【作者】 周颖；

【导师】 李学宝；

【作者基本信息】 华中师范大学 ， 植物学， 2014， 博士

【摘要】 棉花是我国重要的经济作物,是关系国计民生的重要物资。随着工业发展和人民生活水平的提高,人们对高质量纺织品的需求量逐步增大,因而纺织工业对棉纤维品质的要求更高。棉花纤维是由胚珠表皮分化而来的不分枝的单细胞毛状突起,其突起细胞数目及纤维长度等指标直接与棉花产量和品质相关。研究棉纤维分化和发育的分子机制,克隆棉纤维发育相关基因,对于棉纤维品质改良具有重要意义。油菜素内酯(BR)是一种重要的植物激素,对棉纤维细胞起始分化和伸长生长都有重要作用。为探讨影响棉花纤维发育的BR信号通路,本文研究了Gh14-3-3与BZR1等靶蛋白的相互作用及BR信号在棉纤维发育中的调控作用,取得如下主要研究结果：1.Gh14-3-3h基因克隆鉴定及表达分析本实验室在先前的研究中分离鉴定了7个棉花14-3-3基因。在此基础上,利用酵母双杂交技术,从棉花纤维cDNA酵母双杂交文库中筛选到一个新的14-3-3蛋白,命名为Gh14-3-3h。利用PCR扩增技术,克隆了该蛋白基因的DNA全长序列和cDNA序列。序列分析表明,Gh14-3-3h基因含有6个外显子和5个内含子,属于￡型。Gh14-3-3h蛋白与其它物种的14-3-3蛋白氨基酸同源性达到610%-92%,具有14-3-3蛋白典型的结构特征,具有九个保守的α螺旋结构,但其N端和C端都不保守。利用荧光定量PCR技术分析了Gh14-3-3h在棉花组织中的表达谱。结果表明,Gh14-3-3h基因在纤维中优势表达,而在其它组织中表达量较低。在棉纤维发育过程中,Gh14-3-3h基因在棉纤维发育早期(3-10DPA)表达量较高,而在纤维细胞次生壁发育时期表达量逐步下降。并且,在胚珠离体培养的条件下,用2,4-表油菜素内酯(BL)处理棉花10天纤维,发现Gh14-3-3h表达量显著上调。这说明Gh14-3-3h在棉纤维细胞起始和伸长过程中发挥一定的调控作用,而且可能涉及到棉纤维发育中的BR信号应答。2.Gh14-3-3互作蛋白筛选及蛋白质相互作用验证为了研究Gh14-3-3在棉花纤维发育中涉及的调控信号通路,利用酵母双杂交系统,以Gh14-3-3f/g/h为诱饵蛋白,筛选棉花纤维cDNA酵母双杂交文库,获得了32种蛋白。它们可分为6大类,分别是14-3-3蛋白、BR等激素相关蛋白、代谢相关蛋白、信号相关蛋白、生长发育相关蛋白及功能未知蛋白。将这些蛋白序列在棉花EST公共数据库中进行比对分析,发现这些互作蛋白都能在棉纤维EST库中找到其同源序列,说明Gh14-3-3蛋白可能与这些蛋白在棉花纤维细胞中发生相互作用,共同调节棉纤维生长发育。14-3-3蛋白是一类高度保守的小分子调节蛋白,通常以同源或异源二聚体形式存在。在筛选的棉花14-3-3互作蛋白中,有大约2.8%是14-3-3蛋白本身,这表明14-3-3蛋白确实能够形成同源或异源二聚体。因此,我们进一步验证了Gh14-3-3h蛋白与其它5个Gh14-3-3蛋白之间的相互作用。结果表明,Gh14-3-3h能够与其它Gh14-3-3蛋白特异性相互作用,形成异源二聚体。因此,我们推测这些14-3-3蛋白可能以二聚体的形式参与棉花纤维细胞发育的调节。3. GhBZR1与Gh14-3-3蛋白相互作用分析在上述所筛选鉴定的棉花14-3-3互作蛋白中,有一个与拟南芥BZR1具有较高同源性的蛋白,命名为GhBZR1。对棉花BZR1蛋白序列进行分析,发现有22个可能被BIN2磷酸化的位点,并且有PEST区域和可能与14-3-3相互作用的作用位点。而且,蛋白质亚细胞定位分析表明,GhBZR1分布在细胞质和细胞核中,但受BL诱导后,该蛋白能够在细胞核中大量积累。为研究磷酸化对BZRl与14-3-3相互作用的影响,我们克隆了棉花BIN2蛋白基因。该基因表达与GhBZR1表达有其时空的一致性,都在棉花纤维伸长期优势表达。酵母双杂交和pull down实验证明GhBIN2与GhBZR1能够发生相互作用。在体外磷酸化实验中,GhBIN2能够磷酸化GhBZR1。而且,被磷酸化的GhBZR1能够与Gh14-3-3相互作用。进一步实验证明,GhBZR1与Gh14-3-3蛋白相互作用是通过保守的ModeI基序来完成的,其中第163位磷酸化的丝氨酸(Ser)是二者相互作用的关键位点。这表明被GhBIN2磷酸化的GhBZR1与Gh14-3-3相互作用可能对棉纤维细胞伸长发育发挥重要调控作用。4. GhMYB73与14-3-3蛋白相互作用分析在上述所筛选鉴定的棉花14-3-3互作蛋白中,有一个与拟南芥MYB73具有较高同源性的蛋白,命名为GhMYB73。它在棉花纤维伸长期表达量最高,定位在细胞核中,表明该蛋白可能是一个MYB转录因子。实验表明,GhMYB73可以与拟南芥BR通路中关键转录因子AtBES1在酵母系统中相互作用,推测GhMYB73可能参与BR信号通路。而且,酵母双杂交和pull down实验表明,GhBIN2也能够与GhMYB73蛋白发生相互作用,并在体外磷酸化GhMYB73,而磷酸化的GhMYB73与Gh14-3-3蛋白发生相互作用。这表明GhMYB73可能参与了14-3-3调节的BR信号通路,在棉纤维发育过程中发挥作用。5.Gh14-3-3蛋白在棉纤维细胞发育中的功能分析为研究14-3-3蛋白在棉纤维细胞分化和发育中的调节作用,构建了Gh14-3-3L/e/h等3个基因的RNA干扰(RNAi)表达载体和Gh14-3-3L过量表达载体,转化棉花,获得转基因棉花植株(TO代)及其后代株系(T1-T4代)。对转基因棉花后代株系(T1一T4代)表型观察和遗传分析表明,在RNAi转基因棉花中,纤维细胞起始和伸长发育都受到抑制,导致纤维细胞数目减少,长度变短。Gh14-3-3L/e/h RNAi转基因棉花成熟纤维长度仍然短于野生型,而过量表达Gh14-3-3L棉纤维长度则较野生型长,这表明Gh14-3-3蛋白在棉纤维伸长发育中发挥重要作用。离体胚珠培养实验表明,与野生型相比,Gh14-3-3L RNAi转基因棉花纤维长度和TFU均减少,而过量表达Gh14-3-3L转基因棉花纤维长度和TFU均有所增加。当施加外源BL时,Gh14-3-3L RNAi转基因棉花纤维长度和TFU与未加BL相比虽有所增加,但仍没有恢复到野生型的相当水平。在不添加外源BL的情况下,与野生型相比,Gh14-3-3L RNAi棉花纤维中GhCPD和GhDWF4基因表达显著下降,而在过量表达Gh14-3-3L转基因棉花纤维中GhCPD表达量没有发生改变,GhDWF4表达量则上调。在添加BL后,Gh14-3-3L RNAi棉花纤维中只有GhDWF4表达水平轻微下调。当BL施加后,与野生型相比,两个BR合成相关基因在过量表达Gh14-3-3L棉花纤维中表达量迅速下调。以上结果表明,在棉花纤维伸长阶段,Gh14-3-3可能对BR信号中的反馈调节起着一定的调控作用,从而影响棉花纤维伸长生长和TFU产量。为研究Ghl4-3-3L/e/h RNAi和过量表达棉纤维发育与BR信号调控的关系,检测了一些BR信号通路及纤维发育相关基因在转基因棉花纤维中的表达变化情况。结果表明,GhBZR1、 GhXTH1、GhEXP, GhTUB1和GhCesA5在Gh14-3-3L/e/h RNAi转基因棉花纤维中和Gh14-3-3L过量表达棉花纤维中表达都有不同程度的变化。多代(T1-T4)遗传分析表明,这些基因在不同世代的转基因棉花中的表达都发生改变,具有遗传稳定性。以上结果表明Gh14-3-3蛋白参与调节BR信号通路,通过改变BR信号相关基因的表达水平来影响棉花纤维起始和伸长发育过程。更多还原

【Abstract】 Cotton is an important crop in China and it is beneficial to the lives of people. With the development of industry and the improvement of human living standard, industry gradually need high quality textile. Cotton fiber is composed of ovule epidermal differentiation and unbranched single cell hair. The number of projections and cotton fiber length associate with the cotton quality and production efficiency. It is very important to provide the ways to know the molecular mechanism of cotton fiber development. It is significant for improving cotton fiber quality through cloning the gene in the development of cotton fiber.Brassinolide is an important plant hormone. It plays an important role in initiation and elongation of cotton. In order to study the molecular mechanism in brassinosteroid signaling pathway of cotton fiber, we analysised that the interaction between Gh14-3-3and its target protein BZR1. We also studied that the BR signaling pathway had function in regulating the development of cotton fiber. The main results are as follows:1. Identification of Gh14-3-3h gene and its expression profiling in cotton Seven cotton14-3-3cDNA in the lab had been isolated in previously study. We cloned a14-3-3gene which was named Gh14-3-3h from the yeast two-hybrid cDNA library of cotton fiber. We also cloned the gene from the cotton genome. The structure of Gh14-3-3h gene contained six exons and five introns, belonging to the epsilon type14-3-3proteins. Gh14-3-3h with other species of the14-3-3protein amino acid had homology of61%-92%. In addition, Gh14-3-3h had the14-3-3typical structure characteristics and nine conservative alpha helixes. But its N and C terminals were not conserved.Fluorescence quantitative PCR was used to analyze the expression of Gh14-3-3h in cotton organizations. We found the expression of Gh14-3-3h in the fiber was high. However, the expression level of the gene in other organizations was low. Gh14-3-3h gene had high expression level during the early stage of fiber (3-10DPA) and decreased during fiber cell secondary wall development. In addition, the expression level of Gh14-3-3h was up-regulated by addition of BL. These results indicated that Gh14-3-3h might play a role in regulating the initiation and elongation of cotton fiber cell. Moreover, Gh14-3-3might involve in the response to BR signaling pathway.2. Screening the interaction partners of Gh14-3-3proteinsTo study Gh14-3-3involved in regulating the signaling pathway during the growth and development of cotton fiber, we analyzed the interactions between Gh14-3-3f/g/h proteins and their partners by the yeast two-hybrid system. We achieved a total of32kinds of proteins. They were divided into six categories, containing cotton14-3-3proteins, hormones related proteins, metabolic related proteins, signaling related proteins, proteins involved in growth and development, and unknown proteins. We analyzed the sequence of these proteins in the cotton EST database and we found that most of these interaction proteins were presented in cotton fiber. These results indicated that Gh14-3-3proteins might interact with these proteins in the fiber cells to co-regulate the growth and development of cotton fiber.14-3-3is highly conserved protein in eukaryotes, and it usually interacts with other proteins through homogeneous or heterogeneous dimer. In the14-3-3target proteins, we found that2.8%proteins were the14-3-3proteins. It indicated that the14-3-3proteins could form the homogeneous or heterogeneous dimer. Moreover, we checked the interaction between the Ghl4-3-3h and five other Ghl4-3-3s. We found that it had specific interactions between them, and the six Ghl4-3-3s were in the form of heterologous dimers. We speculated that14-3-3played a role as heterodimer to participate in the regulation of cotton fiber elongation.3. Analysis of the interaction of GhBZRl and Gh14-3-3proteins We cloned a target protein of Ghl4-3-3s by yeast two-hybrid. It had high homology comparing with Arabidopsis BZR1and it was named GhBZRl. Analysis of cotton BZR1protein, we found that it had22probable BIN2phosphorylation sites and the PEST area. It also had a motif of interaction with the14-3-3. In addition, GhBZRl located in the cytoplasm and nucleus. When addition of BL, a large number of GhBZRl-GFP proteins accumulated in the nucleus.To study the influence of phosphorylation to the interaction between GhBZR1and Ghl4-3-3protein, we cloned the cotton BIN2gene. The gene expression pattern was the same as expression pattern of GhBZR1. It had the preferential expression in elongation stage of cotton fiber. We found GhBIN2could interact with GhBZRl protein by the experiments of yeast two-hybrid and pull down. GhBIN2phosphorylated GhBZR1in vitro and phosphorylation GhBZRl could interact with Gh14-3-3protein. In addition, GhBZRl interacted with Gh14-3-3protein by conservative Mode I motif and phosphorylation of S (163) was important to the interaction between them. It indicated that GhBIN2phosphorylated GhBZR1to promote the interaction between GhBZR1and Gh14-3-3proteins and the response played an important role in regulating elongation of cotton fiber.4. Analysis of the interaction of GhMYB73and Gh14-3-3proteinsIn the yeast two-hybrid system, it had an interaction partner of Ghl4-3-3.The amino acid of GhMYB73had homology with Arabidopsis thaliana AtMYB73and it was named GhMYB73. The expression of GhMYB73had the highest amount in elongation of cotton fiber and subcellular localization was in the nucleus. GhMYB73interacted with the transcription factor AtBES1in BR pathway of Arabidopsis thaliana. The results indicated that GhMYB73might interact with AtBESl to participate in BR signaling pathway.GhBIN2could interact with GhMYB73in yeast two-hybrid and pull down experiments. GhBIN2could phosphorylate GhMYB73in vitro. In addition, phosphorylated GhMYB73could interact with Gh14-3-3. It indicated that GhMYB73might involve in14-3-3regulating the BR signaling pathway of fiber development.5. Gh14-3-3proteins function in fiber cell development of cottonTo study the regulation of Gh14-3-3proteins in the differentiation and development of cotton fiber cell, we constructed the vectors of Gh14-3-3L/e/h RNAi and Gh14-3-3L overexpression.When the vectors were introduced into cotton by Agrobacterium-mediated transformation, TO generation and their later generations T1-T4were obtained. Analysis of transgenic cotton (T1-T4generation), the number and length of cotton fiber initiation significantly suppressed in35S promoter driven Gh14-3-3L/e/h RNAi transgenic plants. The number of Gh14-3-3L RNAi fiber decreased and the length got shorter. The length of mature fiber in T1-T4generation Gh14-3-3L/e/h RNAi was shorter than wild type, while the length of cotton fiber in overexpression Gh14-3-3L was longer than wild type. It indicated that Gh14-3-3had the important function in the development of cotton fiber.In vitro culture of fiber, the length and TFU of Gh14-3-3L RNAi decreased comparing to wild type. But overexpression Gh14-3-3L increased. When applying with BL, Gh14-3-3L RNAi fiber length and TFU increased compared with in normal condition, but they had not recovered to wild type levels. Expression of GhCPD in Gh14-3-3L RNAi in cotton fiber and the expression level GhDWF4decreased significantly. But expression of GhCPD in overexpression plants was no significant difference and the expression of GhDWF4was up-regulated comparing with wild type. After BL treatment, only GhDWF4expression level was slightly down-regulated in Gh14-3-3L RNAi transgenic plants, compared with control (without BL). But the expression of two cotton BR synthesis genes in overexpression Gh14-3-3L comparing with wild type were rapidly down-regulated. Above results showed that Gh14-3-3might involve in BR signal feedback regulation in the cotton fiber elongation stage and it affect cotton fiber elongation and TFU production.To study the relationship between Gh14-3-3transgenic cotton fibers phenotype and brassinosteroid signal transduction, we studied the genes associated with BR signal transduction and cotton fiber initiation and elongation related gene expression in transgenic cotton fiber. The results showed that GhBZR1, GhXTH1, GhEXP, GhTUB1and GhCesA5in Gh14-3-3L/e/h RNAi transgenic cotton fiber and Gh14-3-3L overexpression of cotton fiber had different degree of changes in the expression. Analysis of T1-T4generation, the expression of these genes were changed in different generations and the hereditary was stable. The results indicated that Gh14-3-3proteins participated in regulating BR signaling pathways to influence the initiation and elongation of cotton fibers through regulating the expression levels of these genes.更多还原