| The pearl oyster, Pinctada fucata is a widely cultivated marine shellfishbecause of its commercial value especially pearl production. Nevertheless, unlikethe vertebrates, growth mechanisms of invertebrates such as molluscs were stillunknown. No gene similar to growth hormone genes of invertebrates has beenidentified to date as well. Activities of digestive enzymes constitute aphysiological parameter affecting the digestive capacity especially in aquaticanimals. Amylase is one of the most important digestive enzymes forphytophagous animals. In the present study, α-amylase gene were cloned.Meanwhile, screening of single nucleotide polymorphisms (SNP) loci inα-amylase gene as well as association analysis of SNPs with growth-related traitswere carried out. And then effects of cultural ecological factors and age on theexpression of α-amylase were analysed using real-time quantitative polymerasechain reaction (Real-time qRT-PCR). The detailed results were as follows:1. The cDNA, genomic DNA and promoter region of α-amylase gene wereidentified by using the reverse transcription-polymerase chain reaction (RT-PCR),rapid-amplification of cDNA ends (RACE) and genome-walking methods.Sequence anylsis indicated that the complete cDNA sequence was1704bp,consisted of a5’-untranslated region of17bp, a3’-untranslated region of118bp,and an open reading frame (ORF) of1569bp. The deduced amino acid sequenceshowed the existence of a522-residue-long pre-enzyme containing a signalpeptide of20amino acids. Sequence alignment revealed that Pinctada fucataα-amylase (pfAMY) shared the highest identity (91.6%) with that of Pinctadamaxima. Consistent with this, the phylogenetic tree showed that it was closedrelated to Pinctada maxima. The genomic DNA was10850bp containing nineexons, eight introns and3932bp promoter region. Various transcription factors such as GATA-1, AP-1, SP1were predicted in the promoter region. Real-timeqRT-PCR assay was performed to estimate tissue-specific expression of pfAMYmRNA. Result indicated that relative level of pfAMY mRNA was significantlyhigher in the digestive gland than that in the others (intestine, gonad, gills, muscle,mantle)(P<0.001). This information could be used to establish the genetic basisof pfAMY activity and therefore facilitated studies of Pinctada fucata geneticbreeding.2. Single nucleotide polymorphisms (SNP) in α-amylase gene of pearl oysterPinctada fucata were isolated and characterized in this study.207SNP loci wereidentifed by polymerase chain reaction (PCR) and DNA sequencing in twentyindividuals from nine crossbred populations. SNP loci were unevenly distributed,with the highest density of one SNP every25bp in the intronic area and thelowest of one SNP every209bp in the regulatory region. All candidate SNP locicould be divided into three types including transition, transversion, insertion ordeletion with the percentage of47.82%,44.93%,7.25%, respectively. Mutationsin the coding region showed the existence of only synonymous polymorphisms orfunctionally equivalent non-synonymous polymorphisms.30SNP loci were usedfor genotyping among which only fourteen loci were dimorphic. Sevenmonomorphic loci and two trimorphic loci were also detected. Allele frequency ofall dimorphic loci had statistically significant difference (P<0.05) between twofull-sib families except locus+4453(A/C). Results of genetic diversity analysisshowed that both average polymorphism information content (PIC) and Shannon’sInformation index of family1were higher compared to those of family2, whichsuggested genetic diversity of family1was richer and therefore had the potentialto be used in breeding project. Association analysis indicated that individualswith different genotypes of locus-201(G/T),+3523(G/A),+4402(A/G),+5025(T/A),+6379(T/A),+6481(T/C),+6523(T/A) showed significant difference(P<0.05) at shell width, shell height or wet flesh weight. Diplotype wereconstructed based on eleven SNP loci and then used for correlation analysis.Results showed that all phenotypic values of diplotype S1, S3, S4, S9weresignificantly higher (P<0.05) than those of S2, S5, S6, S7, S8, S10. Interestingly,the former was only found in family1while the later was only detected in family1, which implied there were strong linkage disequilibrium between the eleven SNP loci. Future studies should examine larger data sets to better determine if theSNP-trait association mentioned above exists.3. Real-time qRT-PCR assay was performed to estimate the relative mRNAexpression level of α-amylase in response to age and gradients of ecologicalfactors including temperture, salinity, food concentration and water depth. Resultindicated that as the temperature rose the mRNA level increased with the highestlevel at21℃and then followed by a decrease within the range21℃～29℃. Asimilar expression pattern was presented responding to salinity variation. ThemRNA level at midrange salinity (27) was significantly higher than both low (15,21) and high (33,39) salinities. In elevated food concentration treatments, mRNAlevel decreased first and then followed by an increase within the range16×10~4cell·ml-1～20×10~4cell·ml-1. The water depth had a great effect on themRNA level. It was significantly higher in deep water (4,5m) compared to thatin shallow water (1,2, and3m). Influence of age on the expression level ofα-amylase was significant as well. Overall, the expression level in young agegroups (8,12,15months of age) were significantly higher (P<0.01) compared tothat of the older age groups (18,22,26,29months of age) which reflected ahigher utilization ratio of carbohydrate in young individuals. |
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