马氏珠母贝TLRP基因克隆及其功能研究

Cloning and study on functions of TLRP gene in Pinctada fucata martensii

【目的】明确马氏珠母贝TLRP基因(PmTLRP)的组织表达特征及哈维氏弧菌(Vibrio harveyi)刺激、植核及脂多糖(LPS)刺激后的时序表达特点,为进一步研究TLRs在马氏珠母贝中的免疫机制打下基础。【方法】采用RACE克隆PmTLRP基因c DNA序列,通过ProtParam、ProtScale、SignalP 4.0、TMHMM v.2.0、SMART、SoftBerry Psite和SOPMA等在线软件进行生物信息学分析,并以实时荧光定量PCR检测PmTLRP基因在马氏珠母贝各组织中及经哈维氏弧菌、植核及LPS刺激后的表达情况。【结果】PmTLRP基因cDNA序列全长2214 bp,包含33 bp的5’非编码区(5’-UTR)、135 bp的3’非编码区(3’-UTR)及2043 bp的开放阅读框(ORF),共编码681个氨基酸残基;其编码蛋白含有信号肽、富含亮氨酸重复序列(LRRs)、跨膜结构域和TIR结构域,符合TLRs家族所具有的特征;PmTLRP氨基酸序列与其他物种的TLRs氨基酸序列存在一定相似性,其中与美洲牡蛎TLP氨基酸序列的相似度较高。PmTLRP基因在马氏珠母贝外套膜、血淋巴、肝胰腺、鳃、性腺和闭壳肌中均有表达,以在肝胰腺中的相对表达量最高,显著高于在其他组织中的相对表达量(P<0.05)。在哈维氏弧菌刺激下,PmTLRP基因在血淋巴中的相对表达量于注射刺激后12 h开始上调,至注射刺激后16 h达最大值,约是注射刺激前(0 h)的19.0倍。经植核刺激后,PmTLRP基因相对表达量在植核后5~20 d呈逐渐上升趋势,于植核后30 d达最大值,约是植核前(0 h)的6.4倍。在LPS刺激下,PmTLRP基因在血淋巴中的相对表达量先上升后下降,至注射刺激后3 h达最大值,约是对照组的5.0倍;PmTLRP基因在肝胰腺中的相对表达量呈升高→降低→升高→降低→升高的波动变化趋势,至注射刺激后24 h相对表达量上调到最大值,约是对照组的6.0倍。【结论】PmTLRP基因在马氏珠母贝外套膜、血淋巴、肝胰腺、鳃、性腺和闭壳肌中均有表达,但表达水平存在差异,经哈维氏弧菌、植核及LPS刺激后呈明显上调表达趋势,说明TLRP在马氏珠母贝的免疫防御反应中扮演重要角色。

【Objective】To explore tissue expression characteristics of TLRP gene(PmTLRP)in Pinctada fucata martensii and temporal expression of PmTLRP under stimulations of Vibrio harveyi,nucleus insertion operation and lipopolysaccharide(LPS),so as to provide a foundation for further exploring immune mechanism of TLRs in P.fucata martensii.【Method】c DNA sequences of PmTLRP gene was cloned by rapid amplification of c DNA ends(RACE),and bioinformatics analysis was carried out by online software such as ProtParam,ProtScale,SignalP 4.0,TMHMM v.2.0,SMART,SoftBerry Psite and SOPMA.Quantitative real-time PCR(qRT-PCR)was used to detect the expression of PmTLRP gene in various tissues of P.fucata martensii and under stimulations of V.harveyi stimulation,nucleus insertion operation and LPS stimulation.【Result】The total length of PmTLRP cDNA was 2214 bp,including a 5’-UTR of 33 bp,a 3’-UTR of 135bp and an open reading frame(ORF)of 2043 bp which encoded 681 amino acid residues.The protein encoded had signal peptide,leucine rich repeats(LRRs),transmembrane domain,and TIR domain,which was consistent with the characteristics of TLRs family.Amino acid sequences of PmTLRP were somehow similar with that of TLRs from other species,with the highest similarity with amino acid sequences of TLP in Crassostrea virginica.PmTLRP was expressed in mantle,hemolymph,hepatopancreas,gills,gonads and adductor muscle,and the highest expression was found hepatopancreas,which was significantly higher than that in other tissues(P<0.05).After stimulation of V.harveyi,expression of Pm TLRP began to increase at 12 h,reached the highest expression at 16 h,about 19.0 times that of the control group before stimulation(0 h).Under stimulation of nucleus insertion operation,the relative expression of PmTLRP gene increased from 5to 20 d.Finally,it reached the maximum expression at 30 d,which was about 6.4 times that of the control group.After LPS stimulation,the expression of PmTLRP in hemolymph was significantly up-regulated,and reached the maximum at3 h,about 5.0 times that of the blank control group(0 h).PmTLRP in hepatopancreas showed a fluctuating trend of increase→decrease→increase→decrease→increase,and the relative expression increased to the maximum at 24 h after injection stimulation,which was about 6.0 times that of the control group.【Conclusion】PmTLRP gene is expressed in mantle,hemolymph,hepatopancreas,gill,gonad and adductor muscle of P.fucata martensii,but the expression are different.The expression of PmTLRP was obviously up-regulated after stimulations of V.harveyi,nucleus insertion operation and LPS,indicating that TLRP plays an important role in the immune defense response of P.fucata martensii.