Isolation And Functional Study Of Four Key Gene Families Associated With Spermatogenesis From Banna Mini-pig Inbred Line

Banna mini-pig inbred line(BMI)is the first successfully cultivated large mammal inbred line in the world and generally considered to be the ideal biomedical laboratory animal,which have extensive applied prospects in biological and medical fields.However,in nearly 35 years of continuous highly inbred,we found some individuals are hardly reproducible because of their impaired fertility which lead many substrain to end.This problem has been an obstacle to expand the numbers of groups,which must be solved.Mammalian spermatogenesis is a long and complex process in which spermatozoa are produced from male primordial germ cells by way of mitosis and meiosis,which is also a highly coordinated process controlled by multiple gene.Currently,the mechanism of BMI male infertile is not clear and the study on BMI spermatogenesis area is blank in China.Thus,identification of the spermatogenesis related genes,understanding the genetic causes of BMI male sterility and elucidating the pathogenesis are essential to the breeding of BMI,These studies will provide a breeding guide for BMI reproduction on molecular level and basic data on research of human male infertility.It has important theoretical significance and application prospects.This study choosed gene family associated with mammalian male infertile based on the literature at home and abroad as candidate gene,obtaind gene CDS sequence using PCR and classical 5'RACE techniques.These functional gene families included testis-specific serine/threonine kinase family(TSSK1-4 and TSSK6),Septin family(Septin1-12 and Septin14),Deleted in azoospermia family(DAZL and BOLL),DEAD-box protein family(DDX25 and DDX3Y).Then we further study on sequence features,characteristics of tissue expressions,subcellular localization and the expression regulation of DDX3Y gene by RNAi and overexpression technologies,which would help to understand the molecular and regulation mechanism of BMI spermatogensis.The main reasearsh results are,as follows:1.Gene cloning,bioinformatic and mRNA tissue expression profile analysis We obtained the total 22 gene cDNA sequences of BMI TSSK family,Septin family,DAZL family and DEAD-box protein family,which have been submitted to Genbank database for certification.In particular,comparison with human and cattle BOLL gene,found in the translation initiation codon ATG,BMI BOLL gene is AAG.The T?A mutation made BMI BOLL gene lost the start codon and not normally translated into protein,suggesting that this gene has lost expression activity,may be a pseudogene.We found similar sitiation in Large white,Landrace and black pigs,speculated that the BOLL gene start codon deletion of the porcine may be universal,but further confirmed.Other 21 BMI gene amino acids were significantly homologous to human according to homologous alignment which were 77%and above,highly conservative.Sequence similarity results suggest that the fuction of these genes is similar to that of the species.We searched in the SMART database for possible motif of BMI 21 proteins and found that TSSK family contains the S_TKc catalysis doamin,Septin family contains the Septin/CDC_Septin function domain,most in the near C end has coiled coil structure;DAZL contains the RRM domain and the DAZ repetition;DDX25 and DDX3Y contain the DEXDc and HELICc domain.Domain is a functional unit that can exist independently in protein molecules.The existence of domains suggests that these genes are functional genes and participate in the life activities of BMI.Molecular phylogenetic tree was constructed based on the homology comparison of multi species amino acid sequences of TSSK and Septin,results showed TSSK family may divide into 3 groups(A,B and C):Group A contains TSSK1 and TSSK2,Group B contains TSSK3 and TSSK6,Group C contains TSSK4;Septin family also may divide into 3 groups(A,B and C),Group A contains Septin6,8,10,11 and Septin14,Group B contains Septinl,2,4,5 and Septin7,Group C contains Septin3,9 and Septin12.Members in the same group has a higher similarity in the structure,and thus has a more similar function.RT-PCR results indicate that above 22 genes were differentially expressed in BMI 34 tissues,14 genes(TSSK6,Septin1-11,DDX25 and DDX3Y)have a wide expression pattern in normal tissues.However,TSSK2,BOLL and DAZL were testis-specific expression genes;TSSK1,TSSK3,TSSK4,Septin12 and Septin14 gene were testis and the seminal vesicle specific expression genes.Testis and seminal vesicle gland are important reproductive organs of male mammal animals.The specific expression of these two tissues suggests that these genes play an important function in the development of BMI.The next step is that using testis specific or highly expressed genes as key candidate genes,in-depth study of its function.2.Development and protein expression profile and promoter analysis The expression of five genes(TSSK2,Septin12,Septin14,DAZL and BOLL)were significantly higher(p<0.01)on Mother2.5 than those on Mother0.There was no significantly difference between the expression on 5-18 months of age,on 36 months were significantly decline(P<0.01),maintain a low level.The expression of the emerged from Mother2.5 when the sperm cells appeared on Mother2.5 testis tissue and cauda epididymis,which showed that the expression of these genes were consistent with the occurrence of boar spermatogensis and indicates that these genes did participate in sperm BMI.To confirm its protein expression,we prepared TSSK2 and DDX3Y prokaryotic protein and immunized the mouse with it.After that,we got the specific antiserum for TSSK2 and DDX3Y.The western blot result has a similar tendency with the RT-PCR result in several tissues,TSSK2,DAZL and DDX3Y protein specifically expressed in testis,Septin12 and 14 protein specifically expressed in testis and seminal vesicle.In addition,using PCR amplification and cloning,we obtained the 5'UTR sequences of BMI TSSK2?Septin12?Septin14?DAZL and DDX3Y genes,found that TSSK2,DAZL and DDX3Y 5'UTR region exist a CpG island in the core region of the promoter,which provides new ideas and experimental basis for the further study of the mechanism of expression and regulation of these genes.3.Subcellular localization analysis and preliminary study on the regulation of DDX3Y gene expressionWe also constructed the pEGFP-C1-TSSK2/Septin12/Septin14/DAZL/DDX3Y plasmid to observe the subcellular localization of the fusion protein.Resluts showed TSSK2 was located in mitochondria and nucleus,mainly in the nucleus;Septin12 and Septin14 were located in mitochondrial surrounding the nucleus,DAZL and DDX3Y were located in the nucleus;which is consistent with the bioinformatic prediction results.Each organelle contains a specific set of proteins,having a specific function,subcellular localization results provide important clues to clarify the biological function of these gene.Using RNAi and overexpression technologies,we preliminary explorated the expression and regulation of DDX3Y gene in swine testicular cell lines.Compared with the control,the expression level of DDX3Y mRNA was significantly increased in overexpression group,and was significantly reduced in interference group,which laid the foundation for further research.