【摘要】：蘭科植物是最進(jìn)化的單子葉植物類群,分布區(qū)域廣,生態(tài)環(huán)境多樣,形態(tài)、結(jié)構(gòu)和生理特性均高度特異,是進(jìn)行花發(fā)育研究的理想材料。寒蘭(Cymbidium kanran)隸屬于蘭科(Orchidaceae)蘭屬(Cymbidium),是國蘭家族的重要成員,具有唇瓣形態(tài)變異多樣,四季開花和花期長(zhǎng)等特點(diǎn),被譽(yù)為“蘭中之王”。然而,包括寒蘭在內(nèi)的蘭科植物從種子到開花所需時(shí)間非常長(zhǎng),嚴(yán)重地制約了花發(fā)育的相關(guān)研究以及資源開發(fā)。目前試管開花的體系在很多蘭科植物中已建立起來,但花芽誘導(dǎo)率及后續(xù)生殖生長(zhǎng)情況還有待改善,并且試管開花的誘導(dǎo)增加了操作的成本和技術(shù)難度,所以寒蘭開花分子機(jī)制的研究對(duì)其發(fā)展具有重要意義。本文擬對(duì)寒蘭進(jìn)行轉(zhuǎn)錄組De Novo測(cè)序,獲得參考序列,然后對(duì)寒蘭開花各個(gè)階段的頂芽進(jìn)行數(shù)字化基因表達(dá)譜(Digital gene expression profiling;DGE)分析,篩選差異表達(dá)的基因,再利用實(shí)時(shí)熒光定量PCR(Quantitative real-time reverse-transcription polymerase chain reaction;RT-qPCR)對(duì)所得的差異基因進(jìn)行驗(yàn)證,初步揭示了寒蘭開花的分子機(jī)制。研究結(jié)果為寒蘭開花的分子調(diào)控機(jī)制奠定了基礎(chǔ),也為蘭科植物的花期調(diào)控提供了依據(jù)。主要結(jié)果包括:1.利用Solexa/Illumina二代高通量測(cè)序平臺(tái),對(duì)寒蘭根、莖、葉和花四部分混合樣品進(jìn)行轉(zhuǎn)錄組測(cè)序,得到有效序列108,927,860 nt,經(jīng)過組裝獲得Unigene 68,699條,平均長(zhǎng)度是867 nt。2.利用寒蘭轉(zhuǎn)錄組文庫中68,699條Unigene進(jìn)行SSR位點(diǎn)分析,共獲得位點(diǎn)11,646個(gè),占16.95%。隨機(jī)選取141對(duì)SSR引物,其中79對(duì)可以擴(kuò)增出特異條帶,擴(kuò)增率達(dá)到56.03%,可以擴(kuò)增出具有多態(tài)性條帶的引物共15對(duì),達(dá)10.64%。3.對(duì)寒蘭三個(gè)時(shí)期的花芽進(jìn)行DGE分析,通過對(duì)三個(gè)轉(zhuǎn)錄組數(shù)據(jù)庫的比較,共獲得23,720個(gè)差異表達(dá)基因,從中篩選出與成花過程有關(guān)的9個(gè)基因,分別為:CkFPA,CkGA2OX,CkGID1,CkCO,CkPHYB,CkELF3,CkFRI,CkFLC和CkAP1。4.通過RT-qPCR技術(shù),驗(yàn)證9個(gè)差異表達(dá)基因在寒蘭成花過程中的表達(dá)模式,發(fā)現(xiàn)其中CkFPA,CkGA2OX,CkCO,CkFLC和CkAP1 5個(gè)基因在寒蘭開花過程呈現(xiàn)顯著差異及規(guī)律的表達(dá)模式,表明其與寒蘭開花密切相關(guān)。綜上所述,寒蘭成花是由自主途徑、赤霉素途徑、光周期途徑和春化途徑共同調(diào)控的,其中CkFPA,CkGA2OX,CkCO,CkFLC耦合CkAP1在成花過程中的高表達(dá),誘導(dǎo)寒蘭開花。本研究構(gòu)建了高質(zhì)量的轉(zhuǎn)錄組數(shù)據(jù)庫,獲得了寒蘭開花候選功能基因,為進(jìn)一步揭示寒蘭開花時(shí)間調(diào)控機(jī)制奠定了基礎(chǔ),并可為寒蘭早花分子育種提供豐富的基因資源,具有重要的理論和實(shí)踐意義。
[Abstract]:Orchidaceae is the most evolved monocotyledonous group with wide distribution, diverse ecological environment, highly specific morphology, structure and physiological characteristics, and is an ideal material for flower development. (Cymbidium kanran) is an important member of the Guolan family, which belongs to the (Orchidaceae) genus (Cymbidium),. It is known as the "king of orchids" because of the variety of labial morphology, the flowering of four seasons and the long flowering period. However, it takes a long time from seed to flowering for the orchids, including cold orchids, which seriously restrict the research of flower development and the exploitation of resources. At present, the system of in vitro flowering has been established in many orchids, but the rate of flower bud induction and subsequent reproductive growth still need to be improved, and the induction of in vitro flowering increases the cost and technical difficulty of operation. Therefore, the study on the molecular mechanism of flowering is of great significance to its development. In this paper, the transcriptome De Novo sequence of cold orchid was sequenced, and the reference sequence was obtained. Then, the terminal buds of cold orchid were analyzed by (Digital gene expression profiling;DGE (digital gene expression profile), and the differentially expressed genes were screened. The differential genes were verified by real-time fluorescence quantitative PCR (Quantitative real-time reverse-transcription polymerase chain reaction;RT-qPCR, which revealed the molecular mechanism of the flowering of cold orchid. The results laid a foundation for the molecular regulation mechanism of the flowering of the orchid, and also provided the basis for the regulation of the flowering period of the orchids. The main results include: 1. Using Solexa/Illumina second generation high-throughput sequencing platform, four mixed samples of cold orchid root, stem, leaf and flower were sequenced by transcriptome sequencing. The effective sequence of 108927860 nt, was assembled to obtain 68699 pieces of Unigene, with an average length of 867 nt.2.. A total of 11 646 loci (16.95%) were obtained from 68699 Unigene loci in the cold orchid transcriptional library. One hundred and forty-one pairs of SSR primers were randomly selected, of which 79 pairs could amplify specific bands, the amplification rate was 56.03 and 15 pairs of primers with polymorphic bands could be amplified, up to 10.64.3. By DGE analysis of flower buds in three stages of cold orchid, a total of 23 720 differentially expressed genes were obtained by comparing the three transcriptional databases. Nine genes related to flower formation were screened out, namely: CkFPAA CkGA _ 2OXCkGID1, CkPHYBCkELF3CkFRICkFLC and CkAP1.4.. The expression patterns of 9 differentially expressed genes in the flowering process of cold orchid were verified by RT-qPCR technique. It was found that 5 genes of CkFPA,CkGA2OX,CkCO,CkFLC and CkAP1 showed significant differences and regular patterns in flowering process of cold orchid, which indicated that they were closely related to the flowering of cold orchid. To sum up, orchid flower is regulated by independent pathway, gibberellin pathway, photoperiod pathway and vernalization pathway, in which CkFPA,CkGA2OX,CkCO,CkFLC coupled with CkAP1 is highly expressed in the process of flower formation, which induces the flowering of cold orchid. In this study, a high quality transcriptome database was constructed, and candidate functional genes were obtained, which laid a foundation for further revealing the mechanism of flowering time regulation of cold orchid, and could provide abundant gene resources for molecular breeding of cold orchid early flower. It has important theoretical and practical significance.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S682.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李梅;陳林波;田易萍;夏麗飛;宋維希;梁名志;江昌俊;;雌蕊缺失茶樹花3個(gè)發(fā)育期的數(shù)字基因表達(dá)譜分析[J];茶葉科學(xué);2017年01期

2 吳超;彭娟;向林;顧敏;秦德輝;丁曉瑜;郭方其;;基于高通量測(cè)序的鐵皮石斛葉片轉(zhuǎn)錄組分析[J];分子植物育種;2016年12期

3 朱uG昊;董誠明;鄭曉珂;馮衛(wèi)生;劉孟奇;趙樂;;基于轉(zhuǎn)錄組測(cè)序的山茱萸次生代謝生物合成相關(guān)基因的挖掘[J];中國中藥雜志;2017年02期

4 劉柳姣;劉震;韋燕;賴碧丹;;外源激素促進(jìn)墨蘭提早開花的試驗(yàn)[J];農(nóng)業(yè)研究與應(yīng)用;2016年06期

5 張慶田;李曉艷;楊義明;范書田;艾軍;;藍(lán)靛果忍冬轉(zhuǎn)錄組SSR信息分析及其分子標(biāo)記開發(fā)[J];園藝學(xué)報(bào);2016年03期

6 李滿堂;張仕林;鄧鵬;侯喜林;王建軍;;洋蔥轉(zhuǎn)錄組SSR信息分析及其多態(tài)性研究[J];園藝學(xué)報(bào);2015年06期

7 鄢秀芹;魯敏;安華明;;刺梨轉(zhuǎn)錄組SSR信息分析及其分子標(biāo)記開發(fā)[J];園藝學(xué)報(bào);2015年02期

8 袁秀云;梁芳;蔣素華;王默霏;劉佳;崔波;;文心蘭一個(gè)PEBP家族基因的克隆、表達(dá)及載體構(gòu)建[J];江西農(nóng)業(yè)大學(xué)學(xué)報(bào);2014年06期

9 李敬;谷慧英;王志敏;湯青林;宋明;;擬南芥成花關(guān)鍵基因調(diào)控網(wǎng)絡(luò)研究進(jìn)展[J];生物技術(shù)通報(bào);2014年12期

10 趙哲霞;蔣珊;王濱花;舒漢鼎;龔瑞s，

本文編號(hào)：2219231