莱氏野村菌Cq菌株几丁质酶基因的克隆与表达分析

Cloning and expression analysis of the chitinase gene Chit1 from entomopathogenic fungus Nomuraea rileyi CQ strain

摘要:

摘要：【目的】为揭示昆虫病原真菌分泌的几丁质酶对宿主感染致病时的作用，对莱氏野村菌Cq菌株几丁质酶基因进行了克隆与表达，并检测了表达产物的活性。【方法】采用CTAB法提取菌体DNA，设计特异性引物，多次PCR扩增克隆莱氏野村菌Cq菌株几丁质酶基因全序列，并克隆基因的ORF片段chit1，与载体pPIC9K相连接，构建表达载体pPIC9K-Chit1，转入毕赤酵母感受态细胞中，然后通过1.5 mg/L浓度的G418筛选及PCR验证，将阳性转化子进行诱导培养，对发酵液分别进行酶活性测定试验、几丁质酶透明圈验证

Abstract:

Abstract: [Objective] In order to deeply understand the role of the entomopathogenic fungal chitinase in invasion and pathogenic process, we cloned and expressed a chitinase gene from Nomuraea rileyi CQ strain, and detected chitinase activity in recombinant Pichia pastori fermentation supernatant. [Methods] Specific primers were used to amplify the complete sequence of chitinase gene and open reading frame (ORF) from genomic DNA of N. rileyi Cq Strain extracted by CTAB. The fragment of ORF was linked with vector plasmid pPIC9K to construct the pPIC9K-chit1 expressing vector and transferred into Pichia pastori. The recombinant yeast P. pastori was screened by 1.5mg/L G418 and PCR check. Activity of chitinase in recombinant P. pastori fermentation liquid was verified by transparent zone test and OD540 determination, and the molecular weight of chitinase was analyzed by SDS-PAGE. [Results] The complete sequence length of chitinase gene of N. rileyi CQ strain is 2756bp (GenBank accession number EU795711) that contains a 1827bp of ORF chit1, a 76bp uncoding region at the 5’end, a 240bp uncoding region at the 3’end, and 3 introns. The ORF chit1 encoding 424 amino acids of chitinase precursor, and the theoretical restriction site of single peptide is between Gly(20) and Leu(21). Activity of chitinase expressed by recombinant P.pastoris increased with fermentation time, and reached the peak of 482.5U/100μL at 72h. Hydrolysis test of chitin showed clear transparent zone at the 1% colloidal chitin plate. SDS-PAGE analysis suggested that the molecular weight of chitinase was 41.0 kDa. [Conclusion] This research cloned the chitinase gene Chit1 from N. rileyi CQ strain and expressed in recombinant P. pastori successfully. It has great significance for in-depth study on the infection and lethal mechanism of pathogenic fungi to insects.