本文選題：辣根素 + 玉米象�。� 參考：《西北農(nóng)林科技大學》2016年博士論文

【摘要】：儲糧害蟲一直是儲糧過程中面臨的重大難題,對農(nóng)產(chǎn)品的數(shù)量以及質(zhì)量都有嚴重影響,有效防治儲糧害蟲的新技術研究一直以來頗受各國科研人員的重視。玉米象作為重要的儲糧害蟲,在全世界范圍內(nèi)都對農(nóng)產(chǎn)品儲藏具有巨大威脅。磷化氫是現(xiàn)今世界范圍內(nèi)應用最廣的熏蒸劑,在減少農(nóng)產(chǎn)品儲藏損失損耗和防治儲糧害蟲方面發(fā)揮了積極的作用。然而由于長期不合理的使用,導致儲糧害蟲對磷化氫的抗性問題已經(jīng)嚴重限制了它們的應用,故尋找新型生物合理性熏蒸劑顯得迫在眉睫。辣根素來源于十字花科植物的,是硫代硫酸酯類化合物的一種水解產(chǎn)物,對儲糧害蟲具有顯著的熏蒸活性,具有進一步開發(fā)利用的價值。為了更高效合理的開發(fā)和利用辣根素作為熏蒸劑,本研究以玉米象為供試昆蟲,從生物活性驗證,組織病理學研究及生化與分子生物學水平,辣根素對玉米象線粒體結構與功能影響,研究了辣根素熏蒸引起的玉米象體內(nèi)轉(zhuǎn)錄組水平的變化,并利用RNAi技術驗證了線粒體基因nd5,nd6,cox1和cox5與辣根素對試蟲熏蒸致死作用的關系,本研究所取得主要結果如下:1.測定了辣根素對玉米象的熏蒸活性,觀察試蟲中毒癥狀進行了及組織病理學水平的變化。結果表明:(1)在三角瓶熏蒸條件下,辣根素對玉米象試蟲具有顯著的熏蒸活性,LC50為0.74μg/ml(72h)。熏蒸放大研究也證明了辣根素具有實際應用價值。(2)辣根素熏蒸后,試蟲的中毒癥狀主要分為:興奮、痙攣、麻痹和死亡4個階段。(3)透射電鏡觀察結果表明中毒試蟲線粒體結構受到顯著破損,線粒體內(nèi)膜結構出現(xiàn)明顯的空泡化。2.比較了辣根素和磷化鈣熏蒸作用對玉米象試蟲線粒體呼吸鏈及下游ROS的影響。結果表明:(1)在相同致死濃度下,辣根素與磷化鈣熏蒸對玉米象試蟲線粒體能產(chǎn)生類似的影響,對線粒體復合物I和IV都有一定的抑制作用。(2)辣根素和磷化鈣熏蒸對試蟲復合體IV的抑制作用基本一致,都伴隨有劑量效應,LC25,LC50和LC75濃度處理下,AITC對線粒體復合體IV的抑制效果分別為20.04%,37.47%和54.32%,而磷化鈣熏蒸所產(chǎn)生的抑制效果則分別為22.61%,50.65%和69.04%。同時兩種化合物對復合體IV的抑制效果也伴隨有時間效應,隨著處理時間的增加,所產(chǎn)生的抑制效果越顯著。而對復合體I的影響卻不盡相同,辣根素熏蒸所產(chǎn)生的抑制作用伴隨有劑量效應,LC25,LC50和LC75濃度處理下,復合體I的活性分別降低14.25%,17.17%和37.68%,而磷化鈣熏蒸所產(chǎn)生的抑制作用卻無明顯的劑量效應。隨著處理時間的增加,兩種化合物對復合體I的抑制效果愈加顯著。(3)辣根素熏蒸對線粒體呼吸鏈的抑制效果會引起試蟲體內(nèi)活性氧(Reactive oxygen species,ROS)的產(chǎn)生和累積,并對ROS相關酶系有不同的影響。其中過氧化氫酶(catalase,CAT)和過氧化物酶(Peroxidase,POD)活性在辣根素熏蒸后會受到抑制,而超氧化物歧化酶(Superoxidase,SOD)被激活。3.對玉米象試蟲的轉(zhuǎn)錄組信息進行了研究,并對辣根素熏蒸引起的試蟲體內(nèi)轉(zhuǎn)錄組水平變化進行了測序及分析。結果表明:(1)在玉米象試蟲轉(zhuǎn)錄組測序研究中,組裝匹配后共得到31,041條獨立基因(unigenes)。對辣根素熏蒸所引起的試蟲體內(nèi)差異表達基因進行了統(tǒng)計,共得到388個差異表達基因,其中表達量上調(diào)的基因有117個,表達量下調(diào)的占271個。(2)對差異表達基因進行GO聚類分析分析發(fā)現(xiàn),這些差異表達基因可分配至38個GO terms,其中大部分集中在“Binding”,“Cell process”和“metabolic”。KEGG聚類分析發(fā)現(xiàn)大部分差異表達基因被富集至“Folding,sorting and degradation”,“Transport and catabolism”,“Energy metabolism”和“Carbohydrate metabolism”。(3)qRT-PCR研究發(fā)現(xiàn),辣根素熏蒸能夠降低試蟲體內(nèi)cat基因表達量,而cuznsod表達量卻有顯著提高。4.為驗證編碼呼吸鏈復合體I和IV的線粒體基因nd5,nd6,cox1和cox5在辣根素熏蒸致死過程中的作用,本研究采用了RNAi技術降低基因的表達量,測定辣根素對基因表達量下降試蟲的熏蒸活性及下游ROS效應。結果表明:(1)nd5,cox1 and cox5 RNAi處理后,玉米象試蟲的生存能力受到明顯抑制,其中抑制試蟲體內(nèi)cox1基因表達量,其生存能力所受抑制最為顯著,而降低試蟲體內(nèi)nd6基因表達量對試蟲生存能力與未進行RNAi處理試蟲相比,則無顯著變化。(2)辣根素對cox1 RNAi處理試蟲熏蒸活性較未經(jīng)RNAi處理試蟲降低19.07%,而對nd6 RNAi處理試蟲熏蒸活性較未經(jīng)RNAi處理試蟲則提高25.53%。(3)辣根素熏蒸對試蟲體內(nèi)線粒體依賴型ROS有明顯提高作用,而AITC熏蒸對RNAi處理試蟲體內(nèi)ROS也有不同的影響:其中cox1 RNAi處理試蟲熏蒸后,體內(nèi)ROS比未經(jīng)RNAi處理試蟲有所下降,但仍比未熏蒸試蟲顯著提高,而nd6RNAi處理試蟲熏蒸后,體內(nèi)ROS比未經(jīng)RNAi處理試蟲會進一步增加。綜上所述,辣根素具有進一步開發(fā)為生物熏蒸劑的潛力,可以作為磷化氫的替代品。本研究推測辣根素對玉米象的熏蒸作用機理為:辣根素與線粒體復合體I和IV結合,并抑制其活性,導致氧還原不徹底,體內(nèi)產(chǎn)生活性氧自由基,在CAT和POD活性受抑制,而SOD活性被提高的情況下,會進一步導致氧自由基的過度累積,從而引起線粒體膜脂過氧化,最終致死。其對各代謝途徑的具體影響,仍有進一步研究價值。
[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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S379.5

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