| Recent advance in porcine skeletal muscle profile obtained a number of differentially expressed ESTs. In a long period from now, the primary issue will be establishing the relationship between these ESTs and phenotypic difference of economic traits, and translating genomic sequences into enhanced value of the phenotypes. The first challenge is to obtain the corresponding gene based on these ESTs, study their function, and discover the mutual regulatory relationship.Based on three differentially expressed ESTs discovered in our previous transcriptome study of porcine skeletal muscle, the cloning, mapping and functional study of candidate gene had been done, and the results obtained were as follows:1. Combining bioinformatics and experiments validation, the cDNA sequences including complete CDS were obtained for porcine PPP2R5A, CSRP1 and CSRP2 respectively. The full-length cDNA sequence of porcine CSRP3 and LDB1 were also obtained based on corresponding differential expressed ESTs.2. The SCHP and RH panel were used to assign five genes mentioned above and another six PP2A-related genes, and obtained their chromosome location information as follows: PPP2CA to SSC 2q21-qter, PPP2CB to SSC 15q, PPP2R1A to SSC 6q12-q21, PPP2R1B to SSC 9p21, PPP2R5A to SSC 9q26, PPP2R5B to SSC 2pl4-pl7, PPP2R5D to SSC 7q11-q12, CSRP1 to SSC 10, CSRP2 to SSC5, CSRP3 to SSC 2p14-p17, and LDB1 to SSC14.3. QPCR assay indicated that PPP2R5A mRNA expressed widely in porcine heart, liver, spleen, lung, kidney, skeletal muscle, adipose, and small intestine, with the most abundance in skeletal muscle, followed by adipose. Furthermore, PPP2R5A mRNA shows an up-regulated trend during fetal skeletal muscle development of Tongcheng and Landrace pigs. The expression level of porcine PPP2R5A mRNA show significant difference between two pig breeds at both stages of 33-day old and 65-day old embryos.4. RT-PCR assay indicated that CSRP1 and CSRP2 mRNA distributed in all the tissues we detected, however, CSRP3 were only expressed in striated muscle. QPCR assay showed that CSRP3 was up-regulated during skeletal muscle development in Tongcheng and Landrace pigs. We deduced the amino acid sequences and constructed phylogenic tree of the CSRP family members. Combing the similarity information of the amino acid sequences and the cluster results, we deduced that the three members originated from the same ancestor. CSRP1 shows much more similarity with CSRP2 in structure and function, and they have a close evolutionary relationship, but CSRP3 is a separated member.5. We obtained 6293 bp DNA sequence of porcine CSRP3, speculated the genomic structure and analyzed the splicing sites information. We further cloned partial promoter sequence of CSRP3, which contained typical "TATA" box, "E-Box", and transcriptional factor binding sites, including MEF2, RAR-beta and HNF3-like. Thirteen potential SNPs and two possible haplotypes were discovered in CSRP3 DNA, Taqâ… PCR-RFLP assay were further conducted to detect the C466T polymorphic site, and significant difference in genotype frequency between indigenous and exotic pig breeds were discovered.6. Alternative splicing in the 5'UTR, 3'UTR and CDS of porcine LDB1 were detected. One alternative splicing in CDS produced a protein lacking 46 amino acids, which was found to be relatively high expressed in lung, followed by skeletal muscle. We successfully constructed two fused-expression vectors of pEGFP-N1 and two variants of porcine LDBl respectively, and found the dual location pattern in nucleus and cytoplasm of these two variants by observation and analyzing using Laser Scanning Confocal Microscope after transfection.7. The 2.6 kb upstream promoter of porcine LDB1 was obtained and used to construct pG13 reporter vectors. Eleven vectors were obtained and transfected into porcine IBRS-2 cell. Through dual-luciferase reporter assay, we identified three conserved regulatory region in LDB1 promoter, which were used for transcription factor binding sites prediction. SP1 and NF-KappaB were speculated to be involving in transcriptional activation, and ARP-1 and TtK in transcriptional repression.8. These unusual alternative splicing sites detected in porcine PPP2R5D and LDB1 were not accordant with the general "GT-AG" rule, but were speculated to be connected with the short repeat elements around the splicing sites.This research is the extension of our previous study in porcine skeletal muscle development, which will add new materials for porcine functional genomic research, and lay a foundation for establish relationship between these ESTs and phenotypic difference of meat production traits. |
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