| As the special mussel resources in China,Hyriopsis cumingii,the pearl produced by that is top-quality and smooth with bright color.Studies have shown that the cultivation ability of male individuals is superior to female mussels.In recent years,researches on protection of germplasm resources,pearl production performance,disease control and artificial breeding of H.cumingii have achieved considerable results,while the mechanism of sex determination of bivalve mussels is still unclear,and research on gender-related genes is rare.Therefore,This study selected three genes related to gonadal development from the existing H.cumingii c DNA transcriptome library: Dmrta2-1,Dmrta2-2 and Lhx9.We isolated the full c DNA length of three genes and analyzed the biological information of sequence structure.By experiments of fluorescence quantitative expression,signal detection of in situ hybridization and RNA interference to study their roles of sex in freshwater pearl mussels.The main results were as follows:1.Cloning and sequence analysis of three genesThe full-length sequence of three genes were obtained by RACE amplification.The two genes of the Dmrta2 subfamily had high homology,named Dmrta2-1 and Dmrta2-2.Dmrta2-1 nucleotide sequence was 1723 bp in length(Genbank accession number: MK372349),ORF(Open reading frame)1158 bp,encoding 385 aa(amino acid),5 'untranslated region(UTR)237 bp,3'UTR is 328 bp.The theoretical theoretical isoelectric point PI / protein relative molecular weight MW was 4.97 / 143293.27,and had no signal peptides or transmembrane regions appeared as the hydrophilic protein by Prot Scale program prediction.Dmrta2-2 had a full length of 1 682 bp(MK372348),an ORF region of 1 170 bp,encoding 389 aa,340 bp 5'UTR and 172 bp 3'UTR.The predicted value of PI was 6.75 and MW was 42 568.48,there were not any contain signal peptides or transmembrane regions and the intracellular protein was hydrophilic(the average value was-0.643).NCBI predicted the conserved sites that both two Dmrta2 genes had a conserved DM domain and a DMA conserved motif.Amino acid sequence alignments and phylogenetic trees indicated that H.cumingii had the high similarity with Hyriopsis schlegelii,clustered into a small branch in the evolutionary tree with a conservative evolutionary status.The full cDNA length of the Lhx9 was 1 931 bp(MN006952),including the ORF of 531 bp,encoding 176 aa,36 bp 5'UTR and 1 364 bp 3'UTR.The predicted p I / Mw was 6.21 / 20174.25.NCBI predicted a conserved Homebox(HM)domain.The prediction index of the hydrophilicity and hydrophobicity of amino acids is negative,that is,hydrophilicity.The amino acid sequence of Lhx9 was similar to that of other species.It clustered with oysters and scallops in the phylogenetic tree,and the structure-function relationship was highly conserved in evolution.2.Expression analysis of three genes by qRT-PCRThe Dmrta2-1 gene expressed in the gonad,gill,liver,kidney,mantle,adductor and foot of 2-year-old mature individuals.Except for the liver,the expression of male and female in other tissues was extremely significant(p<0.01),in which the expression of female gonads was extremely higher than that of males;Early expression of gonads at different developmental stages(0D-8M)showed the highest level at one month old,followed by eight months old,predicting Dmrta2-1 was involved in sexual differentiation and gonadal development.The expression of Dmrta2-2 gene in two years old tissues was highest in both male and female adductors,and the difference between male and female tissues were significantly different(p<0.05)except liver.Early expression had the highest expression at three days,followed by three months.Expression of Dmrta2-2 in one to three years old gonads exhibited that the expression of females were all higher than that of in males,and the difference of one and two years old female gonads was extremely significant.Among them the expression of one year old male and female were both the highest,while males were low and stable at whole stage.We speculated Dmrta2-2 may be related to sexual differentiation,female gonad development and stable maintenance of male gonad function.Lhx9 was highly expressed specifically in two years old female gonads and were higher in various tissues than that of in male except for mantle,and the difference between male and female in gonad and foot was extremely significant.In early gonad expression,there was the highest value on day three,and the distribution was stable at other stages with lower expression.12-36 months gonad expression showed that females were higher than males.From 12 to 19 months,the expression level continued to decline,and then to 36 months of age,the level continued to increase but below the expression value of 12 months of age.In male gonads showed a similar trend but low expression abundance.It was speculated Lhx9 gene may mainly regulate female gonad development,and participate in ovarian differentiation and may also in maintenance of mature gonad function and a role in sexual maturity.3.ISH analysis of three genes in gonadsIn the in situ hybridization experiment of mature testis and ovary at two years old,the expression signal of Dmrta2-1 gene was detected in both testis and ovary,and it was mainly located on the cell membrane of oocytes in the ovary and was stronger,while the male in the follicular wall and was weaker.Similarly,the in situ hybridization experiment of the Dmrta2-2 gene in the mature testis and ovary of two years old detected more obvious expression signal in the ovary,mainly on the cell membrane of the oocyte,and for testis detected in the follicle wall of the testis.It is speculated that both Dmrta2 genes mainly play a role in ovarian differentiation.The expression of Lhx9 in the situ hybridization of mature testis and ovary in two years old was detected obvious signals on the cell membrane of oocytes in the ovary,and extremely weak in follicle wall of the testis.It is speculated that Lhx9 may be mainly related to female gonad development and differentiation process.4.Functional exploration of genes by RNAiIn the pre-experiment the Tra-2a gene ds Tra-1,2,3 chains were synthesized.After 7 days of injection,ds Tra-3 was detected more interference rate for 70.44% in male gonads and 50.98% in females,which was an ideal interference chain.In the formal interference,ds Tra-3 was injected multiple times and the result showed that expression of Dmrta2-2 in male gonads was significantly reduced after silenced Tra-2a,and the silent rate reached 62.20%,while there was no significant decrease in females.We inferred that Tra-2 gene in H.cumingii had a certain regulatory effect on Dmrta2-2,which may played an important role in gonad development.Selecting one year old H.cumingii and injecting double-stranded RNA of Ds-Lhx9 into adductors,the result showed expression of Lhx9 in the female gonad was significantly reduced and comparing with the blank group,the interference efficiency reached 69.16%,while in the male only 12.14%.It was speculated that Lhx9 played an indispensable role in gonadal development especially in females in H.cumingii.The above results provided the data support for the establishment of more effective RNA interference technology of H.cumingii and the study of its sex regulation mechanism. |
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