基于iTRAQ的急性热应激下绍兴鸭肝脏差异表达蛋白的鉴定与分析  

Identification and Analysis of Liver Differentially Expressed Proteins in Shaoxing Duck(Anas platyrhynchos)Under Acute Heat Stress Based on iTRAQ

作　　者：王德前[1] 董捷 徐宁迎 黄敏婕 WANG De-Qian;DONG Jie;XU Ning-Ying;HUANG Min-Jie(Institute of Animal Husbandry and Veterinary Science,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;College of Animal Science,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江省农业科学院畜牧兽医研究所,杭州310021 [2]浙江大学动物科学学院,杭州310058

出　　处：《农业生物技术学报》2021年第9期1752-1760,共9页Journal of Agricultural Biotechnology

基　　金：浙江省畜禽新品种选育专项(2016C02054)。

摘　　要：随着蛋鸭(Anas platyrhyncha)养殖集约化程度不断提高,夏季持续高温引起的热应激问题给蛋鸭安全生产带来严峻挑战。为阐明热应激对蛋鸭肝脏蛋白表达的影响及探索差异蛋白基因的功能,本研究选取产蛋高峰期(245日龄)的绍兴鸭进行热应激处理(40℃),采集肝脏组织,应用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification,iTRAQ)和液相色谱质谱串联(LC-MS/MS)技术,共鉴定到98个热应激肝脏差异表达蛋白,其中热休克蛋白60(heat-shock protein 60,HSP60)表达极显著上调(P<0.01)。KEGG通路分析结果显示,热应激后差异蛋白显著富集于15个代谢通路,其中糖酵解和糖异生、三羧酸循环和丙酮酸等代谢途径可能与热应激反应存在相关性。GO生物学过程富集分析显示翻译延伸、葡萄糖分解、糖酵解等代谢过程相关蛋白显著富集。应用RNA干扰技术,通过筛选和优化HSP60-siRNA干扰条件,将HSP60-siRNA转染入鸭肝细胞。采用实时荧光定量PCR反应检测发现,与阴性对照组相比,处理组HSP60基因表达量降低75.69%(P<0.01),细胞色素C氧化酶基因(cytochrome C oxidase,CCO)和应激活化蛋白激酶基因(stress-activated protein kinase,SAPK)表达极显著升高(P<0.01),蛋白激酶B基因(protein kinase B,PKB)表达极显著降低(P<0.01),肿瘤坏死因子凋亡诱导因子受体基因(tumor necrosis factor-related apoptosis inducing receptors,TRAIL-Rs)表达无显著差异(P>0.05)。结果表明,急性热应激条件下蛋鸭肝脏中HSP60表达量显著增加,通过调控细胞凋亡信号通路关键基因的表达来抑制细胞凋亡。本研究初步探索了蛋鸭热应激发生机理,有助于预防控制蛋鸭热应激发生。In recent years,with the increasing intensification of laying duck(Anas platyrhynchos)cultivation,the effect of heat stress caused by constant high temperature in summer poses a serious challenge to safety production.To elucidate the effect of heat stress on hepatic protein expression and to explore the function of differential protein genes in laying duck,in this study,Shaoxing ducks at the peak laying period(245-day old)were selected for heat stress treatment and liver tissues were collected.A total of 98 differentially expressed proteins(DEPs)were identified by isobaric tags for relative and absolute quantification(iTRAQ)and LC-MS/MS,with significantly upregulation of heat shock protein 60(HSP60)(P<0.01).KEGG analysis showed that DEPs enriched in 15 metabolic pathways after heat stress.Glycolysis and gluconeogenesis,tricarboxylic acid cycle and pyruvate may be related to the effects of heat stress.GO analysis showed that DEPs were involved in translation extension,glucose catabolism process,glycolysis.HSP60-siRNA was transfected into duck hepatocytes after screening and optimization.Compared with negative control(NC)group,HSP60 gene expression was decreased by 75.69%(P<0.01),cytochrome coxidase gene(CCO)(P<0.01),and stressactivated protein kinase gene(SAPK)were significantly increased(P<0.01),protein kinase gene(PKB)was significantly decreased(P<0.01).There was no significant difference in the expression of tumor necrosis factor-related apoptosis inducing receptors gene(TRAIL-Rs)(P>0.05).The results showed that HSP60 was significantly increased in the liver of laying ducks under acute heat stress and inhibited apoptosis by regulating the expression of key genes of apoptotic signaling pathways.It is helpful to prevent and control the occurrence of heat stress by preliminarily exploring the mechanism of heat stress occurrence in laying ducks.

关 键 词：绍兴鸭 热应激 同位素标记相对和绝对定量(iTRAQ) RNAI 热休克蛋白60(HSP60) 

分 类 号：S834[农业科学—畜牧学]