Screening Of The Differentially-expressed Genes Of Lung Tissue In Pigs

Now porcine respiratory disease is a serious problem to many famers, it is one of the most serious questions to the production and management and mades heavy economic loss .There are many factors can result in porcine respiratory disease ,the major factors include genetic factor, infectivity, environment and management. Since the molecular mechanisms, gene systems and expression regulation of lung tissue inflammation resistance in pig remain unclear, The aim of the research was to isolate and clone the lung tissue inflammation resistant genes to look into molecular events associated with lung tissue inflammation resistance and establish fundamental materials and techniques for further study and genetic selection.1:Firstly we screen and clone lung tissue inflammation resistant genes in large white pig, differentially expressed cDNA library of lung tissue were constructed using suppression subtractive hybridization through following protocol: poly (A) RNA were purified using Oligotex mRNA Kits (Qiagen) from lung tissue of 3 large white pig with pathological changes and 20 health large white pig as control;single- and double-stranded cDNA were synthesized from the poly(A)+ RNA using PCR-SelectTM cDNA Subtraction Kit (Clontech) and screened the ESTs of differentially-expressed genes of lung tissue in large white pigs;these ESTs were inserted into PGM-T Easy vector and transformed into E. coli TOP10 competent cells;422 positive clones were obtained;identification of the inserted cDNA fragments in subtractive library was done using PCR. The results showed that there were inserted fragments of 150-500 bp in the 21 randomly selected positive clones.2:Subsequently differentially screening was performed using the reverse northern dot-blotting method. A total of 422 clones were spotted onto MagnaProbe nylon membranes, positively charged (USA Osmonics) and hybridized with tester cDNA and driver cDNA probe which was labeled using DIG High Prime Labeling and Detection Starter Kit I (Roche). 41 strong signals clones were isolated and sequenced resulting in 34 sequences.3:A search for sequence homology in the GenBank nr and EST database by BLASTnrevealed that the distribution of EST clusters for high homology genes and high homology ESTs were 12and 22, respectively, in pig, lung tissue inflammation specific cDNAs. The high homology genes was grouped into 8 clusters based on the best known function of there protein products: signaling transduction/ communication, cell structure/mobility, transporters, apoptosis, metabolism, translation expression regulation, cell/body defence and one cluster that function is unknown or without an ontological theme.4:Roles of lung tissue inflammation resisitance of partial diferentially expreesed genes in large white pig were presumed based on references and our research results. L0C396781,NF-kB^1QC>SMM9, calgranulinB gene^ ACTA2, RPS11, RPS20were considered playing an important role in lung tissue inflammation resistance in large white pig .