Isolation, Identification, Expression And Function Studies Of Growth Performance Candidate Genes In Litopenaeus Vannamei

Litopenaeus vannamei occupies a very important position in the aquaculture in China.The gradual increase of its cultivation area and production has promoted the economicdevelopment in our country. However, due to several factors such as nonstandardized parentfoster, nonstrict screening, widespread inbreeding, and nonstandardized breeding technology,the quality of seedlings declines seriously, mainly in slow growth rate and large differences inindividual growth. Thus, carrying out thorough research on growth related genes of L.vannamei to explore the molecular mechanisms on shrimp growth and development is the keyfoundation for high quality and efficient farming.In this study,7growth performance candidate genes were isolated and identified bydifferential expression profiling screening and candidate gene approaches; correspondingbioinformatic analysis were performand; time and spatial expression patterns of each genewere analysed by quantitative real-time PCR (qRT-PCR); using prokaryotic expressiontechnology sereveal related proteins were successfully expressed and purified and the proteinexpressions of the differential expressed genes in shrimp were further detected byWestern-blot technique; the relationships among mRNA expressions of associated genes andsome of the phenotypic characteristics of shrimp were studied after silence of candidate genesby RNA interference and the potential regulatory pathways for growth-related traits of eachgene were preliminarily identified. The main results obtained were as follows:1. Using GeneFishingTMtechnology combined with qRT-PCR,3differentially expressedsequences were obtained from abdominal muscles of two groups3-month old L. vannamei ofthe same full-sib family with significantly different (P<0.01)body weight(High weight shrimpgroup and Light weight shrimp group). One of them was the3’ end partial cDNA sequence ofactin T2in L.vannamei, and the remaining two were homologous sequences of ribosomalprotein L18gene(RpL18) and small nuclear ribonucleoprotein polypeptide G nuclear gene(SNRPG), all of which showd higher mRNA expression levels in the abdominal muscle ofhigh weight shrimps than that in light weight individuals. With the same method,5differentially expressed sequences were acquired from the abdominal muscles of1,3, and 6-month old L.vannamei of the same full-sib family, and they were3’ end partial cDNAsequences of proliferating cell nuclear antigen (PCNA), heat shock protein90(HSP90), type2myosin heavy chain (MYH2) and calpain B (CalpB) in L.vannamei and the homologoussequence of ribosomal protein L7gene (RpL7). All of the above5sequences showd thehighest expression levels in the abdominal muscle of3-month old L. vannamei.2. The cDNA sequences of differentially expressed genes RpL18, SNRPG and RpL7aswell as other L.vannamei growth performance candidate genes myostatin/growthdifferentiation factor11(MSTN/GDF11), ecdysone receptor (EcR), retinoid X receptor (RXR)and E75were successfully isolated by homology cloning techniques, with8transcripts foundin EcR and2transcripts found in RXR, which contained the whole coding regions.Bioinformatic analyses and predictions of putative proteins for the above sequences wereperformed using various softwares and online programs. The main results were showed asfollows: RpL18, SNRPG and RpL7proteins in crustaceans showed high conservations; theglycosylation sites "PNMTG" at N-terminal, the cleavage site "RXXR" between the peptideand the mature peptide and the nine cysteine residues located in the C-terminus were highlyconserved among species; conserved sites of EcR, RXR and E75found among crustaceansinclude P-box, D-box and eight cysteine residues in C2C2ZFP DNA-binding motifs.Phylogenetic analysis showed close relationships among crustaceans, clustering into onebranch in the evolutionary tree, and especially high sequence similarity were abserved amongshrimps. A phosphorylation site with the sequence of "MPRVHRPPIALSKV" locatedbetween the49thand66thanmino acids was found in RpL18. A signal peptide, two distincttransmembrane helixs and a TGF-β (transforming growth factor β) domain were found inMSTN/GDF11. The ZnF_C4and the HOLI domains were detected in EcRa, RXRa, and E75,respectively, and two distinct transmembrane helixs were found in E75.3. Transcriptional expression patterns of the7genes mentioned above RpL18, SNRPG,RpL7, MSTN/GDF11, EcR, RXR and E75were studied by qRT-PCR. Tissue expessionpatterns indicate the highest mRNA expresson levels of RpL18, SNRPG and RpL7in theabdominal muscle of L.vannamei, and the mRNA expressions of MSTN/GDF11were higherin the thoracic muscle and the heart than that in other tissues; the mRNA expressions of EcRwere higher in the thoracic muscle, ovary and the epidermis than that in other tissues; themRNA expressions of RXR were higher in the thoracic muscle, ovary, epidermis, heart andwalking leg than that in other tissues, and the mRNA expressions of E75were higher in theabdominal muscle, thoracic muscle and the eyestalk than that in other tissues. Geneexpression profiles at different developmental stages showed higher expressions of RpL18,SNRPG, RpL7and MSTN/GDF11at the postlarva stages than that at early larval stages and RpL7and MSTN/GDF11were highly expressed in fertilized eggs; low expression levels ofEcR, RXR and E75were found in fertilized eggs and nauplii and the mRNA expressions wererelatively stable from zoea which were higher than that in the early development. The mRNAexpressions during the molt cycle indicate that, in the abdominal muscle, the highestexpression levels of RpL18, SNRPG and RpL7were found at the intermolt stage, andMSTN/GDF11was mainly expressed at the early postmolt stage; in the hepatopancreas,epidermis, and the muscle, the mRNA expressions of EcR and RXR were mainly detected atthe premolt stage; in the hepatopancreas and epidermis, the expression levels of E75wererelatively high at the premolt stage, while in the muscle, E75was constantly expressed at eachstage during the molt cycle.4. The prokaryotic expression vector of was constructed by inserting the conding regionsof RpL18, SNRPG and RpL7as well as the mature peptide sequence of MSTN/GDF11into thepET32a (+) vector, respectively. Recombinant proteins were successfully expressed in E. coliBL21and the fusion proteins of RpL18, SNRPG, RpL7and MSTN/GDF11-MP withhistidine-tag were purified using different methods of purification. Western-blot techniquewas further used to detect the protein expressions of RpL18and SNRPG in the abdominalmuscle of of3-month old L. vannamei from the same full-sib family with significantlydifferent body weight (P<0.01), and the results showed that the expressions of RpL18andSNRPG were higher in the high weight shrimps than that in light weight individuals. Proteinexpression profiles of RpL7in the abdominal muscle of L.vannamei at differentdevelopmental stages from the same full-sib family indicated highest expression level atthree-month, followed by one-month, with the lowest expression level found at six-month oldshrimp.5. The dsRNA for the7genes RpL18, SNRPG, RpL7, MSTN/GDF11, EcR, RXR and E75were successfully prepared by the method of in vitro transcription, and the mRNA expressionof each gene above can be effectively silenced by injections of its corresponding dsRNA intoshrimp.Silence of RpL18, SNRPG and RpL7, the mRNA expression levels of muscle growthmarker genes Actin and MHC were subsequently decreased. Studies on the relationshipsamong RpL18, SNRPG, RpL7and another ribosomal protein gene QM (RpL10) showed thatsilence of RpL18, the mRNA expression levels of QM and RpL7were increased, and themRNA expression of SNRPG was decreased; silence of SNRPG, the mRNA expression levelsof QM and RpL7were increased, while the mRNA expression of RpL18was decreased;silence of RpL7, the expression of QM was decreased, while the mRNA expression levels ofRpL18and SNRPG were increased. It could be inferred that there existed functional relationships among RpL18, SNRPG, RpL7and QM, and the four genes participate jointly inthe positive regulation of muscle growth through the synergistic or complementaryrelationships among them.Silence of MSTN/GDF11, the mRNA expression levels of muscle growth marker genesActin and MHC were increased, and the mRNA expressions of muscle growth controllinggenes Akirin, Follistatin and cdk2were also increased, indicating negative regulatory roles ofMSTN/GDF11on muscle growth, which may be achieved by its negative regulations on themRNA expressions of Akirin, Follistatin and cdk2.After RNAi downregulated the expressions of EcR, RXR and E75, correspondingexpression changes of molting related genes Cathepsin-L (CHSL) and Hemocyanin (HCyn) inthe hepatopancreas, chitin metabolism related genes chitin synthase (ChS) and Chitinaseisoenzyme2(Chi2) in the epidermis, muscle growth marker genes Actin and MHC in themuscle and EcR, RXR and E75in all of the above three tissues were observed; decreasedaccumulative molting rates, morphological abnormalities, as well as increased mortalitieswere also found. Correspondingly, after in vivo injections of20hydroxyecdysone, specificexpression changes of EcR, RXR, E75, CHSL and HCyn in the hepatopancreas, EcR, RXR,E75, ChS and Chi2in the epidermis, and EcR, RXR, E75, Actin and MHC in the muscle werealso observed, respectively. Results in our study indicate the functional receptor heterodimerEcR-RXR and early responsive gene E75, once activated by20E in vivo, may act throughvarious pathways to perform multiple regulation roles on shrimp molting, chitin metabolism,muscle growth.