【作者】 肖忠意；

【导师】 罗明；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 棉花是世界上一种重要的纤维作物。棉花纤维是棉花胚珠表面突起分化出的单个细胞,能够快速伸长并且进行次生壁的合成。棉花纤维的发育过程分为四个阶段,包括纤维细胞起始、纤维细胞伸长、次生壁合成和脱水成熟。其中在纤维快速伸长时期纤维细胞的每天伸长速度最快可以达到2.0mm。纤维的长度是纤维品质的一个重要指标,同时也是一个对纤维产量具有重要权重的农艺性状。因此,纤维细胞的伸长对成熟纤维的最终长度具有决定作用。传统育种方法曾经为棉花产量和品质的提高做出了巨大贡献,但利用传统育种方法难以打破纤维产量和品质的负相关,难以达到产量和品质的同步提高。基因工程可以实现目标性状的定向改良,但需要了解纤维发育的分子机理和获得相关的目标基因以及适宜的启动子。由于棉花分子生物学的研究的时间尚短,所以目前对棉花纤维伸长的主要机制还不清楚。因此,克隆和了解棉花纤维细胞起始和伸长相关的关键基因,探索纤维伸长的分子机制,将为利用基因工程改良棉花纤维提供理论基础和转基因材料。植物激素是指在植物体内合成的、对植物生长具有显著调节作用的微量有机物质。油菜素类固醇类物质是第六大植物激素。外施油菜素内酯能促进植物生长,达到增产增质效果。虽然前人发现棉花纤维细胞中BRs合成酶基因在纤维起始和伸长过程中具有重要作用,在棉花纤维中超量表达BRs合成酶基因能够改良纤维数量和质量,但是目前尚未见通过转基因棉花手段了解BRs信号途径基因对棉花纤维细胞起始和伸长的影响的报道。因此,从BRs信号途径基因探索棉花纤维发育的分子机制具有重要的意义,对培育新的棉花品种具有重大的潜力。液泡H⁺-ATPase具有够调节细胞的渗透压的功能,并且由于其在纤维伸长时期的酶活性最高,因此前人认为V-ATPase能够调节对棉花纤维的生长和发育。DET3基因编码V-ATPase C亚基基因,对V-ATPase的酶活性具有重要的调节作用,DET3基因的缺失突变体表现为BRs缺失的典型性状,却对外施BRs不敏感,因而认为DET3介导了BRs信号途径的快速反应途径,从而促进的细胞伸长。为了了解V-ATPase C亚基基因棉花纤维发育的影响,本实验克隆了棉花液泡H⁺-ATPase C亚基基因（GhDET3）,并对基因序列和表达特征进行了分析,然后利用转基因烟草和棉花分析了GhDET3基因的功能。主要结果如下:一、GhDEl"3基因克隆和表达分析通过AtDET3探针在NCBI数据库中筛选和整合相关EST序列,并利用3’-RACE克隆了GhDET3基因的3′-端序列,并最终获得了GhDET3的cDNA和gDNA序列。该基因氨基酸序列与其它物种的同源分析结果表明GhDET3与拟南芥、水稻、苜蓿、大麦和玉米等物种的V-ATPase C亚基有较高的同源性。GhDET3的一级结构具有V-ATPase C亚基保守结构域,且其三级结构与酵母同源蛋白质的三级结构高度相似。Quantitative real-time PCR分析各个组织和器官中GhDET3基因的表达特征,结果表明GhDET3基因在所有受测组织和器官中均有表达,但是在不同组织和器官中表达存在明显差异。在基本组织中,GhDET3基因在下胚轴中的表达水平较高,而在子叶和成熟叶片中的表达水平较低;在棉花纤维发育过程中,GhDET3基因的表达水平在12DPA的纤维中达到最高,是0DPA胚珠（含纤维）表达水平的24.8倍,该基因的表达水平随纤维细胞伸长速率的升高而增加、降低而减少。GhDET3在棉花胚珠中的表达变化模式与纤维中的变化模式相似,但表达量比纤维中小。在棉花胚珠离体培养体系中,外源施用油菜素内酯能够提高GhDET3基因的表达水平,同时,在纤维细胞长度增加的转基因纤维中GhDET3基因的表达水平有所提高。这些结果说明GhDET3基因在棉花纤维细胞的伸长过程中具有重要的作用。二、通过转基因烟草分析GhDET3基因的功能以植物组成性启动子控制GhDET3基因在转基因烟草中超量表达。实验结果表明超量表达GhDET3基因能够增加转基因烟草的生物产量,促进营养生长和生殖生长。转基因烟草的高度与野生型相比较增加24.2%;转基因烟草种子千粒重增加了24.4%;当光照条件下萌发时间到4d时,转基因烟草和野生型的种子萌发比例分别为99.5%和81.8%。对T₂代烟草种子进行萌发实验,结果表明转基因烟草种子萌发比例增加17.7%;在光照条件下处理8d,转基因烟草幼苗的下胚轴和根的长度分别增加38.1%和63.6%:在黑暗条件下处理15d,转基因烟黄化苗的下胚轴和根的长度分别增加37.1%和23.6%。另外,还以此转基因烟草为材料分析了植物激素（24-EBL、GA、ABA和KT）和环境胁迫因子NaCl对超量表达GhDET3烟草生长的影响。实验结果表明,200nM/L 24-EBL处理的转基因烟草幼苗下胚轴和根和长度比相同浓度24-EBL处理的野生型烟草幼苗增加强度更大,说明超量表达GhDET3基因增加了幼苗根和下胚轴对24-EBL的敏感度:超量表达GhDET3烟草在促进根和下胚轴生长作用方面与GA表现协同作用,并且说明在促进根生长方面,GAs与BRs具有协同作用;但是KT,在转基因烟草和野生型烟草幼苗主根和下胚轴的抑制趋势表现相似;利用不同浓度ABA处理烟草种子和幼苗,结果表明,超量表达GhDET3烟草能够影响烟草种子对ABA的拮抗作用,促进种子的萌发,并且在下胚轴和根生长方面,BRs能够与ABA相互拮抗,并且可能通过超量表达GhDET3增加植物对ABA的拮抗作用。另外,超量表达GhDET3烟草能够影响烟草种子对NaCl的拮抗作用,促进种子的萌发,并且在下胚轴和根生长方面,通过超量表达GhDET3可能增加植物对NaCl的拮抗作用。三、通过转基因棉花分析GhDET3基因的功能为了探索GhDET3基因对棉花纤维细胞生长发育影响,本实验构建了反义抑制GhDET3基因的表达载体,并通过遗传转化筛选获得转基因植株。通过quantitative real-time PCR检测GhDET3基因的表达水平,结果发现转基因棉花中GhDET3基因的表达水平受到强烈抑制。对转基因棉花的表型变异观察发现,反义抑制GhDET3基因棉花植株表现为矮化、侧枝长度缩短,顶端优势减弱。但是,棉铃一般在4DPA～6DPA时脱落,因此很难得到成熟的种子和纤维进行观察。扫描电镜和石蜡切片观察0DPA～2DPA棉花胚珠,结果发现反义抑制GhDET3的转基因胚珠表面突起的纤维细胞明显减少,而且纤维细胞的伸长受到抑制。同时,进一步通过胚珠体外培养反义抑制转基因棉花的胚珠证明反义抑制GhDET3基因表达能够抑制纤维细胞的起始和伸长。更多还原

【Abstract】 Cotton is the world’s leading fiber crop. Cotton fiber is an elongated single cell of the ovule epidermis that undergoes rapid and synchronous elongation. Cotton fiber development consists of four discrete yet overlapping stages: initiation, elongation, secondary cell wall accumulation and maturation. During the rapid polar-expansion phase of development, fiber cell may elongate at peak rates in excess of 2.0mm per day. Therefore, fiber cell elongation is one of the important physiological processes directly determined final fiber length, which is an important trait in agronomy. However, the molecular base of fiber cell elongation largely unknown.Plant hormones are any of various hormones produced by plants that control or regulate germination, growth, metabolism, or other physiological activities. Brassionsteroids (BRs) is nominated as the "The Sixth Plant Hormone" of plant. Previous studies indicated that applied into the treatment of plant was able to improve the growth and productivity. Although overexpressing the gene involved BRs synthesis improve the quantity and quality of cotton fiber, less studies of the gene involved in BRs signal transduction through transgenic strategies have been reported up to now.Vacuolar H~+-ATPase (V-ATPase) was regarded as a key enzyme promoting the fiber cell elongation through regulating turgor-driven pressure involved in polar-expansion of single cell fiber. The DET3, encoding V-ATPase subunit C, played an important role in assembling the subunits of V-ATPase complex and regulating the complex activity, and involved in Brassinosteroids induced cell elongation. In order to understand the effects of cotton V-ATPase subunit C gene on fiber growth and development and the molecular base of BR action in fiber cell growth, we cloned the V-ATPase subunit C gene. Gene sequence analysis and expression pattern were taken, and then cotton and tobacco, as plant models, were transformed for genetic analysis.1. Identification and Characterization of GhDET3AtDET3 was utilized as electronic probe to screen the candidate sequences in NCBI database, and 3’-RACE technique was applied to clone the 3’-downstream sequence for the full length of GhDET3 cDNA. The full length clones were obtained from cDNA and gDNA, respectively. The deduced amino acid sequence from ORF had a high homology with DET3 from Arabidopsis, rice, as well as maize. Quantitative real-time PCR analysis showed that GhDET3 expressed ubiquitously in all detected tissues and organs. However, obvious differences were investigated between samples detected. The accumulation of GhDET3 mRNA reached the highest level in 12DPA fibers, in which stage fiber cell elongated rapidly. The lowest level was detected in 0DPA ovules (with fibers), in which stage fiber cell initiated from epidermis. The transcript accumulation of GhDET3 mRNA in ovules shared a similar variation with that in in fibers. In addition, in vitro ovule culture experiment demonstrated that applying exogenous 24-EBL treatment to 4DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, while the accumulative of GhDET3 increased in transgenic FBP7∷GhDET2 cotton fibers in vivo. All these results pointed out that the GhDET3 gene played a crucial role in cotton fiber elongation.2. Gene function analysis of GhDET3 in transgenic tobaccoTo understand effects of GhDET3, a homologue of DET3 in cotton, p6-CaMV35S:.senseGhDET3 cassette was constructed and transformed into tobacco through Agrobacterium-mediated transformation. As a result, we found that the transgenic plants overexpressing GhDET3 promoted the vegetative growth. The plant height and leaf size were increased relative to wild-type, and the length of hypocotyl and root of transgenic seedling were longer than that of wild-type either in the light or dark. Moreover, overexpression of GhDET3 also resulted in the achene weight and seed size increased. Furthermore, transgenic seedling that compared with wild-type was more sensitive in response to gibberellin and 24-epibrassinolide. On the contrary, the transgenic seedling was insensitive in response to ABA and NaCl in the manner of seed germination and seedling elongation. Together with our data provided, the results indicated that GhDET3 plays a significant role in plant growth, while it was proposed that GhDET3 might be the most significant subunit that is responsible for BRs-induced cell elongation and acts as a master accounting for regulating stress tolerance.3. Gene function analysis of GhDET3 in transgenic cottonTo understand roles of GhDET3 gene in the development of cotton fiber growth and development, we constructed p6-CaMV35S∷antisenseGhDET3 vector and transformed into cotton plants, by means of Agrobacterium-mediated transformation. And then, the positive lines were screened and confirmed through both histochemical staining of GUS activity and PCR analysis of GUS gene. Comparing with wild-type, quantitative real-time PCR analysis indicated expression levels of GhDET3 in transgenic plants were inhibited dramatically. Suppression of GhDET3 expression also resulted in changes of phenotypes in transgenic cotton plants. Analysis of basic organs and tissues revealed that transgenic plants exhibited dwarf, reduced leaf size, shorten branches, and decreased apical advantages. Cotton boll produced from the antisenseGhDET3 plants were easy to abort between 4DPA to 6DPA. Thus, it is impossible to detect the quantity and quality of mature fiber. Based on the technologies of SEM and paraffin sections, 0DPA-2DPA ovules from wild-type and transgenic plants were analyzed. The results revealed that less fiber cells of 0DPA and 2DPA were detected in transgenic plants, comparing with wild-type, while the elongation of fibercell was also suppressed in transgenic plants. Furthermore, ovules from 0DPA balls were separatedand cultured in BT medium for 10 d in vitro, the resulted demonstrated GhDET3 gene play a crucialrole in fiber cell elongation.更多还原