The Expression Of DUI Genes And Survival Rate Of Sperm In Hyriopsis Cumingii

Many bivalve molluscs have two mitochondrial hereditary methods,one that transfers the mitochondrial genome through spermatozoa,one that transfers the mitochondrial genome through the egg.This phenomenon is known as the double uniparental inheritance of the mitochondrial genome.In the species with the DUI,there are two heterogeneous mitochondrial DNA(mtDNA),called F-type and M-type mtDNA,and F-type mt DNA is transmitted to the male and female offspring with their mother,M-type mt DNA with their father transmission to male offspring.And F,M-type mtDNA are very different,single gene COII and ATP8 difference is greater.In this study,F and M-type ATP8 genes were cloned and their cDNA sequences were obtained.The expression of F and M-type ATP8 gene was studied from the embryonic stage to the three-year-old stage,and the expression of F and M-type COII gene in the early stage of Hyriopsis cumingii was studied.At the same time,the molecular markers of male and female of H.cumingii were established by analyzing the association of SNPs between male and female with F-type ATP8 gene.The research contents are as follows:1.Cloning and structural analysis of F and M-type ATP8 gene of H.cumingiiThe full-length cDNA of H.cumingii F and M-type ATP8 gene was obtained.The F-type ATP8 gene was 275 bp in length,including 54 bp of 5 'untranslated region,71 bp of 3'nd untranslated region,150 bp in ORF region,Encoding 49 amino acids,the encoded amino acid as a whole showed hydrophobic.The M-type ATP8 gene was 222 bp in length and had no UTR at the 5 'end and 45 bp in the 3UTR.The ORF region was 177 bp,encoding 58 amino acids,and the amino acid was encoded as hydrophobic.Their instability coefficients were 46.90 and 56.34,respectively,indicating that their encoded protein was unstable.Homology analysis showed that the F-type ATP8 gene had the highest similarity with the H.schlegelii,followed by some similarity with other freshwater mussel.M-type ATP8 gene is not too similar to that of freshwater mussels,indicating that the M-type ATP8 gene is less conserved in evolution than the F-type ATP8 gene.2.The expression of F-type and M-type COII gene and ATP8 gene in H.cumingiiThe qRT-PCR results of of F-type and M-type COII gene and ATP8 gene in H.cumingii tissues showed that M and F-type COII genes could be detected in the early juvenile(embryonic-5-month-old)H.cumingii,and the expression of F-type COII gene was significantly higher than that of M-type.In the six-month-old tissues,the expression of M-type COII gene was the highest in the gonad,and the expression of F-type COII gene in the same tissue was still at a high level.The M and F-type ATP8 genes were expressed in all stages of the gonad,and the expression of F-type ATP8 gene was significantly higher than that of M-type ATP8 gene in the same stage.The M and F-type ATP8 genes were expressed in all tissues of one-year old H.cumingii,the abundance of F-type ATP8 was higher than that of M-type ATP8,which confirmed the dominant position of F-type ATP8 gene.The M and F-type ATP8 genes were expressed in all tissues of two-year old H.cumingii,the high expression of M-type ATP8 gene only in male and female gonad,the rest of the tissues were micro-expression or not expressed.The F-type ATP8 gene was detected in both male and female tissues of three-year old H.cumingii.The expression of M-type ATP8 in female tissues was very low,and in the male tissues,only high expression in gonad,the rest of the tissues was almost undetectable.3.Screening of SNP of F-type ATP8 gene of H.cumingii and analysis of differences between male and femaleThere were 16 SNPs in the DNA sequence of F-type ATP8 gene,and the single nucleotide polymorphism was abundant.The correlation analysis of SNPs in both male and female found that only a small number of detected SNPs had no statistical differences.There were 9 SNPs with genotype differences and 4 SNPs with different genotype and allele frequencies in male and female.This was significantly different from other genes in SNPs in H.cumingii,which further demonstrated that the mitochondrial genome had higher mutation rates and faster evolutionary rates.The G / A genotype of the E-115-G specificity present in the female and all of the genotypes in the male is G / G.4.Study on sperm survival rate of H.cumingiiEosin Y can only stain the sperm after death,we get the best dye concentration of 0.05% and the optimum dyeing time is 10 min.The survival rate of spermatozoa of H.cumingii was negatively correlated with temperature,and the higher temperature,the higher mortality rate.The survival rate of spermatozoa of H.cumingii was also related to the ion concentration and species in the water,and the mortality rate in saline was slightly higher than that in distilled water,the highest rate in saline was in river.