甘蓝型冬油菜VDAC1基因的克隆、表达分析与亚细胞定位

Cloning,expression analysis and subcellular localization of VDAC1 gene in Brassica napus

为研究甘蓝型冬油菜VDAC1基因在低温胁迫下的功能,以弱抗寒性甘蓝型油菜天油2288和强抗寒性甘蓝型油菜16NTS309为材料,分别以天油2288和16NTS309的cDNA为模板,克隆得到BnVDAC1基因CDS区。生物信息学分析发现,两基因编码区氨基酸数目都为276,等电点分别是7.28和8.46,是稳定性蛋白(<40为稳定),二级结构以无规则卷曲为主,三级结构都是由β-折叠环绕组成两个β-桶状结构。构建pBI121-BnVDAC1-GFP融合表达载体,进行烟草叶片亚细胞定位发现,BnVDAC1主要定位于线粒体或质膜。联系定量结果表明,低温处理下两个品种叶片的相对电导率、叶片相对含水量、H_(2)O_(2)和O_(2)^(⋅-)含量有差异,且与BnVDAC1的表达量有相关关系,BnVDAC1基因具有明显的品种特异性和组织表达特异性。

To study the function of VDAC1 gene in Brassica napus under low temperature stress,CDS region of BnVDAC1 gene was cloned using cDNAs as templates from both Tianyou 2288(weak cold resistance)and 16NTS309(strong cold resistance).Bioinformatics analysis found that their BnVDAC1 genes encoded 276 amino ac-ids.The isoelectric points were 7.28 and 8.46 respectively.The proteins were stable(<40).The secondary struc-tures were mainly random coil,and the tertiary structures were composed of 2β-barrels surrounded byβ-sheets.Through pBI121-BnVDAC1-GFP fusion expression vector,tobacco leaf subcellular localization showed that BnV-DAC1 was mainly located in mitochondria or plasma membrane.The quantitative results showed that the relative conductivity,relative water content,H_(2)O_(2) and O⋅-2 content of the leaves of the 2 varieties were different under low temperature treatment.The expression level of BnVDAC1 was correlated with relative conductivity,relative water content and ROS changes.The differences in relative conductivity,relative water content,H_(2)O_(2) and O⋅-2 content of leaves between the 2 varieties under low temperature treatment were analyzed and compared.The quantitative re-sults showed that the expression level of BnVDAC1 was correlated with relative conductivity,relative water content of leaves and ROS changes.The gene had obvious variety specificity and tissue expression specificity.