家蚕乙醇脱氢酶基因的表达特征及乙醇在蚕体内的代谢分析

乙醇脱氢酶(alcohol dehydrogenase,ADH)是生物体内重要的乙醇代谢酶。生物信息学分析显示家蚕基因组中存在7个ADH编码基因(BmADH1～BmADH7),半定量RT-PCR检测BmADH2、BmADH3、BmADH4和BmADH5在家蚕5龄第3天幼虫的丝腺中表达水平较高,BmADH1、BmADH6、BmADH7在脂肪体中高水平表达。利用直接注射和口器灌喂2种方式,对家蚕5龄第3天幼虫分别以体积分数28%、56%的乙醇进行刺激处理后,调查家蚕体内乙醇的代谢与脂肪体中ADH基因的表达及酶活性变化。半定量RT-PCR检测表明56%乙醇注射处理组家蚕的脂肪体中BmADH1、BmADH6、BmADH7的表达上调,而28%乙醇处理后3个基因的表达基本无变化;酶活性检测表明28%、56%乙醇处理后1 h家蚕脂肪体中的ADH活性均显著升高(P<0.05);气相色谱分析显示家蚕血液中的乙醇会快速转化为乙醛。以上结果表明,家蚕幼虫在受到高浓度乙醇刺激后,通过上调脂肪体内的ADH基因的表达,增强ADH活性,使其参与体内乙醇的代谢过程,以保护蚕体免受高浓度乙醇的伤害。

Expressional Profile of Alcohol Dehydrogenase Genes and Metabolic Analysis of Ethanol in Bombyx mori Larvae

Alcohol dehydrogenase(ADH) is a critical ethanol metabolic enzyme in organisms.Bioinformatics analysis showed that there are 7 ADH coding genes in the silkworm(Bombyx mori) genome(BmADH1～BmADH 7).Semi-quantitative RT-PCR indicated that BmADH 2,BmADH 3,BmADH 4 and BmADH 5 had high expression level in silk gland of day 3 silkworm larvae of the 5th instar and BmADH 1,BmADH 6 and BmADH 7 had high expression level in fat body.After day 3 silkworm larvae of the 5th instar were treated with 28% or 56% ethanol via direct injection and oral feeding,the metabolism of ethanol in silkworm larvae as well as the variations of ADH gene expression and ADH enzyme activity in fat body were investigated.Semi-quantitative RT-PCR analysis indicated that the expressions of BmADH 1,BmADH 6 and BmADH 7 genes were up-regulated in silkworm fat body after being treated by direct injection of 56% ethanol,while the expressions of these three genes remained unchanged after treatment with 28% ethanol.Enzyme activity assay revealed that ADH enzyme activities were predominantly increased in silkworm fat body at 1 h after treatment with 28% or 56% ethanol(P<0.05).Gas chromatography analysis showed that ethanol was quickly converted into acetaldehyde in the larval hemolymph.These results indicate that,after silkworm larvae receive stimulation of high concentration ethanol,the expression of ADH genes is up-regulated in fat body,and the increased ADH enzyme activities participate in ethanol metabolic process to protect the larvae from being harmed by high concentration ethanol.