Molecular Cloning Of Growth Trait-related Genes In Penaeus Monodon And Screening And Verification Of Their SNPs Makers

Penaeus monodon is the largest prawn in mariculture penaeid species, with great economic values. It is one of the three major cultured species in the world and also an important specie in Chinese traditional aquaculture. Although the new variety “South China Sea No. 1” with quick growth and many new strains with good characters had been bred, In the face of present situation that the great difference of regional aquaculture environment and the urgent requirements of prawn species diversity, go on promoting genetic breeding research on P. monodon, and to carry out breeding technology innovation and continuous good varieties breeding, which provided the shrimp culture industry a powerful guarantee to healthy and sustainable development. The growth traits(carapace length, abdominal segment length, body length, body weight etc.) are important economic characters for the quality and yield of P. monodon. The objects of present study on genetic improvement and breeding was that application of molecular genetic marker, to recognize the genetic basis of the growth traits, and further identify the gene or makers in P. monodon associated with good growth characters.Based on the EST sequences of cDNA library of P. monodon, the complete cDNA of cathepsin L gene, trypsin gene, alpha-amylase gene in P. monodon were cloned by rapid amplification of cDNA ends in this paper. In order to explore the regulatory mechanism and function of these gene in P. monodon, the relative expression level was detected in different tissues, different growth periods and different development stages of ovary though RT-PCR reaction. SNP loci were screened in four geographical population though re-sequencing, and the genotyping of candidate SNP were detected by SnaPshot. Then we analyzed the association of these polymorphic locis with growth traits aims to find the SNP marker associated with growth traits and apply to the marker assisted selection, to accelerate the process of genetic improvement and new variety breeding in P. monodon. The major results were as follows:(1) The cathepsin L gene in P. monodon: molecular cloning, expression analysis and identification of SNPs associated with growth traits. The PmCatL cDNA was 1850 bp long and consisted of a 5’-untranslated region(UTR) of 28 bp, a 3’-UTR of 796 bp, and an open reading frame(ORF) of 1026 bp encoding a polypeptide of 341 amino acids, which contained a typical signal peptide sequence, a prodomain and a mature domain. The expression of PmCat L was detected in all tested tissues and with the highest expression level in lymph, second is in hepatopancreas. The highest expression level in post-larval, this may indicate PmCatL was associated with growth and development of P. monodon. While in different stages of ovary development, PmCatL had highest express in Ⅳstage, suggesting that PmCatL may play an important role in ovary development in P. monodon. Totally forty-two SNPs were obtained, fourteen located in the exon region and twenty-eight in the intron region. In PmCatL-E213 locus, shrimps with genotype GG had significantly smaller carapace length(CL), carapace width(CW), carapace height(CH) and weight(W) values than those with genotype GT and TT(P < 0.05). PmCat L-E784 showed no significant association with growth traits. PmCatL-E820 showed significant association with growth traits including CH and FSL.(2) The trypsin gene in P. monodon: molecular cloning, expression analysis and identification of SNPs associated with growth traits. The Pm Try cDNA was 916 bp which encodes for 366 amino acids with the predicted molecular weight of 28.38 KDa and the isoelectric point of 4.58. Homologous analysis of amino acid sequences indicated that the PmTry shared 42%~91% similarity with other species. The phylogenetic tree showed that PmTry was closely related to Litopenaeus vannamei. The expression level of PmTry in hepatopancreas significantly higher than other tested tissues(P < 0.05). The expression level of PmTry in nauplius stage significantly higher than other growth stages, suggesting that PmTry was a growth related gene in P. monodon. 8 SNPs were obtained from PmTry gene, and PmTry-629 had no significant association with growth traits. The locus PmTry-524 showed significant association with growth traits including CL, CW, FSL and SSL. In locus PmTry-798, shrimps with genotype AC had significantly higher values on CW trait than those with genotype AC and CC(P < 0.05), and shrimps with genotype AC had significantly higher CH values than those with genotype CC(P < 0.05).(3) The alpha-amylase gene in P. monodon: molecular cloning, expression analysis and screening of SNPs. The PmAmy cDNA was 2465 bp which encodes for 724 amino acids with the predicted molecular weight of 78.9 KDa and the isoelectric point of 4.66. The expression level of PmAmy in hepatopancreas significantly higher than other tested tissues(P < 0.05). The expression of PmAmy was detected in the whole growth stages, with the highest level in mysis stage significantly differences with other stages. 6 SNPs were obtained from PmAmy gene, which fill the domestic blank in the research of alpha amylase gene polymorphism in P. monodon. The SNPs markers provides a genetic basis for research of alpha amylase gene polymorphism associated with growth traits in the future.