花生AhRab7基因的克隆及其原核表达研究

Cloning and Prokaryotic Expression Character Analysis of AhRab7 Genes from Peanut(Arachis hypogaea L.)

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。

The full length cDNA of AhRab7-1 and AhRab7-2 was isolated from HuaYu 20（Arachis hypogaea L.） by RT-PCR and transferred into Escherichina coli to analyze whether the expression of the small G protein AhRab7 in E.coli was associated with the tolerance to high salt environment for E.coli.AhRab7-1 and AhRab7-2 were 872 bp and 816 bp,containing a 621 bp and 618 bp open reading frame and encoding 206 and 205 amino acids,respectively.AhRab7-1 and AhRab7-2 were constructed into the expression vector pET-28a（＋） with full-length ORF,then transferred into E.coli.After inducing by IPTG,the E.coli were treated with high salinity stresses and the function of the protein AhRab7 could be tested.The tolerance activity assay showed that pET-28a-AhRab7-1 and pET-28a-AhRab7-2 had normal restructure enzyme activities and significantly released the salt tolerance of E.coli in LB medium with high NaCl concentration（5.5%-10%）.The control which had been transferred into the vector pET-28 did not showed the similar enzyme activity.The result suggested the expression of AhRab7 in E.coli could extremely improve the salt tolerance for E.coli.The product proteins of AhRab7-1 and AhRab7-2 were detected by SDS-PAGE and the proteins were 23 kDa as expected.Our researches could be used as a starting point for generation of plants tolerance to saline-alkali and other abiotic stress in the future.