【摘要】：煙粉虱MED隱種自入侵我國以來,迅速擴(kuò)散至我國大部分地區(qū),其在短時(shí)間內(nèi)對不同地理環(huán)境的成功適應(yīng)顯示了其極強(qiáng)的生態(tài)適應(yīng)性。本研究著眼于煙粉虱入侵的生態(tài)適應(yīng)性機(jī)制,從溫度耐受性差異的生態(tài)學(xué)現(xiàn)象出發(fā),從表觀遺傳學(xué)的角度,研究不同溫度脅迫處理下,煙粉虱入侵種MED隱種和本地種AsiaII3隱種各世代之間及雌雄之間的耐受性差異;在此基礎(chǔ)上采用F-MSAP技術(shù),探討甲基化變化與溫度耐受性變化的關(guān)系;最后通過RACE技術(shù)克隆煙粉虱3個(gè)隱種的BtDnmt1基因,并采用RNAi技術(shù)對該基因的溫度耐受性功能進(jìn)行了驗(yàn)證。主要結(jié)果與結(jié)論如下:1.煙粉虱溫度脅迫耐受性具有可遺傳性:(1)溫度脅迫耐受性至少可以遺傳1-3代;高溫對煙粉虱耐受性的影響大于低溫對耐受性的影響;耐熱性的改變幅度高于耐寒性的改變幅度;雌蟲的溫度耐受性的遺傳力高于雄蟲溫度耐受性的遺傳性;(2)MED隱種對高溫脅迫耐受性改變幅度高于本地AsiaII3隱種:其中MED隱種的親一代(F1)的耐熱性和子一代(F11)的耐寒性顯著高于AsiaII3隱種。(3)不同產(chǎn)卵溫度脅迫對煙粉虱耐受性影響顯著:連續(xù)性溫度脅迫誘導(dǎo)的MED隱種耐受性的改變幅度均顯著高于非連續(xù)性的;且連續(xù)性脅迫誘導(dǎo)的MED隱種耐受性的改變在子代中具有更高的遺傳力。2.煙粉虱溫度脅迫的耐受性與DNA甲基化變化密切相關(guān):(1)MED隱種不同溫度脅迫處理組的不同世代的甲基化水平均低于AsiaII3隱種,但MED隱種總甲基化率均高于其對照組;AsiaII3隱種甲基化水平均卻低于其對照組;(2)MED隱種和AsiaII3隱種不同發(fā)育歷期的DNA甲基化水平和模式有差異;(3)溫度脅迫下,卵期DNA甲基化水平顯著高于其它發(fā)育階段。連續(xù)性溫度脅迫卵的甲基化率升高幅度大于非連續(xù)性溫度脅迫。3.克隆得到了煙粉虱MED、MEAM1和AsiaII3三個(gè)隱種Dnmt1基因的全長c DNA序列:其推導(dǎo)氨基酸序列均具有Dnmt1蛋白的典型結(jié)構(gòu),即N端調(diào)控區(qū)的復(fù)制叉作用位點(diǎn)(Dnmt1-RFD)、鋅指結(jié)構(gòu)域(zf-CXXC)、兩個(gè)BAH結(jié)構(gòu)域及C端催化活性結(jié)構(gòu)域Dcm。RNAi結(jié)果發(fā)現(xiàn)該基因表達(dá)量顯著降低能顯著影響煙粉虱響應(yīng)溫度脅迫的存活率,表明Dnmtl對煙粉虱溫度耐受性起關(guān)鍵作用。綜上所述,煙粉虱的耐受性改變可以遺傳,且煙粉虱的耐受性變化與DNA甲基化變化密切相關(guān),其中Dnmt1在煙粉虱的溫度耐受性中起關(guān)鍵作用。
[Abstract]:Bemisia Tabaci MED has spread rapidly to most parts of China since it invaded China. Its successful adaptation to different geographical environments in a short period of time has shown its strong ecological adaptability. This study focused on the ecological adaptive mechanism of whitefly invasion, based on the ecological phenomenon of temperature tolerance difference and epigenetics, studied under different temperature stress. Differences of tolerance between MED saphenous species and native AsiaII3 cryptic species as well as between male and female species of Bemisia Tabaci invasive species; On the basis of this, the relationship between methylation and temperature tolerance was studied by F-MSAP, and the BtDnmt1 gene of three species of whitefly was cloned by RACE, and the function of temperature tolerance of the gene was verified by RNAi. The main results and conclusions are as follows: 1. The tolerance of whitefly to temperature stress is hereditary: (1) tolerance to temperature stress can be inherited for at least 1-3 generations, the effect of high temperature on tolerance of whitefly is greater than that of low temperature. The change of heat tolerance was higher than that of cold tolerance, and the heritability of temperature tolerance of female was higher than that of male. (2) the change of tolerance of MED crypt to high temperature stress was higher than that of native AsiaII3 crypt. Among them, the heat tolerance of parent generation (F1) of MED crypt and the cold tolerance of progeny (F11) were significantly higher than that of AsiaII3 crypt. (3) different spawning temperature. Stress had a significant effect on the tolerance of Bemisia Tabaci: the change of tolerance of MED induced by continuous temperature stress was significantly higher than that of non-continuity. And the change of tolerance of MED cryptic species induced by continuous stress had higher heritability in offspring. 2. The tolerance of Bemisia Tabaci to temperature stress was closely related to the change of DNA methylation: (1) the methylation level of different generations of MED cryptic species was lower than that of AsiaII3 cryptic species, but the total methylation rate of MED cryptic species was higher than that of control group. The methylation level of AsiaII3 was lower than that of the control group, (2) the DNA methylation level and pattern of MED and AsiaII3 were different, and (3) under temperature stress, the level of DNA methylation in egg stage was significantly higher than that in other developmental stages. The methylation rate of continuous temperature stress was higher than that of discontinuous temperature stress. The full-length c DNA sequence of Dnmt1 gene of whitefly MED,MEAM1 and AsiaII3 was cloned. The deduced amino acid sequence of the c DNA sequence has the typical structure of Dnmt1 protein, that is, the Dnmt1-RFD of N-terminal regulatory region. Zinc finger domain (zf-CXXC), two BAH domains and C-terminal catalytic activity domain (Dcm.RNAi) showed that the expression of this gene significantly decreased the survival rate of Bemisia Tabaci in response to temperature stress. The results suggest that Dnmtl plays a key role in the temperature tolerance of Bemisia Tabaci. In conclusion, the change of tolerance of Bemisia Tabaci can be inherited, and the change of tolerance of Bemisia Tabaci is closely related to the change of DNA methylation. Dnmt1 plays a key role in the temperature tolerance of Bemisia Tabaci.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433

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