茶尺蠖丝氨酸蛋白酶基因EoSP1的克隆、时空表达及对饥饿的表达响应

张新; 陈成聪; 杜琴; 李喜旺; 孙晓玲

doi:10.13305/j.cnki.jts.2019.06.006

茶叶科学 >

2019 , Vol. 39 >Issue 6: 669 - 680

DOI: https://doi.org/10.13305/j.cnki.jts.2019.06.006

茶尺蠖丝氨酸蛋白酶基因EoSP1的克隆、时空表达及对饥饿的表达响应

张新 ,
陈成聪 ,
杜琴 ,
李喜旺 ,
孙晓玲

展开

1. 中国农业科学院茶叶研究所,浙江杭州 310008;
2. 农业农村部茶树生物学与资源利用重点实验室,浙江杭州 310008;
3. 国家茶叶质量安全工程技术研究中心,福建泉州 362400

张新,助理研究员,主要从事茶树抗虫机理研究,xinzhang@tricaas.com。

收稿日期: 2019-10-08

修回日期: 2019-10-23

网络出版日期: 2019-12-24

基金资助

国家自然科学基金项目（31272053）、中央级公益性科研院所基本科研业务费专项（1610212017017、1610212018004）国家茶叶质量安全工程技术中心开放课题基金项目（2018NTQS0102）

收起

Cloning and Expression Analysis of Serine Protease EoSP1 in Tea Geometrid (Ectropis obliqua) and Its Response to Starvation

ZHANG Xin ,
Chen Chengcong ,
DU Qin ,
LI Xiwang ,
SUN Xiaoling

Expand

1. Tea Research Institute of the Chinese Academy of Agricultural Sciences, Hangzhou 310008, China;
2. Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture and Rural Affairs, Hangzhou 310008, China;
3. National Research Center of Engineering and Technology of Tea Quality and Safety, Quanzhou 362400, China

Received date: 2019-10-08

Revised date: 2019-10-23

Online published: 2019-12-24

Fold

摘要

丝氨酸蛋白酶是鳞翅目昆虫消化系统中一类重要的蛋白酶。本研究从茶尺蠖（Ectropis obliqua）中克隆到一条丝氨酸蛋白酶基因EoSP1并分析了其结构特征和表达特性。EoSP1基因序列全长858 bp,编码285个氨基酸,预测蛋白分子量为29.53 kDa,等电点为5.44。经与其他丝氨酸蛋白酶比对,EoSP1含有保守的丝氨酸蛋白酶催化位点（H₉₅,A₁₆₁和S₃₂₈）及蛋白互作结构域,与蓓带夜蛾（Mamestra configurata）中SPs亲缘关系较近。进一步获得了与EoSP1-GST融合蛋白大小接近的目的蛋白。qRT-PCR分析发现,EoSP1在幼虫期的表达显著高于成虫、蛹和卵期,并在幼虫中肠中特异性表达;饥饿处理后EoSP1表达下降,恢复饲喂后表达量接近对照组。以上结果为茶尺蠖消化酶功能解析及抗虫新靶点的筛选提供了依据。

关键词： 茶尺蠖; 丝氨酸蛋白酶; 克隆; 表达分析

本文引用格式

张新 , 陈成聪 , 杜琴 , 李喜旺 , 孙晓玲 . 茶尺蠖丝氨酸蛋白酶基因EoSP1的克隆、时空表达及对饥饿的表达响应[J]. 茶叶科学, 2019 , 39(6) : 669 -680 . DOI: 10.13305/j.cnki.jts.2019.06.006

Abstract

Serine protease plays an important role in the digestion process of Lepidoptera insects. In this study, we cloned a serine protease encoding gene EoSP1 from Ectropis obliqua and analyzed its basic characteristics and expression patterns. The coding sequence of EoSP1 is 858 bp, encoding 285 amino acid residues with deduced molecular weight of 29.53 kDa and isoelectric point of 5.44. Compared with other serine proteases, EoSP1 contains conserved serine protease catalytic sites (H₉₅, A₁₆₁ and S₃₂₈) and protein interaction domains, and shows the closest relationship with SPs from Mamestra configurata. Further, EoSP1-GST fusion protein similar to the predicted size was purified from E. coli cells. qRT-PCR analysis showed that the expression level of EoSP1 was much higher in larvae than that in adults, pupae and eggs, and expressed in midgut of larvae specifically. EoSP1 was down-regulated by starvation treatment, and the expression level was change back to that of control group after re-feeding. The above results provide a basis for the function analysis of digestive enzyme and screening of new insect-resistance targets in Ectropis obliqua.

Key words： Ectropis obliqua; serine protease; cloning; expression pattern

参考文献

[1] Jiang H B, Michael R Kanost.The clip-domain family of serine proteinases in arthropods[J]. Insect Biochemistry and Molecular Biology, 2000, 30(2): 95-105.
[2] Srinivasan A, Giri A P, Gupta V S.Structural and functional diversities in lepidopteran serine proteases[J]. Cell Mol Biol Lett, 2006, 11(1): 132-154.
[3] Jeremy Rossa, Michael R Kanostb, Wang Y.Serine proteases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationships[J]. Gene, 2003, 304: 117-131.
[4] Veillard F, Troxler L, Reichhart J M.Drosophila melanogaster clip-domain serine proteases: Structure, function and regulation[J]. Biochimie, 2016, 122: 255-269.
[5] Colebatch G, Cooper P, East P. cDNA cloning of a salivary chymotrypsin-like protease and the identification of six additional cDNAs encoding putative digestive proteases from the green mirid, Creontiadesdilutus (Hemiptera: Miridae)[J]. Insect Biochemistry and Molecular Biology, 2002, 32(9): 1065-1075.
[6] 王镜岩, 朱圣庚, 徐长发. 生物化学[M]. 第三版. 北京: 高等教育出版社, 2002: 405.
[7] Joseph Kraut.Serine proteases: Structure and mechanism of catalysis[J]. Ann. Rev. Biochem, 1977, 46: 331-358.
[8] Page M J, Cera E Di.Serine peptidases: classification, structure and function[J]. Cell Mol Life Sci, 2008. 65(7/8): 1220-1236.
[9] Hedstrom L.Serine Protease Mechanism and Specificity[J]. Chemical Reviews, 2002, 102(12): 4501-4523.
[10] Lomate, P R, Mahajan N S, Kale, et al. Identification and expression profiling of Helicoverpa armigera microRNAs and their possible role in the regulation of digestive protease genes[J]. Insect Biochem Mol Biol, 2014, 54: 129-137.
[11] Anisuzzaman, Islam M K, Alim M A, et al. Longistatin is an unconventional serine protease and induces protective immunity against tick infestation[J]. Mol Biochem Parasitol, 2012, 182(1/2): 45-53.
[12] Srinivasan A, Ashok P Giri1, Abhay M Harsulkar, et al. A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts anti-metabolic effect on podborer (Helicoverpa armigera) larvae[J]. Plant Mol Biol, 2005, 57(3): 359-374.
[13] 李莉莉, 周晓群, 赵奎军, 等. 苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究[J]. 应用昆虫学报, 2016, 53(4): 706-715.
[14] 周晓群, 高艳玲, 赵奎军, 等. 苜蓿夜蛾中肠丝氨酸蛋白酶cDNA 的克隆、序列分析及原核表达[J]. 昆虫学报, 2014, 57(9): 1008-1017.
[15] Broehan G, Kemper M, Driemeier D, et al.Cloning and expression analysis of midgut chymotrypsin-like proteinases in the tobacco hornworm[J]. J Insect Physiol, 2008, 54(8): 1243-1252.
[16] Zhang C, Zhou D H, Zheng S C, et al.A chymotrypsin-like serine protease cDNA involved in food protein digestion in the common cutworm, Spodoptera litura: Cloning, characterization, developmental and induced expression patterns, and localization[J]. J Insect Physiol, 2010, 56(7): 788-799.
[17] 冯一璐, 傅晓斌, 吴帆, 等. 茶尺蠖信息素结合蛋白PBP2的基因克隆、原核表达及其结合功能[J]. 中国农业科学, 2017, 50(3): 504-512.
[18] 赵旭东, 孙宇航, 陈昌宇, 等. 美国白蛾丝氨酸蛋白酶基因HcSP1的克隆、时空表达及对取食不同寄主植物的表达响应[J]. 昆虫学报, 2019, 62(2): 160-169.
[19] Wang R X, Tong X L, Gai T T, et al.A serine protease homologue Bombyx mori scarface induces a short and fat body shape in silkworm[J]. Insect Mol Biol, 2018, 27(3): 319-332.
[20] 张永红, 朱峰, 唐芬芬, 等. 家蚕丝氨酸蛋白酶基因BmSP25转录分析及其免疫响应[J]. 南方农业学报, 2017, 48(6): 1093-1098.
[21] Liu Q Y, Li M G, Liu X F, et al.Characterization of trypsin-like and chymotrypsin-like serine proteases from midgut of Mythimna separata Walker[J]. Arch Insect Biochem Physiol, 2016, 92(3): 173-191.
[22] Fan L M, Rui X R, Yang L, et al.RNA interference mediated serine protease gene (Spbtry1) knockdown affects growth and mortality in the soybean pod borer (Lepidoptera: Olethreutid)[J]. Florida Entomologist, 2017, 100(3): 607-615.
[23] Tamhane V A, Chougule N P, Giri A P, et al.In vivo and in vitro effect of Capsicum annum proteinase inhibitors on Helicoverpa armigera gut proteinases[J]. Biochimica Et Biophysica Acta-General Subjects, 2005, 1722(2): 156-167.
[24] Yang Z W, Duan X N, Jin S, et al.Regurgitant derived from the tea geometrid Ectropis obliqua suppresses wound-induced polyphenol oxidases activity in tea plants[J]. Journal of Chemical Ecology, 2013, 39(6): 744-751.
[25] Huang C, Zhang J, Zhang X, et al.Two new polyphenol oxidase genes of tea plant (Camellia sinensis) respond differentially to the regurgitant of tea geometrid, Ectropis obliqua[J]. Int J Mol Sci, 2018, 19(8): 2414. DOI: 10.3390/ijms19082414.
[26] Sun X L, Li X W, Xin Z J, et al.Development of synthetic volatile attractant for male Ectropis oblique moths[J]. Journal of Integrative Agriculture, 2016, 15(7): 1532-1539.
[27] Gorman M J, Kankanala P, Kanost M R.Bacterial challenge stimulates innate immune responses in extra-embryonic tissues of tobacco hornworm eggs[J]. Insect Molecular Biology, 2004, 13(1): 19-24.
[28] 张娴, 李超林, 郑乔木, 等. 菜青虫胰凝乳蛋白酶基因PrCT1的克隆及表达分析[J]. 河南农业科学, 2019, 48(9): 74-81.
[29] Chen C X, Yan Y, Yang H, et al.Cloning of two clip-domain serine protease genes and their expression in response to exogenous hormone and immune challenge in Lasioderma serricorne (Coleoptera: Anobiidae)[J]. Acta Entomologica Sinica, 2019, 62(5): 535-546.
[30] He W Y, Zheng Y P, Tang L, et al.Cloning, expression and localization of a trypsin-like serine protease in the spruce budworm, Choristoneura fumiferana[J]. Insect Science, 2009, 16(6): 455-464.
[31] Zhan Q L, Zheng S C, Feng Q L, et al.A midgut-specific chymotrypsin cDNA (Slctlp1) from Spodoptera litura: cloning, characterization, localization and expression analysis[J]. Arch Insect Biochem Physiol, 2011, 76(3): 130-143.
[32] Shi M, Zhu N, Yi Y, et al.Four serine protease cDNAs from the midgut of Plutella xylostella and their proteinase activity are influenced by the endoparasitoid, cotesia vestalis[J]. Arch Insect BiochemPhysiol, 2013, 83(2): 101-114.
[33] 高旭晖, 宛晓春, 杨云秋, 等. 茶尺蠖生物学习性研究[J]. 植物保护, 2007, 33(3): 110-113.
[34] 郭萧, 王晓庆, 彭萍, 等. 茶树不同成熟度叶片对茶尺蠖发育适合度的影响[J]. 茶叶科学, 2012, 32(3): 229-235.
[35] Fang R, Sally P Redfern, Don Kirkup, et al.Variation of theanine, phenolic, and methylxanthine compounds in 21 cultivars of Camellia sinensis harvested in different seasons[J]. Food Chemistry, 2017, 220: 517-526.

Options

文章导航

模态框（Modal）标题

摘要

本文引用格式

Abstract

参考文献