羽衣甘蓝柱头酵母双杂交cDNA文库的构建与分析

目的:构建应用于酵母双杂交系统的羽衣甘蓝柱头cDNA文库。方法:以羽衣甘蓝S13-bS13-b自交不亲和系为材料,提取柱头的总RNA,用亲和层析法分离纯化mRNA,利用CytoTrapXR建库试剂盒构建羽衣甘蓝柱头cDNA文库。结果:羽衣甘蓝柱头cDNA原始文库的库容量为2.5×105;扩增后文库的库容量约为4×108,重组率为96%,插入片段大小为0.4～3kb,平均长度在0.8kb左右。结论:构建了应用于酵母双杂交系统的羽衣甘蓝自交不亲和系柱头的cDNA文库,为探讨芸苔属植物自交不亲和的分子机理奠定了基础。

Construction of a Yeast Two-Hybrid cDNA Library from the Stigma of Ornamental Kale (Brassica oleracea var.acephala)

Objective：To construct the CytoTrap cDNA library,applied to yeast two-hybrid screening,from the stigma of ornamental kale（Brassica oleracea var.acephala） homozygous for S13-b haplotype.Methods：Total RNA was isolated from the stigma of ornamental kale S13-b homozygotes,and the mRNA was purified by affinity chromatography.The CytoTrap cDNA library from the stigma,which applied to yeast two-hybrid screening,was constructed.Results：The total size of the library transformed into Escherichia coli was 2.5×105 clones,and the total size of the amplified library was about 4×108 clones.The percentage of vectors with inserts was above 96%.The insert size by PCR from the colony and plasmids from library with Myr-specific primers was 0.4～3 kb,and the average insert size was about 0.8 kb in length.Conclusion：These data indicate that the cDNA Library may be applied to screen proteins involved in the molecular mechanisms of self-incompatibility.