甘蓝型油菜防御素基因的克隆与表达分析

Cloning and Expression Analysis of Defensin Genes from Brassica napus

植物防御素具有广谱抗菌活性,不仅具有抗真菌、抗细菌、蛋白酶抑制和昆虫淀粉酶抑制等活性,而且参与调节植物的生长和发育。本研究根据白菜防御素基因序列设计引物,从甘蓝型油菜中克隆获得5个防御素基因,其c DNA全长325~461 bp,含有177~243 bp开放阅读框,编码58~80个氨基酸,含有8个保守Cys残基,具备Knot1功能域。系统进化分析表明,Bn PDF2.1、Bn PDF2.3、Bn PDF2.5与拟南芥PDF2亲缘关系较近,可能具有蛋白酶抑制活性。荧光定量分析表明,防御素基因具有组织表达特异性,在花蕾和叶中表达量较高,角果中次之;经1 mmol L–1水杨酸处理开花期油菜2 h后,防御素基因在茎、花蕾、角果中的表达量均有不同程度的上调,但在叶中表达有所下调,在根中表达无明显变化。

Plant defensins show a broad spectrum of antimicrobial activity, which not merely have antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity, but play roles in regulating plant growth and development as well. According to the sequences of B. rapa defensin genes, specific primers were designed to clone five B. napus defensin genes. The lengths of c DNA sequence were between 325 and 461 bp, with 177 to 243 bp of open reading frames（ORFs）, encoding polypeptides of 58 to 80 amino acid residues. The amino acid sequences of plant defensins showed a big difference, but the six to eight conservative cysteine residues contained were stable. All the five cloned B. napus defensin genes contained a conservative Knot1 function domain. Phylogenetic analysis showed that Bn PDF2.1, Bn PDF2.3, Bn PDF2.5 and Arabidopsis thaliana PDF2 were clustered into a group, which indicates they may have a protease inhibitory activity. RTFQ PCR analysis indicated that B. napus defensin genes were expressed in various organs, but the levels of expression were obviously different. The higher expression appeared in the bud and leaf, following silique. During the flowering stage, 1 mmol L–1 SA was used to treat B. napus for 2 hours, which caused the expression levels of defensin genes to be increased in varying degrees in the stem, bud, and silique, but decreased in the leaf, and even no remarked change in the root.