摘要
利用抑制性差减杂交方法构建了稻瘟病菌分生孢子接种24 h所形成的附着胞差异表达基因文库。采用复印胶片诱导稻瘟病菌附着胞形成,在孢子浓度为1.0×106个/mL时,附着胞的形成率达到96.5%。分别提取附着胞、菌丝和分生孢子RNA,反转录成cDNA并经A luⅠ酶切、接头连接后,以附着胞cDNA片段为tester,菌丝及分生孢子cDNA为driver进行抑制性差减杂交,构建成差减文库。从文库中分离获得142个基因片段,通过RT-PCR方法鉴定其中的71个为差异表达基因,证实文库高效可靠。构建优质的稻瘟病菌附着胞差异表达基因差减文库,为深入了解附着胞形成过程中基因的表达及功能奠定了基础。
A subtractive libraries in association with appressorium formation expressed genes of M. grisea 24 hours after the conidia inoculated was constructed by suppression subtractive hybridization （SSH）. The experiment was carded out with appressorium induced on projection membrane. The ratio of appressorium formation on projection membrane was 96.5% at 1.0 ×10^6 conidia.mL^-1. Total RNA was extracted from appressorium and mixture of mycelium and conidia and reverse-transcripted into cDNA. After cut and ligation, suppression subtractive hybridization was performed with appressorium cDNA as tester and the mycelium/conidium cDNA as driver to construct subtractive libraries. Reliability of the libraries was evaluated by approach of comparing RT-PCR products with templates from tester and driver cDNAs. The results indicated that 142 target segments were isolated from the library; seventy one of them were verified to be differential expressed genes by RT- PCR, which suggested that the libraries were reliable and efficient. This study founded the basis of further investigation of genes＇ expression and function during the course of appressorium formation by constructing subtractive libraries in associated with appressorium formation differential expression genes.
出处
《植物病理学报》
CAS
CSCD
北大核心
2006年第2期129-134,共6页
Acta Phytopathologica Sinica
基金
国家自然科学基金资助项目(3027004930470064)
国家"八六三"资助项目(2002AA245041)
关键词
稻瘟病菌
附着胞
抑制性差减杂交
Magnaporthe grisea
appressorium
suppression subtractive hybridization
