Molecular Biological Study Of Rongchang Pig's Pulmonary Surfactant Protein A

Pulmonary surfactant protein A is a highly conserved glycoprotein and plays an important role in the maintenance of normal respiratory function, reducing susceptibility to lung and regulation of lung inflammation. So SP-A could be a good candidate gene for disease resistance in lung and will be studied broadly。In this study, the SP-A gene was amplificated and scanned for SNPs in Rongchang pig. Then, an oleic acid—acute lung injury (OA-ALJ) model population was constructed. Throughout this population, the associations of polymorphism and copy number variation (CNV) of SP-A gene with water content in lung were done as the following results:(1) A 5.2 kb fragment as the complete genomic DNA sequence of SP-A gene (including the promoter region) was cloned. Four exons and three introns were detected in this gene by aligned the mRNA sequence with the 5.2 kb fragment. The homology of SP-A gene between Rongchang pig and Large White is 99.43%. Polymorphisms then were screened throughout all the 5.2 kb fragment of the SP-A gene by PCR amplification and sequenced of a pooled DNA sample from 10 Rongchang pigs. A total of 11 SNPs (3 SNPs in exons, 7 in introns and 1 in the promoter region.) were identified.(2) According to the 9 bp insertion/deletion in exon 2, 274 Rongchang pigs were genotyped by -/-,+/- and +/+ . From chi-square testing, the population was not in Hardy-Weinberg equilibrium. However, the polymorphism information content of SP-A gene was moderate in Rongchang pigs.(3) Devided 60 Rongchang pig into two groups (including all 20 pigs of -/-,+/- and +/+), group 1 and 2, were designed by 1 hour and 3 hour after injection .In OA-ALJ population, water content of lung was measured in every individual. After OA injection, water content of test group was markedly higher than control group. In group 1, water content mean of lung for +/+ genotypes was higher than -/- and +/-genotypes (P<0.05). But there was no significant difference in water content of lung among the three genotypes in group 2. The expression level of SP-A gene in lung after OA injection was lower than control group. Although the expression level of +/+ genotype was lower than -/- and +/- genotypes in group 1, the expression levels of three genotypes did not differ in group 2. These results suggested that the correlation of the degree of lung injury and water content was positively, while, the correlation of expression level of SP-A gene in lung and lung injury was negatively. The individuals of +/+ genotype suffered more severe lung injury after OA injection than the other genotypes, so they may be less resistance to the injury.(4) Experiment on SP-A and four CNV fragments which were located on chromosome 14 of Rongchang pig and close to SP-A's location, analised data and showed some result: in this Rongchang pig population, CNV2 showed moderate copy number polymorphism, and may have 5-6 copies. CNV2 associated with lung water content, the difference was not significant.