温度诱导烟粉虱DNA甲基化的特征和Dnmt1基因温度耐受性功能的鉴定
发布时间：2018-11-07 09:08
【摘要】：烟粉虱MED隐种自入侵我国以来,迅速扩散至我国大部分地区,其在短时间内对不同地理环境的成功适应显示了其极强的生态适应性。本研究着眼于烟粉虱入侵的生态适应性机制,从温度耐受性差异的生态学现象出发,从表观遗传学的角度,研究不同温度胁迫处理下,烟粉虱入侵种MED隐种和本地种AsiaII3隐种各世代之间及雌雄之间的耐受性差异;在此基础上采用F-MSAP技术,探讨甲基化变化与温度耐受性变化的关系;最后通过RACE技术克隆烟粉虱3个隐种的BtDnmt1基因,并采用RNAi技术对该基因的温度耐受性功能进行了验证。主要结果与结论如下:1.烟粉虱温度胁迫耐受性具有可遗传性:(1)温度胁迫耐受性至少可以遗传1-3代;高温对烟粉虱耐受性的影响大于低温对耐受性的影响;耐热性的改变幅度高于耐寒性的改变幅度;雌虫的温度耐受性的遗传力高于雄虫温度耐受性的遗传性;(2)MED隐种对高温胁迫耐受性改变幅度高于本地AsiaII3隐种:其中MED隐种的亲一代(F1)的耐热性和子一代(F11)的耐寒性显著高于AsiaII3隐种。(3)不同产卵温度胁迫对烟粉虱耐受性影响显著:连续性温度胁迫诱导的MED隐种耐受性的改变幅度均显著高于非连续性的;且连续性胁迫诱导的MED隐种耐受性的改变在子代中具有更高的遗传力。2.烟粉虱温度胁迫的耐受性与DNA甲基化变化密切相关:(1)MED隐种不同温度胁迫处理组的不同世代的甲基化水平均低于AsiaII3隐种,但MED隐种总甲基化率均高于其对照组;AsiaII3隐种甲基化水平均却低于其对照组;(2)MED隐种和AsiaII3隐种不同发育历期的DNA甲基化水平和模式有差异;(3)温度胁迫下,卵期DNA甲基化水平显著高于其它发育阶段。连续性温度胁迫卵的甲基化率升高幅度大于非连续性温度胁迫。3.克隆得到了烟粉虱MED、MEAM1和AsiaII3三个隐种Dnmt1基因的全长c DNA序列:其推导氨基酸序列均具有Dnmt1蛋白的典型结构,即N端调控区的复制叉作用位点(Dnmt1-RFD)、锌指结构域(zf-CXXC)、两个BAH结构域及C端催化活性结构域Dcm。RNAi结果发现该基因表达量显著降低能显著影响烟粉虱响应温度胁迫的存活率,表明Dnmtl对烟粉虱温度耐受性起关键作用。综上所述,烟粉虱的耐受性改变可以遗传,且烟粉虱的耐受性变化与DNA甲基化变化密切相关,其中Dnmt1在烟粉虱的温度耐受性中起关键作用。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433
本文编号：2315873
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433
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