斜纹夜蛾中肠糖代谢相关基因的克隆及表达模式分析

Cloning and expression profiling of genes involved in glycometabolism in the midgut of Spodoptera litura(Lepidoptera:Noctuidae)

斜纹夜蛾Spodoptera litura是一种世界性分布的重要农业害虫,在生长发育过程中要经历幼虫一蛹的变态发育过程。由于变态发育前后昆虫的食性发生了明显的改变,作为食物消化吸收的中肠也发生了解体和重建。与此相适应,昆虫中肠的各种物质和能量代谢也可能会相应地发生改变。为研究斜纹夜蛾中肠变态发育过程中糖代谢途径的变化情况,我们从斜纹夜蛾中肠EST文库中鉴定出了12个糖代谢相关基因,克隆了其中3个基因的全长cDNA,并应用半定量PCR和定量PCR的办法检测了其在幼虫-蛹变态发育期中肠组织的转录表达以及对激素和饥饿等因素的响应情况。结果表明:这3个基因(α-L-岩藻糖苷酶、N-乙酰葡萄糖胺-6-磷酸去乙酰酶和烯醇化酶基因)的开放阅读框分别为1 461,1 200和1 299 bp,预测的分子量分别为56.3,43.3和46.7 kDa。这12个糖代谢相关的基因在变态发育期的中肠组织中具有5种不同的mRNA表达模式:(Ⅰ)只在幼虫期高表达(唾液麦芽糖酶前体蛋白、糖基水解酶31家族成员蛋白、线粒体乙醛脱氢酶、β-1,3-葡聚糖酶基因);(Ⅱ)只在预蛹期高表达(β-葡萄糖醛酸酶、β-N-酰基氨基葡萄糖苷酶3基因);(Ⅲ)只在蛹期高表达(葡萄糖胺-6-磷酸异构酶基因);(Ⅳ)在预蛹期和蛹期高表达(α-葡萄糖苷酶、α-淀粉酶、N-乙酰葡糖胺-6-磷酸脱乙酰酶和α-L-岩藻糖苷酶基因);(V)在变态发育期恒定表达(烯醇化酶基因)。这说明,为适应变态发育斜纹夜蛾中肠糖代谢途径发生了明显的改变。保幼激素对这些基因的表达没有明显的影响,但蜕皮激素对Ⅰ类基因(如糖基水解酶31家族成员蛋白基因)具有一定的抑制作用,对Ⅲ类基因(如葡萄糖胺-6-磷酸异构酶基因)有显著的上调作用。此外,我们还发现饥饿对几乎所有这些基因的表达都有显著的抑制作用。这些结果表明,昆虫中肠变态发育过程中糖代谢相关基因的动态变化可能受到蜕皮激素以及饥饿相关因素的共同调控。这一研究对从代谢角度揭示昆虫变态发育的分子机理具有重要意义。

The common cutworm, Spodoptera litura is an important agricultural pest, which has spread out worldwide. It undergoes larval-pupal metamorphosis in the life cycle. As a major organ for food digestion and nutrient absorption, the midgut is degraded and reconstructed during the metamorphosis to adapt to the dramatic change in food type between larvae and adults. Therefore, the substances and energy metabolism in the midgut change accordingly. In order to study the sugar metabolic dynamics in the midgut during metamorphosis, twelve glycometabolism-related genes were identified from the midgut EST library of S. litura. Three of them were cloned to obtain their full cDNAs. The mRNA expression profiles of all the 12 genes were examined by realtime or semi-quantitative PCR in the midgut during metamorphosis, and their mRNA levels in response to the induction of hormones and starvation were also quantified. The results indicated that the open reading frames of α-L-fucoside fucohydrolase, N-acetylglucosamine-6-phosphate deacetylase and enolase genes are 1 461, 1 200 and 1 299 bp, and the predicted proteins are 56.3, 43.3 and 46.7 kDa, respectively. The mRNA expression profiles revealed five different patterns： （Ⅰ） High expression only in the larval stages, including salivary maltase precursor, glycosyl hydrolase family 31 protein, mitochondrial aldehyde dehydrogenase and β-1, 3-glucanase genes. （Ⅱ） High expression only in the prepupal stage, including β-glucuronidase and β-N-acetylglucosaminidase 3 genes. （Ⅲ） High expression only in the pupal stage, including glucosamine-6-phosphate isomerase gene. （Ⅳ） High expression both in the prepual and pupal stages, including glucosidase, α-amylase, N-acetylglucosamine-6-phosphate deacetylase and α-L-fucoside fucohydrolase genes. （Ⅴ） Stable expression, including enolase gene. This indicates that significant changes happen in glycometabolism in the midgut during metamorphosis. Juvenile hormone （JH） had no obvious effect on the mRNA level of these genes. However, 20-hydroxyecdysone （20E） suppressed the mRNA level of type I gene such as glycosyl hydrolase family 31 protein gene, and up-regulated the type Ⅲ gene （glucosamine-6-phosphate isomerase gene）. In addition, the mRNA level of all the genes was significantly suppressed by starvation. The results suggest that the dynamics of mRNA level of sugar metabolic genes may be controlled by both 20E and starvation-related factor. These findings in glycometabolism will contribute to elucidating the mechanism of larval-pupal metamorphosis.