Laiwu Pigs Liver Cdna Library Construction And Identification

Laiwu pig (Sus scrofa) is one of excellent local pig breeds in China, which is well known for its good germplasm properties:early sexual maturation, high prolificacy, good stress resistance and fine meat quality, etc.The cDNA library construction of Laiwu pig is essential not only for its preservation of genetic resources,but for the cloning and expressing of functionally known genes as well as the screening and studying of new genes.A plasmid cDNA library and a X phage cDNA library from liver tissue of Laiwu pig were respectively constructed, and small-scale EST sequencing and bioinformatics analyzing were conducted.The accomplishment of the cDNA libraries provided an essential resource for study of the function-known genes and exploration of new genes in Laiwu pig.The main results were as follows:1.A total RNA was extracted from liver tissue of Laiwu pig by using a modified one-step acid guanidinium thiocyanate method. The first-strand cDNA was synthesized through reverse transcription using PowerscriptTM reverse transcriptase and the long-distance PCR (LD-PCR) assay was used to amplify double-strand cDNA based on the SMART (Switching Mechanism At 5'end of RNA Transcript) technique. After digestion by proteinase K and size fractionation, the cDNA fragments longer than 500 bp were ligated to pMD18-T vector. The ligation mixture was transformed into the competent cells of E. coli DH5a to complete the construction of the unamplified cDNA library.Single clones were picked randomly from the library for screening the size of cDNA inserts through double digestion with HindⅢand EcoR I.By evaluation, the titer of unamplified plasmid cDNA library from porcine liver tissue was estimated as 2.8×105cfu/mL with the percentage of recombinant clones about 98%.The insert sizes ranged from 0.5 kb to 2 kb with the average length of 1.0 kb.2. Using the total RNA separated from liver tissue of Laiwu pig as template, the first-strand cDNA was synthesized through reverse transcription using SMARTScribeTM MMLV reverse transcriptase, and LD-PCR assay was used to amplify double-strand cDNA based on the SMART technique.The PCR products were digested by proteinase K.After digestion by Sfi I and size fractionation using CHROMA SPIN-400 columns, the cDNA fragments longer than 500 bp were ligated toλTriplEx2 vector. Thenλphage packaging reaction and library amplification were performed.The qualities of original and amplified cDNA libraries were tested strictly. Single plaques were randomly picked from the library with the purpose of screening the size of cDNA inserts using PCR technique. By evaluation, the titer of unamplifiedλphage cDNA library from porcine liver tissue was estimated as 1.47×106 pfu/mL with the percentage of recombinant clones larger than 98%, and that of the amplified library was 5.8×109 pfu/mL.PCR results showed that the insert sizes varied from 0.75 kb to 2.5 kb with the average length of 1.2 kb.3.λTriplEx2 clones were converted to pTriplEx2 plasmid using E. coli BM25.8,then randomly selected clones of the BM25.8 plasmids were sequenced from 5'end. The obtained four expressed sequence tags (EST) were identified by BLAST searches against NCBI non-redundant nucleotide databases as well as non-redundant protein databases;by bioinformatics analysis these four ESTs showed significant homology to sequences already present in the databases. After detecting the open reading frames and putative conserved domains in these four ESTs corresponding known genes, the functions of translated protein sequences of these ESTs were predicted.The results indicted that the longest ORF of clone LW2 was 171 bp and encoded 56 amino acid residues, after analyzing by using the BLASTp program, the amino acids sequence proved to contain Sushi-2 conserved domain which was capable of binding lipopolysaccharide (LPS);the longest ORF of clone LW9 was 333 bp and encoded 110 amino acid residues with a molecular weight of 12.2 kD,the amino acids sequence contained cytochrome P450 conserved domain which was responsible for secondary metabolites biosynthesis,transport and catabolism;the longest ORF of clone LW11 was 831 bp and encoded 276 amino acid residues with a molecular weight of 30.7 kD,the amino acids sequence proved to contain albumin conserved domain which was related to transport; the longest ORF of clone LW12 was 435 bp and encoded 144 amino acid residues with a molecular weight of 16 kD,the amino acids sequence contained fibrinogen-related domain (FReD) which was involved in blood clotting.These ESTs'functions reflect the significant importance of porcine liver as "metabolization centrum" of the whole organism;it lays foundation for further large-scale ESTs sequencing and analyzing.