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1.
Agrobacterium tumefaciens induces tumors in plants by transferring and integrating oncogenes (T-DNA) into the chromosomes of host plant cells. Agrobacterium strains were used to transfer complementary DNA copies of a potato spindle tuber viroid (PSTV) to plant cells at a wound site on tomato plant stems. Subsequently, infectious viroid RNA was found in the leaves of these plants, indicating systemic PSTV infection. This process utilized the T-DNA transfer mechanisms of Agrobacterium since PSTV infection required most virulence genes (vir) as well as one of the DNA sequences that flank either side of the Agrobacterium T-DNA. However, transfer still occurred from virE mutants of Agrobacterium, strains that fail to induce tumors even though a completely functional T-DNA is present. The virE gene seems to be directly involved in the integration of foreign DNA into plant chromosomes. 相似文献
2.
Beijersbergen A Dulk-Ras AD Schilperoort RA Hooykaas PJ 《Science (New York, N.Y.)》1992,256(5061):1324-1327
Agrobacterium tumefaciens transfers part of its Ti plasmid, the transferred DNA (T-DNA), to plant cells during tumor induction. Expression of this T-DNA in plant cells results in their transformation into tumor cells. There are similarities between the process of T-DNA transfer to plants and the process of bacterial conjugation. Here, the T-DNA transfer machinery mediated conjugation between bacteria. Thus, products of the Vir region of the Ti plasmid of Agrobacterium tumefaciens, normally involved in transfer of DNA from bacteria to plants, can direct the conjugative transfer of an IncQ plasmid between agrobacteria. 相似文献
3.
Caplan A Herrera-Estrella L Inzé D Van Haute E Van Montagu M Schell J Zambryski P 《Science (New York, N.Y.)》1983,222(4625):815-821
The tumor-inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens is the agent of crown gall disease in dicotyledonous plants. The Ti plasmid contains two regions that are essential for the production of transformed cells. One of these regions, termed transfer DNA, induces tumor formation and is found in all established plant tumor lines; the other, termed the virulence region, is essential for the formation but not the maintenance of tumors. Transfer DNA, which transfers to the plant genomes in a somewhat predictable manner, can be increased in size by the insertion of foreign DNA without its transferring ability being affected. The tumor-causing genes can be removed so that they no longer interfere with normal plant growth and differentiation. This modified Ti plasmid can thus be used as a vector for the transfer of foreign genes into plants. 相似文献
4.
Pti5基因植物双元表达载体的构建及鉴定 总被引：1，自引：1，他引：1
Pto基因编码丝氨酸／苏氨酸蛋白激酶,对番茄细菌病具有良好抗性。Pti5是与Pto互作的蛋白质,Pti5蛋白与广泛存在的病程相关蛋白基因（PR基因）中的顺式元件相结合,可增强PR基因的表达,为提高植物抗病性提供了有效的途径。本研究将Pti5基因构建到植物双元表达载体pBI121上,获得pBI121UCH1重组质粒,并将该质粒转到根癌农杆菌LBA4404中,为植物利用根癌农杆菌转化系统转Pti5基因进行抗病基因工程育种奠定了基础。 相似文献
5.
A simple and general method for transferring genes into plants 总被引：9，自引：0，他引：9
《Science (New York, N.Y.)》1985,227(4691):1229-1231
Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration method. Surface-sterilized leaf disks were inoculated with an Agrobacterium tumefaciens strain containing a modified tumor-inducing plasmid (in which the phytohormone biosynthetic genes from transferred DNA had been deleted and replaced with a chimeric gene for kanamycin resistance) and cultured for 2 days. The leaf disks were then transferred to selective medium containing kanamycin. Shoot regeneration occurred within 2 to 4 weeks, and transformants were confirmed by their ability to form roots in medium containing kanamycin. This method for producing transformed plants combines gene transfer, plant regeneration, and effective selection for transformants into a single process and should be applicable to plant species that can be infected by Agrobacterium and regenerated from leaf explants. 相似文献
6.
利用已构建的番木瓜环斑病毒(Papaya ringspot virus,PRSV)基因,通过农杆菌介导法,建立美中红番木瓜(Carica papaya L.)体胚的转化体系。结果表明:共培养时,当农杆菌EHA105携带pCAMBIA2300质粒时,农杆菌的浓度(OD600 nm)应小于或等于0.1,其共培养后抑制农杆菌的羧苄青霉素浓度应以750 mg/L为宜;当农杆菌EHA105携带pBI121质粒时,则农杆菌的浓度(OD600 nm)应小于或等于0.8,其共培养后抑制农杆菌的羧苄青霉素的浓度则为500 mg/L;加上滤纸处理,抗生素洗涤以及洗涤后的干燥处理,可完全抑制体胚表面的农杆菌生长。对再生的番木瓜体胚进行PCR检测,结果表明PRSV基因已转入体胚中,但还需要成苗后的进一步检测。 相似文献
7.
应用RT-PCR技术克隆了猪传染性胃肠炎病毒TGEV-JL株S基因全序列,将其连接到pMD18-T载体。经SacⅠ和BamHⅠ酶切鉴定,其产物全长4320bp。测序后与TH-98等8个TGEV毒株的S基因序列进行比对,同源性为97.6%～99.8%。将该基因插入植物表达载体pBI121的CaMV35S启动子下游,构建高效植物表达载体,转入根癌农杆菌EHA101中。结果表明:成功构建了重组植物表达载体pBI121-S,获得农杆菌工程菌。 相似文献
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[目的]克隆了脯氨酸脱氢酶ProDH基因的全长cDNA,构建了ProDH基因的RNA干扰植物表达载体,并转化到农杆菌.[方法]以“耐运2000番茄”幼苗为试材,根据GenBan中公布的番茄脯氨酸脱氢酶ProDH基因的序列信息设计了一对特异性引物,克隆了该基因的全长cDNA,分析基因序列选择正反向片段并扩增,并通过酶切、连接的方法构建了ProDH基因的RNA干扰植物表达载体PBI121-PDHi利用冻融法将表达载体转化到农杆菌EHA105中.[结果]所克隆到的ProDH基因片段长2 001 bp,其中CDS为1 491 bp,编码380个氨基酸.测序结果与公布序列同源性100％,因此可以用来构建干扰载体;通过酶切与测序鉴定,证明表达载体构建成功.[结论]经特异性引物扩增检测,证明表达载体已转入农杆菌,为进一步的研究该基因奠定了基础. 相似文献
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基于Cre/loxP重组系统的多基因载体构建及烟草转化研究 总被引：3，自引：1，他引：2
利用一套基于Cre/loxP重组系统的植物多基因表达载体系统将苏云杆菌毒蛋白基因(BtCryIAc)、甜菜碱醛脱氢酶基因(BADH)、GA20氧化酶基因(pttGA20ox)和rolB基因构建于植物表达载体pYL1305上,命名为pYL1305BBGR。采用农杆菌介导的叶盘法转化烟草,经PCR和Southern blot检测发现,多基因已成功转入烟草基因组中;经半定量RT-PCR检测证实,4个基因均能正常表达。 相似文献
10.
农杆菌是一种革兰氏阴性土壤病原细菌,携带具有天然转基因功能的Ti质粒或Ri质粒,能将一部分遗传物质插入到寄主植物的染色体上,使其稳定遗传和表达,赋予植物新的性状。所以农杆菌作为最有效的转化媒介,已被广泛应用于多数双子叶植物和部分单子叶植物转基因研究。虽然农杆菌介导的转化技术具有操作简单、成本低廉,转基因沉默几率小,插入基因拷贝数少等优点,但农杆菌介导植物遗传转化是一个复杂的生物学过程,需要一系列农杆菌蛋白和植物蛋白相互作用,共同完成外源基因的转入和整合。植物相关蛋白在转化过程中起着重要作用。其中,阿拉伯半乳聚糖蛋白(AGP)、植物根钙粘附蛋白和类玻连蛋白等参与农杆菌附着于植物细胞表面的过程;鸟苷三磷酸腺苷酶（GTPase）和BTI蛋白协助T-DNA和Vir效应蛋白进入植物细胞;actin、GIP、VIP等蛋白参与T-DNA复合体在细胞质中的运输的过程;与Vir效应蛋白互作的VIP1、VIP2、KAPa、PP2C、Roc等蛋白协助T-DNA定位于植物细胞核;组蛋白、VIP1和VIP2等引导T-DNA在植物基因组上的整合。由于植物种类间存在巨大差异,上述一些植物蛋白基因的过表达虽能提高农杆菌转化某些植物的转化效率,但不能提高另一些植物的转化效率。在容易被农杆菌遗传转化的植物如拟南芥、水稻中的研究表明,VIP1、VIP2、AGP、H2A等蛋白与农杆菌转化关系密切,但这些蛋白在利用农杆菌转化较难的作物如小麦、玉米中的功能还不明确,因而需要在不同植物中继续筛选和鉴定与T-DNA转化相关重要蛋白的编码基因。目前,农杆菌介导的植物遗传转化有2个显著特点,一是农杆菌介导转化烟草、拟南芥、水稻等模式植物的技术日渐成熟,二是农杆菌介导转化小麦、玉米、大豆等重要作物的技术仍然没有本质突破,植物相关蛋白在T-DNA转运、整合等过程中的作用还需要深入研究和进一步明确。文章主要对参与农杆菌介导遗传转化植物整个过程中相关植物蛋白的研究进展进行了综述,以期为提高农杆菌转化顽拗型作物的转化效率提供参考。 相似文献
11.
[目的]探讨将完整的含柑橘cNHX1基因转化为草莓植株。[方法]以弗吉尼亚草莓品种为试材构建了含柑橘cNHX1基因的植物表达载体,并进行了酶切鉴定,然后将表达载体导入农杆菌EHA105中,获得了工程菌株;利用农杆菌转化法,把cNHX1基因转化成草莓植株,并利用PCR方法鉴定了转化植株。[结果]利用农杆菌转化法获得了转cNHX1基因,通过进一步利用含Kam的平板进行筛选,获得了纯合的转基因植株;对获得的转基因草莓植株提取叶片DNA,利用引物F01和R02进行扩增,结果在草莓基因组中可以扩增出1.63kb的目的条带,证明cNHX1基因已经整合到草莓基因组。[结论]为获得耐盐程度显著提高的转基因草莓奠定了初步基础。 相似文献
12.
St Schell J 《Science (New York, N.Y.)》1987,237(4819):1176-1183
Transgenic plants are generated in nature by Agrobacterium tumefaciens, a pathogen that produces disease through the transfer of some of its own DNA into susceptible plants. The genes are carried on a plasmid. Much has been learned about how the plasmid is transferred, how the plasmid-borne genes are organized, regulated, and expressed, and how the bacteria's pathogenic effects are produced. The A. tumefaciens plasmid has been manipulated for use as a general vector for the transfer of specific segments of foreign DNA of interest (from plants and other sources) into plants; the activities of various genes and their regulation by enhancer and silencer sequences have been assessed. Future uses of the vector (or others like it that have different host ranges) by the agriculture industry are expected to aid in moving into vulnerable plants specific genes that will protect them from such killers as nonselective herbicides, insects, and viruses. 相似文献
13.
Tumor DNA structure in plant cells transformed by A. tumefaciens 总被引：25，自引：0，他引：25
P Zambryski M Holsters K Kruger A Depicker J Schell M Van Montagu H M Goodman 《Science (New York, N.Y.)》1980,209(4463):1385-1391
Crown gall tumors are induced in plants by infection with the soil bacterium Agrobacterium tumefaciens. Because the tumor induction involves transfer of a portion of the tumor-inducing (Ti) plasmid DNA from the bacterium to the plant cells, this system is of interest for the study of genetic exchange as well as tumor induction. The boundaries of the transferred DNA (T-DNA) have been cloned from transformed plant cells of tobacco. Detailed mapping with restriction enzymes and nucleotide sequence analysis of two independent clones were used to study the molecular structure of the ends of the T-DNA. One clone contains the two ends of the T-DNA joined together; the other contains one end of the T-DNA joined to repetitive plant DNA sequences. These studies provide direct evidence that the T-DNA can be integrated into the plant genome. In addition, the data suggest that in the plant, T-DNA can be tandemly repeated. Sequence analysis of the junction of crown gall clone 1 reveals several direct repeats as well as an inverted repeat; these structures may be involved in the transfer of the DNA from Agrobacterium to plant cells. 相似文献
14.
以大豆基因组DNA为模板,利用聚合酶链式反应(PCR)技术克隆了大豆胰蛋白酶抑制剂基因SKTI3的全长DNA片段,并将其构建到pMD18-T vector上。核苷酸序列测定结果表明:该基因片段全长654 bp,与已发表的SKTI3基因序列同源性达99%。将目的基因片段插入到pB I121 35S启动子下,构建重组质粒pB ISKTI3,采用冻融法将该重组质粒转入农杆菌EHA105中,获得了植物表达载体。 相似文献
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【目的】应用基因工程技术,获得表达血管内皮生长因子（VEGF）的转基因番茄植株,为以番茄作为生物反应器生产药用蛋白奠定基础。【方法】利用植物偏好的密码子改造合成VEGF基因全长,构建植物表达载体p1390RVEGF,通过农杆菌菌株EHA105介导将其T-DNA区转入到番茄细胞中,再生后获得转基因番茄植株,对其进行分子和蛋白水平检测。【结果】成功构建了植物整株表达载体p1390R-VEGF,建立了高频的番茄再生培养体系;PCR检测、Southern blot和Western blot检测结果表明,VEGF基因已经转入番茄中,并成功得到了表达。【结论】得到了转基因番茄植株和果实,且VEGF蛋白有良好的抗原性。 相似文献
17.
幽门螺杆菌被世界卫生组织认定为一类致癌因子,其热休克蛋白(HSP)具有较强的免疫原性,存在着发展为疫苗成分的可能性.四川省农科院生物技术核技术研究所构建了幽门螺杆菌热休克蛋白B亚单位基因HspB植物表达载体pYHWHRI并转化进入根癌农杆菌(Agrobacterium tumefaciens,EHA105).本文采用农杆菌介导的叶盘法对烟草(Nicotiana tabacum L.)叶片进行遗传转化.在含有卡那霉素(Kan)的MS筛选分化培养基上筛选,获抗性烟草植株.经PCR分子检测,其中6株已成功导入HspB基因. 相似文献
18.
农杆菌介导灰绿藜NHX1基因转化新疆大叶苜蓿的研究 总被引：2，自引：0，他引：2
利用转基因技术能够快速有效地获得抗盐碱苜蓿的新品种,但苜蓿的品种直接影响着其再生能力和遗传转化,因此建立农杆菌介导的大叶苜蓿转基因体系对于大叶苜蓿抗逆新品种培育具有重要的促进作用.将新疆盐生植物灰绿藜(Chenopodium glaucum Linn.)的Na+/H+反向运输体基因Cg-NHX1构建在植物表达载体pBIN438上,并转化入根瘤农杆菌EHA105,以5～7 d苗龄的新疆大叶紫花苜蓿(Medicago.sativa)子叶为转化受体,通过重组EHA105介导的子叶浸染法将Cg-NHX1基因导入新疆大叶紫花苜蓿中,获得了抗卡那霉素的再生植株.提取转基因植株基因组DNA,进行PCR检测和Southern blot分析,结果表明Cg-NHX1基因已经被整合到新疆大叶紫花苜蓿基因组中.研究成功地获得灰绿藜NHX1基因转化的新疆大叶紫花苜蓿植株,并在新疆大叶紫花苜蓿遗传转化影响因素的研究中,发现菌液浓度OD600nm为0.2～0.3,浸染时间20 min适宜于子叶愈伤诱导,有利于基因转化植株的获得. 相似文献
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利用重组PCR技术将香蕉束顶病毒的复制酶基因和黄瓜花叶病毒的衣壳蛋白基因融合重组,并与植物表达载体pBI121连接,构建了抗香蕉束顶病和香蕉花叶病的双价植物表达载体,通过冻融法转化载体人根癌农杆菌LBA4404,获得了工程农杆菌pBI121．FBC,从而为培育抗病转基因香蕉品种奠定了基础． 相似文献