二斑叶螨cDNA文库的构建及鉴定

为研究二斑叶螨发育相关基因的功能,利用TRIzol试剂提取二斑叶螨总RNA,通过SMART技术构建cDNA文库.利用SMART IVTM Oligonucleotide和CDSIII/3忆PCR Primer为引物,在反转录酶作用下,合成cDNA第1链,利用LD-PCR方法合成cDNA第2链。合成的双链cDNA经酶切回收后,再连接pDNR-LIB载体,转化大肠杆菌感受态细胞Trans5琢,检验库容量和重组效率,并采用PCR方法检测插入cDNA片段的大小.结果表明,构建的二斑叶螨cDNA文库的库容量为3.54C6 CFU/mL,重组效率达95.8%,插入片段长度在0.3~2.0 kb之间。构建的二斑叶螨cDNA文库质量良好袁能够为进一步研究二斑叶螨基因的功能奠定基础。

Construction and identification of the cDNA library for Tetranychus urticae

To construct the cDNA library of Tetranychus urticae for further studying its related genes, total RNA was extracted of T.urticae by TRIzol and the construction of cDNA library was used by SMART cDNA library construction kit. A modified CDS III/3''PCR primer and the SMART IV Oligo primed the first-strand synthesis reaction and the second-strand cDNA was synthesized and amplified by LD (longdistance)PCR. Then the PCR products were purified with a universal DNA purification kit, the double-stranded cDNA were ligated to the pDNR-LIB vector. The ligation mixture was transformed into the E.coli competent cells to complete the construction of the cDNA library of T.urticae. The content and recombination rate of the cDNA library were tested and the length of inserted fragments was analyzed by PCR. The content of library was 3.54×106 CFU/mL, the recombinant efficiency was 95.8% and the average of insert size was between 0.3 kb and 2.0 kb.The results showed that this library was of high quality to set the basis for further exploring the genes of T. urticae.