棉铃虫蛹滞育的糖代谢相关基因研究

发布时间：2018-06-22 17:26

本文选题：滞育 + 棉铃虫　；参考：《中山大学》2016年博士论文

【摘要】：自然环境并非总是适合于生物的生长发育,面对恶劣环境时,昆虫往往会选择进入滞育状态以躲过不利环境的影响,待到环境条件适合再重新启动发育。因此,滞育是昆虫赖以生存的重要策略。滞育的主要特征是个体代谢显著下调,抗逆性增强以及发育极其缓慢,由此可以看出滞育是整体变化,十分复杂,涉及昆虫体内许多生理生化调控。一般认为激素是调节滞育的关键因素,且已经得到大量的证据证实,其中蛹滞育是由于促前胸腺激素-蜕皮激素信号下调导致的。虽然滞育过程生理生化变化已经有大量的研究,但是这些生理生化背后的分子机制却尚不十分清楚,因此本研究以蛹滞育的棉铃虫为研究对象,试图阐明棉铃虫滞育过程中代谢的变化原因以及胰岛素受体对滞育的影响。为了调查棉铃虫滞育过程中代谢下调的机制,克隆了糖酵解途径的第一个关键基因己糖激酶(HK),并调查了它在棉铃虫发育或者滞育个体脑中表达和酶活。结果发现HK mRNA和蛋白在发育型蛹脑中表达明显高于滞育型蛹脑,且其活性也与表达水平一致,在发育型蛹脑中活性较高。下调HK表达活性可以引起代谢降低以及ROS增加,从而降低细胞活性并延迟蛹的发育。暗示着HK是一个调节昆虫发育的重要因子。随后我们鉴定了三个转录因子CREB、c-Myc和POU特异性结合到HK启动子上调节HK活性,但有趣的是POU和c-Myc是特异性调节HK表达的转录因子,而CREB却是非特异性的。另外,POU和c-Myc能够响应上游激素信号蜕皮激素的调节。在滞育个体中低水平的蜕皮激素导致低水平的POU和c-Myc表达,从而抑制了HK的表达水平,导致代谢的下调和ROS的积累,抑制蛹继续发育而进入滞育状态。细胞能量代谢的主要场所是线粒体,以前的报道中发现滞育型棉铃虫蛹脑中COX活性下调,暗示着线粒体活性也可能处于较低水平,但是具体机制并不十分清楚。因此,首先调查了线粒体DNA含量以及COX活性,发现线粒体活性在滞育型蛹脑中的确受到抑制。随后的调查结果显示,HIF-1α在滞育型蛹脑中表达较发育型蛹脑增高,且HIF-1α高表达能够抑制线粒体活性。HIF-1α抑制线粒体活性是通过蛋白酶体降解c-Myc蛋白发挥的作用。线粒体转录因子A(TFAM)是一个调节线粒体转录和线粒体DNA复制的重要转录因子,c-Myc能够直接结合到TFAM启动子上并激活其表达,而TFAM活性的缺失是导致线粒体活性下调的重要原因,证实了HIF-1α-c-Myc-TFAM信号参与到调节昆虫滞育过程中线粒体的活性。胰岛素(insulin)信号通路是调节发育的重要途径,有许多研究发现insulin信号参与到昆虫滞育的调节并起到重要作用。为了研究insulin信号通路在棉铃虫滞育中的作用,克隆并调查了胰岛素受体(InR)在棉铃虫蛹脑中的表达。结果发现InR在滞育型蛹脑中高表达,且P-InR水平在滞育型蛹脑中明显高于发育型蛹脑;insulin通过P-InR调节P-ERK表达;ROS能够激活P-InR,P-AKT和P-ERK;且ROS对P-AKT和P-ERK的激活是通过P-InR调控的。结合我们已有的一些结果,发现了ROS-P-InR-P-ERK信号能够参与到棉铃虫滞育过程中蛋白酶体的调控,降解控制发育的相关蛋白,使蛹顺利进入滞育。
[Abstract]:Natural environment is not always suitable for biological growth and development. In the face of bad environment, insects often choose diapause to avoid the effect of adverse environment and adapt to environmental conditions to restart and develop. Therefore, diapause is an important strategy for the survival of insects. It can be seen that diapause is a whole, very complex and involves many physiological and biochemical regulation in the body of insects. It is generally believed that hormone is the key factor regulating diapause, and a large amount of evidence has been obtained, in which the diapause of pupae is caused by the downregulation of the pre thymus hormone - ecdysone. The physiological and biochemical changes in diapause have been studied, but the molecular mechanisms behind these physiological and biochemical factors are still not very clear. Therefore, this study aims to elucidate the causes of metabolic changes and the effect of insulin receptor on diapause during the diapause of the cotton bollworm. In order to investigate the stagnation of cotton bollworm. The mechanism of down regulation of metabolism, the first key gene of hexokinase (HK), the key gene of glycolysis, was cloned, and the expression and enzyme activity in the individual brain of the cotton bollworm and diapause were investigated. The results showed that the expression of HK mRNA and protein in the developmental pupa brain was obviously higher than that of diapause pupa brain, and its activity was consistent with the expression level. The activity of the pupae in the pupae is high. Down regulation of HK expression activity can cause the decrease of metabolism and the increase of ROS, thus reducing cell activity and delayed the development of pupae. It suggests that HK is an important factor regulating the development of insects. Then we identified three transcription factors CREB, c-Myc and POU specific binding to the HK promoter to regulate HK activity, but it is interesting. It is that POU and c-Myc are the transcription factors that specifically regulate the expression of HK, while CREB is nonspecific. In addition, POU and c-Myc can respond to the regulation of the upstream hormone signal ecdysone. Low levels of ecdysone in diapause individuals lead to low levels of POU and c-Myc expression, thus inhibiting the expression level of HK, leading to metabolic downregulation and ROS. Accumulation, inhibition of the continued development of pupae and diapause. The main site of cell energy metabolism is mitochondria. In previous reports, the COX activity in the diapause type Helicoverpa armigera was downregulated in the pupa brain, suggesting that mitochondrial activity may also be at a lower level, but the specific mechanism is not very clear. Therefore, the content of mitochondrial DNA and C are investigated first. OX activity was found to be suppressed in the diapause pupa brain. The results of the subsequent investigation showed that the expression of HIF-1 alpha in diapause pupa brain was higher than that of the developmental pupae, and the high expression of HIF-1 a could inhibit the mitochondrial activity of the mitochondrial activity by inhibiting the mitochondrial activity by protease degradation of the c-Myc protein. Transcriptional factor A (TFAM) is an important transcription factor regulating mitochondrial transcription and mitochondrial DNA replication. C-Myc can directly bind to TFAM promoter and activate its expression, and the absence of TFAM activity is an important cause of mitochondrial activity downregulation. It is confirmed that the HIF-1 alpha -c-Myc-TFAM signal is involved in the regulation of mitochondria in the process of insect diapause. Activity. Insulin (insulin) signaling pathway is an important way to regulate development. Many studies have found that insulin signals play an important role in regulating and regulating insect diapause. In order to study the role of insulin signaling pathway in the diapause of cotton bollworm, the expression of insulin receptor (InR) in the chrysalis of cotton bollworm was cloned and investigated. The results found that In R was highly expressed in the diapause pupa brain, and the level of P-InR was significantly higher in the diapause pupa brain than in the developmental pupa brain; insulin was used to regulate P-ERK expression through P-InR; ROS could activate P-InR, P-AKT and P-ERK; and ROS to P-AKT and P-ERK activation was regulated. The regulation of proteasome during the diapause of Helicoverpa armigera leads to the degradation of related proteins in development and the smooth passage of pupae into diapause.
【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q963

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