龍眼胚性培養(yǎng)物DlRan3A和DlRan3B基因的功能分析
發(fā)布時間：2018-08-08 12:12
【摘要】：龍眼是我國南方具有重要經(jīng)濟價值的熱帶亞熱帶常綠木本果樹,龍眼生產(chǎn)上存在諸多問題制約其產(chǎn)業(yè)發(fā)展,如缺乏具有生產(chǎn)價值的焦核品種、成熟期過于集中以及果實品質、產(chǎn)量和貯藏受到環(huán)境因素影響。目前關于龍眼焦核、結果期調節(jié)以及抗逆性等方面的分子機制研究較少。Ran(Ras-related nuclear protein)小G蛋白廣泛參與生長發(fā)育、抗病和激素敏感性調控等過程。前人研究表明,龍眼Ran可能與龍眼體胚發(fā)生(somatic embryogenesis,SE)過程的細胞分裂有關,但具體機制尚不清楚,目前關于龍眼Ran表達調控機制研究仍有大量空白,尤其是龍眼Ran基因在龍眼胚胎和果實發(fā)育過程的功能特異性以及響應外界環(huán)境的相應機制等。因而本研究首先分離龍眼DlRan3A和DlRan3B基因啟動子并驗證其功能,并通過對龍眼體胚發(fā)生、合子胚發(fā)育以及果肉發(fā)育等過程以及龍眼不同組織器官中DlRan3A和DlRan3B基因的表達模式進行分析,為了解Ran基因在龍眼的胚胎發(fā)育及果實發(fā)育中的作用機制提供新的線索。同時,利用異位表達、原位轉化以及轉錄組分析等手段對龍眼DlRan3A和DlRan3B基因及啟動子進行功能分析,探索Ran在植物生長發(fā)育和抗性調節(jié)等過程中的功能特異性,進一步研究植物Ran表達調控網(wǎng)絡。主要研究結果如下:1龍眼DlRan3A和DlRan3B基因啟動子的克隆與生物信息學分析以龍眼胚性愈傷組織(Embryogenic callus,EC)為材料,利用Tail-PCR法和連接介導染色體步移法分別獲得1256bp和1569bp的DlRan3A和DlRan3B基因啟動子序列;生物信息學分析預測獲得這兩個基因啟動子區(qū)的核心啟動子區(qū)域和可能的轉錄起始位點,且DlRan3A啟動子區(qū)存在一個長度為147bp的CpG島。分析功能元件發(fā)現(xiàn),DlRan3A和DlRan3B基因啟動子區(qū)均含有生長素、水楊酸(salicylic acid,SA)和茉莉酸甲酯(methyl jasmonate,MeJA)響應元件以及厭氧誘導和防御與脅迫響應元件。DlRan3A基因啟動子還含有參與傷害和病原體誘導的響應元件,DlRan3B基因啟動子區(qū)還含有赤霉素和脫落酸(abscisic acid,ABA)響應元件及低溫響應元件,說明龍眼Ran家族不同成員在激素響應和抗逆調節(jié)方面可能存在功能特異性。2龍眼DlRan3A和DlRan3B基因啟動子功能驗證構建龍眼DlRan3A和DlRan3B啟動子的3'和5'不同缺失突變體的表達載體,利用煙草葉片GUS瞬時表達水平進行啟動子缺失分析和啟動子激素應答分析。結果顯示,DlRan3A和DlRan3B基因啟動子中,正調控和負調控元件并存,8.6 μMIAA(indole-3-acetic acid,IAA)、75μM SA或100μM MeJA誘導DlRan3A基因啟動子的轉錄活性,8.6μM IAA 或 34.6 μM CA3(gibberellin A3,GA3)激活DlRan3B基因啟動子的轉錄活性;基于激素應答元件分布于DlRan3A和DlRan3B基因啟動子的區(qū)域,推測生長素元件均為正調控作用元件;MeJA應答元件的廣泛分布預示其復雜的調控作用;推測DlRan3A基因啟動子的SA響應元件、防御和逆境響應元件以及box S元件為正調控元件,且DlRan3B基因啟動子的SA響應元件位于防御和逆境響應元件和低溫和干旱響應元件為負調控元件;DlRan3A和DlRan3B的啟動子在參與抗逆反應中可能發(fā)揮特異的轉錄調控作用。3龍眼DlRan3A和DlRan3B的表達模式分析利用qPCR分析龍眼DlRan3A和DlRan3B基因的表達模式。從龍眼DlRan3A和DlRan3B基因在龍眼不同生長發(fā)育階段以及組織器官中的表達譜可知,龍眼DlRan3A和DlRan3B基因在龍眼不同組織部位中均有表達,其中二者均在龍眼種子和果肉中表達量較高;在體胚發(fā)生的子葉胚時期和合子胚發(fā)育的幼嫩胚胎中表達量較高;DlRan3A和DlRan3B基因表達量隨著種子萌發(fā)過程下降,且隨著果肉的膨大而提高。說明龍眼DlRan3A和DlRan3B基因在龍眼胚胎(種子)發(fā)育以及果肉膨大過程中發(fā)揮更為重要的作用。不同激素及非生物脅迫處理龍眼EC下的DlRan3A和DlRan3B基因的定量表達分析結果顯示,二者的表達均受到一定濃度外源生長素(IAA)、赤霉素(GA3)的誘導而受到高濃度SA和MeJA的抑制,并且一定程度上受到鹽脅迫、滲透脅迫、PEG脅迫和ABA脅迫的誘導。說明龍眼DlRan3A和DlRan3B基因廣泛參與激素應答和非生物脅迫響應。4 35S或特異啟動子驅動下異位表達龍眼DlRan3A與DlRan3B基因的功能分析構建DlRan3A與DlRan3B亞細胞定位載體,瞬時轉化本氏煙葉片;構建35S或特異啟動子驅動DlRan3A或DlRan3B基因過表達載體、特異啟動子驅動GUS基因表達載體及DlRan3A干擾表達載體,轉化本氏煙。亞細胞定位顯示,龍眼DlRan3A和DlRan3B主要定位于細胞核。特異啟動子驅動GUS表達的轉基因本氏煙幼苗(PA-GUS和PB-GUS)的GUS染色結果表明DlRan3A和DlRan3B均在細胞分裂旺盛的部位(根尖和根系分支處)表達量較高,同時,二者在果實和種子中也分布較多,此外DlRan3A相對于DlRan3B在花器官中有更顯著的表達。特異啟動子驅動DlRan3A過表達的株系pCAMBIA1301-pDlRan3A(1256bp)-DlRan3A(PA_A)表現(xiàn)出植株矮壯,開花推遲,果實發(fā)育異常且結籽率低的現(xiàn)象,說明龍眼特異啟動子驅動DlRan3A過表達對植物生長發(fā)育尤其是調節(jié)植物生長周期以及果實和種子發(fā)育有顯著影響。PA_A株系的根系中主根相對不明顯,且根毛發(fā)育旺盛,推測DlRan3A基因與根毛發(fā)育密切相關。干擾表達DlRan3A基因的本氏煙株系(ran3a)表現(xiàn)出生長極其緩慢、花果發(fā)育異常且不結籽的現(xiàn)象,說明龍眼Ran基因在植物生長發(fā)育中發(fā)揮至關重要作用。過表達DlRan3A基因株系pCAMBIA1301-35S-DlRan3A(P35S_A)和 PA_A 相對于野生型植株(WT)有較高的抗非生物脅迫(鹽脅迫、滲透脅迫、干旱脅迫和熱脅迫)能力;相反,過表達DlRan3B基因株系pCAMBIA1301-35S-DlRan3B(P35S_B)和(pCAMBIA1301-pDlRan3B(1569bp)-DlRan3B(PB_B)的抗性相對較弱,其中P35S_B株系對鹽脅迫、滲透脅迫、干旱脅迫和熱脅迫的耐受性均弱于WT,且對鹽脅迫和滲透脅迫高度敏感;相同的是,過表達轉基因株系的抗冷脅迫能力得到提高,且均對外源ABA高度敏感,說明龍眼DlRan3A和DlRan3B基因與冷脅迫抗性和ABA信號通路密切相關。5 3S或特異啟動子驅動下異位表達龍眼DlRan3和DlRan3B的轉錄組學分析利用轉錄組測序技術對35S或特異啟動子驅動下過表達龍眼DlRan3A和DlRan3B的本氏煙株系進行分析,結果顯示,過表達株系中存在顯著上調或下調的生長素信號通路相關差異基因,說明龍眼Ran可能存在復雜作用機制影響生長素信號通路的調控;參與ABA運輸?shù)牟糠植町惢蝻@著上調,結合ABA脅迫表型可知,龍眼Ran過表達顯著影響植株對外源ABA的應答能力。BR(brassinosteroid)正調控基因顯著上調,而負調控BR合成的基因顯著下調,說明轉基因植株內BR含量增多或BR信號輸出增強。過表達龍眼Ran基因的4種轉基因株系中絕大多數(shù)細胞壁蛋白上調表達,可見龍眼DlRan3A和DlRan3B基因可能通過影響龍眼細胞壁物質的積累以參與調控龍眼生長發(fā)育過程,尤其是早期胚胎及果肉發(fā)育過程。過表達龍眼Ran的株系顯著富集碳水化合物代謝和丙氨酸、谷氨酸和天冬酰胺代謝通路;P35S_A株系還存在過氧化物為受體的氧化還原酶活性的通路;PA_A株系還存在參與細胞壁組織、結構成分和生物合成、纖維素生物合成及代謝、葡聚糖代謝、外包結構組織等通路;PA_A與P35S_A株系相比以及PB_B與P35S_B株系相比,均顯著富集細胞壁組織、結構成分和生物合成通路,包含的基因均為3個extensin-like家族的Pollen Ole e 1結構域基因,然而在PA-A株系中為顯著上調基因而在PB_B株系中為顯著下調基因;結合表型分析可知,特異啟動子驅動DlRan3A過表達通過提高部分extensin-like基因水平從而影響植株根毛、果實和種子發(fā)育,說明龍眼Ran特異啟動子在參與器官細胞壁組織結構及生物合成方面可能發(fā)揮特異的轉錄調控作用。過表達龍眼Ran的株系中存在較多編碼過氧化物酶(PER)、水通道蛋白(TIP)、谷氨酰胺合成酶(AS)、谷胱甘肽轉移酶(GST)、熱激蛋白(HSP)及其伴侶蛋白(DNAJ)等的植物抗性相關基因和編碼MYB、NAC、WRKYH ERF、GRAS及bHLH等抗性相關轉錄因子基因,可見龍眼Ran與抗性調節(jié)密切相關。過表達DlRan3A基因的株系呈現(xiàn)比過表達DlRan3B基因的株系更強的耐非生物脅迫能力,一方面在于P35S_A和PA_A株系中部分增強植物抗性的基因(如PER、GST、OLP、TIS、PPO和BG等)顯著上調,另一方面在于P35S_B和PB_B株系有大量植物抗性相關轉錄因子(如ERF、WRKY、bHLH、C3H、C2H2和GRAS等)基因顯著下調。6DlRan3A與DlRan3B基因原位轉化龍眼的功能分析在過表達龍眼DlRan3A或DlRan3B基因的原位轉化株系中,CESA6、EXLA2和EXT3等細胞壁結構和合成相關基因的表達量均顯著提高;MYB59、DlPPO1和TIP1-1等抗性相關基因的表達量也均顯著提高;驗證了過表達龍眼Ran基因引起的細胞壁合蛋白基因和抗性基因差異表達結果,進一步說明龍眼Ran基因與細胞壁合成和抗性調節(jié)等過程密切相關。綜上所述,本研究分離了DlRan3 和DlRan3B基因啟動子并分析了二者在參與激素應答和抗性調節(jié)等過程存在的功能特異性;龍眼Dlan3A和DlRan3B基因是龍眼生長發(fā)育所必需的重要小G蛋白基因,尤其在龍眼胚胎(種子)發(fā)育和果肉發(fā)育過程中發(fā)揮重要作用;龍眼Ran可能參與生長素、細胞分裂素、ABA和BR等激素信號通'路的調控。異位表達龍眼Ran提高植株對外源ABA的敏感性,且提高植株抗寒能力;異位表達龍眼DlRan3A提高植株抗非生物脅迫能力,特異啟動子驅動DlRan3A表達通過提高部分extensin-like基因水平,從而影響根毛、果實和種子發(fā)育;龍眼Ran基因與細胞壁合成和抗性調節(jié)等過程密切相關。
[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.
【學位授予單位】：福建農(nóng)林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S667.2
，
本文編號：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.
【學位授予單位】：福建農(nóng)林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S667.2
，
本文編號：2171755
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