摘要
为探究旋毛虫肌幼虫体内海藻糖酶(TsTRE)和海藻糖在抵抗低温胁迫中的作用,将旋毛虫肌幼虫置于37℃环境中培养1 h后,移至不同温度的环境中,通过qRT-PCR方法筛选出最佳胁迫温度,再在最佳胁迫温度下,应用qRT-PCR和Western-blot方法检测不同培养时间下TsTRE的基因转录水平与蛋白表达水平的变化,用3,5-二硝基水杨酸法检测TsTRE活性,用蒽酮比色法检测旋毛虫肌幼虫体内海藻糖含量的变化,免疫荧光检测TsTRE在虫体内的分布情况。结果显示,在最佳胁迫温度4℃下,可引起TsTRE基因转录水平、蛋白表达水平和酶活性显著下降,均在1 h时达到最低值,其分别下降约64.96%、68.89%和36.98%(P<0.01),随着低温胁迫时间的增加,TsTRE基因转录水平和蛋白表达水平整体呈“W”型趋势;而海藻糖含量则与之相反,呈“M”型趋势,在3 h达到最高值(P<0.01),约是对照组的1.6倍;TsTRE主要分布于旋毛虫肌幼虫全身表皮、中肠、后肠及生殖原基内,且低温处理组较对照组诱导的绿色荧光信号明显减弱(P<0.01)。以上研究揭示,在低温胁迫下,旋毛虫肌幼虫体内TsTRE表达的变化和海藻糖含量的变化呈此消彼长的关系,TsTRE表达水平及其酶活性的降低引起海藻糖迅速积累以保护虫体,本研究为进一步探讨旋毛虫抗低温的分子机制提供了理论参考。
In order to investigate the role of trehalase(TRE)and trehalose in resistance to low-temperature stress in Trichinella spiralis(T.spiralis)muscle larvae(ML),the ML were incubated at 37℃for 1 h,then moved to different temperatures,and the optimal stress temperature was screened using qRT-PCR.After culturing ML for different times at the optimum stress temperature,transcription and protein levels of TRE of T.spiralis(TsTRE)were detected by qRT-PCR and Western-blot.The TsTRE activity was measured by 3,5-dinitrosalicylic acid assay,trehalose content of the ML was detected by the anthrone colorimetric method,and TsTRE distribution in the ML was detected by immunofluorescence.The results showed that 4℃was the optimal stress temperature,which could significantly reduce transcription and protein expression levels,as well as TsTRE enzyme activity,which reached its lowest at 1 h and decreased approximately 64.96%,68.89%,and 36.98%,respectively(P<0.01).With increasing time of low-temperature stress,TsTRE transcription and protein levels showed a“W”trend,while trehalose content showed an“M”trend and peaked at 3 h(P<0.01),about 1.6 times that of the control group.TsTRE was primarily found in the ML’s cuticle,midgut,hindgut,and reproductive primordia.The green fluorescence signal of the low-temperature treatment group was significantly lower compared to that of the control group(P<0.01).TsTRE expression was reciprocated with trehalose content of ML under low-temperature stress,according to this study.When TsTRE expression and enzyme activity are reduced,trehalose accumulates quickly to protect the ML.This study provides a theoretical foundation for future research into the molecular mechanism of low-temperature resistance of T.spiralis.
作者
王雪莹
侯嘉茗
薄禄琪
魏冬旭
阮入琳
张博涵
张鉴慧
宋铭忻
张子群
WANG Xue-ying;HOU Jia-ming;BO Lu-qi;WEI Dong-xu;RUAN Ru-lin;ZHANG Bo-han;ZHANG Jian-hui;SONG Ming-xin;ZHANG Zi-qun(Heilongjiang Key Laboratoryfor Zoonosis,Northeast Agricultural University,Harbin 150030,China;Technology Center of Harbin Customs,Harbin 150008,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第1期78-84,共7页
Chinese Veterinary Science
基金
国家自然科学基金项目(32172883)
国家寄生虫资源库项目(NPRC-2019-194-30)。
关键词
旋毛虫
肌幼虫
海藻糖
海藻糖酶
低温
Trichinella spiralis
muscle larvae
trehalose
trehalase
low-temperature
