棉花GhRGL基因克隆及其原核表达研究

GA信号转导途径是通过DELLA蛋白来抑止的。通过对Genbank进行Blast比较，我们首次克隆了棉花DELLA蛋白基因，长度为1644bp，分离的棉花DELLA蛋白进行生物信息学分析显示,该蛋白具有与拟南芥中的DELLA蛋白一样的保守结构域，我们对克隆的基因进行原核表达研究，将克隆的基因转化原核表达载体pET22b，在E.coli BL21（DE3）菌株中成功表达了与标签蛋白融合的GhRGL蛋白, 大小约为64kDa。首次实现了棉花DELLA蛋白基因在原核系统中的表达, 为棉花DELLA蛋白的抗体制备及其表达定位研究奠定了基础。

Studies on gene cloning of GhRGL from cotton and prokaryotic expression

The GA signaling pathway is repressed by the DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified in the NCBI GenBank database with the NCBI BLAST program, according to the sequence of the cotton nucleotide, full-length cDNAs and genomic coding sequences from upland cotton (Gossypium hirsutum) RGL (GhRGL) were obtained and characterized. Sequence comparisons between the sequence and all of DELLA proteins in Arabidopsis indicted which is a ortholog of AtRGL. And over-express it in prokaryotic systems, The expression construct pET22b carrying the GhRGL coding sequence was preformed, and transformed into E. coli BL21 used as a host. Induced by IPTG, GhRGL-His Tag fusion protein with molecular weight of 64kDa was successfully expressed in transformant. It is the first time to demonstrate recombinant expression of rape GhRGL gene in prokaryotic system, which paves the way for antibody preparation and the study of in situ expression of GhRGL.