羽衣甘蓝Rubisco活化酶基因BoRCA的克隆、生物信息学及表达分析

Cloning,bioinformatics and expression analysis of Rubisco activase gene BoRCA from Brassica oleracea

Rubisco活化酶(RCA)是一种可促进和维持植物光合作用关键酶Rubisco催化活性的伴侣蛋白.为了阐明RCA基因在羽衣甘蓝中的功能特征,根据油菜Rubisco活化酶基因BnRCA的全长序列设计引物,通过RT-PCR方法从羽衣甘蓝中克隆了BnRCA的同源基因,命名为BoRCA(GenBank登录号:JX944042).序列分析表明,BoRCA基因全长1 388 bp,包括1 317 bp的开放阅读框,编码438个氨基酸.该基因与BnRCA在核苷酸和氨基酸水平上的同源性分别为99.57%和99.77%.BoRCA蛋白具有定位于叶绿体基质的N端转运肽,2个保守的ATP-binding结构域和多个磷酸化位点.多重序列比对和系统进化分析显示,BoRCA与其他植物的RCA蛋白具有较高的同源性,并属于RCA的β亚基.半定量RT-PCR表达分析表明,BoRCA基因在成熟叶中表达量最高,根和老叶中没有表达,且BoRCA基因在低温条件下的表达丰度显著低于室温.这些结果为进一步研究BoRCA基因在羽衣甘蓝光合作用中的分子调控机理奠定了基础.

Rubisco activase （RCA） is a chaperone that promote and maintain the catalytic activity of Rubi sco,which is the critical enzyme in plant photosynthesis. To clarify the role of RCA gene in Brassica oleracea, which homologous with the BnRCA gene encoding Rubisco activase of Brassica napus, was cloned from Brassica oleracea. The full length of the BoRCA gene was 1388 bp and contained a 1317 bp open reading frame （ORF） encoding a 438 amino acid precursor protein. The BoRCA shared 99.57 % and 99.77 % identity, respectively, with that of BnRCA at the nucleotide and amino acid level. The BoRCA contained a predicted N - terminal transit peptide to chloroplast stroma, two high conserved ATP - binding domains and multiple phosphorylation sites. Multiple sequence alignment and phylogenetic tree revealed that BoRCA belongs to β isoform of RCA protein and has high identities with other plant RCAs. The results of semi - quantitative RT - PCR showed that the expression of BoRCA was the strongest in mature leaves, but no in roots and old leaves, and the expression under low tem perature was significantly lower than room temperature. These results lay a foundation of further study on the pho tosynthesis mechanism of RCA in Brassica oleracea.