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为了研究花生小G蛋白AhRab7基因在大肠杆菌中的表达模式及与高盐胁迫的关联性,采用RT-PCR技术从花生(Arachis hypogaea L.)品种花育20中克隆了AhRab7(7-1、7-2)的cDNA全长序列,通过核苷酸序列和氨基酸序列分析结果表明AhRab7(7-1、7-2)的cDNA全长分别为872 bp、816 bp,分别含1个621 bp、618 bp大小的开放阅读框(ORF,open readingframe),拟编码氨基酸数分别为206aa、205aa。将携带完整的ORF序列连入表达载体pET-28a(+)中,经IPTG诱导后进行耐盐性测定,结果表明两种全长重组酶(分别含pET-28a-Rab7-1和pET-28a-Rab7-2的重组质粒)均具有正常酶活性,明显缓解了高盐环境(5.5%～10%NaCl溶液)对大肠杆菌的生长胁迫,而对照组(含空载体pET-28a)未能检测出相同的酶活性,结果表明AhRab7基因表达可以显著缓解高盐胁迫影响。目的基因经IPTG诱导后高效表达,SDS-PAGE电泳检测到目标蛋白的大小为23kDa,与预测结果一致。这为后期探讨花生对盐胁迫及其他非生物胁迫的研究奠定了一定的基础。
In order to study the expression pattern of small Arabin gene AhRab7 in Escherichia coli and its relationship with high salt stress, AhRab7 was cloned by RT-PCR from Huayu 20 (Arachis hypogaea L.) The full-length cDNA of AhRab7 (7-1, 7-2) was 872 bp and 816 bp respectively, which contained 1 621 bp, The 618 bp ORF (open reading frame), the number of amino acids to be encoded are 206aa and 205aa, respectively. The complete ORF sequence was ligated into the expression vector pET-28a (+) and induced by IPTG to measure the salt tolerance. The results showed that the two full-length recombinases (containing pET-28a-Rab7-1 and pET-28a -Rab7-2 recombinant plasmid) had normal enzyme activity, significantly alleviated the growth of E. coli in high salt environment (5.5% ~ 10% NaCl solution), while the control group (empty vector pET-28a) failed to detect The same enzyme activity, the results show that AhRab7 gene expression can significantly alleviate the impact of high salt stress. After induced by IPTG, the target gene was highly expressed. The size of the target protein was 23 kDa detected by SDS-PAGE, which was consistent with the predicted result. This laid the foundation for the later study of peanut on salt stress and other abiotic stresses.