棉铃虫,几丁质,Ⅳ型几丁质酶,酶学性质,基因表达分析," /> 棉铃虫,几丁质,Ⅳ型几丁质酶,酶学性质,基因表达分析,"/> 棉铃虫Ⅳ型几丁质酶的基因克隆、酶学性质及表达谱

昆虫学报 ›› 2019, Vol. 62 ›› Issue (2): 170-180.doi: 10.16380/j.kcxb.2019.02.004

• 研究论文 • 上一篇    下一篇

棉铃虫Ⅳ型几丁质酶的基因克隆、酶学性质及表达谱

麦麦提艾力·阿卜杜纳斯尔1, 马纪1, 刘宁2, 王威2, 李梦鸽1, 古新蓉1, 王新1, 刘小宁1,*   

  1.  (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)

     

  • 出版日期:2019-02-20 发布日期:2019-02-28

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

Maimaitiaili ABUDUNASIER1, MA Ji1, LIU Ning2, WANG Wei2, LI Meng-Ge1, GU Xin-Rong1, WANG Xin1, LIU Xiao-Ning1,*   

  1. (1. Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China; 2.InstituteofCropVariety Resources,XinjiangAcademyof Agricultural Sciences,Urumqi830091,China)

  • Online:2019-02-20 Published:2019-02-28

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 armigera Ⅳ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-PCRRACE技术从棉铃虫中肠中克隆Ⅳ型几丁质酶基因,分别运用DNAMANMEGA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cDNA1 624 bpORF1 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507,动力学参数KmVmax值分别为1.76±0.35 mg/mL0.0220±0.0012 μg/mL·sqPCR分析表明,HaCHT41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。

关键词: 棉铃虫')">棉铃虫, 几丁质')">几丁质, Ⅳ型几丁质酶')">Ⅳ型几丁质酶, 酶学性质')">酶学性质, 基因表达分析')">基因表达分析

Abstract: 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 HaCHT4 (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 of50and pH value of 7. The values of kinetic parameters Km and Vmax of the recombinant protein were 1.76±0.35 mg/mL and 0.0220±0.0012 μg/mL·s, respectively. qPCR results revealed that the relative expression levels of HaCHT4 inthe 1st and 2nd larval instars of H.armigera were significantly higher than those in other larval instars and the pre-pupal stage, and HaCHT4 was predominantly expressed in the midgut and fat body of the 6th instar larva, but lowly expressed in the integument and head. Conclusion The results suggest that HaCHT4 may participate in chitin degradation in the peritrophic matrix in H. arrmigera. These results lay a foundation for the further function study of HaCHT4 and provide useful information for pest control.

Key words: Helicoverpa armigera, chitin, group IV chitinase, enzyme characterization, gene expression analysis