The Study Of Genetic Variation,Alternative Splicing And Dna Methylation In Promoter Region Of PSMC Gene Family

Qinchuan cattle is an outstanding local cattle breed of China, with the implement of the of Qinchuan beef cattle improvement programmes. The growth and carcass traits are important economic traits of Qinchuan beef cattle, they are the quantitive traits which controlled by multiple genes too. In this study, we chosed Qinchuan beef cattle as the research object and selected six PSMC family gene as the candidate gene, using DNA pool sequencing,PCR-SSCP, PCR-RFLP, CRS-PCR(the methods of SNP detection) and RT-PCR, QPCR, BSP,TA clone etc modern molecular biology experimental techniques, we did researches on genetic variation,detection of alternative splicing, mRNA expression and DNA methylation in promoter region of PSMC family gene.The main results are as follows:1.The detection of genetic variation in Qinchuan cattle PSMC gene family and thier association with growth and carcass traits: we found 15 mutations in five PSMC family genes and verified 8 SNPs which located on PSMC1 gene exon 9 PSMC1-C69T), PSMC2 gene intron 6 and 8(PSMC2-A131 G and PSMC2-C265G), PSMC3 gene exon 1, intron 10 and 12(PSMC3-A218, PSMC3-T4363 C and PSMC3-G4649 A the missense mutation), PSMC4 gene exon 5(PSMC4-C106G) and PSMC gene intron 4(PSMC5-G369T).The results of association analysis showed that the SNP PSMC1-C69 T, PSMC4-C106 G significantly associated with body length and hip width(P<0.01 or P<0.05), SNP PSMC2-C265 G 、 PSMC3-A218 significantly associated with slaughter weight and carcass weight(P<0.05);in locus PSMC3-T4363,the different genotypes are associated significantly with body lengthand hip width(P<0.05), the PSMC3-G4649 A locus associated significantly with body height(P<0.05),but in PSMC5-G369 T locus, there was not significant differnce between differnet genotypes(P>0.05).2.A new mRNA transcript AS2 of Qinchuan cattle PSMC5 gene was detected, it belongs to skipping exon type which missing128 amino acid. The new transcript expressed extensively in spleen, abdominal muscle, longissimus dorsi, abdominal adipose, front leg fat of adult Qinchuan cattle and abdominal muscle, front leg fat, back fat of young Qinchuan cattle. Due to the differences in age and tissues, the existence of conditions of alternativesplicing is different.3.We detected the expression of 6 PSMC family genes in 15 tissues of Qinchuan adult cattle. It showed that PSMC1 highly expressed in hind leg fat, PSMC2 got high exprssion in abdominal fat and back fat, PSMC3 got high expression in lung and 4 muscle tissues, PSMC4 highly expressed in abdominal muscle and front leg muscle, PSMC5 showed high expression in abdominal adipose and 4 muscle tissues, PSMC6 got high expression in spleen, abdominal muscle and abdominal adipose. As for young Qinchuan cattle, we tested the expression of PSMC2 in abdominal muscle, front leg muscle, longissimus dorsi, abdominal adipose, front leg muscle and back fat. The expression of PSMC2 in abdominal muscle, abdominal adipose and front leg muscle got the top three position, but the expression in front leg fat was the lowest.4.We screened the DNA methylation level in PSMC2 gene promoter region of longissimus dorsi and back fat in Qinchuan adult cattle and young cattle. The results showed there was a higher DNA methylation level in adult cattle group than young group, but the expreesion of PSMC2 in adult cattle group was lower than young group.The results suggested that the important molecular genetic markers of breeding, new transcript were detected of PSMC family gene, the DNA methylation level were various in diffent age and tissues group. These results could be used as a reference of marker assistant breeding program and provide the scientific theory basis for breeding and improvement of Qinchuan cattle. Meanwhile, they expands the knowledge and understanding of PSMC family gene and palys an important role on illuminating the molecular mechanism of growth and development related to genetic traits and expression regulation in Qinchuan cattle.