论文标题:广谱抗稻瘟病基因Pi9的应用和进化研究
Studies on Application and Evolution of the Broad-spectrum Blast Resistance Gene Pi9 in Rice
论文作者
论文导师 戴良英；王国梁,论文学位 博士,论文专业 植物病理学
论文单位 湖南农业大学,点击次数 697,论文页数 105页File Size7298K
2006-11-24论文网 http://www.lw23.com/lunwen_130822/
Agrobacterium-mediated method;; indica rice;; rice blast;;broad-spectrum blast resistance gene Pi9;; Pi2/9 locus
本研究系统地探讨了农杆菌介导籼稻遗传转化的主要影响因素，建立了稳定、高效的农杆菌介导籼稻的转化体系。通过农杆菌介导法，将广谱抗稻瘟病基因Pi9转入8个籼稻品种(丰源B，湘晚籼11号，湘晚籼13号，R996，527，1701，25H003，25H075)和粳稻品种日本晴的愈伤组织中，得到了转Pi9基因的株系，并对T1代和T2代转基因植株进行了GUS、PCR和RT-PCR检测以及T2代植株的稻瘟病抗性鉴定。同时，对3个野生稻中的Pi2／9位点进行了进化分析。本研究主要结果如下： 1 pc1301-Pi9质粒的鉴定 将大肠杆菌和农杆菌中提取的质粒pc1301-Pi9分别用限制性内切酶SalⅠ和KpnⅠ酶切，电泳分析表明质粒pc1301-Pi9可以在大肠杆菌和农杆菌中稳定遗传。 2高效转化体系的建立和Pi9基因对籼稻的转化研究 (1)不同培养基对籼稻愈伤组织诱导的影响 将8个籼稻品种的成熟种子分别接种子NB、N6、MS、CC和L3培养基上，结果表明，NB、N6、MS、CC和L3这5种培养基诱导籼稻的平均出愈率分别为：81.7％、75.4％、68.0％、58.5％和55.5％。因此，NB培养基最适于诱导籼稻胚性愈伤组织。 (2) ABA对愈伤组织诱导的影响 ABA对某些品种愈伤组织的诱导具改善作用，可减少褐化死亡现象。 (3)愈伤组织的继代分析 比较了L3和NB两种培养基对愈伤组织继代效果，发现L3更适于愈伤组织继代。愈伤组织继代8d、15d、25d的分析表明，愈伤组织宜选择继代15d左右做共培养较好，这时愈伤组织大而嫩黄且生命力强。 (4)共培养时间对转化的影响 以1701为受体，共培养时间为1d、2d、3d、4d，获得的抗性愈伤率分别为32.6％、68.2％、75.5％和60.0％。因此，共培养时间为3d的转化效果最好，获得的抗性愈伤率最高，达75.5％。 (5)不同菌液浓度对愈伤组织转化的影响 用OD值为0.5、1.0、1.5、2.0的菌液浓度与1701的愈伤组织进行共培养，获得的抗性愈伤率分别为64.1％、74.7％、62.1％和51.0％。因此，OD值为1.0菌液浓度更适于籼稻愈伤组织的转化。 (6)乙酰丁香酮对转化的影响 共培养中加入200μmol／L的乙酰丁香酮可获得的较高的抗性愈伤率，为75.3％。在菌液、共培养培养基以及同时在菌液和共培养培养基中加入乙酰丁香酮，获得1701的抗性愈伤率分别为59.8％、68.3％和75.7％。表明菌液和共培养培养基同时加入乙酰丁香酮，转化效果较好。 (7)筛选压力的确定 40mg／L的潮霉素B浓度适用于籼稻抗性愈伤组织的筛选；20mg／L的潮霉素B浓度用于生根培养基中幼苗的筛选。 (8)抗性愈伤组织的筛选情况 愈伤组织经S_(40)培养基筛选后，日本晴的抗性愈伤率达89.9％。籼稻的抗性愈伤率在69.4％～82.5％之间，其中R996为69.4％，25H003为82.5％。 (9) 9个品种的转化情况 本研究日本晴的转化率是63.3％，籼稻品种阳性苗的转化率在3.2％～25.9％之间，其中25H075最低为3.2％，R996最高达25.9％。 (10)不同激素配比对愈伤组织分化率的影响 6-BA 2mg／L+KT 2mg／L+NAA 0.5mg／L+IAA 0.5mg／L的激素配比适于抗性愈伤组织的分化。 3 GUS、PCR和RT-PCR检测及稻瘟病抗性鉴定 对获得的T1代和T2代植株经GUS、PCR和RT-PCR检测以及T2代植株的稻瘟病抗性鉴定，表明Pi9基因已整合到受体基因组中，并遗传给子代，在子代中稳定表达。 4 Pi2／9位点的进化分析 对3个野生稻中的Pi2／9位点NBS-LRR基因与Pi2和Pi9位点NBS-LRR基因进行序列比较分析，表明除假基因外的所有这些NBS-LRR基因属于相同的进化树，有共同的祖先，并且大多数NBS-LRR基因都有一个高度保守的内含子。这一研究为稻瘟病抗性基因的进化分析奠定了基础，并为植物中广谱抗性分子机理的阐明提供了线索。
In this study, the factors affecting genetic transformation of indica rice mediated byAgrobacterium tumefaciens were studied systematically, and an efficient indica ricetransformation system was established. The broad-spectrum blast resistance gene Pi9was successfully introduced into the calli of eight indica rice cultivars (Fengyuan B,Xiangwanxian11, Xiangwanxian13, R996, 527, 1701, 25H003,25H075)and the japanicrice variety Nipponbare based on the Agrobacterium-mediated method. T1 and T2transgenic plants were verified by GUS, PCR and RT-PCR, T2 transgenic plants weretested by Magnaporthe grisea. At the same time, the evolution of Pi2/9 loci in the threewild rices was studied. The results were summarized as follows: 1 Identification of the plasmid pc 1301-Pi9 The plasmid pc1301-Pi9 was digested by SalⅠand KpnⅠrespectively, it wasshowed that the plasmid pc1301-Pi9 was steadily inheritable in E.coli andA.tumefaciens by electrophoretic analysis. 2 Establishment of highly efficient transformation system and study on transformationofPi9 gene in indica rice (1) Influence of different media on calli induction in indica rice The calli of 8 indica rice cultivars were induced on NB, N6, MS, CC and L3 media,it was showed that the average rates of calli induction were 81.7%, 75.4%, 68.0%,58.5% and 55.5% respectively. Therefore, NB medium was the best for induction. (2) Influence of ABA on calli induction ABA could improve calli quality for some indica rice cultivars, it could reduce thecalli browning and death. (3) Analysis of calli subculture L3 is better for subculture by compared NB and L3 medium. Analysis showed thatcalli were suitable for co-culture aboutl5 days by compared 8 days, 15 days and 25 daysafter subculture, the calli were big, light-yellow and vigorous. (4) Influence of co-culture duration on transformation Interaction duration of calli and A.tumefaciens was 1d, 2d, 3d and 4d for 1701cultivar, the rates of resistant calli were 32.6%, 68.2%, 75.5% and 60.0% respectively. Itshowed that 3 days of co-culture was fitter for transformation and the resistant calli wasthe highest. (5) Influence of different A. tumefaciens concentration on calli transformation As 1701 cultivar for receptor, the OD values for 0.5, 1.0, 1.5 and 2.0 ofA.tumefaciens concentration were used for co-culture, and the rates of resistant calliwere 64.1%, 74.7%, 62.1% and 51.0% respectively. So the OD value for 1.0 ofA.tumefaciens concentration was suitable for calli transformation in indica rice. (6) Influence of acetosyringone on transformation We got higher resistant calli of 75.3% with 200μmol/L acetosyringone inco-culture. We obtained 59.8%, 68.3% and 75.7% of resistant calli of 1701 by addingacetosyringone in A.tumefaciens liquid, co-culture medium and A.tumefaciens liquidplus co-culture medium. So it was better by adding acetosyringone in A.tumefaciensliquid plus co-culture medium than singly adding. (7) Definition of selection pressure 40mg/L hygromycin B concentration was suitable for calli selection, and 20mg/Lhygromycin B concentration was used for plantlets selection in 1/2MS medium. (8) Selection ofhygromycin-resistant calli Calli were selected under 40mg/L hygromycin B concentration on S_(40) medium, therates of resistant calli of Nipponbare was 89.9%, that of indica rice cultivars wasbetween 69.4%～82.5%, R996 was 69.4%, and 25H003 was 82.5%. (9) Rate of transformation of 9 rice cultivars In this study, the rate of transformation of Nipponbare was 63.3%, that of positiveplantlets in indica rice cultivars was between 3.2%～25.9%, R996 was 25.9% and25H075 was 3.2%. (10) Influence of proportion of different plant hormones on calli differentiation 6-BA 2mg/L+KT 2mg/L+NAA 0.5 mg/L+IAA 0.5 mg/L was fit for resistantcalli differentiation. 3 Detection of GUS, PCR, RT-PCR and resistance test for transgenic plants The broad-spectrum blast resistance gene Pi9 was introgressed into receptorgenome by GUS histochemical assay, PCR, RT-PCR and resistance test, and the aliengene was melt into T2 plant genome and normally expressed. 4 Evolution analysis of Pi2/9 locus By sequence alignment of NBS-LRR genes at Pi2/9 locus in three wild rices withNBS-LRR genes at Pi2 and Pi9 loci, it was showed that those NBS-LRR genes, exceptfor pseudogenes, belonged to the same phylogenetic clade and had the commonprogenitor, and most of NBS-LRR genes had a highly conserved intron. This studyestablishs the basis for evolution analysis of rice blast R gene and provides importantinformation for elucidation the molecular mechanism of the broad spectrum resistance.
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