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拟南芥开花调控基因EFS1的功能研究

标题: 拟南芥开花调控基因EFS1的功能研究
英文标题: Functional Analysis of Flower-Related Gene EFS1 in Arabidopsis Thaliana
作者: 李莉
出版时间: 2009-01-01
所在大学: 河北农业大学
关键词: 拟南芥,早花,GUS,RT-PCR,Real-Time PCR
英文关键词: Arabidopsis thaliana,early flowering,GUS,RT-PCR,Real-Time PCR
论文级别: 硕士
学位: 学位论文
导师: 董金皋%邢继红
专业: 植物学
提交时间: 2009
摘要: 高等植物的开花过程是植物由营养生长向生殖生长转换的最重要的过程,决定了植 物生殖发育的时期和质量,对于植物的生殖发育具有重要的意义,研究植物开花时间调 控的分子机制对于调节植物繁殖和发育、提高作物产量和品质具有重大的意义.实验室 前期研究从拟南芥T-DNA插入突变体库中筛选获得了一株早花突变体efs1(early flowering in short mutant 1),通过Genome Walking技术和生物信息学方法鉴定出一个新 的拟南芥开花调控相关基因EFS1,为进一步明确该基因的功能及其在开花调控途径中的 具体作用,本研究通过互补回复和超表达试验,验证了EFS1基因确为拟南芥开花调控基 因;利用RT-PCR和GUS组织化学染色技术,明确了EFS1基因在不同组织中表达水平及 组织定位;利用RT-PCR和Real-Time PCR技术,初步明确了EFS1基因在开花控制途径中 的位置及其调控开花的模式,为进一步阐明EFS1基因的调控机理奠定良好基础. 1.将已经构建的EFS1基因的表达载体pCAMBIA1300-EFS1(含启动子)-NOS经农 杆菌介导转化efs1突变体和Col-0野生型,获得了回复和超表达转基因株系;经PCR和 Southern blotting鉴定,确定了EFS1基因以多拷贝形式成功整合到efs1突变体基因组中; 经RT-PCR分析,确定了转基因系中EFS1基因的表达高于野生型;表型分析发现,回复 转基因系的开花时间与野生型相似,恢复到了野生型表型,且开花时间与EFS1的表达量 呈正相关,表明了转基因植物的恢复表型确实是由于外源EFS1的表达造成的. 2.利用RT-PCR技术,鉴定了EFS1在莲座叶、茎、茎叶和花中均有表达,尤其在莲 座叶和花中表达较高;构建了双元载体pCAMBIA1300-ProEFS 1∷GUS-NOS分别转化烟 草和拟南芥Col-0进行组织定位,经GUS染色发现EFS1在成熟植株的莲座叶、茎、茎叶、 花及一周龄幼苗的地上部和根中均有表达,且在花中表达最多,与RT-PCR分析结果一 致. 3.利用RT-PCR和Real-Time PCR 技术,检测了EFS1 基因突变对开花调控中重 要基因(SPY、FLC、GI、CO、FT和LFY)的作用,发现EFS1 基因突变(表达量降低) 后,SPY、FLC、GI、CO和FT的表达水平下降,由此可初步推断,该基因对开花起抑 制作用. 4.根据开花调控相关基因的RT-PCR和Real-Time PCR 检测的结果,我们初步明 确了EFS1的基因调控开花的模式.EFS1 基因的低表达量降低了赤霉素途径中的负调 控因子SPY的表达,促进内源GA的传递,同时,春化途径中负调控因子FLC的表达 以及光周期途径GI、CO和FT的表达也降低,从而促进植株早花.
英文摘要: Flowering is the most important result of a plant developmental process that controls the transition from vegetative maturity to the reproductive stage.It plays a key role in the phase and quality of development.Investigation of flowering controlling molecular mechanism is important to regulate development and propagation,improve crop\''s output and quality.In previous research,we had got a mutant from T-DNA insert library named efs1(early flowering in short mutant 1),which had identified based on Genome Walking and bio-information to be a candidate gene involved with Arabidopsis thaliana flowering regulation.To investigate the function and role in the regulation pathway,we designed the complementation and overexpression to confirm that EFS1 certainly belongs to the flowering controlling gene.Semi-quality RT-PCR and GUS histochemical staining were used to confirm the expression level and tissue localization.RT-PCR and Real-Time PCR were used to confirm the location of EFS1 in the regulating pathway.We have provided a foundation to the further regulation mechanism research.1.EFS1 was constructed to the pCAMBIA1300 vector,complementation and overexpression were obtained via Agrobacterium-mediated transformed to efs1 mutant and Col-0.The EFS1 was certainly integrated to the efs1 mutant as multiple copies based on the PCR and Southern blotting.The RT-PCR analysis showed that in the reverse mutant, EFS1 had a higher expression than the wild type.In the complementation,the flowering time was similar to the wild type Col-0,and also the flowering time was positively correlated to the expression level of EFS1.This result showed that the phenotypic reversion was caused by the expression of exogenous EFS1.2.RT-PCR was used to analyze the expression of EFS1.The results showed that the EFS1 might be a constitutive expression in the different tissues such as rosette leaf,stem, cauline leaf and flower.It had a highest expression in the flower.Binary vector pCAMBIA1300-ProEFS1::GUS-NOS was constructed to transform to Nicotiana tabacum and Arabidopsis thaliana to get the tissue localization analysis,EFS1 promoter-driven GUS expression was detectable in rosette leaf,stem,cauline leaf and flower of six-week seedings,and in the whole one-week seedings,this result is consistent with RT-PCR.3.Expressed pattern of some important genes regulated in flowering were detected, such as SPY,FLC,GI,CO,FT and LFY.The results indicated that the expression of LFY was increased,the expression of SPY、FLC、GI、CO and FT was decreased.So a conclusion was determined that the EFS1 might suppresse flowering.4.According to the results,the flowering model of EFS1 was determined.The reduced expression of EFS1 may promote flowering by repressing the expression of SPY via gibberellin pathway,at the same time,the expression of FLC of vernalization pathway and the expression of GI,CO and FT of photoperiod pathway was also decreased.