Cloning, Sequence Analysis And Expression Of Yak(Bos Mutus)SPAG11, TLRs And NODs Gene In The Male Reproductive Organs

SPAG11(Sperm associated Antigen11, SPAG11) gene,which exhibited antimicrobial properties of Beta-defensins and had unique reproductive function, such as promoting the mature sperm,improving sperm motility,promoting the capacitation and passing through the transparent tape and so on,mainly expressed in the male reproductive system.TLRs and NODs belong to PRRs, which can recognize the corresponding PAMPs specifically, initiating the early immune response in view of the invasion of pathogens and inducing acquired immune response by signal transduction. This study revealed the reproduction and immune function of SPAG11, TLRs and NODs in reproductive system of Yak, by means of cloning SPAG11、TLRs、NOD1and NOD2gene and analyzing their expression specificity.1.SPAG11gene(SPAG11C、D、E、U、V and W) sequence was amplified by RT-PCR from caput epididymidis of Yak.Inserting target fragment into pMD19-T simple vector, which indicating building recombinant cloning plasmid pMD19-SPAG11succesfully by PCR identification and gene sequencing. To analyze SPAG11gene sequence using on-line BLAST and software, of which SPAG11C、D and V had3、1and1nucleotide mutation respectively, and leading to amino acid mutations; The similarity of cloned SPAG11sepuence and Bos taurus SPAG11gene sepuence was high,with other mammals SPAG11genes,Bos taurus Beta-defensins, TAP and LAP was low, so genetic relationship was far.SPAG11protein were positive charge, had stronger cationic characteristics, and was rich in arginine and leucine. Its N-terminal contained signal peptide consists of19to27aa. SPAG11protein consisted of extracellular area (laa-3aa), transmembrane region(4aa-26aa) and intracellular area (27-aa), it was hydrophilic protein. SPAG11C, SPAG11V and SPAG11W protein had N-glycosylation site, but SPAG11D and SPAG11E protein had O-glycosylation site and all the SPAG11protein had phosphorylation site.2.To analyze the expression of the mRNA transcribed from SPAG11gene of reproductive tissues taken from Yak, semi-quantitation RT-PCR and mmunohistochemistry were used and they showed that SPAG11C、U、V and W were lower abundance in testis and epididymis, but SPAG11D and SPAG11E were readily observed in testis and epididymis; SPAG11C and SPAG11U were marginally detected in spermaduct but SPAG11E was abundant; The expression of SPAG11D was relatively low in ovary; SPAG11mRNA was not detected in other tissues. The result of immunohistochemical staining indicated that SPAG11E protein expressed in testicular sperm cells, immature sperm head and the inner wall of the epididymis sperm head. It turned out that SPAG11gene expression with species and tissue specificity.3. Semi-quantitation RT-PCR was used for analyzing the expression of the mRNA transcribed from TLRs gene of reproductive tissues taken from Yak, suggesting that TLR1, TLR3-TLR9were readily observed in testis、epididymis、spermaduct and penis, TLR2were abundant in testis, caput and corpus epididymidis, spermaduct and penis, but with relatively lower abundance in cauda epididymidis and spermaduct; TLR10was marginally detected in testis, caput and corpus epididymidis, penis, but not the cauda epididymidis and spermaduct.4.To analyze the expression of NOD1and NOD2gene mRNA in male reproductive tissues of Yak by semi-quantitation RT-PCR, which showed that NOD1was low in the testis, caput epididymidis, corpus epididymidis, cauda epididymidis, spermaduct and penis, but NOD2was abundant. The results demonstrated that NODs of yak had differential expression in male reproductive tissues.