甘蓝型油菜BnPLE2基因克隆及转基因拟南芥过表达分析

Cloning of BnPLE2 from Brassica napus L. and over-expression in transgenic Arabidopsis

为研究油菜中二氢硫辛酸乙酰转移酶(E2)基因的功能,利用甘蓝型油菜品种中双11号参考序列设计引物,克隆E2基因的CDS序列,命名为BnPLE2,序列全长1 503bp,由6个外显子构成,编码500个氨基酸残基,预测蛋白相对分子质量为25.17k D,等电点为9.04。BnPLE2氨基酸序列高度保守,与拟南芥Atpl E2相似性最高。构建BnPLE2超表达载体p35S::BnPLE2转化拟南芥哥伦比亚生态型。转基因拟南芥后代表现出花序簇状分布,花蕾排列紧密呈菊花状。主花序顶端优势消失,侧枝生长发达,果荚畸形,柱头突出。

To better understand the functions of dihydrolipoamide acetyltransferase（ E2） gene in rapeseed（ Brassica napus L.）,CDS sequence of E2 gene from Brassica napus L. cv Zhongshuang 11（ designated as BnPLE2） was cloned and transformed into Arabidopsis thaliana. BnPLE2 full- length sequence（ 1 503bp） included 6 exons encoding 500 amino acids with a predicted molecular mass of 25. 17 k D and p I 9. 04. The amino acids of BnPLE2 were highly conserved. Phylogenetic analysis indicated that BnPLE2 protein showed highest similarity with Atpl E2 in Arabidopsis. Overexpression construct p35S： ： BnPLE2 was performed and transformed into Arabidopsis Columbia. The positive transgenic lines were obtained and the abnormal phenotype was observed,including inflorescence spike like cluster,compact flower arrangement and chrysanthemum shaped. In addition,the disappeared apical dominance of main inflorescence,increased lateral branch,pod deformity and stigma protrusion were also observed.