陕西地区粘虫越冬北界、体内抗寒物质变化及相关基因表达量研究

发布时间：2018-04-22 20:35

本文选题：粘虫 + 越冬北界　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：粘虫Mythimna separata是一种危害严重的多食性、暴食性、迁飞性农业害虫,每年由越冬区向非越冬区之间季节性往返迁飞习性是造成近年来在局部地区突然暴发成灾的主要因素。我国在20世纪六七十年代就确立了粘虫在我国东部地区的越冬北界为1月份0℃等温线(北纬33°),但随着气候的变化、种植结构的改变及我国经纬度跨度大、东西部温度差异大,越冬北界也有可能发生变化,而且粘虫在我国西北部地区的越冬北界也未见研究报道。昆虫能否在一个地方越冬是由环境温度和昆虫体内自身能够抵抗寒冷的生理生化物质决定的,这些抗寒物质也和相关调控基因的表达量有关。本研究于2015年和2016年,连续两年时间在我国陕西省汉中、安康及杨凌地区约北纬33°附近,每个地区间隔固定距离设点,将室内饲养的末龄粘虫采用笼罩实验于每年11月放置在固定点,至翌年3月每月调查粘虫的存活率。同时,每月定时调查取样,用生化技术测定了与越冬有关的粘虫体内水分、糖原、海藻糖和甘油三酯的含量变化;并采用分子生物学技术克隆了与粘虫抗寒物质相关的热激蛋白Hsp70、Hsp90、脂动激素AKH1、AKH2四个基因,通过荧光定量PCR检测了四种基因在越冬期不同时间段的相对表达量。本研究首次确定了粘虫在陕西地区的越冬北界,明确了越冬期间的抗寒物质变化规律和相关基因的表达调控机理,取得的主要研究结果如下:1、通过两年的粘虫越冬成活率调查实验,初步认为粘虫在陕西地区北纬33°是可以越冬的,但越冬存活率较低。2015年,汉中地区粘虫越冬存活率最高3.77%,安康地区粘虫越冬存活率最高4%,杨凌地区粘虫越冬存活率为0。2016年,汉中地区粘虫越冬存活率最高2.33%,安康地区粘虫越冬存活率最高1.83%,杨凌地区粘虫越冬存活率为0.5%。粘虫在越冬过程中存活率呈下降趋势,且在每次越冬结束时,汉中和安康地区均能发现已羽化的粘虫成虫。2、测定了两年间粘虫在整个越冬过程中每月水分、糖原、海藻糖和甘油三酯的含量变化。发现糖原和海藻糖、甘油三酯含量在粘虫越冬过程中迅速消耗。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),后期几乎不变。3、通过PCR克隆技术得到了Hsp70、Hsp90、AKH1和AKH2四个与上述抗寒物质相关的调控基因,基因保守区序列分别为:1962bp、2154bp、343bp和479bp。4、通过Real-time PCR技术检测了2016年越冬期间上述4种基因在越冬过程中的表达情况。发现粘虫热激蛋白Hsp70、Hsp90和脂动激素AKH1、AKH2在越冬过程中均有表达,但表达量不同。Hsp70基因表达量较低而Hsp90基因的表达量较高。在粘虫进入越冬期的12月,体内的AKH1基因表达量较低,1月份之后表达量逐渐升高。AKH2基因在越冬的12月就大量表达,翌年1月表达量下降,2、3月表达量又上升。5、研究发现粘虫越冬期间,Hsp70、Hsp90表达量均呈现升高趋势,体内糖脂含量则呈现下降趋势,说明热激蛋白Hsp70、Hsp90与粘虫的糖脂代谢为负调控模式。体内AKH1、AKH2表达量变化说明了粘虫通过调节自身糖脂代谢以抵御低温逆境,其中AKH2在粘虫越冬前期的大量表达导致了粘虫在越冬前期体内糖原的急剧下降,而AKH1在越冬后期大量表达则说明粘虫在越冬后期是动用甘油三脂作为能源物质的,这说明脂动激素AKH1、AKH2与糖脂代谢为正调控模式。本研究发现粘虫在陕西地区北纬33°能够越冬,明确了粘虫在越冬过程中体内相关抗寒物质以及体内Hsp70、Hsp90、AKH1、AKH2基因在粘虫越冬过程中的表达量变化过程。为今后进一步研究粘虫越冬抗低温逆境的分子机制奠定基础,同时也为从分子水平上揭示粘虫的生态适应性提供思路。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.4

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