低温胁迫下山葡萄叶片酵母cDNA文库构建

Construction of Yeast cDNA Library Using Vitis amurensis Leaves Under Lowtemperature Stress

【目的】葡萄栽培过程中,由低温造成的冷害或冻害严重影响葡萄的生长发育、果实品质和产量。山葡萄是葡萄抗寒育种的重要种质资源,本文通过构建低温胁迫下山葡萄叶片酵母cDNA文库,为葡萄抗寒分子机理研究提供物质基础。【方法】以抗寒山葡萄左山-1(V.amuerensis cv.Zuoshan-1)扦插盆栽苗为材料,提取4℃低温处理0、2、4、8、12、24 h后叶片总RNA,采用SMART技术反转录为cDNA,经纯化和短片段去除后克隆至pGADT7三框质粒载体上,经过纯化后获得初级cDNA文库,对初级文库进行扩增、提取文库质粒后转入酵母Y187菌株中,制作扩增的酵母文库,并对文库质量进行鉴定。【结果】经检测所构建的3个读码框初级文库库容分别为1.7×10^(6)、2.0×10^(6)和1.9×10^(6) cfu,重组率为100%,插入的片段长度主要分布在500~2000 bp;电泳检测和测序结果表明,插入片段所对应的编码蛋白类型丰富,具有良好的多态性。最终获得的Y187酵母文库滴度为4.0×10^(8) cfu·mL^(-1)。【结论】构建的低温胁迫下山葡萄叶片酵母cDNA文库质量较高,为筛选山葡萄低温胁迫下的互作蛋白提供了材料基础。

【Objective】A yeast cDNA library was constructed using the leaves from a cold tolerant Amur grape plant under low-temperature stress to facilitate the study of the resistance mechanism.【Method】As a well-known frost resistant Vitis amurensis Rupr.,V.amuerensis cv.Zuoshan-1 was cultured in a pot experiment for a total RNA extraction from the seedling leaves after 0,2,4,8,12,and 24 h at 4°C.The extract was then reverse transcribed into cDNA using the SMART technology to be purified.Short fragments were removed and ligated into pGADT73-frame plasmid vector to establish a primary cDNA library.After amplification,plasmid was extracted and transformed into yeast Y187 for the library construction,followed by a qualitative verification.【Result】The capacities of the 3 reading frame primary libraries were tested to be 1.7×10^(6) cfu,2.0×10^(6) cfu,and 1.9×10^(6) cfu with a recombination rate of 100%.The lengths of the inserted fragments were largely in the 500-2000 bp region.The electrophoretic detected and sequenced encoded proteins corresponding to the inserted fragments were abundant and highly polymorphic.The titer of the finally obtained Y187 yeast library was approximately 4.0×10^(8) cfu·mL^(-1).【Conclusion】A high quality yeast cDNA library using Amur grape leaves under low temperature was successfully constructed for future studies on the interacting proteins under cold stress that could induce injury impairing the growth,development,fruit quality,and yield of grape vines.