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作物学报 ›› 2015, Vol. 41 ›› Issue (05): 725-732.doi: 10.3724/SP.J.1006.2015.00725

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜防御素基因的克隆与表达分析

郑小敏,郭楠,高天姝,龚慧明,张涛   

  1. 重庆师范大学生命科学学院 / 重庆市植物环境适应分子生物学重点实验室,重庆401331
  • 收稿日期:2014-09-22 修回日期:2015-02-06 出版日期:2015-05-12 网络出版日期:2015-03-13
  • 通讯作者: 张涛, E-mail: zht2188@126.com
  • 基金资助:

    本研究国家自然科学基金项目(31171588),重庆市自然科学基金(cstc2012jjA80010)和重庆市教委科学技术研究项目(KJ1400511)资助。

Cloning and Expression Analysis of Defensin Genes from Brassica napus

ZHENG Xiao-Min,GUO Nan,GAO Tian-Shu,GONG Hui-Ming,ZHANG Tao   

  1. College of Life Sciences / Chongqing Key Laboratory of Plant Molecular Biology Adaptation to the Environment, Chongqing Normal University, Chongqing 401331, China
  • Received:2014-09-22 Revised:2015-02-06 Published:2015-05-12 Published online:2015-03-13
  • Contact: 张涛, E-mail: zht2188@126.com

摘要:

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cDNA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,BnPDF2.1、BnPDF2.3、BnPDF2.5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。

关键词: 甘蓝型油菜, 防御素, 基因克隆, 表达分析, 水杨酸

Abstract:

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 cDNA 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 BnPDF2.1, BnPDF2.3, BnPDF2.5 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.

Key words: Brassica napus, Plant defensin, Gene cloning, Expression analysis, Salicylic acid

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