甘蓝BYPASS1编码基因的克隆与表达分析

Encoding Gene Cloning and Expression Analysis of BYPASS1 in Brassica oleracea L.

通过双向电泳结合MALDI-TOF-TOF/MS质谱技术在甘蓝柱头内鉴定出1个受自花授粉诱导上调表达的BYPASS1蛋白(BPS1)。利用PCR技术扩增甘蓝BPS1基因的编码序列,序列分析结果表明:该基因没有内含子,开放阅读框为1059bp,编码352个氨基酸,蛋白质分子量为38.7kD。氨基酸同源性和系统发生树分析结果表明:甘蓝BPS1与拟南芥BPS1亲缘最近,氨基酸同源相似性达85%;与烟草BPS2关系较远。RT-PCR检测结果表明:甘蓝BPS1基因在开花前1～2d的花瓣、萼片、花药、柱头和叶片中均有表达,柱头中的表达量最高。实时荧光定量PCR分析结果表明:甘蓝柱头内BPS1表达水平在自花授粉后逐渐升高,异花授粉后柱头内BPS1表达水平先升高后降低再升高。

An up-regulated protein in Brassica oleracea L. stigma induced by self-pollination was identified as BYPASS1 protein by 2-DE electrophoresis and MALDI-TOF-TOF/MS. The genomic DNA and cDNA coding sequences of BYPASS1(BPS1)were amplified by PCR.The sequence analysis showed that BPS1 had no intron. Its ORF was 1 059 bp,encoded a polypeptide with 352 amino acids with a predicted molecular mass of 38.7 kD. Phylogenetic tree analysis using MEGA 5.1 showed that Brassica oleracea L. BPS1 was more close to Arabidopsis thaliana BPS1 rather than Nicotiana benthamiana BYPASS1,amino acid homologous similarity is 85%.RT-PCR analysis showed that 1-2 days before flowering,BPS1 expressed in petal,sepal,pollen,stigma and leaf,and the express level in the stigma was higher than the other organs.Real-time fluorescence quantitative PCR analysis showed that the relative expression level of BPS1 in stigma was continually increased after self-pollination,and after cross-pollination its expression level was increased firstly,than decreased and finally increased again.