栉孔扇贝（Chlamys farreri）Toll样受体及其信号传递相关基因的克隆与表达

【作者】 邱丽梅；

【导师】 宋林生；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2006， 博士

【摘要】 扇贝是我国海水养殖的重要品种,但自1994年以来,养殖扇贝陆续爆发的大规模死亡,不但造成了巨大的经济损失,而且直接威胁到现有产业的生存和发展。引起扇贝大规模死亡原因是多方面的,其主要原因是养殖环境恶化、扇贝种质衰退和抗病力下降。因此,深入研究扇贝免疫防御机制,探讨提高机体抗病力的有效途径和方法,改良种质和培育抗病品系,无疑是解决目前困扰扇贝养殖业健康可持续发展的必经之路。Toll样受体(TLRs)家族是新近发现的模式识别受体(PRRs),参与识别病原体相关的分子模式(PAMPs),在天然免疫系统中起着非常重要的作用。哺乳动物中Toll样受体信号通路还参与诱导树枝状细胞成熟、参与免疫耐受、参与凋亡发生发展、介导非感染性因素的识别等,被视为联系天然免疫和获得性免疫的桥梁。同时果蝇的Toll信号通路也是不具备获得性免疫的果蝇赖以抵御病毒、细菌和真菌感染,介导天然免疫反应的重要信号通路。本研究采用大规模EST测序方法,结合Genome Walker库的构建和cDNA末端快速扩增技术,从栉孔扇贝克隆得到CfToll-1、CfMyd88、CfTRAF6和CfCactus这四个Toll样受体信号通路基因的全长cDNA,同时用荧光实时定量PCR技术检测了这些基因的组织分布及在脂多糖(LPS)和肽聚糖(PGN)刺激下的表达规律。栉孔扇贝Toll样受体(CfToll-1)的cDNA序列全长4308 bp,包含5’非翻译区(UTR)211 bp,3597 bp的开放阅读框,500 bp的3’UTR,最后为18个腺嘌呤的ploy A尾巴。开放阅读框编码1198个氨基酸的多肽,该多肽的估计分子量为137.41kd,估计的等电点为5.62,该多肽有信号肽,具有一个预测的跨膜区,因此是一种跨膜蛋白。经BLAST比对,CfToll-1基因与节肢动物多种Toll蛋白高度的相似性。SMART(Simple Modular Architecture Research Tool)软件分析,CfToll-1包含典型的Toll样受体的结构:富含亮氨酸的重复序列的胞外区(leucine-richrepeats, LRR),一段跨膜结构域,以及胞内区的TIR结构域(Toll/IL-1 receptorhomologous region)。利用Real-time RT-PCR发现CfToll-1mRNA在扇贝体内普遍存在于血细胞、肌肉、外套膜、心、性腺和鳃组织中。利用体外培养的原代血细更多还原

【Abstract】 Scallop culture is an important marine aquaculture industry in China. But since 1994,scallop aquaculture in China has been experiencing a continual large-scale mortality,which not only caused a great economic loss but also threatened the existencedevelopment of the culture industry. With the improvement and control of the marineenvironment, enhancing the disease resistance of scallop is commonly believed to beresolution to the control of the disease. The cloning and expression of the genes involvedimmune defense are now considered to be a basic solution in the disease control becauseof their potential use in the development of therapeutic agents, study of immune defensemechanism and genetic improvement to increase the resistance to disease. Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors,which show homology with the Drosophila Toll protein and play key roles in detectingvarious non-self substances and then initiating and activating immune system. Inaddition, TLRs could induce DC maturation, participate in immune tolerance, participatein apoptosis, and mediate recognition of non-infection factors et al, which have beenconsidered as a linkage of innate and adaptive immunity. In Drosophila devoid of anadaptive immune system, Toll proteins also play very important role to mediate innateimmunity for resistance to bacteria and fungal infection. But full length and relatedresearch about Toll pathway have never been reported in bivalve before.In the present study, large scale EST sequencing method together with GenomeWalker and RACE technique was used to isolate and clone the genes involved in Tollpathway signaling from Zhikong scallop. Four full length cDNA sequences wereobtained and the tissue distribution and the temporal expression of these genes with thetreatment of LPS or PGN were measured by Real-time RT-PCR.CfToll-1 was the only Toll-like receptor identified from the six homology ESTfragments from a cDNA library of Zhikong scallop (Chlamys farreri). The full lengthcDNA of CfToll-1 consisted of 4308 nucleotides with a polyA tail, encoding a putativeprotein of 1198 amino acids with a 5’ UTR (untranslated region) of 211bp and a 3’UTRof 500bp. The predicted amino acid sequence comprised an extracellular domain with apotential signal peptide, nineteen leucine-rich repeats (LRR), two LRR-C-terminal(LRRCT) motifs, and a LRR-N-terminal (LRRNT), followed by a transmembranesegment of 20 amino acids, and a cytoplasmic region of 138 amino acids containing theToll/IL-1R domain (TIR). The deduced amino acid sequence of CfToll-1 was homologyto Drosophila melanogaster Tolls (DmTolls) with 23-35% amino acids sequencesimilarity and 30-54% in the TIR domain. Real-time PCR showed that the transcriptswere constitutively expressed in tissue of haemocyte, muscle, mantle, heart, gonad andgill. The expression of CfToll-1 could be regulated by LPS, and this regulation was LPSdose-dependent.The first mollusk Myd88 (CfMyd88) consisted of 1554 nucleotides with a polyA tail,encoding a polypeptide of 367 amino acids containing a typical structure with clearmodular parts of TLR and IL-1R-related (TIR) domain and death domain (DD).Homology analysis revealed that the predicted protein was significant homology to avariety of Myd88 previously submitted to the database. The highest BLASTP identitymatch for CfMyd88 was 33% amino acid identity with the Rattus norvegicus Myd88.The temporal expression of CfMyd88 in mixed primary cultured haemocytes wasmeasured by Real-time RT-PCR system and found that PGN (peptidoglycans)down-regulated the expression of CfMyd88 obviously.The full-length cDNA of CfTRAF6 was 2510bp, including a 5’ UTR of 337 bp, a 3’UTR of 208 bp, a poly (A) tail, and an open reading frame (ORF) of 1965bp encoding apolypeptide of 655 amino acids with a predicted molecular mass of 74.09 kDa. Theputative protein has characteristics motifs of the TRAF family of proteins including oneZinc finger of RING-type, two Zinc finger of TRAF-type, a MATH domain (the meprinand TRAF homology) and a coiled coil region. The highest BLASTP identity match forthis entire scallop TRAF6 was 68% amino acid identity with the squid TRAF6 and 45%amino acid identity with mouse TRAF6. The mRNA transcripts of CfTRAF6 werewidely spread in all the examined tissues with different expression levels. The highestexpression was observed in the gonad, relatively lower expression was observed inmuscle, mantle, heart, gill and haemocyte. After treatment of 500ng.mL-1 PGN, theCfTRAF6 gene transcripts began to diminish at 1.5 h and this trend lasted till 3h. Itrecovered to the original level since 6h. When the concentration of PGN increased to20μg.mL-1, the expression of CfTRAF6 was obviously decreased and never recovered tothe original level in the process of 12h stimulation. And the amount of mRNA transcriptsat 3h reached to the bottom, only about 1/9 times to control.The full-length cDNA of CfCactus was 2488bp, including a 5’ UTR of 181 bp, a 3’UTR of 1467 bp with a poly (A) tail, and an open reading frame (ORF) of 840bpencoding a polypeptide of 279 amino acids with a predicted molecular mass of 31.37kDa. The putative protein has three characteristics motif of ankyrin repeats. The highestBLASTP identity match for this entire scallop Cactus was 35% amino acid identity withthe Crassostrea gigas IκB and 44% amino acid identity with Carcinoscorpiusrotundicauda IκB. Real-time RT-PCR revealed that the mRNA transcripts of CfCactuswere much more than that of CfMyd88 and CfTRAF6 in tissue of gonad, haemocyte andintestine.The data together leads the strong support that the presence of the canonical TLRssignaling pathway presented in Drosophila and mammals is also likely to exist inmollusk. Further, expression profiles of CfToll-1, CfMyd88 and CfTRAF6 mRNAchallenged by genes bacteria LPS and PGN and tissue distribution profiles implied thatthis pathway would be involved in functional immunity and developmental response ofscallops.更多还原