利用cDNA-AFLP技术分离当归间座壳菌诱导的野生车前草差异表达基因  

Detection of differentially expressed genes by the plantain upon Diaporthe angelicae infection using a cDNA-AFLP technique

作　　者：崔朝宇[1] 刘乔丽[1] 蒋军喜[1] 刘登全[1] 刘冰[1] 马建[1] 

机构地区：[1]江西农业大学农学院,江西南昌330045

出　　处：《菌物学报》2014年第1期69-77,共9页Mycosystema

基　　金：国家自然科学基金"穗枯病菌诱导的野生车前草抗性相关基因克隆及功能分析"(No.30960213)

摘　　要：以高抗穗枯病野生车前草种质PPZ14为材料,用引起车前草穗枯病的当归间座壳菌Diaporthe angelicae强致病力真菌菌株DA‐8对其接种,采用cDNA‐AFLP技术分离接种后不同时间点产生的差异表达基因片段。结果从64对引物组合中筛选到4对多态性丰富的引物组合,并用其进行扩增得到202条差异表达的转录衍生片段(transcript‐derived fragment,TDF),其中以接种24h后出现的TDF数量最多,表明在此时间点车前草与当归间座壳菌互作最活跃。对部分TDF进行克隆及序列测定,共获得7个TDF的核苷酸序列和推导的氨基酸序列,经BLAST比对,5个TDF的氨基酸序列在GenBank中找到与其对应的显著同源序列,其中4个为已知功能的基因,对其进行功能分析后,推测认为,决定ACC氧化酶、细胞色素C生成蛋白C、液泡膜H+‐ATPase的TDF与车前草抗穗枯病性相关。A wild type plantain breed PPZ14 with a high resistance to plantain head blight was inoculated with a strong virulent isolate of DA-8 of the pathogenic fungus Diaporthe angelicae. The differentially expressed gene fragments produced at different time intervals after inoculation were isolated by using a cDNA-AFLP technique. Four primer pairs obtaining abundant polymorphic bands were acquired from 64 primer combinations. Using these four primer pairs, a total of 202 differentially expressed transcript-derived fragments （TDFs） were amplified, of which, the number of TDFs was highest after 24h post inoculation, showing that the interaction between the plantain and fungus was most intensively at this time point. Some TDFs were cloned and sequenced, and nucleotide sequences of seven TDFs and their deduced amino acid sequence were obtained. By BLAST analysis, five of them have significant amino acid sequence homologies with those in GenBank database, and four TDFs had their known function. Base on their functions, those TDFs encoded 1-aminocyclopropane-1-carboxylate oxidase, cytochrome C biogenesis C and vacuolar-type H＋-ATPase that were putatively related to the resistance against D. Angelicae by the wild type plantain.

关 键 词：车前草 当归间座壳菌 CDNA-AFLP 差异表达基因 

分 类 号：S567.239[农业科学—中草药栽培]