拟南芥隐花素CRY1在亚细胞结构的功能分析及激活标签突变体库的构建
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摘要
植物生长发育过程受到多种外界环境因素影响,光作为主要的环境因子,不仅提供光合作用所需能量,而且在植物光形态建成等多个过程中起重要调节作用。高等植物进化具备了一套极其精细的光感受和信号转换系统,以监视光信号的方向、量和质,并调节植物的生长发育。大量研究表明,蓝光受体隐花素CRY1在介导蓝光调控植物生长发育过程中发挥了重要作用,包括抑制下胚轴伸长、刺激子叶展开、气孔开启、花青素的积累以及调节相关基因的表达等。随着研究的深入,进一步研究隐花素CRY1在亚细胞结构中的生物学功能以及分离、鉴定与隐花素信号传导相关的组分对阐明其作用机制十分重要。本论文通过不同的光处理,探讨隐花素CRY1在植物亚细胞中的定位及受光诱导转移的过程。在此基础上,以隐花素cry1-304突变体为研究背景构建CRY1-GR/cry1-304转基因植株,采用外源糖皮质激素控制隐花素CRY1在细胞核--细胞质间的分布,以期阐明隐花素CRY1在亚细胞结构中的生物学功能。最后,以隐花素cry1cry2突变体为研究背景构建了激活标签突变体库,分离、鉴定与隐花素下游信号转导相关的组分。通过遗传、分析等方法,主要取得以下结果:
1.将35S::CRY1-GFP转基因植株在黑暗中培养5天后,采用不同的光处理,实时观察CRY1-GFP融合蛋白在亚细胞中的定位及受光诱导转移过程。实验发现:黑暗条件下CRY1-GFP融合蛋白定位在细胞质中且荧光强度非常微弱;随着蓝光处理时间的延长和光强的增大,GFP荧光逐渐增强,在亚细胞结构(细胞膜、细胞质、细胞核)中均有表达,实验表明CRY1蛋白表达受蓝光诱导,且与光照时间和强度成正比。在蓝光处理过程中(0-15 min),CRY1-GFP融合蛋白从细胞质、细胞膜向细胞核内转移聚集;蓝光处理15 min时,细胞核中GFP荧光强度达到最高,且细胞核与细胞GFP平均荧光强度的比值达到峰值。蛋白免疫杂交结果进一步证实,在蓝光下(0-15 min)GFP蛋白表达与蓝光处理时间成正比。实验首次发现:CRY1-GFP融合蛋白在蓝光诱导下,由细胞质、细胞膜向细胞核内转移累积,这种转移与红光无关。
2.将黑暗中培养5天的野生型黄化苗采用不同的光照处理30 min,提取细胞核蛋白。蛋白免疫杂交检测隐花素CRY1蛋白表达量显示:蓝光下,细胞核内CRY1蛋白表达量比黑暗、红光高,黑暗条件下CRY1蛋白表达量最低。结果进一步证实细胞核内隐花素CRY1蛋白特异性受蓝光诱导表达,或CRY1蛋白受蓝光诱导完成由细胞质、细胞膜向细胞核的转移过程。
3.以隐花素cry1-304突变体为研究背景,成功构建了CRY1-GR/cry1-304转基因植株。实验发现:CRY1-GR/cry1-304转基因植株仅在蓝光和外源糖皮质激素同时存在的条件下回复野生型表型,包括抑制下胚轴的伸长、子叶的展开、花青素的累积及气孔的开启等。实验证实:核定位信号(NLS)和光子激活两要素是隐花素CRY1在细胞核中发挥生理功能的充分必要条件。实验首次将糖皮质受体GR作用机理运用到隐花素CRY1基因功能研究,并证实细胞核隐花素CRY1与下胚轴的伸长、气孔的开启相关;细胞质隐花素CRY1与子叶的展开、叶柄的伸长和花青素的积累相关。
4.以隐花素cry1cry2突变体为研究背景,构建了基因功能获得型T-DNA突变体库,获得了近2 000个独立转化株系。从T1代转基因植株中筛选获得35个株系,与cry1cry2突变体表型明显不同。从中筛选得到一组有价值的光周期开花突变体,为进一步研究隐花素与植物光形态建成提供了宝贵实验材料。采用IPCR法,成功获得了Scc101-D突变体基因组T-DNA插入位点旁邻序列。实验发现,Scc101-D突变体晚花的表型是与At5g28237基因超量表达相关。
5.以野生型为研究背景构建AT5G28237过量表达转基因植株,该转基因植株开花延迟,进一步证实Scc101-D突变体晚花表型是由AT5G28237基因超量表达所致。基因枪轰击洋葱表皮实验首次发现该基因定位在细胞质中,且无组织特异性表达。AT5G28237转基因植株发育缓慢,表明At5g28237基因过表达影响了色氨酸的合成。外源色氨酸能部分恢复至野生型表型,暗示At5g28237基因在拟南芥的生长发育过程中还行使了其他功能。此外,转基因植株的光周期途径开花关键基因FT和CO的转录水平显著降低,结果进一步证实其参与调控植物的开花信号途径。
Plant growth and development process are affected by many external factors.Light as a major environmental element, not only provides the energy required for photosynthesis, but also plays an important role in photomorphogenesis. The evolution of higher plants has a fine feeling of light and signal system, to monitor the direction of light signal, quantity and quality to adjust their growth and development. A large number of studies have shown that blue light receptor cryptochrome-1 mediated blue light to regulate plant growth and development, including the inhibition of hypocotyl elongation, cotyledon opening, stomatal opening, anthocyanin accumulation and the regulation of gene expression. With further research, the study of CRY1 biological functions in subcellular, isolation and identification of Up-and down-stream components was very essential to understand the molecular mechanism of CRY mediated light signal pathway. In this work, we used different lights to analyse subcellular localization of CRY1 and light-induced migration process. On this basis, we constructed the CRY1-GR/cry-304 transgenetic plant on cry1-304 background. Control the location of CRY1 in subcell with the glucocorticoid to assess the biological functions. At the same time, we also contructed the activation tag mutant library on cry1cry2 background, isolated and identified the factors associated cryptochrome components. Through biochemical, genetic and other analysis methods, main results were obtained as followed:
1. The 35S:: CRY1-GFP transgenic plants were grown for 7 days in the dark condition, and then dealt with different lights. The results showed that no GFP fluorescence in the dark. Under blue light, GFP fluorescence gradually increased with intensity increasing of the blue light, and expressed in cytoplasm and cell membrane, and had a transfer from cytoplasm to nucleus. As the treatment of blue light during 0-15 min, GFP fluorescence intensity was the highest, and the nuclei and cell GFP fluorescence intensity ratio reached a peak, then the GFP fluorescence was gradually quenched. Western blot results showed that the expression of GFP protein being directly proportional to the intensity of blue light, but not red light.
2. The wild type were grown for 7 days in the dark condition, then dealt with different light qualities for 30 minutes and nuclear protein was extracted. Western blot analysis showed that: the abundance of blue-induced CRY1 protein was higher than it in red light and dark. The results proved the CRY1 protein specifically induced by blue light, and transported from the cytoplasm to nucleus.
3. We obtained CRY1-GR/cry1-304 transgenic plants on the cry1-304 mutant background, and screened multiple independent lines. The results showed that, CRY1-GR/cry1-304 transgenic plants restore WT phenotype only in the absence of blue light and glucocorticoid (+Dex) conditions, including inhibition of hypocotyl elongation, cotyledon opening, anthocyanin accumulation and stomatal opening. The results show that: Nuclear localization signal (NLS) and photon activation are sufficient and necessary for CRY1 to play function in nucleus. The present study found no evidence of a role for cytoplasmic CRY1 in the control of hypocotyl, stomatal opening and cotyledon petiole elongation, but cotyledon expansion, anthocyanin accumulation all displayed a dependence on cytoplasmic CRY1.
4. We got a gain-of-function mutant library on cry1cry2 background and screened nearly 2000 independent transformant lines. 35 transformant lines had obvious phenotype, and some of the mutant showed delayed flowering time. We also defined the flanking sequence of T-DNA insertion site in Scc101-D mutant through I-PCR analysis. The results showed that the phenotype of Scc101-D mutant caused by the overexpression of At5g28237 gene.
5. The At5g28237 overexpression transgenic plant can restore Scc101-D mutant phenotype of delayed flowering, this result confirmed that the phenotype of Scc101-D mutant caused by the At5g28237 gene overexpressed. At5g28237 gene was tryptophan synthase gene belongs to the family ofβ-subunit. And the gene gun bombardment of onion skin experiments show this gene was localized in the cytoplasm, and no tissue-specific expression. At5g28237 transgenic plant growed slowly, and exogenous tryptophan can restore wild type phenotype. The phenotype of At5g28237 transgenic plant was similar with At5g58470 deficient mutant. According to the structure of amino acid sequence analysis, the salt bridge formation between the two sites and substrate binding sites are different. Therefore, we speculated that in Arabidopsis thaliana At5g28237 gene may not exercise TRP synthase beta subunit function. But over-expression, it competed At5g58470 protein complex in the tryptophan synthase locus body, the resulting loss of the At5g58470 similar phenotype. In addition, the transcription levels of FT and CO were significantly reduced in At5g28237 transgenic plant, comfirming their involvement in the regulation of flowering signaling pathways.
1.将35S::CRY1-GFP转基因植株在黑暗中培养5天后,采用不同的光处理,实时观察CRY1-GFP融合蛋白在亚细胞中的定位及受光诱导转移过程。实验发现:黑暗条件下CRY1-GFP融合蛋白定位在细胞质中且荧光强度非常微弱;随着蓝光处理时间的延长和光强的增大,GFP荧光逐渐增强,在亚细胞结构(细胞膜、细胞质、细胞核)中均有表达,实验表明CRY1蛋白表达受蓝光诱导,且与光照时间和强度成正比。在蓝光处理过程中(0-15 min),CRY1-GFP融合蛋白从细胞质、细胞膜向细胞核内转移聚集;蓝光处理15 min时,细胞核中GFP荧光强度达到最高,且细胞核与细胞GFP平均荧光强度的比值达到峰值。蛋白免疫杂交结果进一步证实,在蓝光下(0-15 min)GFP蛋白表达与蓝光处理时间成正比。实验首次发现:CRY1-GFP融合蛋白在蓝光诱导下,由细胞质、细胞膜向细胞核内转移累积,这种转移与红光无关。
2.将黑暗中培养5天的野生型黄化苗采用不同的光照处理30 min,提取细胞核蛋白。蛋白免疫杂交检测隐花素CRY1蛋白表达量显示:蓝光下,细胞核内CRY1蛋白表达量比黑暗、红光高,黑暗条件下CRY1蛋白表达量最低。结果进一步证实细胞核内隐花素CRY1蛋白特异性受蓝光诱导表达,或CRY1蛋白受蓝光诱导完成由细胞质、细胞膜向细胞核的转移过程。
3.以隐花素cry1-304突变体为研究背景,成功构建了CRY1-GR/cry1-304转基因植株。实验发现:CRY1-GR/cry1-304转基因植株仅在蓝光和外源糖皮质激素同时存在的条件下回复野生型表型,包括抑制下胚轴的伸长、子叶的展开、花青素的累积及气孔的开启等。实验证实:核定位信号(NLS)和光子激活两要素是隐花素CRY1在细胞核中发挥生理功能的充分必要条件。实验首次将糖皮质受体GR作用机理运用到隐花素CRY1基因功能研究,并证实细胞核隐花素CRY1与下胚轴的伸长、气孔的开启相关;细胞质隐花素CRY1与子叶的展开、叶柄的伸长和花青素的积累相关。
4.以隐花素cry1cry2突变体为研究背景,构建了基因功能获得型T-DNA突变体库,获得了近2 000个独立转化株系。从T1代转基因植株中筛选获得35个株系,与cry1cry2突变体表型明显不同。从中筛选得到一组有价值的光周期开花突变体,为进一步研究隐花素与植物光形态建成提供了宝贵实验材料。采用IPCR法,成功获得了Scc101-D突变体基因组T-DNA插入位点旁邻序列。实验发现,Scc101-D突变体晚花的表型是与At5g28237基因超量表达相关。
5.以野生型为研究背景构建AT5G28237过量表达转基因植株,该转基因植株开花延迟,进一步证实Scc101-D突变体晚花表型是由AT5G28237基因超量表达所致。基因枪轰击洋葱表皮实验首次发现该基因定位在细胞质中,且无组织特异性表达。AT5G28237转基因植株发育缓慢,表明At5g28237基因过表达影响了色氨酸的合成。外源色氨酸能部分恢复至野生型表型,暗示At5g28237基因在拟南芥的生长发育过程中还行使了其他功能。此外,转基因植株的光周期途径开花关键基因FT和CO的转录水平显著降低,结果进一步证实其参与调控植物的开花信号途径。
Plant growth and development process are affected by many external factors.Light as a major environmental element, not only provides the energy required for photosynthesis, but also plays an important role in photomorphogenesis. The evolution of higher plants has a fine feeling of light and signal system, to monitor the direction of light signal, quantity and quality to adjust their growth and development. A large number of studies have shown that blue light receptor cryptochrome-1 mediated blue light to regulate plant growth and development, including the inhibition of hypocotyl elongation, cotyledon opening, stomatal opening, anthocyanin accumulation and the regulation of gene expression. With further research, the study of CRY1 biological functions in subcellular, isolation and identification of Up-and down-stream components was very essential to understand the molecular mechanism of CRY mediated light signal pathway. In this work, we used different lights to analyse subcellular localization of CRY1 and light-induced migration process. On this basis, we constructed the CRY1-GR/cry-304 transgenetic plant on cry1-304 background. Control the location of CRY1 in subcell with the glucocorticoid to assess the biological functions. At the same time, we also contructed the activation tag mutant library on cry1cry2 background, isolated and identified the factors associated cryptochrome components. Through biochemical, genetic and other analysis methods, main results were obtained as followed:
1. The 35S:: CRY1-GFP transgenic plants were grown for 7 days in the dark condition, and then dealt with different lights. The results showed that no GFP fluorescence in the dark. Under blue light, GFP fluorescence gradually increased with intensity increasing of the blue light, and expressed in cytoplasm and cell membrane, and had a transfer from cytoplasm to nucleus. As the treatment of blue light during 0-15 min, GFP fluorescence intensity was the highest, and the nuclei and cell GFP fluorescence intensity ratio reached a peak, then the GFP fluorescence was gradually quenched. Western blot results showed that the expression of GFP protein being directly proportional to the intensity of blue light, but not red light.
2. The wild type were grown for 7 days in the dark condition, then dealt with different light qualities for 30 minutes and nuclear protein was extracted. Western blot analysis showed that: the abundance of blue-induced CRY1 protein was higher than it in red light and dark. The results proved the CRY1 protein specifically induced by blue light, and transported from the cytoplasm to nucleus.
3. We obtained CRY1-GR/cry1-304 transgenic plants on the cry1-304 mutant background, and screened multiple independent lines. The results showed that, CRY1-GR/cry1-304 transgenic plants restore WT phenotype only in the absence of blue light and glucocorticoid (+Dex) conditions, including inhibition of hypocotyl elongation, cotyledon opening, anthocyanin accumulation and stomatal opening. The results show that: Nuclear localization signal (NLS) and photon activation are sufficient and necessary for CRY1 to play function in nucleus. The present study found no evidence of a role for cytoplasmic CRY1 in the control of hypocotyl, stomatal opening and cotyledon petiole elongation, but cotyledon expansion, anthocyanin accumulation all displayed a dependence on cytoplasmic CRY1.
4. We got a gain-of-function mutant library on cry1cry2 background and screened nearly 2000 independent transformant lines. 35 transformant lines had obvious phenotype, and some of the mutant showed delayed flowering time. We also defined the flanking sequence of T-DNA insertion site in Scc101-D mutant through I-PCR analysis. The results showed that the phenotype of Scc101-D mutant caused by the overexpression of At5g28237 gene.
5. The At5g28237 overexpression transgenic plant can restore Scc101-D mutant phenotype of delayed flowering, this result confirmed that the phenotype of Scc101-D mutant caused by the At5g28237 gene overexpressed. At5g28237 gene was tryptophan synthase gene belongs to the family ofβ-subunit. And the gene gun bombardment of onion skin experiments show this gene was localized in the cytoplasm, and no tissue-specific expression. At5g28237 transgenic plant growed slowly, and exogenous tryptophan can restore wild type phenotype. The phenotype of At5g28237 transgenic plant was similar with At5g58470 deficient mutant. According to the structure of amino acid sequence analysis, the salt bridge formation between the two sites and substrate binding sites are different. Therefore, we speculated that in Arabidopsis thaliana At5g28237 gene may not exercise TRP synthase beta subunit function. But over-expression, it competed At5g58470 protein complex in the tryptophan synthase locus body, the resulting loss of the At5g58470 similar phenotype. In addition, the transcription levels of FT and CO were significantly reduced in At5g28237 transgenic plant, comfirming their involvement in the regulation of flowering signaling pathways.
引文
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[85]郑继刚,李成梅,肖英化等.激活标签法及其在植物基因工程上的应用.遗传,2003,25(4):471-474
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