棉花绒毛蛋白基因GhVLN1的功能分析
发布时间：2019-05-18 17:34
【摘要】：棉花纤维是由胚珠部分表皮细胞发育而来的单细胞毛状体,其长度是直径的1000倍以上,因此是研究植物细胞定向伸长的良好模型,研究纤维伸长发育分子机制对于提高棉花纤维产量和品质形状的遗传改良具有十分重要的意义。细胞骨架在植物细胞形态建成以及植物生长发育中起关键作用。由肌动蛋白构成的微丝参与细胞运动、分裂、信号、细胞形态建成等过程。肌动蛋白结合蛋白通过改变肌动蛋白结构的结构调节微丝的结构和功能。在真核生物中,绒毛蛋白(villin)是一种肌动蛋白结合蛋白,对微丝具有成核、切割、加帽、捆绑成束等作用。拟南芥中绒毛蛋白的研究表明,绒毛蛋白对花粉管、根毛等极性生长细胞的伸长发育起重要作用。本实验室曾在Li-1纤维不伸长突变体中鉴定出一个下调表达的绒毛蛋白,暗示这类肌动蛋白结合蛋白在纤维细胞的定向伸长中可能具有重要的调控作用。克隆了编码该蛋白质的基因并命名为GhVLN1。但GhVLN1的功能研究未见报道。本研究通过GhVLN1亚细胞定位,转GhVLN1酵母表型观察,GhVLN1过表达拟南芥表现型和耐盐性、耐热性鉴定,及其RNA-seq转录组表达分析,构建干扰表达载体转化棉花等试验,分析了 GhVLN1的功能及其分子机制,主要结果如下:1、构建植物表达载体pBINPLUS-GhVLN1标记GhVLN1(绿色荧光),与标记微丝的ABD2-mCherry(红色荧光)共转化烟草表皮细胞,发现两种颜色荧光重合,说明GhVLN1定位于微丝骨架,是微丝结合蛋白。2、将酵母表达载体pREP-5N-GhVLN1转化粟酒裂殖酵母,发现GhVLN1基因表达改变了酵母形态,使转基因酵母长宽比增大34.29%,说明GhVLN1基因促进了细胞的极性伸长。3、通过观察GhVLN1过表达拟南芥表型,发现GhVLN1通过影响微丝骨架改变了细胞形态,使得根部细胞伸长,从而促进拟南芥根长增加。GhVLN1过表达拟南芥株系2和5的根长比野生型分别增加了 36.9%和59.1%;GhVLN1促进根部细胞伸长,株系2和5根部伸长区细胞长度比野生型分别增加了 14.8%和15.6%;GhVLN1促进根毛伸长,株系2和5根毛长度比野生型分别增加了 15.7%和18.2%;GhVLN1增加了根部细胞微丝骨架数量,加剧了微丝骨架捆绑,株系2和5的skewness值分别比野生型增加了 32.2%和38.3%。4、GhVLN1过表达拟南芥在种子萌发期和幼苗期的耐盐性显著增强,其中200 mmol/L NaCl存在时,GhVLN1过表达拟南芥株系2和5的种子萌发率分别是野生型的4.57倍和5.38倍;GhVLN1过表达拟南芥幼苗期的耐热性显著增强,热胁迫后,株系2和5的存活率分别是野生型的3.21倍和3.23倍。5、对GhVLN1过表达拟南芥进行RNA-Seq转录表达分析,发现过表达GhVLN1可使拟南芥多糖降解,谷脱甘肽、黄酮类、类胡萝卜素生物合成,角质和蜡质的生物合成等多个通路发生变化,从而提高拟南芥抵抗非生物胁迫的能力。6、构建了干扰表达载体pHellsgate4-GhVLN1并转化了棉花,已获得再生植株。
[Abstract]:Cotton fiber is a single-celled trichomes developed from some epidermis cells of ovule. Its length is more than 1000 times that of diameter, so it is a good model to study the directional extension of plant cells. It is of great significance to study the molecular mechanism of fiber extension and development in order to improve the yield and quality shape of cotton fiber. Cytoskeleton plays a key role in plant cell morphogenesis and plant growth and development. Microfilaments composed of actin are involved in cell movement, division, signal, cell morphogenesis and so on. Actin binding proteins regulate the structure and function of microfilaments by changing the structure of actin. In eukaryotes, chorionic villi (villin) is a kind of actin binding protein, which has the effects of nucleation, cutting, capping and bundling on microfilaments. The study of chorionic villi protein in Arabidopsis thaliana showed that chorionic villi played an important role in the extension and development of polar growth cells such as pollen tube and root hair. In our laboratory, a down-regulated villi protein was identified in Li-1 fiber unelongated mutant, suggesting that this kind of actin binding protein may play an important role in the directional extension of fiber cells. The gene encoding the protein was cloned and named GhVLN1. However, there is no report on the function of GhVLN1. In this study, the subcellular localization of GhVLN1, the phenotypic observation of transgenic GhVLN1 yeast, the phenotypic observation of GhVLN1 overexpression in Arabidopsis thaliana, the identification of heat tolerance, the expression analysis of RNA-seq transcript group, and the construction of interference expression vector for cotton transformation were carried out. The function and molecular mechanism of GhVLN1 were analyzed. The main results were as follows: 1. The plant expression vector pBINPLUS-GhVLN1 labeled GhVLN1 (green fluorescence) was constructed and co-transformed with the labeled microfilament ABD2-mCherry (red fluorescence) into tobacco epidermis cells. It was found that the two colors coincided with each other, indicating that GhVLN1 was located in the microfilament skeleton and was a microfilament binding protein. 2. The yeast expression vector pREP-5N-GhVLN1 was transformed into Saccharomyces cerevisiae, and it was found that the expression of GhVLN1 gene changed the morphology of Saccharomyces cerevisiae. The aspect ratio of transgenic yeast was increased by 34.29%, which indicated that GhVLN1 gene promoted the polar extension of cells. 3. By observing the overexpression of GhVLN1 phenotype in Arabidopsis thaliana, it was found that GhVLN1 changed the cell morphology and made the root cells elongate by affecting the microfilament skeleton. Thus, the root length of Arabidopsis thaliana was increased by 36.9% and 59.1%, respectively, compared with the wild type, and the root length of the overexpressed strain 2 and 5 of ghVLN1 was 36.9% and 59.1% higher than that of the wild type, respectively. GhVLN1 promoted the extension of root cells. The cell length of root extension area of line 2 and 5 was 14.8% and 15.6% higher than that of wild type, respectively. GhVLN1 promoted root hair elongation, and the length of root hair in line 2 and 5 increased by 15.7% and 18.2%, respectively, compared with wild type, and the length of root hair in line 2 and 5 increased by 15.7% and 18.2%, respectively. GhVLN1 increased the number of microfilament skeletons in root cells and aggravated the binding of microfilament skeletons. The skewness values of lines 2 and 5 were 32.2% and 38.3% higher than those of wild type, respectively. The salt tolerance of Arabidopsis thaliana overexpression of GhVLN1 was significantly enhanced at seed germination stage and seedling stage. In the presence of 200 mmol/L NaCl, the seed germination rate of Arabidopsis thaliana line 2 and 5 was 4.57 times and 5.38 times higher than that of wild type, respectively. The heat tolerance of Arabidopsis thaliana seedlings with overexpression of GhVLN1 was significantly enhanced. After heat stress, the survival rates of line 2 and 5 were 3.21 times and 3.23 times higher than those of wild type, respectively. 5. The RNA-Seq transcriptional expression of overexpression of GhVLN1 in Arabidopsis thaliana was analyzed. It was found that overexpression of GhVLN1 could improve the ability of Arabidopsis thaliana to resist abiotic stress by changing many pathways, such as polysaccharide degradation, glutathion, flavonoids, carotenoid biosynthesis, horniness and waxy biosynthesis. The interference expression vector pHellsgate4-GhVLN1 was constructed and transformed into cotton, and the regenerated plants were obtained.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562
本文编号：2480198
[Abstract]:Cotton fiber is a single-celled trichomes developed from some epidermis cells of ovule. Its length is more than 1000 times that of diameter, so it is a good model to study the directional extension of plant cells. It is of great significance to study the molecular mechanism of fiber extension and development in order to improve the yield and quality shape of cotton fiber. Cytoskeleton plays a key role in plant cell morphogenesis and plant growth and development. Microfilaments composed of actin are involved in cell movement, division, signal, cell morphogenesis and so on. Actin binding proteins regulate the structure and function of microfilaments by changing the structure of actin. In eukaryotes, chorionic villi (villin) is a kind of actin binding protein, which has the effects of nucleation, cutting, capping and bundling on microfilaments. The study of chorionic villi protein in Arabidopsis thaliana showed that chorionic villi played an important role in the extension and development of polar growth cells such as pollen tube and root hair. In our laboratory, a down-regulated villi protein was identified in Li-1 fiber unelongated mutant, suggesting that this kind of actin binding protein may play an important role in the directional extension of fiber cells. The gene encoding the protein was cloned and named GhVLN1. However, there is no report on the function of GhVLN1. In this study, the subcellular localization of GhVLN1, the phenotypic observation of transgenic GhVLN1 yeast, the phenotypic observation of GhVLN1 overexpression in Arabidopsis thaliana, the identification of heat tolerance, the expression analysis of RNA-seq transcript group, and the construction of interference expression vector for cotton transformation were carried out. The function and molecular mechanism of GhVLN1 were analyzed. The main results were as follows: 1. The plant expression vector pBINPLUS-GhVLN1 labeled GhVLN1 (green fluorescence) was constructed and co-transformed with the labeled microfilament ABD2-mCherry (red fluorescence) into tobacco epidermis cells. It was found that the two colors coincided with each other, indicating that GhVLN1 was located in the microfilament skeleton and was a microfilament binding protein. 2. The yeast expression vector pREP-5N-GhVLN1 was transformed into Saccharomyces cerevisiae, and it was found that the expression of GhVLN1 gene changed the morphology of Saccharomyces cerevisiae. The aspect ratio of transgenic yeast was increased by 34.29%, which indicated that GhVLN1 gene promoted the polar extension of cells. 3. By observing the overexpression of GhVLN1 phenotype in Arabidopsis thaliana, it was found that GhVLN1 changed the cell morphology and made the root cells elongate by affecting the microfilament skeleton. Thus, the root length of Arabidopsis thaliana was increased by 36.9% and 59.1%, respectively, compared with the wild type, and the root length of the overexpressed strain 2 and 5 of ghVLN1 was 36.9% and 59.1% higher than that of the wild type, respectively. GhVLN1 promoted the extension of root cells. The cell length of root extension area of line 2 and 5 was 14.8% and 15.6% higher than that of wild type, respectively. GhVLN1 promoted root hair elongation, and the length of root hair in line 2 and 5 increased by 15.7% and 18.2%, respectively, compared with wild type, and the length of root hair in line 2 and 5 increased by 15.7% and 18.2%, respectively. GhVLN1 increased the number of microfilament skeletons in root cells and aggravated the binding of microfilament skeletons. The skewness values of lines 2 and 5 were 32.2% and 38.3% higher than those of wild type, respectively. The salt tolerance of Arabidopsis thaliana overexpression of GhVLN1 was significantly enhanced at seed germination stage and seedling stage. In the presence of 200 mmol/L NaCl, the seed germination rate of Arabidopsis thaliana line 2 and 5 was 4.57 times and 5.38 times higher than that of wild type, respectively. The heat tolerance of Arabidopsis thaliana seedlings with overexpression of GhVLN1 was significantly enhanced. After heat stress, the survival rates of line 2 and 5 were 3.21 times and 3.23 times higher than those of wild type, respectively. 5. The RNA-Seq transcriptional expression of overexpression of GhVLN1 in Arabidopsis thaliana was analyzed. It was found that overexpression of GhVLN1 could improve the ability of Arabidopsis thaliana to resist abiotic stress by changing many pathways, such as polysaccharide degradation, glutathion, flavonoids, carotenoid biosynthesis, horniness and waxy biosynthesis. The interference expression vector pHellsgate4-GhVLN1 was constructed and transformed into cotton, and the regenerated plants were obtained.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562
【参考文献】
相关期刊论文 前1条
1 王娜;张振葆;黄凤珠;李洪有;张素芝;;WRKY转录因子参与植物非生物胁迫应答的研究进展[J];核农学报;2014年10期
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