Isolation, Characterization And Mapping Of Differentially Expressed EST From Porcine Skeletal Muscle In Different Breeds Or Developing Stages

Improvement of muscle growth and meat quality, quantitative traits both controlled by multigenes, is always one of the main goals for livestock genetics and breeding. Different pig breeds, especially Chinese indigenous and exotic pigs, express significant phenotypic differences on the above traits. Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. In order to basically understand the genetic factors causing these differences between Chinese indigenous and exotic pigs, mRNA differential display and cDNA macroarray techniques were used to compare the mRNAs expression profiles in the adult longissimus dorsi muscle tissue between two representative pig breeds - Duroc and Erhualian. Furthermore, to clone potential candidate gene for muscle growth, the full-length cDNA of a novel EST which differentially expressed in porcine skeletal muscle among different developing stages was obtained by in silico cloning combined with rapid amplification of cDNA end method. The main results of this study were presented as following:I . The condition of related techniques was optimized by a series of trial and error and a set of experimental silver-staining mRNA differential display was established. Five anchor primers in combination with 20 arbitrary primers resulting 1 00 primer sets were used to display the total RNA of adult longissimus dorsi muscle between these two above pig breeds. Nearly 5000 cDNAs were examined and 22 reproducible differential displayed cDNA bands were obtained, among which 4 were over-expressed in Duroc pigs and the others over-expressed in Erhualian pigs. All of these 22 bands were recovered, re-amplified, cloned and sequenced. Homologous analysis of these sequences with those available in GenBank indicated that four of 22 bands were novel EST in pig and the others were identical to porcine myosin heavy chain mRNA (MyHC), NADH dehydrogenase subunit 2 (NADH2), NADH dehydrogenase subunit 4 (NADH4), cytochrome c oxidase subunit II (Co II), 12S ribosomal RNA, 16S ribosomal RNA, ATPase subunit 6 (ATPase 6) and phosphoglycerate kinase subunit I (PGK-l) respectively. 22 bands represented 12 different cDNAs because in some cases there was several different primer sets produced EST homologous to one same porcine mRNA. Reverse Northern blot and Northern blot analysis showed that two differential display EST (porcine NADH2 and a novel EST - ESThpl) were remarkably differentially expressed between Duroc and Erhualian pigs. Semi-quantitative RT-PCR were performed to detected the expression profile of two novel EST (ESThpl and ESThp9-l) in porcine main tissue, the results revealed ESThpl were expression only in heart and skeletal muscle whereas ESThp9-l expressed in all the porcine main tissue. Finally, a novel EST (ESThp9-l)was physically mapped on Sscrl2ql.1-1.5 by somatic cell hybrid panel and linked with microsatellite S0090 with LOD score of 11.86 by radiation hybrid analysis.2. Several experimental condition of cDNA macroarray technique was selected. cDNA macroarray membrane containing 327 ESTs originated from porcine embryonic and adult skeletal muscle cDNA library was applied to screen the gene expression patterns in the longissimus dorsi muscle between Duroc and Erhualian pig. Results indicated that no significant differences of gene expression were detected in the 327 ESTs between these two pig breeds.3. The 3'-end sequence of M223 (GenBank accession number: AA063663), a novel EST that differentially expressed in different developing stages of pig, was obtained by using rapid amplification of cDNA end method. The full-length cDNA sequence was further assembled by in silico cloning and verified by laboratory bench-work. The cloned cDNA consisted of 3901bp nucleotides, including 317bp nucleotides of the 5'-untranslated region (UTR), 1650bp of the open reading frame which encoded 550 amino acid and an extraordinarily long 3'-UTR of 1902bp nucleotides followed by a poly(A)32 tail. It was partially homology to a human unnamed protein XM₀72203 an...