家蚕经菊酯类农药诱导后脂肪体蛋白的表达谱及烯醇酶基因的组织转录活性

Protein Expression Profiles of Fat Body in Bombyx mori Induced by Cypermethrin and Enolase Gene Transcription Activity in Various Tissues

为了从蛋白质水平探讨家蚕对菊酯类农药的抗性机制,采用双向电泳结合基质辅助质量飞行时间质谱(MALDI-TOF-MS)技术,分析家蚕5龄幼虫经氯氰菊酯诱导前后脂肪体蛋白的表达特征。采用ImageMaster6.0软件分析表明,对照组共检测到432个蛋白点,诱导实验组检测到369个蛋白点,其中能匹配的蛋白点有342对,匹配率为76.68%。对特征蛋白进行MALDI-TOF-MS分析鉴定的蛋白包括烯醇酶、线粒体醛脱氢酶、伴侣素、肌动蛋白、gag-like蛋白。烯醇酶(enolase)是糖酵解过程中极为重要的酶类。采用半定量RT-PCR对经氯氰菊酯诱导后的家蚕脑、中肠、丝腺、脂肪体、精巢、马氏管、血液等7个组织器官的烯醇酶基因进行表达分析,结果表明,烯醇酶基因转录水平在7个组织器官中都呈现下调趋势,分别下调了13.79%、10.71%、22.48%、7.40%、27.00%、11.50%、14.52%,其中脂肪体的烯醇酶基因表达和蛋白的表达下调一致。受菊酯类农药诱导的影响,家蚕5龄幼虫脂肪体蛋白的数目减少,烯醇酶基因在各组织器官减量表达,提示可能与蚕体的基础生理代谢受到抑制有关。

To study the resistance mechanism of Bombyx mori to cypermethrin at the protein level, high resolution and two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption ionization and time of flight mass spectrometry （MALDI-TOF-MS） was applied. The ImageMaster 6.0 analysis showed that 432 protein spots were detected in the control group, 369 protein spots in the induced experimental group, and the matched protein spots were 342. Comparative analysis showed that the matched value between the two groups was 76.68%. The protein spots of interest were identified by MALDI-TOF-MS. The identified proteins included enolase, mitochondrial aldehyde dehydrogenase, chaperonin containing t-complex polypeptide 1 beta subunit, actin, and gag-like protein respectively. Enolase is an extremely important enzyme in the glycolysis process. Inducement effect of cypermethrin treatment on transcription level of enolase gene in the 5th instar larvae was determined in 7 tissues, brain, midgut, silk gland, fat body, testis, Malpighian tubules and hemolymph by semi-quantitative RT-PCR method, respectively. The result showed that the transcriptional level of enolase gene was reduced in 7 tissues after induction by cypermethrin and the specific value was 13.79%, 10.71%, 22.48%, 7.40%, 27.00%, 11.50% and 14.52%, respectively. Both gene expression and protein expression of fat body enolase showed a downward tendency. Affected by the cypermethrin＇s induction, the number of proteins expressed in 5th instar larvae of Bombyx mori fat body was reduced, and the transcriptional level of enolase gene in various tissues and organs was lowered, showing that the basic physiological metabolism of Bombyx mori body might have been inhibited. The obtained results provide useful reference for studies on resistance mechanism of Bombyx morito pyrethroid pesticide and its relationship with enolase gene.