Effects Of Pig's Stanniocalcin-1 Gene On Mitochon-Drial Function And Detection Of Its Signal Pathwa-Y Related Genes Expressed In Different Sizes Pig Expressing Patterns

Calcitonin,a kind of glycoprotein hormones,was firstly found in fish,adjusting the balance of the calcium/phosphorus.Elevated serum calcium can cause Nissl-body release on STC-1,and the calcium ion flow through the gill and intestine to maintain stability of the calcium concentration in the blood.In recent years,STC has also been found in humans and other mammals,and has been named STC-1 and STC-2,respectively.These two genes are widely expressed in most tissues.They mainly regulate the physiological functions of animal body through autocrine/paracrine.The main function of STC-1 gene is to regulate the calcium/phosphorus balance and regulate the bone metabolism,and through the mitochondrial uncoupling protein block the norarmal production of adenosine triphosphate(ATP),which affects the growth and development of mammals.The expression of STC-1 gene was raise and lower by overexpression vector and double-stranded interfering RNA expression vector of STC-1 gene.The effect of STC-1 gene on mitochondrial function was detected at the cellular level.The STC-1 gene and the mitochondrial-related genes were screened by two different piglets of Bama pig and big white pig.The aim was to study the expression characteristics of these genes.Through the analysis of the above genic physiological functions and their regulatory mechanisms and the identification of new pathways affecting pig body development,it provides a theoretical basis for elucidating the genetic mechanism of different type pig have different development.The main contents and results are as follows:1.The effect of pig's STC-1 gene on cell mitochondrial function was examinedIn this study,three pairs of small interfering RNA(siRNA)and negative control siRNA were designed according to porcine muscle Stanniocalcin(STC)mRNA sequence.After transfection of PK15 cells(porcine kidney cells),the transfection efficiency and gene expression efficiency were observed by fluorescence microscopy.After intracellular siRNA silencing,the expression of STC-1 siRNA and the STC-1 Protein expression were detected by QRT-PCR and Western-blot.The results showed that the primer sequences of STC-1 siRNA was successfully designed and the silencing efficiency of this gene was detected at the cellular level.It was proved that STC-1 siRNA could silence STC-1 gene,and STC 1-132 gene silencing efficiency is the best.The transfected STC-1 siRNA was transfected into PK15 cells by Western blot.The expression of STC-1 siRNA was significantly higher than that of the STC-1 gene overexpressing vector(STC group)STC-1 protein,mitochondrial-associated protein and acetylated expression.Screened out the more silent gene of STC-1 siRNA(siSTC group)and the STC-1 gene overexpressing vector(STC group)were transfected into PK15 cells.The expression of STC-1 protein,mitochondria related proteins and acetylation in STC group and siSTC group were detected by Western blot.The results showed that overexpression or silencing of STC-1 gene could significantly up-regulate or down-regulate the expression of PMP70,OP A,DRP and mfn.The apoptosis,mitochondrial membrane potential,reactive oxygen species(ROS),ATP and other related physical and chemical indexes were measured by flow cytometry,and the changes of mitochondrial structure and morphology were observed by transmission electron microscope.The results showed that overexpression and silence of STC-1 gene could decrease the apoptosis rate and reactive oxygen species production,significantly increase the cell membrane potential,greatly reduce the production of intracellular ATP,but also affect the morphological structure and quantity of mitochondria.The results suggest that STC-1 gene overexpression and silence can cause a certain degree of impaired mitochondria,STC-1 gene might play a role in maintaining the normal functions of mitochondria.2.The detection of temporal and spatial expression patterns that STC-1 gene and regulation pathway related protein in different size body of swine.In this study,the lungs,kidneys,ovaries,skulls,cartilage,liver,spleen,muscle,heart and other tissues of Bama pig and big white pig were collected.The expression of STC-1 protein and mitochondrial fusion protein OPA,Mfn,DRP and mitochondrial energy metabolizing protein PMP70 were detected by Western blot respectively.Analysis of STC-1 gene epigenetic detection and promoter transcription factor binding site analysis.The results showed that the expression of STC-1 protein and mitochondrial fusion protein OPA,Mfn,DRP and mitochondrial energy metabolizing protein PMP70 were significantly different in all-sized pigs,which may be related to the difference of body development.