【作者】 张玉；

【导师】 刘永胜；

【作者基本信息】 合肥工业大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 植物在生长发育过程中可能受到一定的生物胁迫与非生物胁迫。植物响应非生物胁迫时发生一系列生理生化反应,以适应不同的环境变化。DNMTs是植株DNA甲基化过程中重要的甲基转移酶。目前关于植物体内DNA甲基过程中DNMT2的作用机制和功能性分析实验研究较少,因此,研究DNMT2基因在植物生长发育过程中或植物抗逆性过程中的功能和作用,对提高作物抗性和品质具有重要意义。本文以模式植物番茄（Solanum lycopersicum）为实验材料,通过NCBI数据库比对获得11种物种中DNMT2蛋白的氨基酸序列,然后通过DNMAN软件进行蛋白质保守域的分析,发现这些氨基酸序列具有相对比较保守的蛋白结构域。对这11种蛋白质氨基酸序列进行进化树分析和DNA甲基化结构域分析,结果表明番茄SlDNMT2蛋白与马铃薯St DNMT2蛋白的亲缘关系较近,同源性高达95.54%。同时构建SlDNMT2的多个植物表达载体进行一系列生理生化分析。首先,构建表达绿色荧光蛋白的融合表达载体p ART27-SlDNMT2-e GFP观察SlDNMT2蛋白的亚细胞定位情况,结果表明SlDNMT2蛋白定位于细胞核中。利用植物基因工程技术和RNAi干扰技术,构建RNAi转基因表达载体p BI121-SlDNMT2-si RNA,并通过农杆菌介导的侵染法,侵染番茄野生型（AC）受体,进而通过植物组织培养技术与分子生物学技术,最终成功得到转基因阳性植株,并利用Real-time PCR技术确定RNAi转基因植株中SlDNMT2基因已被下调。高盐、重金属污染以及干旱是影响植物生长发育的重要环境因素,为此我们分析了高盐、重金属以及干旱条件下转基因植株的生长发育状态。首先,我们分析了200 mmol/L Na Cl和300 mmol/L甘露醇胁迫处理条件下,野生型与转基因番茄植株的生长状态,结果发现在200 mmol/L盐胁迫和300 mmol/L甘露醇处理条件下,野生型番茄（AC）的生长发育的状态都比转基因植株更好,且鲜重具有显著性差异,这说明SlDNMT2 RNAi转基因植株对盐胁迫和甘露醇胁迫较敏感。其次,我们还分析了SlDNMT2 RNAi转基因植株在不同二价重金属离子胁迫条件下的生长发育情况,结果发现在Zn²⁺胁迫条件下,RNAi转基因植株生长状态较差,该结果表明RNAi转基因植株对Zn²⁺抗性较低。但是,在Fe²⁺胁迫条件下,RNAi转基因植株的根系较野生型番茄（AC）植株更发达,RNAi转基因植株根的总数目较野生型番茄（AC）更多,该结果表明RNAi转基因植株的根系对Fe²⁺抗性较高。最后,我们将土培种植的野生型番茄和SlDNMT2 RNAi转基因植株共同干旱处理10 d,结果发现转基因植株的生长状态与野生型相比更差,叶片卷曲程度更为严重,该实验结果进一步验证:与野生型番茄相比,RNAi转基因植株较不耐旱。进一步通过显微镜观察RNAi转基因植物和野生型植物叶片中气孔的数目,结果发现转基因植株叶片中气孔的分布较野生型更密集,这一结果说明:RNAi转基因植物不耐旱的原因可能与叶片中气孔数目的增多有关。据报道,拟南芥中TINY1基因、SAG1基因可能参与响应干旱胁迫,我们利用半定量技术分析了他们在番茄植株中的同源基因Sl TINY1、Sl SAG1的表达情况,结果显示:与野生型相比,Sl SAG1基因在转基因植株中的含量没有明显变化;但是Sl TINY1基因在转基因植株中的含量下调了。通过以上结果和数据分析发现SlDNMT2基因可能参与植物应对胁迫环境的应答途径,但是,具体调节机制有待后续进一步探究。该文为进一步分析SlDNMT2基因在DNA甲基化过程中的功能作用奠定了良好的科学基础,对进一步探究植物体内DNA甲基转移酶DNMT2基因作用的分子机制具有重要意义。为进一步研究遗传育种和改良作物以及植物对逆境胁迫的抗性提供了科学依据和实验基础。更多还原

【Abstract】 During the growth and development of plants,they may be subjected to certain biological and abiotic stresses.In response to abiotic stresses,plants undergo a series of physiological and biochemical reactions to adapt to different environmental changes.DNMTs are methyltransferases in DNA methylation.DNMT2 has a highly conserved domain in plants,but no catalytic activity.Nowadays,there are few experimental studies on the mechanism and functional analysis of DNMT2 of DNA methylation in plants.Therefore,studying the function and role of DNMT2 in the process of growth and development or stress resistance of plants is important and meaningful to improve crops（tomato）resistance and quality.In this paper,tomatoes,the model plant,are used as the material.And the amino acid sequences of DNMT2 protein in 11 species were obtained through NCBI database comparison.The analysis of the conserved protein domains by DNAMAN software revealed that these amino acid sequences had relatively conserved protein domains.The evolutionary tree analysis and DNA methylation domain analysis of the amino acid sequences of these 11 proteins show that the relationship between SlDNMT2 protein and St DNMT2 protein was close,and the homology was as high as 95.54%.Simultaneously,we construct multiple plant expression vectors of SlDNMT2 for a series of physiological and biochemical analysis.First,the fusion expression vector p ART27-SlDNMT2-e GFP was constructed to observe the subcellular localization of SlDNMT2.The results showed that the SlDNMT2 protein was localized in the nucleus.Using plant genetic engineering technology and RNAi interference technology,we construct the transgenic RNAi expression vector p BI121-SlDNMT2-si RNA,and infect the tomato through Agrobacterium-mediated infection method,and then through plant tissue culture technology and molecular biology technology,we successfully obtain the transgenic positive plants.Next,Real-time PCR technology was used to confirm that the SlDNMT2 gene had been down-regulated in the transgenic RNAi plants.High salt,heavy metal pollution and drought are important environmental factors during growth and development of plants.For this reason,we analyzed the growth and development status of transgenic plants under high salt,heavy metal and drought conditions.First,we treated wild-type tomatoes（AC）and transgenic tomato plants with200 mmol/L Na Cl and 300 mmol/L D-Mannitol,and the results showed that under 200mmol/L salt stress and 300 mmol/L D-Mannitol treatment,the growth and development status of wild type is better than that of transgenic plants,and there is a significant difference in their fresh weight,which indicates that SlDNMT2 RNAi transgenic plants are more sensitive to salt and D-Mannitol stress.Secondly,we also analyzed the growth and development of SlDNMT2 RNAi transgenic plants under different divalent heavy metal ion stress,and the results illustrated that under Zn²⁺stress conditions,the growth status of transgenic RNAi plants was poor compared with wild type.This result demonstrated that the transgenic RNAi plants were more resistant to Zn²⁺.However,under Fe²⁺stress,the roots of transgenic RNAi plants are more developed compared with wild type（AC）,and the total number of roots of transgenic RNAi plants is more than that of wild type（AC）.This result indicates that the roots of transgenic RNAi plants have a higher resistant to Fe²⁺stress.Finally,we treated the wild-type tomato and SlDNMT2 RNAi transgenic plants grown in soil culture for drought treatment for 10days,and the results showed that the growth status of the transgenic plants was worse and the leaves are more curled.These experimental results further verified,transgenic RNAi plants are less tolerant to drought stress than wild type.Observing the number of stoma in the leaves of transgenic RNAi plants through a microscope,it was found that the distribution of stoma in the leaves of transgenic plants was more densely compared with wild type.According to reports,the TINY1 and SAG1 genes may be involved in response to drought stress in Arabidopsis.We used semi-quantitative techniques to analyze the expression of their homologous genes named Sl TINY1 and Sl SAG1 in tomato.The results demonstrated that,compared with AC,there was no significant change about the content of Sl SAG1 gene in transgenic plants.However,the content of Sl TINY1 gene was down-regulated in transgenic plants.In summary,thanks to the above results and analysis of related data,it is found that the SlDNMT2 gene is likely to involve in the response pathway to deal with stress in plants,but the specific regulatory mechanism needs to be further investigated.This article lays a good scientific foundation for further analysis of the functional role of the SlDNMT2 gene in the process of DNA methylation,and is of great significance for further exploring the molecular mechanism of DNMT2 in plants.Meanwhile,it also provides a scientific and experimental basis for further studying genetic breeding and resistance of crop to stress.更多还原