IGFBP5and IGF2BP2Genes In Yesso Scallop(Patinopecten Yessoensis): Molecular Cloning, Expression Analysis And Identification Of SNPs Associated With Growth Traits

The Yesso scallop (Patinopecten yessoensis) is an economically importantshellfish species in China. Identification of growth-related genes and gene loci, andanalyzing the expression patterns of these genes would benefit our understanding inthe molecular mechanisms underlying scallop growth traits, and also provide usefulinformation for high-yielding scallop breeding. In this study, reference genes forreal-time quantitative RT-PCR (qRT-PCR) were screened in Yesso scallop. Then twogenes, insulin-like growth factor binding protein5(PyIGFBP5) and insulin-likegrowth factor2mRNA binding protein2(PyIGF2BP2) were cloned, and theirexpression patterns were analyzed. A SNP (single nucleotide polymorphysm) whichwas significantly associated with growth traits including shell length, shell height,body weight, soft tissue weight and striated muscle weight, was found in PyIGFBP5and PyIGF2BP2, respectively. Our data will be helpful to further understanding thefunction of IGFs related genes in scallops and provide candidate loci for Yesso scallopbreeding aiming at production improvement.1. Reference genes selection for qRT-PCR in Yesso scallopThe mRNA sequences of12candidate housekeeping genes in Yesso scallop wereobtained from the transcriptome data of this species and Genbank. The12geneswere β-actin (ACT), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH),Cytochrom C (CC), Nicotinamide adenine dinucleotide (NADH), elongationfactor1-α (EF-1-α), Ubiquitin (UBQ), TATA-box bindingprotein (TBP),60Sribosomalprotein L16(RPL16), DEAD-box RNAhelicase-like protein (HELI),β-Tubulin (TUB), Cyclophilin (CYP) and Histon H3.3(His3.3). Expression stabilitiesof these genes in scallop embryos/larvae, and adult tissues before and after heat shock were evaluated, respectively, using three algorithms, geNorm, NormFinderand ΔCt method. The results showed that NADH, CC, His3.3and GAPDH were theoptimal reference genes combination for different embryo/larva stages, while fordifferent tissues HELI, UBQ and RPL16were recommended for qRT-PCRnormalization. And after heat shock, no cross-all-tissue type reference gene wasidentified among the examined panel of candidate reference genes, and differentreference genes combination was recommended for different tissues: EF-1-α, RPL16and UBQ for striated muscle; His3.3, UBQ, HELI and CYP for gonad; HELI, RPL16and UBQ for mantle; CC, GAPDH, NADH and TBP for gill; HELI and EF-1-α forhepatopancrease; and CC, GAPDH and NADH for kidney. The most commonly usedreference gene ACT was proved not the ideal one in most conditions.2. Molecular cloning, expression analysis of PyIGFBP5gene, and theassociation of allelic variants with growth traitsThe full-length cDNA and DNA sequences of PyIGFBP5gene were obtained.The gene consisted of3exons and2introns and the deduced amino acid sequencescontained signal peptide, N-terminal domain signal, two conserved amino sequencesPCRCCXXC and GICXXV, and some other conserved domains reported in vertebrateIGFPB5gene. Conserved regulatory elements in IGFBP5, including NFIL6,TATA-box, Myb, EBP, CAAT-box and two muscle-specific regulatory elements E-boxwere identified in the5’ flanking region of the gene. Expression analysis revealed thatPyIGFBP5transcripts were detected in each sampled developmental stages (fertilizedeggs, blustulae, gastrulae, trochophore larvae and D-shaped larvae), among whichhigher expression levels of PyIGFBP5were detected in blastulae and trochophorelarvae, and lower in D shaped larvae. PyIGFBP5mRNA was also detected in all thetissues analyzed, including striated muscle, gonad, mantle, gill, hepatopancrease andkidney, with significantly higher expression levels in mantle than in other tissues. Thelowest expression level was detected in kidney. Four SNPs, C.-117T>C, C.859A>T,C.876C>T and C.1051C>A were obtained in UTR region of PyIGFBP5genes, andC.1051A>G showed significant association with growth traits including shell length,shell height, body weight, soft tissue weight, striated muscle weight (P<0.05). Scallops with genotype AA and GG had significantly higher growth traits values thanthose with genotype AG in two independent groups (P<0.05). Principal componentanalysis (PCA) showed that there was a positive correlation between the fivegrowth-related traits. An expression analysis detected higher PyIGFBP5transcripts inthe mantle of scallops with genotype AG compared to those with genotype AA or GG.Our results suggested the negative regulatory function of PyIGFBP5in scallop shellgrowth3. Molecular cloning and expression analysis of PyIGF2BP2gene, andidentification of growth traits-associated SNPThe full-length cDNA sequence and DNA sequence of PyIGF2BP2wereobtained. The gene contained17exons and16introns, and encoded a591-amino acidpeptide with two RNA recognition motifs and four K-homology RNA-bindingdomains which were all conserved in IGF2BPs family. TATA-box, CAAT-box, E-boxand some other regulatory elements were identified in the5’ flanking region of thisgene. Expression of PyIGF2BP2was detected in all developmental stages and tissuesexamined. In embryos and larvae, higher expression levels of PyIGF2BP2was ingastrulae while the lowest was in D-shaped larvae. Among adult tissues, gonadshowed significantly higher PyIGF2BP2expression level than the other five tissuesand the expression in kidney was lowest. One synonymous SNP C.852G>A wasfound significantly associated with the growth traits, including shell length, shellheight, body weight, soft tissue weight, and striated muscle weight (P<0.05). Scallopswith genotype GG showed significantly higher values of growth traits than those withgenotype AA (P<0.05) in two wild populations.