史黎黎,周婷婷,李逸群,曾坤辉,陈兆明,王成桂,章双

• 1:广东海洋大学水产学院

摘要(Abstract)：

克隆并鉴定墨吉明对虾(Fenneropenaeus merguiensis)的一种c型溶菌酶基因(Fm-Lyz),其cDNA全长为770 bp,包括71 bp的5′非编码区(UTR)、222 bp的3′UTR和477 bp完整开放阅读框,编码158个氨基酸。SMART分析显示,Fm-Lyz氨基酸序列包含1个LYZ1结构域和1个含18个氨基酸的信号肽。同源性分析发现,Fm-Lyz与斑节对虾(Penaeus monodon)溶菌酶同源性最高;进化树结果显示,Fm-Lyz与不同对虾的c型溶菌酶聚为一类。实时荧光定量PCR结果显示,Fm-Lyz在所测组织中均有表达,其中在血细胞中表达量最高。副溶血弧菌(Vibrio parahaemolyticus)和溶壁微球菌(Micrococcus lysodeikticus)感染后,Fm-Lyz表达量比对照组明显上调(P<0.05),最高表达量时间分别为感染后24、12 h,暗示Fm-Lyz参与了墨吉明对虾的抗菌免疫防御。

关键词(KeyWords)： 墨吉明对虾;c型溶菌酶;基因;克隆;表达

Abstract:

Keywords:

基金项目(Foundation): 广东省自然科学基金-博士启动项目(2016A030310334);广东省自然科学基金-自由申请项目(2016A030313757);; 广东海洋大学优秀青年骨干教师特别资助计划项目(HDYQ2017003,HDYQ2015004)

作者(Author): 史黎黎,周婷婷,李逸群,曾坤辉,陈兆明,王成桂,章双

参考文献(References)：

• [1]陈曌.对虾的免疫系统及免疫机制[J].海洋与渔业,2011(8):49-50.
• [2]YANG D L,WANG Q,CAO R W,et al.Molecular characterization,expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum[J].Developmental&Comparative Immunology,2017,73:109-118.
• [3]CHEN T,REN C H,WANG Y H,et al.Molecular cloning,inducible expression and antibacterial analysis of a novel i-type lysozyme(lyz-i2)in Pacific white shrimp,Litopenaeus vannamei[J].Fish&Shellfish Immunology,2016,54:197-203.
• [4]XING Y,GAO F Y,ZHENG Q M,et al.Cloning and characterization of the tiger shrimp lysozyme[J].Molecular Biology Reports,2009,36(6):1239-1246.
• [5]LIU H T,WANG J,MAO Y,et al.Identification and expression analysis of a new invertebrate lysozyme in Kuruma shrimp(Marsupenaeus japonicus)[J].Fish&Shellfish Immunology,2016,49:336-343.
• [6]YAO C L,WU C G,XIANG J H,et al.The lysosome and lysozyme response in Chinese shrimp Fenneropenaeus chinensis,to Vibrio anguillarum,and laminarin stimulation[J].Journal of Experimental Marine Biology and Ecology,2008,363(1/2):124-129.
• [7]杨杰.重组对虾溶菌酶基因在原核细胞中的表达及其产物分析[D].大连:大连工业大学,2012.
• [8]张海波.凡纳滨对虾溶菌酶基因在大肠杆菌和酵母中的重组表达和活性检测[D].上海:上海海洋大学,2009.
• [9]杨世平,王成桂,黄海立,等.环境温度和盐度对墨吉明对虾(Fenneropenaeus merguiensis)胚胎发育的影响[J].海洋与湖沼,2014,45(4):817-822.
• [10]赵吉臣,王刚,邓中宁,等.不同条件下哈维氏弧菌(Vibrio harvey)对墨吉明对虾(Fenneropenaeus merguiensis)致病性研究[J].海洋与湖沼,2015,46(6):1413-1419.
• [11]MAI W J,HU C Q.Molecular cloning,characterization,expression and antibacterial analysis of a lysozyme homologue from Fenneropenaeus merguiensis[J].Molecular Biology Reports,2009,36(6):1587-1595.
• [12]ZHUO R Q,ZHOU T T,YANG S P,et al.Characterization of a molt-related myostatin gene(Fm Mstn)from the banana shrimp Fenneropenaeus merguiensis[J].General and Comparative Endocrinology,2017,248:55-68.
• [13]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
• [14]BALCáZAR J L,ROJAS-LUNA T,CUNNINGHAM D P.Effect of the addition of four potential probiotic strains on the survival of pacific white shrimp(Litopenaeus vannamei)following immersion challenge with Vibrio parahaemolyticus[J].Journal of Invertebrate Pathology,2007,96(2):147-150.
• [15]ZHANG S,SHI L L,LüK,et al.Cloning,identification and functional analysis of aβ-catenin homologue from Pacific white shrimp,Litopenaeus vannamei[J].Fish&Shellfish Immunology,2016,54:411-418.
• [16]SUN C B,WANG G,CHAN S F.Effects of artificial infection of Litopenaeus vannamei,by Micrococcus lysodeikticus and WSSV on the activity of immunity related enzymes[J].Fish&Shellfish Immunology,2015,46(2):778-786.
• [17]OTERO-GONZáLEZ A J,MAGALH?ES B S,GARCIA-VILLARINO M,et al.Antimicrobial peptides from marine invertebrates as a new frontier for microbial infection control[J].FASEB Journal,2010,24(5):1320-1334.
• [18]SUPUNGUL P,RIMPHANITCHAYAKIT V,AOKI T,et al.Molecular characterization and expression analysis of a c-type and two novel muramidase-deficient i-type lysozymes from Penaeus monodon[J].Fish&Shellfish Immunology,2010,28(3):490-498.
• [19]王美娟.淇河鲫C型和g型溶菌酶基因克隆及表达分析[D].新乡:河南师范大学,2015.
• [20]DAUTIGNY A,PRAGER E M,PHAM-DINH D,et al.c DNA and amino acid sequences of rainbow trout(Oncorhynchus mykiss)lysozymes and their implications for the evolution of lysozyme and lactalbumin[J].Journal of Molecular Evolution,1991,32(2):187-198.
• [21]UMASUTHAN N,BATHIGE S D N K,KASTHURI S R,et al.Two duplicated chicken-type lysozyme genes in disc abalone Haliotis discus discus:molecular aspects in relevance to structure,genomic organization,m RNA expression and bacteriolytic function[J].Fish&Shellfish Immunology,2013,35(2):284-299.
• [22]NG S H S,ARTIERI C G,BOSDET I E,et al.A physical map of the genome of Atlantic salmon,Salmo salar[J].Genomics,2005,86(4):396-404.
• [23]KARTHIK V,KAMALAKANNAN V,THOMAS A,et al.Functional Characterization of a c-type Lysozyme from Indian Shrimp Fenneropenaeus indicus[J].Probiotics and Antimicrobial Proteins,2014,6(2):114-121.
• [24]IRWIN D M,GONG Z M.Molecular evolution of vertebrate goose-type lysozyme genes[J].Journal of Molecular Evolution,2003,56(2):234-242.
• [25]GAO F Y,QU L,YU S G,et al.Identification and expression analysis of three c-type lysozymes in Oreochromis aureus[J].Fish&Shellfish Immunology,2012,32(5):779-788.
• [26]刘洪涛.日本囊对虾stylicin抗菌肽和i型溶菌酶的基因克隆与表达研究[D].厦门:厦门大学,2016.