杂色鲍C型溶菌酶基因的克隆及表达分析

Molecular Cloning and Expression Analysis of C-Type Lysozyme Gene in Haliotis diversicolor

为了了解C型溶菌酶与杂色鲍(Haliotis diversicolor)的抗菌效应关系,通过SMART-RACE技术克隆到一条杂色鲍C型溶菌酶基因(HdLysC)全长cDNA,经生物信息学分析及应激表达分析得到以下结果:1)基因全长591 bp,开放阅读框441 bp,编码146个氨基酸,有1个LYZ1功能域(20—146aa)、1个保守基序(G/T)SSDYGLFQINS和两个催化位点(Glu 52和Asp 70)。2)多重比对和系统发育分析表明,HdLysC与亲缘关系较近的盘鲍(Haliotis discus discus)C 2型溶菌酶基因的同源性最高。3)组织表达差异实验表明,HdLysC基因在各组织中均有表达且差异显著(P<0.01),在外套膜组织中的表达量最高,而在血淋巴中的表达量最低。4)实时荧光定量PCR(qRT-PCR)实验表明,HdLysC能够响应副溶血弧菌感染,其表达水平随着感染的不同时相而变动。在副溶血弧菌感染4 h后,HdLysC在血淋巴组织中表达量增加(未达到显著差异),而在鳃组织中表达量有所下降;感染12 h后,HdLysC在各组织中的表达量都有明显的增加,呈极显著差异(P<0.01),尤其在血淋巴中增加最多;感染24 h后,其表达量在各组织中都有所降低。

To understand the relationship between C-type lysozyme in the Haliotis diversicolor and the antibacterial effect of heterosorbide,the full-length of C-type lysozyme gene cDNA sequence was cloned by SMART RACE in H.diversicolor.The results showed that the full-length cDNA sequence is 591bp,with a 441bp open reading frame.The deduced protein contained 146 amino acids,with a LYZ1 domain(20-146aa),a conserved motif(G/T)SSDYGLFQINS and two catalytic sites(Glu 52 and Asp 70).Multiple alignments and phylogenetic analysis showed that the C-type lysozyme gene of H.diversicolor had the highest homology the C-2 type lysozyme gene of H.discus discus.The experimental results showed the C-type lysozyme gene in H.diversicolor was expressed in all detected tissues.The highest expression of C-type lysozyme gene was detected in the mantle(P<0.01),and the lowest expression in hemolymph.Real-time quantitative PCR results showed that the expression of C-type lysozyme gene in H.diversicolor increased in blood lymphoid,the expression in gill was declined at 4 h post challenged by Vibrio parahaemolyticus.The expression of C-type lysozyme gene in different tissues increased significantly(P<0.01)at 12 h post challenged,especially in the hemolymph.The expression decreased in various tissues at 24 h post challenged.These results showed C-type lysozyme gene in H.diversicolor could respond to the infection of V.parahaemolyticus,while the expression fluctuated in different time after infection.