利用酵母双杂交系统筛选与BnSmD1相互作用的蛋白

Screening of the proteins interacting with the BnSmD1 by Yeast Two-Hybrid System

本研究以油菜BnSmD1为诱饵蛋白,利用酵母双杂交技术,从甘蓝型油菜cDNA文库中筛选出与之有相互作用的Arf-GTP酶激活蛋白1(Brassica napus ADP-ribosylation factorGTPase activating proteinl,BnArf GAP1),采用RT-PCR扩增并克隆到3个油菜ArfGAP基因的开放阅读框全长序列,BnArf GAP1、BnArf GAP2和BnArfGAP3.BnArfGAP1和BnArfGAP2都拥有1个507bp的开放阅读框,编码168个氨基酸残基;与前两个基因相比,BnArfGAP3核苷酸序列中插入了一个93bp的带有终止密码子的外源片段,导致翻译的提前终止,它拥有1个249bp的开放阅读框,编码82个氨基酸残基.BnArfGAP1和BnArfGAP2核苷酸序列一致性达到98.22%;BnArfGAP1与BnArfGAP3核苷酸序列一致性达到82.83%,BnArf GAP2与BnArf GAP3核苷酸序列一致性达到84.17%.BnArfGAP1、BnArf GAP2和BnArf GAP3都含有一个C2结构域.

From the Brassica napus eDNA library, BnArf GAP1 （Brassica napus ADP-ribosylation factor GTPase activating protein1）, wihch interacted with the bait protein, BnSmD1, was obtained by yeast two-hybrid system screening. By reverse transcription PCR, the open reading frames of three Arf GAP genes in Brassica napus were cloned, which were named as BnArf GAP1, BnArf GAP2 and BnArf GAP3, respectively. Both BnArf GAP1 and BnArf GAP2 possessed an open reading flame of 507 bp, encoding a protein of 168 amino acid residues. BnArfGAP3 possessed an open reading frame of 249 bp, encoding a protein of 82 amino acid residues, because its nucleotide sequence was inserted by an unhomologous 93bp sequence with a stop codon, which caused the early termination of translation. The nucleotide sequences of BnArf GAP1 and BnArf GAP2 had 98.22% similarity. The nucleotide sequences of BnArfGAP3 and the other two genes had 82.83% and 84. 17% similarity, respectively. All of the three proteins contained a C2 domain.