甘蓝型油菜热激转录因子的克隆及表达分析

Cloning and Expression Analysis of BnHSFA4a Gene in Brassica napus

以甘蓝型纯系油菜为试材,用RT-PCR和RACE方法首次从油菜种子中获得油菜热激转录因子的cDNA全长序列,命名为BnHSFA4a,GenBank登录号为HQ435241。结果表明:该cDNA长1 667bp,编码1个具有389aa的蛋白质,该基因编码的氨基酸序列与拟南芥热激转录因子AtHSFA4a编码的氨基酸序列间相似度为82%。对34条编码不同热激转录因子的氨基酸序列进行多重比对,结果在100～190aa处序列间氨基酸总相似性超过60%,显示出较高的保守性。构建原核表达载体并成功表达出预期蛋白。半定量RT-PCR结果显示,该基因的表达先于油菜肌醇半乳糖苷合成酶(BnGOLS1)基因,符合该基因调控BnGOLS1的假设。推测AtHSFA4a基因可能通过调控BnGOLS1的表达量进而在油菜种子脱水耐性获得过程中发挥一定的作用。

A full-length sequence of BnHSFA4a was obtained from Brassica napus by RT-PCR and RACE for the first time,named BnHSFA4a （GenBank accession No. HQ435241）. The full length of BnHSFA4a is 1 667 bp,encoding 389 amino acids. Homology analysis of amino acid sequence of BnHSFA4a showed 82% similarity with AtHSFA4a. Multi-alignments of 33 different kinds of HSFs showed high similarity more than 60% at 100 to 190 aa in their amino acid sequences. Prokaryotic expression showed the recombinant vector can express the prospective protein. The semi-quantitative RT-PCR indicated that the expression of BnHSFA4a was followed by the expression of BnGOLSt, which is in accord with hypothesis that BnHSFA4a regulates BnGOLS1. It suggests that BnHSFA4a may play an important role in the mechanisms of seed desiccation tolerance through regulating the expression of BnGOLS1 .