Studies On Application And Evolution Of The Broad-spectrum Blast Resistance Gene Pi9 In Rice

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-Pi9The plasmid pc1301-Pi9 was digested by Salâ… and Kpnâ… respectively, 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 riceThe 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 inductionABA could improve calli quality for some indica rice cultivars, it could reduce thecalli browning and death.(3) Analysis of calli subcultureL3 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 transformationInteraction 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 transformationAs 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 transformationWe 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 pressure40mg/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 calliCalli were selected under 40mg/L hygromycin B concentration on S₄₀ 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 cultivarsIn 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 differentiation6-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 locusBy 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.