熊蜂抗冻蛋白基因序列分析及表达特性研究  

Sequencing analysis and expression characteristcs of AFP gene in Bombus terriestris

作　　者：徐凯 陈东海[1] 王志[1] 刘玉玲[1] 王振伟[1] 牛犇 蔡向强 牛庆生[1] Xu Kai;Chen Donghai;Wang Zhi;Liu Yuling;Wang Zhenwei;Niu Ben;Cai Xiangqiang;Niu Qingsheng(Apiculture Science Institute of Jilin Province,Jilin 132108,China)

机构地区：[1]吉林省养蜂科学研究所

出　　处：《山西农业大学学报（自然科学版）》2019年第5期74-81,共8页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：国家蜂产业技术体系专项(CARS-44-SYZ 4);吉林省中青年科技创新领军人才及团队项目(20180519009JH);吉林省科技创新中心-基地与科技服务平台建设(20180623003TC);吉林省重点科技攻关项目(20160204014NY)

摘　　要：[目的]本研究旨在探究熊蜂 Bombus terriestris 抗冻蛋白基因(Antifreeze protein, BtAFP )的序列特征和表达规律,为该基因功能的研究提供理论基础。[方法]利用多种生物信息学软件对 BtAFP 序列进行分析,并采用荧光定量PCR方法对熊蜂不同发育阶段、不同组织以及不同低温胁迫下的mRNA表达量进行检测。[结果]序列分析结果表明,BtAFP包含1 143 bp的开放阅读框区域,编码380个氨基酸,理论蛋白分子量为38 kDa。该蛋白包含信号肽结构但无跨膜结构域,其二级和三级结构主要为α螺旋,与鱼type-I型AFPs的结构相似。 BtAFP 基因的5’UTR区域包含1个启动子序列和多个与环境变化相关的转录因子。进化树分析结果表明,BtAFP与蜜蜂科昆虫抗冻蛋白亲缘关系最近,氨基酸序列一致性为74%。荧光定量PCR结果表明, BtAFP 表达量在熊蜂不同的发育阶段和组织中存在显著差异( P <0.05), BtAFP mRNA在熊蜂褐眼蛹期的表达量最高,该基因在熊蜂工蜂成虫触角、翅膀和足中的表达量较高。 BtAFP 的表达受低温胁迫的诱导,其在-20 ℃和0℃低温下分别持续20 min、16 h的mRNA表达量显著高于其它时间点( P <0.05)。[结论] BtAFP 基因在熊蜂生长发育和抵御寒冷时发挥着重要的生理作用,可作为熊蜂抗寒研究的候选基因。[Objective]The objective of this study was to analyze the sequence properties of AFP gene from Bombus terriestris, and to investigate its expression patterns at different developmental stages,tissues and cold stresses.[Methods]Physiochemical properties and structure characteristics of BtAFP were predicted using multiple bioinformatics approaches.The expression levels of BtAFP mRNA at different developmental stages,tissues and under different cold stresses of B.terriestris were performed by qRT-PCR.[Results]The open reading frame (ORF) of BtAFP is 1 143 bp,encoding a protein of 380 amino acids with an estimated molecular mass of 38 kDa.BtAFP possesses a signal peptide and no transmembrane domains.α-helix was an important part of the secondary and tertiary structures of BtAFP,which was similar to the structure of fish typeI AFPs.A promoter sequence and several transcription factors associated with environmental changes were identified in 5’UTR region of BtAFP. Phylogenetic tree analysis showed that BtAFP has 74% amino acid sequence identify and the closest evolutionary relationship with the antifreeze proteins of Apidae.The result of qRT-PCR revealed that the BtAFP transcripts were differentially expressed in different tissues and developmental stages of B.terriestris ( P <0.05).Pupal with brown eyes had the highest expression level of BtAFP. The expression profiles of BtAFP in antenna,wings and legs of adult workers were higher than that in other tissues.Under cold stresses,the mRNA expression of BtAFP was changed by the increase of process time.The expression level of BtAFP at -20℃for 20 min and 0 ℃ for 16 h were significantly higher than other given time,respectively.[Conclusion]These result suggested that BtAFP plays an essential role in growth and response to cold stresses of Bombus terriestris, which might be used as a candidate gene for further studies on cold resistance of bumble bees.

关 键 词：熊蜂 抗冻蛋白 发育表达 低温胁迫 

分 类 号：S891[农业科学—特种经济动物饲养]