植酸酶基因表达与调控机制研究(Ⅱ)──番茄幼苗cDNA文库构建与植酸酶基因筛选

Studies on Expression and Regulation of the Phytase Gene(Ⅱ) Construction of cDNA Library of Tomato Seedlings and Screening of a Phatase Gene

采用异硫氰酸胍法从发芽３～４ｄ的番茄幼苗中提取出总ＲＮＡ，用Ｏｌｉｇｏ（ｄＴ）纤维素亲和层析分离纯化ｍＲＮＡ．以ｍＲＮＡ为模板，Ｏｌｉｇｏ（ｄＴ）为引物用逆转录法合成双链ｃＤＮＡ．将ｃＤＮＡ与ＥｃｏＲＩ接头连接后克隆到表达载体λｇｔ１１的单一ＥｃｏＲＩ位点，经体外包装后感染宿主菌Ｙ１０９０，成功地构建了完整的番茄幼苗ｃＤＮＡ文库．用颜色筛选法筛选重组子，然后用植酸酶抗体做探针进行免疫筛选，得到两个阳性克隆．挑取阳性克隆噬菌斑扩增后纯化ＤＮＡ，经琼脂糖凝胶电泳鉴定，显示确有外源ＤＮＡ存在．该插入片断序列测定正在进行．

Total RNA is isolated from fresh embryos of 3to 4dayold tomato seedlings, using guanidine thiocyanate. From total RNA, mRNA is selected and purified through Oligo(dT) cellulose affinity chromatography. The cDNA is synthesized by the cDNA synthesis system(Promega) with Oligo(dT) as primer according to the protocols. After the ligation of Eco RI adaptors, the cDNA is cloned directly into the single Eco RI site of the expression vector Lambda gt11. About 500 independent recombinants are obtained through color selection after packaging and infection of E.coli stain Y1090. The Lambda gt11 cDNA library is amplified in E.coli Y1090 strain. Immunological screening of phage plaques is performed using the ProtoBlot D○R Immunosreening System(Promega), with a crude rabbit antiserum directed against the tomato phytase. Two positive recombinant phages are isolated after screening 5×10 3 plaques. The size of phytase cDNA insert is estimated to be about 1.5 kb, and the sequence of that insert is conducting in our laboratory.