两个水稻品种(系)酵母双杂交cDNA文库的构建和比较分析

Construction and Comparative Analysis of Yeast Two-hybrid cDNA Libraries of Two Rice Cultivars(Strains)

"武育粳3号"和"KT95-418"为两个遗传背景基本一致而对水稻条纹叶枯病表现为明显抗性差异的粳稻(Oryza sativa L.ssp.japonica)品种(系)。利用SMART技术合成双链cDNA后,通过SfiⅠ酶切位点将cDNA片段定向插入到改造的载体NpGADT7中,构建了两品种(系)水稻的酵母双杂交cDNA文库。检测结果表明:所构建的两个文库库容量均大于1.0×106cfu;初始文库滴度分别为1.0×1010cfu/mL和5.0×1010cfu/mL,扩增文库滴度均为1.0×1011cfu/mL;两文库重组率均大于95%;"武育粳3号"文库cDNA插入片段长度集中分布在700bp^800bp之间,"KT95-418"集中分布在750bp^1000bp之间;小规模测序结果表明两文库中全长基因的比例均超过60%。两品种(系)水稻酵母双杂交cDNA文库的构建为筛选分离抗病相关的变异基因及开展寄主与水稻条纹病毒(Rice stripe virus,RSV)互作的研究奠定了基础。

＂Wuyujing 3＂ and ＂ KT95-418＂ are two rice cuhivars （strains） which have similar genetic background while have notable difference in resistance to rice stripe disease. In this study, SMART technology was used to synthesis double-strand cDNA, and then cDNA were inserted into modified vector NpGADT7 through Sill enzyme sites, and yeast two-hybrid cDNA libraries of two rice cuhivars （strains） were constructed. The results of detection showed that the two libraries both contained 1.0 × 10^6 independent clones; The titer of primary library were 1.0 × 10^10 cfu/mL and 5.0 × 10^10 cfu/mL respectively, and the titer of amplified library were 1.0 × 10^11 cfu/mL; The recombination rates were both above 95 % ; The size of most inserts were 700 bp - 800 bp in the cDNA library of ＂Wuyujing 3＂, and 750 bp - 1 000 bp in the cDNA library of ＂KT95-418＂ ; Small-scale sequencing results and bioinformatics analysis showed that the percentage of full-length cDNA both above 60 % ; Construction of yeast two hybrid cDNA libraries of two rice cuhivars （strains） laid the foundation for screening defense-responsive mutant genes and carrying out the research on the interaction of host and Rice stripe virus through yeast two-hybrid system.