CDNA Clone, SNPs And Tissues Expression Analysis Of 8 Genes On Cattle Reproduction Trait

Reproduction traits are an important economic trait. Calving trait, which is controlled by polygene, is a mostly factor affecting production efficiency. This trait was affected by add-apparent gene model and it has low heredity. The heredity of twinning and ovary are 0.03 and 0.07, respectively. At present, the rate of twinning is increased by hormone inducement, hormone immunity and embryo transfer, but there are some defects, such as slow choose evolve, numerous means and high cost.How to choose some genes which can improve the sperm quality and/or increase the number of calving is a goog aspect. To open out mechanism of reproduction trait and quicken the course of animal breeding, the genes were cloned about sperm quality. Therefore, 8 genes including ACTN1,Dmrt7,Mina53,Trf2,Tbxa2r,CyclinA1,GDNF and CIB1 were served as candidate genes, theses genes were cloned by RT-PCR technology and bioinformatics means. Tissue expression was employed by RT-PCR technique and real-time PCR .SNP was detected by sequencing and association between the SNPs and the number of calving.1. The complete CDS sequence of ACTN1, Dmrt7, Mina53, Trf2, Tbxa2r, CyclinA1, GDNF and CIB1 cDNA were cloned by RT-PCR, EST, Genescan and analyzed by bioinformatics methods.2. The entire 3'UTR sequence of Dmrt7 gene was cloned by 3'rapid amplified .And the CDS sequence of ACTN1, Dmrt7 and Trf2 genes were submitted to Genbank, the accession numbers were Ef512630, EF634775 and EU140625, respectively.3. The 5 candidate genes were detected in by RT-PCR and the results showed that 4 candidate genes have expression in various tissues except that Dmrt7 gene in specific expressed in testicle.4. The syntheses of the first-strand cDNA was followed by PCR using SYBR Green I was combined with dsDNA , twelve different tissues including liver, testicle, lung, rumen, womb, small intestine, spleen, heart, fat ,ovary, kidney and muscle were collected from a mature Simmental bull and cow for genes expression study of Trf2 and Mina53. SYBR Green I analysis was performed to determine the relative mRNA level of Trf2 and Mina53 in various tissues. The housekeeping geneβ-actin was used for endogenous control. Expression data demonstrate that the bovine Trf2 gene expressed in all 12 tissues of interest, it is expressed predominantly in testis, and very low rumen and spleen. Mina53 gene expressed in 12 tissues of interest, it is expressed predominantly in testis, and very low in ovary.5. Identification for mutation in the amplified fragments of Dmrt7gene was performed in intron1, exon 2 and intron2, intron4, intron5 and exon5, intron7, exon8 and intron8 area. 5 SNPs including G/A, A/T, C/G/, C/T, C/G mutation in different introns were detected by sequencing. Allele frequencies of 1 SNP that can be detected by PCR-SSCP were analyzed among different bovine population (Luxi cattle, Simmental, buffalo, Jinnan cattle and Hereford) and the results showed that the CC genotype frequencies were high in all population exception Simmental group; 3 populations were located in middle polymorphism.6. Identification for mutation in the amplified fragments of ACTN1 gene was performed in exon8 and intron9, exon9 and intron10, exon10 and intron11, exon12 and intron13, exon14 and intron15 area. 7 SNPs including G/A, A/G, G/A, G/C, A/G, G/A, A/G mutation sites in different introns were detected by sequencing. These some domestic breed including Luxi cattle, Luxi twinning cattle, Nanyang cattle, Jinnan cattle, Chinese Simmental and Holstein were used to dectect SNPs. The analysis of least square was used to employ to association between these SNPs and the number of calving by SAS 9.1 software. A/G mutation site in intron10 was detected by ApaⅠrestrition enzyme in domestic breeds. The result showed that, all population was located in low polymorphism. intron10-3124 nt was significant association between the SNPs and the number of calving(P<0.01).A/G mutation in intron13 was detected by HaeⅢrestriction enzyme in domestic breeds. The result showed that there were no association between intron 13-669 SNP and the number of calving (P>0.05).G /A mutation in intron15 was detected by RsaⅠrestriction enzyme in domestic breeds. The result showed that all population was located in middle polymorphism. There were no association between intron15 -227 SNP and the number of calving (P>0.05).7. The prokaryotic expression vector of Dmrt7 genes which was expressed only in testicle was structured using RT-PCR and pET28a+vector, and the Dmrt7 fusion protein was expressed in the BL21 strain.