甘蓝型油菜BnADH3基因的克隆及转BnADH3拟南芥的耐淹性

Cloning of Bn ADH3 Gene from Brassica napus L. and Submergence Tolerance of Bn ADH3 Transgenic Arabidopsis

利用RT-PCR技术从甘蓝型油菜耐淹品系WR-4中克隆获得Bn ADH3基因,其完整开放阅读框为1137 bp。该基因编码379个氨基酸,与甘蓝Bo ADH3基因和拟南芥At ADH3基因高度同源,同源性分别达到96%和91%。利用定量RT-PCR检测Bn ADH3基因在油菜耐淹系WR-4和不耐淹系WR-24中的表达表明,在淹水处理下该基因表达受到一定的诱导,淹水处理6 h后表达开始上调,说明Bn ADH3基因在油菜耐淹机制中发挥作用;将其转化到模式生物拟南芥中,采用幼苗淹水3 d后去水处理,测定表明转基因株系叶片和根系中乙醇脱氢酶活性均高于野生型;生长4周和6周的拟南芥植株淹水3 d后的表型显示,Bn ADH3的表达可增强拟南芥对淹水胁迫的耐性,处理的T2代转基因幼苗大部分恢复,但野生型幼苗枯死;调查表明,淹水5 d后野生型植株的存活率为26.7%,转基因株系ADH33和ADH44的存活率分别为80.0%和66.7%。

BnADH3 gene was cloned from submergence-tolerant line WR-4 of Brassica napus L. using RT-PCR technique. The full-length open reading frame is 1137 bp, encoding 379 amino acids. Homology analysis showed that BnADH3 gene was highly homologous to BoADH3 from Brassica oleracea and AtADH3 from Arabidopsis, with the 96% and 91% similarity, respectively. Quantitative RT-PCR assay was carried out to compare BnADH3 expression between the submergence-tolerant line WR-4 and the submergence-susceptible line WR-24. The result showed that the BnADH3 expression was induced by submergence and the up-regulation occurred since 6-hour post treatment. The BnADH3 transgenic Arabidopsis was obtained and the transgenic seed- lings were exposed to three-day submergence stress. Overexpression of BnADH3 resulted in higher ADH activity in leaf and root of transgenic Arabidopsis compared to that of the wild type. The four- and six-week seedlings of Tz generation showed higher tolerance to submergence stress after three-day submergence treatment and most Tz seedlings were recovered with nor- mal growth when the stress was relieved for three days. However, the wild-type seedlings withered until death. After five-day submergence, the survival ratios were 26.7% for the wild type, 80.0% for transgenic line ADH33, and 66.7% for transgenic line ADH44.