油菜防卫素和草酸氧化酶基因的克隆与诱导表达水平研究  

Cloning of plant defensin and oxalic acid oxidase genes and expressions of these genes induced by pathogens and chemicals in Brassica napus

作　　者：刘胜毅[1] B.D.L.Fitt 刘仁虎 N.Evans 董彩华[1] 黄永菊[1] 

机构地区：[1]中国农业科学院油料作物研究所农业部油料作物重点开放实验室,湖北武汉430062 [2]Plant Pathogen Interactions Division, Rothamsted Research

出　　处：《中国油料作物学报》2004年第3期43-49,共7页Chinese Journal of Oil Crop Sciences

基　　金：国家人事部回国留学人员资助项目;"948"项目;洛桑国际(RothamstedInternational)资助项目

摘　　要：基于拟南芥、甘蓝及油菜等的植物防卫素(PDF)相关基因序列和已克隆的油菜草酸氧化酶(OXO)基因序列设计PCR引物,对甘蓝型油菜基因组DNA进行扩增,获得了序列大小分别为470bp和629bp的PDF1.2和OXO。生物信息学分析表明,该PDF1.2和GenBank中的相关基因同源性在42%～71%,OXO和GenBank中的相关基因同源性在70%以上。在油菜苗期,油菜黑胫病病原接种后基因PDF1.2表达量增加3倍以上,基因OXO也有不同程度的增强表达;诱导剂acibenzolar-s-methyl诱导基因OXO增强表达,但不能诱导PDF1.2增强表达。诱导的系统表达(非处理叶)高于局部表达(处理叶)。这是首次报道油菜PDF1.2克隆和诱导表达结果。PDF1.2(plant defensin 1.2)and oxalic acid oxidase (OXO) genes were cloned using PCR-based methods. Primers for PCR were designed based on sequences of PDF genes of Arabidopsis thaliana and Brassica oleracea, an antifungal protein of B. napus for PDF1.2 amplification, and OXO gene of B. napus for OXO amplification. Sequences of 470bp for PDF1.2 and 629bp for OXO were obtained by the amplification based on genomic DNA of B.napus cv. Maddrigal, and cloned separately into a vector. There were 42%～71% identity between the cloned PDF1.2 and any of relevant sequences in the GenBank and above 70% identity between the cloned OXO and any of relevant sequences in the GenBank. Expressions of the two genes were investigated in B.napus using real-time quantitative PCR. B. napus seedlings were induced by inoculation with Leptosphaeria biglobosa or spraying with acibenzolar-s-methyl (ASM) in the first true leaves, and 24h later were challenge inoculated with L. maculans in the first and second true leaves. Twenty-four hours after challenge inoculation, expression of the (PDF1.2) increased 3 times more in local (induced leaf 1) and systemic leaves (non-induced leaf 2,3) than in the un-inoculated control, and that of OXO increased 1.5-fold in the systemic leaves. There was no increase in PDF1.2 expression after spraying with ASM, but a 2-fold increase in expression of OXO in the systemic leaves. Levels of expression of the two genes were greater in the systemic leaves than that in the local leaves.

关 键 词：油菜 防卫素基因 草酸氧化酶基因 克隆 真菌侵染 基因表达水平 

分 类 号：S565.4[农业科学—作物学] Q78[生物学—分子生物学]