干旱区生态环境知识资源中心(Arid): 荒漠甲虫小胸鳖甲几丁质酶基因MpCht8c的克隆、鉴定与低温表达

Arid

	荒漠甲虫小胸鳖甲几丁质酶基因MpCht8c的克隆、鉴定与低温表达
其他题名	Cloning,characterization and expression profiling at low temperature of chitinase gene MpCht8c from the desert beetle Microdera punctipennis
	郭晓星; 蔡卿龄; 刘小宁; 马纪
来源期刊	环境昆虫学报
ISSN	1674-0858
出版年	2019
卷号	41 期号:1 页码:138-147
中文摘要	为了挖掘荒漠昆虫小胸鳖甲Microdera punctipennis的耐寒性相关基因,从其4℃转录组数据库筛选出差异表达的几丁质酶基因片段394seq2,检测其编码蛋白的几丁质酶活性,研究该基因的表达对低温胁迫的响应情况。采用RACE技术扩增394seq2的5’端和3’端,克隆全长序列,将其ORF构建至原核表达载体pET28a,导入Transetta (DE3)感受态细胞,诱导表达融合蛋白,Western blot法检测表达蛋白的正确性;二硝基水杨酸法测定几丁质酶活性,qRT-PCR技术探究低温表达谱。结果表明,394seq2的5’-UTR为39 bp,3’-UTR为181 bp; ORF为1 140 bp。系统进化树显示该序列与赤拟谷盗几丁质酶8 (TcCHT8)聚为一支,命名为MpCht8c。MpCht8c编码379个氨基酸,分子量为41.2 kDa,理论等电点pI为4.67。MpCHT8c含有完整的几丁质酶结构基序,其N端含有几丁质酶催化域,内有一个类18家族几丁质酶的保守基序KXXXXXGGW,中部是一段富PEST的连接区, C端是几丁质酶结合域,属于IV型昆虫几丁质酶。Western blot结果表明His-MpCHT8c在大肠杆菌中正确表达;融合蛋白粗酶液的几丁质酶活性为1.89 U/mL。在4℃冷胁迫0.5 h和5-9 h时Mpcht8c出现两个上调表达峰值,约为对照的2倍。研究表明荒漠昆虫小胸鳖甲的几丁质酶MpCHT8c具有几丁质酶活性,MpCht8c基因的表达可快速响应4℃低温胁迫。研究结果有助于深入研究几丁质酶在小胸鳖甲耐寒性方面的作用机理。
英文摘要	In order to discover more cold related genes from the desert beetle Microdera punctipennis,a gene fragment of a chitinase,394seq2,was selected from the transcriptome database of M. punctipennis treated at 4℃. The chitinase activity of its encoding protein was determined,and its expression profiling at low temperature was explored. The 5’-end and 3’-end of 394seq2 were amplified by RACEs,and the full-length sequence was cloned. The ORF was inserted into plasmid pET28a,and then transformed into Transetta (DE3) competent cells. The cells were induced by IPTG to over express the fusion protein. The protein was detected by SDS-PAGE and by Western blot. The chitinase activity of the protein was determined by DNS method. qRT-PCR was used to explore its expression profile at 4℃. The results showed that 394seq2 had 39 bp 5’-UTR,181 bp 3’-UTR,and 1 140 bp ORF. Phylogenetic tree analysis showed that 394seq2 and chitinase 8 from Tribolium castaneum (TcCHT8) were clustered together,so 394seq2 was named as MpCht8c. MpCht8c encoded a protein of 379 amino acids with a molecular mass of 41.2 kDa and a theoretical pI of 4.67. MpCHT8 contained complete motifs of a chitinase,with a chitinase catalytic domain in the N-terminal,a conserved KXXXXXGGW motif of 18 family chitinase in this region. The C-terminal had a chitinase binding domain. A junction region rich in proline (P) , glutamic acid (E) ,serine (S) ,and threonine (T) connects the chitinase binding domain and the catalytic domain. It belonged to IV insect chitinase. Western blot result showed that His-MpCHT8c was correctly expressed in E. coli. The chitinase activity of the crude protein was 1.89 U/mL. In conclusion, MpCHT8c from the desert beetle M. punctipennis has chitinase activity,the expression of its encoding gene rapidly responds to 4℃ low temperature stress. These results will facilitate the study on the mechanism of chitinase in the cold resistance of M. punctipennis.
中文关键词	荒漠昆虫 ; 几丁质酶 ; 低温表达 ; 融合蛋白
英文关键词	Desert beetle chitinase low temperature expression fusion protein
语种	中文
国家	中国
收录类别	CSCD
WOS类目	ENTOMOLOGY
WOS研究方向	Entomology
CSCD记录号	CSCD:6451675
来源机构	新疆大学
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/239287
作者单位	新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐, 新疆 830046, 中国
推荐引用方式 GB/T 7714	郭晓星,蔡卿龄,刘小宁,等. 荒漠甲虫小胸鳖甲几丁质酶基因MpCht8c的克隆、鉴定与低温表达[J]. 新疆大学,2019,41(1):138-147.
APA	郭晓星,蔡卿龄,刘小宁,&马纪.(2019).荒漠甲虫小胸鳖甲几丁质酶基因MpCht8c的克隆、鉴定与低温表达.环境昆虫学报,41(1),138-147.
MLA	郭晓星,et al."荒漠甲虫小胸鳖甲几丁质酶基因MpCht8c的克隆、鉴定与低温表达".环境昆虫学报 41.1(2019):138-147.