巴西橡胶树HbMADS4的克隆及原核表达分析

Cloning and Prokaryotic Expression Analysis of HbMADS4 from Hevea brasiliensis

为了探讨巴西橡胶树自根幼态无性系高产的分子机制,通过抑制缩减杂交获得了在巴西橡胶树自根幼态无性系和老态无性系胶乳中差异表达的基因片段。为进一步鉴定差异表达基因的功能,对相关基因进行克隆。通过RACE方法克隆一个新的橡胶树MADS-box转录因子基因(命名为Hb MADS4),Hb MADS4全长984 bp,含有633 bp的阅读框,编码230个氨基酸。推测Hb MADS4蛋白分子量为23.71 ku,等电点为7.61,在N端含有典型的MADS-box结构域。构建Hb MADS4原核表达载体p Hb MADS4,将其转化E.coli,经优化条件后,在20℃,0.1 mmol/L IPTG诱导4 h的条件下,获得Hb MADS4融合蛋白。Hb MADS4的克隆和Hb MADS4融合蛋白的获得为深入研究Hb MADS4的功能奠定基础。

In order to understand the mechanism underlying the difference between self-rooting juvenile clone and donor clones, the suppression subtractive hybridization（SSH） method was used to profile gene changes in the latex between self-rooting juvenile clones and donor clones. To study differentially expressed genes, a full-length c DNA encoding a MADS-box transcription factor, designated as Hb MADS4, was isolated from Hevea brasiliensis by RACE method. The Hb MADS4 was 984 bp containing a 633 bp open reading frame. Sequence analysis revealed that Hb MADS4 encoded 230 amino acid residues with a total predicted molecular mass of 23.71 ku and isoelectric point of 7.61. The putative protein products of Hb MADS4 contain the conserved MADS-box motif at the N-terminus, which is typical for the MADS-box transcription factors. The p Hb MADS4 prokaryotic expression vector was constructed and transferred into Escherichia coli. The recombinant protein was produced under a condition of IPTG treatment at 20 ℃. This result contributes to further study the function of Hb MADS4.