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以巴西橡胶树无性系‘热研7-33-97’胶乳为材料,采用SMART~cDNA合成技术反转录合成cDNA第一链,并通过LDPCR合成双链cDNA(ds-cDNA),采用双链特异性核酸酶(DSN)对ds-cDNA进行均一化处理,并经过CHROMA SPIN+TE-400柱子去除短片段的cDNA,纯化后的cDNA和线性化载体p GADT7-Rec共转化酵母Y187感受态细胞构建均一化酵母双杂交cDNA文库。结果显示:均一化之前橡胶树胶乳cDNA片段分布较广,丰度分布不均匀,经DSN均一化处理和CHROMA SPIN+TE-400柱纯化后,小于500 bp的片段得到有效去除,高丰度cDNA明显下降,而且RT-PCR扩增结果显示,均一化处理后,18S rRNA和β-actin两个管家基因的表达丰度均明显降低。最终获得初始文库的独立克隆为1.26×10~6 cfu,文库滴度达到3.23×107 cfu·mL~(-1),重组率为87%,平均插入片段大于1.0 kb。本研究构建了一个橡胶树胶乳均一化酵母双杂交cDNA文库,为进一步研究天然橡胶生物合成途径及其调控的分子机制提供借鉴。
The first strand of cDNA was reverse transcribed using SMART ~ cDNA synthesis technology and the double-stranded cDNA (ds-cDNA) was synthesized by LDPCR. The double strand cDNA The ds-cDNA was homogenized by specific nuclease (DSN), and the cDNA of the short fragment was removed by CHROMA SPIN + TE-400 column. The purified cDNA and the linearized vector pGADT7-Rec were co-transformed into yeast Y187 competent cells Constructs a homogenous yeast two-hybrid cDNA library. The results showed that the cDNA fragments of rubber latex were homogeneously distributed and the abundance distribution was not uniform. After homogenized by DSN and purified by CHROMA SPIN + TE-400, the fragments less than 500 bp were effectively removed and the abundant cDNAs were significantly , And the results of RT-PCR amplification showed that the abundance of 18S rRNA and β-actin genes were significantly decreased after homogenization. The final clone was 1.26 × 10 ~ 6 cfu, the titer of the library was 3.23 × 107 cfu · mL -1, and the recombination rate was 87%. The average insert size was greater than 1.0 kb. In this study, we constructed a yeast rubber yeast two-hybrid cDNA library of rubber latex for further study of the molecular mechanism of natural rubber biosynthesis and its regulation.