| Laiwu pig is one of our local pig varieties for five thousand years. It has the characters of high reproductive capacity, disease-resistance, good meat quality, resistance to coarse grain, heterosisr. A serial of the porcine products have been developed including popular cold fresh meat, roast meat, sausage.Laiwu pig has formed a good brand effect in our country. It is hopeful of its having a big market and a good future. Therefore, it is a very important value in theory and practice to develop and protect this porcine rare genetic resource.Total RNA was extracted form the heart tissue of Laiwu pig using improved isothiocyanate method. The total RNA was reverse-transcribed into single-strand cDNA by SMART (the Switch Mechanism At the 5'end of RNA Templates) technique. Long distance PCR (LD-PCR) was then carried out to synthesize the double-strand cDNA that were digested by Sfi I, and the digested fragments were fractionated by CHROMA SPIN-400. Then cDNA fragments longer than 0.5 kb were collected and ligated toλTriplEx2 vector. Ligation products were packed in vitro intoλphage particulates in order to gain unamplified cDNA library. The titer of primary cDNA library was 2.0×105 pfu/ml checked strictly by conventional titer determination and the rate of recombinant was about 94.4%. The titer of the amplified library was 3.0×108 pfu/ml. The recombinantλTriplEx2 clones picked from the amplified library infected E.coli BM25.8 to convert into pTriplEx2 plasmids. Plaques picked randomly from transformed pTriplEx2 plasmids were tested using PCR with universal primers derived from the sequence flanking the vector. The length of the most inserted fragments was above 0.75 kb.The recombinant pTriplEx2 plasmids carried the inserts more than 500 bp were picked from the transformed plates with E.coli BM25.8 and sequenced from 5' end and 3'end. Eight pieces of effective ESTs were obtained totally between 244 and 711 bp in length. The putative open reading frames (ORFs) of these 8 ESTs were analyzed through Open Reading Frame Finder in NCBI and by using the BLASTp program, and the functions of amino acid sequences translated from these ORF of the eight ESTs were predicted. The results indicated that the longest ORF of LW13,LW15,LW16 and LW17 clones were not complete, and their amino acid sequences translated from their ESTs had unmatched with the protein sequences in database, they might be one part of some new genes;the longest ORF of LW19 clone was was 609 bp in length and encoded a 202 amino acid residues,22.4 kD in molecular weight. The amino acid sequences had a matching protein sequence in database, its containing thioredoxin-like superfamily conserved domain which was related with oxidation-reduction levels in vivo. The longest ORF of LW20 clone was 198 bp in length and encoded 65 amino acid residues. The longest ORF of LW22 clone was 105 bp in length and encoded only 34 amino acid residues. The longest ORF of LW24 clone was not complete. Three clones of LW20, LW22 and LW24 had unmatched protein sequences in database, they might be representative of one part of a unknown gene.By combined alignment of BLASTx and BLASTn programs in NCBI, it was shown that the amino acid sequences encoded by two ESTs of the clones LW19 and LW22 functioned in foundmental researches of cell differentiation, cell cycle control, cell aging and death, and body immunity, and might play a very important role in the expression mechanism of some favourite characters in Laiwu pig. These also laid some foundations for further research.The cDNA library from the heart tissue of Laiwu pig constructed by SMART technique might be helpful to preservation of local rare porcine resources, but also provided a valuable clues for cloning and expression of functional genes and for screening of unknown genes. |
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