【摘要】 【目的】海藻糖酶(trehalase,Tre)作为昆虫体内海藻糖代谢的关键性酶,在昆虫能量调节和生长发育中具有重要作用。本研究旨在克隆获得沙葱萤叶甲Galeruca daurica海藻糖酶基因,并对其表达模式进行定量分析,以期探究海藻糖酶在沙葱萤叶甲生长发育和成虫夏滞育中的作用。【方法】根据沙葱萤叶甲转录组数据,采用RACE技术,克隆得到Tre基因的c DNA全长,并进行生物信息学分析;应用实时荧光定量PCR(RT-q PCR)技术检测其在沙葱萤叶甲不同发育阶段[卵、1-3龄幼虫、预蛹、蛹、成虫(羽化后3,7,10,15,25,40,60,80和100 d)]、成虫不同组织(头、胸和腹)以及3日龄成虫在不同温度(15,20,25,30,35和40℃)胁迫下的表达水平;采用3,5-二硝基水杨酸法测定不同日龄成虫体内海藻糖酶活性。【结果】克隆获得了沙葱萤叶甲可溶性海藻糖酶基因,并命名为Gd Tre1(Gen Bank登录号:KY697913),该基因全长1 933 bp,开放阅读框1 704bp,编码567个氨基酸;蛋白预测分子量为66.56 k D,等电点为6.62;编码蛋白具有海藻糖酶超基因家族典型的功能结构域,包含1条信号肽,不具有跨膜结构。同源序列比对和系统发育分析表明,Gd Tre1与马铃薯甲虫Leptinotarsa decemlineata Tre1b的同源性最高,氨基酸序列一致性达70.25%。RT-q PCR检测结果表明,Gd Tre1在沙葱萤叶甲各发育阶段均有表达,其中在卵期和成虫滞育期间高表达,而在幼虫、预蛹、蛹及成虫滞育前低表达;在成虫不同组织中,通常在腹部中表达量最高,其次为胸部,最低为头部;Gd Tre1表达量随着温度升高而上升,30℃达最高值,而后随温度升高略有下降。沙葱萤叶甲不同日龄成虫体内Gd Tre1表达量及Tre活性存在显著差异,且Gd Tre1表达量与Tre活性变化趋势一致。【结论】海藻糖酶与沙葱萤叶甲生长发育和成虫夏滞育有密切的关系。该结果为进一步揭示该虫的夏滞育分子机理提供了必要的基础。更多还原

【Abstract】 【Aim】Trehalase( Tre),a key enzyme in trehalose metabolism of insects,plays important roles in the energy regulation and development of insects. This study aims to clone a trehalase gene fromGaleruca daurica,and to analyze its expression patterns in order to investigate the functions of trehalase in the development of G. daleruca and summer diapause of its adults. 【Methods 】 Based on the transcriptome data of G. daleruca,the full-length c DNA of the Tre gene was cloned using the rapid amplification of c DNA ends( RACE)-PCR and analyzed by bioinformatics. Real-time quantitative PCR( RT-q PCR) was performed to detect the expression levels of the Tre gene in different developmental stages( egg,1 st-3 rd instar larva,prepupa,pupa,and 3,7,10,15,25,40,60,80 and 100 day-old adult) and various adult tissues( head,thorax and abdomen) of G. daleruca,and the 3 day-old adults under different stress temperature( 15,20,25,30,35 and 40℃). The Tre activity in different day-old adults was assayed by the 3,5-dinitrosalicylic acid colorimetry. 【Results】A soluble trehalase gene was cloned from G. daleruca,and named Gd Tre1( Gen Bank accession no.: KY697913). Its full-length c DNA is 1 933 bp containing a 1 704 bp open reading frame( ORF) that encodes 567 amino acids with the predicted molecular weight of 66. 56 k D and p I of 6. 62. The coded protein contains typical functional domains in the trehalase superfamily with a signal peptide but without transmembrane domain. Homology and phylogenetic analyses showed that Gd Tre1 has the highest amino acid sequence identity( 70. 25%)with Leptinotarsa decemlineata Tre1 b. RT-q PCR results revealed that Gd Tre1 was expressed throughout various developmental stages of G. daleruca,with higher expression levels in the egg and diapause adult,and lower expression levels in the larva,pre-pupa,pupa and pre-diapause adult. Tissue expression profiles revealed that the expression level of Gd Tre1 was the highest in the abdomen,second high in the thorax and the lowest in the head of adults. The expression level of Gd Tre1 in 3 day-old adults increased as the stress temperature increased,with the highest level at 30℃,and afterwards decreased a little with the temperature increasing. The expression levels of Gd Tre1 and the Tre activity were significantly different among different day-old adults and showed the same change trend. 【Conclusion】Trehalase has a close relationship with the development and summer diapause in G. daleruca. The results provide a necessary foundation for further investigation on the molecular mechanisms of summer diapause in this insect.更多还原