低温胁迫下香梨优斑螟内参筛选及HSP70、HSP90基因表达分析  

Screening of Reference Genes and Analysis of HSP70 and HSP90 Gene Expression of Euzophera pyriella Under Low Temperature Stress

作　　者：王德钢 贺鹏鹏 马光皇 肖海兵[1,3] 熊仁次 韩旭[1,3] 杨明禄 WANG De-gang;HE Peng-peng;MA Guang-huang;XIAO Hai-bing;XIONG Ren-ci;HAN Xu;YANG Ming-lu(College of Plant Science,Tarim Univeraity,Xinjiang Alar 843300,China;The National and Local Joint Engineering Laboratory of High Efficiency and Superior-Quality Cultivation and Fruit Deep Processing Technology of Characteristic Fruit Trees in South Xinjiang,Xinjiang Alar 843300,China;Scientific Observing and Experiment Station of Crop Pests in Alar,Ministry of Agriculture and Rural Affaire,P.R.,Xinjiang Alar 843300,China)

机构地区：[1]塔里木大学植物科学学院,新疆阿拉尔843300 [2]南疆特色果树高效优质栽培与深加工技术国家地方联合工程实验室,新疆阿拉尔843300 [3]农业农村部阿拉尔作物有害生物科学观测实验站,新疆阿拉尔843300

出　　处：《西南农业学报》2020年第11期2503-2508,共6页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金(31560512,31760516)。

摘　　要：【目的】为筛选出香梨优斑螟(Euzophera pyriella)在低温胁迫下合适的内参基因,并明确低温胁迫对热激蛋白基因表达的影响。【方法】从梨园采集香梨优斑螟5龄幼虫,于4℃处理0、0.25、1、3、8、12、18、24 h,以实时荧光定量技术测定内参基因及热激蛋白基因的表达丰度,利用ΔCt法、GeNorm、NormFinder、BestKeeper和RefFinder软件综合评价5个内参基因的稳定性,进一步分析低温处理后热激蛋白(HSP70和HSP90)基因表达规律。【结果】△Ct和Normfinder方法的内参基因稳定性为UBQ>GAPDH>Actin>18s>TUB,BestKeeper方法中18s和UBQ基因表现最为稳定,GeNorm认为Actin和GAPDH稳定性好,其次是UBQ,RefFinder综合评价稳定性UBQ最高,其次为GAPDH,而稳定性最差的是TUB。以UBQ为内参基因,发现热激蛋白家族中HSP70和HSP90基因相对表达量都是在1 h处理时达到峰值、然后下降,在18 h后有小幅度上升,维持在较高的水平,总体上表达呈现显著的先上升后下降的趋势。【结论】香梨优斑螟在温度胁迫中UBQ和GAPDH基因较为稳定。HSP70和HSP90基因对低温胁迫响应曲线相似,24 h内基因表达显著高于对照,这可能有助于提升幼虫对低温的耐受力。【Objective】The suitable reference genes of Euzophera pyriella under low temperature stress were screened out,and the effect of low temperature stress on the expression of heat shock protein gene was determined.【Method】The 5th instar larvae of E.pyriella was collected from pear orchard and treated at 4℃for 0,0.25,1,8,12,18,24 hours.Using real-time quantitative PCR technique to determine the expression abundance of reference genes and heat shock protein genes,the stability of five candidate reference genes was evaluated byΔCt method,geNorm,NormFinder,BestKeeper and RefFinder software,then made further analysis of heat shock protein(HSP70 and HSP90)genes expression law under low temperature treatment.【Result】ΔCt method and Normfinder software analysis showed that the stability of the five reference genes were UBQ>GAPDH>Actin>18 s>TUB,the results of BestKeeper analysis showed that 18 s and UBQ had the best stability,while Genorm software showed that Actin and GAPDH had the best stability.According to RefFinder synthesis,it was concluded that UBQ had the highest stability,followed by GAPDH,with the worst TUB.Using UBQ as reference Gene,it was founded that the relative expression of HSP70 and HSP90 genes in the heat shock protein family reached the peak at 1 hours treatment,and then decreased.After 18 hours,there was a small increase and maintained at a high level.On the whole,the expression showed a significant trend of first rise and then fall.【Conclusion】UBQ and GAPDH genes of E.pyriella were stable under temperature stress.The response curves of HSP70 and HSP90 genes at low temperature stress were similar,it was significantly higher than that of the control within 24 hours,this may help to improve the tolerance of larvae at low temperatures.

关 键 词：香梨优斑螟 低温胁迫 内参基因 热激蛋白 基因表达 

分 类 号：S436.619[农业科学—农业昆虫与害虫防治]