Transcriptome Analysis Using454Pyrosequencing And Cloning And Expression Of Growth-related Genes For The Blood Clam Tegillarca Granosa (linnaeus,1758)

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 using454pyrosequencingDe 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 subcrenataEST-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-Smad3Smad3could 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. granosaBMP7, 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. granosaERK2, 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. granosaGRB2, 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. granosaLAP3, 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.