Expression, Alternative Splicing Variants And Promoter Methylation Status Of Dead-Box Family In Yak And Cattle-Yak

DEAD-box protein family is an ATP-dependent RNA helicase family which was found involved in a variety of RNA metabolic processes and widely existed in most organisms including bacteria of prokaryotes and yeast、plants and animals of eukaryotes DDX4、 DDX3y and DDX25are the members of DEAD-box family which were suggest related to spermatogenesis.The deletion of DDX4、DDX3y and DDX25in most animals as mouse、 human and bovine leads to different forms of spermatogenetic malfunction.The male infertility such as spermatogenetic malfunction always accompanied with deletion of DDX4、DDX3y and DDX25.we uesed cattle-yak as model of male infertility.to study the relationship between these two genes and spermatogenetic malfunction,The mRNA expression level of DEAD-box family genes(DDX4、DDX3y and DDX25) were detected by real-time PCR;The coding region of DDX4gene of yak and cattle-yak was cloned and the structure、evolution and phylogenetic were analyzed by bioinformatics method;Alternative splicing variants of DDX4were screened by gene clone and the mRNA expression level of each splicing variant were detected by real-time PCR;The5’flanking region of DDX4gene of yak and cattle-yak was exemplified and the methylation statute of promoter region was detected by bisulfite sequence PCR.The result was hoped to explore relationship of DDX4gene and male infertility.Results as below:1. gene expression analysis of DDX、DDX3y and DDX25in yak and cattle-yakDDX4and DDX25gene expression level in testis of cattle-yak with character of male sterilize is significantly lower (P<0.01) than that in yak with normal reproductive capability. The expression pattern was nearly same as in other mammals with similar phenotype of meiotic arrest in human and mouse, suggesting that DDX4gene play an important role in spermatogenesis and male sterility of cattle-yak.The expression level of DDX3y gene in cattle-yak testis is significantly higher than that in cattle testis (P<0.01).On account of different transcripts found in human,mouse and bovine,we conjecture that the high expression of DDX3y gene in cattle-yak was result of interaction between each transcription and feedback regulation between mRNA and protein.2. Gene clone and bioinformatics analysis of DDX4in yak and cattle-yakThe translation region of DDX4gene were2190bp both in yak and cattle-yak and the nucleotide sequence was high homology with those reported in other mammalians.The sequences identity value of DDX4in yak and cattle-yak reached99.95%with only one transition of T to C at1202bp,which lead to an amino acid mutation of Ile to The DDX4was located at chromosome20in bovine by chromosomal location and consisted17exons and16introns.The start code located in the second exon.The result of phylogenetic tree of mammalian DDX4is consisitent with the classical systematics. Protein DDX4consists729amino acid residues and contains all the conserved motifs(Q、Ⅲ、Ⅴ、GG and so on)and classical structural domains(DEADc、DEXDc) of DEAD-box family,suggesting that DDX4gene of yak and cattle-yak exercises the similar biological function of other mammals and participates in the process of meiosis and spermatogenesis3. clone and expression analysis of alternative splicing variants for DDX4gene in yak and cattle-yakWe found three different transcriptions of DDX4gene in yak and cattle-yak testis.One is whole long sequence DDX4gene and another two were alternative splicing variants named as DDX4svl and DDX4sv2.Compared with normal DDX4genesequence,DDX4sv1loss whole exon4and part3’end of exon5(72bp) corresponding50amino acid and DDX4sv2loss whole exon4(78bp) corresponding26amino acid.Splicing of DDX4svl and DDX4sv2both followed the "GT-AG" splicing rule and the absence part both located N terminal beyond the consistent region. DDX4svl and DDX4sv2contain all the consistent domains and foundional motifs,suggesting that DDX4sv1and DDX4sv2still remain the foundational biological function of DDX4protein.The expression pattern of the three transcriptions is DDX4sv2>DDX4>DDX4sv1in testis of yak(P>0.05);The comparison of each trancripion respectively in yak and cattle showed that the expression of DDX4in Yak testis is significant higher than that in cattle-yak(0.01<P<0.05),expression level of transcription DDX4svl and DDX4sv2are both great significant higher in Yak testis than that in cattle-yak(P<0.01).The results infers that presence of DDX4sv2and DDX4svl is an arranged biological process and participate in the process of meiosis and spermatogenesis of male bovine together with whole long DDX4 gene.4. methylation level of DDX4gene promoter analysis in yak and cattle-yakWe cloned1369bp of the5’flanking region sequence of DDX4in yak and cattle-yak and predicted the promoter region and CpG island.The result showed that the core promoter region is251bp located-394bp～-134bp(A in start codeon as+1)containing methylation sensitive of SP1sites,and CpG island is345bp located-268bp～12bp containing20CpG sites.The methylated level of DDX4promoter region is67.0%(134/200) in cattle-yak testis which is greatly significant higher (P<0.01) than compare to86.5%(173/200) in yak testis,which is consistent with the low expression of DDX4in cattle-yak.No significantly (P>0.05) difference of single CpG site was observed between cattle and cattle-yak. We conjectured that the DNA methylation of DDX4promoter plays an important role in regulation of DDX4mRNA,meiosis process and male-sterilize of cattle-yak.