Screening, Cloning, Identifying And Function Analyzing Of Genes Involved In Body Weight Regular In Abdominal Muscles Of White Shrimp

Litopenaeus vannamei has become a significant economic species as the development of shrimp culture industry worldwide. Enhancement of growth rate by genetic improvement is an important technique due to the wide development of breeding technology and resistant isolates, which may decrease the length of grow-out cycles, resulting in the reduce of production cost and culture cycle of shrimp. Shrimp muscles are primarily present in the abdominal part of its body, but the genes related to muscle growth, especially regulatory genes is not well understood.In the present study, abdominal muscle samples of the female-shrimp between large female (LF; body weight>90 percentile of weight distribution curve) and small female (SF; body weight<10 percentile of weight distribution curve)were selected as the target tissue to construct forward and reverse subtracted cDNA libraries of abdominal muscle , aiming to identify and clone the differently expressed genes, and predict their structure and function by bioinformatics, and analyze their expression pattern by real-time reverse transcriptase-polymerase chain reaction, The expression levels of the selected body weight regulation genes in abdominal muscle were also analyzed. The main results were showed as follows:1. Suppression subtractive hybridization (SSH) is used to construct forward and reverse subtracted cDNA libraries of abdominal muscle of the female-shrimp between large female S-L(LF as tester, SF as driver) and small female S-L(SF as tester, LF as driver). The subtraction efficiency was estimated by a housekeeping gene signated asβ-actin, and the results showed thatβ-actin was subtracted efficiently at 2¹⁰ and 2⁵ folds for SF and LF subtracted cDNA library respectively which demonstrated that differentially expressed genes were also enriched at the same folds.2. 50 clones were isolated from S-L subtracted cDNA library, and 30 clones were randomly selected from the library which different insert fragments were sequenced. The results showed that there exist 18 ESTs in S-L subtracted cDNA library,and 14 of them are known in L. vannamei, 4 are unknown in L. vannamei but have more than 95% homology with other species. These genes are categoried as: elongation factor 1-alpha (EF1A) , NADH dehydrogenase subunit 2 , NADH dehydrogenase subunit 1 , Cytochrome c oxidase subunitâ…¡, Cytochrome c oxidase subunitâ…¢, Cytochrome b , Heat shock protein 90 , crustacean hyperglycemic hormone B1, Arginine kinase, Tropomyosin slow isoform (sTm1), Ribosomal protein 31 , Ribosomal protein S24 , Cytochrome c oxidase , subunit 2 B, troponin C;150 clones were isolated from L-S subtracted cDNA library, and 100 clones were randomly selected from the library which different insert fragments were sequenced. Searching GenBank by using these nucleotide sequences indicated that 49 cDNA fragments could not find their homologous sequences in the database. The results indicated that there exist 49 ESTs in L-S subtracted cDNA library,and 19 of them are known in L. vannamei, 19 are unknown in L. vannamei but could find their homologous sequences with other species in the database, and 11 of them is the sequence of mitochondrion, these differentially gene mainly include 5 groups: cDNA sequence that expressed with synthesis of tissue or cell components occupied 37.21% of total unigenes,Genes that expressed with growth or remediation of cell account for 13.935% of the total unigenes, Among the unigenes with molecular function classification,Binding protein, biological regulation binding activity and catalytic acitivity related proteins took up to 4.65%, 4.65% and 4.65%, respectively.3. We designed primers based on the high homology sequence in gene coding region among different species and existed sequence for L. vannamei, and successfully cloned the coding region of RAS,P23 and Troponin of L. vannamei for the first time, namely, GenBank:RAS(ORF 564bp ,GenBank No:JF806618),P23(ORF 495bp ,GenBank No:JF806619)and Troponin(ORF 480bp, GenBank No:JF806620),which laid a foundation for further functional research.The structure and potential function of RAS,P23 and Troponin,as well as the secondary structure and tertiary structure of the three genes, were analyzed by using bioinformatics software. The obtained results demonstrated that partial Sequence of the PAS gene had strong hydrophobicity with high structural domain. Partial Sequence of the P23 gene also had strong hydrophobicity but with no transmembrane helices structure. The signal peptide prediction in Troponin gene showed that there was a signal peptide with strong amino acid residues in the partial Sequence of the P23 gene, whose possibility was 90.2%. Hydrophobicity profile analysis and signal peptide prediction in Troponin gene demonstrated that there were strong transmembrane regions and no signal peptide with strong amino acid residues (6-24), but with no structural domain.4.In the present study, analysis on 12 gene expression in Litopenaeus vannamei of different gender were performed and the results indicated that VTG gene expression was found in muscles of males sand females, which was inconsistent with the previous research. Study on mRNA expression of 12 genes in five tissues (eyestalk, heart, liver and pancreas, stomach and digestive tract) showed that high expression of gene AK was found in the heart and stomach where CHH gene expression was the lowest, While the highest expression of Troponin gene was found in the muscle, which were related to their functions. Correlation analysis on 9 genes and 10 characters of weight indicated that the relative expression of genes AK, PK, and TM was not associated with that of other genes, but only VTG gene was negatively correlated with body weight, and the highest correlation coefficient were observed in CHH and ER genes which are related with the shrimp molt (R²=0.795).5. The coding region of the RAS,P23 and Troponin were constructed into pET32a (+) vector for proeukaryotic expression. The obtained results showed that RAS and Troponin were expressed successfully in BL21 E.coli and detected. The expression condition of RAS and Troponin recombinant protein was optimized at 30℃which was induced for 6 h by IPTG(1 mmol/L).The recombinant protein pET32a (+)-P23 was not expressed in BL21 E.coli.