棉枯萎病菌异核体及其不同表型分离子在基因转录水平上的差异  

Transcriptional Differences Between a Heterokaryon and Its Segregants of Fusarium oxysporum f.sp.vasinfectum

作　　者：杨红[1] 李颖[1] 关国华[1] 李秀玉[1] 

机构地区：[1]中国农业大学生物学院,农业部农业微生物资源与应用重点开放实验室,北京1OOO94,北京100094

出　　处：《Acta Genetica Sinica》2004年第2期166-170,共5页

基　　金：国家自然科学基金资助项目(编号:39770395)~~

摘　　要：采用银染mRNA差异显示技术 ,研究了棉花枯萎病菌异核体菌株 (Ag14 9)及其两个不同表型分离子(Ag14 9 Ⅰ和Ag14 9 Ⅲ )之间差异表达的基因 ,观察到在 3 0 0～ 70 0bp之间出现了 19个差异条带 ,经反向RNA印迹 ,证实其中 2条差异带为阳性 :编号为C6的差异条带在Ag14 9和Ag14 9 Ⅰ菌株中呈高表达 ,而编号为G5的差异条带在菌株Ag14 9和Ag14 9 Ⅲ中呈高表达。这两条差异片段经测序后在GenBank中分别进行同源比较 ,发现由C6片段编码的氨基酸序列与多种生物 (包括动物、植物和微生物 )NADH脱氢酶的第 6个亚基有不同程度的同源性 (3 0 %～70 % ) ;而编码G5片段的氨基酸序列与龟裂链霉菌 (Streptomycesrimosus)的OtrB(tetracyclineeffluxprotein)蛋白有 3 5%的同源性。首次证明在异核体菌株及其不同表型分离子之间存在基因转录水平差异 。In order to elucidate the mechanism of fungal heterokayosis,a wild type strain of Fusarium oxysporum f.sp.vasinfectum was isolated from the cotton field in Anyang,Henan Province.Through single hyphal-tip isolation,a heterokaryon,Ag149,was obtained,and its two different phenotypic segregants,Ag149-Ⅰ and Ag149-Ⅲ,were separated from the mutated sectors on colony of the heterokaryon.They have remarkable differences on color of colony,morphology of hypha and pathogenicity.After analyzing by RAPD on their nuclear DNA with 100 random primers,no polymorphic difference was found among them.On going to find the different expressed gene,the mRNA differential display method was performed.Two kinds of reverse transcriptase AMV and MMLV,and a kit which consists of three kinds of 3′ terminal anchor primers and eight kinds of 5′ terminal arbitrary primers were used in differential display PCR (DD-PCR).Total RNA as template was reverse transcribed into corresponding cDNA by 3′ terminal anchor primers,and the cDNA were amplified by polymerase chain reaction with a set of one same 3′ anchor primer and one 5′ arbitrary primer.The PCR products were then resolved on denaturing polyacylamimide gel,and the cDNA bands were visualized by silver staining.Among the 144 PCR products,19 differentially expressed cDNA fragments ranged from 300 bp to 700 bp were purified.All of them were ligated to pGEM-T vector respectively for sequencing and Rev-Northern blotting.Two cDNA fragments (G5 and C6) were observed to be positive after Rev-Northern blotting.The C6 was highly expressed in the heterokaryon Ag149 and its segregant Ag149-Ⅰ.It is 564 bp in length and can be predicted 77 amino acids from the beginning of the 3rd to 233th base,and then searched from GenBank.The amino acid sequence of C6 shared homologies with the 6th subunit of NADH dehydrogenase found in some bacteria,plants and animals at 30%～70% level.While the G5 was highly expressed in Ag149 and its segregant Ag149-Ⅲ.It is 432 bp in length and can be predicted 101 am

关 键 词：棉花枯萎病菌 异核体 MRNA差异显示 

分 类 号：Q943[生物学—植物学]