玉米籽粒酵母双杂交cDNA文库的构建及ZmSCL1互r作因子筛选

摘要

ZmSCL1 is a member of GRAS gene family. Although highly expressed in maize seeds in the key period for the filling period of maize,especially in the endosperm,the molecular function of ZmSCL1 for maize seeds development remains unclear. Therefore,to screen interaction factors for ZmSCL1,yeast two-hybrid cDNA libraries of maize inbred line(B73)developing seeds(12 and 20 days after pollination)were constructed in yeast strain AH109 using homologous recombination-mediated SMART technology. The qualities of the cDNA libraries were evaluated as the followings :the titers were 1.10×105 CFU/mL,the capacity of cDNA library was 1.32×106,the recombinant rate was 81.90%,and the protein length encoded by the inserted cDNA ranged from 133 aa to 500 aa. Finally,109 clones were obtained for sequencing. GO enrichment analysis showed that,ZmSCL1 may be involved in the regulation of starch and nutrition accumulation.%ZmSCL1属于GRAS家族基因,在玉米籽粒,尤其是胚乳中高表达,而这个时期是玉米灌浆的关键时期,鉴于ZmSCL1的分子功能以及其与玉米籽粒发育的关系尚不明确.利用SMART同源重组交换技术,在酵母菌株AH109中构建了玉米自交系B73授粉后不同时期籽粒的cDNA文库,利用此文库筛选了ZmSCL1的互作因子.文库质量评定结果显示:cDNA文库的滴度为1.10×105 CFU/mL,文库的库容量为1.32×106,文库的重组率为81.90%,插入片段所编码的蛋白质长度在133-500个氨基酸之间.最终,获得了190个阳性克隆.对这些克隆进行测序,利用GO注释对所筛选的互作因子进行初步分类,并进行功能富集分析.结果显示ZmSCL1可能参与结合、催化以及营养储备相关生物过程的调控.