拟南芥光周期调控开花突变体的筛选及基因和蛋白质的鉴定分析
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摘要
近几年来,植物的开花成为了研究的热点。现在发现已经确定至少存在4条调控植物开花时间的信号途径:即光周期途径、春化途径、自主途径和赤霉素途径。日照长度(即光周期)是影响开花时间的主要因素之一。光周期中隐花素CRY1、CRY2等光受体位于光周期途径的上游,直接感受光信号后诱导生物钟成分及转录因子的表达而促进开花。研究表明cry1突变体在各种光照条件下都比野生型更迟开花;cry2突变体在长日照下延迟开花,对光周期敏感。cry1cry2双突变体的晚花的表型更为明显。
为了更深入地研究隐花素调控光周期开花的作用机制,本文以拟南芥蓝光受体突变体cry1cry2为实验材料,采用激活标签技术构建了T-DNA插入突变体库,并从中筛选到与开花时间相关的突变体,其中SCC106-D、SCC60-D为晚花突变体;SCC105-4-D、SCC147-3-D为早花突变体,并采用IPCR和TAIL-PCR等方法得到了突变体T-DNA插入的基因组旁邻序列。
采用半定量RT-PCR对突变体T-DNA插入位点两侧的候选基因的mRNA水平进行了初步分析,结果发现在SCC106-D突变体中,T-DNA左边界附近的At3g57760基因的转录量比野生型和cry1cry2突变体中均高;在SCC60-D突变体中,T-DNA右边界的AT4G27240和AT4G27260两个基因的转录量比野生型和cry1cry2突变体中均高;在SCC105-4-D突变体中, T-DNA右边界的AT5G19950基因的转录量比野生型和cry1cry2突变体中均高;在SCC147-3-D突变体中, T-DNA两个插入位点的左边界AT2G34160、AT4G18197、AT4G18205和AT4G18210基因的转录量比野生型和cry1cry2突变体中均高。获得了一些与开花相关的候选基因。
通过不同光照下突变体下胚轴和根长的测量与分析,结果发现无论是在暗处理、红光还是蓝光下,SCC106-D突变体的下胚轴和主根都要明显的短于cry1cry2。说明SCC106-D突变体表现为组成型光形态建成。而SCC60-D突变体在蓝光下下胚轴和主根没有明显的变化,而在红光下下胚轴和主根变化较为明显,说明SCC60-D突变体对红光比较敏感。
通过双向电泳分离T-DNA插入突变体SCC106-D与拟南芥蓝光受体突变体cry1cry2的总蛋白,得到了高重复性的双向凝胶电泳图谱。通过初步的斑点配比,获得了两种材料的差异蛋白质点图谱;并选取37个差异较明显的蛋白质点进行MALDI-TOF-TOF-MS质谱分析,利用肽质量指纹图数据在数据库中进行检索,有22个蛋白质点得到了可靠的鉴定。通过鉴定得到一些与能量代谢,次生代谢和调控等相关的蛋白。这些差异蛋白与突变体的表型形成有一定的关系,包括晚开花的表型。本文的研究结果,为进一步研究这些隐花素介导的光周期开花信号传导途径奠定了基础。
In recent years,the flowering of plants has been the research focus.4 pathways that regulate flowering time were discovered:photoperiod pathway,jarovization pathway,self-determination pathway and GA pathway.Photoperiod is one of the major factors which control the flowering time.The CRY1,CRY2 that located on the upstream of photoperiod pathway take the light signal directly,induce the biological clock factors and expression of transcription factors to accelerate the flowering.The result showed mutant cry1 took more time to come into flower than the col-4 under several illumination conditions;the flowering time of mutant cry2 was delayed with the treatment of longtime illumination;the phenotype of mutant cry1cry2 was especially obviously.
In order to further study the mechanism which cyptochrome regulate the photoperiod for flowering,the mutant cry1cry2 were used as the material in this article. A library of Arabidopsis mutants was constructed by activation tagging method, which several mutants that related to flowering time were obtained from. SCC106-D,SCC60-D was late flowering mutant and SCC105-4-D,SCC147-3-D was early flowering mutant.IPCR and TAIL-PCR were used to identify the flanking genomic sequences of mutated target genes.
After the identification of the T-DNA insertion site,the semi-quantitative RT-PCR was used to analyse expression of the mutant genes as the first step towards the function of mutant genes,the results indicated that the expression of the flanking genomic sequences was enhanced compared with the col-4 and cry1cry2 mutant,for instance,the At3g57760 gene in mutant SCC106-D,the AT4G27240 and AT4G27260 genes in mutant SCC60-D,the AT5G19950 gene in mutant SCC105-4-D,the AT2G34160,AT4G18197,AT4G18205 and AT4G18210 genes in mutant SCC147-3-D. some candidate genes related with flowering were obtained.
By measuring and analyzing the length of hypocotyl and root under different wavelengths of light we found that mutant SCC106-D was not hypersensitive to different wavelengths of light,but mutant SCC60-D was hypersensitive to red light. These results showed the mutant SCC106-D was constitutively photomorphogenic. The protein of T-DNA insertion mutant SCC106-D and mutant cry1cry2 was separated by two-dimensional electrophoresis,different spots were obtained and identified.Thirty seven protein spots,which were differentially expressed in the cry1cry2 and SCC106-D,were identified by the method:MALDI-TOF-TOF-MS peptide fingerprint analysis of the protein spots and protein database searching. Twenty two of them were identified succesfully.By identification of these spots,some protein related with energy metabolism,secondary metabolism and regulation were obtained.The result showed in the paper laid the foundation for further study of photoperiod regulation by cyptochrome.
为了更深入地研究隐花素调控光周期开花的作用机制,本文以拟南芥蓝光受体突变体cry1cry2为实验材料,采用激活标签技术构建了T-DNA插入突变体库,并从中筛选到与开花时间相关的突变体,其中SCC106-D、SCC60-D为晚花突变体;SCC105-4-D、SCC147-3-D为早花突变体,并采用IPCR和TAIL-PCR等方法得到了突变体T-DNA插入的基因组旁邻序列。
采用半定量RT-PCR对突变体T-DNA插入位点两侧的候选基因的mRNA水平进行了初步分析,结果发现在SCC106-D突变体中,T-DNA左边界附近的At3g57760基因的转录量比野生型和cry1cry2突变体中均高;在SCC60-D突变体中,T-DNA右边界的AT4G27240和AT4G27260两个基因的转录量比野生型和cry1cry2突变体中均高;在SCC105-4-D突变体中, T-DNA右边界的AT5G19950基因的转录量比野生型和cry1cry2突变体中均高;在SCC147-3-D突变体中, T-DNA两个插入位点的左边界AT2G34160、AT4G18197、AT4G18205和AT4G18210基因的转录量比野生型和cry1cry2突变体中均高。获得了一些与开花相关的候选基因。
通过不同光照下突变体下胚轴和根长的测量与分析,结果发现无论是在暗处理、红光还是蓝光下,SCC106-D突变体的下胚轴和主根都要明显的短于cry1cry2。说明SCC106-D突变体表现为组成型光形态建成。而SCC60-D突变体在蓝光下下胚轴和主根没有明显的变化,而在红光下下胚轴和主根变化较为明显,说明SCC60-D突变体对红光比较敏感。
通过双向电泳分离T-DNA插入突变体SCC106-D与拟南芥蓝光受体突变体cry1cry2的总蛋白,得到了高重复性的双向凝胶电泳图谱。通过初步的斑点配比,获得了两种材料的差异蛋白质点图谱;并选取37个差异较明显的蛋白质点进行MALDI-TOF-TOF-MS质谱分析,利用肽质量指纹图数据在数据库中进行检索,有22个蛋白质点得到了可靠的鉴定。通过鉴定得到一些与能量代谢,次生代谢和调控等相关的蛋白。这些差异蛋白与突变体的表型形成有一定的关系,包括晚开花的表型。本文的研究结果,为进一步研究这些隐花素介导的光周期开花信号传导途径奠定了基础。
In recent years,the flowering of plants has been the research focus.4 pathways that regulate flowering time were discovered:photoperiod pathway,jarovization pathway,self-determination pathway and GA pathway.Photoperiod is one of the major factors which control the flowering time.The CRY1,CRY2 that located on the upstream of photoperiod pathway take the light signal directly,induce the biological clock factors and expression of transcription factors to accelerate the flowering.The result showed mutant cry1 took more time to come into flower than the col-4 under several illumination conditions;the flowering time of mutant cry2 was delayed with the treatment of longtime illumination;the phenotype of mutant cry1cry2 was especially obviously.
In order to further study the mechanism which cyptochrome regulate the photoperiod for flowering,the mutant cry1cry2 were used as the material in this article. A library of Arabidopsis mutants was constructed by activation tagging method, which several mutants that related to flowering time were obtained from. SCC106-D,SCC60-D was late flowering mutant and SCC105-4-D,SCC147-3-D was early flowering mutant.IPCR and TAIL-PCR were used to identify the flanking genomic sequences of mutated target genes.
After the identification of the T-DNA insertion site,the semi-quantitative RT-PCR was used to analyse expression of the mutant genes as the first step towards the function of mutant genes,the results indicated that the expression of the flanking genomic sequences was enhanced compared with the col-4 and cry1cry2 mutant,for instance,the At3g57760 gene in mutant SCC106-D,the AT4G27240 and AT4G27260 genes in mutant SCC60-D,the AT5G19950 gene in mutant SCC105-4-D,the AT2G34160,AT4G18197,AT4G18205 and AT4G18210 genes in mutant SCC147-3-D. some candidate genes related with flowering were obtained.
By measuring and analyzing the length of hypocotyl and root under different wavelengths of light we found that mutant SCC106-D was not hypersensitive to different wavelengths of light,but mutant SCC60-D was hypersensitive to red light. These results showed the mutant SCC106-D was constitutively photomorphogenic. The protein of T-DNA insertion mutant SCC106-D and mutant cry1cry2 was separated by two-dimensional electrophoresis,different spots were obtained and identified.Thirty seven protein spots,which were differentially expressed in the cry1cry2 and SCC106-D,were identified by the method:MALDI-TOF-TOF-MS peptide fingerprint analysis of the protein spots and protein database searching. Twenty two of them were identified succesfully.By identification of these spots,some protein related with energy metabolism,secondary metabolism and regulation were obtained.The result showed in the paper laid the foundation for further study of photoperiod regulation by cyptochrome.
引文
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