Identification,Expression And Function Of Sex Differentiation Genes(fem-1b、fem-1c And Ank)in Hyriopsis Cumingii

Hyriopsis cumingii is an important freshwater pearl culture in China,Its cultivated pearl market share is high.however,in the low-level organisms such as bivalves and shellfish,the research on sex is far from enough,which hinders the research on breeding of shellfish.The fem-1b,fem-1c and ank genes of Hyriopsis cumingii were cloned by RACE.Gene function was explored by fluorescence quantification,in situ hybridization and RNA interference.This will provide the basis for a better follow-up study of the gender studies of the H.cumingii.1.Full-length cDNA and structural analysisThe full-length cDNA of fem-1b gene is 2105 bp and consists of 50 bp and147 bp 5 ’and 3’ UTRs with an ORF of 1908 bp and 635 amino acids.GenBank number is KY465662.It contains 5 ankyrin repeat regions,no signal peptide and transmembrane region,amino acid residues are hydrophilic.The amino acid sequence is highly similar to other species and has a high degree of conservatism during its evolution.The full-length cDNA of fem-1c gene is 2196 bp in length and contains 5 bp and 320 bp of 5 ’and 3’ UTRs.The ORF is 1866 bp and encodes 622 amino acids.GenBank access number is KY465663.It contains seven ankyrin repeat regions,no signal peptide and transmembrane region,acid residues are hydrophilic.It is highly similar to other species and has a high degree of conservatism during its evolution.The full-length cDNA of ank gene is 2509 bp in length and contains 420 bp and 277 bp 5 ’and 3’ UTRs.The ORF contains 1812 bp and encodes 603 amino acids.GenBank access number is KY465664.It contains 13 ankyrin repeat regions,no signal peptide and transmembrane region,amino acid residues are hydrophilic.Amino acid sequences are less similar to other species and have a high rate of mutation during evolution.2.Quantitative real-time PCR(qPCR)expressionThe fem-1b gene was highly expressed in female gonads and male kidney of Hypriopsis cumingii,and the female expression of fem-1b gene was significantly higher than that of males.The expression of fem-1b in the gonads from embryonic to 8-month-old was the highest on day 3 of embryo development.The fem-1c gene was highly expressed in female gonads and male mantle of H.cumingii,and the expression level.In early gonadal development,fem-1c expression was significantly increased at 5 months of age.The overall expression of ank gene in both sexes was consistent,and was highly expressed in the female and male adductor muscles and foot.In early gonadal development,ank significantly increased at 5 months of age.It is speculated that the fem-1b gene may be maternally inherited.fem-1b and fem-1c may be involved in female sexual differentiation or gonadal development.ank gene may be related to the gonadal development of H.cumingii.3.Analysis of fem-1c gene in situ hybridizationThe expression of fem-1c in male and female gonads of H.cumingii was analyzed.ISH showed strong expression of fem-1c in the spermatocytes of cytoplasm and gonad of oocytes of female gonads,indicating that fem-1c gene is involved in the formation of male and female gametes.4.RNA interference of fem-1c geneThe interfering chain of fem-1c-i2 was screened by RNA interference chain and experimental samples,and the 5-month-old H.cumingii was selected as the experimental sample.The samples were injected every 4 days for 5 times,a total of 24 days of experiments.The results were observed by fluorescence quantitative and frozen section staining.The results showed that gonadal development in the sample with interfering RNA chains slowed down and the growth rate was inhibited.By calculation,the average interference efficiency of the interference chain is 64.15%.It shows that this interference experiment has a certain effect and provides the basis for further research on the gender study of H.cumingii.