泥蚶高通量转录组分析及生长相关基因的克隆与表达研究
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摘要
泥蚶（Tegillarca granosa）俗称血蚶，是江、浙、闽沿海居民喜食的海鲜佳品，是我国传统四大养殖贝类之一，也是我国滩涂贝类养殖的主要对象。近年来，随着泥蚶人工育苗技术的突破和规模化养殖技术的成熟，泥蚶养殖业呈快速发展趋势，产生了巨大的经济效益和社会效益。然而，与之不相适宜的是，目前泥蚶分子遗传基础薄弱、基因组序列信息匮乏、遗传育种工作刚刚起步，严重影响功能基因开发的进程和分子育种实践的开展。为此，本研究利用新一代高通量454测序技术对泥蚶不同组织器官和不同发育阶段样品进行转录组测序，通过对高质量序列的拼接组装和基因注释，发掘与生长、营养代谢、繁殖、抗病等主要经济性状相关的候选基因，为后续开展功能基因研究和分子育种实践提供有价值信息；利用RACE和RT-PCR技术，对泥蚶生长调控基因Smad3、BMP7、ERK2、GRB2和蛋白质代谢基因LAP3进行基因克隆和表达分析，研究它们与生长的密切关系，筛选可作泥蚶良种选育的候选基因；从泥蚶转录组中批量开发和验证SSR分子标记，为群体遗传分析、遗传连锁图谱构建、生长性状主效QTLs的精确定位等研究提供有力工具。主要结果和结论如下：
1.基于454GS FLX高通量测序的泥蚶转录组分析
应用454高通量测序技术，对泥蚶4个组织和3个发育阶段样品进行了转录组测序，获得了1170337条高质量序列，通过序列拼接得到17940个重叠群（isotig）和131980个单拷贝序列（singleton），isotig平均长度达到934bp，测序深度为5.6倍。对泥蚶拼接得到的isotig与Swiss-prot、Nr等公共蛋白质数据库比对，通过GO注释对基因进行分类，KEGG注释了解基因间的联系和生物体的生化代谢网络。从注释的基因中鉴别出大量调控泥蚶肌肉、上皮、神经、纤维等的生长因子及受体，找到了与生长相关的TGFβ信号通路、MAPK信号通路中的多个重要基因；查找到大量与生长、发育、繁殖和免疫等生产相关的候选基因，为后续开展功能基因研究和分子育种实践有重要价值。在泥蚶转录组中，发现血红素合成过程中的大部分关键酶，推测贝类血红蛋白合成的基本过程可能与脊椎动物十分相似。另外，从泥蚶转录组中检测到6338个SSR和20038个SNP候选标记，为分子标记的大规模开发提供了重要资源。
2.利用泥蚶转录组序列大量开发微卫星标记，并研究其引物在毛蚶中的通用性
EST文库筛查法是目前大量开发微卫星标记的重要方法，具有方法简便、花费低廉等优点。本研究从泥蚶454转录组测序得到的大量isotig和singleton序列中搜索到6338个SSR位点，可用于大规模开发SSR标记。在3828条isotig序列里，共检测到138条适合设计引物的序列，扩增得到81个稳定扩增的位点，这些位点在泥蚶奉化群体中进行多态性检测，共扩增出242个等位基因，各位点的等位基因数为2~9个，平均每个位点产生3.90个等位基因，观测杂合度为0.040~1.000，期望杂合度为0.040~0.844，多态信息含量为0.038~0.806。33个SSR来自注释基因序列，对这些基因的准确定位有很大帮助。用62对泥蚶多态性SSR引物在毛蚶中进行了通用性验证，结果有22对成功扩增，16对表现为多态，这些多态位点可作为毛蚶和泥蚶的通用性标记。
3.泥蚶Smad3基因的克隆与表达分析
Smad3是TGF-β通路的重要成员，可将信号分子从细胞表面受体传导至细胞核，是重要的生长调控因子。利用RACE技术扩增得到泥蚶Smad3基因2341bp的cDNA全长，编码423个氨基酸。蛋白质功能域分析表明，Tg-Smad3含有两个保守功能域MH1和MH2。氨基酸序列比对发现，它与脊椎动物的同源性达到80%以上，说明Tg-Smad3氨基酸序列十分保守。RT-PCR结果表明，Tg-Smad3在泥蚶各成体组织都有表达，其中足和血液中表达量最高，可能与细胞增殖和免疫过程有关；在早期发育阶段，Tg-Smad3的表达量呈逐渐升高的趋势，在D形幼虫时期表达量最高，推测其参与贝壳的形成过程。
4.泥蚶BMP7基因的克隆与表达分析
BMP7也是TGF-β通路中的重要成员，在生物体内具有重要且广泛的生物功能，参与骨骼的生长、代谢，调节细胞的分化、增殖和凋亡，并在生殖系统的生长、发育中发挥重要作用。本研究克隆得到Tg-BMP7基因的cDNA全长，开放阅读框为1275bp，编码425个氨基酸。功能域预测表明，Tg-BMP7是一个糖蛋白，包含由29个氨基酸组成的信号肽，3个二硫键，并有一个TGF-β家族信号。RT-PCR结果显示，Tg-BMP7在成贝外套膜和鳃中表达量高；在早期发育阶段中，D形幼虫期的表达量显著高于原肠胚、担轮幼虫和幼贝，推测Tg-BMP7可能参与贝壳的形成过程，并具有组织分化、调控胚胎生长发育等生理功能。
5.泥蚶ERK2基因的克隆与表达分析
ERK2是MAPK家族重要的反应激酶系统，主要参与调控细胞生长、分化和细胞周期调控等生理功能。泥蚶ERK2基因cDNA全长1673bp，编码359个氨基酸，功能域预测找到一个丝氨酸/苏氨酸蛋白激酶的活性区域，这是ERK家族蛋白的特征性区域。氨基酸序列比对和系统进化分析发现，Tg-ERK2蛋白与脊椎动物的同源性达到80%以上，说明该基因十分保守，可能功能与脊椎动物有类似之处。RT-PCR结果表明，Tg-ERK2在泥蚶各组织、各个发育阶段都有表达，说明Tg-ERK2功能强大，参与到多种生命活动中去；相对而言，Tg-ERK2在泥蚶成体的血液中表达量最高；而在早期发育阶段中表达量逐渐升高，D形幼虫期达到最高，这可能与此期细胞分化加剧、神经细胞形成较多有关。
6.泥蚶GRB2基因的克隆与表达分析
GRB2蛋白是一个重要的接头分子，参与细胞内各种受体激活后的下游调节，调控细胞的增殖和分化。Tg-GRB2基因全长1275bp，编码236个氨基酸。Tg-GRB2蛋白包含3个功能域，1个SH2和2个SH3，这是GRB2发挥生理功能的活性区域。Tg-GRB2在泥蚶不同组织中的表达量由大到小依次为：血液＞足＞鳃＞外套膜＞闭壳肌＞内脏团；在早期发育阶段中，GRB2基因在原肠胚、担轮幼虫、D形幼虫中均有较高的表达量，原因可能是随着胚胎发育过程的进行，细胞分裂速度加快，生命活动旺盛，信号传导需要较多的接头蛋白
7.泥蚶蛋白质代谢基因LAP3的克隆与表达分析
LAP3是M17类蛋白家族的一员，在细胞成熟、细胞生长、调节损伤防御、维持细胞动态平衡方面发挥重要作用。Tg-LAP3基因全长为1590bp，编码530个氨基酸，包括Peptidase_M17超家族序列的N-端结构域（41-174bp）和Peptidase_M17催化结构域（209-522bp），缺失HEXXH锌结合体motif，充分证明了该蛋白为LAP3蛋白。RT-PCR结果表明，Tg-LAP3在足中表达量最高，而在早期发育阶段中表达量逐渐升高，D形幼虫期达到最高，在幼贝中表达量最低。
The blood clam, Tegillarca granosa, is one of the most popular commercially-important maricultural shellfish which is extensively cultured for seafood along thesouthern coastline of China, especially in Zhejiang province and Jiangsu Province. Inrecent years, with the technological breakthroughs of artificial breeding andlarge-scale farming, the aquaculture industry of T. granosa presented the trend ofrapid development, resulting in a huge economic and social benefits. Despite theeconomic value and research importance, knowledge of genetics and genome in T.granosa has been largely limited, and breeding work has just begun up to date. As aneconomically important aquacultural species, the lack of genomic resources would beunfavorable to understand the genetic mechanisms that involved in the growth,reproduction and immunity. In order to develop molecular resources and expeditegene discovery, we have undertaken454pyrosequencing of diverse cDNA libraries tofirst produce a comprehensive transcriptome for the blood clam T. granosa. Ourresults are as follows.
1. Transcriptomic analysis for T. granosa using454pyrosequencing
De novo assembly and transcriptomic analysis for T. granosa were conducted using454GS FLX technology.1,170,337quality-filtered and trimmed reads were obtainedfrom1,190,945raw reads and then assembled into17,940transcripts and131,981singletons, with5.6×sequencing depth. Average length of the assembled transcriptswas934bp, and33.6%of the transcripts were longer than1,000bp. More than9,000unique protein-coding genes were identified from a variety of developmental stagesand adult tissues based on sequence similarities with known protein databases. By GOannotation and KEGG pathway mapping, functional annotation of the unigenesrecovered diverse biological functions and processes. Of which, a lot of candidategenes and signal pathways putatively involved in growth and Hb synthesis wereidentified. Furthermore,6,338single nucleotide polymorphisms (SNPs) and20,038simple sequence repeats (SSRs) were also detected. In conclusion, A considerableamount of promising candidate genes potentially involved in growth and Hb synthesiswere identified, and are considered as an invaluable genetic resource for functional analysis and further production improvement.
2. Characterization of polymorphic EST-SSR markers in T. granosaand their cross-amplification in Scapharca subcrenata
EST-SSR is one of important methods to isolate microsatellite loci, which issimple and low cost chices. Next-generation sequencing technologies provide anexcellent opportunity to mining large numbers of EST-SSR markers quickly andcost-effectively for non-model organisms. Here we isolated and characterized62polymorphic EST-SSR markers in the blood clam, Tegillarca granosa, from the ESTsgenerated by454sequencing. The number of alleles for the62SSR markers in25individuals varied from2to9, with an average of3.90alleles per locus. The observedheterozygosity ranged from0.040to1.000, while the expected heterozygosity variedfrom0.040to0.844. Polymorphic information content ranged from0.038to0.806.Interspecific transferability of the62markers revealed that16were polymorphic inScapharca subcrenata, resulting in a transferability rate of25.81%. To our knowledge,this is the first report on the survey of SSR transferability in blood clams. These novelpolymorphic EST-SSR markers should be particularly useful for further investigationof population and conservation genetics.
3. The full length cDNA cloning and gene expression analysis of Tg-Smad3
Smad3could conduct the TGF--β signaling from cell surface to nucleus andpalys important crucial roles on singling transduction. By RACE method, the fulllength of cDNA is2341bp, encoding423amino acids. The amino sequences ofTg-Smad3is very conservatice with functional domains MH1and MH2，and sharedmore than80%homology with verbrate. By RT-PCR，Tg-Smad3were detected in alltissues, and tissues of blood and foot showed higher expression, and the Tg-Smad3expression with development of embryos, which implied that Tg-Smad3playedimportant role on embryo development. All of results suggested that Tg-Smad3mighthave functions of controlling growth, immune and shell formation.
4. Cloning and expression of BMP7gene from T. granosa
BMP7, a member of TGF-β pathway, was involved in many importantbiological functions, such as bone skeletal growth, metabolism and regulating celldifferention, proliferation and apoptosis. Additionally, BMP7plays important role onanimal reproductive system and embryo development. The full length of Tg-BMP7has a ORF fragment of1275bp, encoding425amino acids. Tg-BMP7was a glycoprotein that contains a signal peptide of29amino acids, three disulfide bonds,and a TGF-beta family signaling, which fully suggested that Tg-BMP7was a memberof the TGF-beta family. The results obtained by RT-PCR implied that Tg-BMP7wasshowed higher expression levels in the gills and mantle, relatively high expression inD-larval stage, suggesting that the Tg-BMP7was may be involved in the formationof the shell, tissue differentiation, regulation of embryonic growth and development.
5. Gene cloning and expression of ERK2from T. granosa
ERK2, extRACEllular signal-regulated protein kianse, an important member ofMAPK, is mainly involved in the regulation cell growth, differentiation and cell cycleregulation. By studying Tg-ERK2, it may lay solid foundation to investigate themechanism of TGF-β pathway. The full length of Tg-ERK2was1673bp encoding359amio acids. One functional domain，a serine/threonine protein kinase active sitewas found, which is the characteristic region of the ERK family proteins. By aminoacid sequence alignment and constructing phylogenetic tree, Tg-ERK2shared morethan80%homology with vertebrate， which indicated that the gene is veryconservative and may have similiar function with vertebrate. The results of RT-PCRshowed that Tg-ERK2was detected in all tissues and developmental stages, indicatingthat the function of Tg-ERK2was powerful to participate in various life activities.
6. Gene cloning and expression of GRB2from T. granosa
GRB2, growth factor receptor-bound protein2, is an important adaptor involvedin a variety of receptor activator of intRACEllular. It plays important roles inregulation of cell proliferation and differentiation. The full length of Tg-GRB2was1275bp, encoding236amino acids. There were three functional domains in Tg-GRB2protein, one SH2and two SH3, which were active region of physiological function.Tissue-specific expression of the Tg-GRB2showed an trend of blood> Foot> gill>mantle> adductor muscle> visceral mass, and developmental stage-specificexpression of the Tg-GRB2suggested that the Tg-GRB2had the function ofpromoting embryonic development, cell differentiation.
7. Cloning and expression of metabolism gene LAP3from T. granosa
LAP3, leucine aminopeptidase, one of the M17family of proteins, play animportant role in cell maturation, cell growth, regulating injury defense andmaintenance of cell dynamic equilibrium. In this study, the full length of Tg-LAP3gene was1590bp encoding530amino acids, including the sequence of Peptidase_M17superfamily N-terminal domain (41-174) and Peptidase_M17catalytic domain (209-522). By RT-PCR, we investigated the tissue anddevelopmental specific expression to study the roles of leucine aminopeptidase inphysical activity. The results suggested that LAP3played important role in embryodevelopment，promoting cell growth and metabolism. The study of Tg-LAP3couldlay theoretical foundation for protein metabolism studies and provide a guidance forfine varieties breeding of T. granosa.
1.基于454GS FLX高通量测序的泥蚶转录组分析
应用454高通量测序技术，对泥蚶4个组织和3个发育阶段样品进行了转录组测序，获得了1170337条高质量序列，通过序列拼接得到17940个重叠群（isotig）和131980个单拷贝序列（singleton），isotig平均长度达到934bp，测序深度为5.6倍。对泥蚶拼接得到的isotig与Swiss-prot、Nr等公共蛋白质数据库比对，通过GO注释对基因进行分类，KEGG注释了解基因间的联系和生物体的生化代谢网络。从注释的基因中鉴别出大量调控泥蚶肌肉、上皮、神经、纤维等的生长因子及受体，找到了与生长相关的TGFβ信号通路、MAPK信号通路中的多个重要基因；查找到大量与生长、发育、繁殖和免疫等生产相关的候选基因，为后续开展功能基因研究和分子育种实践有重要价值。在泥蚶转录组中，发现血红素合成过程中的大部分关键酶，推测贝类血红蛋白合成的基本过程可能与脊椎动物十分相似。另外，从泥蚶转录组中检测到6338个SSR和20038个SNP候选标记，为分子标记的大规模开发提供了重要资源。
2.利用泥蚶转录组序列大量开发微卫星标记，并研究其引物在毛蚶中的通用性
EST文库筛查法是目前大量开发微卫星标记的重要方法，具有方法简便、花费低廉等优点。本研究从泥蚶454转录组测序得到的大量isotig和singleton序列中搜索到6338个SSR位点，可用于大规模开发SSR标记。在3828条isotig序列里，共检测到138条适合设计引物的序列，扩增得到81个稳定扩增的位点，这些位点在泥蚶奉化群体中进行多态性检测，共扩增出242个等位基因，各位点的等位基因数为2~9个，平均每个位点产生3.90个等位基因，观测杂合度为0.040~1.000，期望杂合度为0.040~0.844，多态信息含量为0.038~0.806。33个SSR来自注释基因序列，对这些基因的准确定位有很大帮助。用62对泥蚶多态性SSR引物在毛蚶中进行了通用性验证，结果有22对成功扩增，16对表现为多态，这些多态位点可作为毛蚶和泥蚶的通用性标记。
3.泥蚶Smad3基因的克隆与表达分析
Smad3是TGF-β通路的重要成员，可将信号分子从细胞表面受体传导至细胞核，是重要的生长调控因子。利用RACE技术扩增得到泥蚶Smad3基因2341bp的cDNA全长，编码423个氨基酸。蛋白质功能域分析表明，Tg-Smad3含有两个保守功能域MH1和MH2。氨基酸序列比对发现，它与脊椎动物的同源性达到80%以上，说明Tg-Smad3氨基酸序列十分保守。RT-PCR结果表明，Tg-Smad3在泥蚶各成体组织都有表达，其中足和血液中表达量最高，可能与细胞增殖和免疫过程有关；在早期发育阶段，Tg-Smad3的表达量呈逐渐升高的趋势，在D形幼虫时期表达量最高，推测其参与贝壳的形成过程。
4.泥蚶BMP7基因的克隆与表达分析
BMP7也是TGF-β通路中的重要成员，在生物体内具有重要且广泛的生物功能，参与骨骼的生长、代谢，调节细胞的分化、增殖和凋亡，并在生殖系统的生长、发育中发挥重要作用。本研究克隆得到Tg-BMP7基因的cDNA全长，开放阅读框为1275bp，编码425个氨基酸。功能域预测表明，Tg-BMP7是一个糖蛋白，包含由29个氨基酸组成的信号肽，3个二硫键，并有一个TGF-β家族信号。RT-PCR结果显示，Tg-BMP7在成贝外套膜和鳃中表达量高；在早期发育阶段中，D形幼虫期的表达量显著高于原肠胚、担轮幼虫和幼贝，推测Tg-BMP7可能参与贝壳的形成过程，并具有组织分化、调控胚胎生长发育等生理功能。
5.泥蚶ERK2基因的克隆与表达分析
ERK2是MAPK家族重要的反应激酶系统，主要参与调控细胞生长、分化和细胞周期调控等生理功能。泥蚶ERK2基因cDNA全长1673bp，编码359个氨基酸，功能域预测找到一个丝氨酸/苏氨酸蛋白激酶的活性区域，这是ERK家族蛋白的特征性区域。氨基酸序列比对和系统进化分析发现，Tg-ERK2蛋白与脊椎动物的同源性达到80%以上，说明该基因十分保守，可能功能与脊椎动物有类似之处。RT-PCR结果表明，Tg-ERK2在泥蚶各组织、各个发育阶段都有表达，说明Tg-ERK2功能强大，参与到多种生命活动中去；相对而言，Tg-ERK2在泥蚶成体的血液中表达量最高；而在早期发育阶段中表达量逐渐升高，D形幼虫期达到最高，这可能与此期细胞分化加剧、神经细胞形成较多有关。
6.泥蚶GRB2基因的克隆与表达分析
GRB2蛋白是一个重要的接头分子，参与细胞内各种受体激活后的下游调节，调控细胞的增殖和分化。Tg-GRB2基因全长1275bp，编码236个氨基酸。Tg-GRB2蛋白包含3个功能域，1个SH2和2个SH3，这是GRB2发挥生理功能的活性区域。Tg-GRB2在泥蚶不同组织中的表达量由大到小依次为：血液＞足＞鳃＞外套膜＞闭壳肌＞内脏团；在早期发育阶段中，GRB2基因在原肠胚、担轮幼虫、D形幼虫中均有较高的表达量，原因可能是随着胚胎发育过程的进行，细胞分裂速度加快，生命活动旺盛，信号传导需要较多的接头蛋白
7.泥蚶蛋白质代谢基因LAP3的克隆与表达分析
LAP3是M17类蛋白家族的一员，在细胞成熟、细胞生长、调节损伤防御、维持细胞动态平衡方面发挥重要作用。Tg-LAP3基因全长为1590bp，编码530个氨基酸，包括Peptidase_M17超家族序列的N-端结构域（41-174bp）和Peptidase_M17催化结构域（209-522bp），缺失HEXXH锌结合体motif，充分证明了该蛋白为LAP3蛋白。RT-PCR结果表明，Tg-LAP3在足中表达量最高，而在早期发育阶段中表达量逐渐升高，D形幼虫期达到最高，在幼贝中表达量最低。
The blood clam, Tegillarca granosa, is one of the most popular commercially-important maricultural shellfish which is extensively cultured for seafood along thesouthern coastline of China, especially in Zhejiang province and Jiangsu Province. Inrecent years, with the technological breakthroughs of artificial breeding andlarge-scale farming, the aquaculture industry of T. granosa presented the trend ofrapid development, resulting in a huge economic and social benefits. Despite theeconomic value and research importance, knowledge of genetics and genome in T.granosa has been largely limited, and breeding work has just begun up to date. As aneconomically important aquacultural species, the lack of genomic resources would beunfavorable to understand the genetic mechanisms that involved in the growth,reproduction and immunity. In order to develop molecular resources and expeditegene discovery, we have undertaken454pyrosequencing of diverse cDNA libraries tofirst produce a comprehensive transcriptome for the blood clam T. granosa. Ourresults are as follows.
1. Transcriptomic analysis for T. granosa using454pyrosequencing
De novo assembly and transcriptomic analysis for T. granosa were conducted using454GS FLX technology.1,170,337quality-filtered and trimmed reads were obtainedfrom1,190,945raw reads and then assembled into17,940transcripts and131,981singletons, with5.6×sequencing depth. Average length of the assembled transcriptswas934bp, and33.6%of the transcripts were longer than1,000bp. More than9,000unique protein-coding genes were identified from a variety of developmental stagesand adult tissues based on sequence similarities with known protein databases. By GOannotation and KEGG pathway mapping, functional annotation of the unigenesrecovered diverse biological functions and processes. Of which, a lot of candidategenes and signal pathways putatively involved in growth and Hb synthesis wereidentified. Furthermore,6,338single nucleotide polymorphisms (SNPs) and20,038simple sequence repeats (SSRs) were also detected. In conclusion, A considerableamount of promising candidate genes potentially involved in growth and Hb synthesiswere identified, and are considered as an invaluable genetic resource for functional analysis and further production improvement.
2. Characterization of polymorphic EST-SSR markers in T. granosaand their cross-amplification in Scapharca subcrenata
EST-SSR is one of important methods to isolate microsatellite loci, which issimple and low cost chices. Next-generation sequencing technologies provide anexcellent opportunity to mining large numbers of EST-SSR markers quickly andcost-effectively for non-model organisms. Here we isolated and characterized62polymorphic EST-SSR markers in the blood clam, Tegillarca granosa, from the ESTsgenerated by454sequencing. The number of alleles for the62SSR markers in25individuals varied from2to9, with an average of3.90alleles per locus. The observedheterozygosity ranged from0.040to1.000, while the expected heterozygosity variedfrom0.040to0.844. Polymorphic information content ranged from0.038to0.806.Interspecific transferability of the62markers revealed that16were polymorphic inScapharca subcrenata, resulting in a transferability rate of25.81%. To our knowledge,this is the first report on the survey of SSR transferability in blood clams. These novelpolymorphic EST-SSR markers should be particularly useful for further investigationof population and conservation genetics.
3. The full length cDNA cloning and gene expression analysis of Tg-Smad3
Smad3could conduct the TGF--β signaling from cell surface to nucleus andpalys important crucial roles on singling transduction. By RACE method, the fulllength of cDNA is2341bp, encoding423amino acids. The amino sequences ofTg-Smad3is very conservatice with functional domains MH1and MH2，and sharedmore than80%homology with verbrate. By RT-PCR，Tg-Smad3were detected in alltissues, and tissues of blood and foot showed higher expression, and the Tg-Smad3expression with development of embryos, which implied that Tg-Smad3playedimportant role on embryo development. All of results suggested that Tg-Smad3mighthave functions of controlling growth, immune and shell formation.
4. Cloning and expression of BMP7gene from T. granosa
BMP7, a member of TGF-β pathway, was involved in many importantbiological functions, such as bone skeletal growth, metabolism and regulating celldifferention, proliferation and apoptosis. Additionally, BMP7plays important role onanimal reproductive system and embryo development. The full length of Tg-BMP7has a ORF fragment of1275bp, encoding425amino acids. Tg-BMP7was a glycoprotein that contains a signal peptide of29amino acids, three disulfide bonds,and a TGF-beta family signaling, which fully suggested that Tg-BMP7was a memberof the TGF-beta family. The results obtained by RT-PCR implied that Tg-BMP7wasshowed higher expression levels in the gills and mantle, relatively high expression inD-larval stage, suggesting that the Tg-BMP7was may be involved in the formationof the shell, tissue differentiation, regulation of embryonic growth and development.
5. Gene cloning and expression of ERK2from T. granosa
ERK2, extRACEllular signal-regulated protein kianse, an important member ofMAPK, is mainly involved in the regulation cell growth, differentiation and cell cycleregulation. By studying Tg-ERK2, it may lay solid foundation to investigate themechanism of TGF-β pathway. The full length of Tg-ERK2was1673bp encoding359amio acids. One functional domain，a serine/threonine protein kinase active sitewas found, which is the characteristic region of the ERK family proteins. By aminoacid sequence alignment and constructing phylogenetic tree, Tg-ERK2shared morethan80%homology with vertebrate， which indicated that the gene is veryconservative and may have similiar function with vertebrate. The results of RT-PCRshowed that Tg-ERK2was detected in all tissues and developmental stages, indicatingthat the function of Tg-ERK2was powerful to participate in various life activities.
6. Gene cloning and expression of GRB2from T. granosa
GRB2, growth factor receptor-bound protein2, is an important adaptor involvedin a variety of receptor activator of intRACEllular. It plays important roles inregulation of cell proliferation and differentiation. The full length of Tg-GRB2was1275bp, encoding236amino acids. There were three functional domains in Tg-GRB2protein, one SH2and two SH3, which were active region of physiological function.Tissue-specific expression of the Tg-GRB2showed an trend of blood> Foot> gill>mantle> adductor muscle> visceral mass, and developmental stage-specificexpression of the Tg-GRB2suggested that the Tg-GRB2had the function ofpromoting embryonic development, cell differentiation.
7. Cloning and expression of metabolism gene LAP3from T. granosa
LAP3, leucine aminopeptidase, one of the M17family of proteins, play animportant role in cell maturation, cell growth, regulating injury defense andmaintenance of cell dynamic equilibrium. In this study, the full length of Tg-LAP3gene was1590bp encoding530amino acids, including the sequence of Peptidase_M17superfamily N-terminal domain (41-174) and Peptidase_M17catalytic domain (209-522). By RT-PCR, we investigated the tissue anddevelopmental specific expression to study the roles of leucine aminopeptidase inphysical activity. The results suggested that LAP3played important role in embryodevelopment，promoting cell growth and metabolism. The study of Tg-LAP3couldlay theoretical foundation for protein metabolism studies and provide a guidance forfine varieties breeding of T. granosa.
引文
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