马氏珠母贝TLRP基因克隆及其功能研究

【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...

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

【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】cDNA sequences of PmTLRP gene was cloned by rapid amplification of cDNA 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 135 bp 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 PmTLRP 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 5 to 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 at 3 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.