本文選題：鐵皮石斛　切入點(diǎn)：試管開花　出處：《西南交通大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：通過(guò)組培以及一些分子生物學(xué)方面的技術(shù),優(yōu)化鐵皮石斛開花條件。其主要目的是通過(guò)試管苗開花授粉得到更多的果莢,為后續(xù)胚胎及相關(guān)的實(shí)驗(yàn)提供物質(zhì)基礎(chǔ)。通過(guò)5種不同激素的單因子實(shí)驗(yàn),得到更高的成花植株,并確定花與苗的質(zhì)量?jī)?yōu)異。通過(guò)對(duì)鐵皮石斛進(jìn)行植物組織培養(yǎng),優(yōu)化鐵皮石斛的開花條件,促進(jìn)鐵皮石斛提前成花;通過(guò)石蠟切片技術(shù),觀察鐵皮石斛在由營(yíng)養(yǎng)生長(zhǎng)到生殖生長(zhǎng)的轉(zhuǎn)變過(guò)程中,最具代表性的莖尖的細(xì)胞切片圖,觀察細(xì)胞發(fā)育變化:通過(guò)RACE技術(shù)克隆出與開花相關(guān)的基因序列;采用生物信息學(xué)的方法初步分析了這些序列的一些基本性質(zhì),包括長(zhǎng)度、亞細(xì)胞定位、等電點(diǎn)、二級(jí)結(jié)構(gòu)等;采用實(shí)時(shí)熒光定量PCR(Quantitative real-time reverse transcription-PCR,RT-qPCR)對(duì)這些基因在不同組織,不同激素條件下的表達(dá)模式進(jìn)行分析,與優(yōu)化條件相結(jié)合并進(jìn)行分析,可以進(jìn)一步得到哪些基因與激素促進(jìn)條件有關(guān)。ABA(Abscisic Acid)促進(jìn)鐵皮石斛的試管開花,當(dāng)在栽培在ABA開花培養(yǎng)基中15-20天,再轉(zhuǎn)移到含0.2mg·L-1 NAA和0.2mg·L-1 6-BA的誘導(dǎo)后培養(yǎng)基中,發(fā)芽數(shù)量和發(fā)芽率顯著增加。當(dāng)Spermidine HCL濃度為0.1 mg·L-1時(shí),其最適宜培養(yǎng)的天數(shù)為20天左右,在Spermidine HCL濃度為1.5 mg·L-1時(shí),其最適宜培養(yǎng)的天數(shù)為15天左右。花芽誘導(dǎo)率在PP333(Paclobtrazo1)0.6mg·L-1時(shí)最高,為24%�；ㄑ空T導(dǎo)率在乙烯利1 mg·L1時(shí)最高,為13.3%,其致死濃度一般在2.5 mg·Ll1左右,在此時(shí)刻,鐵皮石斛在誘導(dǎo)30d后會(huì)伴隨著莖變黃,葉子枯萎凋零,并最終死亡。TDZ(Thidiazuron)可以縮短營(yíng)養(yǎng)生長(zhǎng)期2-3年,乙烯處理會(huì)加速營(yíng)養(yǎng)生長(zhǎng)到生殖生長(zhǎng)的轉(zhuǎn)變。根對(duì)鐵皮石斛的花芽分化具有抑制作用。采用RACE技術(shù),基于轉(zhuǎn)錄組數(shù)據(jù),得到了 16條開花相關(guān)基因的完整cds序列。通過(guò)生物信息學(xué)的方法,得到了基因的一些基本性質(zhì)。氨基酸序列長(zhǎng)度從237bp到2958bp,分子量從19304.06ku到245735.14ku,所有序列的G+C含量都在0.44-0.54之間。通過(guò)實(shí)時(shí)熒光定量PCR技術(shù),得到基因在不同組織、不同激素處理下的表達(dá)情況�；蛟诩に谹BA、spermidine HCL、TDZ等處理下,與組培優(yōu)化的實(shí)驗(yàn)結(jié)果相吻合。在用ABA預(yù)處理15天左右,鐵皮石解的開花率會(huì)達(dá)到比較高的峰值;基因在激素spermidine HCL處理下,大部分的基因都在21d左右的表達(dá)量最高;基因在激素TDZ處理下,大部分的基因都在18d左右的表達(dá)量最高。4號(hào)基因Flowering Locus T、5 號(hào)基因 Flowering time control protein FCA、12 號(hào) Flowering time control protein FPA、14號(hào)基因Flowering time control protein FC4和16號(hào)基因LEAFY表達(dá)模式比較特殊,可以篩選作為花特異性基因。這些實(shí)驗(yàn)結(jié)果為后續(xù)的分子機(jī)理的研究打下了基礎(chǔ)。
[Abstract]:The flowering conditions of Dendrobium candidum were optimized by tissue culture and some molecular biological techniques. To provide the material basis for the subsequent embryo and related experiments. Through the single factor experiment of five different hormones, the higher flowering plants were obtained, and the quality of the flowers and seedlings was determined to be excellent. The plant tissue culture was carried out on Dendrobium candidum. To optimize the flowering conditions of Dendrobium candidum, to promote the early flowering of Dendrobium candidum; to observe the most representative cell sections of the stem tip of Dendrobium candidum during the transition from vegetative growth to reproductive growth through paraffin section technology. Observe the changes of cell development: clone the gene sequence related to flowering by RACE, analyze the basic properties of these sequences by bioinformatics, including length, subcellular localization, isoelectric point, secondary structure, etc. The expression patterns of these genes in different tissues and different hormone conditions were analyzed by real-time fluorescence quantitative PCR(Quantitative real-time reverse transcription-PCRR-RT-qPCR. Which genes can be further obtained to promote the flowering of Dendrobium candidum in vitro, which are related to hormone promoting conditions. When cultured in ABA flowering medium for 15 to 20 days, it was transferred to the induced medium containing 0.2 mg 路L -1 NAA and 0.2 mg 路L -1 6-BA. When the concentration of Spermidine HCL was 0.1 mg 路L -1, the most suitable culture days were about 20 days, and when the concentration of Spermidine HCL was 1.5 mg 路L -1, the most suitable culture days were about 15 days. The flower bud induction rate was the highest at PP333(Paclobtrazo1)0.6mg 路L -1. When ethephon was 1 mg 路L ~ (-1), the highest rate of flower bud induction was 13.33.The lethal concentration was about 2.5 mg 路Ll1. At this time, Dendrobium candidum would become yellow with the stem after 30 days of induction, and the leaves would wither and wither. And finally death. TDZ Thidiazuron can shorten the vegetative growth period of 2-3 years, ethylene treatment can accelerate the transformation of vegetative growth to reproductive growth. Root has inhibitory effect on flower bud differentiation of Dendrobium candidum. Using RACE technique, based on transcriptional data, The complete cds sequence of 16 flowering related genes was obtained. Some basic properties of the gene were obtained. The length of amino acid sequence ranged from 237bp to 2958bp, the molecular weight ranged from 19304.06ku to 245735.14ku. the G C content of all sequences ranged from 0.44-0.54. By real-time fluorescence quantitative PCR, the gene was obtained in different tissues. The expression of the gene under different hormone treatments was consistent with the results of tissue culture optimization under the treatment of the hormone ABA Spermidine HCLmidine TDZ etc. The flowering rate of ferrite pyrolysis reached a relatively high peak after 15 days of pretreatment with ABA. Under the treatment of hormone spermidine HCL, the expression of most genes was the highest at 21 days, and the gene was treated with hormone TDZ. Most of the genes were expressed at about 18 days. The expression patterns of Flowering time control protein Flowering time control protein FPA12, Flowering time control protein FC4 and LEAFY 16 of 4 gene Flowering Locus TG5 and 14 gene Flowering time protein FC4 and 16 gene were very special. These results provide a basis for the further study of molecular mechanism.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S567.239

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 寧英海;顧敬文;劉志萍;銀勝高;;鐵皮石斛活性成分及藥理作用研究進(jìn)展[J];亞太傳統(tǒng)醫(yī)藥;2016年13期

2 張孝廉;張吉順;鄒頡;趙杰宏;任學(xué)良;;蛋白泛素化和類泛素化修飾在植物開花時(shí)間調(diào)控中的作用[J];浙江大學(xué)學(xué)報(bào)(農(nóng)業(yè)與生命科學(xué)版);2015年04期

3 龔慶芳;何金祥;黃寧珍;付傳明;石云平;唐鳳鸞;;鐵皮石斛花化學(xué)成分及抗氧化活性研究[J];食品科技;2014年12期

4 黃勇;;鐵皮石斛組織培養(yǎng)技術(shù)研究進(jìn)展[J];文山學(xué)院學(xué)報(bào);2013年06期

5 張崗;趙明明;張大為;郭順星;;鐵皮石斛實(shí)時(shí)定量PCR內(nèi)參基因的篩選[J];中國(guó)藥學(xué)雜志;2013年19期

6 辛明;張娥珍;李楠;黃振勇;蘇燕竹;黃茂康;何全光;;不同干燥工藝對(duì)鐵皮石斛多糖及石斛堿的影響[J];南方農(nóng)業(yè)學(xué)報(bào);2013年08期

7 聶少平;蔡海蘭;;鐵皮石斛活性成分及其功能研究進(jìn)展[J];食品科學(xué);2012年23期

8 蔡振鋒;平軍嬌;張珍;湯賢春;白威峰;錢剛;;千里光泛素(Ubiquitin)基因生物信息學(xué)與功能分析[J];生物技術(shù)通報(bào);2012年10期

9 吳高杰;李璐;賴鐘雄;;蘭花試管開花研究進(jìn)展[J];中國(guó)農(nóng)學(xué)通報(bào);2011年10期

10 王偉英;鄒暉;陳永快;林江波;;鐵皮石斛的綜合利用與展望[J];中國(guó)園藝文摘;2011年01期

相關(guān)碩士學(xué)位論文 前3條

1 曾水玉;萬(wàn)壽菊試管開花及其分子機(jī)制的研究[D];福建農(nóng)林大學(xué);2012年

2 姜順日;孔雀草試管開花及蛋白質(zhì)組學(xué)研究[D];福建農(nóng)林大學(xué);2011年

3 李璐;石斛蘭試管開花及其分子機(jī)制研究[D];福建農(nóng)林大學(xué);2010年

，

本文編號(hào)：1612588