杂色鲍两种perlucin基因的克隆与表达分析

摘要/Abstract

摘要： 利用SMART RACE技术克隆杂色鲍两个甘露聚糖结合型凝集素（mannan-binding lectin，MBL）成员perlucin1和perlucin4基因的全长cDNA序列，使用Real time PCR分析这些基因在杂色鲍不同组织中的表达。结果显示，杂色鲍perlucin1 cDNA序列全长为668 bp，165个氨基酸，经分析该基因含有2个二硫键、2个糖基化位点和13个磷酸化位点，预测蛋白分子量约为18.8 kD，等电点约为4.57；perlucin4 cDNA序列全长为587 bp，156个氨基酸，含有2个二硫键、1个糖基化位点和7个磷酸化位点，预测蛋白分子量约为17.8 kD，等电点约为4.43；鳃、肝胰腺、血淋巴、上足、外套膜、黏液腺、肾脏7个组织中，perlucin1基因和perlucin4基因只有在肝胰腺中表达，在其它组织中没有检测到表达。结果表明，perlucin基因参与了杂色鲍天然免疫防御机制，在免疫识别中发挥着重要作用。

关键词: perlucin凝集素, 甘露聚糖结合型凝集素, 实时定量PCR

Abstract: SMART RACE technology was employed to clone the full-length cDNA sequences of 2 Manna-binding lectin（MBL）members of perlucin1 and perlucin4 in Haliotis diversicolor. Real-time PCR were used to analyze their expression patterns in the different tissues of H. diversicolor. As results，the full length cDNA of perlucin1 was 668 bp，and the deduced protein was composed of 165 amino acids，with two disulfide bonds，two glycosylation sites and 13 phosphorylation sites；its predicted molecular weight was about 18.8 kD，and pI was about 4.57. The full length cDNA of perlucin4 was 587 bp and the deduced protein was composed of 156 amino acids，with two disulfide bonds，one glycosylation sites and 7 phosphorylation sites；its predicted molecular weight was about 17.8 kD，and pI was about 4.43. Gene perlucin1 and perlucin4 exclusively expressed only in hepatopancreas among 7 tissues of gill，hepatopancreas，blood lymph，up foot，mantle，mucous gland，and kidney. The above results indicate that perlucin genes involved in innate immune defense mechanisms and play a key role in immune recognition of H. diversicolor.

Key words: perlucin lectin, mannan-binding lectin, Real-time PCR

中图分类号:

10.13560/j.cnki.biotech.bull.1985.2016.12.019

林师,张丽莉,王国栋. 杂色鲍两种perlucin基因的克隆与表达分析[J]. 生物技术通报, 2016, 32(12): 113-123.

LIN Shi,ZHANG Li-li,WANG Guo-dong. Molecular Cloning and Expression Analysis of Two Types of perlucin Gene from Small Abalone Haliotis diversicolor[J]. Biotechnology Bulletin, 2016, 32(12): 113-123.

参考文献

[1] Loker ES. Gastropod immunobiology[J] . Advances in Experimental Medicine and Biology, 2010, 708：17-43.
[2] Hoffmann JA, Kafatos FC, Janeway CA, et al. Phylogenetic perspectives in innate immunity[J] . Science, 1999, 284（5418）：1313-1318.
[3] 吴蔚, 陶然, 尚世强. 甘露糖结合凝集素研究进展[J] . 中华微生物学和免疫学杂志, 2009（12）：1148-1152.
[4] Taylor ME, Brickell P, Craig R, et al. Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein[J] . Biochem J, 1989, 262：763-771.
[5] Presanis J, Kojima M, Sim R. Biochemistry and genetics of mannan-binding lectin（MBL）[J] . Biochemical Society Transactions, 2003, 31（4）：748-752.
[6] Endo Y, Nonaka M, Saiga H, et al. Origin of mannose-binding lectin-associated serine protease（MASP）-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate, amphioxus[J] . The Journal of Immunology, 2003, 170（9）：4701-4707.
[7] Ottinger CA, Johnson SC, Ewart KV, et al. Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon（Salmo salar）macrophages by a mannose-binding lectin[J] . Comparative Biochemistry and Physiology Part C：Pharmacology, Toxicology and Endocrinology, 1999, 123（1）：53-59.
[8] Ewart KV, Johnson SC, Ross NW. Identification of a pathogen-binding lectin in salmon serum[J] . Comparative Biochemistry and Physiology Part C：Pharmacology, Toxicology and Endocrinology, 1999, 123（1）：9-15.
[9] Jackson AN, McLure CA, Dawkins RL, et al. Mannose binding lectin（MBL）copy number polymorphism in Zebrafish（D. rerio）and identification of haplotypes resistant to L. anguillarum[J] . Immunogenetics, 2007, 59（11）：861-872.
[10] Ourth DD, Narra MB, Simco BA. Comparative study of mannose-binding C-type lectin isolated from channel catfish（Ictalurus punctatus）and blue catfish（Ictalurus furcatus）[J] . Fish&Shellfish Immunology, 2007, 23（6）：1152-1160.
[11] Zhang H, Peatman E, Liu H, et al. Characterization of a mannose-binding lectin from channel catfish（Ictalurus punctatus）[J] . Research in Veterinary Science, 2012, 92（3）：408-413.
[12] Huang H, Huang S, Yu Y, et al. Functional characterization of a ficolin-mediated complement pathway in amphioxus[J] . Journal of Biological Chemistry, 2011, 286（42）：36739-36748.
[13] 王江勇, 郭志勋, 冯娟, 等. 养殖杂色鲍暴发病超微病理学研究[J] . 海洋科学, 2007, 31（3）：28-32.
[14] 杨蕊, 姜敬哲, 王江勇, 等. 杂色鲍低温病毒病研究进展[J] . 广东农业科学, 2013（2）：114-117.
[15] 陈佰义, 何礼贤, 胡必杰. 中国鲍曼不动杆菌感染诊治与防控专家共识[J] . 中华医学杂志, 2012, 92（2）：76-85.
[16] 葛辉. 杂色鲍Toll样受体信号通路分子的克隆及表达分析[D] . 厦门：集美大学, 2011.
[17] Chomczynski P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples[J] . Biotechniques, 1993, 15（3）：532-534, 536-537.
[18] 彭怀明, 周书林. 软体动物免疫功能研究进展[J] . 动物医学进展, 2010, 31（8）：79-83.
[19] 谢广成, 李丹彤, 周立敬. 刺参甘露糖结合凝集素的生物信息学分析[J] . 生物技术通报, 2011（2）：136-140.
[20] Worthley D, Bardy P, Mullighan C. Mannose-binding lectin：biology and clinical implications[J] . Internal Medicine Journal, 2005, 35（9）：548-555.
[21] 卜令真, 张士璀. 甘露糖结合凝集素在先天性免疫中的作用[J] . 鲁东大学学报：自然科学版, 2013, 29（3）：229-233.
[22] Weis WI, Taylor ME, Drickamer K. The C-type lectin superfamily in the immune system[J] . Immunological Reviews, 1998, 163（1）：19-34.
[23] Drickamer K. Engineering galactose-binding activity into a C-type mannose-binding protein[J] . Nature, 1992, 360（6400）：183-186.
[24] Nakao M, Kajiya T, Sato Y, et al. Lectin pathway of bony fish complement：identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp（Cyprinus carpio）[J] . The Journal of Immunology, 2006, 177（8）：5471-5479.
[25] Taylor ME, Bezouska K, Drickamer K. Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor[J] . Journal of Biological Chemistry, 1992, 267（3）：1719-1726.
[26] Skjoedt MO, Palarasah Y, Rasmussen K, et al. Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis[J] . Developmental & Comparative Immunology, 2010, 34（1）：59-68.
[27] Huang M, Song X, Zhao J, et al. A C-type lectin（AiCTL-3）from bay scallop Argopecten irradians with mannose/galactose binding ability to bind various bacteria[J] . Gene, 2013, 531（1）：31-38.
[28] Jin XK, Li WW, Cheng L, et al. Two novel short C-type lectin from Chinese mitten crab, Eriocheir sinensis, are induced in response to LPS challenged[J] . Fish & Shellfish Immunology, 2012, 33（5）：1149-1158.
[29] Costa F, Valen?a N, Silva A, et al. Cloning and molecular modeling of Litopenaeus vannamei（Penaeidae）C-type lectin homologs with mutated mannose binding domain-2[J] . Genet Mol Res, 2011, 10：650-664.
[30] Mann K, Weiss IM, Andre S, et al. The amino-acid sequence of the abalone（Haliotis laevigata）nacre protein perlucin[J] . European Journal of Biochemistry, 2000, 267（16）：5257-5264.
[31] Kania PW, Sorensen RR, Koch C, et al. Evolutionary conservation of mannan-binding lectin（MBL）in bony fish：Identification, characterization and expression analysis of three bona fide collectin homologues of MBL in the rainbow trout（Onchorhynchus mykiss）[J] . Fish & Shellfish Immunology, 2010, 29（6）：910-920.
[32] Wang GD, Zhang KF, Zhang ZP, et al. Molecular cloning and responsive expression of macrophage expressed gene from small abalone Haliotis diversicolor supertexta[J] . Fish & Shellfish Immunology, 2008, 24（3）：346-359.
[33] Ge H, Wang GD, Zhang LL, et al. Characterization of interleukin-1 receptor-associated kinase 1 binding protein 1 gene in small abalone Haliotis diversicolor[J] . Gene, 2012, 506（2）：417-422.
[34] Ge H, Wang GD, Zhang LL, et al. Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor[J] . Fish & Shellfish Immunology, 2011, 30（4）：1138-1146.
[35] Wang GD, Zhang ZP, Lin S, et al. A homologue of dermatopontin from Haliotis diversicolor and its response to pathogenic infection[J] . Aquaculture Research, 2015, 46（7）：1537-1549.
[36] 王国栋, 和四梅, 张丽莉, 等. 杂色鲍AP-1的克隆及在发育和弧菌感染后的表达分析[J] . 水产科学, 2016, 35（3）：227-233.
[37] Wang L, Yue F, Song X, et al. Maternal immune transfer in mollusc[J] . Developmental & Comparative Immunology, 2015, 48（2）：354-359.
[38] Yue F, Zhou Z, Wang L, et al. Maternal transfer of immunity in scallop Chlamys farreri and its trans-generational immune protection to offspring against bacterial challenge[J] . Developmental & Comparative Immunology, 2013, 41（4）：569-577.
[39] Yue F, Shi X, Zhou Z, et al. The expression of immune-related genes during the ontogenesis of scallop Chlamys farreri and their response to bacterial challenge[J] . Fish & Shellfish Immunology, 2013, 34（3）：855-864.
[40] Tirapé A, Bacque C, Brizard R, et al. Expression of immune-related genes in the oyster Crassostrea gigas during ontogenesis[J] . Developmental & Comparative Immunology, 2007, 31（9）：859-873.
[41] 王长法, 张士璀, 王昌留. 水生无脊椎动物凝集素研究概述[J] . 海洋科学, 2005, 29（4）：63-67.
[42] 张涛. 海洋无脊椎动物幼虫附着变态研究进展[J] . 海洋科学, 2000, 24（1）：25-29.
[43] 杨金龙, 王冲, 顾忠旗, 等. 微生物膜对海洋无脊椎动物幼体附着变态的影响研究[J] . 海洋科学, 2012, 36（8）：116-121.
[44] Kirchman D, Graham S, Reish D, et al. Lectins may mediate in the settlement and metamorphosis of Janua（Dexiospira）brasiliensis Grube（Polychaeta：Spirorbidae）[J] . Marine Biology Letters, 1982, 3（3）：131-142.