诱导根癌土壤杆菌vir区基因表达信号分子的构效研究

以根癌土壤杆菌为介导的外源基因转化系统是目前应用最为广泛和有效的植物基因转化系统。根癌土壤杆菌D质粒毒性区。irA、virB、。irC、。irD。。irE、。irG6个基因位点,在外源诱导性信号分子的作用下,表达出转移DNA（T－DNA）复合物,整合人植物细胞核基因组中。利用根癌土壤杆菌这种天然的转化机制,已获得双子叶转基因植株。但单子叶植物难以被根癌土壤杆菌转化,某些研究者认为这是由于单子叶植物缺乏促使根癌土壤杆菌产生趋化运动以及诱导。ir区基因表达的信号分子［‘」。我们从幼穗分化期至抽穗扬花期水稻中分离获得2种高效…     

Identification of rice (Oryza sativa L.) signal factors capable of inducing Agrobacterium vir gene expression

Two kinds of signal factors capable of inducing Agrobaorerium vir gene expression were purified and identified from leaf extracts of panicle-differentiating to flowering stage of rice (Oryza saliva L. cv. IR 72) detected by Agrobacterium vir(?) lacZ. fusion genes. The induction was similar to that observed with 5 μm actosyringone (AS). Based on the comprehensive analysis of the data by UV, IR, NMR, MS, HMQC and HMBC, the structures of these two signal factors are identified as 5, 7, 4'-trihydroxy-3', 5'-dimethoxy-flavone (named tricin) and 5, 4' -dihydroxy-3', 5' -dimethoxy-7- (β-D-glucosyloxy) -flavone, respectively. These results demonstrate that monocotyledonous plants do contain highly efficient vir gene inducing factors of Agrobacterium, and the reason why monocotyledonous plants are difficult to transform by Ayrobacterium is not due to absence of vir gene inducing factors, but due to the signal factors only produced in specific stage and tissue of monocotyledonous plants     

农杆菌介导法在橡胶树遗传转化中的应用与展望

综述农杆菌介导法在巴西橡胶树遗传转化中的应用进展,分析影响农杆菌转化的关键因素,如植物基因型与外植体、菌株与载体类型、菌液浓度与侵染时间、vir诱导物、筛选剂与抑菌剂、培养基的组成和附加成分等,并对提高巴西橡胶树转化效率的策略进行探讨。     

农杆菌vir基因诱导因子研究进展

在众多遗传转化法中,农杆菌(Agrobacterium tumefaciens)介导法以易操作、低费用、插入片段明确、拷贝数低等独特优点成为植物遗传转化的首选。然而,至今仍有许多物种不能被农杆菌转化。研究表明,农杆菌的转化能力是由位于染色体基因组之外Ti质粒上的vir基因决定的。在所有vir基因中,除virA和virG组成型表达外,其它vir基因的表达均需酚类化合物的诱导;糖类物质可增强酚类化合物对vir基因的诱导;低磷酸和酸性pH环境也可促进vir基因的诱导表达。文章论述了酚类化合物、糖类物质、低磷酸、酸性pH和培养温度等因素对农杆菌vir基因诱导表达的影响,以期为更好地利用这一天然载体及为提高转化效率提供依据。     

Agrobacterium-mediated transformation of sea urchin embryos

Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. In the present work, we studied whether Agrobacterium can transfer genetic information to animal (sea urchin) embryos. Sea urchin embryos were co-cultivated with A. tumefaciens strains carrying binary vectors containing the nptII marker gene and agrobacterial rolC and rolB oncogenes. Bacterial plasmid T-DNA-sea urchin DNA junction sites were identified in the genome of these embryos, thus indicating successful transformation. The nptII and both rol genes were expressed in the transformed embryos. The processes of transgene integration and transgene expression were suppressed when Agrobacteria contained mutated virA, virB or virG genes, suggesting that Agrobacterium transforms sea urchin cells by a mechanism similar to that which mediates T-DNA transfer to plants. Some of the embryos co-cultivated with Agrobacterium developed teratoma-like structures. The ability of Agrobacterium strains to trigger formation of teratoma-like structures was diminished when they contained the mutated vir genes. In summary, our results demonstrate that Agrobacterium is able to transform animal (sea urchin) embryonic cells, thus indicating a potential of this natural system for gene delivery to animal hosts. We also discuss the possibility of horizontal gene transfer from Agrobacterium to marine invertebrates.     

根癌农杆菌介导转化水稻成熟胚及转Metr基因植株的获得

以粳稻品种广陵香糯为材料,用携带有双元载体pBB的根癌农杆菌EHA105为载体,研究了根癌农杆菌介导转化粳稻成熟胚愈伤组织的几个影响因素,建立了合适的粳稻成熟胚愈伤组织转化系统。一种基于MS为基本培养基的商品培养基(HRM)较适合于作为粳稻成熟胚愈伤组织诱导培养基,合适的愈伤组织诱导培养天数为7~8 d,合适的筛选培养基为CC培养基。在此基础上,将Metr基因导入粳稻品种广陵香糯中,获得了一批转基因水稻植株,PCR分析表明外源基因已经整合进了水稻的基因组中。部分转基因植株后代遗传分析表明外源基因的分离符合3∶1的理论比例。     

根癌农杆菌介导的水稻转化研究进展

农杆菌介导的水稻基因转化是水稻基因转化的热门。本文对由农杆菌介导转化获得的水稻品系（品种）,影响农杆菌介导转化的因素,农村菌浸染的方法,外源基因的检测和遗传等方面作综合论述,并提出了农杆菌介导转化水稻的前景。     

Enhanced Induction of Vir Genes Results in the Improvement of Agrobacterium - Mediated Transformation of Eggplant

Induction of Agrobacterium vir genes is one of the basic requirements for T-DNA transfer and integration into plant genome. Here we study the vir gene induction by various explant types of eggplant in order to develop a transformation protocol with improved efficiency using binary vector constructs - harbouring a hygromycin phosphotransferase gene (hpt) as a selection marker and a gfp:gus fusion gene as a reporter. A protocol for efficient Agrobacterium-mediated transformation of eggplant (Solanum melongena L cv Pusa Purple Long) has been developed by optimizing factors. Leaf, cotyledon and hypocotyl explants were tested for their ability to induce Agrobacterium vir-genes using a VirE:lacZ fusion construct and were shown to be poor inducers of the same. Addition of 100 µM acetosyringone during infection and co-cultivation steps of transformation could enhance the vir gene induction as well as a 2–3 fold increase in transformation frequency. Transformed explants showed the expression of reporter genes gus and gfp. The transgenics were analysed by peR and Southern blot hybridization, and were shown to have T-DNA integrated into their genome. The data suggest that eggplant is a relatively poor inducer of Agrobacterium vir genes, probably due to minimal phenolic production, and by modulating vir gene induction using phenolics like acetosyringone eggplant transformation can be improved.     

根癌农杆菌转化禾谷类作物及影响其转化的因素

综述了根癌农杆菌转化禾谷类作物的研究现状,根癌农杆菌与禾谷类作物间的相互作用研究,根癌农杆菌成功转化禾谷类的例子;影响根癌农杆菌转化成功的因素,如菌株类型,感受态细胞的选择,Vir基因的活化,选择合适的转化途径等。这些将为利用根癌农杆菌介导的方法,将外源基因导入禾谷类作物提供有益的帮助。     

Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid.

Upon incubation of Agrobacterium tumefaciens A348 with acetosyringone, the vir genes encoded by the Ti (tumor-inducing) plasmid are induced. The addition of certain opines, including octopine, nopaline, leucinopine, and succinamopine, enhanced this induction 2- to 10-fold. The compounds mannopine, acetopine, arginine, pyruvate, and leucine did not stimulate the induction of the vir genes to such an extent. The enhancement of vir gene induction by opines depended on acetosyringone and the genes virA and virG. Opines stimulated the activity of the vir genes, the double-stranded cleavage of the T (transferred)-DNA at the border repeat sequences, and the production of T-strands by the bacterium. The transformation efficiency of cotton shoot tips was markedly increased by the addition of acetosyringone and nopaline at the time of infection.     

水稻双元细菌人工染色体载体系统转化体系的建立

普通双元载体己被广泛碰用于农杆菌介导的植物转化,但这类载体通常只能转移5～20kb的外源DNA片段;而双元细菌人工染色体（BIBAC）载休可以弥补普通双元裁体的不足,通过它已在烟草、番茄等双子叶植物中实现了大片段DNA（150kb）的转移。BIBAC载体在单子叶植物转化中的应用尚未见报道。面于单、双子叶植物间以及大、小片段转化间的转化体系存在明显差异,常规的农杆菌介导的水稻转化体系不能适应BIBAC系统转化的要求。因此,建立适于BIBAC系统的水稻转化体系是十分必要的。通过比较不同的受体材料,不同的预培养、其培养条件,不同的去除农杆菌及选择阳性愈伤的方式等对转化效率的影响,建矿了适合水稻BIBAC系统的转化体系。该体系的技术要点包括：以水稻品种H1493为转化受体：以含毒性辅助质粒pCH32的LBA4404菌株（HP4404）为侵染菌株;预培养的培养拱pH5．6：以N6A代替AAM悬浮农杆菌：侵染菌液浓度为OD600=1．0;共培养温度为24℃;采用过渡（Resting）培养除去农杆菌;采用二步法进行选择等。基于PCR检测、Southern印迹分析的结果表明,BIBAC载体所携带的插入片段及标记基因已整合到转化植株的基因组中。这个体系的建立为在水稻中利用BIBAC系统进行大片段DNA转化奠定基础。     

Mutants of the Agrobacterium tumefaciens virA gene exhibiting acetosyringone-independent expression of the vir regulon.

Hydroxylamine-induced mutations in the virA gene of Agrobacterium tumefaciens that do not require the plant phenolic-inducing compound acetosyringone for vir regulon induction were isolated. The isolation was based on the activation of both virB::lacZ and virE::cat fusions by mutant virA loci in the absence of acetosyringone. Three of these virA(Ais) (acetosyringone-independent signaling) mutants were characterized. All three mutants expressed a virB::lacZ fusion at high levels in the absence of acetosyringone. One virA (Ais) mutant, virA112, exhibited vir gene expression in the absence of inducing monosaccharides and acidic growth conditions, both of which are normally required for vir gene induction. The phenotype of the virA112 mutant resulted from a glycine to glutamic acid change near His-474, the site of VirA autophosphorylation.     

Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens

The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants. VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG. Inducing signals include phenols, monosaccharides, and acidic pH. While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low. Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA. Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit. However, sugar enhancement of vir gene expression was vector dependent. virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA. Inhibition was conditional, depending on the induction medium and the virA allele tested. Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism.     

Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides.

The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA, in press). In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined. Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible. The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested. Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides. A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed.     

High Efficiency of Genetic Transformation of Rice Using Agrobacterium Mediated Procedure

Four japonica varieties and two indica varieties were used for the genetic transformation of rice （Oryza sativa L.） by using Agrobacterium tumefaciens (Smith et Townsend) Conn EHA101 harboring binary vector containing GUS gene and selectable marker gene of NPTⅡ and HPT. Calli derived from mature and immature embryos of rice were infected and cocultured with Agrobacterium at logarithmic phase. The highest transformation frequency was 55.1% (indica) and 85.2% (japonica) respectively according to the estimation of hygromycin resistant calli produced. The ratio of transgenic plants regenerated from the calli of indica and japonica varieties was 37.8% and 69.0% respectively. The putative transformed plants were confirmed by GUS assay, PCR analysis and Southern blotting. The segregation of foreign genes in T1 progeny corresponded to the Mendelian ratio. This transformation procedure of rice will provide an efficient model for the transformation of monocots.