.However,increased tolerance to low temperature was observed only in plants overexpressing AP37.These AP37overexpres-sors showed signi?cantly enhanced drought tolerance in the?eld,increasing grain yield by16%to57%over the controls under severe drought conditions,yet they displayed no signi?cant difference in yield under normal growth conditions.In contrast,grain yield in OsCc1:AP59plants was reduced by23%to43%under both normal and drought stress conditions.We discuss the similarities and differences between the functions of AP37and AP59with respect to stress tolerance and grain yield.RESULTS

Identi?cation of Stress-Inducible AP2Transcription Factors in Rice

Previously,the rice genome was predicted to contain 139AP2domain genes(Nakano et al.,2006).To iden-tify stress-inducible AP2genes,we performed expres-sion pro?ling with the Rice3#-Tiling microarray (GreenGene Biotech)using RNAs from14-d-old leaves of rice seedlings subjected to drought,high salinity, abscisic acid(ABA),and low temperature.When three replicates were averaged and compared with un-treated leaves,a total of42genes were found to be up-regulated by1.6-fold or greater(P,0.05)by one or more of these stress conditions(Table I).Phylogenic analysis of the amino acid sequences of42factors revealed the presence of six subgroups(I–VI),with AP37assigned to subgroup I,AP59to subgroup II, OsDREB1A to subgroup V,and OsDREB2A to sub-group VI(Fig.1;Dubouzet et al.,2003).Thirteen of42 factors are not classi?ed into any of the six subgroups (Table I;Supplemental Fig.S1).Comparison of the amino acid sequence spanning the AP2domain iden-ti?ed signature motifs by which these subgroups can be distinguished(Fig.1).For example,signature mo-tifs Ia,Ic,IIb,and IIc are speci?c to subgroups I and II, respectively,and motif Ib(IIa)is common to both.In addition to sequence similarity,members of each sub-group are closely related in terms of their response to stress.For example,expression of the genes in sub-groups I,II,and V is not induced by ABA,whereas that of the members of subgroups III and VI is not induced by ABA or low temperature.

Two genes,AP37(AK061380)and AP59(AK073812), representing subgroups I and II,respectively,were functionally characterized in this study.The transcript levels of AP37and AP59were measured by RNA gel-blot analysis using total RNAs from leaf tissues of 14-d-old seedlings exposed to high salinity,drought, ABA,and low temperature(Fig.2).The expression of both AP37and AP59was found to be induced after2h of exposure to high-salinity and drought stress.AP37 differs from AP59in its response to low temperature and ABA.The expression of the former responded rapidly to low temperature and was induced by ABA, whereas the latter responded slowly to low tempera-ture and was not induced by ABA.This is somewhat inconsistent with the microarray results,which indi-cated that AP37is not induced by ABA.This discrep-ancy may be due to variation in the stress treatments. Overall,our results showed that AP37and AP59are stress-inducible AP2genes that are closely related yet different in their expression pro?les.

Stress Tolerance of OsCc1:AP37and OsCc1:AP59Plants at the Vegetative Stage

To enable the overexpression of the AP37and AP59 genes in rice,their full-length cDNAs were isolated and linked to the OsCc1promoter for constitutive

Rice AP37Gene Confers Drought Tolerance

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expression (Jang et al.,2002),generating the constructs

OsCc1:AP37and OsCc1:AP59(Fig.3A).These con-structs were then introduced into rice by Agrobacterium tumefaciens -mediated transformation (Hiei et al.,1994),which yielded 15to 20independent transgenic lines per construct.Transgenic T 1-4seeds were collected,and three independent T 4-5homozygous lines of both OsCc1:AP37and OsCc1:AP59plants were selected for further analysis.All of the transgenic lines grew

normally with no stunting.The transcript levels of AP37and AP59in the OsCc1:AP37and OsCc1:AP59plants were determined by RNA gel-blot analysis.For this purpose,total RNAs were extracted from leaf tissues of 14-d-old seedlings grown under normal growth conditions (Fig.3B).Transcript levels of AP37and AP59were clearly enhanced at various levels in different transgenic lines as compared with those in the nontransgenic (NT)controls.To investigate whether

Table I.Rice AP2transcription factor genes up-regulated under stress conditions

Numbers in boldface indicate up-regulation by more than 1.6-fold (P ,0.05)in plants grown under stress conditions.

Subgroups

Sequence Identi?er a

Drought High Salinity ABA Low Temperature Mean b

P c

Mean b

P c

Mean b

P c

Mean b

P c

AP37

3.80.00 3.40.00 1.10.72 1.90.10Os04g0610400 2.10.01 1.50.1721.20.65 2.90.02Os04g0669200 2.80.01 2.60.02 1.30.59 2.90.04Os05g0497300 2.70.00 1.80.05 1.10.86 2.50.04II

AP59

7.70.00 6.10.00 1.30.438.60.00Os02g0655200 1.40.27 2.80.01 1.20.6321.10.91Os10g0562900 1.30.27 1.10.92 1.30.5210.10.00Os03g0860100 3.60.00 2.40.0221.40.39 1.20.72III

Os03g019190012.70.00 4.90.0021.30.42 1.20.56Os05g0572000 3.50.00 3.20.00 1.40.2921.10.87Os06g0222400 1.80.03 1.20.5821.60.1321.30.43Os08g04085009.10.00 5.20.00 1.00.98 1.20.61IV

Os01g0313300 1.50.29 1.30.69 3.20.02 4.20.03Os02g0782700 1.20.41 1.50.14 1.90.0421.00.93Os03g0183000 2.00.01 2.60.0012.80.00 1.40.30Os06g0194000 1.60.04 1.70.04 1.60.1721.30.51Os07g0617000 2.30.01 2.70.01 4.00.00 2.10.07Os07g0674800 1.30.33 1.80.0321.00.96 1.80.09Os09g028700021.90.1921.20.8922.00.00 2.00.30Os09g0286600 1.20.5521.00.99 2.30.02 1.20.59V

OsDREB1A 2.40.0121.10.9121.80.077.70.00OsDREB1B 9.50.00 6.00.00 1.20.67 3.10.04Os01g0968800 6.30.00 5.70.0021.30.627.90.00Os08g0545500 2.20.01 1.50.2421.00.99 1.50.33Os02g0677300 1.60.19 1.10.95 1.00.988.20.00Os09g052200021.20.4021.90.0223.20.0051.40.00Os09g0522100 1.60.1721.10.93 1.10.90 6.40.01VI OsDREB2A 3.00.01 3.80.01 2.20.0721.60.41Os05g0346200 2.90.00 2.20.00 1.50.09 1.20.50Others

Os06g0166400 2.80.00 2.50.01 1.80.05 1.10.83Os02g0797100 3.30.01 1.50.3721.00.95 1.50.48Os01g0131600 1.80.01 1.20.53 1.10.70 1.00.99Os09g0309700 1.20.43 1.50.12 2.10.0121.00.98Os01g0868000 2.60.02 1.80.16 1.60.30 1.70.32Os02g05466009.70.01 3.90.07 1.40.75 2.30.38Os04g0398000 2.00.01 2.30.01 1.50.1721.00.99Os12g0168100 5.10.00 3.90.00 1.70.26 1.30.67Os02g07647009.40.00 4.20.0021.00.96 6.90.01Os08g047400021.20.0014.10.00 2.20.0526.70.00Os09g045790015.20.007.70.00 1.20.6762.50.00Os08g0537900 3.10.00 3.20.01 5.10.0021.20.65Os08g0521600 4.70.00 3.00.0121.10.8521.10.84

Numbers for full-length cDNA sequences of the corresponding genes.b

Numbers represent means of three independent biological replicates.These microarray data sets can be found at http://www.ncbi.

https://www.wendangku.net/doc/789263834.html,/geo/(Gene Expression Omnibus).c

P values were analyzed by one-way ANOVA.

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the overexpression of AP37and AP59correlated with stress tolerance in rice,4-week-old transgenic plants

and NT controls were exposed to drought stress (Fig.3C).The NT plants started to show visual symptoms of drought-induced damage,such as leaf rolling and wilting with a concomitant loss of chlorophylls,at an earlier stage than the OsCc1:AP37and OsCc1:AP59plants.The transgenic plants also recovered faster than the NT plants upon rewatering.Consequently,the NT plants remained severely affected by the time at which all of the transgenic lines had fully recovered except

for the OsCc1:AP37-9line.This line recovered slower than the other transgenic lines,probably due to a lower level of transgene expression than in the others (Fig.3,B and C).Levels of transgene expression in the OsCc1:AP59-2line were also lower than those of others,while its phenotype was comparable to those of the other lines (Fig.3,B and C).Thus,the difference in transgenic phenotype does not always re?ect dif-ferent levels of mRNA.

To further verify the stress-tolerance phenotype,we measured the F v /F m values of the transgenic and NT

Figure 1.Alignment of AP2domain sequences from 29stress-inducible rice genes.Deduced amino acid sequences of the AP2domains of the 29genes listed in Table I were aligned using the ClustalW program.Identical and conserved residues are highlighted in gray.Signature motifs are indicated by boxes identi?ed on the top as follows:Ia to Ic,IIa to IIc,IIIa to IIId,IVa,Va to Vc,and VIa to VId for subgroups I to VI,respectively.

Figure 2.Expression of AP37and AP59in response to stress conditions in rice.Ten micrograms of total RNA was prepared from leaf tissues of 14-d-old seedlings exposed to drought,high salinity,ABA,or low temperature for the indicated time points:for drought stress,the seedlings were air dried at 28°C;for high-salinity stress,seedlings were exposed to 400m M NaCl at 28°C;for low-temperature stress,seedlings were exposed to 4°C;for ABA stress,seedlings were exposed to a solution containing 100m M ABA.Total RNAs were blotted and hybridized with the AP37and AP59gene-speci?c probes.The blots were then reprobed for the Dip1(Oh et al.,2005b)and rbcS (Jang et al.,1999)genes,which were used as markers for up-and down-regulation,respectively,of key genes following stress treatments.Ethidium bromide (EtBr)staining was used to determine equal loading of RNAs.

Rice AP37Gene Confers Drought Tolerance

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control plants,all at the vegetative stage (Fig.4).The F v /F m values represent the maximum photochemical ef?ciency of PSII in a dark-adapted state,where F v stands for variable ?uorescence and F m stands for maximum ?uorescence.The F v /F m levels were about 30%and 20%higher in the OsCc1:AP37and OsCc1:AP59plants,respectively,than in the NT plants un-der drought and high-salinity conditions.Under low-temperature conditions,in contrast,the F v /F m levels were 15%higher in the OsCc1:AP37plants than in the NT plants,whereas the levels in the OsCc1:AP59plants were at similar levels to those of the NT plants.Hence,our results indicate that the overexpression of AP37and AP59in transgenic rice increases the tolerance of these plants to drought and high-salinity stress con-ditions during the vegetative stage but that an in-creased tolerance to low temperature occurs only in plants overexpressing AP37.

Identi?cation of Genes Up-Regulated by Overexpressed AP37and AP59

To identify genes that are up-regulated by the over-expression of AP37and AP59,we performed expres-sion pro?ling of OsCc1:AP37and OsCc1:AP59plants in comparison with NT controls under normal growth conditions.Expression pro?ling with the Rice 3#-Tiling microarray was conducted using RNA samples ex-tracted from 14-d-old leaves of these transgenic plants and NT controls,all grown under normal growth conditions.Each data set was obtained from three biological replicates.As listed in Supplemental Table S1,statistical analysis of each data set using one-way ANOVA identi?ed 85genes that are up-regulated by AP37and/or AP59with 3-fold or greater induction in the transgenic plants than in NT plants (P ,0.05).Speci?cally,a total of 37genes were found to be commonly activated by AP37and AP59,whereas 10and 38genes are speci?c to AP37and AP59,respec-tively.Based on our microarray data shown in Table I and Supplemental Table S1,we selected eight stress-inducible genes out of 37common target genes and veri?ed their AP37-and AP59-dependent expression patterns under normal growth conditions by reverse transcription (RT)-PCR (Fig.5A,control).To test whether their expression levels increased further un-der stress conditions,we measured the transcript levels of the eight genes in OsCc1:AP37,OsCc1:AP59,and NT plants after exposure to drought,high-salinity,and low-temperature conditions (Fig.5).In NT plants,all of the target genes were induced at various levels within 2h of the stress treatments.In the transgenic plants,in contrast,the transcript levels of many genes are lower under stress conditions in comparison with

Figure 3.Production of OsCc1:AP37and OsCc1:AP59transgenic rice plants.A,The OsCc1:AP37and OsCc1:AP59plasmids consist of OsCc1promoter (Jang et al.,2002),a constitutive promoter linked to the AP37and AP59coding region,respectively,the 3#region of the potato (Solanum tuberosum )proteinase inhibitor II gene (3#PinII ),and a gene expression cassette that contains the 35S promoter,the bar coding region,and the 3#region of the nopaline synthase gene (3#nos ).The entire expression cassette is ?anked by the 5#matrix attachment region (MAR)of the chicken lysozyme gene (Oh et al.,2005a).BL,Left border;BR,right border.B,RNA gel-blot analysis was performed using total RNAs from young leaves of three homozygous T4lines of OsCc1:AP37and OsCc1:AP59plants and of NT control plants.The blots were hybridized with the AP37and AP59gene-speci?c probes and reprobed for rbcS .Ethidium bromide (EtBr)staining was used to determine equal loading of RNAs.C,Appearance of transgenic plants during drought stress .Three independent homozygous T4lines of OsCc1:AP37and OsCc1:AP59plants and NT controls were grown in a greenhouse for 4weeks and subjected to drought stress treatments.Four-week-old transgenic and NT plants were subjected to 3d of drought stress,followed by 2and 7d of rewatering in the greenhouse,respectively.Photographs were taken at the indicated time points.+denotes the number of rewatering days under normal growth conditions.

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stress-treated NT plants.The same is true for the AP59gene in OsCc1:AP37plants exposed to drought,high-salinity,and low-temperature conditions and also for AP37in OsCc1:AP59plants grown under low-temperature conditions.Such lower levels of target gene transcripts in OsCc1:AP37and OsCc1:AP59plants than in NT plants under stress conditions were not unexpected,because the transgenic plants were less affected by stress damage compared with the NT controls.The transgenic plants were more tolerant to stress than the NT controls at the time of stress treatments;hence,stress-induced expression levels of target genes were smaller in the transgenic plants than in NT controls.We repeated the experiments shown in Figure 5A

using real-time PCR,obtaining results similar to those of RT-PCR (Fig.5B;Supplemental Fig.S2).Overall,our results suggest that the AP37and AP59genes enhance stress tolerance differently by activating distinct groups of stress-regulated genes.

Overexpression of AP37Increases Rice Grain Yield under Drought Conditions

A phenotypic evaluation of OsCc1:AP37,OsCc1:AP59,and NT control plants revealed no major differ-ences in the vegetative growth of the entire plants.To then investigate whether the overexpression of AP37and AP59improved the rice grain yield of transgenic plants under ?eld conditions,we transplanted three independent T5homozygous lines of the OsCc1:AP37and OsCc1:AP59plants,together with their respective NT controls,to a paddy ?eld and grew them to matu-rity.A completely randomized design with two repli-cates was employed.The subsequent evaluation of the yield parameters of these plants revealed that the grain

yield of OsCc1:AP37plants remained similar to that of the NT controls under normal ?eld conditions (Fig.6;Table II;Supplemental Table S2).In the OsCc1:AP59plants under the same ?eld conditions,however,total grain weight was reduced by 23%to 43%compared with the NT controls,which appears to be due to decreases in the number of spikelets.These observa-tions prompted us to examine the yield components of the transgenic rice plants grown in the ?eld under drought conditions.Three independent lines of the OsCc1:AP37and OsCc1:AP59plants and NT controls were transplanted to a paddy ?eld with a removable rain-off shelter and exposed to drought stress at the panicle heading stage (from 10d before heading to 20d after heading).Statistical analysis of the yield parame-ters showed that the decrease in grain yield under drought conditions was signi?cantly smaller in the OsCc1:AP37plants than in the controls.Speci?cally,in the drought-treated OsCc1:AP37plants,the ?lling rate was higher than in the drought-treated NT plants by 17%to 36%,which resulted in an increase in the total grain weight by 16%to 57%,depending on the trans-genic line (Fig.6;Table III).In the drought-treated OsCc1:AP59plants,in contrast,the total grain weight was reduced by about 30%when compared with the drought-treated NT controls,which was similar to the yields observed for these plants under normal growth conditions.Despite the similar levels of drought toler-ance at the vegetative stage of the OsCc1:AP37and OsCc1:AP59plants,a sharp difference in grain yield under drought conditions is actually not surprising.This is because,unlike in OsCc1:AP37plants,spikelet development of OsCc1:AP59plants was signi?cantly affected by the constitutive overexpression of AP59under both normal and drought conditions,which

Figure 4.Changes in chlorophyll ?uorescence (F v /F m )of rice plants under drought,high-salinity,and low-temperature stress conditions.Three independent homozygous T4lines of OsCc1:AP37and OsCc1:AP59plants and NT controls grown in MS medium for 14d were subjected to various stress conditions as described in “Materials and Methods.”After stress treatments,the F v /F m values were measured using a pulse modulation ?uorometer (mini-PAM;Walz).All plants were grown under continuous light of 150m mol m 22s 21prior to stress treatments.Each data point represents the mean 6SE of triplicate determinations (n =10).

Rice AP37Gene Confers Drought Tolerance

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resulted in a loss of grain yield.Taken together,these results suggest that the overexpression of AP37confers drought tolerance in rice at the reproductive stage and improves grain yield signi?cantly.

DISCUSSION

In this study,expression pro?ling using RNAs from stress-treated rice plants identi?ed 42AP2domain

factors that are stress inducible (Table I).Alignment of these stress-inducible factors revealed six subgroups within which the members are more closely related,suggesting a common function during the stress re-sponse.The overexpression of AP37(OsCc1:AP37)and AP59(OsCc1:AP59),representative members of sub-groups I and II,respectively,increased the rice plant tolerance to drought and high salinity at the vegetative stage.Increased tolerance to low temperature was

Figure 5.Regulated expression of stress-related genes in OsCc1:AP37,OsCc1:AP59,and NT plants under normal and stress conditions.Homo-zygous T4lines of OsCc1:AP37,OsCc1:AP59,and NT control rice plants were grown in a greenhouse for 14d.Transgenic and NT plants were then treated with various stress conditions as described in the legend to Figure 2.A,Transcript levels of AP37,AP59,and eight target genes were determined by RT -PCR (using primers listed in Supplemental Table S3).B,Transcript levels of AP37,AP59,and three target genes were deter-mined by quantitative RT-PCR (using primers listed in Supplemental Table S3).Dip1(Oh et al.,2005b)was used as a marker for the up-regulation of key genes following stress treatments.The rice ubiquitin gene (OsUbi )was used as an equal loading control.

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observed only in OsCc1:AP37plants,suggesting a functional difference between the two closely related AP2factors in the stress response.Given the different numbers of target genes up-regulated in OsCc1:AP37and OsCc1:AP59plants,the difference in their re-sponse to low temperature was not unexpected.Our microarray experiments identi?ed 10and 38putative target genes that are speci?c to the AP37and AP59proteins,respectively,in addition to a further 37target genes that appeared to be common to both factors (Supplemental Table S1).Similarly contrasting results were previously obtained for two closely related AP2factors,HvCBF4from barley and CBF3/DREB1A from Arabidopsis.The overexpression of HvCBF4and CBF3/DREB1A in rice confers increased tolerance to drought,high salinity,and low temperature,and in the case of low temperature this effect was more pro-nounced in plants overexpressing HvCBF4(Oh et al.,2007).The composition of the target rice genes was also different between the HvCBF4and CBF3/DREB1A plants.

The expression patterns of the AP37and AP59genes are similar under drought and high-salinity conditions but different under low-temperature and ABA stress,consistent with the observed differences between these genes in conferring low-temperature tolerance.The AP37transcript levels were rapidly increased in rice plants within 30min of exposure to stress condi-tions,similar to the reported observations for the OsDREB1A transcripts (Dubouzet et al.,2003).The OsDREB1A gene was classi?ed in subgroup V in this analysis,the members of which have a similar expres-sion pattern to the genes in subgroups I and II (Table I).

Consistent with our results for OsCc1:AP37rice plants,the overexpression of OsDREB1A in rice (Ito et al.,2006)and in Arabidopsis (Dubouzet et al.,2003)has been shown previously to confer tolerance to low temperature,in addition to drought and high-salinity resistance.The overexpression of OsDREB1A in rice induced the accumulation of soluble sugars,including raf?nose,Suc,Glc,and Fru,which may act as osmo-protectants (Ito et al.,2006).The target genes we identi?ed in OsCc1:AP37plants included genes that function in carbon metabolism such as phosphoglu-conate aldolase,phosphoglucomutase,UDP-glucosyl transferase,and isocitrate lyase.These genes may similarly increase the content of soluble sugars.It is also generally accepted that excessive amounts of reactive oxygen species are generated upon exposure of plants to stress stimuli,and these must be removed in order to maintain cellular homeostasis.Overexpres-sion of JERF3,a tomato ortholog of our subgroup IV (Table I),was shown previously to enhance stress tolerance by increasing the expression of genes encod-ing antioxidant enzymes (Wu et al.,2008).Similarly,the expression levels of several antioxidant genes,such as thioredoxin,peroxidase,and ascorbate oxido-reductase,were found to be increased in our OsCc1:AP37and OsCc1:AP59transgenic rice plants,which

may indicate the activation of a reactive oxygen species-scavenging system.Loss-of-function mutants on AP37and AP59may provide us with further evidence of molecular mechanisms for stress tolerance,al-though the presence of many homologous AP2do-main genes may not allow knockout phenotypes to be displayed.

Figure 6.Agronomic traits for OsCc1:AP37and OsCc1:AP59rice plants grown in the ?eld under normal and stress conditions.Spider plots of agro-nomic traits of three independent ho-mozygous T5lines of OsCc1:AP37and OsCc1:AP59and corresponding NT controls under normal and drought conditions,respectively,were drawn using Microsoft Excel software.Each data point represents a percentage of the mean values (n =20)listed in Tables II and III.Mean values from NT controls were set at 100%as a refer-ence.CL,Culm length;PL,panicle length;NP ,number of panicles per hill;NSP ,number of spikelets per panicle;TNS,total number of spike-lets;FR,?lling rate;NFG,number of ?lled grains;TGW,total grain weight;1,000GW,1,000grain weight.

Rice AP37Gene Confers Drought Tolerance

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Grain yield from rice plants is severely affected when they are exposed to drought stress at the repro-ductive stage.Therefore,it was important to examine the effects of drought stress on grain yield at this stage of growth in our transgenic plants.It was also impor-tant to use transgenic lines that were not genetically segregating under?eld conditions.It is relatively straightforward to identify segregating families of transgenic rice plants up to the T4generation in the ?eld,even though they are homozygous for a trans-gene.To evaluate whether any improvements in grain yield had occurred in our transgenic rice under

Table II.Analysis of seed production parameters in OsCc1:AP37and OsCc1:AP59plants under normal growth conditions

Each parameter value represents the mean6SD(n=20)for OsCc1:AP37and OsCc1:AP59and the respective NT control plants.The percentage differences(%D)between the values for the OsCc1:AP37or OsCc1:AP59plants and for the respective NT controls were calculated.P values were determined according to the LSD test.

Constructs Panicle

Length

No.of

Spikelets

Total No.of

Spikelets

Filling

Rate

No.of

Filled Grain

Total Grain

Weight cm per panicle per hill%per hill g

NT18.3261.3796.04612.741,209.706137.5590.8063.841,098.456133.2622.5163.03 AP37-419.7061.2595.8668.191,294.556244.7591.2362.821,177.656202.9324.5665.28 %D7.5320.197.010.477.219.11 P0.0110.9510.1510.8860.1440.129 AP37-518.1761.1890.7669.501,136.956152.6687.1563.68992.806151.3921.6063.83 %D20.8225.5026.0124.0229.6224.04 P0.7760.0860.2180.6650.0530.500 AP37-919.1062.4883.6867.101,205.956186.4988.2462.861,066.506182.7822.3164.39 %D 4.26212.8720.3122.8222.9120.89 P0.1440.0000.9490.0750.5530.884 NT18.8061.1998.9066.991,327.106191.1692.8463.011,232.556185.7325.7864.39 AP59-116.9561.1471.39611.76996.106181.0773.9067.57734.756156.9114.7264.34 %D29.84227.81224.94220.39240.38242.88 P0.0000.0000.0000.0000.0000.000 AP59-218.6061.5071.7065.371,044.206241.5478.3066.08811.206167.0917.9764.42 %D21.06227.50221.31215.65234.18230.27 P0.6220.0000.0000.0000.0000.000 AP59-618.4561.2380.13610.631,045.106186.8588.0163.60919.256165.6219.7064.19 %D21.87218.97221.2425.19225.41223.58 P0.3890.0000.0000.0060.0000.000 Table III.Analysis of seed production parameters in OsCc1:AP37and OsCc1:AP59plants under drought stress conditions

Constructs Panicle

Length

No.of

Spikelets

Total No.of

Spikelets

Filling

Rate

No.of

Filled Grain

Total Grain

Weight cm per panicle per hill%per hill g

NT19.5062.3898.34614.521,526.766753.8860.15616.39871.756349.0915.2067.29 AP37-419.1061.5192.49611.711,443.806662.6182.24610.531,144.406472.4820.6069.67 %D22.0525.9425.4336.7131.2735.52 P0.7030.4580.8190.0200.2610.273 AP37-517.5060.9193.9166.731,741.256212.7872.88610.671,262.506203.7623.8763.75 %D210.2524.5014.0421.1544.8257.07 P0.0870.5920.5760.1710.1340.105 AP37-916.8560.7587.29611.101,483.756106.2270.92611.661,059.756240.4917.7064.75 %D213.46211.2322.8117.8921.5616.44 P0.0300.1940.9100.2420.4550.624 NT19.7562.0697.01612.271,538.256737.3160.31616.38895.256307.0015.4566.82 AP59-115.3762.4954.86620.711,001.256589.5263.20610.24663.756481.759.93610.41 %D222.15243.45234.90 4.78225.85235.76 P0.0080.0300.1990.7810.3560.324 AP59-218.3760.9492.25637.811,133.756431.6353.66615.71579.256170.1210.4064.35 %D26.9624.89226.29211.01235.29232.68 P0.3310.7850.3250.5260.2150.366 AP59-617.1761.7660.89612.581,137.336280.3278.86614.19902.336317.8615.3767.36 %D213.08237.23226.0630.760.7920.53 P0.1050.0750.3650.1190.9790.989

Oh et al.

drought conditions,we transplanted T5homozygous lines of OsCc1:AP37and OsCc1:AP59plants to the?eld in2008,which had been prescreened in the?eld for segregation in2007.The plants were exposed to drought stress at the panicle heading stage from10d prior to heading to20d after heading in?eld condi-tions.The OsCc1:AP37plants showed signi?cantly enhanced drought tolerance in the?eld,with a grain yield of16%to57%higher than the controls under severe drought conditions yet displayed no signi?cant differences under normal growth conditions(Fig.6; Tables II and III).In contrast,grain yield from the OsCc1:AP59plants in the?eld was reduced by23%to 43%compared with the controls under both normal and drought stress conditions.The decrease in grain yield in OsCc1:AP59plants was primarily due to the disrupted spikelet development caused by augmented AP59expression.The vegetative growth of both the OsCc1:AP37and OsCc1:AP59plants was visually in-distinguishable from that of the NT controls.Impor-tantly,the overexpression of AP37in rice was effective against drought stress at the reproductive stage as well as at the vegetative stage.In addition,the overexpres-sion of AP37does not seem to affect the development of reproductive organs while conferring stress toler-ance in transgenic plants.Development of the panicle and/or spikelet meristem is repressed in rice under drought conditions,resulting in a reduction in the number of panicles and/or spikelets(Boonjung and Fukai,1996;Wopereis et al.,1996;Asch et al.,2005). The lower decreases in the?lling rate and in the number of spikelets of OsCc1:AP37plants under drought conditions implied that the developmental processes for panicles and spikelets had been protected from drought stress,indicating drought tolerance at the reproductive stage.The overexpression of AP59,in contrast,was ineffective in increasing grain produc-tivity,yet it conferred stress tolerance in rice plants at the vegetative stage.To date,the potential impact of homeotic genes like the AP2factors upon grain yield have received relatively little attention,because of their negative effects on fertility,plant growth,and development.It is thus important to evaluate agro-nomic traits in transgenic crops throughout all stages of plant growth to address the advantages of using such homeotic genes for improving stress tolerance. Overall,our results here demonstrate that AP37can improve rice grain yield under drought conditions without conferring an undesirable growth phenotype.

MATERIALS AND METHODS

Plasmid Construction and Transformation of Rice The expression plasmids OsCc1:AP37and OsCc1:AP59contained the bar gene under the control of the cauli?ower mosaic virus35S promoter to enable herbicide-based plant selection.The OsCc1promoter was used to drive constitutive plasmid gene expression(Jang et al.,2002).The coding regions of AP37and AP59were ampli?ed from rice(Oryza sativa)total RNA using an RT-PCR system(Promega)according to the manufacturer’s instructions.Primer pairs were as follows:AP37forward(5#-ATGGCGCCCAGAGCAGCTAC-3#)and AP37reverse(5#-CTAGTTCTCTACCGGCGGCG-3#);and AP59forward (5#-ATGCTGCTTAATCCGGCGTC-3#)and AP59reverse(5#-TTAGCTCAC-CAGCTGCTGGA-3#).Plasmids were introduced into Agrobacterium tumefa-ciens LBA4404by triparental mating,and embryogenic calli from mature seeds (cv Nakdong)were transformed as described previously(Jang et al.,1999). Drought Treatments at the Vegetative Stage

Transgenic and NT rice seeds were germinated in half-strength Murashige and Skoog(MS)solid medium in a growth chamber in the dark at28°C for4d, transplanted into soil,and then grown in a greenhouse(16-h-light/8-h-dark cycles)at28°C to30°C.Eighteen seedlings from each transgenic and NT line were grown in pots(33335cm;one plant per pot)for4weeks before undertaking the drought stress experiments.To induce drought stress, 4-week-old transgenic and NT seedlings were unwatered for3d followed by7d of watering.The numbers of plants that survived or continued to grow were then scored.

Chlorophyll Fluorescence under Conditions of Drought, High Salinity,and Low Temperature

Transgenic and NT rice seeds were germinated and grown in half-strength MS solid medium for14d in a growth chamber(16-h-light[150m mol m22s–1]/ 8-h-dark cycles at28°C).The green portions of approximately10seedlings were cut using a scissors prior to stress treatments in vitro.To induce low-temperature stress,the seedlings were incubated at4°C in water for up to6h under continuous150m mol m22s–1light.For high-salinity stress treatments, they were incubated in400m M NaCl for2h at28°C under continuous150 m mol m22s–1,and to simulate drought stress,they were air dried for2h at 28°C under continuous150m mol m22s–1light.The F v/F m values were then measured as described previously(Oh et al.,2005b).

Rice3#-Tiling Microarray Analysis

Expression pro?ling was conducted using the Rice3#-Tiling microarray manufactured by NimbleGen(https://www.wendangku.net/doc/789263834.html,/),which con-tains27,448genes deposited at the International Rice Genome Sequencing Project Rice Annotation Project1database(https://www.wendangku.net/doc/789263834.html,b.nig.ac.jp).Fur-ther information on this microarray,including statistical analysis,can be found at https://www.wendangku.net/doc/789263834.html,(GreenGene Biotech).Among the genes on the microarray,20,507are from representative Rice Annotation Project1se-quences with cDNA/EST supports,and6,941genes have been predicted without cDNA/EST supports.Ten60-nucleotide-long probes were designed from each gene starting at60bp ahead of the stop codon and with10-bp shifts in position,so that10probes covered150bp within the3#region of the gene. In total,270,000probes were designed in this way(average size,60nucleo-tides)to have melting temperature values of between75°C and85°C.Random GC probes(38,000)were used to monitor the hybridization ef?ciency,and ?ducial markers at the four corners(225)were included to assist with overlaying of the grid on the image.

To identify stress-inducible AP2genes in rice,total RNA(100m g)was prepared using14-d-old rice leaves from plants subjected to drought,high-salinity,ABA,and low-temperature stress conditions.For the high-salinity and ABA treatments,the14-d-old seedlings were transferred to a nutrient solution containing400m M NaCl or100m M ABA for2h in the greenhouse under continuous light of approximately1,000m mol m22s21.For drought treatment,14-d-old seedlings were air dried for2h under continuous light of approximately1,000m mol m22s21.For low-temperature treatment,14-d-old seedlings were exposed at4°C in a cold chamber for6h under continuous light of150m mol m22s21.For identi?cation of genes up-regulated in OsCc1: AP37and OsCa1:AP59plants,total RNA(100m g)was prepared from leaf tissues of14-d-old transgenic and NT rice seedlings grown under normal growth conditions.The mRNA was puri?ed using the Qiagen Oligotex column according to the manufacturer’s instructions.For normalization, data were processed with cubic alpine normalization using quartiles to adjust signal variation between chips and with robust multi-chip analysis using a median polish algorithm implemented in NimbleScan(Workman et al.,2002; Irizarry et al.,2003).To assess the reproducibility of the microarray analysis, we repeated the experiments three times with independently prepared total RNAs and analyzed each data set statistically using one-way ANOVA.

Rice AP37Gene Confers Drought Tolerance

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RT-PCR and Quantitative PCR Analysis

Total RNA was prepared as reported previously(Oh et al.,2008).For the analysis of target by RT-PCR,a cDNA synthesis system(Invitrogen)was used according to the manufacturer’s instructions.PCR products were ampli?ed using primers designed with Primer Designer4software(Sci-ed Software).RT-PCR was carried out using the primer pairs listed in Supplemental Table S3at a ?nal concentration of10p M each and2m L(equivalent to5ng of total RNA)of cDNA as the template.PCR was performed at95°C for10min,followed by20to 25cycles of94°C for30s,57°C for30s,and68°C for1min.Ampli?ed products were resolved on a2%agarose gel.To validate our RT-PCR results,we repeated each experiment twice with independently prepared total RNA.

For quantitative real-time PCR experiments,the SuperScript III Platinum One-Step Quantitative RT-PCR system(Invitrogen)was used.For PCR,a master mix of reaction components was prepared according to the manufac-turer’s protocol for Platinum SYBR Green qPCR SuperMix-UDG(Invitrogen). Thermocycling and?uorescence detection were performed using the Mx3000p Real-Time PCR machine(Stratagene).PCR was performed at95°C for10min,followed by20to25cycles of94°C for30s,57°C for30s,and68°C for1min.To validate our quantitative PCR results,we repeated each experiment three times.

Drought Treatments in the Field for Reproductive-Stage Rice Plants

To evaluate yield components of transgenic plants under normal?eld conditions,three independent T5homozygous lines of the OsCc1:AP37and OsCc1:AP59plants,together with NT controls,were transplanted to a paddy ?eld at the Rural Development Administration,Suwon,Korea.A completely randomized design was employed with two replicates,each consisting of four plots of5m2.At25d after sowing,the seedlings were randomly transplanted within a15-330-cm distance.Fertilizer was applied at70:40:70(nitrogen: phosphorus:potassium)kg ha21after the last paddling and45d after trans-plantation.Yield parameters were scored for10plants per plot and20plants per line.

To evaluate yield components of transgenic plants under drought?eld conditions,three independent T5homozygous lines of the OsCc1:AP37and OsCc1:AP59plants and NT controls were transplanted to a removable rain-off shelter with a1-m-deep container?lled with natural paddy soil located at Myongji University,Yongin,https://www.wendangku.net/doc/789263834.html,pletely randomized design,trans-planting distance,and use of fertilizer were employed as described above for normal?eld conditions.Drought stress was applied at the panicle heading stage(from10d before heading to20d after heading)by?owing water through a drain at the bottom of the container.To prevent plants from dying, we irrigated twice when the plant leaves rolled during drought stress.After exposure to drought stress conditions,the polyvinyl roofs were removed and plants were irrigated until harvesting.

When the plants grown under normal and drought conditions had reached maturity and the grains had ripened,they were harvested and threshed by hand(separation of seeds from the vegetative parts).The un?lled and?lled grains were taken apart,independently counted using a Countmate MC1000H (Prince),and weighed.The following agronomic traits were scored:?owering date,panicle length,number of tillers,number of panicles,spikelets per panicle,?lling rate(%),total grain weight(g),and1,000grain weight(g).The results from three independent lines were separately analyzed by one-way ANOVA and compared with those of the NT controls.The ANOVA was used to reject the null hypothesis of equal means of transgenic lines and NTcontrols (P,0.05).SPSS version16.0was used to perform statistical analysis.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AK061380(AP37)and AK073812(AP59). Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1.Phylogenic relationship of the rice AP2family.

Supplemental Figure S2.Up-regulation of?ve target genes in OsCc1:AP37 and OsCc1:AP59plants in comparison with NT controls.

Supplemental Table S1.Up-regulated genes in OsCc1:AP37and OsCc1: AP59plants in comparison with NT controls.

Supplemental Table S2.Agronomic traits of the OsCc1:AP37and OsCc1: AP59transgenic rice plants under normal growth and drought stress conditions in the?eld.

Supplemental Table S3.Primers used for RT-PCR.

ACKNOWLEDGMENT

We are grateful to Dr.Soon Jong Kweon at the National Academy of Agricultural Sciences,Rural Development Administration,of Korea for making critical comments on the?eld experiments and analyses. Received February23,2009;accepted May3,2009;published May8,2009.

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必须懂的53个电脑英文缩写

·PC：个人计算机Personal Computer ·CPU：中央处理器Central Processing Unit ·CPU Fan：中央处理器的“散热器”(Fan) ·MB：主机板MotherBoard ·RAM：内存Random Access Memory，以PC-代号划分规格，如PC-133，PC-1066，PC-2700 ·HDD：硬盘Hard Disk Drive ·FDD：软盘Floopy Disk Drive ·CD-ROM：光驱Compact Disk Read Only Memory ·DVD-ROM：DVD光驱Digital Versatile Disk Read Only Memory ·CD-RW：刻录机Compact Disk ReWriter ·VGA：显示卡(显示卡正式用语应为Display Card) ·AUD：声卡(声卡正式用语应为Sound Card) ·LAN：网卡(网卡正式用语应为Network Card) ·MODM：数据卡或调制解调器Modem ·HUB：集线器

The way常见用法

The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法）举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法：在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because

The way的用法及其含义(二)

The way的用法及其含义（二）二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中

(完整版)the的用法

定冠词the的用法：定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...

学电脑必懂的53个英文单词和缩写

学电脑必懂的53个英文单词和缩写（我不信你都认识）潜龙飞天发表于2010年06月28日 09:36 阅读(1) 评论(0) 分类：个人日记举报 PC：个人计算机Personal Computer ·CPU：中央处理器Central Processing Unit ·CPU Fan：中央处理器的“散热器”(Fan) ·MB：主机板MotherBoard ·RAM：内存Random Access Memory，以PC-代号划分规格，如PC-133，PC-1066，PC-2700 ·HDD：硬盘Hard Disk Drive ·FDD：软盘Floopy Disk Drive ·CD-ROM：光驱Compact Disk Read Only Memory ·DVD-ROM：DVD光驱Digital Versatile Disk Read Only Memory ·CD-RW：刻录机Compact Disk ReWriter ·VGA：显示卡(显示卡正式用语应为Display Card) ·AUD：声卡(声卡正式用语应为Sound Card) ·LAN：网卡(网卡正式用语应为Network Card) ·MODM：数据卡或调制解调器Modem ·HUB：集线器

·WebCam：网络摄影机 ·Capture：影音采集卡 ·Case：机箱 ·Power：电源 ·Moniter：屏幕，CRT为显像管屏幕，LCD为液晶屏幕 ·USB：通用串行总线Universal Serial Bus，用来连接外围装置 ·IEEE1394：新的高速序列总线规格Institute of Electrical Ａnd Electronic Engineers ·Mouse：鼠标，常见接口规格为PS/2与USB ·KB：键盘，常见接口规格为PS/2与USB ·Speaker：喇叭 ·Printer：打印机 ·Scanner：扫描仪 ·UPS：不断电系统 ·IDE：指IDE接口规格Integrated Device Electronics，IDE接口装置泛指采用IDE接口的各种设备 ·SCSI：指SCSI接口规格Small Computer System Interface，SCSI 接口装置泛指采用SCSI接口的各种设备 ·GHz：(中央处理器运算速度达)Gega赫兹/每秒 ·FSB：指“前端总线(Front Side Bus)”频率，以MHz为单位 ·ATA：指硬盘传输速率ATAttachment，ATA-133表示传输速率为133MB/sec

“the way+从句”结构的意义及用法

“tｈｅwａｙ+从句”结构的意义及用法首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法

不对。Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.

使用电脑必懂的53个英文单词和缩写

使用电脑必懂的53个英文单词和缩写 ·PC：个人计算机Personal Computer ·CPU：中央处理器Central Processing Unit ·CPU Fan：中央处理器的“散热器”(Fan) ·MB：主机板MotherBoard ·RAM：内存Random Access Memory，以PC-代号划分规格，如PC-133，PC-1066，PC-2700 ·HDD：硬盘Hard Disk Drive ·FDD：软盘Floopy Disk Drive ·CD-ROM：光驱Compact Disk Read Only Memory ·DVD-ROM：DVD光驱Digital Versatile Disk Read Only Memory ·CD-RW：刻录机Compact Disk ReWriter ·VGA：显示卡(显示卡正式用语应为Display Card) ·AUD：声卡(声卡正式用语应为Sound Card) ·LAN：网卡(网卡正式用语应为Network Card) ·MODM：数据卡或调制解调器Modem ·HUB：集线器 ·WebCam：网络摄影机 ·Capture：影音采集卡 ·Case：机箱 ·Power：电源

·Moniter：屏幕，CRT为显像管屏幕，LCD为液晶屏幕 ·USB：通用串行总线Universal Serial Bus，用来连接外围装置·IEEE1394：新的高速序列总线规格Institute of Electrical Ａnd Electronic Engineers ·Mouse：鼠标，常见接口规格为PS/2与USB ·KB：键盘，常见接口规格为PS/2与USB ·Speaker：喇叭 ·Printer：打印机 ·Scanner：扫描仪 ·UPS：不断电系统 ·IDE：指IDE接口规格Integrated Device Electronics，IDE接口装置泛指采用IDE接口的各种设备 ·SCSI：指SCSI接口规格Small Computer System Interface，SCSI接口装置泛指采用SCSI接口的各种设备 ·GHz：(中央处理器运算速度达)Gega赫兹/每秒 ·FSB：指“前端总线(Front Side Bus)”频率，以MHz为单位·ATA：指硬盘传输速率AT Attachment，ATA-133表示传输速率为133MB/sec ·AGP：显示总线Accelerated Graphics Port，以2X，4X，8X表示传输频宽模式 ·PCI：外围装置连接端口Peripheral Component Interconnect ·ATX：指目前电源供应器的规格，也指主机板标准大小尺寸·BIOS：硬件(输入/输出)基本设置程序Basic Input Output System

way 用法

表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如：我不喜欢他说话的态度。正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。一、in a way的用法表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。三、by the way的用法

The way的用法及其含义(一)

The way的用法及其含义（一）有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析：一、the way作先行词，后接定语从句以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.

53个英文缩写

53个英文缩写 PC：个人计算机Personal Computer ·CPU：中央处理器Central Processing Unit ·CPU Fan：中央处理器的“散热器”(Fan) ·MB：主机板MotherBoard ·RAM：内存Random Access Memory，以PC-代号划分规格，如PC-133，PC-1066，PC-2700 ·HDD：硬盘Hard Disk Drive ·FDD：软盘Floopy Disk Drive ·CD-ROM：光驱Compact Disk Read Only Memory ·DVD-ROM：DVD光驱Digital Versatile Disk Read Only Memory ·CD-RW：刻录机Compact Disk ReWriter ·VGA：显示卡(显示卡正式用语应为Display Card) ·AUD：声卡(声卡正式用语应为Sound Card) ·LAN：网卡(网卡正式用语应为Network Card) ·MODM：数据卡或调制解调器Modem ·HUB：集线器 ·WebCam：网络摄影机 ·Capture：影音采集卡 ·Case：机箱 ·Power：电源 ·Moniter：屏幕，CRT为显像管屏幕，LCD为液晶屏幕 ·USB：通用串行总线Universal Serial Bus，用来连接外围装置 ·IEEE1394：新的高速序列总线规格Institute of Electrical and Electronic Engineers ·Mouse：鼠标，常见接口规格为PS/2与USB ·KB：键盘，常见接口规格为PS/2与USB ·Speaker：喇叭 ·Printer：打印机 ·Scanner：扫描仪 ·UPS：不断电系统 ·IDE：指IDE接口规格Integrated Device Electronics，IDE接口装置泛指采用IDE接口的各种设备 ·SCSI：指SCSI接口规格Small Computer System Interface，SCSI接口装置泛指采用SCSI接口的各种设备 ·GHz：(中央处理器运算速度达)Gega赫兹/每秒 ·FSB：指“前端总线(Front Side Bus)”频率，以MHz为单位 ·A TA：指硬盘传输速率AT Attachment，ATA-133表示传输速率为133MB/sec ·AGP：显示总线Accelerated Graphics Port，以2X，4X，8X表示传输频宽模式

way 的用法

way 的用法【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗？ 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。【归纳总结】 ●way作“方法，方式”讲时，如表示“以……方式”，前面常加介词in。如例1； ●way作“方法，方式”讲时，其后可接不定式to do sth.，也可接of doing sth. 作定语，表示做某事的方法。如例2，例3；

计算机组装必懂的53个英文单词和缩写

计算机组装必懂的53个英文单词和缩写 PC：个人计算机Personal Computer CPU：中央处理器Central Processing Unit CPU Fan：中央处理器的“散热器”(Fan) MB：主机板MotherBoard RAM：内存Random Access Memory，以PC-代号划分规格，如PC-133，PC-1066，PC-2700 HDD：硬盘Hard Disk Drive FDD：软盘Floopy Disk Drive CD-ROM：光驱Compact Disk Read Only Memory DVD-ROM：DVD光驱Digital Versatile Disk Read Only Memory CD-RW：刻录机Compact Disk ReWriter VGA：显示卡(显示卡正式用语应为Display Card) AUD：声卡(声卡正式用语应为Sound Card) LAN：网卡(网卡正式用语应为Network Card) MODM：数据卡或调制解调器Modem HUB：集线器 WebCam：网络摄影机 Capture：影音采集卡 Case：机箱 Power：电源 Moniter：屏幕，CRT为显像管屏幕，LCD为液晶屏幕

USB：通用串行总线Universal Serial Bus，用来连接外围装置IEEE1394：新的高速序列总线规格Institute of Electrical and Electronic Engineers Mouse：鼠标，常见接口规格为PS/2与USB KB：键盘，常见接口规格为PS/2与USB Speaker：喇叭 Printer：打印机 Scanner：扫描仪 UPS：不断电系统 IDE：指IDE接口规格Integrated Device Electronics，IDE接口装置泛指采用IDE接口的各种设备 SCSI：指SCSI接口规格Small Computer System Interface，SCSI 接口装置泛指采用SCSI接口的各种设备 GHz：(中央处理器运算速度达)Gega赫兹/每秒 FSB：指“前端总线(Front Side Bus)”频率，以MHz为单位 ATA：指硬盘传输速率AT Attachment，ATA-133表示传输速率为133MB/sec AGP：显示总线Accelerated Graphics Port，以2X，4X，8X表示传输频宽模式 PCI：外围装置连接端口Peripheral Component Interconnect ATX：指目前电源供应器的规格，也指主机板标准大小尺寸BIOS：硬件(输入/输出)基本设置程序Basic Input Output System

the-way-的用法讲解学习

t h e-w a y-的用法

The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.

(完整版)必须懂的53个电脑英文缩写

必须懂的53 个电脑英文缩写 PC:个人计算机Personal Computer CPU 中央处理器Central Processing Unit CPU Fan：中央处理器的散热器”Fan） MB 主机板MotherBoard RAM 内存Random Access Memory,以PC-代号划分规格，如PC-133, PC-1066，PC-2700 HDD 硬盘Hard Disk Drive FDD 软盘Floopy Disk Drive CD-ROM 光驱Compact Disk Read Only Memory DVD-ROM DVD光驱Digital Versatile Disk Read Only Memory CD-RW 刻录机Compact Disk ReWriter VGA显示卡（显示卡正式用语应为Display Card） AUD声卡（声卡正式用语应为Sou nd Card） LAN网卡（网卡正式用语应为Network Card） MODM数据卡或调制解调器Modem HUB集线器 WebCam网络摄影机 Capture :影音采集卡 Case :机箱 Power：电源 Mo niter :屏幕，CRT为显像管屏幕，LCD为液晶屏幕 USB :通用串行总线Universal Serial Bus ，用来连接外围装置 IEEE1394 :新的高速序列总线规格Institute of Electrical and Electronic Engineers Mouse :鼠标，常见接口规格为PS/2与USB KB :键盘，常见接口规格为PS/2与USB Speaker :喇叭 Printer :打印机 Scanner :扫描仪 UPS :不断电系统

way的用法总结大全

way的用法总结大全 way的用法你知道多少，今天给大家带来way的用法，希望能够帮助到大家，下面就和大家分享，来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地，大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。

way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句

使用电脑必懂的53个英文单词和缩写

·Moniter：屏幕，CRT为显像管屏幕，LCD为液晶屏幕 ·USB：通用串行总线Universal Serial Bus，用来连接外围装置·IEEE1394：新的高速序列总线规格Institute of Electrical Ａnd Electronic Engineers ·Mouse：鼠标，常见接口规格为PS/2与USB ·KB：键盘，常见接口规格为PS/2与USB ·Speaker：喇叭 ·Printer：打印机 ·Scanner：扫描仪 ·UPS：不断电系统 ·IDE：指IDE接口规格Integrated Device Electronics，IDE接口装置泛指采用IDE接口的各种设备·SCSI：指SCSI接口规格Small Computer System Interface，SCSI接口装置泛指采用SCSI接口的各种设备 ·GHz：(中央处理器运算速度达)Gega赫兹/每秒 ·FSB：指“前端总线(Front Side Bus)”频率，以MHz为单位·ATA：指硬盘传输速率AT Attachment，ATA-133表示传输速率为133MB/sec ·AGP：显示总线Accelerated Graphics Port，以2X，4X，8X表示传输频宽模式 ·PCI：外围装置连接端口Peripheral Component Interconnect ·ATX：指目前电源供应器的规格，也指主机板标准大小尺寸

the_way的用法大全教案资料

t h e_w a y的用法大全

The way 在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的

英文单词缩写规则

英文单词的缩写规则 1.单词缩写应省略在辅音之后，元音之前英文单词缩写一般以辅音结尾，而不以元音结尾。如American省略为Am，而不省略为Ame或Amer，Medicine或Medical缩写为Med，European缩写为Eur 等。但Science例外，缩写为Sci，可能是因为元音I之后又是元音E的缘故。缩写刊名每个词首字母必须大写，而不可全部都用大写或小写。 2.压缩字母法仅个别单词采用压缩字母方式缩写，如Japanese缩写为Jpn而不是Jan，National应缩写为Natl而不是Nat等。经常有读者将Japanese写成Jan是参考文献著录中常见的错误。如Japanese Journal of Ophthalmology，应缩写为Jpn J Ophthalmol，National Cancer Institute Research Report缩写为Natl Cancer Inst Res Rep。而Nat是Nature和Natural的缩写，如Nature Medicine，Nature biotechnology 分别缩写为Nat Med，Nat Biotechnol。另外CN是中国的国别代码，期刊缩写刊名中，China和Chinese不得缩写为CN，而应缩写为Chin。采用压缩写法是为了避免与其他常用缩写混淆。如Japanese不能缩写为Jan，可能是Jan是January的固定缩写形式，National缩写为Natl而不缩写为Nat，可能是Nat是Nature和Natural 的缩写。 3.学科名称缩写刊名中学科名称缩写很常见，因而了解学科名缩写规则非常必要。凡以-ogy 结尾的单词，一律将词尾-ogy去掉，如Cardiology缩写为Cariol，Biology缩写为

the way 的用法

The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.

the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms. 从那天起,其他同学是夹着书本来上课,而他们却带着"失败"的思想负担来上课.