两种质粒中gus基因在植物细胞和根癌农杆菌中的表达特性研究_英文_

基因组学与应用生物学，2009年，第28卷，第1期，第34－38页Genomics and Applied Biology,2009,Vol.28,No.1,34－38Research Report研究报告Study on Expression Character of gus Gene Contained in two Different Plas-mids on Plant Cell and Agrobacterium tumefaciensHuang Yonghong1Mei Mei2Zeng Jiwu1Zhou Birong1Xia Rui1Yi Ganjun1*1Fruit Research Institute,Guangdong Academy of Agricultural Science,Guangzhou,510640;2Jiaozhou Bureau of Agriculture,Qingdao,266300*Corresponding author,yiganjun@Abstract Using the Agrobacterium tumefaciens EHA105containing two different plasmids pBI121and pCAM-BIA2301to transform cucumis melon cotyledons respectively,and using a histochemical essay was used to identify the transient expression of gus gene on the cotyledons,callus induced from the cotyledons and the bacterium suspension,the expression characters of gus gene included in different plasmid victors on plant cells and bacterium cells were studied.The results showed different expression characters were possessed in different plasmid vectors. The gus gene contained in the pBI121was expressed not only in the plant cells,but also in the Agrobacterium tumefaciens cells;while the one included in the pCAMBIA2301was not expressed in the Agrobacterium tumefa－ciens cells but in the plant cells.Keywords gus gene,Plant cell,Agrobacterium tumefaciens,Plasmid vector两种质粒中gus基因在植物细胞和根癌农杆菌中的表达特性研究黄永红1梅眉2曾继吾1周碧容1夏瑞1易干军1*1广东省农业科学院果树研究所,广州,510640;2山东省胶州市农业局,青岛,266300*通讯作者,yiganjun@摘要以含有基因转化操作过程中常用的两种质粒载体pBI121和pCAMBIA2301的根癌农杆菌EHA105为材料，分别转化甜瓜子叶，应用组织化学方法检测了甜瓜子叶和子叶培养后的愈伤组织及根癌农杆菌菌液的瞬时转化效果，研究了两种不同的质粒载体上所含的gus基因在根癌农杆菌中和植物细胞中的表达特性。

结果表明，不同质粒载体上所含的gus基因的表达特性不同，质粒载体pBI121上所含的gus基因既能在植物细胞中能表达，也能在根癌农杆菌细胞中表达，而质粒载体pCAMBIA2301上所含的gus基因能在植物细胞中表达，但是不能在根癌农杆菌细胞中表达。

关键词gus基因,植物细胞,根癌农抗菌,质粒载体GUS(β－glucuronidase),a kind of hydrolase that catalyzes many chemical reactions of GUS-like zy-molytes,is encoded by gus gene.The advantages of it are mainly in followings:(1)It possesses very stable quality;(2)It needs no coenzyme to express activity;(3)It has no special requirement for ions;(4)The opti-mum pH is5.2~8.0;(5)It has certain heat stability(it's half life is2h at55℃).Moreover,it can be made quanti-tative assay by fluorescence spectrophotometer or used for localization of gene with histochemical method.It is with far-ranging applicability because there is a little or little content of intrinsic GUS-like activities in m any life-form systems and it has high sensitivity(jia and wang,1998).Until now,the gene has still being widely used for gene engineering and genetic transformation, especially in the experiments made use of its transient expression to determine and optimize various factors that influence transformation efficiency.The gus gene has been successfully applied in genetic transformation of plants such as flowers(Chin et al.,2007;Mishiba et al., 2005),rice(Lüet al.,2007),Cotton(Tohidfar et al.,2005), fruits(Kayim et al.,2004),Grass(Ge et al.,2007;Nan-dakumar et al.,2007),medical use plant(Tiwari et al., 2008)and even algae(Tan et al.,2005).However,the reports on the different expression char-acters of gus gene contained in different plasmid vec-tors were been seen for some reasons,which led to false results sometimes and cause the waste of time and resources in the end in plant genetic transforma-tion practices.The aim of the investigation reported here is to study different expression characters of gus genes con-tained in different plasmid vectors using two plasmid vectors in common use named pBI121and pCAMBIA-2301(Figure1)to find out the real expression characters and make good use of the reporter gene in plant genetic transformation.1Materials and Methods1.1Plant materialThe seeds of Cucumis melon GT－1were bought at the seed market.Agrobacterium tumegaciens strain At. -EHA105、engineering bacterium pBI121/At.EHA105 and pCAMBIA2301/At.EHA105were stored atprovincFigure1Gene diagram of T-DNA region of plasmid vector pBI121and pCAMBIA2301ial key laboratory on new technology of fruit and veg-etable.The seeds were shelled and surface-sterilized in 70%ethanol for15s,followed by a treatment with 0.1%corrosive sublimate for7min.After being rinsed in sterile distilled water for4times,they were placed on the sterile filter paper to dry and inoculated on the MS medium containing no plant hormone.Finally they were cultured in a simulate climate box which was con-trolled25℃under a(16/8h)(day/night)photoperiod with light intensity of3000Lx.All MS mediums were supplemented with3%sucrose,and solidified with0.7% agar.The pH of the mediums was adjusted to7.0with 1mol/L NaOH before autoclaving for25min at121℃.1.2Bacterium preparationAt.EHA105from a fresh plate colony was grown at 28℃overnight in liquid YEPmedium containing25mg/L Rifampicin and25mg/L Streptomycin.And pBI121/ At.EHA105and pCAMBIA2301/At.EHA105picked from a single colony on a fresh plate were cultured at 28℃overnight in liquid YEP medium containing25mg/L Rifampicin,25mg/L Streptomycin and50mg/L Kanamycin.All bacterial cells were collected by cen-trifugation and resuspended in MS liquid medium to a final OD560＝0.15.All the bacterial suspensions were di-vided in two parts,one for the transformation of Cucumis melon and the other for the staining reaction of GUS.1.3Cucumis melon transformationCotyledon explants were excised from5－day-old germfree seedlings and the cuts were1mm away from hypocotyls.Then the excised cotyledons were cut trans-versely at the middle into two pieces,the adaxial halives of which were used for transformation.The prepared cotyledon explants were immersed in the three sorts of bacterial suspensions for15min respectively,blotted up on a filter paper and then placed on co-culture medium. On the selection induction medium supplemented with 75mg/L Kana,callus was inducted after about2weeks. The activity of gus gene expressed in the cotyledon was determined after they were co-cultured with the bacteri-um for4days and that in the callus were tested when they were inducted on the induction medium using hisStudy on Expression Character of gus Gene Contained in two Different Plasmids on Plant Cell两种质粒中gus基因在植物细胞和根癌农杆菌中的表达特性研究35基因组学与应用生物学Genomics and Applied Biology tochemical method according to Jefferson(1987).And GUS in the bacterial cells was detected at the same time by the same way.1.4GUS assayA histochemical assay to detect GUS activity was performed on the cotyledons co-cultured with the bac-terium for4days,the callius inducted on the induction medium and one of the bacterial suspensions.These tis-sues were immersed in X-Gluc solution(Jefferson, 1987),placed under a mild vacuum for10min,and then incubated overnight at37℃.The tissues were soaked in70%ethanol for several hours to remove chlorophyll before observation.2Results and Analysis2.1The expression character of gus gene expressed in the plant cellsIn the experiment,the activities of gus gene in the Cucumis melon callus explants immersed in the three sorts of bacterial suspension were determined by histo-chemical method after co-culturing for4days.And the results were showed in Figure2.There was no blue color on the explants with Agrobacterium tumefaciens At.E-HA105,while there were lots of them on those im-mersed in the engineering bacterium pCAMBIA2301/ At.EHA105and pBI121/At.EHA105.Agrobacterium tumegaciens strain At.EHA105was a vector without gus gene,so the explants with it also contained no gus gene.The X-gluc was not hydrolyzed when it met the explants treated with the bacterium.Simultaneously, the fact that staining reaction could not happen on these explants suggested there was no endogenous gus gene background in the Cucumis melon plant,that was to say,it was feasible that gus gene was used as a reporter gene for the transformation of Cucumis melon.The blue colors on the explants immersed in engineering bacterium pCAMBIA2301/At.EHA105or pBI121/At. EHA105showed that the activities of gus genes in the both engineering bacterium were expressed and the GUS was produced.However,it had no enough evi-dence to say the blue color was in the plant cells and the materials were transformed bacause that the bacterial cells was still in the explants,and it was not safe to say whether the gus gene can be expressed in the bacterial cells or not.This resulted in inconvenience in the iden-tification of putative transformants in gene engineering and genetic transformation.2.2The expression character of gus gene expressed in the bacterial cellsTo fin d out that the activities of gus genes were expressed in the plant cells or in the bacterial cells,an-ther experiment was designed to make sure the exact location where the indigo blue dye was.The result was showed in following(Figure2G):The staining reaction happened in the bacterial suspension pBI121/At.E-HA105instead of At.EHA105or pCAMBIA2301/At.Figure2The expression of gus gene contained in the different plasmid vectorsNote:A,D:The cotyledon(A)and calli(D)immersed in engineer-ing bacterium pBI121/At.EHA105had staining reaction;B,E:The cotyledon(B)and calli(E)immersed in engineering bacterium pCAMBIA2301/At.EHA105had staining reaction too;C,F:The cotyledon(C)and calli(F)immersed in Agrobacterium tumega－ciens At.EHA105had no staining reaction;G:The expression of gus gene contained in the different plasmid vectors on the bacteri-um cells;Left:Staining reaction does not happen in Agrobacterium tumegaciens At.EHA105;Middle:Staining reaction does not hap-pen in engineering bacterium pCAMBIA2301/At.EHA105, Right:Staining reaction happens in engineering bacteriumpBI121/At.EHA10536EHA105.That the Indigo blue dye did not exist in the bacterial suspension At.EHA105suggested there was no endogenous gus gene background in the Agrobacteri－um tumegaciens At.EHA105.And the color response in engineering bacterium pCAMBIA2301/At.EHA105 and pBI121/At.EHA105showed the expression charac-ters of the gus genes in the two plant expression vectors were different bining the results2.1 with2.2,a conclusion could be drawn that all the stain-ing reactions in the Cucumis melon callus explants were not resulted f rom Agrobacterium tumegaciens At.E-HA105and the plant materials but from the plant ex-pression vectors pCAMBIA2301and pBI121.The ac-tivity of gus gene in the plant expression vectors pCAMBIA2301could be expressed in plant cells,but not in the bacterial cells.However,that in the plant ex-pression vectors pBI121could be expressed not only in the plant cells,but also in the bacterial cells.Therefore, when the gus genes the expression characters of which are like that in the plant expression vectors pCAMBI-A2301are used for gene engineering and genetic trans-formation,it is certain that the blue color is in plant cells and the transformants are obtained if the staining reaction happens in the explants,adventitious buds or com plete shoots.On the contrary,if the gus genes whose expression characters are the same as that in the plant expression vectors pBI121are applied for the ge-netic transformation,it is not sure that the transgenic plants are recovered even if the plant cells or tissues turn blue.The reason is that it doesn't make certain the blue color is in the plant cells or in the bacterial cells if the bacterial cells are not sure to be thoroughly elimi-nated from the plant materials.In addition,it doesn't guarantee the transgenic materials are obtained also in the situation that the instruments used in the experiment are contaminated by the bacterium.So the phenomenon of false positive should be considered when the gus gene as that in plant expression vector pBI121is used in the experiment and the putative transgenic plants are supposed to be further determined by other means.3DiscussionThe gus gene has been used more and more in the field of the biology engineering since Jefferson(1987) firstly applied it as a report gene in the plant genetic transformation in1987.From the experiment,we know there are two types of gus gene expression character: the one like that within pBI121can be expressed both in the plant cells and in the bacterial cells;and the other one such as that contained in the pCAMBIA2301is only expressed in plant cells.Therefore,it is important to know well which type of gus gene is used in experi-ment.An advisable protocol can be made and ideal re-sults can be gotten without too many time and sources wasted when we are familiar with the gus gene expres-sion character.Since the gus gene can be expressed in Agrobac－terium tumegaciens cells,it is necessary to make certain whether there are Agrobacterium tumegaciens cells in the materials or not before the putative materials would be tested by the method to avoid false positive.The methods to check whether Agrobacterium tumegaciens exists in the callus or the transgenic buds are showed as followings:(1)To homogenize the putative transgenic buds,spread it on a YEP medium plate,and observe whether bacterial cells grown on the plate(Kaneyoshi et al.,1994);(2)To stab several several times in and around the putatively transformed callus with a inocula-tion loop and streak on a LB medium plate,then ob-serve whether bacterial cells can grow on the plate (Franklin et al.,1993);(3)To plasmolyze the putative transgenic callus and dye them with X-gluc,and then observe the blue color was inside the cells or in the in-tercellular spaces of the callus cultures(Franklin et al., 1993).According to the principle that the shearing char-acter of intron doesn't exist in prokaryotes but in the eu-karyote,a section of plant intron was introduced into gus gene by Vancanneyt et al.(1990)to make gus gene expressed in eukaryotic cells instead of prokaryotic cells.The gus gene containing a section of intron can e-liminate false positive resulted from the Agrobacterium tumegaciens,so the problem of false positive phe-nomenon can be solved thoroughly.From our experi-ment,we recognize the gus gene in the pCAMBI-A2301is an intron-containing one while that in the pBI121is not.Therefore,the former can guarantee theStudy on Expression Character of gus Gene Contained in two Different Plasmids on Plant Cell两种质粒中gus基因在植物细胞和根癌农杆菌中的表达特性研究37基因组学与应用生物学Genomics and Applied Biology accuracy in the experiment easily.However,we suggest through the experiment above that at least two elemen-tary problems should be solved before the gus gene is to be used in the transgenic transformation:the one is to know whether the endogenous gus gene background ex-ists in the materials including both the plant and bacteri-um;the other one is to know well which type of gus gene is to be used in the experiment,namely the gus gene is expressed in both plant cells and bacterial cells or only in plant cells.Thus,the corresponding measures can be taken to ensure the experiment to go on well in the sub-sequent identification of the putative transgenic plants.ReferencesChin 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