板栗疫病菌cpsr1基因与cphyma基因的克隆及其功能研究
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摘要
低毒病毒／板栗疫病菌系统是一个具有独特优点的、崭新的研究病毒与宿主相互作用的模型系统。在前期的cDNA芯片杂交试验中，本实验室发现2个受低毒病毒调控，可能与分生孢子形成相关的基因cpsr1与cphyma。在此基础上，本论文克隆了这2个基因，用RNA干扰及基因敲除技术分别研究了这2个基因在板栗疫病菌中的功能。荧光定量PCR分析表明，在cpsr1基因的RNA干扰株中，cpsr1基因表达量明显降低，最高干扰程度达到了90％，干扰株的分生孢子数量呈下降趋势，与对照株△cpku80相比，敲除cphyma基因的缺失株分生孢子量显著减少，菌落生长缓慢，菌落边缘不规则，菌株颜色偏浅。本研究证明了基因cpsr1与cphyma在板栗疫病菌分生孢子形成中起作用。
Hypovirus/Cryphonectria parasitica system is a novel model inthe investigation of virus-host interaction. By cDNA array analysis,two hypovirus-regulated C. parasitica genes cpsr1 and cphyma wereimplied in conidial production. Here we report the cloning andfunctional identification of cpsr1 gene by RNA interference (RNAi)and cphyma by gene disruption. RNA quantification by real-timePCR of the transformants derived from the transformation of cpsr1RNAi construct revealed that they exhibited RNA interference withup to 90% reduction in RNA accumulation for cpsr1. Compared withthe parental strain△cpku80 and the EP155, the selectedtransformants with cpsr1 interfered have significant fewer number ofconidial spores. Deletion of cphyma gene in C. parasitica resulted in aphenotype of lowered asexual sporulation, reduced growth rate,irregular colony edge and reduced orange pigment. This workprovides evidence that both cpsr1 and cphyma genes are involved in asexual spore development in C. parasitica.
Hypovirus/Cryphonectria parasitica system is a novel model inthe investigation of virus-host interaction. By cDNA array analysis,two hypovirus-regulated C. parasitica genes cpsr1 and cphyma wereimplied in conidial production. Here we report the cloning andfunctional identification of cpsr1 gene by RNA interference (RNAi)and cphyma by gene disruption. RNA quantification by real-timePCR of the transformants derived from the transformation of cpsr1RNAi construct revealed that they exhibited RNA interference withup to 90% reduction in RNA accumulation for cpsr1. Compared withthe parental strain△cpku80 and the EP155, the selectedtransformants with cpsr1 interfered have significant fewer number ofconidial spores. Deletion of cphyma gene in C. parasitica resulted in aphenotype of lowered asexual sporulation, reduced growth rate,irregular colony edge and reduced orange pigment. This workprovides evidence that both cpsr1 and cphyma genes are involved in asexual spore development in C. parasitica.
引文
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