辣根素熏蒸对玉米象线粒体作用机理研究

发布时间：2018-06-06 00:06

本文选题：辣根素 + 玉米象　；参考：《西北农林科技大学》2016年博士论文

【摘要】：储粮害虫一直是储粮过程中面临的重大难题,对农产品的数量以及质量都有严重影响,有效防治储粮害虫的新技术研究一直以来颇受各国科研人员的重视。玉米象作为重要的储粮害虫,在全世界范围内都对农产品储藏具有巨大威胁。磷化氢是现今世界范围内应用最广的熏蒸剂,在减少农产品储藏损失损耗和防治储粮害虫方面发挥了积极的作用。然而由于长期不合理的使用,导致储粮害虫对磷化氢的抗性问题已经严重限制了它们的应用,故寻找新型生物合理性熏蒸剂显得迫在眉睫。辣根素来源于十字花科植物的,是硫代硫酸酯类化合物的一种水解产物,对储粮害虫具有显著的熏蒸活性,具有进一步开发利用的价值。为了更高效合理的开发和利用辣根素作为熏蒸剂,本研究以玉米象为供试昆虫,从生物活性验证,组织病理学研究及生化与分子生物学水平,辣根素对玉米象线粒体结构与功能影响,研究了辣根素熏蒸引起的玉米象体内转录组水平的变化,并利用RNAi技术验证了线粒体基因nd5,nd6,cox1和cox5与辣根素对试虫熏蒸致死作用的关系,本研究所取得主要结果如下:1.测定了辣根素对玉米象的熏蒸活性,观察试虫中毒症状进行了及组织病理学水平的变化。结果表明:(1)在三角瓶熏蒸条件下,辣根素对玉米象试虫具有显著的熏蒸活性,LC50为0.74μg/ml(72h)。熏蒸放大研究也证明了辣根素具有实际应用价值。(2)辣根素熏蒸后,试虫的中毒症状主要分为:兴奋、痉挛、麻痹和死亡4个阶段。(3)透射电镜观察结果表明中毒试虫线粒体结构受到显著破损,线粒体内膜结构出现明显的空泡化。2.比较了辣根素和磷化钙熏蒸作用对玉米象试虫线粒体呼吸链及下游ROS的影响。结果表明:(1)在相同致死浓度下,辣根素与磷化钙熏蒸对玉米象试虫线粒体能产生类似的影响,对线粒体复合物I和IV都有一定的抑制作用。(2)辣根素和磷化钙熏蒸对试虫复合体IV的抑制作用基本一致,都伴随有剂量效应,LC25,LC50和LC75浓度处理下,AITC对线粒体复合体IV的抑制效果分别为20.04%,37.47%和54.32%,而磷化钙熏蒸所产生的抑制效果则分别为22.61%,50.65%和69.04%。同时两种化合物对复合体IV的抑制效果也伴随有时间效应,随着处理时间的增加,所产生的抑制效果越显著。而对复合体I的影响却不尽相同,辣根素熏蒸所产生的抑制作用伴随有剂量效应,LC25,LC50和LC75浓度处理下,复合体I的活性分别降低14.25%,17.17%和37.68%,而磷化钙熏蒸所产生的抑制作用却无明显的剂量效应。随着处理时间的增加,两种化合物对复合体I的抑制效果愈加显著。(3)辣根素熏蒸对线粒体呼吸链的抑制效果会引起试虫体内活性氧(Reactive oxygen species,ROS)的产生和累积,并对ROS相关酶系有不同的影响。其中过氧化氢酶(catalase,CAT)和过氧化物酶(Peroxidase,POD)活性在辣根素熏蒸后会受到抑制,而超氧化物歧化酶(Superoxidase,SOD)被激活。3.对玉米象试虫的转录组信息进行了研究,并对辣根素熏蒸引起的试虫体内转录组水平变化进行了测序及分析。结果表明:(1)在玉米象试虫转录组测序研究中,组装匹配后共得到31,041条独立基因(unigenes)。对辣根素熏蒸所引起的试虫体内差异表达基因进行了统计,共得到388个差异表达基因,其中表达量上调的基因有117个,表达量下调的占271个。(2)对差异表达基因进行GO聚类分析分析发现,这些差异表达基因可分配至38个GO terms,其中大部分集中在“Binding”,“Cell process”和“metabolic”。KEGG聚类分析发现大部分差异表达基因被富集至“Folding,sorting and degradation”,“Transport and catabolism”,“Energy metabolism”和“Carbohydrate metabolism”。(3)qRT-PCR研究发现,辣根素熏蒸能够降低试虫体内cat基因表达量,而cuznsod表达量却有显著提高。4.为验证编码呼吸链复合体I和IV的线粒体基因nd5,nd6,cox1和cox5在辣根素熏蒸致死过程中的作用,本研究采用了RNAi技术降低基因的表达量,测定辣根素对基因表达量下降试虫的熏蒸活性及下游ROS效应。结果表明:(1)nd5,cox1 and cox5 RNAi处理后,玉米象试虫的生存能力受到明显抑制,其中抑制试虫体内cox1基因表达量,其生存能力所受抑制最为显著,而降低试虫体内nd6基因表达量对试虫生存能力与未进行RNAi处理试虫相比,则无显著变化。(2)辣根素对cox1 RNAi处理试虫熏蒸活性较未经RNAi处理试虫降低19.07%,而对nd6 RNAi处理试虫熏蒸活性较未经RNAi处理试虫则提高25.53%。(3)辣根素熏蒸对试虫体内线粒体依赖型ROS有明显提高作用,而AITC熏蒸对RNAi处理试虫体内ROS也有不同的影响:其中cox1 RNAi处理试虫熏蒸后,体内ROS比未经RNAi处理试虫有所下降,但仍比未熏蒸试虫显著提高,而nd6RNAi处理试虫熏蒸后,体内ROS比未经RNAi处理试虫会进一步增加。综上所述,辣根素具有进一步开发为生物熏蒸剂的潜力,可以作为磷化氢的替代品。本研究推测辣根素对玉米象的熏蒸作用机理为:辣根素与线粒体复合体I和IV结合,并抑制其活性,导致氧还原不彻底,体内产生活性氧自由基,在CAT和POD活性受抑制,而SOD活性被提高的情况下,会进一步导致氧自由基的过度累积,从而引起线粒体膜脂过氧化,最终致死。其对各代谢途径的具体影响,仍有进一步研究价值。
[Abstract]:Grain storage pest has been a major problem in the process of grain storage. It has a serious impact on the quantity and quality of agricultural products. Research on the effective prevention and control of stored grain pests has been paid much attention by researchers in various countries. Corn, as an important stored grain pest, has a great threat to the storage of agricultural products worldwide. Hydrogen is the most widely used fumigant in the world today. It plays a positive role in reducing the loss of storage loss of agricultural products and preventing stored grain pests. However, because of the long-term irrational use, the resistance of stored grain pests to phosphine has seriously restricted its application. Therefore, a new biological fumigation is sought. It appears imminent. Horseradish is derived from the cruciferous plants and is a hydrolysate of thiosulfate compounds. It has significant fumigating activity to stored grain pests and has the value of further exploitation and utilization. In order to develop and utilize horseradish as a fumigant more efficiently and rationally, the corn elephant was used as a test insect. Biological activity verification, histopathological studies and biochemical and molecular biological levels, the effects of horseradish on the structure and function of maize mitochondria, and the changes in the transcriptional group level in maize images caused by horseradish fumigation were studied. The mitochondrial gene ND5, ND6, cox1, cox5 and horseradish were used to test the death of insect fumigation. The main results obtained in this study were as follows: 1. the fumigating activity of horseradish to corn was measured, and the symptoms of insect pests were observed and the histopathological changes were observed. The results showed that (1) the fumigating activity of horseradish to maize elephant test worms was significant under the condition of the fumigation of the triangulation, and LC50 was 0.74 mu g/ml (72h). The study also proved that horseradish has practical application value. (2) after the fumigating of horseradish, the poisoning symptoms of the insects are mainly divided into 4 stages: excitement, spasm, paralysis and death. (3) transmission electron microscope observation shows that the mitochondria structure of the poisoned insects is significantly damaged, the mitochondria structure of the mitochondria is obviously vacuolated and.2. is compared with horseradish and phosphorus The effect of calcium fumigation on the mitochondrial respiratory chain and downstream ROS of maize elephant insect. The results showed that: (1) at the same lethal concentration, horseradish and calcium phosphide fumigation could have a similar effect on the mitochondria of maize elephants, and had certain inhibitory effects on the mitochondrial complex I and IV. (2) horseradish and calcium phosphide fumigation for insect test complex The inhibitory effects of IV were basically consistent with the dose effect. The inhibitory effects of AITC on the mitochondrial complex IV were 20.04%, 37.47% and 54.32% respectively under the treatment of LC25, LC50 and LC75, while the inhibitory effects of calcium phosphide fumigation were 22.61%, 50.65% and 69.04%. at the same time, and the inhibition effect of two compounds on complex IV was also accompanied by the inhibition. With time effect, the inhibition effect is more significant with the increase of treatment time. The effect on complex I is not the same. The inhibitory effect of horseradish fumigation is accompanied by a dose effect. The activity of LC25, LC50 and LC75 decreases by 14.25%, 17.17% and 37.68%, respectively, and the inhibition of calcium phosphide fumigation. There was no obvious dose effect in the preparation. With the increase of treatment time, the inhibitory effect of two compounds on the complex I was more significant. (3) the inhibition effect of horseradish fumigation on mitochondrial respiratory chain could cause the production and accumulation of Reactive oxygen species (ROS) in the body, and had different effects on the related enzymes of ROS. The activity of catalase (CAT) and peroxidase (Peroxidase, POD) was inhibited after horseradish fumigation, and superoxide dismutase (Superoxidase, SOD) was activated by.3. to study the transcriptional group information of maize elephant worm, and the changes in the transcriptional level of the test worm induced by horseradish fumigation were sequenced and analyzed. The results were as follows: (1) 31041 independent genes (unigenes) were obtained after assembly and matching in the sequencing study of the maize elephant test insect transcriptional group. A total of 388 differentially expressed genes were obtained by horseradish fumigation, of which 117 genes were up-regulated and 271 were down regulated. (2) GO cluster analysis of differentially expressed genes found that these differentially expressed genes could be assigned to 38 GO terms, most of which were concentrated in "Binding", "Cell process" and "metabolic".KEGG clustering analysis found that most of the differentially expressed genes were enriched to "Folding, sorting and degradation". Bolism, "Energy metabolism" and "Carbohydrate metabolism". (3) the study of qRT-PCR found that horseradish fumigation could reduce the expression of cat gene in the body of the test worm, but the expression of CuZnSOD was significantly increased in.4. to verify the mitochondrial gene ND5 of I and IV in the respiratory chain complex. In this study, RNAi technique was used to reduce the expression of gene and determine the fumigating activity and downstream ROS effect of horseradish gene on the decrease of gene expression. The results showed that: (1) ND5, cox1 and cox5 RNAi treatment, the survival ability of maize elephant test worm was obviously inhibited, which inhibited the expression of cox1 gene in the body of the insect and its viability. There was no significant change in the inhibition of ND6 gene expression on the survival ability of the insect tested insect compared with that of the non RNAi treatment insect. (2) the fumigating activity of horseradish to cox1 RNAi treatment was 19.07% less than that without RNAi treatment, while the fumigation activity for ND6 RNAi treatment increased by 25.53 than that of the non RNAi treated worms. (3) (3) horseradish fumigation could significantly improve the mitochondrial dependent type of the worm in the body, and AITC fumigation had different effects on the ROS in the RNAi treated insect. After the cox1 RNAi treatment was fumigated with insect fumigation, the ROS in the body was lower than that without the RNAi treatment, but it was still significantly higher than that of the non fumigated insect, while the nd6RNAi treatment was fumigated with the insect. In this study, the mechanism of horseradish fumigation for maize images is that horseradish is combined with mitochondrial complex I and IV, and inhibits its activity, which leads to oxygen reduction. At the end, living oxygen free radicals in the body are inhibited in CAT and POD activities, while the activity of SOD is increased, which will further lead to excessive accumulation of oxygen free radicals, resulting in mitochondrial membrane lipid peroxidation and ultimately fatal. The specific effects on various metabolic pathways are still of further research value.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S379.5

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