桑树FT基因启动子的克隆和功能分析

发布时间：2018-07-04 23:17

本文选题：桑树 + 成花　；参考：《西北农林科技大学》2016年硕士论文

【摘要】：成花是高等植物生命周期的一个重要生长阶段,由基因和环境共同调控。FT(Flowering Locus T)基因编码的蛋白质可由叶片韧皮部长距离运输至茎顶端分生组织,在成花诱导上起着关键的调控作用。研究FT基因的表达调控机制,对加快桑树遗传改良进程有重要的意义。本试验利用PCR这一技术手段从桑树基因组中克隆得到了FT基因的启动子序列,并通过5,缺失突变对启动子的功能进行了初步分析,为研究桑树FT基因的表达调控机制、进一步探索开花调控机制奠定了理论基础,为缩短桑树童期、实现桑果的早实丰产提供了理论依据。本试验的主要内容和结论如下:1.桑树叶片不定芽的诱导对新一之濑组培苗的离体叶片进行不定芽的诱导,在本实验室的环境下,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%琼脂为分化培养基,成功诱导出了不定芽,为桑树再生体系的建立提供了参考。2.桑树MaFT基因启动子的克隆及功能分析根据川桑的全基因组序列,通过PCR扩增技术从白桑中获得921 bp FT基因启动子序列,命名为MaFTP,GenBank登录号为KU550086。MaFTP上不存在CpG岛,序列与川桑相比,尽管相似性达到92%,但存在大片段的缺失和插入。多序列比对结果显示,桑树、杨树、苹果、拟南芥、水稻等不同物种的FT启动子序列相似性极低。以上结果表明,FT启动子作为调控基因时空表达的重要工具,在不同物种中的差异较大,甚至即使是在同一物种不同品种中也不完全相同。Plant CARE和PLACE启动子预测结果表明,MaFTP上除含有TATA-box、CAAT-box等真核生物启动子的核心元件外,还分布有光照、干旱、节律性、脱落酸、生长素、水杨酸等多种顺式作用应答元件。根据MaFTP上预测元件的分布情况,设计引物,得到MaFTP的5,缺失片段,分别命名为MaFTP903、MaFTP762、MaFTP542、MaFTP289、MaFTP162。将五个缺失片段分别替换pBE2113的35S CaMV启动子,与GUS基因融合构建植物表达载体,并通过农杆菌介导的方式转化烟草,GUS染色结果表明,除了MaFTP903活性较弱外,其余片段均能驱动GUS基因高效的表达,且表达量相近,表明FT基因启动子的核心区段位于162bp内。通过研究不同缺失启动子的活性,从而确定启动子的核心功能位点,对于深入研究FT基因的转录调控机制具有指导意义。3.桑树KdsA在干旱胁迫中的功能分析构建pBE2113-KdsA-GFP的融合表达载体,通过农杆菌介导的方式转化烟草叶片,KdsA的亚细胞定位结果表明,KdsA蛋白在整个细胞中都有分布,主要分布在细胞膜和细胞核。干旱胁迫下,瞬时表达KdsA的桑树叶片脯氨酸含量和干旱相关基因P5CS、DREB2B的表达量显著上升。结果表明,KdsA基因表达量的增加能够引起干旱相关基因的表达量及干旱相关指标发生改变。可见,KdsA基因在干旱胁迫中发挥着一定的作用,试验为KdsA基因在干旱胁迫中功能的研究奠定了基础。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S888;Q943.2

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