浅黄根须腹菌γ-actin基因的克隆及表达分析

Cloning and Expression Analysis of γ-actin Gene from Rhizopogon luteolus

以油松的优良外生菌根真菌即浅黄根须腹菌为对象,用简并PCR法和RACE技术分离其γ-肌动蛋白基因(Rl-act)的全长cDNA序列。该全长序列为1 339 bp,包含一个1 128 bp的开放阅读框(ORF),编码375个氨基酸,5'端非翻译区(5'UTR)95 bp,3'UTR长度116 bp。Port Param软件在线分析结果表明,该cDNA所编码的蛋白质理论等电点为5.01,相对分子质量为94.929 kD,具有真菌γ-actin基因3个保守特征序列。Blast同源性检索结果表明,Rl-act序列与12种担子菌actin序列同源性均在97%以上,与同为外生菌根真菌的双色蜡蘑actin的亲缘关系最近。Rl-act基因在不同碳源及磷水平培养条件下表达量基本一致,验证了该基因作为分子内标的可靠性。

In this paper,a superior ectomycorrhizal fungus,Rhizopogon luteolus of Pinus tabulaeformis＇ was used as the research object. The fungus γ-actin gene（ Rl-act） was isolated by using homology based method and RACE technique.The results showed that the full-length sequences of Rl-act cDNA 1 339 bp which was consisted of a 1 128 bp opening reading frame（ ORF）,encoding a protein of 375 amino acids,a 5＇-UTR with 95 bp and a 3＇-UTR with 116 bp. The online analysis of Port Param software displayed that the putative amino acids had an isoelectric point of 5. 01,a molecular weight of 94. 929 kD,and 3 highly conserved regions of γ-actin gene of fungi. Homology search indicated that the homogeneity was more than 97% between Rl-act cDNA and 12 basidiomycetes actin sequence. Phylogenetic tree indicated that it had closer relationship with Laccaria bicolor,an ectomycorrhizal fungus. In different culture conditions of carbon and phosphorus level,the quantity expression of Rl-act was almost same,which validated the reliability of Rl-act as reference gene in quantitative analysis of mRNA expression. The research provided an internal standard for studying other stress resistance genes＇ expression and regulation in symbiosis of R. luteolus and P. tabulaeformis,and gave a theoretical foundation for exploring the molecular mechanism in stress resistance.