The Studies On Growth Related Genes In Tibet Mini-pigs

Animal growth is a complex physiological process,affected by many factors in vivo and in vitro.Including the animal species and gender,nutrition level,living environment,maternal effect etc..Many factors play important role by ultimately integration and regulation of neuroendocrine.And the key of regulation is nerve endocrine growth axis,which is consisted of growth hormone releasing hormone,growth hormone and insulin like growth factor.The neuro endocrine growth axis including growth hormone releasing hormone,somatostatin,growth hormone,insulin like growth factor,hormone receptors and binding proteins.Mammals are stimulated by many factors from internal and external,hypothalamus begin to secrete growth hormone releasing hormone and somatostatin,growth hormone secretion is regulated by both growth hormone releasing hormone and somatostatin.After the combination of growth hormone and growth hormone binding protein,transported to the organs by blood circulation,combined with target organ of the growth hormone receptor.The process initiate the transduction of intracellular signal,promote the expression of insulin like growth factor-1.Insulin like growth factor-1 release into the blood after synthesized by the liver,conbinated with insulin like growth factor binding protein,and then transported to variety of tissues in vivo,promoting protein synthesis,promoting cell proliferation.The combination mainly promoting growth of skeletal muscle of mammals.Growth hormone also play important role in extrahepatic tissues,the expression of insulin-like growth factor by autocrine or paracrine.Growth hormone play growth promoting effect,was mediated by insulin like growth factor-1,The increasing of the liver of insulin like growth factor-1 secretion along with the increases of growth hormone in blood;when the blood insulin like growth factor-1 concentration exceeds a certain concentration,it will inhibit the expression of growth hormone of pituitary,promote the secretion of somatostatin in the hypothalamus.Miniature pig,belong to the class of Mammalia,Artiodactyla,not Pecora,Suidae,pig animal.Because of the swine and human is very similar in anatomy,physiology.In 1700,the British JohnAuther had proposed the swine can be the study model of human circulatory system.But the pig's oversize?The body weight of some adult swine in 400kg above?,it is very inconvenient.In American,small pig breeding suitable for experimental were bred in early 1950s.The same as the porker in anatomy,physiology,but smaller,more convenient for doing scientific experiment.The widespread application of these miniature pig in the biomedical field,especially in the study of cardiovascular system disease,digestive system disease,endocrine system diseases,skin diseases,as well as xenotransplantation,medical devices for the detection and drug dynamic metabolism research,miniature pig is the value of experimental animal.And it can be used for the experiment of Dental Medicine.The resources of miniature pigs are rich and unique in our country,and are closed colony as long-term inbreeding.They have the characteristics of small size,genetic stability,have obvious advantages compared with foreign varieties of hybrid swine.The resources of The miniature pigs as the base,by blocking breeding,not only saves precious varieties,and the genetic and phenotypic characteristics of varieties is more stable,more in accordance with the requirements of science research,the purposes of conservation and breeding.In China,we have these miniature pig breeds:Tibet min-pig,Wuzhishan miniature pig,Banna miniature pig,Guizhou miniature pig,Guangxi Bama miniature pig and Diannan small ear pig.The study of the growth related genes axis of Wuzhishan miniature pig,Banna miniature pig and Guangxi Bama miniature pig have been done,but few studies on Tibet minipig.Tibet mini-pig,comes from the Qinghai Tibet Plateau.The altitude of 2500-4300 meters farming and farming and pastoral areas,is the only one who can adapt to high altitude climate.The closed geographical environment makes Tibet mini-pig kept very pure breed resources.The research group imported Tibet mini-pig to Guangzhou from Tibet in 2004,the current study on acclimatization and experimental animal has been completed,and carried out the research of related animal model,drug experiment and transgenic cloning.Base on the study of immunology,genetics and its unique shape,Tibet mini-pig breed is excellent experimental animal.The adult weight of Tibet mini-pig is only 25-40kg,15-20%of normal porker body weight.This experiment is the growth related GH,GHR and IGF-1 gene of Tibet miniature pig,it can also provide theoretical basis for molecular breeding of small size Tibet mini-pig.In this studies:?1?We found the SNP sites of GH,GHR gene by the method of PCR products sequencing,and analized the relationship of different genotype and some growth trait.Results:Five single nucleotide polymorphisms?A12G,T45C,G84A,G93A,C133T?were found in exon 2 of GH gene.There was no single nucleotide polymorphisms found in exon 9 of GHR gene.Statistical results show that Tibet-minipig withTC?AA?GG genotype at T45C?G84A?G93A of GH gene maybe related to the smaller body size.?2?The cloned GHR,IGF-1 gene cDNA sequence of Tibet miniature pig.The results showed that:Tibet mini-pig GHR gene fragment was 1912bp,containing the coding region was 1721bp,encoding 572 amino acids,99%homology were compare with Banna miniature pig?JF276446.1?and Wuzhishan miniature pig?DQ422962.1?.Compare with pig?NM₂14254.2?,we found that the mutation of T G at 1225pb,the mutation loci cause changes of amino acids encoded serine?alanine;compare with Wuzhishan mini-pig,the results showed that in 1248bp,1287bp were G?A mutated,but the site did not cause a corresponding change in amino acids;compared with Banna miniature pig,T?C mutation in 1656bp,the site also did not cause the corresponding changes of amino acids.Tibet mini-pig IGF-1 gene fragment containing the coding region of 567bp,the coding region encodes 186 amino acids,99%homology were compare with the pig?NM₂14256.1?.Compare with pig?NM₂14256.1?found that the mutation in the 440bp,455pb,occurred at G?A,C?T mutation,and the corresponding amino acids changed,histidine?arginine,leucine?serine respectively.The mutation of the site changed the protein function or not need to be further studied.?3?Real-time PCR was used to analyze GH,GHR and IGF-1 gene expression of Tibet mini-pig heart,liver,spleen,lung,kidney,muscle,skin tissue in 0 years old?1 days old?,0.1 years?36 days?,0.25 years?90 days?,0.5 years?180 days?,1 years?360 days?,2 years?720 days?,3 years?1080 days old?.The results showed that:three genes expressed in 0,0.1,0.25,0.5,1,2,3,of all ages,and heart,liver,spleen,lung,kidney,muscle,skin of various organs.The GH gene,the highest expression of the skin and the lowest expression of muscle at the age of 0,0.5;the highest expression of lung tissue and the lowest expression of muscle tissue at the age of 0.25,1;the highest expression of liver at the age of 0.1,3.The GHR gene,the highest expression in lung and the lowest in muscle at the age of 1,2;the lowest expression in muscle tissue at the age of 0.25,0.5,3;the lowest expression in the heart at the age of 0,0.1;the highest expression level in the kidney tissue at the age of 0,0.25.The IGF-1 gene,the highest expression in skin and the lowest expression in muscle tissue at the age of 0,0.25,0.5,the highest expression in liver at the age of 0.1,2,3,the lowest expression in muscle tissue at the age of 1,2.In different age period,GH and GHR genes and reached the peak at the age of 0.1,while the IGF-1 gene and reached the peak at the age of 0.25.The expression of Tibet mini-pig growth axis related genes showed a temporal and spatial specificity obviously.?4?The prokaryotic expression vector of Tibet mini-pig's GHR gene was constructed,named GHR-pIRES2-EGFP,was transfected into Tibet mini-pig's fetal fibroblasts.The relative quantitative RT-PCR method to detect the expression of the IGF-1 gene.The results showed that:IGF-1 mRNA levels in the cells transfected were upregulated.