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缺磷是影响橡胶树产量的主要因素之一,蛋白泛素化在其中发挥了重要作用。为研究泛素化在橡胶树低磷胁迫中的调控作用,本研究分别以巴西橡胶树“热研7-33-97”组培苗地上部和地下部为材料,构建酵母双杂交文库,并对其进行了评价。首先用CTAB法大量提取橡胶树地上部和地下部总RNA,再用PROMEGA试剂盒处理获得纯化的m RNA,然后采用SMART誖技术和LD-PCR合成双链cDNA(ds-cDNA),继而通过双链特异性核酸酶(DSN)对ds-cDNA进行均一化处理,再经CHROMA SPIN+TE-400柱子纯化长片段的cDNA,最后将获得的长片段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文库。结果显示:地上部文库滴度8×107cfu/m L,插入片段≥0.5 kb的重组率为100%;地下部文库滴度7×107cfu/m L,插入片段≥0.5 kb的重组率为87%。该结果表明文库已成功构建,可以用于进一步实验。
Phosphorus deficiency is one of the main factors that affect the yield of rubber trees, and protein ubiquitination plays an important role. In order to study the regulatory effect of ubiquitination on low-phosphorus stress in rubber trees, two-hybrid yeast library was constructed in this study. It was evaluated. First, a large amount of total RNA was extracted from the upper and lower parts of the rubber tree by CTAB method. Then, the purified mRNA was obtained by using PROMEGA kit. Then double-stranded cDNA (ds-cDNA) was synthesized by SMART paradox and LD- The ds-cDNA was homogenized by specific nuclease (DSN), and the cDNA of the long fragment was purified by CHROMA SPIN + TE-400 column. Finally, the long cDNA fragment and the linearized vector pGADT7-Rec were co-transformed into yeast Y187 Competent cells, construct a homozygous yeast two-hybrid library. The results showed that the recombination rate of the aboveground library was 10 × 107 cfu / m L, the recombination rate of the inserted fragment ≥ 0.5 kb was 100%, that of the underground library was 7 × 107 cfu / m L, that of the inserted fragment ≥ 0.5 kb was 87% . This result indicates that the library has been successfully constructed and can be used for further experiments.