棉花絨毛蛋白基因GhVLN1的功能分析
發(fā)布時間：2019-05-18 17:34
【摘要】：棉花纖維是由胚珠部分表皮細(xì)胞發(fā)育而來的單細(xì)胞毛狀體,其長度是直徑的1000倍以上,因此是研究植物細(xì)胞定向伸長的良好模型,研究纖維伸長發(fā)育分子機制對于提高棉花纖維產(chǎn)量和品質(zhì)形狀的遺傳改良具有十分重要的意義。細(xì)胞骨架在植物細(xì)胞形態(tài)建成以及植物生長發(fā)育中起關(guān)鍵作用。由肌動蛋白構(gòu)成的微絲參與細(xì)胞運動、分裂、信號、細(xì)胞形態(tài)建成等過程。肌動蛋白結(jié)合蛋白通過改變肌動蛋白結(jié)構(gòu)的結(jié)構(gòu)調(diào)節(jié)微絲的結(jié)構(gòu)和功能。在真核生物中,絨毛蛋白(villin)是一種肌動蛋白結(jié)合蛋白,對微絲具有成核、切割、加帽、捆綁成束等作用。擬南芥中絨毛蛋白的研究表明,絨毛蛋白對花粉管、根毛等極性生長細(xì)胞的伸長發(fā)育起重要作用。本實驗室曾在Li-1纖維不伸長突變體中鑒定出一個下調(diào)表達(dá)的絨毛蛋白,暗示這類肌動蛋白結(jié)合蛋白在纖維細(xì)胞的定向伸長中可能具有重要的調(diào)控作用�？寺×司幋a該蛋白質(zhì)的基因并命名為GhVLN1。但GhVLN1的功能研究未見報道。本研究通過GhVLN1亞細(xì)胞定位,轉(zhuǎn)GhVLN1酵母表型觀察,GhVLN1過表達(dá)擬南芥表現(xiàn)型和耐鹽性、耐熱性鑒定,及其RNA-seq轉(zhuǎn)錄組表達(dá)分析,構(gòu)建干擾表達(dá)載體轉(zhuǎn)化棉花等試驗,分析了 GhVLN1的功能及其分子機制,主要結(jié)果如下:1、構(gòu)建植物表達(dá)載體pBINPLUS-GhVLN1標(biāo)記GhVLN1(綠色熒光),與標(biāo)記微絲的ABD2-mCherry(紅色熒光)共轉(zhuǎn)化煙草表皮細(xì)胞,發(fā)現(xiàn)兩種顏色熒光重合,說明GhVLN1定位于微絲骨架,是微絲結(jié)合蛋白。2、將酵母表達(dá)載體pREP-5N-GhVLN1轉(zhuǎn)化粟酒裂殖酵母,發(fā)現(xiàn)GhVLN1基因表達(dá)改變了酵母形態(tài),使轉(zhuǎn)基因酵母長寬比增大34.29%,說明GhVLN1基因促進了細(xì)胞的極性伸長。3、通過觀察GhVLN1過表達(dá)擬南芥表型,發(fā)現(xiàn)GhVLN1通過影響微絲骨架改變了細(xì)胞形態(tài),使得根部細(xì)胞伸長,從而促進擬南芥根長增加。GhVLN1過表達(dá)擬南芥株系2和5的根長比野生型分別增加了 36.9%和59.1%;GhVLN1促進根部細(xì)胞伸長,株系2和5根部伸長區(qū)細(xì)胞長度比野生型分別增加了 14.8%和15.6%;GhVLN1促進根毛伸長,株系2和5根毛長度比野生型分別增加了 15.7%和18.2%;GhVLN1增加了根部細(xì)胞微絲骨架數(shù)量,加劇了微絲骨架捆綁,株系2和5的skewness值分別比野生型增加了 32.2%和38.3%。4、GhVLN1過表達(dá)擬南芥在種子萌發(fā)期和幼苗期的耐鹽性顯著增強,其中200 mmol/L NaCl存在時,GhVLN1過表達(dá)擬南芥株系2和5的種子萌發(fā)率分別是野生型的4.57倍和5.38倍;GhVLN1過表達(dá)擬南芥幼苗期的耐熱性顯著增強,熱脅迫后,株系2和5的存活率分別是野生型的3.21倍和3.23倍。5、對GhVLN1過表達(dá)擬南芥進行RNA-Seq轉(zhuǎn)錄表達(dá)分析,發(fā)現(xiàn)過表達(dá)GhVLN1可使擬南芥多糖降解,谷脫甘肽、黃酮類、類胡蘿卜素生物合成,角質(zhì)和蠟質(zhì)的生物合成等多個通路發(fā)生變化,從而提高擬南芥抵抗非生物脅迫的能力。6、構(gòu)建了干擾表達(dá)載體pHellsgate4-GhVLN1并轉(zhuǎn)化了棉花,已獲得再生植株。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S562
本文編號：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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S562
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 王娜;張振葆;黃鳳珠;李洪有;張素芝;;WRKY轉(zhuǎn)錄因子參與植物非生物脅迫應(yīng)答的研究進展[J];核農(nóng)學(xué)報;2014年10期
，本文編號：2480198
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2480198.html
最近更新
教材專著
