Distribution And Change Rule Of HSP70 And GSDMA In Yak Intestine

ObjectsYak is also known as the "boat of the plateau.It is the only type of cattle that has been able to adapt to the harsh natural environment of the Qinghai-Tibet Plateau,such as alpine,anoxic,and subtilis.It is the main support for the survival of the Tibetan people and is a valuable livestock resource in our country.Under the influence of environmental factors,the intestinal tract of the animal is easily damaged by the congenital barrier of the intestinal tract,causing disorder of the intestinal flora,which in turn causes various diseases and leads to a decline in its production performance.Calves live under conditions of high cold and oxygen deprivation,and large forage grasses(such as tender grass and subtilis),and they are still able to survive in normal conditions.This is inseparable from their unique intestinal environment and digestive function.It is very important to study the molecular regulation mechanism of calf’s intestinal adaptability.Heat shock proteins(HSPs)were initially identified as stress proteins of the Drosophila thermostimulatory response and later confirmed the widespread expression of HSP70 in eukaryotic and prokaryotic organisms.The HSPs family found so far can be divided into the following 6 families according to their molecular weight: small HSP(15-30 k Da),HSP40,HSP60,HSP70,HSP90,and HSP100.HSP70 is an important molecular chaperone,and HSP70 in cells can Helps other proteins in the cell to undergo proper translocation,folding,assembly,and trafficking;it can also be combined with apoptosis-inducing factors through multiple pathways,thereby inhibiting cell apoptosis.The Gasdermin gene family is a new gene family.The family contains a conserved Gasdermin domain of approximately 400 amino acids in length.The N-terminal domain of the Gasdermin family protein has been reported to have the function of inducing cell apoptosis.As a member of this gene family,GSDMA has reported that GSDMA gene can induce growth inhibition and promote apoptosis.In this study,immunohistochemistry,Western blotting(WB)and real-time fluorescence quantification(QRT-PCR)were used to detect changes inHSP70 and GSDMA localization and quantification in the intestine of adult yak 6 segments from the level of protein and gene for further study of HSP70 and GSDMA provides a morphological basis for the regulatory mechanism of yak’s digestive tract.Results1.Immunohistochemical method was used to find that GSDMA was expressed in both large and small intestines of yak and the expression was similar.GSDMA is mainly expressed in the mucous layer of calf intestinal wall and is not expressed in the submucosa and muscularis.The positive expression of GSDMA in the lamina propria is mainly located in the scattered fusiform cells in the connective tissue between the intestinal glands.The positive reaction is mainly distributed in the cytoplasm of the above cells.in.The expression of GSDMA in duodenum,jejunum,ileum,cecum,colon and rectum of adult calves was detected by Western blotting.GSDMA had a similar expression pattern in calf’s intestine,and GSDMA had a tendency of increasing first and then decreasing.Among the highest levels in the jejunum and colon2.Immunohistochemical method was used to find that HSP70 was expressed in the mucous layer,submucosa and muscular layer of the small intestine of calf.Among them,columnar cells of the small intestine mucosal epithelium showed strong positive expression,while no expression was observed in goblet cells;intestinal gland cells of small intestine glands.Muscle and smooth muscle cells also showed positive expression,positive reactions mainly distributed in the cytoplasm and nucleus of the above cells.The expression of HSP70 in the large intestine is similar to that in the small intestine.It is also expressed in the mucosal,submucosal and muscular layers of the large intestine.However,the positive expression of HSP70 in the large intestine of calves is mainly located in the connective tissue cells distributed in the mucosal epithelium and lamina propria.Intrinsic intracerebroventricular gland cells were only expressed in the nucleus and cytoplasm negative.The analysis of the average integrated optical density from each segment of the intestine showed that HSP70 showed a trend of first increase and then decrease in the proximal small intestine to the distal part of the large intestine and reached the maximum in the cecum,exceptthat the difference between the cecum and colon was not significant(P > 0.05),the difference in other intestinal segments was extremely significant(P < 0.01).The expression of HSP70 in duodenum,jejunum,ileum,cecum,colon and rectum of adult calves was detected by Western blotting.It was found that the expression of HSP70 increased firstly and then decreased from the anterior end of small intestine to the end of large intestine.Cecal expression reached a peak.In addition,the expression of HSP70 was significantly higher in the large intestine than in the small intestine(P < 0.01).QRT-PCR results showed that HSP70 m RNA was expressed in the 6 intestinal segments of calves and the relative expression levels were significantly different(P < 0.05),and the expression of HSP70 in the cecum was significantly higher than other intestinal segments(P < 0.05).),2,1.68,1.06,1.43 and 1.52 times of the twelve-finger,jejunum,ileum,colon and rectum,respectively.Conclusion1.This experiment successfully localized the expression site of GSDMA protein in calf’s intestine,and found the axial expression of GSDMA in the intestine,indicating that yak’s GSDMA specific expression profile is different from other species.2.This experiment confirmed the existence of widespread positive reaction of HSP70 in the calf’s intestine.It was confirmed from the gene and protein level that HSP70 was significantly different in calf’s small intestine and large intestine,and its expression in the blind colon was significantly higher than other intestinal segments.It shows that HSP70 is closely related to the digestion and absorption function of yak’s intestines,and its regulation in the large intestine is stronger than that in the small intestine.