Sharifloo Ali, Zibaee Arash, Sendi Jalal J, Jahroumi Khalil Talebi
Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan Rasht, Iran.
Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran Karaj, Iran.
Front Physiol. 2016 Mar 15;7:96. doi: 10.3389/fphys.2016.00096. eCollection 2016.
The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had V max values of 4.64 and 3.02 U/mg protein and K m values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N', N'-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones.
本研究通过纯化、酶学特性分析、基因表达以及特定抑制剂阿卡波糖的体内效应，对甘蓝粉蝶幼虫体内的一种消化性α-淀粉酶展开研究。尽管α-淀粉酶活性在幼虫的全肠匀浆中最高，但淀粉分解活性的区室化显示中肠后部（PM）和中肠前部（AM）的活性相当。采用硫酸铵、Sepharyl G - 100和DEAE - 纤维素快速流动进行三步纯化，得到一种比活性为5.18 U/mg、回收率为13.20、纯化倍数为19.25且分子量为88 kDa的酶。纯化后的α-淀粉酶在最适pH 8和温度35°C时活性最高。此外，以淀粉和糖原作为底物时，该酶的Vmax值分别为4.64和3.02 U/mg蛋白质，Km值分别为1.37和1.74%。不同浓度的阿卡波糖、乙二胺四乙酸和乙二醇双（β-氨基乙基醚）N,N,N',N'-四乙酸显著降低了纯化后α-淀粉酶的活性。用0.22 mM阿卡波糖处理萝卜叶片后喂养甘蓝粉蝶四龄幼虫，以研究其对营养指标、α-淀粉酶活性和基因表达的体内效应。仅在对照幼虫和用阿卡波糖喂养的幼虫的消化食物转化效率、相对生长率和代谢成本方面发现了显著差异。此外，通过生化实验和天然聚丙烯酰胺凝胶电泳实验发现，处理后的幼虫淀粉分解活性显著降低。逆转录-聚合酶链反应（RT-PCR）结果显示，一个长度为621 bp的基因负责α-淀粉酶的表达，该基因与柑橘凤蝶和达摩凤蝶具有75%的同源性。最后，实时定量RT-PCR显示，与用阿卡波糖喂养的幼虫相比，对照幼虫中α-淀粉酶的表达更高。
