Isolation, Expression And Function Of Porcine Suv39h2, G9α And LSD1 Gene

Epigenetic regulation is a major aspect of gene control by which heritable changes in gene expression occur without an alteration in DNA sequence, which mainly includes histone code, DNA methylation, RNA interference and genomic imprinting.The study of epigentics in porcine lagged behind. In this study, Suv39h2, G9a and LSD1 gene were selected to be as candidate genes which encoding histone modification enzymes. They laid important basis on further study of G9a,Suv39h2 and LSD1 gene on the impacting of pig production and development, trait formation or regulation through isolating, cloning, expression pattern analysis, prokaryotic cloning, polymorphism identification and association with production traits. The main results are as follows:1. The full-length sequences of coding region of the porcine Suv39h2, G9a and LSD1 (including two transcript variants) gene were cloned using the pig specific primers which were designed from the pig EST contig obtained by Blast according to the corresponding human cDNA sequence from NCBI. They have been deposited in GenBank database and the GenBank accession number were EU219913, EU219914, EU219915(transcript variant 1), EU219916(transcript variant 2), respectively. There function and character also predicted by bioinformatic sofeware.2. Gene tissue distribution of Suv39h2, G9a and LSD1 were analyzed by the semi-quantitative RT-PCR in 11 different tissues of heart, liver, spleen, lung, kidney, endometrium, overy, muscle, adipose, stomach and small intestine from mature Meishan Pigs. The results of RT-PCR displayed that three genes were expressed in all eleven detected tissues. In the tissues of muscle, overy and spleen, Suv39h2 gene expressed relatively higher; in the tissues of overy, kidney and spleen, LSD1 gene expressed relatively higher, moreover the two transcript variants of LSD1 gene expressed at the same time.3. Using semi-quantitative RT-PCR, Suv39h2, G9a and LSD1 gene expression pattern in the Meishan pig skeletal muscle of 3dp (day postnatal), 35dp, 60dp, 120dp and 180dp were detected .The expression trend of three genes were similar: they expressed higher in the growth nonage. G9a gene expressed highest on 35dp, but Suv39h2 and LSD1 gene expressed highest on 3dp, furthermore the two transcript variants of LSD1 gene expressed at the same time.4. Recombinant prokaryotic expression vector pGEX-KG-Suv39h2 and pGEX-KG-G9a were constructed and their expressive products were successfully detected by SDS-PAGE and Western blotting. 5. The DNA sequence of the porcine G9a gene was cloned by the method of bioinformatics and comparative genomics according to the corresponding human cDNA and DNA sequence. It has been deposited in GenBank database and the GenBank accession number was EU709015. Screening of SNP of the porcine G9a gene in Large white and Meishan, 41 SNP were obtained. Of 7 are transversion mutations, 32 are transition mutations, 2 are insertion/deletion mutation.6. PCR-RFLP and PCR-PAGE were employed to detect a base pair mutation in the intron 23, intron 25 and 3' untranslated region of the G9a gene respectively. The polymorphism for the mutations were analyzed by PCR-RFLP and PCR-PAGE among the different pig groups, and allele frequencies and the allele distribution were determined among these populations.7. The polymorphism for the mutations of G9a gene were analyzed in Large White×Meishan F2 population. The results showed: the mutation in the intron 23 is significant association with dress percentage (p<0.05), it is also significant association with skin percentage, bone percentage, fat percentage, ration of lean meat versus fat meat, carcass length to 1st spondyle, carcass length to 1st rib, shoulder fat thickness, 6-7 rib fat thicknesses, thorax-waist fat thickness, buttock fat thickness and average backfat thickness (p<0.01). The mutation in the intron 25 is significant association with skin percentage, dress percentage, internal fat rate, pH(BF) and pH(SC) (p<0.05). The mutation in the 3' untranslated region is significant association with shoulder fat thickness, thorax-waist fat thickness, average backfat thickness, pH(BF) and intramuscular fat (p<0.05), it is also significant with skin percentage and 6-7 rib fat thicknesses (p<0.01).