Hemolin, a unique immune protein, belongs to the immunoglobulin superfamily of Lepidoptera insects. A full length cDNA sequence of hemolin gene was cloned by RACE-PCR from tea geometrid (Ectropis obliqua Prout), which was named as EoHML (GenBank Accession No. KM885983). The results of bioinformatics characterization showed that the full length cDNA of EoHML was 1β772βbp, which contained an intact CDS of 1β239βbp and encoded 412 amino acid residues with a putative molecular mass of 45.8βkD and an isoionic point of 8.297. The deduced amino acid sequence demonstrates it belongs to a typical secretory protein that contains 4 Ig functional conservative areas and 2 N-glycosylation sites. Phylogenetic analysis showed that EoHML share the far genetic distance with published hemolin gene and the highest similarity with that of Manduca sexta was only 53%. The results of quantitative real-time PCR analysis further revealed a significant up-regulated expression about 36.5 times in the larvae infected E.obliqua Nucleopolyhedrovirus (EoNPV) than the controls. The study indicated that the EoHML gene may participate in the immune response to EoNPV in tea geometrid.
YU Yugeng
,
YUAN Zhijun
,
YIN Kunshan
,
FU Jianyu
,
XIAO Qiang
. Molecular Cloning and Expression Analysis of Hemolin Gene in Tea Geometrid (Ectropis obliqua)[J]. Journal of Tea Science, 2015
, 35(4)
: 307
-315
.
DOI: 10.13305/j.cnki.jts.2015.04.001
[1] Faye I, Pye A, Rasmuson T, et al. Insect immunity Ⅱ Simultaneous induction of antibacterial activity and selection synthesis of some hemolymph proteins in diapausing pupae of Hyalophora cecropia and Samia cynthia[J]. Infection and Immunity, 1975, 6(12): 1426-1438.
[2] Sun S, Lindstrom I, Boman H G, et al. Hemolin: an insect-immune protein belonging to the immunoglobulin superfamily[J]. Science, 1990, 250(4988): 1729-1732.
[3] Lee K Y, Horodyski F M, Valaitis A P, et al. Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth, Lymantria dispar[J]. Insect Biochemistry and Molecular Biology, 2002, 11(32): 1457-1467.
[4] Bao Y, Yamano Y, Morishima I.Induction of hemolin gene expression by bacterial cell wall components in eri-silkworm, Samia cynthia ricini [J]. Comparative Biochemistry and Physiology: Part B, 2007, 146(1): 147-151.
[5] 何芳青, 陈琳, 姚慧鹏, 等. 家蚕类免疫球蛋白(hemolin)基因的克隆及表达特征和抗菌活性研究[J]. 蚕业科学, 2010, 36(1): 40-45.
[6] 姚慧鹏, 吴小锋. 昆虫类免疫球蛋白结构和功能研究进展[J]. 昆虫学报, 2008, 51(1): 530-536.
[7] 张益民, 王学兰, 张世敏, 等. 茶尺蠖核型多角体病毒超微结构的初步研究[J]. 科学通报, 1985(24): 1918-1920.
[8] 殷坤山, 陈华才, 肖强, 等. 茶尺蠖核型多角体病毒制剂的试制与推广应用[J]. 中国病毒学, 2000, 15(S1): 81-84.
[9] 席羽, 殷坤山, 肖强. 不同地理种群茶尺蠖对EoNPV的敏感性差异研究[J]. 茶叶科学, 2011, 31(2): 100-104.
[10] 罗聪, 何新华, 陈虎, 等. 一种高效获取基因5′末端的RACE方法[J]. 植物生理学报, 2011, 47(4): 409-414.
[11] Petersen Tn, B S V H. SignalP 4.0: discriminating signal peptides from transmembrane regions[J]. Nature Methods, 2011, 340(8): 785-786.
[12] Tamura K, Peterson D, Peterson N, et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.
[13] Lu Y, Yuan M, Gao X, et al. Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)[J]. PloS ONE, 2013, 8(7): e68059.
[14] Teng XL, Zhang Z, He GL, et al. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects[J]. Journal of Insect Science, 2012, 12(60): 1-17.
[15] Bao Y, Lv Z, Liu Z, et al. Comparative analysis of Bombyx mori nucleopolyhedrovirus responsive genes in fat body and haemocyte of B. mori resistant and susceptible strains[J]. Insect Molecular Biology, 2010, 19(3): 347-358.
[16] 袁志军, 张传溪, 肖强, 等. 茶尺蠖核型多角体病毒(EoNPV)实时荧光定量PCR检测方法的优化及应用[J]. 茶叶科学, 2013, 33(3): 229-236.
[17] Olle Terenius H J R P. Bacterial, but not baculoviral infections stimulate Hemolin expression in noctuid moths[J]. Developmental and Comparative Immunology, 2009(33): 1176-1185.
[18] Hirai M, Terenius O, Li W, et al. Baculovirus and dsRNA induce Hemolin, but no antibacterial activity, in Antheraea pernyi[J]. Insect Molecular Biology, 2004, 13(4): 399-405.