苹果属植物独脚金内酯的鉴定及相关基因的克隆与功能分析

发布时间：2018-05-19 09:48

  本文选题：苹果 + 独脚金内酯　； 参考：《西北农林科技大学》2016年博士论文

【摘要】：独脚金内酯是一类新发现的植物激素,在植物的生长发育和逆境抵御等多个方面具有显著作用;同时独脚金内酯也具有环境信号物质的作用,在寄主与丛枝真菌的共生和根寄生植物的寄生关系中起着不可替代的作用。苹果(Malus domestica)是一类世界广泛栽植的水果作物,其生长和繁育主要依赖嫁接方式。砧木能够影响苹果接穗的生长发育以及对逆境的抵御。绝大多数苹果砧木与栽培苹果属于同一个属(Malus),亲缘关系相近。目前几乎未见独脚金内酯在苹果属植物中的相关报道。本文以苹果属野生砧木资源为材料,对独脚金内酯含量、外源独脚金内酯作用和独脚金内酯相关基因进行了研究。获得如下主要结果:1.苹果属植物中含有独脚金内酯,但不同种间含量有差异。利用苹果属植物组织提取液刺激列当(Orobanche)种子萌发来测定独脚金内酯含量。共选取苹果属9个种中24个品种或株系进行测试,植株被分为叶片、茎木质部、茎韧皮部和根系四个部分。所有测试品种或株系的组织提取液均能刺激列当种子萌发,表明测定的苹果属植物中都含有独脚金内酯。山定子(M.baccata)和小金海棠(M.xiaojinensis)诱导的列当种子萌发率高于其它物种诱导的萌发率,说明在这两个种中独脚金内酯含量要高于苹果属其他种。四种组织提取液中,木质部诱导的萌发率最高,根系与叶片次之,韧皮部最低,说明苹果属植物中独脚金内酯在木质部中含量最高。2.外源独脚金内酯(GR24)影响平邑甜茶(M.hupehensis)和M26(M.pumila)幼苗的多个生理过程。侧枝发育:去除平邑甜茶顶芽后,GR24能够直接抑制侧芽的生长;根系发育:GR24能够增加平邑甜茶主根的长度和根毛的数量,减少侧根的数量;种子萌发:GR24能够缓解高蔗糖浓度(30 g·L-1)下对平邑甜茶种子萌发的抑制,但不能提高无糖条件下的种子萌发率;叶片衰老:在平邑甜茶叶片发育晚期及受到苹果褐斑病菌侵染条件下,GR24加速叶片的衰老;组培苗发育:GR24能够提高M26组培苗植株高度,鲜重和叶绿素含量,减少叶片气孔数量,降低气孔的开张程度;丛枝真菌共生:在干旱条件下,GR24能够提高平邑甜茶与丛枝真菌(Glomus mosseae)的共生率。3.克隆与分析了平邑甜茶等苹果属植物的独脚金内酯相关基因。以平邑甜茶为主要材料,分别克隆了负责独脚金内酯合成的四个基因与和信号转导的两个基因。四个合成基因分别为类胡萝卜素顺反异构酶D27,类胡萝卜素裂解双加氧酶CCD7,类胡萝卜素裂解双加氧酶CCD8和细胞色素P450单加氧酶MAX1;两个信号转导基因分别为F-box泛素连接酶MAX2和α/β水解酶D14。通过基因克隆与基因组比对确定平邑甜茶中CCD8、MAX2和D14至少具有两个旁系同源基因,而MAX1至少存在三个旁系同源基因。组织表达分析表明两个合成基因CCD7与CCD8a主要在根系中表达,而信号转导基因MAX2在不同组织表达水平差异不大。此外我们克隆了CCD7、CCD8和MAX2基因的启动子区序列,并在启动子序列中预测到多种响应不同逆境的顺式作用元件。幼苗在干旱、水涝或茉莉酸甲酯处理后,CCD7与CCD8a表达发生了明显的下调;而在缺磷条件下,两个基因表现为表达上调;甲基紫精(MV)诱导的光氧化胁迫降低了MAX2b基因在叶片中的表达,但使其在根系中的表达水平增加。4.筛选了可能与独脚金内酯相关的基因或蛋白。利用酵母单杂交技术,确定了转录因子BPC1能够与CCD7启动子相结合;瞬时过量表达BPC1基因能够降低CCD7启动子的启动活性。转录因子WRKY75能够与CCD8a启动子相结合,可能参与对缺磷响应的调控。通过酵母双杂交与双分子荧光互补分析,确定MAX2b能够与CYP38蛋白相互作用,且CYP38又与质体中的多个蛋白具有相互作用。外源GR24处理后根系中多个质体蛋白的含量也发生了明显的上调。综上所述,我们确定苹果属植物中独脚金内酯合成与信号转导基因均能够转录,并且最终合成独脚金内酯。独脚金内酯调控苹果属植物的侧枝与根系发育、种子萌发、气孔发育、叶片衰老和丛枝真菌共生等多个方面。独脚金内酯合成基因受到干旱,水涝和茉莉酸甲酯等逆境的抑制表达和缺磷的促进表达。独脚金内酯可能对根系中质体内的一些蛋白具有调控作用。上述结果为今后独脚金内酯在苹果中的研究奠定了基础,也为独脚金内酯与逆境的相互作用提供了新的线索。
[Abstract]:The single legged gold lactone is a new type of plant hormone, which plays a significant role in plant growth and stress resistance, and it also plays an irreplaceable role in the symbiotic relationship between host and arbuscular mycorrhizal fungi and the parasitism of root parasites. Malus domestica ) it is a kind of widely cultivated fruit crop in the world. Its growth and breeding are mainly dependent on grafting. The rootstock can affect the growth and development of Apple scion and resistance to adversity. Most apple rootstocks and cultivated apples belong to the same genus (Malus). Related reports. In this paper, the content of single foot gold lactone, the action of exogenous gold lactones and the related genes of the single foot gold lactone were studied with the wild rootstock resources of the genus Malus. The main results were as follows: 1. the apple genus contained the single foot gold lactone, but the content of different species was different. Redon (Orobanche) seed germination was used to determine the content of goldianide. A total of 24 varieties or strains of 9 species in the genus Malus were selected. The plants were divided into leaves, stem xylem, stem phloem and root four parts. All the tissue extracts of the tested varieties or strains could stimulate the germination of Redon seeds, indicating the determination of the apple plants. The seed germination rate induced by M.baccata and M.xiaojinensis was higher than that of other species, indicating that the content of the single foot gold lactone in these two species is higher than that of other species of the genus Malus. In the four tissue extracts, the germination rate of the xylem is the highest and the root and the leaves are the same. The phloem is the lowest, indicating that the highest.2. content in the xylem of the genus Malus is the highest.2. exogenous phlol lactone (GR24) affects the multiple physiological processes of Pingyi sweet tea (M.hupehensis) and M26 (M.pumila) seedlings. Lateral branch development: after the removal of the top bud of Pingyi sweet tea, GR24 can directly inhibit the growth of the lateral buds; the root development: GR24 can increase the level of the plant. The length of the root and the number of root hairs to reduce the number of lateral roots; seed germination: GR24 can alleviate the inhibition of the germination of Pingyi sweet tea seeds under the high sucrose concentration (30 g. L-1), but can not improve the seed germination rate under the sugar free condition; leaf senescence: in the late development of the leaves of Pingyi sweet tea and under the infection condition of the apple brown spot pathogen, G R24 accelerated leaf senescence and tissue culture: GR24 could improve plant height, fresh weight and chlorophyll content in M26 tissue culture, reduce the number of leaf stomata, reduce the opening degree of stomata, and symbiosis of arbuscular mycorrhiza: under drought conditions, GR24 can improve the symbiotic rate of Pingyi sweet tea and Glomus mosseae,.3. cloning and analysis of Pingyi sweet Four genes responsible for the synthesis of monofoot gold lactones and two genes of signal transduction were cloned respectively in Pingyi sweet tea, respectively. The four synthetic genes were carotenoid CIS anti isomerase D27, carotenoid splitting double oxygenase CCD7, and carotenoid lysis. Oxygenase CCD8 and cytochrome P450 monooxygenase MAX1; two signal transduction genes are F-box ubiquitin ligase MAX2 and alpha / beta hydrolase D14. through gene cloning and genome alignment to determine CCD8, MAX2 and D14 in Pingyi sweet tea with at least two homologous genes, and MAX1 at least three homologous genes. Tissue expression analysis The expression of two synthetic genes, CCD7 and CCD8a, was mainly expressed in the root system, and the expression level of signal transduction gene MAX2 was not very different in different tissues. In addition, we cloned the sequence of the promoter region of CCD7, CCD8 and MAX2 genes, and predicted a variety of cis acting elements in the promoter sequence. The expression of CCD7 and CCD8a was obviously downregulated after the treatment of methyl lisinate, and the expression of the two genes was up regulated under the condition of phosphorus deficiency, and the expression of MAX2b gene in leaves was reduced by the oxidative stress induced by MV, but the expression level in the root system was increased by.4. to screen the genes associated with the monololide. Protein. Using yeast single hybridization technique, the transcriptional factor BPC1 can be combined with the CCD7 promoter; transient overexpression of BPC1 gene can reduce the promoter activity of the CCD7 promoter. The transcription factor WRKY75 can be combined with the CCD8a promoter and may participate in the regulation of the response to the phosphorus deficiency. It is determined that MAX2b can interact with CYP38 protein and that CYP38 is interacted with many proteins in the plastids. After exogenous GR24 treatment, the content of multiple plastid proteins in the root system has also been significantly up-regulated. The synthesis of mono difoot gold lactone. Monodontate regulates the development of lateral branches and roots, seed germination, stomatal development, leaf senescence and the symbiosis of arbuscular mycorrhizal fungi in the genus Malus. Some proteins in the root mass in the root system have a regulatory effect. These results provide a basis for the future research of the single foot gold lactone in apple, and provide a new clue for the interaction of the single foot gold lactone with adversity.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S661.1
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本文编号：1909634