【作者】 张则婷；

【导师】 李学宝；

【作者基本信息】 华中师范大学 ， 植物学， 2010， 博士

【摘要】 棉花是世界上最重要的纤维作物,我国是重要的棉花生产国和消费国,棉花在我国国民经济中占有十分重要的地位。棉纤维细胞是由胚珠外表皮分化而成的不分枝单细胞毛状突起,是棉花生产的主要产品,其产量和品质直接决定了棉花生产的产值和效益。因此,提高棉花纤维的产量和品质一直以来都是棉花育种的主要目标。利用基因工程的方法可以实现优良基因的定向转移进而实现棉花纤维产量与品质的改良,因此研究棉花纤维发育的分子机制、克隆与棉花纤维发育相关的基因,对棉花纤维品质改良具有重要意义。14-3-3蛋白是一类广泛存在于真核生物中高度保守的调控蛋白,它们通过识别特异的磷酸化序列与靶蛋白相互作用,参与一系列信号传导和调控过程,在植物生长发育中起着重要的作用。为了研究14-3-3蛋白在棉花纤维发育过程中的调控作用,我们分离克隆了棉花14-3-3基因,并对这些基因的结构、表达模式及其编码蛋白的亚细胞定位、蛋白质之间的相互作用及功能进行了研究。主要结果如下：1.Gh14-3-3基因的分离克隆及氨基酸序列分析从棉花cDNA文库中分离克隆了5个编码14-3-3蛋白基因的cDNA,分别被命名为Gh14-3-3L、Gh14-3-3a、Gh14-3-3e、Gh14-3-3f, Gh14-3-3g。同时,利用PCR技术从棉花基因组中克隆了4个Gh14-3-3基因的全长序列,通过与cDNA序列比较分析发现,Gh14-3-3L和Gh14-3-3a两个基因的结构相似,均由6个外显子和5个内含子组成;Gh14-3-3e和Gh14-3-3f两个基因结构相似,由4个外显子和3个内含子组成,且内含子的插入部位和大小都具有保守性。序列比较和进化分析结果表明,这5个Gh14-3-3基因的编码蛋白可以被分为两类,其中3个(Gh14-3-3e、Gh14-3-3f和Gh14-3-3g)属于非ε型14-3-3蛋白,另外2个(Gh14-3-3L和Gh14-3-3a)蛋白属于ε型14-3-3蛋白。2.Gh14-3-3蛋白的亚细胞定位为了研究Gh14-3-3蛋白在细胞中的分布情况,利用GFP荧光蛋白共定位技术研究了Gh14-3-3蛋白的亚细胞定位。构建了Gh14-3-3L、Gh14-3-3a及Gh14-3-3e基因与eGFP的融合表达载体,并在农杆菌介导下转化棉花下胚轴。经筛选培养后获得抗性愈伤组织,激光共聚焦显微镜观察表明,Gh14-3-3L蛋白主要定位于细胞质以及细胞核中,Gh14-3-3a和Gh14-3-3e蛋白主要分布于细胞质中。3.Gh14-3-3基因在棉纤维发育过程中高水平表达RT-PCR结果表明,5个Gh14-3-3基因都在棉花纤维中优势或者有较高水平的表达,而在其他组织中的表达水平相对较低。为了研究Gh14-3-3基因与棉花纤维起始发育阶段的关系,比较了Ghl4-3-3基因在无绒无絮棉和野生型棉花开花当天胚珠中的差异表达,结果显示有2个Gh14-3-3基因(Gh14-3-3e和Ghl4-3-3f)在野生型开花当天胚珠中的表达水平显著高于同期无绒无絮棉胚珠中的表达水平,其在野生型中的表达量分别为无绒无絮棉中的2.5倍和2.6倍；另外RT-PCR结果还表明,5个Gh14-3-3基因在棉花纤维中的表达都受到纤维发育时期的调控,其转录产物主要集中在伸长期的棉花纤维中,表达水平从开花后逐渐上升至开花后十天达到最高,之后逐步下降。此外,为了研究Gh14-3-3基因与影响棉花纤维发育的各种激素之间的关系,通过在胚珠离体培养条件下添加各种激素对纤维进行处理并比较了Gh14-3-3基因的表达水平变化,结果显示,Gh14-3-3基因在棉花纤维中的表达水平受到生长素、赤霉素、油菜素内酯等激素水平的影响。以上结果说明,Gh14-3-3基因在棉花纤维起始、伸长以及激素调控过程中可能具有重要的作用。4.Gh14-3-3蛋白之间的相互作用具有特异选择性为了研究分离得到的5个Gh14-3-3蛋白之间能否形成同源或者异源二聚体,利用酵母双杂交系统分析了5个Gh14-3-3蛋白之间的相互作用。结果显示这5个Gh14-3-3蛋白中有4组蛋白之间可以发生相互作用,然而这种相互作用都发生于不同的Gh14-3-3蛋白之间,各Gh14-3-3蛋白本身都不能形成同源二聚体。这表明Gh14-3-3蛋白相互作用具有特异性,可能以形成异源二聚体的形式作用于棉花纤维的发育过程。5.Gh14-3-3蛋白的互作蛋白筛选为了研究Gh14-3-3蛋白参与棉花纤维发育调控的机制,选择了酵母双杂交系统从棉花纤维中筛选Gh14-3-3蛋白的互作蛋白。构建了棉花纤维酵母双杂交cDNA文库,分别依次以每个Gh14-3-3蛋白作为诱饵蛋白从棉花纤维酵母双杂交cDNA文库中筛选互作蛋白。通过对阳性克隆的测序和比较分析发现,Gh14-3-3蛋白能够与BZR1、MYB.H+-ATP酶等16种蛋白质发生相互作用。另外,我们还挑选了部分互作蛋白检测了它们与Gh14-3-3蛋白之间互作的选择性,发现多数互作蛋白都能与多个Gh14-3-3蛋白发生相互作用,并且这种相互作用是具有选择性的。提示Gh14-3-3蛋白可能在棉花纤维发育过程中的激素调控、细胞膜调节、基因转录水平调节等过程中发挥一定的作用。6.在拟南芥中过量表达Gh14-3-3e提高植株对Brz的敏感性为了研究Gh14-3-3蛋白对植物生长发育的影响,构建了Gh14-3-3e基因过量表达载体并转化模式植物拟南芥,获得转基因植株。利用RT-PCR和Western-blot实验分别对Gh14-3-3e基因及其编码蛋白的表达进行了分析。结果表明,在RNA水平和蛋白水平均检测到了Gh14-3-3e在转基因拟南芥中的表达。通过对过量表达纯合体小苗的表型分析发现,与野生型相比,在暗培养条件下转基因拟南芥增强了对BR合成抑制剂Brz的敏感性,说明Gh14-3-3e可能在棉花BR信号转导途径中起到调控作用。对转基因拟南芥中BR信号通路相关基因表达水平的分析有待进一步研究。7.Gh14-3-3L和Gh14-3-3e过量表达和RNA i对转基因棉花的影响为了研究Gh14-3-3蛋白在棉花纤维发育过程中的功能,首先将Gh14-3-3L、Gh14-3-3a和Gh14-3-3d在酵母细胞中进行过量表达,研究Gh14-3-3基因对单细胞生长发育及形态方面的影响,发现这三个基因的过量表达都导致酵母细胞出现显著的纵向伸长。同时,构建了Gh14-3-3L、Gh14-3-3e基因的过量表达和RNAi载体并用农杆菌介导的方法转化棉花,共获得了108株转基因植株。分析表明,过量表达转基因棉花中Gh14-3-3基因的表达水平明显上升,大多数Gh14-3-3L和Gh14-3-3e过量表达转基因棉花均出现植株矮小,节间变短,叶片成簇生长以及不育等现象,棉铃在开花后3-5天脱落,无法得到转基因成熟种子和纤维。对RNAi转基因棉花中的Gh1l4-3-3基因表达水平分析表明,在RNAi转基因棉花中,Gh14-3-3L和Ghl4-3-3e基因表达水平都受到极大程度的抑制。同时,对转基因棉花纤维中对与纤维发育相关基因的表达情况分析结果表明,Gh14-3-3基因的沉默导致纤维发育相关基因的表达受到一定程度的抑制。另外,对Gh14-3-3e基因RNAi转基因棉花胚珠的切片分析表明,Gh14-3-3e基因沉默导致转基因棉花纤维的起始和伸长迟缓,纤维细胞数量减少,长度变短。更多还原

【Abstract】 Cotton is one of the most important natural fiber crops in the world, and plays an important role in the national economy of China which is a major cotton producing and consuming country. Cotton fibers, the major product of cotton, are single-celled trichomes differentiated from the ovule epidermal cells. Due to the dependence of the output of production and efficiency on the yield as well as the quality of cotton fibers, improvement of the yield and the quality becomes the critical point of cotton breeding. However, the negative linkage between the yield and quality of cotton fibers limits the synchronous improvement of both of them by traditional methods. Gene engineering provides effective strategies such as transference of target genes to improve the yield and quality of cotton fibers. Therefore, it is significant to figure out the molecular basis for the development of cotton fibers. In addition, cloning the genes related to the yield and quality of cotton fibers, as well as studying the relationship between gene products, is of great theoretical and practical value for the improvement of both the yield and the quality of cotton fibers.Proteins of the 14-3-3 family are acid regulatory proteins that are highly conserved in all eukaryotes. They participate in various signal transduction and regulatory processes through binding to a wide range of target proteins, and play important roles in plant growth, development and signal transduction. In order to study the role of 14-3-3 proteins in cotton fiber development, Gh14-3-3 genes were isolated and characterized. The gene structures, expression patterns, as well as subcellular localizations, interactions and functions of the proteins encoded by Gh14-3-3 genes were further studied. The main results are as follows:1.Five cDNAs encoding 14-3-3 proteins were isolated from the cotton cDNA library, and designated as Gh14-3-3L, Gh14-3-3a, Gh14-3-3e, Gh14-3-3f and Gh14-3-3 g. Sequence comparison and phylogenetic analysis revealed that the five 14-3-3 proteins can be divided into two groups. Gh14-3-3L and Gh14-3-3a belong to theε-like group, and Gh14-3-3e and Gh14-3-3f belong to the non-εgroup. Four Gh14-3-3 genes were amplified from the cotton genome by PCR. Sequence comparison between the cDNAs and their corresponding genes revealed that all the introns in Gh14-3-3 genes were positioned in conserved residues. Gh14-3-3L and Gh14-3-3a which belong toε-like group contained five introns splitting their ORF into six exons. In contrast, Gh14-3-3e and Gh14-3-3f belonging to the non-εgroup contain three introns splitting their ORF into four exons.2. In order to investigate the subcellular localizations of Gh14-3-3 proteins, Gh14-3-3L:eGFP, Gh14-3-3a:eGFP and Gh14-3-3e:eGFP fusion expression vectors were constructed and introduced into cotton cells by transformation. The transformed cotton callus cells were observed under the confocal laser scanning microscopy. The results indicated that Gh14-3-3L was localized in the nucleis and cytoplasm, and Gh14-3-3a and Gh14-3-3e were localized in the cytoplasm.3. The results of RT-PCR indicated that the five Gh14-3-3 genes showed fiber-preferential expression patterns or exhibited its relatively high expression level in fibers. The expression levels of Gh14-3-3e and Gh14-3-3f were higher in ODPA ovules of wild type than those in fuzzless-fiberless mutant. The expression of the five Gh14-3-3 genes in fibers exhibit a developmentally regulated pattern, and their transcripts reached the highest levels in the elongation stage of fiber development. In addition, the expression levels of Gh14-3-3 genes were regulated by phytohormones such as IAA, GA3 and eBL. These results indicated that Gh14-3-3 genes might play a role in the differentiation, elongation and phtohormone response of cotton fiber development.4. The interactions between Gh14-3-3 proteins were analyzed by yeast two-hybrid. The results indicated that interactions were found in four cotton 14-3-3 protein pairs, but the interaction of the same 14-3-3 proteins was not detected in this study. The results indicated that each of the five Gh14-3-3 proteins might display isoform selectivity in the formation of heterodimers, and played a role as heterodimer in fiber development.5. To identify the interaction partners of Gh14-3-3 proteins, yeast two-hybrid analysis was performed using the three of the isolated Gh14-3-3 proteins (Gh14-3-3L、Gh14-3-3a、Gh14-3-3e) as bait to screen the two-hybrid library of cotton fiber cDNAs constructed on the prey vector. Each of the three 14-3-3 proteins was used in a separate screen.16 unique proteins were identified as target proteins of Gh14-3-3s. The targets of Gh14-3-3 proteins were related to various aspects of plant development, metabolism, and signal transduction. The results indicated that Gh14-3-3 proteins may function in the regulation of phytohormone response, plasma membrance and gene expression in fiber development.6. Overexpression of the Gh14-3-3e gene in Arabidopsis enhances the plant sensitivity to Brz. Gh14-3-3e was introduced into Arabidopsis plant, and several transgenic plants were obtained. RT-PCR and Western-blot analysis showed that Gh14-3-3e was expressed in plants of each transgenic line, respectively. Overexpression of Gh14-3-3e resulted in the seedlings displaying hypersensitivity to Brz in darkness. In addition, the expression level analysis of the BR-responsive genes in the transgenic lines is under way.7. In order to study the role of Gh14-3-3 proteins in cotton fiber development, overexpression and RNAi vectors of Gh14-3-3L and Gh14-3-3e were constructed and introduced into cotton by Agrobacterium-mediated transformation. Over 100 transgenic cotton plants were obtained. RT-PCR analysis showed that the expression level of Gh14-3-3 gene was higher in over-expression transgentic plants than that in wild type plants, and was knocked down in RNAi transgentic plants. The over-expression transgentic plants showed a drawf phenotype, and the branches were shorter, while the leaves were smaller than controls. The boll of over-expression transgentic plants drop within a few days after anthesis. Our results indicated that the expression levels of the genes related to fiber development were declined, resulting in slower initiation and elongation of fiber cells, compared with the wild type.更多还原