春大豆花芽cDNA文库的构建及质量分析

Construction and quality analysis of cDNA library from flower buds of spring soybean cultivars

【目的】为了解大豆多荚、多粒相关基因的调控机制，构建了大豆花芽eDNA文库，根据要求初步鉴定了所构建文库的质量．【方法】以大豆吉农18突变体的幼嫩花芽为材料提取总RNA，采用SMART技术合成双链cDNA．经蛋白酶K的消化及靳I酶切后，将所得eDNA克隆到λTriplEx质粒载体中，成功构建了大豆花芽全长cDNA文库．【结果和结论】将初始文库经扩增后保存，检测扩增文库滴度为2．13×10^8pfu／mL，重组率接近95．3％，菌落PCR鉴定插入片段主要分布在0．5～2．0kb．插入片段平均大小在1．0kb左右．表明本研究所构建的文库既满足了目的基因的分离筛选，又可保证全长eDNA文库的获得，该文库的构建为进一步开展相关基因的克隆及分子生物学研究奠定基础．

[ Objective ] To understand soybean pods and muhigrain gene regulation mechanisms of impro- ving soybean production, a soybean flower bud cDNA library was constructed. The quality of library con- struction was initially identified according to the requirements. [ Method ] In order to study the novel genes from flower bud mutants of soybean, a full-length cDNA library from flower bud mutants of soybean Jinong 18 was constructed. Total RNA from young flower bud mutants of soybean was extracted. Double strand cDNA was synthesized by SMART method. After proteinase K digestion and Sfi I digestion, the ds cDNA fragments were ligated to the kTriplEx vector. A cDNA library of soybean was successfully con- structed. [ Result and conclusion ] With the unamplified library stored after amplification, the titer of the amplified library was estimated as 2. 13 x 10s pfu/mL and the recombination rate was approximately 95.3%. PCR results showed that the inserts varied from 0.5 to 2.0 kb with an average size of 1.0 kb or so. It indicated that this library could be used for full-length genes screening and cloning of low abundance genes. The library will lay a foundation for the genes clon and molecular biological research.