【摘要】：賴草是多年生禾本科根莖(根狀莖)型植物,是異形四倍體物種(2n=4x=28)。分蘗是單子葉植物一種獨(dú)特的分枝現(xiàn)象,在發(fā)育生物學(xué)上具有特殊的意義。通過(guò)根莖逐級(jí)分蘗即克隆生長(zhǎng)進(jìn)行無(wú)性繁殖的根莖類禾草是草原牧草種群及群落結(jié)構(gòu)的重要組成部分。很多優(yōu)良牧草都具有地下根莖,通過(guò)不斷分蘗進(jìn)而形成縱橫交錯(cuò)的地下根莖網(wǎng)及與之對(duì)應(yīng)的地上子代分株。賴草根莖頂芽可呈現(xiàn)水平和垂直兩種生長(zhǎng)方向性,本研究主要運(yùn)用RNA-seq技術(shù)探討了根莖頂芽不同生長(zhǎng)狀態(tài)下發(fā)育調(diào)控關(guān)聯(lián)基因及可能的調(diào)控通路。結(jié)合形態(tài)解剖學(xué)分析和關(guān)鍵基因驗(yàn)證檢測(cè)分析,試圖初步揭示賴草根莖頂芽發(fā)育基因調(diào)控機(jī)理及內(nèi)在分子機(jī)制。主要研究結(jié)果如下：(1)賴草水平根莖頂芽和垂直根莖頂芽轉(zhuǎn)錄組測(cè)序總共測(cè)得了49.01Gb的數(shù)據(jù),每個(gè)樣品Clean Data值都在7.84Gb以上,Q30堿基百分比均≥94.76%。組裝后共獲得了76098條Unigene。在各個(gè)數(shù)據(jù)庫(kù)共注釋到44769條Unigene。對(duì)Unigene庫(kù)的基因結(jié)構(gòu)分析,發(fā)現(xiàn)SSR標(biāo)記5397個(gè)。KEGG注釋結(jié)果提示垂直生長(zhǎng)根莖頂芽在生長(zhǎng)方向由水平延伸轉(zhuǎn)為逆地生長(zhǎng)后(垂直),盡管還未伸出地面,但光合相關(guān)功能基因已經(jīng)提早啟動(dòng)表達(dá)。(2)結(jié)果表明賴草水平根莖尖和垂直根莖尖在基因表達(dá)水平上總共有4906個(gè)差異基因,其中1970個(gè)基因在水平根莖中是上調(diào),2936個(gè)基因在垂直根莖中是上調(diào)。經(jīng)過(guò)KEGG注釋發(fā)現(xiàn)這些差異基因在以下幾個(gè)通路中差異表達(dá)顯著:光合作用;光合作用天線蛋白;亮氨酸代謝;谷胱甘肽代謝;生物素代謝;谷胱甘肽生物合成過(guò)程。在賴草水平根莖和垂直根莖4906個(gè)差異基因里,通過(guò)基因注釋,發(fā)現(xiàn)其中含有32個(gè)轉(zhuǎn)錄因子,132個(gè)激酶。其中,有很多轉(zhuǎn)錄因子和激酶都與根莖的生長(zhǎng)發(fā)育和植物抗逆性相關(guān),這一預(yù)測(cè)結(jié)果提示:賴草水平根莖頂芽在株系擴(kuò)展、延伸生長(zhǎng)過(guò)程中,可能首先感知到生物或非生物脅,并啟動(dòng)相應(yīng)應(yīng)激代謝過(guò)程;同時(shí),這一結(jié)果也反映出水平延伸根莖頂芽與逆地生長(zhǎng)的垂直根莖頂芽相比,應(yīng)激反饋更復(fù)雜。(3)為驗(yàn)證RNA-seq的結(jié)果,我們利用實(shí)時(shí)熒光定量PCR技術(shù)對(duì)其進(jìn)行了檢測(cè),我們隨機(jī)挑選了12個(gè)差異基因進(jìn)行qRT-PCR分析,測(cè)得的結(jié)果跟RNA-Seq測(cè)序結(jié)果高度一致,說(shuō)明測(cè)序所得的數(shù)據(jù)準(zhǔn)確性很高,信息學(xué)分析的結(jié)果可信。(4)通過(guò)對(duì)賴草根莖尖進(jìn)行樹(shù)脂切片發(fā)現(xiàn):賴草水平根莖跟垂直根莖橫切片相比,水平根莖表皮層細(xì)胞較大,細(xì)胞壁較厚,組成薄壁組織環(huán)的薄壁細(xì)胞層數(shù)較多,維管束環(huán)數(shù)較多。這可能跟它們即將行使的生理功能有關(guān),水平根莖在沙土中生長(zhǎng),需要穿過(guò)層層阻礙向前方生長(zhǎng),所以水平根莖尖有芽被保護(hù)。目前,利用生物信息學(xué)技術(shù)如RNA-seq對(duì)賴草屬的根莖生長(zhǎng)狀況的研究幾乎還處于零階段。研究賴草轉(zhuǎn)錄組學(xué)為全面了解賴草生長(zhǎng)發(fā)育、生理功能和抗逆相關(guān)機(jī)制提供了基礎(chǔ)。本課題通過(guò)Illumina Hiseq2500對(duì)賴草水平生長(zhǎng)根莖和垂直生長(zhǎng)根莖進(jìn)行轉(zhuǎn)錄組測(cè)序,給挖掘賴草根莖生長(zhǎng)發(fā)育相關(guān)基因、相關(guān)代謝通路和抗逆相關(guān)基因等奠定了基礎(chǔ)。
[Abstract]:Rhizomes of perennial grasses (rhizomes) and heteromorphic species (2n=4x=28). The tillering is a unique branching phenomenon of the mono cotyledon, and has special significance in developmental biology. The Rhizomatous Grasses of the rhizomatous growth of clonal growth through the rhizomatous tillering and clone growth are the grassland herbage population and community structure. An important part. Many excellent herbage have underground rhizomes, through continuous tillering to form a crisscross subterranean rhizome net and corresponding subsurface subplant. The top bud of Leymus rhizome can present two kinds of growth direction. This study mainly uses RNA-seq technique to explore the different growth conditions of the root bud. The regulation mechanism and internal molecular mechanism of the apical bud development of Leymus Leymus were preliminarily revealed by morphological and anatomical analysis and key gene validation detection analysis. The main results were as follows: (1) a total of 49.01 of the root buds of rhizomes and vertical root buds of Leymus Leymus were measured in total. Gb data, each sample Clean Data value is more than 7.84Gb, Q30 base percentage is more than 94.76%. assembly, a total of 76098 Unigene. in each database commented to 44769 Unigene. to the Unigene library gene structure analysis, found SSR marker 5397.KEGG annotation results show that the vertical growth root bud in the direction of growth from the level The extension turned back to the back growth (vertical), although the ground was not extended yet, the photosynthetic function gene had been started early. (2) the results showed that there were 4906 different genes in the level of rhizome and vertical rhizome of Leymus Leymus at the level of gene expression, of which 1970 genes were up-regulated in the horizontal rhizomes and 2936 genes were in the vertical rhizomes. It was up to up. After KEGG annotation, it was found that these differentially expressed genes were significantly expressed in the following pathways: photosynthesis; photosynthetic antenna protein; leucine metabolism; glutathione metabolism; biotin metabolism; glutathione biosynthesis. In the 4906 differential genes in the rhizomes and vertical rhizomes of Leymus Leymus, the findings were found by gene annotation. There are 32 transcription factors and 132 kinases. Among them, many of the transcription factors and kinases are related to the growth and stress resistance of the rhizomes. This prediction suggests that the apical bud of the rhizome of Leymus Leymus is extended in the plant line, and may first perceive raw or abiotic stresses and initiate corresponding stress metabolic processes. The results also showed that the stress feedback was more complex compared to the vertical rhizome buds of the horizontal extension rhizome and the reverse ground. (3) to test the results of RNA-seq, we used real-time fluorescence quantitative PCR to detect it. We randomly selected 12 differential causes for qRT-PCR analysis, and the results were sequenced with RNA-Seq sequencing. The results of the results were very high, indicating that the data obtained by sequencing were very accurate and the results of informatics analysis were credible. (4) through the resin section of the stem apex of the root, it was found that the horizontal rhizomes of the Leymus Leymus were larger, the cell wall was thicker than the vertical rhizome section, and the number of parenchyma cells in the parenchyma ring was more, and the vascular bundle was in the vascular bundle. There are more rings. This may be related to the physiological functions they are about to exercise. The horizontal rhizomes grow in the sandy soil and need to pass through the layers of hindrance to the front. So the tip of the horizontal rhizome is protected. At present, the study of the root growth state of the rhizome of the genus lyris by bioinformatics, such as RNA-seq, is almost zero. The transcript provides a basis for a comprehensive understanding of the growth, physiological function and anti inverse mechanism of Leymus Leymus. Through Illumina Hiseq2500, the transcriptional sequence of the rhizomes and vertical growth rhizomes of Leymus Leymus was carried out, which laid the foundation for the mining of the related genes, related metabolic pathways and resistance related genes in the rhizome of Leymus.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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