Wang Jinfang, Liu Guangying, Zhai Yi, Wan Yongji
College of Biotechnology, Southwest University, Chongqing, Beibei 400716, China.
Wei Sheng Wu Xue Bao. 2010 Apr;50(4):493-9.
In order to 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.
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 1.5 mg/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.
The complete sequence length of chitinase gene of N. rileyi CQ strain is 2756 bp (GenBank accession number EU795711) that contains a 1827bp of ORF chit1, a 76 bp uncoding region at the 5' end, a 240 bp 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 482.5 U/100 microL at 72 h. 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.
We cloned the chitinase gene Chit1 from N. rileyi CQ strain and expressed in recombinant P. pastori successfully to study the infection and lethal mechanism of pathogenic fungi to insects.
为了解昆虫病原真菌几丁质酶在侵染和致病过程中的作用，我们克隆并表达了莱氏野村菌CQ菌株的一个几丁质酶基因，并检测了重组毕赤酵母发酵上清液中的几丁质酶活性。
使用特异性引物从经CTAB提取的莱氏野村菌CQ菌株基因组DNA中扩增几丁质酶基因的完整序列和开放阅读框（ORF）。将ORF片段与载体质粒pPIC9K连接构建pPIC9K-chit1表达载体，并转入毕赤酵母。通过1.5 mg/L G418和PCR检测筛选重组酵母毕赤酵母。通过透明圈试验和OD540测定验证重组毕赤酵母发酵液中几丁质酶的活性，并通过SDS-PAGE分析几丁质酶的分子量。
莱氏野村菌CQ菌株几丁质酶基因的完整序列长度为2756 bp（GenBank登录号EU795711），其中包含1827 bp的ORF chit1、5'端76 bp的非编码区、3'端240 bp的非编码区以及3个内含子。ORF chit1编码424个氨基酸的几丁质酶前体，单肽的理论切割位点在Gly（20）和Leu（21）之间。重组毕赤酵母表达的几丁质酶活性随发酵时间增加，在72 h达到峰值482.5 U/100 μL。几丁质水解试验在1%胶体几丁质平板上显示出清晰的透明圈。SDS-PAGE分析表明几丁质酶的分子量为41.0 kDa。
我们成功从莱氏野村菌CQ菌株中克隆了几丁质酶基因Chit1并在重组毕赤酵母中表达，以研究病原真菌对昆虫的感染和致死机制。
