棉铃虫Ⅳ型几丁质酶的基因克隆、酶学性质及表达谱  

Gene cloning, characterization and expression profiling of a group Ⅳ chitinase from Helicoverpa armigera (Lepidoptera: Noctuidae)

作　　者：麦麦提艾力.阿卜杜纳斯尔 马纪[1] 刘宁[2] 王威[2] 李梦鸽 古新蓉 王新 刘小宁[1] Maimaitiaili ABUDUNASIER;MA Ji;LIU Ning;WANG Wei;LI Meng-Ge;GU Xin-Rong;WANG Xin;LIU Xiao-Ning(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046,China;Institute of Crop Variety Resources,Xinjiang Academy of Agricultural Sciences,Urumqi 830091,China)

机构地区：[1]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830046 [2]新疆农业科学院作物品种资源研究所,乌鲁木齐830091

出　　处：《昆虫学报》2019年第2期170-180,共11页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31471781);新疆维吾尔自治区科学基金项目(2016D01C042);大学生实训项目

摘　　要：【目的】昆虫几丁质酶(chitinase, CHT)主要参与蜕皮、围食膜的降解和机体免疫防御等重要生理生长发育过程。本研究旨在对棉铃虫Helicoverpa armigeraⅣ型(group)几丁质酶基因进行克隆和表达分析,为以该基因作为棉铃虫防控的分子靶标提供理论依据。【方法】采用RT-PCR和RACE技术从棉铃虫中肠中克隆Ⅳ型几丁质酶基因,分别运用DNAMAN和MEGA软件进行多序列比对和构建系统发育树。在大肠杆菌Escherichia coli(DE3)中诱导表达其体外重组蛋白,利用Western blot进一步验证;用Ni-NTA纯化柱纯化重组蛋白,之后研究该蛋白的酶学性质。qPCR分析该基因的在棉铃虫不同发育阶段和6龄幼虫不同组织中的表达谱。【结果】克隆获得棉铃虫几丁质酶基因HaCHT4(GenBank登录号:MH500771),其cDNA长1 624 bp,ORF长1 527 bp,编码509个氨基酸,预测的分子量为55.2 kD。蛋白质序列的N末端具有信号肽,中间序列部分含有一个催化结构域(catalytic domain, CAD), C末端含有一个几丁质结合结构域(chitin binding domain, CBD)。多序列比对显示,HaCHT4具有几丁质酶的保守区域;系统发育分析表明,HaCHT4属于Ⅳ型几丁质酶。重组蛋白His-HaCHT4在大肠杆菌中成功表达。纯化的重组蛋白对胶体几丁质底物具有降解活性,最适温度和pH分别为50℃和7,动力学参数K_m和V_(max)值分别为1.76±0.35 mg/mL和0.0220±0.0012μg/mL·s。qPCR分析表明,HaCHT4在1龄和2龄幼虫期的表达量显著高于其他幼虫龄期及预蛹期;主要在中肠和脂肪体中高度表达,体壁和头部中低表达。【结论】结果提示棉铃虫HaCHT4可能参与围食膜中几丁质降解过程。这些结果为深入研究HaCHT4的功能奠定了基础,并为害虫防治提供了有用的信息。【Aim】Insect chitinases are mainly involved in such important physiological processes as molting,degradation of peritrophic membranes and immune defense.This study aims to clone a group IV chitinase gene from Helicoverpa armigera and to analyze its expression profiles so as to provide the theoretical basis for the control of this insect using the gene as a molecular target.【Methods】A group IV chitinase gene was cloned from the midgut of H.armigera using RT-PCR and RACE techniques and subjected to multiple sequence alignment by DNAMAN,and the phylogenetic tree was constructed by MEGA.Its recombinant protein was expressed in Escherichia coli(DE3)and verified by Western-blot.The enzymatic properties of the recombinant protein were characterized after purification with Ni-NTA.The expression levels of this gene in different developmental stages and tissues of the 6th instar larvae of H.armigera were analyzed by qPCR.【Results】A chitinase gene was cloned from H.armigera and designated as HaCHT 4(GenBank accession no.:MH500771).Its full-length cDNA is 1 624 bp in length and contains an ORF of 1 527 bp that encodes 509 amino acids with a predicted molecular weight of 55.2 kD.Its encoded protein contains a signal peptide,one catalytic domain(CAD)and one chitin binding domain(CBD).Both multiple sequence alignment and phylogenetic analysis showed that HaCHT4 has the conserved domain of chitinase and belongs to group IV chitinases.The recombinant His-HaCHT4 was successfully expressed in E.coli.The purified recombinant protein exhibited the activity of digesting chitin,with the optimum reaction temperature of 50℃and pH value of 7.The values of kinetic parameters K m and V max of the recombinant protein were 1.76±0.35 mg/mL and 0.0220±0.0012μg/mL·s,respectively.qPCR results revealed that the relative expression levels of HaCHT 4 in the 1st and 2nd larval instars of H.armigera were significantly higher than those in other larval instars and the pre-pupal stage,and HaCHT 4 was predominantly expressed in the midgu

关 键 词：棉铃虫 几丁质 Ⅳ型几丁质酶 酶学性质 基因表达分析 

分 类 号：Q966[生物学—昆虫学]