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构建大豆幼荚全长cDNA文库,可为克隆与大豆产量相关基因,培育高产大豆新品种奠定基础。以吉农18大豆幼荚突变体为材料,提取大豆幼荚总RNA,反转录成单链cDNA,LD-PCR方法合成双链cDNA;PCR产物经蛋白酶K消化、Sfil酶切后,采用CHROM SPIN-400柱分级分离,回收500 bp以上的cDNA组分。以λTriplE×2载体连接并进行体外包装,构建吉农18大豆幼荚全长cDNA文库。经检测研究构建的大豆幼荚cDNA原始文库滴度达到1.5×106pfu.mL-1,重组率达92%。扩增的文库滴定达到2.6×108pfu.mL-1,重组cDNA片段长度在500 bp以上。结果显示构建的大豆幼荚全长cDNA文库符合高质量文库的标准,可用于进一步开展相关基因的克隆及分子生物学研究。
The construction of a full-length cDNA library of soybean young pods can lay a foundation for cloning genes related to soybean yield and cultivating new varieties of high yield soybean. The total RNA of soybean young pod was extracted from the young soybean pod mutants of Ji-Nong 18 and reverse transcribed into single-stranded cDNA. The double-stranded cDNA was synthesized by LD-PCR. After digested with proteinase K and digested with Sfil, SPIN-400 column fractionation, recovery of cDNA components more than 500 bp. The λTriplE × 2 vector was ligated and packaged in vitro to construct a full-length cDNA library of jujube 18 soybean young pods. The titers of original cDNA library of soybean young pod reached 1.5 × 106pfu.mL-1 and the recombination rate reached 92%. The titer of amplified library reached 2.6 × 108pfu.mL-1, and the length of recombinant cDNA was over 500 bp. The results showed that the full-length cDNA library of soybean seedlings accorded with the standards of high-quality library and could be used for further cloning and molecular biology of related genes.