Study On The Growth And Development Index And Related Gene Expression Of GHR Gene Editing Pigs

Growth hormone(GH)for animal growth,development and reproductive lactation plays a very important role in immune function,but it must be on the target organs of growth hormone receptor(GHR)combine again after the mediated signal into the can play a role in the cell.GHR is a single-stranded transmembrane protein composed of 620 amino acids and is one of the receptor superfamily members.GH activates downstream signaling pathways such as JAK-STAT PKC and IRS by binding growth hormone receptor GHR,participate in the regulation of animal growth,metabolism,reproduction and many other physiological and pathological processes.JAK2 and STAT3 are important genes in the regulation of JAK/STAT signaling pathway.They play an important role in the biological functions of signal transduction in animal growth and cell proliferation.This study choose guizhou university breeding pig breeding field of F2 generation of GHR gene editing pig as test object,through positive heterozygote pig hybrid F2 generation,and propagation get F3;acquisition F3 generation of piglet ear tissue and extract DNA,PCR amplification,through DNA sequencing,sequencing analysis graph method,screening of F3 generation GHR gene editing positive and negative pigs,and measure its 1-6 months body body weight,body length,body height,chest circumference and growth indicators.By using the method of real-time fluorescent quantitative PCR for GHR,JAK2 and STAT3 gene in guizhou congjiang pig and F2 generation homozygous GHR gene editing the pig heart,liver,lungs,kidneys and back's muscle groups such as amount of mRNA expression were analyzed.The coding region sequence of GHR gene was cloned by PCR,and the expression vector of pEGFP-N3-GHR eukaryotic was constructed and analyzed.The aim of this study was to explore the effects of GHR gene on growth and development at the phenotypic and molecular level of Xiang pig,and to provide a theoretical basis for the molecular regulation mechanism of growth traits in Xiang pig.It has important theoretical value and practical significance for further breeding experimental animal miniature pig and its development andapplication.The research results are as follows:(1)Genotyping identification of GHR gene editing pigs: Analysis of sequencing atlas,base insertion of 2 bp and 4 bp bases at the insertion site,and individuals with a bimodal at the beginning of sequencing atlas are heterozygous negative pigs(GHR+/-);single Individuals with peaks were wild-type,homozygous positive pigs(GHR+/+),homozygous positive pigs had more than 2 GT or 4 GATG bases compared with wild-type piglets.(2)Comparison of body weight,body length,height and chest circumference growth index between positive and negative pigs.There was no significant difference in every index at 1,2 months(P>0.05);There were significant differences in body length,body weight and body height at the age of 3 months(P < 0.05).At the age of 4,5 and 6 months,there were significant differences in body height,body length and body weight in F3 generation GHR gene knockout pigs(P < 0.01).Explain:GHR gene editing pigs can grow normally after base deletion or insertion,but F3 generation positive homozygous pigs are significantly smaller than negative pigs in somatotype.(3)Based on the different generations of GHR gene editing hybrid positive pigs growth index analysis,F3 generations of positive pigs are significantly less than F1 and F2 generation,explain GHR gene is the key to influence the fragrant pig growth.(4)GHR,JAK2,and STAT3 genes were expressed in different tissues in Congjiang pig and GHR gene editing pigs.The expression of GHR gene was the highest in liver tissue,with significant difference(P<0.01).The expression of JAK2 gene was the highest in liver tissue,with significant difference(P < 0.01).The expression of STAT3 gene was the highest in renal tissue,with significant difference(P<0.05).In the same tissue,the expression levels of GHR,JAK2 and STAT3 genes were all higher than that of GHR gene editing pigs in Congjiang xiang pigs.It is inferred that the knockout of GHR may hinder the expression of JAK2 and STAT3 in JAK-STAT signaling pathway,and ultimately affect the growth and development of pigs.It is suggested that the expression of GHR gene plays an important role in regulating the growth and development of animals..(5)PEGFP-N3-GHR eukaryotic expression vector was constructed and the coding region of GHR gene was analyzed.The results showed that:GHR gene encodes 634 Aminoacids,the molecular weight of protein is 68.14 KD,the isoelectric point is pI=9.51,and the protein is unstable,and the transmembrane helical structure of the protein is quite different.The sequence of coding region was different from 548 base.The amino acid sequence was different from the 182 position,and the change was great.After the mutation,there were many terminating codon in the amino acid sequence.The mutation of GHR gene leads to the structural variation of its encoded protein,which may affect the function of protein and directly affect the growth and development of animals.