生长素响应因子GhARF6对棉花生长发育的影响
发布时间：2019-05-30 03:37
【摘要】：棉花是关系国计民生的重要物资,在国民经济发展中占有重要的地位。生长素可以调控细胞的伸长、分裂和分化,不定根的形成、果实的发育和胚的形成等。生长素通过其信号途径对功能基因进行调控,从而影响植物的生长发育。因此,生长素信号传导途径及其对下游基因的调控机理是近年来植物生物学研究的热点。棉花中生长素信号传导相关基因的生物学功能进行研究,有助于解生长素在棉花生长发育以及形态建成过程中所发挥的作用,在阐明棉花中生长素信号传导的复杂分子机制方面具有重要的理论意义。ARFs(Auxin response factors)基因家族的发现,使人们对生长素信号传导途径的认识有了突破性的进展。ARF6基因与植物顶端分生组织的生长、维管组织的发育、解除顶芽休眠和花器官的发育相关。为了进一步解析ARF6基因在陆地棉发育过程中的功能,本论文首先克隆了GhARF6基因,分析其启动子的表达特性。通过调控GhARF6基因在棉花中的表达水平,来探究GhARF6基因对棉花生长发育的影响。具体研究结果如下:1.GhARF6基因的克隆与表达特性分析首先从陆地棉中克隆了ARF6基因的同源基因GhARF6基因。该基因位于26条染色体中的5号染色体上,其含有一个2715 bp的ORF框,编码904个氨基酸残基,蛋白分子量为99.78 kDa,p I=6.33。拟南芥中ARFs被分为5类,将检索到的96个陆地棉ARF基因与拟南芥ARF基因各个分类中具有代表性的10个基因氨基酸序列进行进化树分析显示:陆地棉中含有8个ARF亚家族,与GhARF6基因相似度高的GhARF6-1和GhARF6-2与AtARF6、AtARF8分在了同一个亚家族,且与AtARF6基因同源性较高。对GhARF6基因氨基酸序列结构分析显示其具有典型的ARF蛋白结构,并且在440-620氨基酸之间富集了谷氨酰胺(Q),推测其为转录激活子。在棉花中进行表达分析发现GhARF6基因在棉花植株的各个部位均有表达,茎中表达水平相对最高,是叶片中表达水平的10.1倍。在纤维和胚珠生长发育过程中表达分析显示该基因在0-4 DPA的纤维中相对表达水平较高,0-6 DPA的胚珠中相对于其他阶段表达水平较高,并在开花后6天的胚珠中达到顶峰。预示该基因在胚珠发育起始阶段发挥重要作用。用不同激素对棉花幼苗进行处理,结果显示:GhARF6基因在子叶、下胚轴和根部均有表达。GhARF6基因在IAA、NPA、NAA处理的子叶中表达水平相对较高,相对表达水平分别是未处理的12.7倍、11.5倍、9.0倍;用IAA、NAA、NPA处理后下胚轴中GhARF6基因的相对表达水平是未处理的8.3倍、3.4倍、4.2倍;在根部的表达水平在处理前后无明显变化。用不同浓度的Al3+处理棉花幼苗发现:0?M、25?M、50?M Al3+浓度下GhARF6基因随着Al3+浓度的增加表达量增加,Al3+浓度为50?M时GhARF6基因表达水平达到最高。50?M、100?M、200?M Al3+浓度下GhARF6基因的表达水平随着Al3+浓度的增加而下降。将棉花幼苗在50?M Al3+的培养液中进行了不同时间段的处理,发现GhARF6基因的表达量在0-3 h内上调,3 h后开始下降,9 h后表达水平恢复到未处理前的水平。2.GhARF6基因启动子克隆及表达特性分析为了进一步了解GhARF6基因的功能,课题组前期克隆了该基因上游2100 bp的启动子序列,经PlantCARE网站分析发现该序列含有众多的植物顺式元件,包含生长素响应元件、光响应相关元件、组织表达相关元件、水杨酸响应元件、防御及胁迫响应的相关元件、特定部位表达相关的元件等。将该启动子与GUS基因融合后分别转化棉花和拟南芥,组织化学分析发现:拟南芥中该启动子主要在转基因植株的莲座叶、角果、花柄、花丝、柱头顶部、主根、侧根和根毛中表达。其中在花柄、主根和侧根中表达活性相对较强,GUS基因在转基因棉花植株的各个部位均有表达,主要集中在茎、下胚轴、根的维管束组织中。该基因启动子在拟南芥与棉花中的表达模式基本相一致。对PGhARF6转基因拟南芥用Al3+处理后发现:用50?M Al3+处理9 h后拟南芥的根尖、下胚轴、子叶相对未处理着色深。3.调控GhARF6基因影响棉花生长发育为了进一步探究陆地棉中GhARF6基因的生物学功能,构建CaMV35S组成型启动子控制的GhARF6基因反义抑制表达载体、超量表达载体、RNA干扰表达载体,并分别对棉花进行遗传转化。对转基因棉花植株进行表型观察发现:下调GhARF6基因后,棉花花器官呈现特异表型:花瓣小,雄蕊短且花药延迟释放。同时伴随着落铃的现象,RNA干扰植株落铃较为严重,有的达到了100%。上调GhARF6基因后,棉花花器官发育无明显变化。下调GhARF6基因后,种子变得不饱满并且单株单个棉铃中种子明显减少:反义转基因植株3-4-3、41-1-1和干扰转基因植株2-2、2-4分别减少了42%、33.5%、39.6%、29.7%;对转基因棉花植株单株成熟纤维的长度测量发现:成熟纤维变短,这种表型在RNAi转基因植株中更为明显。与野生型相比,反义转基因植株3-4-3、41-1-1和干扰转基因植株2-2、2-4转基因棉花成熟棉纤维长度分别减少了12%、14.5%、15.4%、19.3%。然而,上调GhARF6基因对棉花种子和纤维的生长发育无明显影响。
[Abstract]:The cotton is an important material for the national economy and the people's livelihood, and plays an important role in the development of the national economy. The auxin can regulate the elongation, division and differentiation of the cell, the formation of the adventitious root, the development of the fruit, the formation of the embryo, and the like. The auxin regulates the function gene through its signal pathway, thus affecting the growth and development of the plant. Therefore, the pathway of auxin signaling and the regulation mechanism of the downstream gene are the hot spots in the research of plant biology in recent years. The study on the biological function of auxin signaling related genes in cotton is of great theoretical significance in elucidating the complex molecular mechanism of auxin signaling in cotton. The discovery of the ARFs gene family has made a breakthrough in the understanding of the pathway of auxin signaling. The growth of the ARF6 gene and the apical meristem of the plant, the development of vascular tissue, the release of the dormancy of the apical bud and the development of the flower organ. In order to further analyze the function of the ARF6 gene in the development of the upland cotton, the GhARF6 gene was first cloned and the expression of the promoter was analyzed. The effects of the GhARF6 gene on the growth and development of cotton were investigated by regulating the expression level of the GhARF6 gene in the cotton. The specific results are as follows:1. The cloning and expression of the GhARF6 gene is the first to clone the gene GhARF6 of the ARF6 gene from the upland cotton. The gene is located on chromosome 5 of the 26 chromosomes, which contains a 2715 bp ORF frame encoding 904 amino acid residues with a protein molecular weight of 99.78 kDa and p I = 6.33. The ARFs in Arabidopsis were divided into 5 classes, and the retrieved 96 upland cotton ARF genes were compared with the representative 10 gene amino acid sequences in each classification of the Arabidopsis ARF gene to show that the upland cotton contains 8 ARF subfamilies, GhARF6-1 and GhARF6-2 and AARF6 with high similarity to the GhARF6 gene, AtARF8 is in the same subfamily and has a high homology to the AARF6 gene. The structural analysis of the amino acid sequence of the GhARF6 gene shows that it has a typical ARF protein structure and is enriched in the 440-620 amino acid with a glutaamine (Q), which is presumed to be a transcriptional activator. The expression of the GhARF6 gene in various parts of the cotton plant was found in the cotton, and the expression level of the stem was relatively high, which was 10.1 times that of the expression level in the leaves. The expression of the gene in the growth and development of the fiber and the ovule showed that the relative expression level of the gene in the fiber of 0-4DPA was high, the expression level of the ovule of 0-6DPA was high relative to the other stage, and the peak was reached in the ovule of 6 days after flowering. It indicates that the gene plays an important role in the initiation stage of the development of the ovule. The results showed that the GhARF6 gene was expressed in the cotyledons, hypocotyl and root. The expression level of the GhARF6 gene in the cotyledons treated with IAA, NPA and NAA was 12.7-fold, 11.5-fold and 9.0-fold, respectively. The relative expression level of the GhARF6 gene in hypocotyl was 8.3-fold, 3.4-fold and 4.2-fold with IAA, NAA and NPA. The expression level of the roots did not change significantly before and after treatment. The expression level of GhARF6 gene increased with the increase of Al3 + concentration and the expression level of GhARF6 was the highest when the concentration of Al3 + was 50? M, with the increase of Al3 + concentration in the cotton seedlings treated with Al3 + treated with different concentration. The expression level of GhARF6 gene in the concentration of 200? M Al3 + decreased with the increase of Al3 + concentration. the cotton seedlings are treated in a culture solution of 50-M Al3 + for different time periods, The expression of the GhARF6 gene was up-regulated within 0-3 h, decreased after 3 h, and the expression level after 9 h was restored to the pre-treated level. The PlantCARRE site analysis found that the sequence contained numerous plant cis-elements, including auxin response elements, light response related elements, tissue expression related elements, salicylic acid response elements, defence and stress response related elements, specific site expression related elements, and the like. After the promoter and the GUS gene were fused, the cotton and the Arabidopsis were respectively transformed, and the chemical analysis of the tissue found that the promoter is mainly expressed in the lotus leaf, the corner fruit, the flower stem, the flower filament, the head of the stigma, the main root, the lateral root and the root hairs of the transgenic plant. Wherein the expression activity of the GUS gene in the stem, the main root and the lateral root is relatively strong, and the GUS gene is expressed in various parts of the transgenic cotton plant, and is mainly concentrated in the vascular bundle tissues of the stem, the hypocotyl and the root. The gene promoter is basically consistent with the expression pattern of the Arabidopsis and the cotton. It was found that the root tip, hypocotyl and cotyledons of Arabidopsis were treated with 50? M Al3 + for 9 h. In ord to further study that biological function of the GhARF6 gene in the Gossypium hirsutum L., the GhARF6 gene antisense expression vector, the over-expression vector and the RNA interference expression vector control by the CaMV35S constitutive promoter are constructed, and the cotton is respectively genetically transformed. Phenotypic observation of transgenic cotton plants showed that, after the GhARF6 gene was down-regulated, the cotton flower organs exhibited a specific phenotype: the petals were small, the stamens were short, and the anthers were delayed release. At the same time, with the phenomenon of the landing bell, the ring of the RNA interference plant is more serious and has reached 100%. After up-regulation of the GhARF6 gene, there was no significant change in the development of the floral organ of the cotton. After the GhARF6 gene was down-regulated, the seed became less plump and the seed of single boll in single plant was significantly reduced:3-4-3,41-1-1 and 2-2,2-4 of the antisense transgenic plants were reduced by 42%, 33.5%, 39.6% and 29.7%, respectively; The mature fiber becomes shorter, and the phenotype is more obvious in the RNAi transgenic plant. Compared with the wild type, the length of the mature cotton fiber of the transgenic cotton 3-4-3,41-1-1 and the transgenic plants 2-2,2-4 decreased by 12%, 14.5%, 15.4% and 19.3%, respectively. However, the up-regulation of the GhARF6 gene has no significant effect on the growth and development of cotton seeds and fibers.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562
本文编号：2488488
[Abstract]:The cotton is an important material for the national economy and the people's livelihood, and plays an important role in the development of the national economy. The auxin can regulate the elongation, division and differentiation of the cell, the formation of the adventitious root, the development of the fruit, the formation of the embryo, and the like. The auxin regulates the function gene through its signal pathway, thus affecting the growth and development of the plant. Therefore, the pathway of auxin signaling and the regulation mechanism of the downstream gene are the hot spots in the research of plant biology in recent years. The study on the biological function of auxin signaling related genes in cotton is of great theoretical significance in elucidating the complex molecular mechanism of auxin signaling in cotton. The discovery of the ARFs gene family has made a breakthrough in the understanding of the pathway of auxin signaling. The growth of the ARF6 gene and the apical meristem of the plant, the development of vascular tissue, the release of the dormancy of the apical bud and the development of the flower organ. In order to further analyze the function of the ARF6 gene in the development of the upland cotton, the GhARF6 gene was first cloned and the expression of the promoter was analyzed. The effects of the GhARF6 gene on the growth and development of cotton were investigated by regulating the expression level of the GhARF6 gene in the cotton. The specific results are as follows:1. The cloning and expression of the GhARF6 gene is the first to clone the gene GhARF6 of the ARF6 gene from the upland cotton. The gene is located on chromosome 5 of the 26 chromosomes, which contains a 2715 bp ORF frame encoding 904 amino acid residues with a protein molecular weight of 99.78 kDa and p I = 6.33. The ARFs in Arabidopsis were divided into 5 classes, and the retrieved 96 upland cotton ARF genes were compared with the representative 10 gene amino acid sequences in each classification of the Arabidopsis ARF gene to show that the upland cotton contains 8 ARF subfamilies, GhARF6-1 and GhARF6-2 and AARF6 with high similarity to the GhARF6 gene, AtARF8 is in the same subfamily and has a high homology to the AARF6 gene. The structural analysis of the amino acid sequence of the GhARF6 gene shows that it has a typical ARF protein structure and is enriched in the 440-620 amino acid with a glutaamine (Q), which is presumed to be a transcriptional activator. The expression of the GhARF6 gene in various parts of the cotton plant was found in the cotton, and the expression level of the stem was relatively high, which was 10.1 times that of the expression level in the leaves. The expression of the gene in the growth and development of the fiber and the ovule showed that the relative expression level of the gene in the fiber of 0-4DPA was high, the expression level of the ovule of 0-6DPA was high relative to the other stage, and the peak was reached in the ovule of 6 days after flowering. It indicates that the gene plays an important role in the initiation stage of the development of the ovule. The results showed that the GhARF6 gene was expressed in the cotyledons, hypocotyl and root. The expression level of the GhARF6 gene in the cotyledons treated with IAA, NPA and NAA was 12.7-fold, 11.5-fold and 9.0-fold, respectively. The relative expression level of the GhARF6 gene in hypocotyl was 8.3-fold, 3.4-fold and 4.2-fold with IAA, NAA and NPA. The expression level of the roots did not change significantly before and after treatment. The expression level of GhARF6 gene increased with the increase of Al3 + concentration and the expression level of GhARF6 was the highest when the concentration of Al3 + was 50? M, with the increase of Al3 + concentration in the cotton seedlings treated with Al3 + treated with different concentration. The expression level of GhARF6 gene in the concentration of 200? M Al3 + decreased with the increase of Al3 + concentration. the cotton seedlings are treated in a culture solution of 50-M Al3 + for different time periods, The expression of the GhARF6 gene was up-regulated within 0-3 h, decreased after 3 h, and the expression level after 9 h was restored to the pre-treated level. The PlantCARRE site analysis found that the sequence contained numerous plant cis-elements, including auxin response elements, light response related elements, tissue expression related elements, salicylic acid response elements, defence and stress response related elements, specific site expression related elements, and the like. After the promoter and the GUS gene were fused, the cotton and the Arabidopsis were respectively transformed, and the chemical analysis of the tissue found that the promoter is mainly expressed in the lotus leaf, the corner fruit, the flower stem, the flower filament, the head of the stigma, the main root, the lateral root and the root hairs of the transgenic plant. Wherein the expression activity of the GUS gene in the stem, the main root and the lateral root is relatively strong, and the GUS gene is expressed in various parts of the transgenic cotton plant, and is mainly concentrated in the vascular bundle tissues of the stem, the hypocotyl and the root. The gene promoter is basically consistent with the expression pattern of the Arabidopsis and the cotton. It was found that the root tip, hypocotyl and cotyledons of Arabidopsis were treated with 50? M Al3 + for 9 h. In ord to further study that biological function of the GhARF6 gene in the Gossypium hirsutum L., the GhARF6 gene antisense expression vector, the over-expression vector and the RNA interference expression vector control by the CaMV35S constitutive promoter are constructed, and the cotton is respectively genetically transformed. Phenotypic observation of transgenic cotton plants showed that, after the GhARF6 gene was down-regulated, the cotton flower organs exhibited a specific phenotype: the petals were small, the stamens were short, and the anthers were delayed release. At the same time, with the phenomenon of the landing bell, the ring of the RNA interference plant is more serious and has reached 100%. After up-regulation of the GhARF6 gene, there was no significant change in the development of the floral organ of the cotton. After the GhARF6 gene was down-regulated, the seed became less plump and the seed of single boll in single plant was significantly reduced:3-4-3,41-1-1 and 2-2,2-4 of the antisense transgenic plants were reduced by 42%, 33.5%, 39.6% and 29.7%, respectively; The mature fiber becomes shorter, and the phenotype is more obvious in the RNAi transgenic plant. Compared with the wild type, the length of the mature cotton fiber of the transgenic cotton 3-4-3,41-1-1 and the transgenic plants 2-2,2-4 decreased by 12%, 14.5%, 15.4% and 19.3%, respectively. However, the up-regulation of the GhARF6 gene has no significant effect on the growth and development of cotton seeds and fibers.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562
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