龙眼胚性培养物DlRan3A和DlRan3B基因的功能分析
发布时间：2018-08-08 12:12
【摘要】：龙眼是我国南方具有重要经济价值的热带亚热带常绿木本果树,龙眼生产上存在诸多问题制约其产业发展,如缺乏具有生产价值的焦核品种、成熟期过于集中以及果实品质、产量和贮藏受到环境因素影响。目前关于龙眼焦核、结果期调节以及抗逆性等方面的分子机制研究较少。Ran(Ras-related nuclear protein)小G蛋白广泛参与生长发育、抗病和激素敏感性调控等过程。前人研究表明,龙眼Ran可能与龙眼体胚发生(somatic embryogenesis,SE)过程的细胞分裂有关,但具体机制尚不清楚,目前关于龙眼Ran表达调控机制研究仍有大量空白,尤其是龙眼Ran基因在龙眼胚胎和果实发育过程的功能特异性以及响应外界环境的相应机制等。因而本研究首先分离龙眼DlRan3A和DlRan3B基因启动子并验证其功能,并通过对龙眼体胚发生、合子胚发育以及果肉发育等过程以及龙眼不同组织器官中DlRan3A和DlRan3B基因的表达模式进行分析,为了解Ran基因在龙眼的胚胎发育及果实发育中的作用机制提供新的线索。同时,利用异位表达、原位转化以及转录组分析等手段对龙眼DlRan3A和DlRan3B基因及启动子进行功能分析,探索Ran在植物生长发育和抗性调节等过程中的功能特异性,进一步研究植物Ran表达调控网络。主要研究结果如下:1龙眼DlRan3A和DlRan3B基因启动子的克隆与生物信息学分析以龙眼胚性愈伤组织(Embryogenic callus,EC)为材料,利用Tail-PCR法和连接介导染色体步移法分别获得1256bp和1569bp的DlRan3A和DlRan3B基因启动子序列;生物信息学分析预测获得这两个基因启动子区的核心启动子区域和可能的转录起始位点,且DlRan3A启动子区存在一个长度为147bp的CpG岛。分析功能元件发现,DlRan3A和DlRan3B基因启动子区均含有生长素、水杨酸(salicylic acid,SA)和茉莉酸甲酯(methyl jasmonate,MeJA)响应元件以及厌氧诱导和防御与胁迫响应元件。DlRan3A基因启动子还含有参与伤害和病原体诱导的响应元件,DlRan3B基因启动子区还含有赤霉素和脱落酸(abscisic acid,ABA)响应元件及低温响应元件,说明龙眼Ran家族不同成员在激素响应和抗逆调节方面可能存在功能特异性。2龙眼DlRan3A和DlRan3B基因启动子功能验证构建龙眼DlRan3A和DlRan3B启动子的3'和5'不同缺失突变体的表达载体,利用烟草叶片GUS瞬时表达水平进行启动子缺失分析和启动子激素应答分析。结果显示,DlRan3A和DlRan3B基因启动子中,正调控和负调控元件并存,8.6 μMIAA(indole-3-acetic acid,IAA)、75μM SA或100μM MeJA诱导DlRan3A基因启动子的转录活性,8.6μM IAA 或 34.6 μM CA3(gibberellin A3,GA3)激活DlRan3B基因启动子的转录活性;基于激素应答元件分布于DlRan3A和DlRan3B基因启动子的区域,推测生长素元件均为正调控作用元件;MeJA应答元件的广泛分布预示其复杂的调控作用;推测DlRan3A基因启动子的SA响应元件、防御和逆境响应元件以及box S元件为正调控元件,且DlRan3B基因启动子的SA响应元件位于防御和逆境响应元件和低温和干旱响应元件为负调控元件;DlRan3A和DlRan3B的启动子在参与抗逆反应中可能发挥特异的转录调控作用。3龙眼DlRan3A和DlRan3B的表达模式分析利用qPCR分析龙眼DlRan3A和DlRan3B基因的表达模式。从龙眼DlRan3A和DlRan3B基因在龙眼不同生长发育阶段以及组织器官中的表达谱可知,龙眼DlRan3A和DlRan3B基因在龙眼不同组织部位中均有表达,其中二者均在龙眼种子和果肉中表达量较高;在体胚发生的子叶胚时期和合子胚发育的幼嫩胚胎中表达量较高;DlRan3A和DlRan3B基因表达量随着种子萌发过程下降,且随着果肉的膨大而提高。说明龙眼DlRan3A和DlRan3B基因在龙眼胚胎(种子)发育以及果肉膨大过程中发挥更为重要的作用。不同激素及非生物胁迫处理龙眼EC下的DlRan3A和DlRan3B基因的定量表达分析结果显示,二者的表达均受到一定浓度外源生长素(IAA)、赤霉素(GA3)的诱导而受到高浓度SA和MeJA的抑制,并且一定程度上受到盐胁迫、渗透胁迫、PEG胁迫和ABA胁迫的诱导。说明龙眼DlRan3A和DlRan3B基因广泛参与激素应答和非生物胁迫响应。4 35S或特异启动子驱动下异位表达龙眼DlRan3A与DlRan3B基因的功能分析构建DlRan3A与DlRan3B亚细胞定位载体,瞬时转化本氏烟叶片;构建35S或特异启动子驱动DlRan3A或DlRan3B基因过表达载体、特异启动子驱动GUS基因表达载体及DlRan3A干扰表达载体,转化本氏烟。亚细胞定位显示,龙眼DlRan3A和DlRan3B主要定位于细胞核。特异启动子驱动GUS表达的转基因本氏烟幼苗(PA-GUS和PB-GUS)的GUS染色结果表明DlRan3A和DlRan3B均在细胞分裂旺盛的部位(根尖和根系分支处)表达量较高,同时,二者在果实和种子中也分布较多,此外DlRan3A相对于DlRan3B在花器官中有更显著的表达。特异启动子驱动DlRan3A过表达的株系pCAMBIA1301-pDlRan3A(1256bp)-DlRan3A(PA_A)表现出植株矮壮,开花推迟,果实发育异常且结籽率低的现象,说明龙眼特异启动子驱动DlRan3A过表达对植物生长发育尤其是调节植物生长周期以及果实和种子发育有显著影响。PA_A株系的根系中主根相对不明显,且根毛发育旺盛,推测DlRan3A基因与根毛发育密切相关。干扰表达DlRan3A基因的本氏烟株系(ran3a)表现出生长极其缓慢、花果发育异常且不结籽的现象,说明龙眼Ran基因在植物生长发育中发挥至关重要作用。过表达DlRan3A基因株系pCAMBIA1301-35S-DlRan3A(P35S_A)和 PA_A 相对于野生型植株(WT)有较高的抗非生物胁迫(盐胁迫、渗透胁迫、干旱胁迫和热胁迫)能力;相反,过表达DlRan3B基因株系pCAMBIA1301-35S-DlRan3B(P35S_B)和(pCAMBIA1301-pDlRan3B(1569bp)-DlRan3B(PB_B)的抗性相对较弱,其中P35S_B株系对盐胁迫、渗透胁迫、干旱胁迫和热胁迫的耐受性均弱于WT,且对盐胁迫和渗透胁迫高度敏感;相同的是,过表达转基因株系的抗冷胁迫能力得到提高,且均对外源ABA高度敏感,说明龙眼DlRan3A和DlRan3B基因与冷胁迫抗性和ABA信号通路密切相关。5 3S或特异启动子驱动下异位表达龙眼DlRan3和DlRan3B的转录组学分析利用转录组测序技术对35S或特异启动子驱动下过表达龙眼DlRan3A和DlRan3B的本氏烟株系进行分析,结果显示,过表达株系中存在显著上调或下调的生长素信号通路相关差异基因,说明龙眼Ran可能存在复杂作用机制影响生长素信号通路的调控;参与ABA运输的部分差异基因显著上调,结合ABA胁迫表型可知,龙眼Ran过表达显著影响植株对外源ABA的应答能力。BR(brassinosteroid)正调控基因显著上调,而负调控BR合成的基因显著下调,说明转基因植株内BR含量增多或BR信号输出增强。过表达龙眼Ran基因的4种转基因株系中绝大多数细胞壁蛋白上调表达,可见龙眼DlRan3A和DlRan3B基因可能通过影响龙眼细胞壁物质的积累以参与调控龙眼生长发育过程,尤其是早期胚胎及果肉发育过程。过表达龙眼Ran的株系显著富集碳水化合物代谢和丙氨酸、谷氨酸和天冬酰胺代谢通路;P35S_A株系还存在过氧化物为受体的氧化还原酶活性的通路;PA_A株系还存在参与细胞壁组织、结构成分和生物合成、纤维素生物合成及代谢、葡聚糖代谢、外包结构组织等通路;PA_A与P35S_A株系相比以及PB_B与P35S_B株系相比,均显著富集细胞壁组织、结构成分和生物合成通路,包含的基因均为3个extensin-like家族的Pollen Ole e 1结构域基因,然而在PA-A株系中为显著上调基因而在PB_B株系中为显著下调基因;结合表型分析可知,特异启动子驱动DlRan3A过表达通过提高部分extensin-like基因水平从而影响植株根毛、果实和种子发育,说明龙眼Ran特异启动子在参与器官细胞壁组织结构及生物合成方面可能发挥特异的转录调控作用。过表达龙眼Ran的株系中存在较多编码过氧化物酶(PER)、水通道蛋白(TIP)、谷氨酰胺合成酶(AS)、谷胱甘肽转移酶(GST)、热激蛋白(HSP)及其伴侣蛋白(DNAJ)等的植物抗性相关基因和编码MYB、NAC、WRKYH ERF、GRAS及bHLH等抗性相关转录因子基因,可见龙眼Ran与抗性调节密切相关。过表达DlRan3A基因的株系呈现比过表达DlRan3B基因的株系更强的耐非生物胁迫能力,一方面在于P35S_A和PA_A株系中部分增强植物抗性的基因(如PER、GST、OLP、TIS、PPO和BG等)显著上调,另一方面在于P35S_B和PB_B株系有大量植物抗性相关转录因子(如ERF、WRKY、bHLH、C3H、C2H2和GRAS等)基因显著下调。6DlRan3A与DlRan3B基因原位转化龙眼的功能分析在过表达龙眼DlRan3A或DlRan3B基因的原位转化株系中,CESA6、EXLA2和EXT3等细胞壁结构和合成相关基因的表达量均显著提高;MYB59、DlPPO1和TIP1-1等抗性相关基因的表达量也均显著提高;验证了过表达龙眼Ran基因引起的细胞壁合蛋白基因和抗性基因差异表达结果,进一步说明龙眼Ran基因与细胞壁合成和抗性调节等过程密切相关。综上所述,本研究分离了DlRan3 和DlRan3B基因启动子并分析了二者在参与激素应答和抗性调节等过程存在的功能特异性;龙眼Dlan3A和DlRan3B基因是龙眼生长发育所必需的重要小G蛋白基因,尤其在龙眼胚胎(种子)发育和果肉发育过程中发挥重要作用;龙眼Ran可能参与生长素、细胞分裂素、ABA和BR等激素信号通'路的调控。异位表达龙眼Ran提高植株对外源ABA的敏感性,且提高植株抗寒能力;异位表达龙眼DlRan3A提高植株抗非生物胁迫能力,特异启动子驱动DlRan3A表达通过提高部分extensin-like基因水平,从而影响根毛、果实和种子发育;龙眼Ran基因与细胞壁合成和抗性调节等过程密切相关。
[Abstract]:Longan is a tropical and subtropical evergreen fruit tree with important economic value in the south of China. There are many problems in the production of longan, which restrict its industrial development, such as the lack of the production value of the coke, the maturity period is too concentrated and the fruit quality, the yield and storage are affected by the environmental factors. Studies on molecular mechanisms, such as nodes and resistance, less.Ran (Ras-related nuclear protein) small G protein is widely involved in the process of growth, disease resistance and hormone sensitivity regulation. Previous studies have shown that longan Ran may be related to the cell division of the longan somatic embryogenesis (somatic embryogenesis, SE), but the specific mechanism is not yet clear. At present, there are still a lot of gaps in the study of the regulation mechanism of Ran expression in longan, especially the functional specificity of longan Ran gene in the development of longan embryo and fruit and the corresponding mechanism to respond to the external environment. The development of zygotic embryo and the development of pulp and the expression patterns of DlRan3A and DlRan3B genes in different tissues of longan were analyzed to provide a new clue to understand the mechanism of Ran gene in the development of the longan embryo and fruit development. At the same time, the method of heterotopic expression, in situ transformation and transcriptional analysis were used for the D of longan. Functional analysis of lRan3A and DlRan3B genes and promoters was carried out to explore the functional specificity of Ran in plant growth and resistance regulation and to further study the regulatory network of plant Ran expression. The main results are as follows: 1 the cloning and bioinformatics analysis of DlRan3A and DlRan3B gene promoters in longan and bioinformatics of the longan Embryogenic callus (Em Bryogenic callus, EC), as the material, obtained the DlRan3A and DlRan3B gene promoter sequences of 1256bp and 1569bp by Tail-PCR method and connection mediated chromosome step method, respectively. Bioinformatics analyses and predicts the core promoter region and possible transcriptional starting site of the two gene promoter regions, and there is a single promoter region in the DlRan3A promoter region. CpG Island, with a length of 147bp. Analysis functional components found that both DlRan3A and DlRan3B promoter regions contain auxin, salicylic acid, SA, and methyl jasmonate (methyl jasmonate, MeJA) response elements, and the anaerobic induction and defense and stress response element.DlRan3A gene promoter are also involved in injury and pathogen induction. Response element, DlRan3B gene promoter region also contains gibberellin and abscisic acid (abscisic acid, ABA) response element and low temperature response element, indicating that the different members of the longan Ran family may have functional specific.2 longan DlRan3A and DlRan3B based promoter function verification in the response to hormone response and anti inverse regulation of the longan Ran family to construct longan DlRan3A and DlRan3B. The promoter 3'and 5' expressed different deletion mutants. The promoter deletion analysis and promoter response analysis were carried out using the transient expression level of tobacco leaves GUS. The results showed that both positive and negative regulatory elements coexist in the DlRan3A and DlRan3B gene promoters, 8.6 mu MIAA (indole-3-acetic acid, IAA), 75 mu M SA or 100 micron M. The transcriptional activity of the promoter of the DlRan3A gene, 8.6 mu M IAA or 34.6 mu M CA3 (gibberellin A3, GA3) activates the transcriptional activity of the DlRan3B promoter; based on the distribution of the hormone response element in the region of the DlRan3A and DlRan3B gene promoter, it is speculated that the auxin element is a positive regulatory element; the wide distribution of the MeJA response element indicates its recovery. The SA response element of the DlRan3A gene promoter, the defense and adversity response element and the box S element are positive regulators, and the SA response elements of the DlRan3B gene promoter are located in the defensive and adversity response elements and the low temperature and drought response elements as negative regulatory elements; the promoter of DlRan3A and DlRan3B is involved in anti inversion. The expression pattern of DlRan3A and DlRan3B in longan,.3, may be used to analyze the expression patterns of the DlRan3A and DlRan3B genes of longan. From the expression profiles of the longan DlRan3A and DlRan3B genes in the different growth and development stages and tissues of longan, the longan DlRan3A and DlRan3B genes are not found in longan. All of them were expressed in the same tissue, two of which were expressed in the seeds and flesh of longan. The expression of the cotyledon embryo in the somatic embryo and the young embryo of the zygotic embryo was higher. The expression of DlRan3A and DlRan3B genes decreased with the germination of the seeds, and increased with the expansion of the flesh. It explained the DlRan3A and DlRan3 of longan. B gene plays a more important role in the development of longan embryo (seed) and the process of pulp expansion. The quantitative expression analysis of DlRan3A and DlRan3B genes under different hormone and abiotic stress treatment under the DlRan3A and DlRan3B of longan EC shows that the expression of the two is subjected to a certain concentration of exogenous auxin (IAA), gibberellin (GA3) and the high concentration of SA And MeJA inhibition, and to some extent, it is induced by salt stress, osmotic stress, PEG stress and ABA stress. It shows that the DlRan3A and DlRan3B genes of longan are widely involved in hormone response and abiotic stress response to.4 35S or specific promoter driven by heterotopic expression of the DlRan3A and DlRan3B genes of longan, DlRan3A and DlRan3B subfining. 35S or specific promoter driven DlRan3A or DlRan3B gene overexpression vector, specific promoter driven GUS gene expression vector and DlRan3A interference expression vector, and transformation of Benedict smoke. Subcellular localization showed that longan DlRan3A and DlRan3B were mainly located in the nucleus. Specific promoter driven GUS The GUS staining results of the transgenic tobacco seedlings (PA-GUS and PB-GUS) showed that both DlRan3A and DlRan3B were highly expressed in the exuberant part of the cell division (root tip and root branch). At the same time, the two were also more distributed in the fruits and seeds. In addition, DlRan3A had a more significant expression in the flower organs compared to the DlRan3B. The overexpressed plant line pCAMBIA1301-pDlRan3A (1256bp) -DlRan3A (PA_A) showed that the plant was short and strong, the flowering was delayed, the fruit development was abnormal and the seed rate was low. It showed that the overexpression of the longan specific promoter driven DlRan3A had significant influence on the growth and development of plant, especially the growth cycle of plant growth and the development of fruit and seed, and.PA. The root of the _A strain was not obvious in the root system, and the root hair was vigorous. It was suggested that the DlRan3A gene was closely related to the root hair development. The ran3a, which interfered with the expression of DlRan3A gene, showed that the growth was extremely slow and the flower and fruit development was abnormal and did not seed. It showed that the longan Ran gene played a vital role in the plant growth and development. Overexpressed DlRan3A gene pCAMBIA1301-35S-DlRan3A (P35S_A) and PA_A have higher anti abiotic stress (salt stress, osmotic stress, drought stress and heat stress) compared with wild type plants (WT). On the contrary, overexpression of DlRan3B gene line pCAMBIA1301-35S-DlRan3B (P35S_B) and (pCAMBIA1301-pDlRan3B (1569bp) -DlRan3B (PB_B)) Resistance was relatively weak, and the tolerance of P35S_B strains to salt stress, osmotic stress, drought stress and heat stress was weaker than WT, and was highly sensitive to salt stress and osmotic stress. The same was that the resistance to cold stress of overexpressed transgenic lines was improved, and all of them were highly sensitive to exogenous ABA, indicating that DlRan3A and DlRan3B genes of longan and DlRan3B were cold and cold. The transcriptional analysis of heterotopic expression of longan DlRan3 and DlRan3B under.5 3S or specific promoter driven by stress resistance and ABA signaling pathway was analyzed by transcriptional sequencing technology to analyze the tobacco strain of longan DlRan3A and DlRan3B in 35S or specific promoter driven by DlRan3A and DlRan3B. The results showed that the overexpressed strain was significantly higher. The regulation or downregulation of auxin signaling pathway related differentially genes indicates that longan Ran may have a complex mechanism that affects the regulation of auxin signaling pathway, and the partial differential genes involved in ABA transport are significantly up-regulated, and that the Ran overexpression of longan has a significant effect on the response ability of plant to exogenous ABA.BR (brassinosteroid). The positive regulation gene was significantly up-regulated, while the negative regulation of BR synthesis was significantly down, indicating the increase of BR content in the transgenic plants or the enhancement of BR signal output. Most of the cell wall proteins in the 4 transgenic lines expressing longan Ran gene are up-regulated, and the DlRan3A and DlRan3B genes of longan may be affected by the substance of the longan cell wall. Accumulation is involved in regulating the growth and development of longan, especially in the development of early embryo and pulp. Over expressed longan Ran lines significantly enriched carbohydrate metabolism and alanine, glutamic acid and asparagine metabolism pathway, and the P35S_A line also existed as a pathway for the oxygenation reductase activity of the peroxide as the receptor; the PA_A strain also existed the ginseng. With cell wall tissue, structural composition and biosynthesis, cellulose biosynthesis and metabolism, glucan metabolism, and outsourced structural tissue, PA_A, compared with P35S_A strains and PB_B and P35S_B lines, significantly enriched cell wall tissue, structural components and biosynthetic pathway, including the Pollen O of 3 extensin-like families. The Le E 1 domain gene, however, is a significant up-regulated gene in the PA-A strain and a significant down-regulation in the PB_B strain. In combination with phenotypic analysis, the specific promoter driven DlRan3A overexpression affects the root hair, fruit and seed development of the plant by raising the level of some extensin-like genes, indicating that the specific promoter of the longan Ran is involved. The tissue structure and biosynthesis of the organ cell wall may play a specific regulatory role. In the strains of longan Ran, there are many plant resistance related groups encoding peroxidase (PER), water channel protein (TIP), glutamine synthetase (AS), glutathione transferase (GST), heat shock protein (HSP) and its chaperone protein (DNAJ). The resistance related transcription factor genes such as MYB, NAC, WRKYH ERF, GRAS and bHLH are closely related to the resistance regulation of longan Ran. The strains that overexpress the DlRan3A gene show a stronger abiotic stress resistance than the strains overexpressing the DlRan3B gene, and on the one hand, the genes that enhance the plant resistance in P35S_A and PA_A strains (such as PE) R, GST, OLP, TIS, PPO and BG are significantly up-regulated, on the other hand, the functional analysis of a large number of plant resistance related transcriptional factors (ERF, WRKY, bHLH, C3H, C3H, etc.) in the P35S_B and PB_B strains The expression of cell wall structure and synthesis related genes of EXLA2 and EXT3 increased significantly, and the expression of resistance related genes, such as MYB59, DlPPO1 and TIP1-1, were also significantly improved, and the results of the differential expression of the cell wall protein gene and resistance genes caused by the overexpressed longan Ran gene were verified, and the Ran gene of longan was further explained by the combination of the Ran gene and cell wall. It is closely related to the process of resistance regulation. In summary, this study isolated the DlRan3 and DlRan3B gene promoters and analyzed the functional specificity of the two participants in the process of participating in hormone response and resistance regulation. The longan Dlan3A and DlRan3B genes are the important small G protein genes necessary for the growth and development of longan, especially in the longan embryo ( Seed) plays an important role in development and flesh development; longan Ran may be involved in the regulation of hormones such as auxin, cytokinin, ABA and BR. Ectopic expression of longan Ran enhances the sensitivity of plant to exogenous ABA and improves plant cold resistance; heterotopic expression of longan DlRan3A to increase plant resistance to abiotic stress is specific. The promoter driven DlRan3A expression can affect the root hair, fruit and seed development by increasing the extensin-like gene level, and the longan Ran gene is closely related to the process of cell wall synthesis and resistance regulation.
【学位授予单位】：福建农林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S667.2
，
本文编号：2171755
[Abstract]:Longan is a tropical and subtropical evergreen fruit tree with important economic value in the south of China. There are many problems in the production of longan, which restrict its industrial development, such as the lack of the production value of the coke, the maturity period is too concentrated and the fruit quality, the yield and storage are affected by the environmental factors. Studies on molecular mechanisms, such as nodes and resistance, less.Ran (Ras-related nuclear protein) small G protein is widely involved in the process of growth, disease resistance and hormone sensitivity regulation. Previous studies have shown that longan Ran may be related to the cell division of the longan somatic embryogenesis (somatic embryogenesis, SE), but the specific mechanism is not yet clear. At present, there are still a lot of gaps in the study of the regulation mechanism of Ran expression in longan, especially the functional specificity of longan Ran gene in the development of longan embryo and fruit and the corresponding mechanism to respond to the external environment. The development of zygotic embryo and the development of pulp and the expression patterns of DlRan3A and DlRan3B genes in different tissues of longan were analyzed to provide a new clue to understand the mechanism of Ran gene in the development of the longan embryo and fruit development. At the same time, the method of heterotopic expression, in situ transformation and transcriptional analysis were used for the D of longan. Functional analysis of lRan3A and DlRan3B genes and promoters was carried out to explore the functional specificity of Ran in plant growth and resistance regulation and to further study the regulatory network of plant Ran expression. The main results are as follows: 1 the cloning and bioinformatics analysis of DlRan3A and DlRan3B gene promoters in longan and bioinformatics of the longan Embryogenic callus (Em Bryogenic callus, EC), as the material, obtained the DlRan3A and DlRan3B gene promoter sequences of 1256bp and 1569bp by Tail-PCR method and connection mediated chromosome step method, respectively. Bioinformatics analyses and predicts the core promoter region and possible transcriptional starting site of the two gene promoter regions, and there is a single promoter region in the DlRan3A promoter region. CpG Island, with a length of 147bp. Analysis functional components found that both DlRan3A and DlRan3B promoter regions contain auxin, salicylic acid, SA, and methyl jasmonate (methyl jasmonate, MeJA) response elements, and the anaerobic induction and defense and stress response element.DlRan3A gene promoter are also involved in injury and pathogen induction. Response element, DlRan3B gene promoter region also contains gibberellin and abscisic acid (abscisic acid, ABA) response element and low temperature response element, indicating that the different members of the longan Ran family may have functional specific.2 longan DlRan3A and DlRan3B based promoter function verification in the response to hormone response and anti inverse regulation of the longan Ran family to construct longan DlRan3A and DlRan3B. The promoter 3'and 5' expressed different deletion mutants. The promoter deletion analysis and promoter response analysis were carried out using the transient expression level of tobacco leaves GUS. The results showed that both positive and negative regulatory elements coexist in the DlRan3A and DlRan3B gene promoters, 8.6 mu MIAA (indole-3-acetic acid, IAA), 75 mu M SA or 100 micron M. The transcriptional activity of the promoter of the DlRan3A gene, 8.6 mu M IAA or 34.6 mu M CA3 (gibberellin A3, GA3) activates the transcriptional activity of the DlRan3B promoter; based on the distribution of the hormone response element in the region of the DlRan3A and DlRan3B gene promoter, it is speculated that the auxin element is a positive regulatory element; the wide distribution of the MeJA response element indicates its recovery. The SA response element of the DlRan3A gene promoter, the defense and adversity response element and the box S element are positive regulators, and the SA response elements of the DlRan3B gene promoter are located in the defensive and adversity response elements and the low temperature and drought response elements as negative regulatory elements; the promoter of DlRan3A and DlRan3B is involved in anti inversion. The expression pattern of DlRan3A and DlRan3B in longan,.3, may be used to analyze the expression patterns of the DlRan3A and DlRan3B genes of longan. From the expression profiles of the longan DlRan3A and DlRan3B genes in the different growth and development stages and tissues of longan, the longan DlRan3A and DlRan3B genes are not found in longan. All of them were expressed in the same tissue, two of which were expressed in the seeds and flesh of longan. The expression of the cotyledon embryo in the somatic embryo and the young embryo of the zygotic embryo was higher. The expression of DlRan3A and DlRan3B genes decreased with the germination of the seeds, and increased with the expansion of the flesh. It explained the DlRan3A and DlRan3 of longan. B gene plays a more important role in the development of longan embryo (seed) and the process of pulp expansion. The quantitative expression analysis of DlRan3A and DlRan3B genes under different hormone and abiotic stress treatment under the DlRan3A and DlRan3B of longan EC shows that the expression of the two is subjected to a certain concentration of exogenous auxin (IAA), gibberellin (GA3) and the high concentration of SA And MeJA inhibition, and to some extent, it is induced by salt stress, osmotic stress, PEG stress and ABA stress. It shows that the DlRan3A and DlRan3B genes of longan are widely involved in hormone response and abiotic stress response to.4 35S or specific promoter driven by heterotopic expression of the DlRan3A and DlRan3B genes of longan, DlRan3A and DlRan3B subfining. 35S or specific promoter driven DlRan3A or DlRan3B gene overexpression vector, specific promoter driven GUS gene expression vector and DlRan3A interference expression vector, and transformation of Benedict smoke. Subcellular localization showed that longan DlRan3A and DlRan3B were mainly located in the nucleus. Specific promoter driven GUS The GUS staining results of the transgenic tobacco seedlings (PA-GUS and PB-GUS) showed that both DlRan3A and DlRan3B were highly expressed in the exuberant part of the cell division (root tip and root branch). At the same time, the two were also more distributed in the fruits and seeds. In addition, DlRan3A had a more significant expression in the flower organs compared to the DlRan3B. The overexpressed plant line pCAMBIA1301-pDlRan3A (1256bp) -DlRan3A (PA_A) showed that the plant was short and strong, the flowering was delayed, the fruit development was abnormal and the seed rate was low. It showed that the overexpression of the longan specific promoter driven DlRan3A had significant influence on the growth and development of plant, especially the growth cycle of plant growth and the development of fruit and seed, and.PA. The root of the _A strain was not obvious in the root system, and the root hair was vigorous. It was suggested that the DlRan3A gene was closely related to the root hair development. The ran3a, which interfered with the expression of DlRan3A gene, showed that the growth was extremely slow and the flower and fruit development was abnormal and did not seed. It showed that the longan Ran gene played a vital role in the plant growth and development. Overexpressed DlRan3A gene pCAMBIA1301-35S-DlRan3A (P35S_A) and PA_A have higher anti abiotic stress (salt stress, osmotic stress, drought stress and heat stress) compared with wild type plants (WT). On the contrary, overexpression of DlRan3B gene line pCAMBIA1301-35S-DlRan3B (P35S_B) and (pCAMBIA1301-pDlRan3B (1569bp) -DlRan3B (PB_B)) Resistance was relatively weak, and the tolerance of P35S_B strains to salt stress, osmotic stress, drought stress and heat stress was weaker than WT, and was highly sensitive to salt stress and osmotic stress. The same was that the resistance to cold stress of overexpressed transgenic lines was improved, and all of them were highly sensitive to exogenous ABA, indicating that DlRan3A and DlRan3B genes of longan and DlRan3B were cold and cold. The transcriptional analysis of heterotopic expression of longan DlRan3 and DlRan3B under.5 3S or specific promoter driven by stress resistance and ABA signaling pathway was analyzed by transcriptional sequencing technology to analyze the tobacco strain of longan DlRan3A and DlRan3B in 35S or specific promoter driven by DlRan3A and DlRan3B. The results showed that the overexpressed strain was significantly higher. The regulation or downregulation of auxin signaling pathway related differentially genes indicates that longan Ran may have a complex mechanism that affects the regulation of auxin signaling pathway, and the partial differential genes involved in ABA transport are significantly up-regulated, and that the Ran overexpression of longan has a significant effect on the response ability of plant to exogenous ABA.BR (brassinosteroid). The positive regulation gene was significantly up-regulated, while the negative regulation of BR synthesis was significantly down, indicating the increase of BR content in the transgenic plants or the enhancement of BR signal output. Most of the cell wall proteins in the 4 transgenic lines expressing longan Ran gene are up-regulated, and the DlRan3A and DlRan3B genes of longan may be affected by the substance of the longan cell wall. Accumulation is involved in regulating the growth and development of longan, especially in the development of early embryo and pulp. Over expressed longan Ran lines significantly enriched carbohydrate metabolism and alanine, glutamic acid and asparagine metabolism pathway, and the P35S_A line also existed as a pathway for the oxygenation reductase activity of the peroxide as the receptor; the PA_A strain also existed the ginseng. With cell wall tissue, structural composition and biosynthesis, cellulose biosynthesis and metabolism, glucan metabolism, and outsourced structural tissue, PA_A, compared with P35S_A strains and PB_B and P35S_B lines, significantly enriched cell wall tissue, structural components and biosynthetic pathway, including the Pollen O of 3 extensin-like families. The Le E 1 domain gene, however, is a significant up-regulated gene in the PA-A strain and a significant down-regulation in the PB_B strain. In combination with phenotypic analysis, the specific promoter driven DlRan3A overexpression affects the root hair, fruit and seed development of the plant by raising the level of some extensin-like genes, indicating that the specific promoter of the longan Ran is involved. The tissue structure and biosynthesis of the organ cell wall may play a specific regulatory role. In the strains of longan Ran, there are many plant resistance related groups encoding peroxidase (PER), water channel protein (TIP), glutamine synthetase (AS), glutathione transferase (GST), heat shock protein (HSP) and its chaperone protein (DNAJ). The resistance related transcription factor genes such as MYB, NAC, WRKYH ERF, GRAS and bHLH are closely related to the resistance regulation of longan Ran. The strains that overexpress the DlRan3A gene show a stronger abiotic stress resistance than the strains overexpressing the DlRan3B gene, and on the one hand, the genes that enhance the plant resistance in P35S_A and PA_A strains (such as PE) R, GST, OLP, TIS, PPO and BG are significantly up-regulated, on the other hand, the functional analysis of a large number of plant resistance related transcriptional factors (ERF, WRKY, bHLH, C3H, C3H, etc.) in the P35S_B and PB_B strains The expression of cell wall structure and synthesis related genes of EXLA2 and EXT3 increased significantly, and the expression of resistance related genes, such as MYB59, DlPPO1 and TIP1-1, were also significantly improved, and the results of the differential expression of the cell wall protein gene and resistance genes caused by the overexpressed longan Ran gene were verified, and the Ran gene of longan was further explained by the combination of the Ran gene and cell wall. It is closely related to the process of resistance regulation. In summary, this study isolated the DlRan3 and DlRan3B gene promoters and analyzed the functional specificity of the two participants in the process of participating in hormone response and resistance regulation. The longan Dlan3A and DlRan3B genes are the important small G protein genes necessary for the growth and development of longan, especially in the longan embryo ( Seed) plays an important role in development and flesh development; longan Ran may be involved in the regulation of hormones such as auxin, cytokinin, ABA and BR. Ectopic expression of longan Ran enhances the sensitivity of plant to exogenous ABA and improves plant cold resistance; heterotopic expression of longan DlRan3A to increase plant resistance to abiotic stress is specific. The promoter driven DlRan3A expression can affect the root hair, fruit and seed development by increasing the extensin-like gene level, and the longan Ran gene is closely related to the process of cell wall synthesis and resistance regulation.
【学位授予单位】：福建农林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S667.2
，
本文编号：2171755
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