番茄论文：人工miR159介导的番茄抗黄瓜花叶病毒遗传转化研究

番茄 论文 政研论文下载论文大学下载论文大学下载关于长拳的论文浙大论文封面下载 ：人工miR159介导的番茄抗黄瓜花叶病毒遗传转化研究 番茄论文:人工miR159介导的番茄抗黄瓜花叶病毒遗传转化研 究 【中文摘要】MicroRNA(miRNA)是一类长度在21,24nt的、广泛存在于真核生物中的非编码小RNA,它们通过与靶标mRNA的结合来发挥相应的负控调节等作用。miR159是一类高度保守的miRNA,因此人们推测它可能存在于所有的陆地植物中。初步研究表明,miR159可能与一些植物花药和种子的产生相关。已有报道发现番茄中的miR159在叶片中的本底表达量较高,但是它在番茄中的具体功能目前还是未知的。近年来关于小RNA介导的基因沉默相关研究较为热门,相关研究显示虽然与miRNA结构和功能相似的siRNA具有一定的基因沉默效应,但是由于其表达不够稳定、对靶标序列识别的专一性不足等缺点,其应用前景具有较大局限性和不确定性。而miRNA则由于其内源性、表达量比较高、与靶标结合更稳定和专一等优点,被认为是基因沉默研究的新热点。黄瓜花叶病毒(Cucumber mosaic virus,CMV)是较为典型的三分体正义单链(positive single strand RNA, +ssRNA)病毒。它具有较为广泛的宿主,主要引起寄主花叶、矮化、绿岛、褪绿、黄化等症状,从而严重影响了植物的正常生长发育。番茄(Lycopersicon esculentum Mill.)是人们日常生活中不可或缺的蔬菜型水果,提高其产量和品质的相关研究长期以来一直是热点。CMV也是引起的番茄病毒病的主要病因之一,每年因CMV造成的农业减产给经济发展和工农业生产带来了严重影响,因而研究与抗CMV相关的番茄遗传转化具 有十分重要的科研价值和经济意义。本研究结合上述miRNA介导的基因沉默、番茄抗病毒相关的转基因等研究热点,主要通过人工改造的miR159对番茄进行遗传转化,并在此过程中对各个操作条件进行优化,从而初步建立了人工miR159介导的番茄抗黄瓜花叶病毒遗传转化体系。本文的主要内容分为三部分:一、通过对受体番茄Micro Tom的组培快繁及抗生素耐受性培养条件的筛选,确定了最适的分化外植体、培养条件以及分化、生根培养基配方,从而建立了Micro Tom遗传转化研究的基础。其中分化培养基配方为:MS+0.25 mg/L IAA+2mg/L 6-BA;生根培养基配方为:MS+0.5 mg/L IBA;抗生素耐受性植株高效筛选浓度配比为:50mg/L卡那霉素+200mg/L头孢霉素。二、构建含人工miR159前体(pre-amiR159)的质粒pBI121- (pre-amiR159),再用该质粒分别转化农杆菌EHA105和番茄Micro Tom,筛得到抗性番茄再生植株若干,从而初步建立了番茄amiR159介导的遗传转化体系。其中质粒的构建 方法 快递客服问题件处理详细方法山木方法pdf计算方法pdf华与华方法下载八字理论方法下载 为:从CMV-Fny的RNA1上的ORF片段中筛选一段21bp的序列作为amiR159的靶标,根据此序列的互补片段 设计 领导形象设计圆作业设计ao工艺污水处理厂设计附属工程施工组织设计清扫机器人结构设计 出前体amiR159序列,人工合成前体amiR159的cDNA序列后,再将此序列连接到质粒pBI121上相应的位点。番茄的遗传转化过程主要为:通过用上述质粒转化的农杆菌侵染番茄外植体、在筛选性培养上筛选抗性再生植株等过程,初步筛选出抗性植株。在此实验过程中同时研究并优化了外植体预处理方法、农杆菌侵染浓度、农杆菌侵染时间等处理条件。三、通过对抗性番茄植株进行分子鉴定和相应的攻毒实验,得到转基因阳性植株抗CMV的综合数据,从而从抗CMV的功能性上验证了 amiR159介导的番茄遗传转化体系的有效性。分子鉴定主要是通过 PCR、Southern杂交方法鉴定抗性植株基因组DNA中的片段、RT-PCR 方法鉴定抗性植株中RNA的表达情况,从而确定转基因阳性番茄。攻 毒实验主要是对炼苗成功的转基因阳性番茄接种靶标病毒CMV-Fny, 并于一定时间后进行抗病效果的检测。通过与未接病毒的番茄比较, 发现转基因阳性番茄对于靶标病毒CMV-Fny具有一定的抗性,主要表 现在与普通番茄相比症状减轻或不感病、植株整体生长情况良好,等 等。综合这三部分的研究成果,本文初步筛选并建立了番茄amiR159 抗黄瓜花叶病毒的遗传转化体系。 【英文摘要】MicroRNAs (miRNAs) are 21,24 nucleotide(nt) long, non-coding small RNAs that exist in almost all eukaryotes. They act by binding to the target mRNAs to negatively regulate gene expression. MiR159 is a kind of highly conserved miRNA which may exist in almost all land plants. Preliminary studies showed that miR159 may have certain correlation with anther and seed germation in some plants. Some studies have already showed that the quantity of miR159 expressed in tomato leaves is relatively higher than other miRNAs, but its factual function in tomato is still unknown. In recently years, researches on siRNA-mediated gene silencing became a hot point, and related studies implied that although siRNAs had some effect in gene-silencing, but owe to its lack of stability in expression and insufficient accuracy in identifying its target sequence, there is still much deficiency and uncertainty in its further application. miRNAs are endogenous, which differ from the siRNAs in their origin, and they have the advantage such as high expression quantity, more stable and unique to the target RNAs compared with siRNAs, so they are thought to be the new focal point in gene silencing related researches.Cucumber mosaic virus (CMV) is a typical tripartite positive single strand RNA (+ssRNA) virus. It has a broad range of hosts and mainly causes host symptoms such as mosaic, dwarf, green islands, chlorisis, yellowing, etc. So it can disturb the normal growth and development of plants. Tomato (Lycopersicon esculentum Mill.) is an indispensable vegetable and fruit in people’s daily life, studies on improving its output and quality have been a hot point for a long time. CMV is one of the main causes that lead to the virus diseases in tomatoes, every year the reduction of output caused by CMV has brought serious influence on the nation’s economic development and on the agriculture and industry production, so the researches on tomatoes’genetic transformation related with CMV resistance has both important significance on scientific research and economics.In this research we intergrated the above researches such as miRNA mediated gene-silencing and anti-virus related transgene studies in tomato, and transformed the tomato by the mediation of artificial miR159. In this way we preliminarily established the anti-CMV transformation system mediated by amiR159 in tomato. The main work in this paper can be separated into three parts. Firstly, by studies on the tissue culture, and antibiotic sensitive selection culture concentration for the fast regeneration of transgenic receptor tomato—Micro Tom, , we found out the proper explant and culture conditions for callus and shoot induction, and we also found out the optimal culture media for callus and root induction. By these results we set up foundations for Micro Tom’s genetic transformation. The ingredient of the callus induction medium is: MS+0.25 mg/L IAA+2mg/L 6-BA, and for the root induction is: MS+0.5 mg/L IBA, while the antibiotic sensitive contration combinations for efficient selecting antibiotic-resistant seedlings is 50 mg/L kanamycin+200mg/L cephamycin. Secondly, we construct the plasmid containing the pre-artificial miR159 (pre-amiR159) named pBI121-(pre-amiR159), then we used the plasmid to transform agrobacterium EHA105 and tomato Micro Tom, and got some antibiotic plantlets, so that we build up the preliminary genetic transformation system mediated by amiR159 in tomato. The process for plasmid constructing is, choose a 21bp long sequence from the ORF in the RNA1 of CMV-Fny as the target of amiR159, and design the pre-amiR159 according to the sequence complementary to it, then synthesize the cDNA of the pre-amiR159 and connect it to the corresponding sites in the plasmid pBI121. The main process for the genetic transformation of tomatoes is, use the transformed agrobacterium to infect the explant , and select the antibiotic-resistant plantlets in antibiotic selection culture medium, etc. We got antibiotic-resistant plantlets in this process. In the course of antibiotic-resistant plantlets selections we investigated and optimized treatment conditions such as the pretreatment of the explant, the concentration of agrobacterium used in infection, the time used in agrobacterium infection, etc. Thirdly, by identifying the successfully transformed tomato from the antibiotic-resistant ones through molecular biology methods, then inoculate them with target virus, we got the comprehensive results for the transformed tomatoes’anti-CMV property. So we validated the effectiveness of the tomato transformation system in resisting CMV guided by amiR159. The molecular biology identifying process mainly refers to the PCR and Southern Bloting techniques in detecting the target sequence in the genome from antibiotic-resistant seedlings, the RT-PCR technique refers to the identifying of the target RNA in the antibiotic-resistant seedlings. In these processes we identified several transformed tomatoes. The virus inoculation experiment is to inoculate CMV-Fny to the transformed tomatoes, and test their anti-virus functions a few days later. By comparing with tomatoes not inoculated, we found that the transformed tomatoes showed resistance to the target virus CMV-Fny to some extent , and their main traits compared with the common tomatoes in that they had less symptoms or even not infected, the growth conditions of the whole plants are relatively better, etc. By integrating these three main results, we preliminarily selected and erected the genetic transformation system for the amiR159 mediated CMV resistance in tomato. 【备注】索购全文在线加好友:1.3.9.9.3.8848 同时提供论文写作一对一指导和论文发表委托服务 【关键词】番茄 组培扩繁 人工miRNA 黄瓜花叶病毒 遗传转化 【英文关键词】Lycopersicon esculentum Mill. regeneration through tissue culture artificial miRNA cucumber mosaic virus genetic transform 【目录】人工miR159介导的番茄抗黄瓜花叶病毒遗传转化研究 摘要 8-10 Abstract 10-12 第一章 文献综述 16-25 1.1 mi RNA 简介及植物miR159 的研究概况 16-19 1.2 植物的抗病毒及相关转基因研究进展 19-22 1.2.1 植物的病毒病以及抗病毒研究 19 1.2.2 植物的转基因研究概况及番茄遗传转化研究进展 19-21 1.2.3 植物抗病毒相关的转基因研究进展 21-22 1.3 植物转基因研 究的方法 22-23 1.3.1 农杆菌介导的遗传转化 22 1.3.2 DNA 直接导入的基因转化技术 22-23 1.3.3 花粉管通道转基因法 23 1.4 miRNA 在植物基因工程上的应用 研究进展 23-25 1.4.1 miRNA 与基因沉默 23-24 1.4.2 miRNA 与植物的转基因研究 24-25 1.5 研究目的及内容 25 第二章 番茄Micro Tom 品种的组培扩繁及抗生素耐受性 体系筛选 25-38 2.1 材料与实验方法 26-30 2.1.1 实 验材料 26 2.1.2 实验方法 26-30 2.1.2.1 外植体及种 子灭菌 26-27 2.1.2.2 外植体的筛选及愈伤、不定芽的诱导 27-28 2.1.2.3 再生植株的生根培养 28 2.1.2.4 再生 植株的炼苗移栽 28 2.1.2.5 再生植株的抗生素耐受性培养条 件筛选 28-29 2.1.2.6 Micro Tom 组培扩繁及抗生素筛选体系 设计 29 2.1.2.7 数据统计方法 29-30 2.2 结果与 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 30-37 2.2.1 适合于分化的外植体类型 30-31 2.2.2 诱导愈伤及不定芽的培养基配方 31-32 2.2.3 愈伤及不定芽 诱导方式 32-33 2.2.4 诱导生根的培养基配方 33-34 2.2.5 不同高度再生植株的生根情况 34 2.2.6 生根植株的炼苗移栽 34-35 2.2.7 适合本研究的抗生素耐受 性植株筛选培养基 35-37 2.3 小结与讨论 37-38 第三 章 番茄artificial miR159 遗传转化体系的建立 38-54 3.1 材料与实验方法 39-45 3.1.1 实验材料 39 3.1.2 实验 方法 39-45 3.1.2.1 CMV-Fny 上靶标序列的筛选 39-40 3.1.2.2 番茄amiR159 的初步构建 40 3.1.2.3 载体的选择及引物的设计 40-41 3.1.2.4 pBI121-amiR159 质 粒载体的构建 41-42 3.1.2.5 对人工改造的质粒载体的双酶 切鉴定 42 3.1.2.6 农杆菌的转化及扩增 42-44 3.1.2.7 质粒的提取及鉴定 44 3.1.2.8 植物受 体材料的准备 44 3.1.2.9 农杆菌侵染外植体及其共培养 44-45 3.1.2.10 再生植株的抗生素选择性筛选 45 3.2 结果与分析 45-53 3.2.1 番茄amiR159 的设计 45-46 3.2.2 番茄amiR159 的二级结构图 46 3.2.3 pBI1121-amiR159 的重组质粒的鉴定 46-47 3.2.4 质粒的双 酶切鉴定结果 47-48 3.2.5 农杆菌转化的鉴定结果 48 3.2.6 农杆菌侵染外植体条件的选择 48-51 3.2.6.1 外植体预培养处理方法 48-49 3.2.6.2 农杆菌浓度及菌液处理方法 49-50 3.2.6.3 农杆菌侵染时间 50 3.2.6.4 外植体与农杆菌的共培养 50-51 3.2.7 再 生植株的抗性表型筛选结果 51-53 3.3 小结与讨论 53-54 第四章 番茄遗传转化的分子鉴定及抗病毒功能分析 54-68 4.1 材料与实验方法 54-60 4.1.1 实验材料 54-55 4.1.2 实验方法 55-60 4.1.2.1 抗性再生植株的分子生物学鉴定 55 4.1.2.2 引物设计 55 4.1.2.3 CTAB 或试剂盒法提取植物基因组DNA 55-56 4.1.2.5 DNA 的PCR 扩增 56 4.1.2.4 总RNA 的提取 56-57 4.1.2.6 RT-PCR 扩增 57-58 4.1.2.7 Southern 斑点杂交 58-59 4.1.2.8 病毒接种 59 4.1.2.9 阳性植株接毒后生长情况统计 59 4.1.2.10 病情指数统计 59-60 4.2 结果与分析 60-66 4.2.1 抗性再生植株的分子鉴定结果 60-63 4.2.1.1 基因组DNA 提取及目的片段PCR 的结果 60-61 4.2.1.2 RT-PCR 结果 61 4.2.1.3 点杂交灵敏度检测结果 61-62 4.2.1.4 点杂交定性检测结果 62-63 4.2.2 阳性植株的综合抗病效果统计 63-66 4.2.2.1 植株生长情况 63-65 4.2.2.2 感病情况及病情指数统计 65-66 4.3 小结与讨论 66-68 总结 初级经济法重点总结下载党员个人总结TXt高中句型全总结.doc高中句型全总结.doc理论力学知识点总结pdf 68-70 参考文献 70-75 致谢 75-76 附录 76-82 攻读学位期间的研究成果 82