maize

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Agrobacterium tumefaciens-Mediated Transformation of Maize Endosperm as a Tool to Study Endosperm

Cell Biology1[OA]

Francisca C.Reyes2,Beimeng Sun2,Hena Guo,Darren(Fred)Gruis,and Marisa S.Otegui*

Department of Botany,University of Wisconsin,Madison,Wisconsin53706(F.C.R.,M.S.O.);and

Pioneer Hi-Bred International,a Dupont Business,Johnston,Iowa50131(B.S.,H.G.,D.G.)

Developing maize(Zea mays)endosperms can be excised from the maternal tissues and undergo tissue/cell-type differen-tiation under in vitro conditions.We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors.We show that both aleurone and starchy endosperm cells can be successfully transformed using a short cocultivation with A.tumefaciens cells.The highest transformation rates were obtained with the A. tumefaciens EHA101strain and the pTF101.1binary vector.The percentage of aleurone cells transformed following this method varied between10%and22%whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells.Cultured endosperms undergo normal cell type(aleurone and starchy endosperm)differentiation and storage protein accumulation,making them suitable for cell biology and biochemical studies.In addition,transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.

The endosperm is a unique plant tissue that arises from a second fertilization event between a male gamete and the central cell.Its main function is to provide nutrients to the embryo either during seed development or during germination.In cereals,the endosperm consists of three main cell types:the starchy endosperm cells,which constitute the bulk of the endosperm and accumulate large quantities of storage proteins and starch;the epidermal aleurone cells;and the transfer cells,which are in contact with the maternal vascular tissues(Olsen,2004).The cereal endosperm is important as a model system to study plant development,cell differentiation,programmed cell death,and synthesis,traf?cking,and accumula-tion of storage compounds.In addition,it is a major source of carbohydrate and proteins for human and animal nutrition.

In spite of its importance,cell biology studies on the cereal endosperm using modern imaging approaches such as expression of?uorescent subcellular markers are very scarce because:(1)the endosperm is deeply immersed in maternal tissues and therefore,not read-ily available for imaging analysis and(2)the long time required for transformation and regeneration of stable transgenic plants.Although several approaches for culturing maize(Zea mays)endosperm in vitro have been reported in the past years(Shimamoto et al., 1983),only recently a novel method developed by Odd-Arne Olsen and colleagues(Gruis et al.,2006)has proven to be successful in retaining endosperm tissue and cell type identity in in vitro conditions.Cultures derived from transgenic maize lines in which endo-sperm cell types are identi?ed by the activity of speci?c promoters have shown that aleurone and starchy endosperm cell identity continues to be estab-lished in vitro(Gruis et al.,2006).

Although Agrobacterium tumefaciens is not a natural pathogen of most monocots(Cleene,1985;Binns and Thomashow,1988),it has been successfully used to transform many cereals,including maize,wheat(Tri-ticum aestivum),Sorghum,barley(Hordeum vulgare), and rice(Oryza sativa;Grimsley et al.,1989;Gould et al.,1991;Chan et al.,1993;Ishida et al.,1996,2007; Gurel et al.,2009;Harwood et al.,2009;Hensel et al., 2009).In the case of maize,stable transgenic plants can be obtained by A.tumefaciens-mediated transformation using either super-binary or standard-binary vectors (Frame et al.,2002;Mohanty et al.,2009a,2009b). However,transformation of isolated maize endo-sperms have been only possible using transient trans-formation approaches such as biolistic methods (Torrent et al.,1997;Gruis et al.,2006)and protoplast transfection(Gallie and Young,1994).Unfortunately, these two methods are not always ideal for cell biology studies.On one hand,biolistic methods often result

1This work was supported by a National Research Initiative competitive grant(no.2008–35304–18672)from the U.S.Department of Agriculture National Institute of Food and Agriculture(to M.S.O.).

2These authors contributed equally to the article.

*Corresponding author;e-mail otegui@https://www.wendangku.net/doc/ae4608162.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/ae4608162.html,)is: Marisa S.Otegui(otegui@https://www.wendangku.net/doc/ae4608162.html,).

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

https://www.wendangku.net/doc/ae4608162.html,/cgi/doi/10.1104/pp.110.154930

in high-copy number transgenic events and on the other,protoplasts are usually highly stressed cells not suitable for detailed protein localization studies.A. tumefaciens-mediated transformation methods circum-vent these disadvantages by resulting in a low-copy number of transgenes in intact tissues.

We have developed a method to transform in vitro-grown endosperms using a brief incubation time with A.tumefaciens cells carrying standard binary vectors. We present here a detailed explanation of the method and quantitative information on the transformation ef?ciency using different A.tumefaciens strains,culture density,and incubation time.We also provide evi-dence that the in vitro-differentiated aleurone and starchy endosperm cells are comparable to the corre-sponding cell types differentiated in planta and there-fore,suitable for cell biology studies.In addition,we show that transgenic cultured endosperms are able to express and accumulate epitope-tagged storage pro-teins that can be isolated for biochemical assays or used for immunolabeling imaging techniques.

RESULTS AND DISCUSSION

In Vitro-Cultured Endosperms Show Normal Cell Structural Features and Storage Protein

Accumulation Patterns

Endosperms from maize inbred lines with good culture growth properties,such as A636,are able to proliferate very well under in vitro conditions.More-over,endosperms grown according to the endosperm in vitro culture system(EICS)developed by Gruis et

al. Figure1.Structural features of aleurone and starchy endosperm cells.A to D,Overviews and cross sections of cultured endosperms at different stages of development.Developing endosperms were excised at6DAPand kept in culture for1(A),4(B),

6(C),and8(D)d.Note the differentiating aleurone(Al)and starchy endosperm(St E)cells in6+6(6DAP+6DIC)and6+8 endosperms.E,Longitudinal section of an A636maize kernel at23DAPand the corresponding endosperm cross section showing

the aleurone and starchy endosperm cells.E,Embryo;End,endosperm.F,Accumulation of endogenous22-kD a,15-kD b,and

27-kD g zeins in developing kernels(K)at6,9,12,16,23,and30DAP and in cultured endosperms(E)at the corresponding developmental stages(6+0,6+3,6+6,6+10,6+17,6+24).G to L,Ultrastructural features of aleurone and starchy endosperm cells from in vitro-and in planta-grown endosperms.Protein storage vacuoles(PSV)and lipid bodies(LB)developed

in aleurone cells of both systems(G and J).Based on the results from immunolabeling experiments with speci?c antibodies,the deposition patterns of the22-kD a(H and K)and27-kD g zeins(I and L)in protein bodies of starchy endosperm cells are also identical in both systems.Bars=0.5mm in A,B,C,D,and E;50m m in A’,B’,C’,D’,and E’;1m m in G and J;and200nm in H,I,

K,and L.

Agrobacterium-Mediated Transformation of Maize Endosperm

(2006)are able not only to proliferate but also to establish aleurone and starchy endosperm cell iden-tity.This has been shown by monitoring the activation of starchy endosperm-and aleurone-speci?c pro-moters.Endosperms are excised from the kernels6d after pollination(DAP)and kept on agar medium containing Murashige and Skoog basal medium,vita-mins,amino acids,cytokinin,and15%Suc.In vitro-grown endosperms do not reach the same?nal size as endosperms developed in planta(Fig.1);in addition, tissue differentiation is accelerated in in vitro-com-pared to in planta-grown endosperms.We observed that the epidermal layer started to acquire structural features of aleurone cells around6d in culture(DIC), that is6DAP plus6DIC(or6+6endosperms;Fig.1C [C’]).By the6+8stage,aleurone cells and starchy endosperm cells were clearly differentiated,the sur-face of the cultured endosperms had acquired a yel-lowish color due to the accumulation of lipids bodies in the aleurone cells,and the starchy endosperm cells contained large starch granules(Fig.1D[D’]).In terms of endosperm differentiation,the6+8endosperm stage was equivalent to approximately22to25DAP endosperms developed in planta(Fig.1E[E’]).

In addition,we also compared the steady-state levels of the22-kD a,15-kD b,and27-kD g zeins between in vitro-and in planta-grown endosperms (Fig.1F).Developing kernels at6,9,12,16,23,and30 DAP and cultured endosperms at the equivalent time points(6DAP+0DIC or6+0,6+3,6+6,6+10, 6+17,6+24)were collected and assayed for storage protein expression.Western-blot analysis of the27-kD g zein showed that the earliest detectable expression starts at9DAP(6+3stage for cultured endosperms) with steadily increasing expression through30DAP (6+24stage for cultured endosperms)both in cultured and in planta-grown endosperms.Twenty-seven kil-dalton g zein accumulation appears higher in in vitro-grown endosperm compared to developing kernels at the equivalent developmental stage,which is consis-tent with the zein transcript pro?les reported previ-ously by Gruis et al.(2006)and our own observations that cultured endosperm show accelerated differen-tiation(Fig.1,D and E[D’and E’]).Comparable expression patterns were also observed for other en-dosperm storage proteins,such as22-kD a and15-kD b zeins(Fig.1F).Thus,the endogenous storage pro-teins follow similar expression patterns in cultured and in planta-developed endosperms.

At the ultrastructural level,aleurone and starchy en-dosperm cells from in vitro-and in planta-developed endosperms were similar.Protein storage vacuoles and lipid bodies formed in aleurone cells(Fig.1,G and J) and protein bodies and starch granules?lled the starchy endosperm https://www.wendangku.net/doc/ae4608162.html,ing speci?c antibodies (Woo et al.,2001;Holding et al.,2007),we also checked the deposition patterns of the22-kD a and the27-kD g zeins in protein bodies of starchy endosperm cells from6+8endosperms and23-DAP kernels and found that they were identical,with27-kD g zein located in the outer zone and22-kD a zein occupying the central core of the protein bodies(Fig.1,H,I,K,and L). Previous analysis on cell-type-speci?c promoter activity and transcript pro?les(Gruis et al.,2006) together with our structural and immunolabeling analysis indicate that EICS is a suitable system for studying cell biology aspects of endosperm develop-ment and differentiation in maize.

A.tumefaciens-Mediated Endosperm Transformation Using Standard Binary Vectors

To analyze membrane dynamics or any other traf-?cking process it is often necessary to introduce trans-genes,such as subcellular?uorescent markers,into the cell/tissues under study.We developed a protocol to transform in vitro-grown maize endosperms using A. tumefaciens-mediated transformation.This method al-lows for the incorporation of a low-copy number of transgenes in intact endosperm tissues.We used the A. tumefaciens strain EHA101(Hood et al.,1986)and a pTF101.1binary vector(Frame et al.,2002)containing GFP coding sequence fused to the endoplasmic retic-ulum(ER)retention signal KDEL under the control of the rice actin1promoter,OsAct1prom(McElroy et al., 1991).This promoter has been shown to be active in both aleurone and starchy endosperm cells of cereals (Cho et al.,

2002).

Figure2.Confocal images of in vitro-grown endosperms(6+8 developmental stage)transformed with an ER-targeted GFP construct. Endosperm tissues were stained with propidium iodide to visualize cell walls.A,Paradermal overview of the aleurone layer showing aleurone cells expressing ER-targeted GFP(asterisks).B,Detail of an aleurone cell expressing ER-targeted GFP.C,Overview of an endosperm cross section showing transformed starchy endosperm cells(asterisks).D, Detail of a starchy endosperm cell expressing ER-targeted GFP.Bars= 50m m in A and C;5m m in B;20m m in D.Al,Aleurone layer;St E, starchy endosperm.

Reyes et al.

For transformation,6DAP excised developing en-dosperms were cocultivated for 3min with different culture densities of A.tumefaciens and placed on solid EICS culture medium supplemented with 500m g/mL carbenicillin.

EICS Transformation Ef?ciency Is Highly Dependent on A.tumefaciens Culture Density

Transformation ef?ciency was evaluated using two parameters:(1)the percentage of transformed epider-mal/aleurone cells and (2)the number of cell layers underneath the epidermis/aleurone layer containing transformed cells (Fig.2).Since auto?uorescence is a common phenomenon in the maize endosperm,only those cells that showed a ?uorescence ER pattern and emission spectra corresponding to GFP (measured with the meta detection system of the Zeiss 510LSM)were scored as transformed cells (Fig.2).The number

of transformed cells was analyzed at 4,6,and 8d after cocultivation (Fig.3A).

We found that the transformation ef?ciency in the epidermal/aleurone layer was directly correlated to the density of A.tumefaciens cultures used to trans-form the endosperms.With the lowest A.tumefaciens concentration we tested (optical density at 600nm [OD 600]=0.2)the percentage of transformed epider-mal/aleurone cells varied between 5%and 15%,whereas with the highest concentration (OD 600=0.8),the transformation ef?ciency in the epidermal/aleu-rone layer varied between 10%and 22%(Fig.3A).No major changes were found in the percentage of trans-formed cells at the different time points checked in this study (Fig.3A).

We also analyzed the number of transformed starchy endosperm cells in endosperm cross sections.Trans-formed cells were detected from the ?rst layer (the starchy endosperm cells right beneath the aleurone layer)up to the eighth layer of starchy endosperm cells (Table I).

Active periclinal (parallel to the surface of the epi-dermal layer)divisions have been reported to occur in in vitro-grown endosperms (Gruis et al.,2006).There-fore,at least some transformed starchy endosperm cells found in deeper areas of the endosperms are likely to be derived from transformed epidermal/aleurone cells.

Transformation Ef?ciency Is Higher with the A.tumefaciens Strain EHA101

To evaluate the ability of a different A.tumefaciens strain to transform maize endosperms,the same bi-nary vector containing the GFP reporter was intro-duced into A.tumefaciens GV3101cells.We used both A.tumefaciens EHA101and GV3101strains for side-by-side endosperm transformation at a concentration of 0.8OD 600and during 3-or 10-min cocultivation time (Fig.3B).We analyzed the transformation ef?ciency in the aleurone layer 8d after cocultivation.We found that the transformation ef?ciency was much higher when the EHA101strain was used.In fact,no trans-formed cells were detected after the 3-min incubation treatment with the GV3101strain (Fig.

3B).

Figure 3.Transformation ef?ciency of epidermal/aleurone cells in in vitro-grown endosperms.Endosperms were transformed with A.tume-faciens cells carrying a pTF101.1-derived binary vector containing an ER-targeted GFP under the control of the OsAct1promoter.A,Trans-formation ef?ciencies recorded at different time points after 3min cocultivation with different cell culture densities of A.tumefaciens EHA101.B,Transformation ef?ciencies using two A.tumefaciens strains (EHA101and GV3101)and different cocultivation times.Trans-formation ef?ciency was measured in the epidermal/aleurone layer 8d after cocultivation.The data depicted in both graphs correspond to the average of three independent experiments with three biological repli-cates in each.ND,Not detected.

Table I.Transformation ef?ciency of starchy endosperm cells Number of starchy endosperm cell layers showing transformed cells after cocultivation with different cell densities of A.tumefaciens EHA101.The results correspond to the average and the SE for three independent experiments with three biological replicates each.

OD 600

No.of Starchy Endosperm Cell Layers with Transformed Cells

Day 4

Day 6

Day 8

0.23602613610.43613603600.63614614610.8561462563

Agrobacterium-Mediated Transformation of Maize Endosperm

Transgenic in Vitro-Cultured Endosperms Express and Accumulate Tagged Storage Proteins

To assess the accumulation levels of proteins en-coded by transgenes transformed into in vitro-grown endosperms,we expressed the 22-kD a ,15-kD b ,and the 27-kD g zeins fused to the HSV ,FLAG,and HA tags,respectively.For driving high expression in the

starchy endosperm,we used the Floury2promoter (FL2prom ;World Intellectual Property Organization patent publication no.9802563)for the expression of 22-kD a zein-HSV and the maize 27-kD g zein promoter (27kDZein prom ;Ueda and Messing,1991;Russell and Fromm,1997)for the expression of 15-kD b zein-FLAG and 27-kD g zein-HA.The CZ19B1prom :DsRed trans-gene was built in the same epitope-tagged zein

con-

Figure 4.Expression of epitope-tagged storage pro-teins in in vitro-grown endosperms.Endosperms were transformed with C-terminal HSV-tagged 22-kD a zein (A and A’),FLAG-tagged 15-kD b zein (B and B’),or HA-tagged 27-kD g zein constructs containing also the CZ19B1prom :DsRed reporter gene.Light (A–C)and ?uorescence images (A’–C’)of transformed endosperms.D,Immunoblot analysis of storage protein expression in in vitro-grown endo-sperms using epitope tag antibodies and antibodies against the native storage

proteins.

Figure 5.Chart showing the main steps and timeline of the protocol to transform in vitro-grown endo-sperms using A.tumefaciens EHA101cells carrying pTF101.1-derived binary vectors.

Reyes et al.

structs to monitor endosperm transformation since 19-kD b zein1promoter(CZ19B1prom;U.S.patent 6225529)shows speci?c expression in the starchy endosperm.Sectors with strong red?uorescence were observed in the transformed cultured endo-sperms(Fig.4,A–C[A’–C’])whereas no?uorescence was detected in control cultures(Agrobacterium incu-bation omitted),con?rming successful endosperm transformation.

Fifteen days after transformation,endosperms showing red?uorescence were collected and ground for protein extraction.The protein extracts were sub-jected to SDS-PAGE followed by immunoblot analysis using antibodies against the epitope tags as well antibodies against the corresponding endogenous storage zein proteins(Woo et al.,2001;Fig.4D).Im-munoblot detection with antibodies against the HSV, FLAG,and HA tags con?rmed the expression and accumulation of22-kD a zein-HSV,15-kD b zein-FLAG,and27-kD g zein-HA in transgenic cultured endosperms,indicating that in vitro-grown endo-sperms can be used for expression and isolation of transgenic endosperm storage proteins or for immu-nolabeling imaging.

CONCLUSION

We have developed a protocol to transform endo-sperm tissue grown in vitro.Since in vitro-cultured endosperms undergo normal differentiation of aleu-rone and starchy endosperm cells,the possibility to introduce transgenes such as?uorescent subcellular markers,allows for easy imaging access to different endosperm cell types that are usually deeply im-mersed within other tissues.

The ability to introduce transgenes into in vitro-cultured developing endosperms provides biochemi-cal and molecular means to study cereal endosperm cell fate differentiation and early endosperm develop-ment.The possibility of imaging?uorescent subcellu-lar markers in both aleurone and starchy endosperm cells following a very simple and short protocol(Fig.5) represents an important technical advance in cell biology studies of differentiating cereal endosperm cells.In addition,this system offers the possibility of expressing and isolating tagged/modi?ed endosperm storage proteins that cannot be successfully expressed in other tissues/systems.

MATERIALS AND METHODS

Isolation and in Vitro Growth of Endosperms Maize(Zea mays;inbred line A636)were grown in a greenhouse under a 14-h light/10-h dark photoperiod,supplemental lighting(700m mol m22s21), and average temperature of28°C during the day and21°C during the night. Endosperms were isolated and cultured in vitro as described by Gruis et al. (2006).Brie?y6DAP ears were harvested and surface https://www.wendangku.net/doc/ae4608162.html,ing a scalpel the kernels were dissected,the maternal tissue removed,and the fertilized embryo sacs isolated to obtain clean endosperms.Developing endosperms were immediately placed on liquid EICS culture medium until cocultivation with Agrobacterium tumefaciens.After the cocultivation,the isolated endosperms were placed on solid EICS culture medium(4.3g/L Murashige and Skoog media;0.5%v/v Murashige Skoog vitamins stock solu-tion;5mg/L thiamine HCl;400mg/L Asn;10m g/L6-benzylaminopurine;15% Suc;and3g/L Gelrite,pH to5.8)supplemented with500m g/mL carbenicillin and kept on dark at25°C.Isolated endosperm not exposed to A.tumefaciens cultures were used as a control.

Plasmids

A DNA fragment containing OsActin1prom:GFP-KDEL was cloned into the pTF101.1vector using a two-step cloning strategy.The GFP-KDEL sequence was ampli?ed from the CD3-955vector(Nelson et al.,2007)using the forward primer5#-TCTAGAATGAAGGTACAGGAGGGT-3#and the reverse primer 5#-CCCGGGTTACAGCTCGTCATG-3#,containing Xba I and Xma I restriction sites(underlined),respectively,and cloned into pTF101.1.The OsActin1 promoter was ampli?ed from the pDM302vector using the following primers: forward5#-AAGCTTGAAGAGAGTCGGGATAGTC-3#containing a Hin dIII restriction5#-TCTAGACAGAAATATATAAAAATATAAAC-CAT-3#containing an Xba I restriction site.The vector pDM302carrying the OsActin1promoter was kindly donated by Ajay Garg(Cornell University)and the GFP coding region fused to the ER retention signal KDEL(CD3-955; Nelson et al.,2007)was obtained from the Arabidopsis Biological Resource Center at Ohio State University.The resulting plasmid was introduced into competent A.tumefaciens cells by freeze-thaw transformation(Chen et al., 1994).

Additional binary vectors used in this study contained CZ19B1prom(U.S. patent6225529)fused to DsRed(CLONTECH),FL2prom:22kD a zein fused to the HSV tag(QPELAPEDPED),27kDZein prom:15kD b zein gene fused to FLAG (DYKDDDDK),and the27kDZein prom:27kD g zein gene fused to HA(YPYDVP-DYA).Sequence data for the zein genes used in this article can be found in the GenBank/EMBL data libraries under accession numbers AF371261, AF371264,and AF371274.

A.tumefaciens-Mediated Transformation

A.tumefaciens EHA101or GV3101strains carrying the different constructs were grown at25°C for2d in Luria-Bertani medium supplemented with the appropriate antibiotics for strain(100m g/mL kanamycin for EHA101or100 m g/mL gentamicin plus10m g/mL rifampicin for GV3101)and plasmid selection(100m g/mL spectinomycin for pTF101.1).After2d of growth,the cultures were centrifuged and washed twice with the in?ltration media(EICS culture media supplemented with100m M acetosyringone).Finally the bacte-rial suspension was diluted with in?ltration media to adjust the inoculum concentration to the?nal OD600value.

Transformation was performed by cocultivating the isolated endosperms with the bacterial suspensions under gentle agitation.After cocultivation with A.tumefaciens cultures,the endosperms were washed three times with EICS culture medium supplemented with carbenicillin,plated,and kept in the dark.

Confocal Imaging of Fluorescent Proteins

Cross or paradermal sections of the transformed endosperms were imaged using a510Zeiss laser-scanning confocal microscope.The transformed tissues were excited with488nm and the GFP emission was detected using a500to 530band-pass?lter.The GFP emission spectrum was collected for every image using the spectral meta detector.Only those cells that showed a positive signal for GFP were scored as transformed cells.

The percentage of transformed cells was calculated by determining the number of transformed cells over the total of cells per?eld.The number of total cells per?eld ranged from45to160in the different images obtained.At least three?elds were analyzed for each section and a total of three indepen-dent in vitro-grown endosperms were analyzed.

For determining the number of starchy endosperm layers(layers under-neath the epidermal/aleurone layer)containing transformed cells,we used transverse sections of the endosperms.

The images were analyzed using the LSM image browser(www.zeiss. com/lsm)and edited using Adobe Photoshop CS4.

Agrobacterium-Mediated Transformation of Maize Endosperm

Structural Characterization and Gold Immunolabeling In vitro-grown endosperms at different stages of development(6+1,6+3, 6+6,and6+8)and thin slices of endosperm tissue from developing kernels were processed by high-pressure freezing/freeze substitution.Pieces of tissue were transferred to freezing planchettes containing0.1M of Suc and high-pressure frozen in a Baltec HPM010unit(Technotrade).Substitution was performed in2%OsO4in anhydrous acetone at280°C for72h,and followed by slow warming to room temperature over a period of2d.After several acetone rinses,samples were removed from the holders and in?ltrated in Epon resin(Ted Pella Inc.)according to the following schedule:5%resin in acetone(4h),10%resin(12h),25%resin(12h),50%,75%,and100%(24h each concentration).Polymerization was carried out at60°C.Sections were stained with2%uranyl acetate in70%methanol for10min followed by Reynold’s lead citrate(2.6%lead nitrate and3.5%sodium citrate,pH12)and observed in a FEI CM120electron microscope.

For gold immunolabeling,high-pressure frozen samples were substituted in0.2%uranyl acetate(Electron Microscopy Sciences)plus0.2%glutaralde-hyde(Electron Microscopy Sciences)in acetone at280°C for72h,and warmed to250°C for24h.After several acetone rinses these samples were in?ltrated with Lowicryl HM20(Electron Microscopy Sciences)for72h and polymerized at250°C under UV light for48h.Sections were mounted on formvar-coated nickel grids and blocked for20min with a10%(w/v)solution of nonfat milk in phosphate-buffered saline(PBS)containing0.1%Tween20. The sections were incubated with the primary antibodies(1:10in PBS-Tween 20)for1h,rinsed in PBS containing0.5%Tween20,and then transferred to the secondary antibody(anti-rabbit IgG1:10)conjugated to15-nm gold particles for1h.Controls omitted the primary antibodies.The antibodies against22-kD a zeins and27-kD g zeins have been characterized elsewhere(Woo et al.,2001; Holding et al.,2007).

Immunoblot Analysis

In vitro-grown endosperms were ground with a plastic pestle in extraction buffer(50m M Tris-HCl,5m M EDTA,and2%SDS;pH8.0)containing a protease inhibitor cocktail(Sigma-Aldrich)at a1:2(w/v)ratio.Cellular debris was removed by centrifugation at140,000rpm for10min.Protein extracts were diluted1:2(v/v)in sample buffer and boiled for5min before separation through a4%to12%(w/v)SDS polyacrylamide gel(Invitrogen)and then transferred to a cellulose membrane in a submerged blotting system(Mini-Trans Blot;Invitrogen).Membranes were blocked for1h with Tris-buffered saline containing5%(w/v)nonfat dry milk.Immunoblotting was performed using the corresponding epitope tag antibodies(HA,FLAG,HSV;Sigma-Aldrich),or the corresponding storage protein antibodies(22-kD a,15-kD b, and27-kD g zeins;Woo et al.,2001),and the anti-rabbit/anti-mouse secondary antibodies(Bio-Rad)in Tris-buffered saline containing0.1%(v/v)Tween20. Proteins that cross-reacted with antibodies were detected with chemilumi-nescent substrates(Pierce)and visualized on?lm.

The anti-22-kD a zein antibody was used at a dilution of1:3,000,the anti-15-kD b zein antibody was used at1:3,500,and the anti-27-kD g zein antibody, at1:3,500.Antibodies against HA,FLAG,and HSV were used at dilutions of 1:8,000,1:8,000,and1:4,000,respectively.

Sequence data for the zein genes used in this article can be found in the GenBank/EMBL data libraries under accession numbers AF371261, AF371264,and AF371274.

ACKNOWLEDGMENTS

We would like to thank Kan Wang(Iowa State University)for providing the A.tumefaciens EHA101strain and the pTF101.1binary vector,Ajay Gang (Cornell University)for providing the OsAct1promoter,and the Arabidopsis Biological Resource Center for the CD3-955plasmid.We also thank Rafael Buono(University of Wisconsin-Madison)for his photography assistance, Gabriele Monshausen(Pennsylvania State University)for her comments on the manuscript,Jerry Ranch,Kimberly Glassman,and Guo Hena(Pioneer Hi-Bred International)for their technical assistance,and Odd-Arne Olsen (University of Norway)for useful discussions about the data presented in this study.

Received February15,2010;accepted March29,2010;published March31, 2010.LITERATURE CITED

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Agrobacterium-Mediated Transformation of Maize Endosperm

电力系统中目前使用的变压器 电抗器多含有有 、.

简 介 电力系统中目前使用的变压器、电抗器多含有有载调压机构，分接头的位置是变压器、电抗器的重要信息。测控单元在采集分接头位置信号时，通常提供的开关量位置较少，因此通常对分接头位置进行编码，转换成与测控系统相适应的 BCD 方式输出。 该装置是配合变电站实现电力调度自动化、无人值班化的一种自动监测仪器。它将来自主变压器有载调压分接开关的升、降、停调压控制、档位机械分接点位置监测、远方/就地控制等功能集于一体。可以在就地位置实现升、降、停操作，也可以与综合自动化系统的测控装置接口，进行远方遥控操作，并且遥测档位位置。 该装置可以满足三种输入方式：（1）一对一（每个档位对应一付空接点）；（2）编码方式（1-9分别对应一付空接点，10位对应一付空接点）；（3）BCD 输入方式。 输出方式：BCD 或HEX 输出。 结构上采用了屏柜安装方便快捷。 技术参数 额定工作电压： DC220V/110V 编码输出类型： BCD 或HEX 输出 输入最大档位数：19档(更多档位订货时注明) 档位输入类型： 一对一的输入、编码输入、BCD 输入 装置端子定义图 输出方式： 空接点输出 输出接点容量： 载流容量 5A 接点断弧容量： 60W(220VDC)；2000VAC 安装方式： 柜面开孔安装

装置电原理图 装置典型使用接线 接线图如下： 1一对一输入的接线方式 2 编码输入的接线方式（仅适用于BCD输出方式时） 3 BCD输入的接线方式（仅适用于BCD输出方式时） 装置操作说明

运行指示灯：档位控制器上电，运行正常时运行灯点亮(绿色)。 远方、就地选择开关 远方位置：允许测控装置通过档位控制器进行调压机构遥控操作。 就地位置：允许通过装置面板上的升、降按钮进行调压机构操作。 升、降、停按钮 升、降按钮：就地操作时，通过面板上的升、降按钮可以实现调压机构的就地升降；档位控制器面板上的按钮只在就地位置时，升、降才有效。 停按钮：按下停按钮时，切断调压机构电源，禁止调压操作；停接点不受远方就地的控制。 码制转换（√表示输入相应档位时该接点与BCOM为通路） BCD码输出：用跳帽将J2、J4、J8、JA跳至“BCD”位置 BCD码输出逻辑23~44 输入档位数码管显示 1 2 4 8 A 无输入00 档位1 01 √ 档位2 02 √ 档位3 03 √√ 档位4 04 √ 档位5 05 √√ 档位6 06 √√ 档位7 07 √√√ 档位8 08 √ 档位9 09 √√ 档位10 10 √ 档位11 11 √√ 档位12 12 √√ 档位13 13 √√√ 档位14 14 √√ 档位15 15 √√√

电抗器的基本结构

电抗器的基本结构 一、铁心式电抗器的结构 铁心式电抗器的结构与变压器的结构相似，但只有一个线圈——激磁线圈；其铁心由若干个铁心饼叠置而成，铁心饼之间用绝缘板（或纸板、酚醛纸板、环氧玻璃布板）隔开，形成间隙；其铁轭结构与变压器相同，铁心饼与铁轭由压缩装置通过螺杆拉紧，形成一个整体，铁轭和所有的铁心饼均应接地。铁心结构，铁心饼由硅钢片叠成，叠片方式有以下几种： （a）单相电抗器铁心；（b）三相电抗器铁心 （1）平行叠片 其叠片方式，与一般变压器相同，每片中间冲孔，用螺杆、压板夹紧成整体，适用于较小容量的电抗器。 （2）渐开线状叠片 其叠片方式，与渐开线变压器的叠片方式相同，中间形成一个内孔，外圆与内孔直径之比约为4:1至5:1，适用于中等容量的电抗器。 （3）辐射状叠片 其叠片方式，硅钢片由中心孔向外辐射排列，适用于大容量电抗器。 （a）平行叠片；（b）渐开线状叠片；（c）辐射状叠片 在平行叠片铁心中，由于气隙附近的边缘效应，使铁心中向外扩散的磁通的一部分在进入相邻的铁心饼叠片时，与硅钢片平面垂直，这样会引起很大的涡流损耗，可能形成严重的局部过热，故只有小容量电抗器才采用这种叠片方式。在辐射形铁心中，其向外扩散的磁通在进入相邻的铁心饼叠片时，与硅钢片平面平行，因而涡流损耗减少，故大容量电抗器采用这种叠片方式。 铁心式电抗器的铁轭结构与变压器相似，一般都是平行叠片，中小型电抗器经常将两端的铁心柱与铁轭叠片交错地叠在一起，为压紧方便，铁轭截面总是做成矩形或丁形。 二、空心式电抗嚣的结构 空心式电抗器就是一个电感线圈，其结构与变压器线圈相同。空心电抗器的特点是直径大、高度低，而且由于没有铁心柱，对地电容小，线圈内串联电容较大，因此冲击电压的初始电位分布良好，即使采用连续式线圈也是十分安全的。空心

电抗器与变压器是一样的产品吗

电抗器与变压器是一样的产品吗 电抗器也叫电感器，一个导体通电时就会在其所占据的一定空间范围产生磁场，所以所有能载流的电导体都有一般意义上的感性。然而通电长直导体的电感较小，所产生的磁场不强，因此实际的电抗器是导线绕成螺线管形式，称空心电抗器；有时为了让这只螺线管具有更大的电感，便在螺线管中插入铁心，称铁心电抗器。电抗分为感抗和容抗，比较科学的归类是感抗器（电感器）和容抗器（电容器）统称为电抗器，然而由于过去先有了电感器，并且被称谓电抗器，所以现在人们所说的电容器就是容抗器，而电抗器专指电感器。 什么叫变压器？ 变压器是一种用于电能转换的电器设备，它可以把一种电压、电流的交流电能转换成相同频率的另一种电压、电流的交流电能。 变压器几乎在所有的电子产品中都要用到，它原理简单但根据不同的使用场合（不同的用途）变压器的绕制工艺会有所不同的要求。变压器的功能主要有：电压变换；阻抗变换；隔离；稳压（磁饱和变压器）等，变压器常用的铁心形状一般有E型和C型铁心。 一、变压器的基本原理 当一个正弦交流电压U1加在初级线圈两端时，导线中就有交变电流I1并产生交变磁通ф1，它沿着铁心穿过初级线圈和次级线圈形

成闭合的磁路。在次级线圈中感应出互感电势U2，同时ф1也会在初级线圈上感应出一个自感电势E1，E1的方向与所加电压U1方向相反而幅度相近，从而限制了I1的大小。为了保持磁通ф1的存在就需要有一定的电能消耗，并且变压器本身也有一定的损耗，尽管此时次级没接负载，初级线圈中仍有一定的电流，这个电流我们称为"空载电流"。 如果次级接上负载，次级线圈就产生电流I2，并因此而产生磁通ф2，ф2的方向与ф1相反，起了互相抵消的作用，使铁心中总的磁通量有所减少，从而使初级自感电压E1减少，其结果使I1增大，可见初级电流与次级负载有密切关系。当次级负载电流加大时I1增加，ф1也增加，并且ф1增加部分正好补充了被ф2 所抵消的那部分磁通，以保持铁心里总磁通量不变。如果不考虑变压器的损耗，可以认为一个理想的变压器次级负载消耗的功率也就是初级从电源取得的电功率。变压器能根据需要通过改变次级线圈的圈数而改变次级电压，但是不能改变允许负载消耗的功率。 二、变压器的损耗 当变压器的初级绕组通电后，线圈所产生的磁通在铁心流动，因为铁心本身也是导体，在垂直于磁力线的平面上就会感应电势，这个电势在铁心的断面上形成闭合回路并产生电流，好象一个旋涡所以称为"涡流"。这个"涡流"使变压器的损耗增加，并且使变压器的铁心发

电源电压为1 100 V及以下的变压器、电抗器、电源装置和类似产品的

I C S29.180 K41 中华人民共和国国家标准 G B/T19212.17 2019 代替G B/T19212.17 2013 电源电压为1100V及以下的变压器二电抗器二电源装置和类似产品的安全第17部分:开关型电源装置和开关型电源装置用变压器的特殊要求和试验 S a f e t y o f t r a n s f o r m e r s,r e a c t o r s,p o w e r s u p p l y u n i t s a n d s i m i l a r p r o d u c t s f o r s u p p l y v o l t a g e s u p t o1100V P a r t17:P a r t i c u l a r r e q u i r e m e n t s a n d t e s t s f o r s w i t c hm o d e p o w e r s u p p l y u n i t s a n d t r a n s f o r m e r s f o r s w i t c hm o d e p o w e r s u p p l y u n i t s (I E C61558-2-16:2013,S a f e t y o f t r a n s f o r m e r s,r e a c t o r s,p o w e r s u p p l y u n i t s a n d s i m i l a r p r o d u c t s f o r s u p p l y v o l t a g e su p t o 1100V P a r t2-16:P a r t i c u l a r r e q u i r e m e n t s a n d t e s t s f o r s w i t c hm o d e p o w e r s u p p l y u n i t s a n d t r a n s f o r m e r s f o r s w i t c hm o d e p o w e r s u p p l y u n i t s,MO D) 2019-10-18发布2020-05-01实施 国家市场监督管理总局

电抗器与变压器异同

电抗器与变压器异同 maychang 电抗器(电感)与变压器最大的不同之处，是变压器并不存储能量，仅传输能量，而电抗器尤其是滤波电抗器必须存储能量。 变压器并不存储能量，空载时一次电流非常小，理想变压器二次空载时一次电流为零。一次之所以有电流，完全是二次电流反射到一次的结果。因此，变压器铁心的作用仅仅是使一次二次达到完全的耦合，也就是一次电流产生的磁场完全穿过二次绕组，二次电流产生的磁场也完全穿过一次绕组。对变压器来说，加在铁心上的限制只有一条：铁心中的磁通密度不得太大以致铁心达到深度饱和。因此，变压器铁心一般不留气隙，纯交流工作的变压器更是如此。 滤波电抗器则不然，它必须存储能量，无论是谐振回路中的电抗器，还是整流电路中的电抗器都必须存储能量。为使电抗器能够存储足够的能量，绝大多数电抗器(电感)中都留有气隙。当然，铁心中磁通密度仍不能太大以致铁心达到深度饱和这一限制条件在电抗器中仍存在，甚至比在变压器中更甚，因铁心中磁通密度即使浅饱和也将使电感量减小而使谐振频率发生变化。故谐振工作的电抗器中铁心磁通密度往往选择得比直流滤波电感中的磁通密度更小。 这一点可以从开关电源中使用的变压器看出来。正激方式工作的开关电源，无论是单端正激、推挽、半桥、全桥，其变压器一般不留气隙。而反激工作的开关电源，在开关管导通期间直流电源输出的能量存储在变压器中，开关管关断期间变压器向负载输出能量，故反激工作的开关电源变压器必留有气隙。留气隙之目的是在体积重量限制条件下存储最大的能量。 磁场强度、磁通密度和存储能量的关系如下

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GZYQ与电抗器及自耦变压器比较

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