KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 新疆野苹果 MsMYB44 转录因子响应腐烂病侵染分子机制初探 | |
| Alternative Title | The molecular mechanism of MsMYB44 in Xinjiang wild apple （Malus sieversii） in response to Valsa mali |
| 丁雨 | |
| Subtype | 硕士 |
| Thesis Advisor | 张道远 |
| 2020-06-30 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 北京 |
| Degree Discipline | 工程硕士 |
| Keyword | 新疆野苹果 MsMYB44 转录因子 腐烂病 功能鉴定 Xinjiang wild apple MsMYB44 transcript factor valsa canker functional identification |
| Abstract | 新疆野苹果（Malus sieversii(Ledeb.) Roem，又名塞威士苹果）是现代栽培苹果的祖先， 种质资源丰富，对生物及非生物胁迫具有较强抗性，不仅是珍贵的第三纪孑遗植物，更是栽培苹果品质改良的宝贵基因库。近二十年来， 我国新疆伊犁地区的新疆野苹果遭受过度人类干扰及病虫害的危害，种群面积急剧减少，而苹果腐烂病的爆发是导致该现象的重要原因之一。因此，系统深入研究新疆野苹果抗腐烂病机制，挖掘抗病优质基因并用于后续抗逆新种质培育有较为重要的意义。 本文主要是揭示新疆野苹果 MsMYB44 转录因子响应腐烂病侵染的分子机制。研究结果如下：（1） MsMYB 转录因子家族生物信息学分析及 MsMYB44 筛选克隆。 对腐烂病侵染的新疆野苹果转录组数据进行分析， 发现 MYB 转录因子在差异表达的转录因子中数量最多， 说明该转因子家族在野苹果响应腐烂病入侵过程中起重要作用。进一步对 MsMYBs 转录因子进行生物信息学分析，共发现 249 个 MsMYBs，其中受腐烂病诱导表达的 16 个，抑制表达的 23 个。对腐烂病菌侵染后上调表达的 MsMYBs 进行定量验证，结果与 RNA-seq 结果高度一致，说明了 RNA-seq 结果的准确性。克隆得到一条 MYB 转录因子，因其与拟南芥 AtMYB44 同源，命名为 MsMYB44。进化树分析显示该转录因子属于 MYB 转录因子家族中的 R2R3亚家族。RNA-seq 和 q-PCR 结果表明该转录因子在腐烂病侵染 0dpi 、1dpi、 2dpi、5dpi 的过程中持续上调表达，说明其可能在野苹果响应腐烂病侵染过程中发挥作用。对 MsMYB44 基因特性及抗病调控分子机制进行初步研究。利用酵母双杂实验证明 MsMYB44 不具有自激活活性， 分析其可能与其他蛋白质形成复合体发挥转录调控功能。进一步构建了腐烂病侵染的新疆野苹果核蛋白酵母文库， 并初步筛选得到与 MsMYB44 产生互作反应的 512 个阳性克隆。（3） 建立了基于新疆野苹果整株瞬时侵染方法的抗病基因快速筛选鉴定技术平台。该研究平台包括目标基因瞬时遗传转化、腐烂病原菌接菌、抗病相关表型统计（叶片和茎段）、 生理生化指标（叶绿素和 MDA）和抗病相关基因定量检测等一系列实验步骤， 并细化了实验方案、规范化了实验参数，使抗病相关基因功能筛选鉴定可缩短至 2 个月，具有短时、 高效的特点，为强抗病基因筛选获得及抗病分子机制研究奠定良好基础。申请发明专利 1 项。基于本平台，鉴定出在新疆野苹果响应腐烂病侵染的过程中， MsMYB44 转录因子起到负调控作用，是一个具有负调功能的抗病相关转录因子。 |
| Other Abstract | Xinjiang wild apple (Malus sieversii(Ledeb.) Roem, also called Seville apple),the ancestor of domesticated apple, possessed rich adverse-resistance genes andlooked as unique genepool for apple genetic improvement. It also looked as a precioustertiary relic plant and listed in the red book of China. However, the naturalpopulations of Xinjiang wild apples in Xinjiang Yili region had been experiencedgreatlly degradation due to outbreak of diseases and insect pests and over humaninterference in the past two decades. The valsa canker caused by Valsa mali pathogenis one of the most important reasons. Therefore, understanding the genes response andregulation, and its molecular resistance function after valsa canker infection is of greatimportance for resistant apple tree breeding and ecological restoration in the future.Here, we focus on MsMYB44 transcription factor and detected its response to valsacanker infection, and try to reveal the molecular regulation mechanism in Xinjiangwild apple tree. The research results are as follows:(1) Analysis of the transcriptome data of Xinjiang wild apples infected by valsacanker found that MsMYB transcription factors covers the largest number ofdifferentially expressed transcription factors, indicating that this family plays animportant role in the response of wild apples to valsa canker invasion. Bioinformaticsanalysis of the MYB family of Xinjiang wild apples found a total of 249 MsMYBstranscription factors, of which, 16 were induced by V. mali and 23 were suppressed.The MsMYBs transcription factor up-regulated after pathogen infection wasquantitatively verified, and the results were highly consistent with the RNA-seqresults, indicating the accuracy of the RNA-seq results. We cloned a MYBtranscription factor and named it MsMYB44 for its homology with ArabidopsisMYB44. Phylogenetic tree analysis showed that the transcription factor belongs to theR2R3 subfamily. MsMYB44 gene continue to up-regulated expression during theinfection of 0dpi, 1dpi, 2dpi, and 5dpi, suggesting that the gene may plays animportant role in the resistance of wild apples after pathogen infection.(2) The yeast two-hybrid experiment proved that MsMYB44 does not haveself-activating activity. Baiting through the wild apple nucleoprotein yeast librarywhich constructed after infecting by pathogen, more than 512 potential proteinsinteraction with MsMYB44 have been preliminary targeted. .(3) A rapid screening and disease-resistant gene function identification platformwas established (an patent of invention had been applied, with No:202010150434.5).The experimental procedures include transient genetic transformation of target genes,inoculation of valsa mali pathogens, disease-related phenotype statistics (leaf andstem segments), physiological and biochemical indicators detecting (chlorophyll andMDA) and disease-resistant genes marker correlation monitoring. After establishmentof platform, the disease-resistant genes rapid identification could be accelerated to 2monthes, with the characteristics of high-throughput and efficiency Use this platform,we identified a negatively-regulated transcript factors, MsMYB44, and probablellydownstream disease resistance genes regulated by MsMYB44, including ERF1B andMIEL1, were targeted by quantitative PT-PCR analysis |
| Subject Area | 生物工程 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/15414 |
| Collection | 中国科学院新疆生态与地理研究所 研究系统 |
| Affiliation | 中国科学院新疆生态与地理研究所 |
| First Author Affilication | 中国科学院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | 丁雨. 新疆野苹果 MsMYB44 转录因子响应腐烂病侵染分子机制初探[D]. 北京. 中国科学院大学,2020. |
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