Comparative Analysis Of Transcriptomics Between Hibernating And Non-hibernating And Whether They Mated Or Not Mating In Different Parts Of Oviduct Of The Chinese Pound Turtles,Mauremys Reevesii

The Chinese pond turtle(Mauremys reevesii)is a member of reptiles,which has crucial values in medicine and edible.In recent years,the number of wild Chinese pond turtle presented significantly reduced,due to the overfishing and destruction of habitat.In order to alleviate the contradiction between the huge market demand and the scarcity of wild Chinese pond turtles,the artificial breeding industry of Chinese pond turtles developed rapidly.However,the healthy development of artificial breeding was limited due to several factors,primarily containing few studies on the artificial breeding,nonstandard breeding technology,and confused genealogical files of parents.This study aimed to explore the potential molecular mechanism involved in differently expressed gene in oviduct of Chinese pond turtle under different time phasing,before and after fertilization,which can provide theoretical basis for artificial breeding and formulation of protection procedures for wild turtle.In this study,we sequenced and analyzed related data in the oviduct of Chinese pond turtle at hibernation and reproduction period after fertilization,and fertilized and unfertilized oviduct via second generation transcriptome sequencing.And then we further investigated and verified the sequencing results using qRT-PCR method,and comprehensively analyzed the potential molecular mechanism involved in differently expressed gene in oviduct of Chinese pond turtle.The specific contents and results are as follows:1.Analysis of the transcriptome in oviduct of Chinese pond turtle during hibernation and reproduction.Three parts of oviduct(vagina,uterus and isthmus)in fertilized Chinese pond turtle at hibernation and idiophase were sequenced.The results of sequencing are as follows: the total data size of 18 samples is 67.3 Gb,in which the content of GC is in the range of 47.95% to 59.05%,while the percentage of Q30 base is over 94.19%.Bowtie2 was used to compare the effective data of the samples to the transcripts,and then the mapping information was counted;Salmon was used to calculate the gene expression levels,and then statistical analysis of differently expressed gene wasperformed by DESeq2;and finally,101 differently expressed genes involved in apoptosis in oviduct between hibernation and idiophase period were screened out,which included72 up-regulated and 29 down-regulated genes.There has been well demonstrated that these differentially expressed genes(DEGs)were involved in p53 signaling pathway,apoptosis signaling pathway,Fas signaling pathway and cAMP/PKA signaling pathway.Further study on differently expressed genes in the oviduct of Chinese pond turtle at hibernation and idiophase revealed several differently expressed genes,which related to cell apoptosis and sperm life,especially Bax,BCL2,BCL10.When compared between different parts of oviduct,the results disclosed that there are most differently expressed genes in the uterine part,especially,CASP3 and CASP9 were only expressed in the uterus.This provides a reliable reference data for the future study of Chinese pond turtle reproduction and sperm storage.2.Analysis of transcriptome of oviduct in fertilized and unfertilized Chinese pond turtle.In order to identify the differently expressed genes involved in sperm storage,the oviduct(vagina,uterus and isthmus)of fertilized and unfertilized Chinese pond turtle,respectively,were used for transcriptome sequencing,and then the data were analyzed for differently expressed genes.The results are as follows: the total data size of 18 samples is 46.35 Gb,in which the content of GC is within 46.45% to 59.05%,while the percentage of Q30 base is more than 93.92%.The data of fertilization and unfertilized oviduct were analyzed,and 246 DEGs related to fertilization were screened out,including 112 up-regulated genes and 134 down-regulated genes.Further analysis revealed that these DEGs were involved in p53 signaling pathway,apoptosis signaling pathway and ER signaling pathway.And then,differently expressed genes were analyzed,the results revealed that there are several genes(ESR,PGR,DAZAP1,etc.),which were related to fertilization and sperm storage.Furthermore,we compared the different parts of the oviduct,the results revealed that there are most differently expressed genes in uterus.All these results provided theoretical basis for the subsequent study on fertilization process of Chinese pond turtle.