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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 Nrhyd coding region of the hydrophobin gene was cloned from Mitochondria larvae by SMART RACE RT-PCR. The expression pattern of Nrhyd gene was analyzed by RT-qPCR under different growth conditions The effects of different culture conditions on the expression of Nrhyd gene were explored from the mRNA transcription level. The results showed that Nrhyd gene was 733bp in length, 132bp in 5 ’untranslated region, 262bp in 3’ untranslated region and 339bp in open reading frame (ORF) encoding 111 amino acids. The theoretical molecular weight of precursor protein was 10.6kDa and the theoretical isoelectric point was 6.19 . Under liquid shake flask culture, the expression of Nrhyd gene tended to decrease gradually. Under solid culture conditions, the expression level of Nrhyd gene increased with conidia production, and reached the highest level on the 8th day when the sporulation amount reached the maximum. Afterwards, the gene expression decreased with the decrease of sporulation amount. Therefore, it is speculated that the Nrhyd gene plays an important role in the conidiospore formation of N. thuringiensis. Phylogenetic analysis of the different fungal hydrophobin genes revealed that the Nrhyd gene is most closely related to the homologous gene from Metarhizium anisopliae.