高温和干旱胁迫下茶树转录组分析及Histone H1基因的功能鉴定

发布时间：2018-05-04 00:28

本文选题：茶树 + 高温胁迫　；参考：《南京农业大学》2016年博士论文

【摘要】：茶树[Camellia sinensis (L.) O. Kuntze]是一种多年生常绿木本植物,在全球范围内广泛种植,由其叶片加工而成的'茶,饮品被誉为“世界三大非酒精饮料”之一。茶树在其生命周期中经常遭受各种环境胁迫的影响,其中高温和干旱胁迫是影响茶树生长发育、茶叶产量和品质的重要因素,且随着全球气候的变暖以及水资源的日益匮乏,高温和干旱胁迫对茶树造成的影响日趋严重。因此,提高茶树高温、干旱抗性、选育抗逆性茶树品种是目前茶树科研工作者面前艰巨而亟待解决的课题。然而,传统的茶树育种方法选育周期漫长且难以控制育种方向以获得理想表型,使茶树遗传改良工作进展缓慢。所以,了解茶树响应高温和干旱胁迫的分子机制,筛选抗性候选基因,进而通过生物技术手段有针对性地改善茶树目的性状,对选育茶树高温、干旱抗性优良品种具有重要意义。针对上述科学问题,本论文以茶树良种'龙井长叶'为试验材料,利用第二代高通量测序、生物信息学和分子生物学等技术手段深入研究了高温、干旱胁迫下茶树叶片在转录水平上的响应机制,干旱胁迫影响茶树叶片主要生物活性成分积累的分子机制,以及茶树HistoneH1基因(CsHsis-H1)在逆境响应中的功能机制,详细的研究内容及结论如下:1.利用RNA-Seq技术分析比较了正常条件和高温、干旱胁迫下茶树叶片的转录响应情况。结果显示,高温胁迫下大量编码Ca2+结合蛋白的基因被下调表达,而干旱胁迫处理下相应差异表达基因(DEGs)中上调表达的比例较高,尤其是CIPK和CML家族基因,表明Ca2+信号途径在茶树响应高温和干旱胁迫过程中均发挥重要作用,但其分子调控机制存在一定差异。另外,六类主要的逆境响应转录因子(HSF、WRKY、AP2/EREBP、bZIP、NAC、MYB)在茶树响应高温和干旱胁迫过程中亦存在不同的调控响应模式,其中差异表达的HSF家族转录因子在高温胁迫下几乎均为上调表达,而在干旱胁迫下更多的为下调表达;差异表达的WRKY、bZIP、MYB、NAC家族转录因子在干旱胁迫下大量为上调表达,而在高温胁迫下几乎均为下调表达。此外,高温胁迫下HSPs基因被大量上调表达,其主要依赖于HSFs的调控;干旱胁迫则诱导了大量AQPs基因的表达,及部分HSPs基因表达;而LEAs蛋白作为植物逆境胁迫中一类重要功能蛋白,高温干旱胁迫下其编码的DEGs几乎均为下调表达。2.观察分析了干旱胁迫对茶树表型、生理特性及主要生物活性成分积累的影响,结果表明干旱胁迫引起茶树叶片脱水和枯萎,并导致叶片总多酚、游离氨基酸含量显著下降及总黄酮含量的增加。同时,HPLC分析表明,干旱胁迫下,茶树叶片中儿茶素、咖啡碱、茶氨酸和某些游离氨基酸的含量均显著减少,进一步表明干旱胁迫严重影响了茶树叶片中主要生物活性成分的积累。此外,基于RNA-Seq的分析数据,我们获得了干旱胁迫下茶树叶片中与氨基酸代谢和次生物质代谢相关的DEGs,特别是与儿茶素、咖啡碱和茶氨酸合成途径相关的关键调控基因,而且这些关键调控基因的表达水平与HPLC分析的结果相一致,揭示了干旱胁迫影响茶树叶片中主要生物活性成分积累的分子调控机制。3.通过qRT-PCR分析了低温、高盐、ABA、干旱和氧化胁迫下茶树叶片中CsHis-H1基因的表达水平,结果显示CsHis-H1被上述非生物胁迫诱导表达,表明其参与了茶树对多种非生物胁迫的响应。通过农杆菌介导法将CsHis-H1导入烟草植株中,经PCR检测和RT-PCR分析获得两个高表达量转基因烟草植株。透射电镜观察显示转基因烟草的染色质发生一定程度的凝聚,而这种变化没有影响转基因烟草正常的生长和发育,结合系统发育树分析结果表明CsHis-H1属于Histone H1家族中的H1C和H1D变体,其主要参与了植物体对胁迫的响应过程。对转基因烟草的抗逆性分析显示,低温、高盐、ABA、干旱和氧化胁迫处理下,转基因烟草较野生型烟草具有更高的抗逆性,主要表现为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可能是通过维持正常的光合效率以提高转基因烟草的抗逆性。
[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.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S571.1

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