本文選題：粘蟲 + 越冬北界　； 參考：《西北農(nóng)林科技大學》2017年碩士論文

【摘要】：粘蟲Mythimna separata是一種危害嚴重的多食性、暴食性、遷飛性農(nóng)業(yè)害蟲,每年由越冬區(qū)向非越冬區(qū)之間季節(jié)性往返遷飛習性是造成近年來在局部地區(qū)突然暴發(fā)成災的主要因素。我國在20世紀六七十年代就確立了粘蟲在我國東部地區(qū)的越冬北界為1月份0℃等溫線(北緯33°),但隨著氣候的變化、種植結(jié)構(gòu)的改變及我國經(jīng)緯度跨度大、東西部溫度差異大,越冬北界也有可能發(fā)生變化,而且粘蟲在我國西北部地區(qū)的越冬北界也未見研究報道。昆蟲能否在一個地方越冬是由環(huán)境溫度和昆蟲體內(nèi)自身能夠抵抗寒冷的生理生化物質(zhì)決定的,這些抗寒物質(zhì)也和相關(guān)調(diào)控基因的表達量有關(guān)。本研究于2015年和2016年,連續(xù)兩年時間在我國陜西省漢中、安康及楊凌地區(qū)約北緯33°附近,每個地區(qū)間隔固定距離設(shè)點,將室內(nèi)飼養(yǎng)的末齡粘蟲采用籠罩實驗于每年11月放置在固定點,至翌年3月每月調(diào)查粘蟲的存活率。同時,每月定時調(diào)查取樣,用生化技術(shù)測定了與越冬有關(guān)的粘蟲體內(nèi)水分、糖原、海藻糖和甘油三酯的含量變化;并采用分子生物學技術(shù)克隆了與粘蟲抗寒物質(zhì)相關(guān)的熱激蛋白Hsp70、Hsp90、脂動激素AKH1、AKH2四個基因,通過熒光定量PCR檢測了四種基因在越冬期不同時間段的相對表達量。本研究首次確定了粘蟲在陜西地區(qū)的越冬北界,明確了越冬期間的抗寒物質(zhì)變化規(guī)律和相關(guān)基因的表達調(diào)控機理,取得的主要研究結(jié)果如下:1、通過兩年的粘蟲越冬成活率調(diào)查實驗,初步認為粘蟲在陜西地區(qū)北緯33°是可以越冬的,但越冬存活率較低。2015年,漢中地區(qū)粘蟲越冬存活率最高3.77%,安康地區(qū)粘蟲越冬存活率最高4%,楊凌地區(qū)粘蟲越冬存活率為0。2016年,漢中地區(qū)粘蟲越冬存活率最高2.33%,安康地區(qū)粘蟲越冬存活率最高1.83%,楊凌地區(qū)粘蟲越冬存活率為0.5%。粘蟲在越冬過程中存活率呈下降趨勢,且在每次越冬結(jié)束時,漢中和安康地區(qū)均能發(fā)現(xiàn)已羽化的粘蟲成蟲。2、測定了兩年間粘蟲在整個越冬過程中每月水分、糖原、海藻糖和甘油三酯的含量變化。發(fā)現(xiàn)糖原和海藻糖、甘油三酯含量在粘蟲越冬過程中迅速消耗。但每年的越冬前期(12月到翌年2月)主要消耗糖原和海藻糖,且糖原消耗要比海藻糖快,每年的越冬后期(翌年2月至3月)主要消耗甘油三酯;水分含量在越冬前期就迅速下降(12月至翌年2月),后期幾乎不變。3、通過PCR克隆技術(shù)得到了Hsp70、Hsp90、AKH1和AKH2四個與上述抗寒物質(zhì)相關(guān)的調(diào)控基因,基因保守區(qū)序列分別為:1962bp、2154bp、343bp和479bp。4、通過Real-time PCR技術(shù)檢測了2016年越冬期間上述4種基因在越冬過程中的表達情況。發(fā)現(xiàn)粘蟲熱激蛋白Hsp70、Hsp90和脂動激素AKH1、AKH2在越冬過程中均有表達,但表達量不同。Hsp70基因表達量較低而Hsp90基因的表達量較高。在粘蟲進入越冬期的12月,體內(nèi)的AKH1基因表達量較低,1月份之后表達量逐漸升高。AKH2基因在越冬的12月就大量表達,翌年1月表達量下降,2、3月表達量又上升。5、研究發(fā)現(xiàn)粘蟲越冬期間,Hsp70、Hsp90表達量均呈現(xiàn)升高趨勢,體內(nèi)糖脂含量則呈現(xiàn)下降趨勢,說明熱激蛋白Hsp70、Hsp90與粘蟲的糖脂代謝為負調(diào)控模式。體內(nèi)AKH1、AKH2表達量變化說明了粘蟲通過調(diào)節(jié)自身糖脂代謝以抵御低溫逆境,其中AKH2在粘蟲越冬前期的大量表達導致了粘蟲在越冬前期體內(nèi)糖原的急劇下降,而AKH1在越冬后期大量表達則說明粘蟲在越冬后期是動用甘油三脂作為能源物質(zhì)的,這說明脂動激素AKH1、AKH2與糖脂代謝為正調(diào)控模式。本研究發(fā)現(xiàn)粘蟲在陜西地區(qū)北緯33°能夠越冬,明確了粘蟲在越冬過程中體內(nèi)相關(guān)抗寒物質(zhì)以及體內(nèi)Hsp70、Hsp90、AKH1、AKH2基因在粘蟲越冬過程中的表達量變化過程。為今后進一步研究粘蟲越冬抗低溫逆境的分子機制奠定基礎(chǔ),同時也為從分子水平上揭示粘蟲的生態(tài)適應性提供思路。
[Abstract]:The Mythimna separata is a major pest of multiple food, gluttony, and migratory agricultural pests, and the seasonal return and migration from the overwintering area to the non wintering area is the main factor causing sudden outbreaks in local areas in recent years. In China, in 60s and 70s twentieth Century, the more it was established in the eastern part of our country. The winter north boundary is 0 degrees centigrade isotherm in January (33 degrees north latitude), but with the change of the climate, the change of the planting structure and the longitude and latitude of our country are large, the temperature difference between the East and the west is great, and the north boundary of the wintering is likely to change. The temperature and the body itself can resist cold physiological and biochemical substances, and these cold resistant substances are also related to the expression of related regulatory genes. In 2015 and 2016, this study was attached for two years in Hanzhoung, Shaanxi, Shaanxi, Ankang and Yangling. The last aged armyworm was kept in a cage at a fixed point in November and investigated the survival rate of the armyworm every month in March the following year. At the same time, a monthly survey was conducted to determine the changes in the content of water, glycogen, trehalose and triglyceride in the body of the wintering related armyworm, and molecular biological techniques were cloned. Four genes of heat shock protein Hsp70, Hsp90, lipoid AKH1, AKH2, were detected by fluorescence quantitative PCR, and the relative expression of four genes in different periods of wintering period was detected by fluorescence quantitative PCR. The main research results are as follows: 1, through the investigation of the survival rate of the overwintering in Shaanxi, it is preliminarily believed that the 33 degree of the wintering can be overwintered in the north latitude of Shaanxi, but the survival rate of the overwintering in the Hanzhoung area is 3.77%, and the survival rate of the wintering in the Ankang area is 4%, Yang Lingdi The survival rate of wintering is 0.2016 years, the highest survival rate of wintering in Hanzhoung area is 2.33%, and the survival rate of wintering in Ankang area is 1.83%. The survival rate of 0.5%. in Yangling area is decreasing during the winter, and at the end of each winter, both Hanzhoung and Ankang can find the formed armyworm. .2 was used to determine the changes in the content of water, glycogen, trehalose and triglyceride during the whole winter of the whole overwintering process. It was found that the content of glycogen and trehalose and triglyceride was consumed rapidly during the overwintering process. But each year was used to consume glycogen and trehalose in early winter (from December to February), and the consumption of glycogen was more than trehalose. Quickly, the main consumption of triglyceride in the late winter of the next year (February to March), the content of water decreased rapidly in the early period of the winter (December to February), and the latter almost invariable.3. Through PCR cloning technology, Hsp70, Hsp90, AKH1 and AKH2 related genes related to the cold resistant substances were obtained, and the sequence of gene conservative region was 1962bp, 2154bp, respectively. 343bp and 479bp.4, Real-time PCR technique was used to detect the expression of the above 4 genes during the wintering period during the winter of 2016. It was found that the heat shock protein Hsp70, Hsp90 and lipohormone AKH1, AKH2 were expressed during the overwintering process, but the expression amount was low and the expression of Hsp90 gene was higher. In the winter of December, the expression of AKH1 gene in the body was low. After January, the expression of.AKH2 gene increased gradually in the December of the winter. The expression in January of the next year decreased and the expression of 2,3 rose by.5. The study found that the expression of Hsp70 and Hsp90 showed an increasing trend during the winter of the next winter, and the content of glycolipid in the body showed a decline. It shows that the Hsp70, Hsp90 and the glycolipid metabolism of the heat shock protein are negative regulation mode. The changes in the expression of AKH1 and AKH2 in the body indicate that the armyworm resists low temperature stress by regulating its own glycolipid metabolism, and the large amount of expression of AKH2 in the early winter of the armyworm leads to the sharp decline of the glycogen in the body before the overwintering period, while AKH1 is in the late winter of the winter. A large number of expressions indicate that the insects use glycerol three fat as energy material in the late winter of the winter, which indicates that the lipoid AKH1, AKH2 and glycolipid metabolism are the positive regulation mode. This study found that the armyworm can be overwintered at 33 degrees north latitude in Shaanxi area, and clearly defined the cold resistant substances in the body and the body Hsp70, Hsp90, AKH1, AKH2 in the body during the overwintering process. The change process of the gene expression in the overwintering process of the armyworm lays the foundation for further study on the molecular mechanism of the anti low temperature stress of the wintering of the armyworm in the future, and also provides a way for revealing the ecological adaptability of the armyworm from the molecular level.

【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S433.4

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