【作者】 郝荣华；

【导师】 赵彦修； 冯献忠；

【作者基本信息】 山东师范大学 ， 植物学， 2012， 博士

【摘要】 大豆（Glycine max (L.) Merr）属于豆科、蝶形花亚科、大豆属，是世界上重要的粮食、油料和饲料作物。大豆是古四倍体植物，其基因组的复制至少发生了两次，由此引起了整个基因组的高度重复，大豆的全基因组测序结果发现约75%的基因以多拷贝形式出现，因此在传统模式植物中发现的重要遗传调控机制在大豆中可能会由更多的遗传调控因子所控制或者演化为更为精细的调控方式。本论文主要对于大豆两侧对称花型发育的分子机制进行了研究，分析了模式植物控制两侧对称花型遗传因子在大豆中功能的演化，以期为实现大豆花型的人工改造提供基础。长期以来的研究表明，模式植物金鱼草两侧对称花型的发育受CYCLOIDEA（CYC）、DICHOTOMA（DICH）、RADIALIS（RAD）和DIVARICATA（DIV）四个基因的控制，其中CYC和DICH属于TCP转录因子家族，控制背部花瓣属性；RAD和DIV属于MYB转录因子家族，RAD决定背部花瓣的属性，DIV决定腹部花瓣的属性；CYC和DICH在花原基的背部表达，激活RAD基因的表达，而RAD基因抑制了DIV基因在背部和侧部花原基中的作用，从而使花瓣的背部属性在近轴的背部花瓣和侧部花瓣得到不同程度的体现，形成具有三种不同花瓣形态的两侧对称花型。本文利用生物信息学手段在全基因组水平上分类和鉴定了大豆中CYC/DICH、RAD和DIV基因的同源基因，分析了其表达模式。从大豆中分离了与金鱼草CYC基因亲缘关系较近的3个TCP基因（GmTCP1、GmTCP2、GmTCP4基因），分别通过这三个基因在大豆中过量表达和基因沉默实验表明：大豆中GmTCPs、GmRADs和GmDIVs基因共同参与了两侧对称花型发育的控制，其参与两侧对称花型发育的因子比金鱼草数目更多，调控机制更为复杂。在模式植物拟南芥中沉默和过量表达此三个基因得到不同的表型，提示它们功能之间的差异性。同时，本研究分别建立了农杆菌介导的大豆子叶节转化体系及基因枪法转化体系并进行了条件优化，为分析大豆基因的功能奠定了基础。具体结果如下：1.农杆菌介导的大豆子叶节转化体系及大豆胚尖的基因枪转化体系的建立首先以13种不同基因型的大豆为材料，研究了子叶节外植体再生体系中大豆基因型对丛生芽数目的影响，选取优势基因型为受体材料，比较了草丁膦和潮霉素筛选压力下外植体的再生情况，并确定了合适的筛选浓度，在对农杆菌介导的子叶节转化体系优化的基础上进行了大豆转化效率的研究。结果表明：相同的芽诱导条件下，山宁14产生的丛生芽最多，适合用于大豆子叶节转化；采用3~8mg L-1潮霉素的梯度筛选更有利于抗性苗的筛选，转化效率可达到3.2%。同时建立了大豆胚尖的基因枪转化体系，获得了具有抗性的植株，转化效率可达到9.3%。2.大豆TCP同源基因的克隆利用大豆数据库phytozome v7.0和Mega5软件分析，在大豆基因组中检索到58个CYC/DICH同源基因，5个RAD同源基因和5个DIV同源基因，对金鱼草、玉米、水稻、拟南芥、蒺藜苜蓿和大豆中的TCP、RAD和DIV同源基因进行系统进化分析。分离了大豆中与金鱼草CYC亲缘关系较近的3个TCP基因（GmTCP1、GmTCP2和GmTCP4基因），构建了其正反义表达载体和VIGS沉默表达载体。3. GmTCP1、GmTCP2和GmTCP4基因的功能分析1）转基因大豆的表型分析：利用农杆菌介导的遗传转化法和基因枪法，将GmTCP1、GmTCP2和GmTCP4基因的过量表达载体分别转化大豆品种山宁14，获得具表型的过量表达转基因株系，同时将GmTCP1、GmTCP2和GmTCP4基因的VIGS基因沉默载体转染大豆后获得基因表达降低的植株。GmTCP2基因过量表达的转基因植株表现为侧部或腹部花瓣旗瓣化，扫描电镜观察发现旗瓣化的侧部和腹部花瓣表皮细胞形态具旗瓣属性。GmTCP4基因过量表达的转基因植株出现2个大小不同的旗瓣相互融合的表型，扫描电镜观察发现2个大小不同的旗瓣表皮细胞均为旗瓣属性，表皮细胞体积大小与旗瓣大小相关。而GmTCP1基因过量表达株系无明显表型。VIGS方法获得的GmTCP1、GmTCP2和GmTCP4基因沉默植株均表现为旗瓣呈闭合筒状结构，扫描电镜结果显示这些植株的旗瓣表皮细胞形状并未发生变化，但较野生型细胞的体积减小，细胞数目增多。2）转基因拟南芥的表型分析：为了进一步研究GmTCPs基因的功能，将GmTCP1、GmTCP2和GmTCP4基因的正反义表达载体转化了拟南芥，过量表达GmTCP2的转基因株系表现为植株生长速度快，株型大，叶片和花器官增大，而反义转基因株系则表现为植株生长慢，株型小，叶片和花器官减小。过量表达GmTCP4的转基因株系出现了叶形变圆的表型，反义株系无明显表型。而GmTCP1转基因株系均无明显表型。4. GmTCPs、GmRADs和GmDIVs基因相互关系的研究利用Real time-PCR方法分析了GmTCPs、GmRADs和GmDIVs基因的表达，结果表明：在野生型大豆中，GmTCPs和GmRADs主要在旗瓣中表达，GmDIVs主要在翼瓣和龙骨瓣中表达；在GmTCP2和GmTCP4过量表达的转基因大豆中，GmTCPs和GmRADs的表达水平均有所升高，而GmDIVs基因的表达量有所降低；在GmTCP1、GmTCP2和GmTCP4-VIGS基因沉默大豆中，GmTCPs和GmRADs基因的表达水平均有明显下降，而GmDIVs基因的表达量有所升高。上述结果表明，GmTCPs、GmRADs和GmDIVs基因共同参与了大豆两侧对称花型发育的控制，GmRADs基因可能是GmTCPs基因的下游调控基因，而GmRADs基因和GmDIVs基因之间可能存在相互拮抗的关系。本论文的主要创新点：1.首次发现改变TCP基因的表达，可以将大豆旗瓣的形状改变为闭合筒状，这为研究TCP基因功能的分化和分析TCP基因与其它控制器官形态遗传因子未知的相互作用方式提供了线索。2.证实了大豆中有更多的转录调控因子参与了两侧对称花型的发育，大豆中GmTCPs、GmRADs和GmDIVs基因共同参与了两侧对称花型发育的控制，其参与两侧花型发育的因子比金鱼草数目更多，调控机制更为复杂。3. GmTCP1、GmTCP2和GmTCP4基因在拟南芥中表达改变时，获得不同的表型，提示它们作用方式的特异性之间存在差异：GmTCP2转基因植株的表型变化最强，GmTCP1最弱；GmTCP2可以通过影响细胞的增殖和生长更广泛地调控植株器官的发育。4.建立和优化了大豆农杆菌介导的大豆子叶节转化体系及大豆胚尖的基因枪转化体系，为研究大豆基因功能提供了基础。更多还原

【Abstract】 Soybean (Glycine max (L.) Merr) belongs to Glycine genus, Faboideaesubfamily, Leguminosae family. It is one of most important food, oil and feed crops inthe world. Soybean is an ancient polyploidy, its genome duplications occurred twice,resulting in a highly duplicated genome with nearly75%of the genes present inmultiple copies. The molecular mechanism from model plant may vary with moregene copies emerging from soybean. In this study, we investigated the molecularmechanism controlling floral symmetry in soybean, analyzed the function divergencyof genes in the network to pave the path for soybean genetic modification.Previous studies on the model plant, Antirrhinum majus, demonstrated that therewere mainly four cardinal genes in the development of dorsoventral asymmetry:CYCLOIDEA(CYC), DICHOTOMA(DICH), RADIALIS(RAD)and DIVARICATA(DIV).CYC and DICH genes which belonged to the TCP transcription factor family wereinvolved to the dorsal identity of flower development. Both RAD and DIV genesbelonged to the MYB transcription factor family, and RAD gene promoted dorsalindentity while DIV to the ventral identity. CYC and DICH genes were expressed indorsal floral primordia, and then activated the expression of RAD gene. DIV gene wasinhibited by the RAD gene expression in the dorsal and lateral petals. Therefore thedorsal identity was expressed in adaxial domain of dorsal and lateral petals indifferent degree, forming three different morphology petals.Based on genome database and bioinformatics method, homologous genes ofCYC/DICH, RAD and DIV were characterized from soybean in this study. Three TCPgenes, GmTCP1, GmTCP2and GmTCP4which were closer to the CYC gene wereisolated from soybean. By transgenic technology, the overexpression and gene silence of these three genes experiments illustrated that GmTCPs genes, GmRADs genes andGmDIVs genes were involved in the control of flower bilaterally symmetry in soybean.More factors were involved in soybean floral symmetry regulation comparing withmodel plant. Altering expression of these genes in Arabidopsis gave rise to dissimilarphenotypes of transformants, which suggested that their funtions were divergent. Wealso set up and optimized the system of Agrobacterium-mediated soybeantransformation using the cotyledonary node and the system of biolisticstransformation using the embryonic tips of soybean, which provided a good platformfor soybean function analysis. The results were as follows:1. Establish the system of Agrobacterium-mediated soybean transformation usingthe cotyledonary node and the system of biolistics transformation using the embryonictips of soybeanThe effects of13cultivars and two selection agents were studied during theprocess of shoot regeneration and transformation. The results showed that the cultivar‘Shanning14’ induced highest number of multiple shoots among tested13cultivars,and hygromycin B was better than glufosinate during transformant selection ofcultivar ‘Shanning14’. The optimal transformant selection scheme was a gradientconcentration of hygromycin B from3mg L-1to8mg L-1. Transformation efficiencywas3.2%based on the number of transformed events. By biolistics transformation ofsoybean embryonic tips, we acquired a number of transgenic plants, and thetransformation efficiency of this method was9.3%based on the number oftransformed events.2. Cloning of the TCP homologous genesFifty-eight CYC homologous genes, five RAD homologous genes and five DIVhomologous genes were characterized from soybean phytozome v7.0database. Thephylogenetic analyisof TCP, RAD and DIV homologous genes were constructed bythe MEGA5software from Antirrhinum majus, Zea mays, Oryza sativa, Arabidopsisthaliana, Medicago truncatula and Glycine max. Three TCP genes, GmTCP1, GmTCP2and GmTCP4, closer to the CYC gene were cloned from soybean, and theirsense expression vectors, antisense expression vectors and VIGS vectors wereconstructed in this study.3. Function analysis of GmTCP1, GmTCP2and GmTCP41) Phenotyping of transgenic soybean of GmTCP1, GmTCP2and GmTCP4: ByAgrobacterium-mediated genetic transformation and biolistics transformation, thesense expression vectors of GmTCP1, GmTCP2and GmTCP4were transformed intosoybean cultivar ‘Shanning14’. Meanwhile, a series of VIGS transgenic lines ofGmTCP1, GmTCP2and GmTCP4were also generated in this study. Theoverexpression of GmTCP2and GmTCP4conferred to ectopic dorsal indentityshifting in other petals, concurring with morphological phenotypes of epidermal cells.No obvious phenotype was oberserved in GmTCP1overexpression transgenic lines.The VIGS transgenic lines of GmTCP1, GmTCP2and GmTCP4were all withabnormal tubular cup dorsal petals to various extent. The phenotypes of scanningelectron microscope on epidermal cells were coincident with their morphologicalvariations.2) Phenotyping of transgenic Arabidopsis of GmTCP1, GmTCP2and GmTCP4:To further study the function of GmTCPs genes, the sense and antisense vectors ofGmTCP1, GmTCP2and GmTCP4were transformed into Arabidopsis thaliana. TheGmTCP2overexpression transgenic lines grew quickly with larger leaves and floralorgans, while the antisense transgenic lines showed opposite phenotype. TheGmTCP4overexpression transgenic lines induced more round leaves, while theantisense transgenic lines had no difference with wild type. There was no obviousphenotype of GmTCP1transgenic lines with both overexpression and antisensevectors.4Interactions of GmTCPs, GmRADs and GmDIVsReal time-PCR analysis result suggested that GmTCPs and GmRADs genes wereexpressed in the dorsal petal, while the GmDIVs genes were expressed in the ventral petal. The transcription levels of GmTCPs genes and GmRADs genes wereconstitutively up-regulated in the GmTCP2and GmTCP4overexpression transgenicplants, but the transcription levels of GmDIVs genes were reduced in the sametransgenic plants. On the contrary, the VIGS lines gave rise to the opposite result. Theabove results suggested that GmTCPs genes, GmRADs genes and GmDIVs genesinvolved in the control of flower bilaterally symmetry in soybean. GmRADs genesmay be downstream of GmTCPs genes. GmRADs may antagonise GmDIVs genes inthe control of flower symmetry.The innovations of this thesis:1. It was the first research to illustrate that alteration expression of TCP genecould cause the tubular cup dorsal petal. This provided the clues for studying thefunction divergency of TCP gene and indentifying their new interaction factors.2. It was proved that more factors were involved in soybean floral symmetryregulation comparing with model plant. GmTCPs, GmRADs and GmDIVs genesinvolved in the control of flower bilaterally symmetry in soybean, which is morecomplicated than Antirrhinum majus as more factors imported.3. The divergent phenotypes were observed from transgenic Arabidopsis ofGmTCP1, GmTCP2and GmTCP4genes, which suggested that their specifities aredifferent. GmTCP2could function in both soybean and Arabidopsis via regulting cellproliferation and differentiation.4. We established and optimized Agrobacterium-mediated transformation andbiolistics transformation platforms of soybean, which availed to soybean genefunction indentification in the future.更多还原