莱氏野村菌疏水蛋白基因Nrhyd的克隆及其表达特征分析

Cloning and expression analysis of an hydrophobin gene Nrhyd in Nomuraea rileyi

运用SMART RACE RT-PCR技术,从虫生真菌莱氏野村菌中克隆出完整的疏水蛋白基因Nrhyd编码区序列,以RT-qPCR技术,对莱氏野村菌Nrhyd基因在不同生长条件下的表达特征进行分析,从mRNA转录水平上探讨了不同培养条件对Nrhyd基因表达的影响。结果表明:Nrhyd基因全长733bp,5′非翻译区132bp,3′非翻译区262bp,开放阅读框(ORF)339bp,编码111个氨基酸,前体蛋白理论分子量10.6kDa,理论等电点为6.19。液体摇瓶培养条件下,Nrhyd基因的表达受抑制呈逐渐降低的趋势。固体培养条件下,Nrhyd基因表达量随着分生孢子的产生而升高,到产孢量达到最大的第8天时,表达量最高,以后随着产孢量的下降,基因的表达量降低。因此推测,Nrhyd基因在莱氏野村菌分生孢子形成过程中起重要作用。与不同真菌疏水蛋白基因的系统进化分析表明,Nrhyd基因与绿僵菌来源的同源基因关系最近。

The full-length cDNA of a hydrophobin gene Nrhyd gene was cloned from the insect pathogenic fungus Nomuraea rileyi using SMART RACE RT-PCR. The expression of Nrhyd gene by N. rileyi under different culture conditions was analyzed using Realtime-PCR, and the effects of different culture conditions on expression of Nrhyd gene on mRNA level were explored. Sequence analysis shows that the cDNA contains an open reading frame of 339bp which encoding a polypeptide with 111 amino acids. The precursor protein has a molecular mass of 10.6 and a calculated pI of 6.19. TheRT-qPCR results showed that the expression ofNrhyd gene was suppressed under liquid culture condition. While under solid agar culture condition, the expression level of Nrhyd gene increased along with the formation of conidia and reached the highest level at day 8 of post-inoculation, and then decreased gradually. It is inferred therefore that the Nrhydgene might play an important role in the formation of conidia in N. rileyi. Phylogenetics analysis of Nrhyd gene with other fungal hydrophobin indicates the gene is most closely related to the homologues from Metarhizium spp.