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

【摘要】：成花是高等植物生命周期的一個(gè)重要生長(zhǎng)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(zhǎng)距離運(yùn)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(dá)調(diào)控機(jī)制,對(duì)加快桑樹遺傳改良進(jìn)程有重要的意義。本試驗(yàn)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(dòng)子序列,并通過(guò)5,缺失突變對(duì)啟動(dòng)子的功能進(jìn)行了初步分析,為研究桑樹FT基因的表達(dá)調(diào)控機(jī)制、進(jìn)一步探索開花調(diào)控機(jī)制奠定了理論基礎(chǔ),為縮短桑樹童期、實(shí)現(xiàn)桑果的早實(shí)豐產(chǎn)提供了理論依據(jù)。本試驗(yàn)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(duì)新一之瀨組培苗的離體葉片進(jìn)行不定芽的誘導(dǎo),在本實(shí)驗(yàn)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(dòng)子的克隆及功能分析根據(jù)川桑的全基因組序列,通過(guò)PCR擴(kuò)增技術(shù)從白桑中獲得921 bp FT基因啟動(dòng)子序列,命名為MaFTP,GenBank登錄號(hào)為KU550086。MaFTP上不存在CpG島,序列與川桑相比,盡管相似性達(dá)到92%,但存在大片段的缺失和插入。多序列比對(duì)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(dòng)子序列相似性極低。以上結(jié)果表明,FT啟動(dòng)子作為調(diào)控基因時(shí)空表達(dá)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(dòng)子預(yù)測(cè)結(jié)果表明,MaFTP上除含有TATA-box、CAAT-box等真核生物啟動(dòng)子的核心元件外,還分布有光照、干旱、節(jié)律性、脫落酸、生長(zhǎng)素、水楊酸等多種順式作用應(yīng)答元件。根據(jù)MaFTP上預(yù)測(cè)元件的分布情況,設(shè)計(jì)引物,得到MaFTP的5,缺失片段,分別命名為MaFTP903、MaFTP762、MaFTP542、MaFTP289、MaFTP162。將五個(gè)缺失片段分別替換pBE2113的35S CaMV啟動(dòng)子,與GUS基因融合構(gòu)建植物表達(dá)載體,并通過(guò)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(dòng)GUS基因高效的表達(dá),且表達(dá)量相近,表明FT基因啟動(dòng)子的核心區(qū)段位于162bp內(nèi)。通過(guò)研究不同缺失啟動(dòng)子的活性,從而確定啟動(dòng)子的核心功能位點(diǎn),對(duì)于深入研究FT基因的轉(zhuǎn)錄調(diào)控機(jī)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(dá)載體,通過(guò)農(nóng)桿菌介導(dǎo)的方式轉(zhuǎn)化煙草葉片,KdsA的亞細(xì)胞定位結(jié)果表明,KdsA蛋白在整個(gè)細(xì)胞中都有分布,主要分布在細(xì)胞膜和細(xì)胞核。干旱脅迫下,瞬時(shí)表達(dá)KdsA的桑樹葉片脯氨酸含量和干旱相關(guān)基因P5CS、DREB2B的表達(dá)量顯著上升。結(jié)果表明,KdsA基因表達(dá)量的增加能夠引起干旱相關(guān)基因的表達(dá)量及干旱相關(guān)指標(biāo)發(fā)生改變�？梢�(jiàn),KdsA基因在干旱脅迫中發(fā)揮著一定的作用,試驗(yàn)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(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S888;Q943.2

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