CDNA Clone, SNPs And Tissues Expression Analysis Of 9 Genes On Cattle Reproduction Trait

Most members of TGF-βsuperfamily were expressed in genminal tissues and had relationship with mammal reproduction and they had spatio-temporal expression pattern in ovary especially during the follicle development and selection. The members of TGF-βsuperfamily can bind to the type-I and type-II receptors on the cells surfaces, then activate the downstream Smads proteins to transfer the TGF-βsignal. In this study, there were 8 cDNA in the TGF-βsignal transfer pathway and 1 cDNA which was specially expressed in testis to be cloned, the SNPs and tissue specific expression pattern were analyzed. This study was the base to understand TGF-βsuperfamily members and their function in cattle. The main results as following.1. The complete CDS sequences of Smad1,Smad4,Smad9,BMPRⅡ,BMPR1A,ZFP474,BMP2,BMP3 and BMP10 cDNA were cloned by RT-PCR, EST, Genescan and analyzed by bioinformatics methods.2. The entire 3'UTR of Smad1,Smad4,Smad9,BMPRⅡand BMPR1A cDNA were cloned by 3'RACE and the entire 5'UTR of Smad4,Smad9,BMPR1A and ZFP474 cDNA were cloned by 5'RACE. The CDS sequences of Smad1, Smad4, Smad9 and BMPR1A were submitted to GenBank and the accession numbers were DQ922947, DQ494856, DQ922948 and DQ922946, respectively.3. The 9 candidate genes were detected in ovary, liver, skeleton muscle, small intestine, fat, uterus, kidney, heart, lung, pancreas, testis and galactophore by RT-PCR and the results showed that 8 candidate genes have broad expression except that ZFP474 gene is specific expressed in testis.4. The Smad4,Smad9 and BMPR1A were detected in ovary, liver, skeleton muscle, small intestine, fat, uterus, kidney, heart, lung, pancreas and testis by dot-blot hybridization using single strand cDNA with p32-dCTP as probe and the results showed that the bovine Smad4 gene had the highest expression in heart, moderate expression in skeleton muscle and lung, low expression in small intestine, uterus and pancreas. Bovine Smad9 gene had the highest expression in heart, moderate expression in lung, live, ovary and testis, low expression in small intestine and uterus. Bovine BMPR1A gene had the highest expression in ovary, moderate expression in heart, low expression in small intestine, pancreas and uterus.5. there were 3 mutations, which were one"GC"insert mutation, one"T"insert mutation and one"A/T"mutation were found in 3'URT of Smad4 gene by asymmetric PCR-SSCP and sequencing. These mutations were detected in Luxi cattle, Luxi twin cattle, Simmental cattle, Sanhe cattle and Holstein cows, the results showed that the"GC"insert mutation and"T"insert mutation had very low frequency in herds. The AA genotype was dominance in all herds but BB genotype was not found. The A allele was dominance in"A/T"mutation. The Sanhe herd had low polymorphism but the other three herds had moderate polymorphism. No significant difference was found in genotype among four herds byχ2 test.6. The"GC"insert mutation was detected in 116 Luxi cattle and 75 Holstein cows by asymmetric PCR-SSCP and HhaⅠ-RFLP and got the same results. This indicated asymmetric PCR-SSCP was accurate in gene typing.7. The secondary structure of ssDNA were predicted by bioinformatics software and the results were compared with the detections by asymmetric PCR-SSCP. The advantages and disadvantages of this method was analyzed.