本文選題：桑樹 + 成花��； 參考：《西北農(nóng)林科技大學》2016年碩士論文

【摘要】：成花是高等植物生命周期的一個重要生長階段,由基因和環(huán)境共同調(diào)控。FT(Flowering Locus T)基因編碼的蛋白質(zhì)可由葉片韌皮部長距離運輸至莖頂端分生組織,在成花誘導(dǎo)上起著關(guān)鍵的調(diào)控作用。研究FT基因的表達調(diào)控機制,對加快桑樹遺傳改良進程有重要的意義。本試驗利用PCR這一技術(shù)手段從桑樹基因組中克隆得到了FT基因的啟動子序列,并通過5,缺失突變對啟動子的功能進行了初步分析,為研究桑樹FT基因的表達調(diào)控機制、進一步探索開花調(diào)控機制奠定了理論基礎(chǔ),為縮短桑樹童期、實現(xiàn)桑果的早實豐產(chǎn)提供了理論依據(jù)。本試驗的主要內(nèi)容和結(jié)論如下:1.桑樹葉片不定芽的誘導(dǎo)對新一之瀨組培苗的離體葉片進行不定芽的誘導(dǎo),在本實驗室的環(huán)境下,以MS+6-BA 5.0 mg/L+NAA 0.2 mg/L+1.0%蔗糖+1.0%葡萄糖+1.0%果糖+0.75%瓊脂為分化培養(yǎng)基,成功誘導(dǎo)出了不定芽,為桑樹再生體系的建立提供了參考。2.桑樹MaFT基因啟動子的克隆及功能分析根據(jù)川桑的全基因組序列,通過PCR擴增技術(shù)從白桑中獲得921 bp FT基因啟動子序列,命名為MaFTP,GenBank登錄號為KU550086。MaFTP上不存在CpG島,序列與川桑相比,盡管相似性達到92%,但存在大片段的缺失和插入。多序列比對結(jié)果顯示,桑樹、楊樹、蘋果、擬南芥、水稻等不同物種的FT啟動子序列相似性極低。以上結(jié)果表明,FT啟動子作為調(diào)控基因時空表達的重要工具,在不同物種中的差異較大,甚至即使是在同一物種不同品種中也不完全相同。Plant CARE和PLACE啟動子預(yù)測結(jié)果表明,MaFTP上除含有TATA-box、CAAT-box等真核生物啟動子的核心元件外,還分布有光照、干旱、節(jié)律性、脫落酸、生長素、水楊酸等多種順式作用應(yīng)答元件。根據(jù)MaFTP上預(yù)測元件的分布情況,設(shè)計引物,得到MaFTP的5,缺失片段,分別命名為MaFTP903、MaFTP762、MaFTP542、MaFTP289、MaFTP162。將五個缺失片段分別替換pBE2113的35S CaMV啟動子,與GUS基因融合構(gòu)建植物表達載體,并通過農(nóng)桿菌介導(dǎo)的方式轉(zhuǎn)化煙草,GUS染色結(jié)果表明,除了MaFTP903活性較弱外,其余片段均能驅(qū)動GUS基因高效的表達,且表達量相近,表明FT基因啟動子的核心區(qū)段位于162bp內(nèi)。通過研究不同缺失啟動子的活性,從而確定啟動子的核心功能位點,對于深入研究FT基因的轉(zhuǎn)錄調(diào)控機制具有指導(dǎo)意義。3.桑樹KdsA在干旱脅迫中的功能分析構(gòu)建pBE2113-KdsA-GFP的融合表達載體,通過農(nóng)桿菌介導(dǎo)的方式轉(zhuǎn)化煙草葉片,KdsA的亞細胞定位結(jié)果表明,KdsA蛋白在整個細胞中都有分布,主要分布在細胞膜和細胞核。干旱脅迫下,瞬時表達KdsA的桑樹葉片脯氨酸含量和干旱相關(guān)基因P5CS、DREB2B的表達量顯著上升。結(jié)果表明,KdsA基因表達量的增加能夠引起干旱相關(guān)基因的表達量及干旱相關(guān)指標發(fā)生改變�？梢�,KdsA基因在干旱脅迫中發(fā)揮著一定的作用,試驗為KdsA基因在干旱脅迫中功能的研究奠定了基礎(chǔ)。
[Abstract]:Flowering is an important growth stage in the life cycle of higher plants. The protein encoded by the Flowering Locus T gene can be transported from the phloem of leaves to the meristem apical meristem. It plays a key role in flower induction. It is important to study the regulation mechanism of FT gene expression for speeding up the process of mulberry genetic improvement. In this experiment, the promoter sequence of FT gene was cloned from mulberry genome by PCR, and the function of the promoter was preliminarily analyzed by 5% deletion mutation in order to study the regulation mechanism of FT gene expression in mulberry. The further exploration of the mechanism of flowering regulation laid a theoretical foundation for shortening the young period of mulberry trees and realizing the early fruiting and high yield of mulberry fruit. The main contents and conclusions of this experiment are as follows: 1. Adventitious buds were induced by adventitious buds of Mulberry leaves. In our laboratory, MS 6-BA 5.0 mg / L NAA 0.2 mg / L 1.0% sucrose 1.0% glucose 1.0% fructose 0.75% Agar was used as the differentiation medium. Adventitious buds were successfully induced, which provided a reference for the establishment of mulberry regeneration system. Cloning and functional Analysis of Mulberry MaFT Gene Promoter 921 BP FT Gene Promoter sequence was obtained from White mulberry by PCR amplification according to the whole genome sequence of Mulberry, named MaFTP GenBank accession number KU550086. MaFTP does not exist CpG island, and the sequence is compared with that of Mulberry. Despite the similarity of 92, large fragments are missing and inserted. The results of multiple sequence alignment showed that the FT promoter sequence similarity of mulberry, poplar, apple, Arabidopsis thaliana, rice and other species was very low. These results suggest that the FT promoter, as an important tool for regulating gene expression in time and space, varies greatly in different species. Even in different varieties of the same species, the prediction results of .Plant care and PLACE promoter showed that in addition to the core elements of eukaryote promoter such as TATA-box, CAAT-box, there were also light, drought, rhythm, abscisic acid, auxin, abscisic acid and auxin on MaFTP. Salicylic acid and other cis-responsive elements. According to the distribution of predictive elements on MaFTP, a primer was designed to obtain 5 deletion fragments of MaFTP, which were named MaFTP903, MaFTP762MFTP542and MaFTP289MaFTP162were named MaFTP903, MaFTP762, MaFTP542, MaFTP289and MaFTP162respectively. Five deletions were replaced by 35s CaMV promoter of pBE2113, and the plant expression vector was constructed by fusion with Gus gene. The results of Gus staining showed that the activity of MaFTP903 was weak except MaFTP903, which was transformed into tobacco by Agrobacterium tumefaciens. All the other fragments could drive the expression of Gus gene efficiently, and the expression was similar, indicating that the core region of FT gene promoter was located in 162bp. By studying the activity of different deletion promoters, the core functional sites of the promoters are determined, which is of great significance for the further study of the transcriptional regulation mechanism of FT gene. The expression vector pBE2113-KdsA-GFP was constructed by functional analysis of mulberry KdsA under drought stress. The results of subcellular localization of KdsA in tobacco leaves mediated by Agrobacterium tumefaciens showed that KdsA protein was distributed in the whole cell. Mainly distributed in cell membrane and nucleus. Under drought stress, the proline content of mulberry leaves and the expression of drought related gene P5CSDREB2B increased significantly. The results showed that the increase of KdsA gene expression could cause the change of drought related gene expression and drought related index. It can be seen that KdsA gene plays a certain role in drought stress, and the experiment lays a foundation for the study of the function of KdsA gene in drought stress.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S888;Q943.2

【參考文獻】

相關(guān)期刊論文 前10條

1 崔喜艷;陳眾峰;范貝;韓琳;劉曉慶;董雅致;張治安;;大豆rbcS基因啟動子的克隆及在轉(zhuǎn)基因煙草中的功能缺失分析[J];西北農(nóng)林科技大學學報(自然科學版);2015年05期

2 姚遠;王曉萍;;擬南芥開花相關(guān)的分子調(diào)控機制的研究[J];安徽農(nóng)業(yè)科學;2015年09期

3 張大燕;賈凌;向仲懷;何寧佳;;農(nóng)桿菌介導(dǎo)的桑樹瞬時表達體系構(gòu)建及miRn51與其靶基因的表達分析[J];蠶業(yè)科學;2015年01期

4 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)研究進展[J];生物技術(shù)通報;2014年12期

5 劉雪梅;王蕾;文添龍;何鵬;俞嘉寧;;農(nóng)桿菌介導(dǎo)的棉花子葉瞬時表達系統(tǒng)的建立[J];植物學報;2014年05期

6 虞莎;王佳偉;;miR156介導(dǎo)的高等植物年齡途徑研究進展[J];科學通報;2014年15期

7 張定國;黃先忠;東銳;鄭銀英;崔百明;;棉花GhFTL1基因啟動子的克隆及序列分析[J];石河子大學學報(自然科學版);2012年05期

8 張睿;陶建敏;蔡斌華;章鎮(zhèn);;藤稔葡萄花發(fā)育相關(guān)基因啟動子的克隆及功能分析[J];植物資源與環(huán)境學報;2011年04期

9 胡廷章;肖國生;黃小云;陳再剛;;水稻OsNRT1-d啟動子的分離和功能分析[J];西北植物學報;2010年07期

10 于延沖;喬孟;劉振華;向鳳寧;;WRKY轉(zhuǎn)錄因子功能的多樣化[J];生命科學;2010年04期

相關(guān)碩士學位論文 前4條

1 劉立群;FT基因在桑樹生長狀態(tài)轉(zhuǎn)變中的表達分析[D];西北農(nóng)林科技大學;2014年

2 韓莎;干旱脅迫下桑樹蛋白質(zhì)的差異表達分析[D];西北農(nóng)林科技大學;2013年

3 李四軍;桑樹FT基因的克隆及表達分析[D];西北農(nóng)林科技大學;2013年

4 鄭小一;蘋果Flowering locus T (FT)基因及其啟動子的克隆及表達分析[D];南京農(nóng)業(yè)大學;2010年

，

本文編號：2097877