本文選題：茶樹(shù) + 高溫脅迫　； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：茶樹(shù)[Camellia sinensis (L.) O. Kuntze]是一種多年生常綠木本植物,在全球范圍內(nèi)廣泛種植,由其葉片加工而成的'茶,飲品被譽(yù)為“世界三大非酒精飲料”之一。茶樹(shù)在其生命周期中經(jīng)常遭受各種環(huán)境脅迫的影響,其中高溫和干旱脅迫是影響茶樹(shù)生長(zhǎng)發(fā)育、茶葉產(chǎn)量和品質(zhì)的重要因素,且隨著全球氣候的變暖以及水資源的日益匱乏,高溫和干旱脅迫對(duì)茶樹(shù)造成的影響日趨嚴(yán)重。因此,提高茶樹(shù)高溫、干旱抗性、選育抗逆性茶樹(shù)品種是目前茶樹(shù)科研工作者面前艱巨而亟待解決的課題。然而,傳統(tǒng)的茶樹(shù)育種方法選育周期漫長(zhǎng)且難以控制育種方向以獲得理想表型,使茶樹(shù)遺傳改良工作進(jìn)展緩慢。所以,了解茶樹(shù)響應(yīng)高溫和干旱脅迫的分子機(jī)制,篩選抗性候選基因,進(jìn)而通過(guò)生物技術(shù)手段有針對(duì)性地改善茶樹(shù)目的性狀,對(duì)選育茶樹(shù)高溫、干旱抗性?xún)?yōu)良品種具有重要意義。針對(duì)上述科學(xué)問(wèn)題,本論文以茶樹(shù)良種'龍井長(zhǎng)葉'為試驗(yàn)材料,利用第二代高通量測(cè)序、生物信息學(xué)和分子生物學(xué)等技術(shù)手段深入研究了高溫、干旱脅迫下茶樹(shù)葉片在轉(zhuǎn)錄水平上的響應(yīng)機(jī)制,干旱脅迫影響茶樹(shù)葉片主要生物活性成分積累的分子機(jī)制,以及茶樹(shù)HistoneH1基因(CsHsis-H1)在逆境響應(yīng)中的功能機(jī)制,詳細(xì)的研究?jī)?nèi)容及結(jié)論如下:1.利用RNA-Seq技術(shù)分析比較了正常條件和高溫、干旱脅迫下茶樹(shù)葉片的轉(zhuǎn)錄響應(yīng)情況。結(jié)果顯示,高溫脅迫下大量編碼Ca2+結(jié)合蛋白的基因被下調(diào)表達(dá),而干旱脅迫處理下相應(yīng)差異表達(dá)基因(DEGs)中上調(diào)表達(dá)的比例較高,尤其是CIPK和CML家族基因,表明Ca2+信號(hào)途徑在茶樹(shù)響應(yīng)高溫和干旱脅迫過(guò)程中均發(fā)揮重要作用,但其分子調(diào)控機(jī)制存在一定差異。另外,六類(lèi)主要的逆境響應(yīng)轉(zhuǎn)錄因子(HSF、WRKY、AP2/EREBP、bZIP、NAC、MYB)在茶樹(shù)響應(yīng)高溫和干旱脅迫過(guò)程中亦存在不同的調(diào)控響應(yīng)模式,其中差異表達(dá)的HSF家族轉(zhuǎn)錄因子在高溫脅迫下幾乎均為上調(diào)表達(dá),而在干旱脅迫下更多的為下調(diào)表達(dá);差異表達(dá)的WRKY、bZIP、MYB、NAC家族轉(zhuǎn)錄因子在干旱脅迫下大量為上調(diào)表達(dá),而在高溫脅迫下幾乎均為下調(diào)表達(dá)。此外,高溫脅迫下HSPs基因被大量上調(diào)表達(dá),其主要依賴(lài)于HSFs的調(diào)控;干旱脅迫則誘導(dǎo)了大量AQPs基因的表達(dá),及部分HSPs基因表達(dá);而LEAs蛋白作為植物逆境脅迫中一類(lèi)重要功能蛋白,高溫干旱脅迫下其編碼的DEGs幾乎均為下調(diào)表達(dá)。2.觀(guān)察分析了干旱脅迫對(duì)茶樹(shù)表型、生理特性及主要生物活性成分積累的影響,結(jié)果表明干旱脅迫引起茶樹(shù)葉片脫水和枯萎,并導(dǎo)致葉片總多酚、游離氨基酸含量顯著下降及總黃酮含量的增加。同時(shí),HPLC分析表明,干旱脅迫下,茶樹(shù)葉片中兒茶素、咖啡堿、茶氨酸和某些游離氨基酸的含量均顯著減少,進(jìn)一步表明干旱脅迫嚴(yán)重影響了茶樹(shù)葉片中主要生物活性成分的積累。此外,基于RNA-Seq的分析數(shù)據(jù),我們獲得了干旱脅迫下茶樹(shù)葉片中與氨基酸代謝和次生物質(zhì)代謝相關(guān)的DEGs,特別是與兒茶素、咖啡堿和茶氨酸合成途徑相關(guān)的關(guān)鍵調(diào)控基因,而且這些關(guān)鍵調(diào)控基因的表達(dá)水平與HPLC分析的結(jié)果相一致,揭示了干旱脅迫影響茶樹(shù)葉片中主要生物活性成分積累的分子調(diào)控機(jī)制。3.通過(guò)qRT-PCR分析了低溫、高鹽、ABA、干旱和氧化脅迫下茶樹(shù)葉片中CsHis-H1基因的表達(dá)水平,結(jié)果顯示CsHis-H1被上述非生物脅迫誘導(dǎo)表達(dá),表明其參與了茶樹(shù)對(duì)多種非生物脅迫的響應(yīng)。通過(guò)農(nóng)桿菌介導(dǎo)法將CsHis-H1導(dǎo)入煙草植株中,經(jīng)PCR檢測(cè)和RT-PCR分析獲得兩個(gè)高表達(dá)量轉(zhuǎn)基因煙草植株。透射電鏡觀(guān)察顯示轉(zhuǎn)基因煙草的染色質(zhì)發(fā)生一定程度的凝聚,而這種變化沒(méi)有影響轉(zhuǎn)基因煙草正常的生長(zhǎng)和發(fā)育,結(jié)合系統(tǒng)發(fā)育樹(shù)分析結(jié)果表明CsHis-H1屬于Histone H1家族中的H1C和H1D變體,其主要參與了植物體對(duì)脅迫的響應(yīng)過(guò)程。對(duì)轉(zhuǎn)基因煙草的抗逆性分析顯示,低溫、高鹽、ABA、干旱和氧化脅迫處理下,轉(zhuǎn)基因煙草較野生型煙草具有更高的抗逆性,主要表現(xiàn)為更低程度上的枯萎和衰老,以及更高的Pn、Gs和Fv/Fm值,表明CsHis-H1可能是通過(guò)維持正常的光合效率以提高轉(zhuǎn)基因煙草的抗逆性。
[Abstract]:Tea tree [Camellia sinensis (L.) O. Kuntze] is a perennial evergreen woody plant, widely cultivated around the world and processed by its leaves. The tea is known as one of the world's three major non-alcoholic beverages. The tea tree is often affected by various environmental stresses during its life cycle, which is affected by high temperature and drought stress. Tea tree growth and development, tea yield and quality are important factors, and with the global warming and the increasing shortage of water resources, the influence of high temperature and drought stress on the tea tree is becoming more and more serious. Therefore, it is an arduous and urgent solution to improve the high temperature and drought resistance of tea trees and to select the resistance tea varieties. However, the traditional tea breeding method has a long period of breeding and difficult to control the breeding direction to obtain ideal phenotype, which makes the progress of genetic improvement of tea tree slow. Therefore, to understand the molecular mechanism of tea tree response to high temperature and drought stress and to screen the candidate genes for resistance, and then to improve the tea tree target by biological techniques. The characters are of great significance to the selection of high temperature and drought resistant varieties of tea trees. In this paper, the paper studies the transcriptional level of tea leaves under high temperature and drought stress by using the second generation high throughput sequencing, bioinformatics and molecular biology, using the good seed of Longjing long leaf as the test material. The molecular mechanism of drought stress on the accumulation of main bioactive components in tea leaves and the functional mechanism of the tea tree HistoneH1 gene (CsHsis-H1) in the response to adversity were studied in the response mechanism. The detailed contents and conclusions were as follows: 1. the transcription of tea leaves under normal conditions and high temperature and drought stress was analyzed by RNA-Seq technique. The results showed that the genes encoding a large number of Ca2+ binding proteins were downregulated under high temperature stress, while the proportion of the corresponding differential expression genes (DEGs) under drought stress was higher, especially the CIPK and CML family genes, indicating that the Ca2+ signaling pathway played an important role in the process of tea tree response to high temperature and drought stress. However, there are some differences in the molecular regulation mechanism. In addition, the six main types of stress response transcription factors (HSF, WRKY, AP2/EREBP, bZIP, NAC, MYB) also have different regulatory response modes in the process of tea tree response to high temperature and drought stress, in which the differential expression of HSF family transcription factors are almost up expression under high temperature stress, but in dry. Under drought stress, more expression of WRKY, bZIP, MYB, NAC family transcriptional factors were up-regulated under drought stress, but under the stress of high temperature stress, the expression of HSPs was almost down expression. In addition, the expression of HSPs gene was largely up-regulated under high temperature stress, which mainly depended on the regulation of HSFs, while drought stress induced a large number of AQPs genes. Expression, and some HSPs gene expression, and LEAs protein as a kind of important functional protein in plant stress stress, under the stress of high temperature and drought stress, almost all of the encoded DEGs was down regulated.2., and the effect of drought stress on the tea tree phenotype, physiological characteristics and accumulation of main bioactive components was analyzed. The results showed that the tea tree caused by drought stress. The contents of total polyphenols, free amino acids and total flavonoids were significantly decreased and the content of total flavonoids increased. Meanwhile, HPLC analysis showed that the content of catechin, caffeine, theanine and some free amino acids in the leaves of tea tree decreased significantly under drought stress, which further indicated that drought stress seriously affected the leaves of tea tree leaves. Accumulation of major bioactive components. In addition, based on RNA-Seq analysis, we obtained DEGs related to the metabolism of amino acids and secondary metabolites in tea leaves under drought stress, especially the key regulatory factors associated with catechin, caffeine and theanine synthesis pathway, and the expression level of these key regulatory genes. The results of HPLC analysis revealed the molecular regulation mechanism of drought stress on the accumulation of main bioactive components in tea leaves.3.. The expression level of CsHis-H1 gene in tea leaves under low temperature, high salt, ABA, drought and oxidative stress was analyzed by qRT-PCR. The results showed that CsHis-H1 was induced by aforementioned abiotic stress and indicated that CsHis-H1 was expressed by the aforementioned abiotic stress. It participates in the response of tea tree to a variety of abiotic stresses. By introducing CsHis-H1 into tobacco plants by Agrobacterium mediated method, two transgenic tobacco plants with high expression are obtained by PCR detection and RT-PCR analysis. Transmission electron microscopy shows that the chromatin of transgenic tobacco has a certain degree of condensation, and this change does not affect the transgenic tobacco. The normal growth and development of tobacco, combined with phylogenetic tree analysis, shows that CsHis-H1 belongs to the H1C and H1D variants in the Histone H1 family, which mainly participates in the response process of plant body to stress. Higher resistance, mainly manifested in a lower degree of Wilt and aging, and a higher Pn, Gs and Fv/Fm value, indicates that CsHis-H1 may improve the resistance of transgenic tobacco by maintaining normal photosynthetic efficiency.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S571.1

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