接种PRSV番木瓜种苗胞质酵母双杂交cDNA文库的构建与分析

Construction and Primary Analysis of Yeast Cytoplasmic Two-hybrid cDNA Library of Carica papaya Seedlings Inoculated with Papaya Ringspot Virus

利用Trizol法提取接种PRSV 4 d的番木瓜种苗总RNA,分离纯化mRNA,反转录合成双链cDNA,双链cDNA末端经Pfu-DNA聚合酶补平,与EcoR I接头连接,XhoI酶切消化产生粘端。用Sepharose CL-2B柱分离纯化去除小分子cDNA片段,再与pMyr酵母表达载体连接,转化受体菌XL10-Gold,构建了接种PRSV番木瓜种苗胞质酵母双杂交cDNA文库。所获得的原始文库的克隆总数为1.8×106cfu,重组率为100%,文库滴度为2.6×109cfu/mL。对随机选取的34个克隆进行PCR鉴定,结果表明插入片段长度均大于0.5 kb且集中在1 kb左右。文库质量鉴定结果表明,该文库具有较好的库容量、较高的重组率以及较大的插入片段。

A high-quality yeast cytoplasmic two-hybrid cDNA library of Carica papaya seedlings inoculated with papaya ringspot virus （PRSV） about 4 days was constructed to investigate the interactions between PRS／ and Carica papaya proteins by using yeast two hybrid, and mRNA from papaya seedlings inoculated with PRSV was purified. The first and second strand cDNA were synthesized by reverse transcriptase and DNA polymerase I, respectively. The ds-DNA termini were blunted with Pfu DNA polymerase. The blunted cDNAs were added with EcoRI adaptor and then digested by Xho I. The eDNA fragments longer than 0.5 kb were collected by Sepharose CL-2B chromatography and ligated into pMyr yeast expression vector. The recombinant plasmids were transformed into the host strain XL10- Gold. A primary eDNA library of Caric, papaya seedlings inoculated with PRSV was constructed. The result showed the primary cDNA library had a total clones of 1.8× 10^6 cfu with an average insertion size of about 1 000 bp, while the titer of amplified was 2.6×10^9 cfu/mL. This indicates that this two-hybrid library has a good quality and facilitates screening Carica papaya proteins interacting with PRSV from cDNA library.