链孢粘帚霉寄生核盘菌菌核差异表达基因的筛选及分析  

Screening and Analysis of Differentially Expressed Genes in Gliocladium catenulatum Parasitizing on Sclerotia of Sclerotinia sclerotiorum

作　　者：高会兰[1] 李世东[1] 郭荣君[1] 张拥华[1] 

机构地区：[1]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室,北京100081

出　　处：《中国生物防治》2009年第1期48-54,共7页Chinese Journal of Biological Control

基　　金：国家863计划项目(2006AA10A211;2006AA06Z357);国家科技支撑计划项目(2006BAD08A02;2006BAI09B03-1);公益性行业(农业)科研专项(nyhyzx-049)

摘　　要：为克隆和研究链孢粘帚霉Gliocladium catenulatum寄生核盘菌菌核的相关基因，应用抑制消减杂交技术构建了cDNA消减文库并进行了筛选。通过PCR技术从文库中共筛选到1315个阳性克隆，克隆中插入片段大小主要集中于300～600bp之间。随机挑取120个克隆，经测序和同源性分析，获得60条有效序列，其中部分序列所编码的血红素加氧酶、核糖体蛋白L11、细胞色素P450及热激蛋白等均参与机体对胁迫条件的应答反应。11条序列在NCBI数据库中未找到显著匹配的序列，可能为新基因片段。分别将寄生于核盘菌菌核上的粘帚霉cDNA和粘帚霉与核盘菌纯培养的cDNA混合物经RasⅠ酶切后进行标记作为探针，利用反向Northern杂交技术验证了所选取的25条序列全部为差异表达基因片段。Subtractive cDNA library of Gliocladium catenulatum parasitizing on sclerotia of Sclerotinia sclerotiorum were constructed using suppression subtractive hybridization （SSH） technique. Mycoparasitism-associated genes were cloned and analyzed. 1315 positive clones were confirmed and selected with PCR from the cDNA library, and size of the inserts in all plasmids in the clones was between 300-600bp. 60 sequences were obtained by performing a sequencing and homology searching from 120 randomly selected clones. Among them, some sequences code proteins similar to peroxidase, ribosomal protein L11, cytochrome P450, and heat shock proteins that expressed under certain stresses. 11 gene fragments had no homology with the sequences deposited in NCBI, and therefore were supposed to be novel genes. To ensure the validity of the subtractive hybridization, cDNA from G. catenulatum parasitizing on sclerotia of S. sclerotiorum and mixture of cDNAs from pure cultures of G. catenulatum and S. sclerotiorum were used as probes respectively to perform reverse-Northern blotting, and all of the 25 sequences selected were demonstrated as differently expressed gene fragments.

关 键 词：链孢粘帚霉 菌寄生 核盘菌菌核 抑制消减杂交 差异表达基因 

分 类 号：S476.8[农业科学—农业昆虫与害虫防治] Q785.6[农业科学—植物保护]