草菇冷诱导相关基因的克隆及序列分析

Cloning and sequence analysis of cold induced genes in Chinese straw mushroom, Volvariella volvacea

利用差异显示技术分离获得草菇低温特异DNA片段,经与正常草菇和低温诱导草菇cDNA分别southern杂交验证后,得到低温特异性片段。采用PCR标记技术对获得的低温特异性片段进行DIG标记,以此为探针,对低温处理的草菇cDNA文库进行筛选,获得4个阳性克隆,分别进行测序。序列同源性比较分析发现,Cor3基因与s-腺苷-L-高半胱氨酸水解酶有很高的同源性,Cor4基因与40S核糖体蛋白S9有很高的同源性,这两个基因可能与草菇的低温自溶现象有关。Cor1基因与脉孢菌的保守假设蛋白(conservedhypotheticalprotein)有同源性,Cor2基因与辅酶A连接酶有同源性。半定量RT-PCR验证发现Cor1和Cor2基因在正常情况下没有表达,低温处理后有表达,Cor3和Cor4基因在正常情况下有表达,低温处理后表达量增加。

Cold induced fragments were obtained by mRNA differential display, after cold specific reconfirmation by southern DNA hybridization with dig-labeled cDNA from normal and cold induced straw mushroom. Four cold specific fragments were chosen to label dig-dUTP by PCR methods for scanning 4℃ V23 cDNA library. Four positive clones were obtained. The results of DNA sequence and blast in the Genbank indicated that Cor3 gene showed high homology with putative S-adenosyl-L-homocysteine hydrolase gene, Cor4 gene showed high homology with 40S ribosome protein S9 gene. These two genes may be related with mycelium autolyzed. Cor1 and Cor2 genes showed homology with conserved hypothetical protein of Neurospora crassa and Co-A ligase. Semi-quantitative RT-PCR showed that the expression level of Cor3 and Cor4 genes was higher in 4℃ than in normal, and that Cor1 and Cor2 genes only expressed in 4℃.