發(fā)布時(shí)間：2018-05-19 09:48

  本文選題：蘋果 + 獨(dú)腳金內(nèi)酯��； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：獨(dú)腳金內(nèi)酯是一類新發(fā)現(xiàn)的植物激素,在植物的生長(zhǎng)發(fā)育和逆境抵御等多個(gè)方面具有顯著作用;同時(shí)獨(dú)腳金內(nèi)酯也具有環(huán)境信號(hào)物質(zhì)的作用,在寄主與叢枝真菌的共生和根寄生植物的寄生關(guān)系中起著不可替代的作用。蘋果(Malus domestica)是一類世界廣泛栽植的水果作物,其生長(zhǎng)和繁育主要依賴嫁接方式。砧木能夠影響蘋果接穗的生長(zhǎng)發(fā)育以及對(duì)逆境的抵御。絕大多數(shù)蘋果砧木與栽培蘋果屬于同一個(gè)屬(Malus),親緣關(guān)系相近。目前幾乎未見獨(dú)腳金內(nèi)酯在蘋果屬植物中的相關(guān)報(bào)道。本文以蘋果屬野生砧木資源為材料,對(duì)獨(dú)腳金內(nèi)酯含量、外源獨(dú)腳金內(nèi)酯作用和獨(dú)腳金內(nèi)酯相關(guān)基因進(jìn)行了研究。獲得如下主要結(jié)果:1.蘋果屬植物中含有獨(dú)腳金內(nèi)酯,但不同種間含量有差異。利用蘋果屬植物組織提取液刺激列當(dāng)(Orobanche)種子萌發(fā)來測(cè)定獨(dú)腳金內(nèi)酯含量。共選取蘋果屬9個(gè)種中24個(gè)品種或株系進(jìn)行測(cè)試,植株被分為葉片、莖木質(zhì)部、莖韌皮部和根系四個(gè)部分。所有測(cè)試品種或株系的組織提取液均能刺激列當(dāng)種子萌發(fā),表明測(cè)定的蘋果屬植物中都含有獨(dú)腳金內(nèi)酯。山定子(M.baccata)和小金海棠(M.xiaojinensis)誘導(dǎo)的列當(dāng)種子萌發(fā)率高于其它物種誘導(dǎo)的萌發(fā)率,說明在這兩個(gè)種中獨(dú)腳金內(nèi)酯含量要高于蘋果屬其他種。四種組織提取液中,木質(zhì)部誘導(dǎo)的萌發(fā)率最高,根系與葉片次之,韌皮部最低,說明蘋果屬植物中獨(dú)腳金內(nèi)酯在木質(zhì)部中含量最高。2.外源獨(dú)腳金內(nèi)酯(GR24)影響平邑甜茶(M.hupehensis)和M26(M.pumila)幼苗的多個(gè)生理過程。側(cè)枝發(fā)育:去除平邑甜茶頂芽后,GR24能夠直接抑制側(cè)芽的生長(zhǎng);根系發(fā)育:GR24能夠增加平邑甜茶主根的長(zhǎng)度和根毛的數(shù)量,減少側(cè)根的數(shù)量;種子萌發(fā):GR24能夠緩解高蔗糖濃度(30 g·L-1)下對(duì)平邑甜茶種子萌發(fā)的抑制,但不能提高無糖條件下的種子萌發(fā)率;葉片衰老:在平邑甜茶葉片發(fā)育晚期及受到蘋果褐斑病菌侵染條件下,GR24加速葉片的衰老;組培苗發(fā)育:GR24能夠提高M(jìn)26組培苗植株高度,鮮重和葉綠素含量,減少葉片氣孔數(shù)量,降低氣孔的開張程度;叢枝真菌共生:在干旱條件下,GR24能夠提高平邑甜茶與叢枝真菌(Glomus mosseae)的共生率。3.克隆與分析了平邑甜茶等蘋果屬植物的獨(dú)腳金內(nèi)酯相關(guān)基因。以平邑甜茶為主要材料,分別克隆了負(fù)責(zé)獨(dú)腳金內(nèi)酯合成的四個(gè)基因與和信號(hào)轉(zhuǎn)導(dǎo)的兩個(gè)基因。四個(gè)合成基因分別為類胡蘿卜素順反異構(gòu)酶D27,類胡蘿卜素裂解雙加氧酶CCD7,類胡蘿卜素裂解雙加氧酶CCD8和細(xì)胞色素P450單加氧酶MAX1;兩個(gè)信號(hào)轉(zhuǎn)導(dǎo)基因分別為F-box泛素連接酶MAX2和α/β水解酶D14。通過基因克隆與基因組比對(duì)確定平邑甜茶中CCD8、MAX2和D14至少具有兩個(gè)旁系同源基因,而MAX1至少存在三個(gè)旁系同源基因。組織表達(dá)分析表明兩個(gè)合成基因CCD7與CCD8a主要在根系中表達(dá),而信號(hào)轉(zhuǎn)導(dǎo)基因MAX2在不同組織表達(dá)水平差異不大。此外我們克隆了CCD7、CCD8和MAX2基因的啟動(dòng)子區(qū)序列,并在啟動(dòng)子序列中預(yù)測(cè)到多種響應(yīng)不同逆境的順式作用元件。幼苗在干旱、水澇或茉莉酸甲酯處理后,CCD7與CCD8a表達(dá)發(fā)生了明顯的下調(diào);而在缺磷條件下,兩個(gè)基因表現(xiàn)為表達(dá)上調(diào);甲基紫精(MV)誘導(dǎo)的光氧化脅迫降低了MAX2b基因在葉片中的表達(dá),但使其在根系中的表達(dá)水平增加。4.篩選了可能與獨(dú)腳金內(nèi)酯相關(guān)的基因或蛋白。利用酵母單雜交技術(shù),確定了轉(zhuǎn)錄因子BPC1能夠與CCD7啟動(dòng)子相結(jié)合;瞬時(shí)過量表達(dá)BPC1基因能夠降低CCD7啟動(dòng)子的啟動(dòng)活性。轉(zhuǎn)錄因子WRKY75能夠與CCD8a啟動(dòng)子相結(jié)合,可能參與對(duì)缺磷響應(yīng)的調(diào)控。通過酵母雙雜交與雙分子熒光互補(bǔ)分析,確定MAX2b能夠與CYP38蛋白相互作用,且CYP38又與質(zhì)體中的多個(gè)蛋白具有相互作用。外源GR24處理后根系中多個(gè)質(zhì)體蛋白的含量也發(fā)生了明顯的上調(diào)。綜上所述,我們確定蘋果屬植物中獨(dú)腳金內(nèi)酯合成與信號(hào)轉(zhuǎn)導(dǎo)基因均能夠轉(zhuǎn)錄,并且最終合成獨(dú)腳金內(nèi)酯。獨(dú)腳金內(nèi)酯調(diào)控蘋果屬植物的側(cè)枝與根系發(fā)育、種子萌發(fā)、氣孔發(fā)育、葉片衰老和叢枝真菌共生等多個(gè)方面。獨(dú)腳金內(nèi)酯合成基因受到干旱,水澇和茉莉酸甲酯等逆境的抑制表達(dá)和缺磷的促進(jìn)表達(dá)。獨(dú)腳金內(nèi)酯可能對(duì)根系中質(zhì)體內(nèi)的一些蛋白具有調(diào)控作用。上述結(jié)果為今后獨(dú)腳金內(nèi)酯在蘋果中的研究奠定了基礎(chǔ),也為獨(dú)腳金內(nèi)酯與逆境的相互作用提供了新的線索。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S661.1
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本文編號(hào)：1909634