盐生杜氏藻(Dunaliella salina)cDNA文库构建及功能基因筛选

摘要

采用Qiagen公司的植物总RNA提取技术、Clontech公司的CreatorTM技术平台以及SMARTTM技术进行cDNA文库构建.从杜氏藻中提取出了高质量的总RNA,通过PowerScript反转录酶反转录杜氏藻的总RNA,采用LD-PCR、酶处理等方法对cDNA进行等比例扩增、纯化,同时使用CHROMA SPIN-400柱子将cDNA分段化,最后将长片段连入pDNR-LIB质粒,1.5 kV,25 μ F电转化大肠杆菌JM109,得到含1.5×106个克隆子的原始文库,滴度为1.5×106cfu ml-1.结合酶切和PCR,对该文库的质量进行了鉴定和统计,文库的平均片段插入长度为1.5kb.采用烯醇酶和UDP葡萄糖脱氢酶的EST作为同源探针,对文库中的功能基因进行筛选,并采用放射性原位杂交法,对扩增文库进行了初筛和复筛,得到了含这两条基因全编码序列的cDNA,烯醇酶为1.8kb,UDP葡萄糖脱氢酶为1.9kb,为今后对该种进行大规模功能基因组学研究奠定基础.%A cDNA plasmid library of Dunaliella salina has been constructed using plasmid pDNR-LIB from Clontech for the first time in order to analyze the gene expression under salt stress of this species. Total RNA was isolated with Qiagen RNeasy Mini Kit for isolation of total RNA from plant cells. The library was then constructed with the CreatorTM SMARTTM cDNA Library Construction Kit of Clontech. The original library consisted of 1.5 ×106 clones with an average insert size of about 1.5 kb. Enolase gene and UDP-glucose dehydrogenase gene were selected from this library to identify the qualification of the library using radiative in situ hybridization. This study has built a base for the functional genornic research ofDunaliella salina.