發(fā)布時(shí)間：2018-05-06 15:04

  本文選題：茶樹(shù) + 休眠��； 參考：《西北農(nóng)林科技大學(xué)》2015年博士論文

【摘要】：茶是世界上消費(fèi)量最大的飲料,越來(lái)越多的研究發(fā)現(xiàn)適當(dāng)飲茶有助于身體健康。茶樹(shù)是多年生常綠木本植物,除熱帶茶區(qū)外,茶樹(shù)在秋季開(kāi)始形成休眠芽以應(yīng)對(duì)冬季的低溫脅迫。冬季休眠是茶樹(shù)的重要生存策略,并影響著茶葉生產(chǎn)及茶葉的經(jīng)濟(jì)價(jià)值。目前,對(duì)茶樹(shù)休眠機(jī)理的研究還鮮有報(bào)道。本研究以不同休眠狀態(tài)的茶樹(shù)腋芽轉(zhuǎn)錄組分析為中心,以生長(zhǎng)素和休眠相關(guān)基因在茶樹(shù)休眠中的作用為切入點(diǎn),深入揭示了茶樹(shù)越冬芽休眠的分子機(jī)理。主要研究結(jié)果如下:1.鑒定和篩選了在休眠誘導(dǎo)及激素處理等條件下表達(dá)穩(wěn)定的內(nèi)參基因。本研究利用6個(gè)試驗(yàn)處理共94個(gè)試驗(yàn)樣品檢測(cè)了11個(gè)內(nèi)參基因的表達(dá)穩(wěn)定性,并利用4種不同的分析軟件對(duì)所選基因的表達(dá)穩(wěn)定性進(jìn)行了綜合排序。結(jié)果顯示,茶樹(shù)目前常用的內(nèi)參基因Cs TUBULIN1、Cs ACTIN1、Cs18S r RNA1和Cs UBQ1在幾乎所有的試驗(yàn)處理中都表現(xiàn)較差。而Cs PTB1、Cs EF1、Cs SAND1、Cs CLATHRIN1和Cs UBC1是5個(gè)最穩(wěn)定的內(nèi)參基因,適合在不同試驗(yàn)處理中作為q RT-PCR分析的穩(wěn)定內(nèi)參。2.利用RNA-seq技術(shù)對(duì)處于不同休眠狀態(tài)的腋芽進(jìn)行了轉(zhuǎn)錄組分析。在不同休眠狀態(tài)腋芽轉(zhuǎn)錄組對(duì)比分析中獲得了16,125個(gè)差異表達(dá)基因,在生物學(xué)過(guò)程聚類中,這些差異表達(dá)基因主要集中在細(xì)胞過(guò)程、單有機(jī)體過(guò)程、代謝過(guò)程、生物學(xué)調(diào)控、應(yīng)激反應(yīng)等類別;在細(xì)胞成分聚類中,主要集中在細(xì)胞、細(xì)胞器、膜和小分子復(fù)合體等類別;在分子功能聚類中,主要集中在結(jié)合和催化活性、核酸結(jié)合轉(zhuǎn)錄因子活性、運(yùn)輸活性和結(jié)構(gòu)分子活性等類別。茶樹(shù)腋芽休眠轉(zhuǎn)換過(guò)程中顯著差異表達(dá)基因鑒定、基因探針富集分析及基因調(diào)控網(wǎng)絡(luò)關(guān)聯(lián)分析顯示,植物激素及休眠相關(guān)的MADS-BOXs和PEBP家族基因在茶樹(shù)越冬芽休眠形成和解除中發(fā)揮重要作用。3.研究了生長(zhǎng)素響應(yīng)因子Cs ARF1在茶樹(shù)越冬芽休眠中的作用。利用RACE技術(shù)克隆了一編碼820個(gè)氨基酸的生長(zhǎng)素響應(yīng)因子Cs ARF1。生物信息學(xué)分析顯示,Cs ARF1編碼蛋白具有ARFs家族保守的N端DNA結(jié)合域和C端二聚化結(jié)構(gòu)域,且中間區(qū)域富含谷氨酸、絲氨酸和亮氨酸,是一個(gè)定位于細(xì)胞質(zhì)的具有激活轉(zhuǎn)錄功能的可溶性蛋白。Cs ARF1在茶樹(shù)越冬芽深休眠和萌動(dòng)期表達(dá)量較高,表明該基因可能與茶樹(shù)越冬芽的休眠維持及解除密切相關(guān)。4.揭示了生長(zhǎng)素相關(guān)基因表達(dá)變化與茶樹(shù)越冬芽休眠形成及解除的聯(lián)系。為全面了解生長(zhǎng)素在茶樹(shù)休眠中的作用,我們選取12個(gè)具有代表性的生長(zhǎng)素相關(guān)基因,并檢測(cè)了他們?cè)诓铇?shù)不同休眠和生長(zhǎng)階段,以及在休眠誘導(dǎo)和生長(zhǎng)素拮抗劑處理?xiàng)l件下的表達(dá)變化。結(jié)果顯示,這些基因在不同條件或處理下都有顯著的表達(dá)差異。生長(zhǎng)素濃度測(cè)定結(jié)果也表明,茶樹(shù)頂芽和腋芽中的生長(zhǎng)素含量均在休眠形成時(shí)期明顯降低,而在萌芽階段迅速升高。以上結(jié)果進(jìn)一步證實(shí)生長(zhǎng)素信號(hào)通路在茶樹(shù)冬季芽休眠轉(zhuǎn)換過(guò)程中發(fā)揮重要作用。5.茶樹(shù)Cs FLOWERING LOCUS T(Cs FT)基因具有促進(jìn)開(kāi)花和調(diào)控生長(zhǎng)的雙重功能。Cs FT基因是PEBP家族中的重要成員,茶樹(shù)Cs FT基因的兩種轉(zhuǎn)錄本(Cs FTa,Cs FTb)被克隆,表達(dá)分析顯示該基因與茶樹(shù)休眠轉(zhuǎn)換密切相關(guān)。進(jìn)一步的功能驗(yàn)證表明,在楊樹(shù)中過(guò)表達(dá)Cs FTa基因可以強(qiáng)烈地促進(jìn)轉(zhuǎn)化植株早開(kāi)花,而Cs FTb基因過(guò)表達(dá)植株則抑制短日照誘導(dǎo)引起的生長(zhǎng)停止和休眠芽的形成。對(duì)蛋白結(jié)構(gòu)進(jìn)行分析發(fā)現(xiàn),Cs FT中的第100位氨基酸可能是與其他調(diào)控因子結(jié)合,進(jìn)而調(diào)控植物開(kāi)花的關(guān)鍵因子。6.茶樹(shù)Cs MADS-BOX基因參與茶樹(shù)休眠調(diào)控。茶樹(shù)中兩個(gè)Cs MADS-BOX基因被克隆,并進(jìn)行了表達(dá)分析和功能驗(yàn)證。結(jié)果顯示,Cs MADS-BOX1基因在休眠形成階段下調(diào)表達(dá),在休眠解除階段上調(diào)表達(dá)。在楊樹(shù)中過(guò)表達(dá)該基因抑制短日照誘導(dǎo)下的頂端生長(zhǎng)點(diǎn)的生長(zhǎng)停滯,但在低溫誘導(dǎo)條件下過(guò)表達(dá)植株快速停止生長(zhǎng)。這表明MADS-BOX1基因可能是調(diào)控茶樹(shù)頂端生長(zhǎng)點(diǎn)生長(zhǎng)且對(duì)低溫敏感的重要基因。
[Abstract]:Tea is the largest consumption of beverages in the world. More and more studies have found that proper tea drink helps health. Tea trees are perennial evergreen woody plants. Apart from the tropical tea area, tea trees begin to form dormant buds in autumn to cope with the low temperature stress in winter. At present, there are few reports on the mechanism of the dormancy of tea trees. In this study, the study of tea tree axillary buds in different dormant states is the center, the role of auxin and dormancy related genes in the dormancy of tea trees is the breakthrough point, and the molecular mechanism of the dormancy of the tea tree is revealed. The main results are as follows: 1. A stable internal reference gene was determined and screened under the conditions of dormancy induction and hormone treatment. In this study, 94 experimental samples were used to detect the stability of the expression of 11 internal reference genes, and the expression stability of the selected genes was synthetically ordered by 4 different analysis software. The results showed that the tea tree was present at present. The internal reference genes Cs TUBULIN1, Cs ACTIN1, Cs18S R RNA1 and Cs UBQ1 are poor in almost all of the experimental treatments. Cs PTB1. A transcriptional analysis of the axillary buds in the state of sleep. 16125 differentially expressed genes were obtained in the contrastive analysis of the axillary buds in different dormancy states. In the biological process clustering, these differentially expressed genes were mainly concentrated in the cell process, single organism process, metabolic process, biological control, stress response and so on. In the class, mainly concentrated in the categories of cells, organelles, membranes and small molecular complexes; in molecular functional clustering, the main focus is on binding and catalytic activity, nucleic acid binding transcription factor activity, transport activity, and structural molecular activity. The identification of significant differential expression in the process of axillary bud dormancy conversion in tea tree and gene probe enrichment analysis And gene regulation network association analysis showed that plant hormones and dormancy related MADS-BOXs and PEBP family genes play an important role in the formation and release of winter bud dormancy of tea tree.3. study the role of auxin response factor Cs ARF1 in the dormancy of tea tree overwintering buds. The use of RACE technology to clone a 820 amino acid auxin response The bioinformatics analysis of Cs ARF1. showed that the Cs ARF1 encoded protein had the conservative N terminal DNA binding domain and the C terminal dimerization domain of the ARFs family, and the intermediate region was rich in glutamate, serine and leucine, which was a soluble protein.Cs ARF1 with the activation function of the cytoplasm in the deep dormancy and germination of the tea tree overwintering buds. The gene expression was high, indicating that the gene may be closely related to the dormancy maintenance and removal of the winter buds of the tea tree..4. revealed the relationship between the changes of auxin related gene expression and the formation and release of the dormancy of the overwintering bud of the tea tree. In order to fully understand the role of auxin in the dormancy of tea tree, we select 12 representative auxin related genes. The results showed that there were significant differences in the expression of these genes under the conditions of dormancy induction and auxin antagonist treatment. The results also showed that the auxin concentration in the apical buds and axillary buds of the tea tree were all closed. The period of dormancy formation was significantly reduced, but it increased rapidly in the bud stage. The above results further confirmed that the auxin signaling pathway plays an important role in the winter bud dormancy transformation of tea tree..5. tea tree Cs FLOWERING LOCUS T (Cs FT) gene has dual function of.Cs FT gene, which promotes flowering and regulation growth, which is an important member of the PEBP family. Two transcripts of the Cs FT gene (Cs FTa, Cs FTb) of the tea tree were cloned. The expression analysis showed that the gene was closely related to the dormancy transformation of the tea tree. Further functional verification indicated that the overexpression of Cs FTa gene in Yang Shuzhong could strongly promote early flowering of the transformed plants, while the Cs FTb gene overexpressed plants inhibited the growth induced by short day illumination. The formation of stopping and dormant buds was analyzed. The analysis of protein structure showed that the 100th amino acids in Cs FT may be combined with other regulatory factors, and then regulate the key factor of plant flowering,.6. tea tree Cs MADS-BOX gene to participate in the regulation of tea dormancy. Two Cs MADS-BOX genes in the tea tree were cloned, and the expression analysis and functional verification were carried out. The results showed that the expression of Cs MADS-BOX1 gene was down regulated at the stage of dormancy formation, and the expression was up-regulated at the stage of dormancy release. The growth of the top growth point under the inhibition of Short Daylight induced by Yang Shuzhong overexpression was stagnant, but the overexpressed plant stopped growing rapidly under the low temperature induction. This suggested that the MADS-BOX1 gene might be the control of the top of the tea tree. An important gene that grows at the tip and is sensitive to low temperature.

【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】：2015
【分類號(hào)】：S571.1

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本文編號(hào)：1852782