【作者】 刘旭新；

【导师】 程智慧； Stephen Jackson； Yiguo Hong；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2008， 博士

【摘要】 拟南芥开花诱导基因网络主要是通过光周期途径(photoperiod pathway)、春化途径(vernalization PathWay)、自发途径(autonomous pathway)和赤霉素途径(GA pathway)等4个途径进行的，有很多相关基因起作用，其中拟南芥FLOWERING LOCUS T(FT)基因在其成花信号基因网络中起很重要的整合作用，在光周期诱导途径中，拟南芥叶片中光受体与相关感光节律基因构成的生物节律钟感受光周期信号，调控CONSTANS(CO)基因节律表达，CO基因在合适光周期调控下，将开花信号传导至FT基因，引起叶片中FT基因表达，FT基因表达产物、FT mRNA单独或相互作用，经韧皮部长距离运移到顶端分生组织，调节下游分生组织及花器官决定基因形成花芽开花。FT基因在光周期及其它开花诱导途径中起开花信号关键整合作用，也是现在已知在叶片中感受开花信号而长距离作用于顶端分生组织的基因。本试验对拟南芥FT基因的研究取得了如下研究结果：1．采集16h长日照条件下始花期野生型拟南芥叶片为样本，采用异硫氰酸胍法提取总RNA，利用RT-PCR法成功克隆得到拟南芥开花诱导遗传网络中关键的枢纽基因FLOWERING LOCUST(FT)，分析得到完整FT基因阅读框序列，完整阅读框架长度为528bp，经与NCBI GeneBank数据库比对序列完全正确。2．利用植物病毒载体PVX(Potato Virus X)，将FT基因和FT起始子突变为终止子(TAG)的基因序列mFT连接到PVX的基因组中，人工接种烟草，成功侵染烟草的后均在烟草细胞中检测到了复制出的FT RNA和mFT RNA，其中FT RNA成功诱导了短日照品种烟草(Maryland Mammoth)在长日照条件下开花。研究证明了FT基因作为开花诱导途径重要整合基因的生物功能及其开花诱导遗传保守性，探索了利用病毒载体表达外源基因验证基因功能的方法。3．进一步研究FT mRNA长距离运移的能力，利用植物病毒载体TCV(TurnipCrinkle Virus)载体，将刀基因序列连接到TCV△CP(去除病毒壳蛋白编码的TCV)的基因组上，体外转录成TCV△CP-FL、TCV△CP-mFT RNA，接种到拟南芥野生种和突变体ft-1叶片上，利用TCV△CP在拟南芥细胞中的侵染及复制特性，在拟南芥接种叶片细胞中转录出FT mRNA和mFT mRNA，接种7天后，采用RT-PCR法检测未接种新生上部叶片及芽，发现FT mRNA和mFT mRNA能够在新生叶片及新芽中检测到，并且在芽中检测条带要强于下层叶片，说明由没有系统侵染能力与长距离移动能力的TCV△CP介导表达的FT mRNA和mFT mRNA能够在拟南芥植物体内长距离向上移动，并有助于恢复TCV△CP病毒长距离移动能力，由试验结果可以推测FT mRNA参与传导开花诱导信号的可能性。4．通过分割mFT序列，将mFT序列DNA片段分为8段，分别在mFTn(n=1，2，3，4，5，6，7，8)，分别连入PVX△CP载体中，构建PVX△CP-mFTn，经体外转录，用其在人工侵染烟草的试验中，采用RT-PCR法没有在接种叶片外检测到PVX△CP-mFTn人工侵染形成的RNA序列片段，说明在人为选择的分割点上形成的8个片段序列，由病毒复制系统产生mFTn RNA在烟草未接种叶片中不具有移动能力，也说明mFT RNA经分割后，可能会失去移动的能力。5．采用生物信息学手段，利用结构分析软件分析FT mRNA的二级结构，分析可能导致其移动的结构基础，以及其结构中可与蛋白结合的结构位点与特别序列结构，分析结果表明FT mRNA二级结构中有稳定的二级结构，有可能是与蛋白结合或作用的特殊结构，这些结果提供了进一步寻找FT mRNA具有系统移动能力结构域的研究方向。研究证明拟南芥FLOWERING LOCUS T(FT)基因的开花诱导生物功能，证明FT基因高等植物间的遗传保守性，同时初步证明FT mRNA在拟南芥中具有的长距离移动能力，能为开花诱导本质作用机理及开花诱导物质探寻打下基础，也探索了采用病毒载体研究开花机理问题以及外源基因表达问题的试验方法。更多还原

【Abstract】 The flowering pathway in Arabidopsis thaliana includes photoperiod pathway、vernalization PathWay、autonomous pathway and GA pathway,Flowering Locus T (FT)playsa very important role in four pathways,not only is FT a key role in photoperiod pathway,butplays a integrated gene in gene network of others pathways;In photoperiod pathway,theporper signal of photoperiod regulates expression of CONSTANS (CO),CO expressionregulates expression of FT,ultimately FT activates the floral meristem identity gene,therebyinducing flowering.FT is known to function as floral integrator in Arabidopsis.Our researchwe get some research results from FT experiments:The details were as follows:1.Total RNA was isolated from Arabidopsis leaves,FT is isolated by using RT-PCR fromtotal RNA,FT gene whole fragment is 528bp.The result is tested by comparision withdatabase ofNBCI GeneBank.2.Using vector of PVX,full length sequence of FT and mFT(start codon replaced by stopcodon) is cloned in wild type PVX vector,to form two constructs PVX-FT and PVX-mFT,PVX-FT and PVX-mFT RNA transcripts was produced by in vitro transcription,inoculatedonto short day tobacco plant(Maryland Mammoth),PVX-FT RNA successfully triggerflowring on Maryland Mammoth in long day condition.This result shows the bio-fuction ofArabidopsis FT gene on different plant and conservation ability of FT in different speciese ofplant,and explore the way of expression of alien gene by using virus vector.3.Using vector of TCV,to know moving ability of FT,we coloned and fused FT and mFTin TCV△CP(coat protein sequence truncated) vector,TCV△CP-FT RNA andTCV△CP-mFT RNA was produced by in vitro transcription,inoculated onto wild type ofArabidopesis wild type andfi-1 mutanted Arabidopesis,FT and mFT RNA will produce afterinfection,later growing leaves harvested from inoculated Arabidopsis after 7 days inoculation,analyzed by RT-PCR,we get positive results from upper leaves and apex shoot,the resultsshow FT mRNA and mFT mRNA have ability of trafficking,indicate them can movingfrom inoculated leaves through phloem to shoot apex,and TCV△CP-mFT RNA andTCV△CP-FT RNA have ability of long-distance moving in Arabidopsis,and recover the moving ability of TCV△CP.4.Whole sequence of FT sequence trancated at four points to formmFTn(n=1,2,3,4,5,6,7,8),we got 8 fragments from FT sequence,and cloned and fused theminto PVX△CP vector,which coat protein sequence was truncated and lose cell to cell movingability and systemic infection ability;8 transcripts of mFTn was produced by in vitrotranscription,inoculated onto N.benthamiana leaves,after 7 days inoculation we use RT-PCRmethod to detect moving signal from later growing leaves and apex shoot,the result showstruncated mFTn RNA losed the ability of long-distance moving,and FT and mFT RNAsequence lose moving ability when it is cut at four points we chosen.5.Using bio-information tools to analyze the secondary structure of FT mRNA,to find theimpossible specific sequence that can bind protein to obtain moving ability for FT mRNA.The result shows there are several special areas on FT mRNA secondary structure that arevery possible areas to bind protein,the results can point out the way to find the motif on FTmRNA,which are key area that make FT mRNA can have moving ability.We get results from research,it shows Arabidopsis Flowering Locus T arewell-conserved,can induce flowering on tobacco plant(Maryland Mammoth) different plantspecies from Arabidopsis,and FT mRNA has a moving ability from leaf to apex shoot,maybeit plays a very important role in floral signal transmission.Our search set up a good meathodon research of function and mechanism of floral gene by using plant viral vector.更多还原