【作者】 程云清；

【导师】 安利佳； 杨君；

【作者基本信息】 大连理工大学 ， 生物化工， 2009， 博士

【摘要】 本研究主要以大豆（Glycine max（L.）merrill）品种铁丰31为试验材料,研究了大豆生长发育过程中内源乙烯释放规律和乙烯生物合成限速酶1-氨基环丙烷-1-羧酸合酶（ACS）基因的表达特征,并通过乙烯生成的促进剂1-氨基环丙烷1-羧酸（ACC）和抑制剂氨基乙氧基乙烯甘氨酸（AVG）的处理,以及利用线性基因的瞬时表达体系和RNA干扰技术研究了乙烯在大豆生长发育过程中的生理功能。（1）对大豆不同生长发育时期和盛花期不同组织器官乙烯释放量及其生物合成限速酶ACS基因的表达特征的研究表明,ACS基因在大豆的整个生命周期和不同组织器官中都有表达,但其表达量存在着明显的差异。乙烯释放特征研究的结果表明,乙烯释放量在大豆开花后期高于大豆营养生长期,在大豆花荚脱落期,乙烯释放量明显增加,在大豆的完熟期,乙烯的释放量达到最大值;大豆植株相对衰老的组织较幼嫩的组织或器官乙烯释放量更大。乙烯生物合成限速酶ACS基因的表达丰度和乙烯生成呈明显正相关。利用乙烯合成的促进剂ACC和抑制剂AVG对大豆苗期喷施的结果表明,AVG可促进大豆植株的营养生长,且大豆的生育期被提前。AVG处理明显提高了大豆幼苗叶片净光合速率、蒸腾速率和气孔导度,降低了胞间CO₂浓度;明显提高了光饱和点、光饱和光合速率、表观量子效率、CO₂饱和点、羧化效率、RuBP最大再生速率,同时,降低了光补偿点和CO₂补偿点。此外,AVG处理还提高了生物量和叶面积,但降低了叶绿素含量与气孔密度。以上结果表明,外源AVG处理能改善大豆幼苗叶片的光合性能。ACC处理的效果则与AVG相反。（2）在大豆种子萌发过程中,胚根突破种皮和下胚轴伸长的两个关键时期,ACS基因大量表达,酶活相应提高,乙烯大量释放。乙烯促进剂和抑制剂对大豆种子萌发的研究结果表明,AVG处理后乙烯的释放被显著抑制,同时能够促进下胚轴的伸长生长,但是对于大豆的干重没有显著影响,同时下胚轴组织中Zn²⁺的含量明显增加;而ACC处理的效果与AVG相反。（3）通过不同浓度乙烯抑制剂AVG和促进剂ACC对大豆花形态、花粉性状及单株产量特征等的研究结果表明,供试品种经AVG处理后,与对照相比,成花量、花的长度、花瓣长度、雄蕊柱长、花粉量、花粉管长度、秸杆重、单株荚数和单株产量有明显增加,花粉败育率下降,ACC处理的效果与AVG相反。两种处理对大豆花粉萌发率无明显影响。花粉扫描电镜结果显示,AVG处理的花粉长度增加,ACC处理的花粉部分出现扭曲与变形。（4）通过洋葱下表皮和大豆子叶节部位最小线性表达框的直接转化,实现了线性元件在洋葱下表皮和大豆子叶节部位的基因瞬时表达。GUS基因的瞬时表达和5’-FITC标记DNA的示踪,表明外植体的高渗预处理有利于线性DNA的直接转化和瞬时表达;向转化缓冲液中添加包括Ca²⁺,表面活性剂和农杆菌LBA4404的细胞培养物也有利于提高基因的瞬时表达效率。FITC-DNA的荧光显微镜观察结果表明,外植体于黑暗培养3h后通过线性DNA基本转化缓冲液的转化,共培养72h后,细胞核中能观察到较强的荧光强度。流式细胞技术检测表明在以MS液体培养基为基本转化缓冲液的条件下,线性DNA的入核效率仅为0.02%,当基本缓冲液同时添加Ca²⁺、Dow Corning Q2-5211和农杆菌细胞培养物时,入核效率由0.02%增加至54.85%。（5）构建大豆ACC合酶基因GMACS保守区域的RNA干扰（RNAi）载体,进行了大豆子叶节部位线性ACS-RNAi基因表达框的转化和瞬时表达研究,PCR、RT-PCR检测和瞬时表达结果表明,线性ACS基因的表达框转化进入大豆子叶节部位。转化后子叶节部位乙烯的释放和ACS基因的表达被抑制,形态学观察发现,与对照相比,转化处理的大豆子叶节部位能够分化出更多花芽。综上结果表明,乙烯直接影响大豆生长发育的全过程。大豆种子萌发过程中,乙烯的三重反应主要与乙烯调控的无机离子特别是Zn²⁺的转运和生物有效性相关。抑制乙烯生成可以增强大豆的光合性能、促进营养生长、增加大豆的成花数量、提高大豆的产量,为大豆的高产栽培和育种提供了重要依据。更多还原

【Abstract】 Plant growth and development have been affected by a wide range of environmental conditions.In order to meet the complex and ever-changing external conditions,the plant in the long-term evolution has developed a variety of gene expression system to regulate and adopt to cope with the challenge from environment.Modulation of ethylene biosynthesis of the plant is a fundamental physiological response to the fluctuations in the internal or external cues.The soybean cultivar Tiefeng-31 was used to study the effects of ethylene on the growth and development of soybean.Ethylene emission and the relative gene expression of 1-aminocyclopropane- 1-carboxylate synthase gene（ACS） were detected during the soybean growth and development;on the other hand,RNAi technology as well as ethylene regulator aminoethoxyvinylglycine（AVG） and 1-aminocyclopropane-1-carboxylic acid（ACC） were used to study the physiological function of ethylene during soybean seed germination and plant growth.（1） The ethylene production and the ACS gene expression were detected at different development stages and multiple soybean tissues,including vegetative and reproductive abscission zones and flanking tissues during full flowering stage of soybean.There is a significantly differences of ACS gene expression and ethylene emission at the different stages and different organs of soybean.The results showed that ethylene emission was at the late stage of the plant life cycle,and mainly produced by the aging organ.There is a maximum emission of ethylene at the soybean ripening period,especially before soybean harvest.The abundance of expression of ACS showed similar rules.Different concentrations of ethylene inhibitor AVG and promoter ACC were sprayed to soybean cultivar Tiefeng 31,and their effects on plant photosynthesis and CO₂ response curve were measured.The results showed that through the treatments of AVG,net photosynthetic rate,transpiration rate and stomatal conductance were significantly increased;intercellular CO₂ concentration were markedly decreased;light saturation point,photosynthetic rate at light saturation point,apparent quantum efficiency,CO₂ saturation point,carboxylation efficiency and maximum RuBP regeneration rate were markedly increased;light compensation point,CO₂ compensation point were significantly decreased.ACC,the precursor of ethylene showed an opposite effects. In addition,the chlorophyll contents were significantly enhanced.The results above indicated that AVG treatment could improve photosynthesis characteristics of soybean seedlings.And with the improvement of photosythesis characteristics,the plant height and the dry weight of seedlings were significantly enhanced by AVG treatment.The ACC treatment showed the opposite effects.（2） The relationship between ethylene production and metal mobilization and transport was investigated.Soybean seeds were incubated with or without inhibitors of ethylene synthesis AVG or in the presence of the natural ethylene precursor ACC,the ddH₂O as control. The kinetics of ethylene emissions,the ethylene key enzyme gene ACS expression,the ACS enzyme activity and the concentration and extractability of metal in axis tissues were measured.The results showed that the ACS gene relative expression,the ACS enzyme activity and ethylene evolution reached the peak value during the visible stage of germination （pericarp splitting） and the prolonging stage of the hypocotyls.Correspondingly,the Zn²⁺ concentration and extractability in axis tissues significantly increased.The concentration of Fe²⁺ and Mn²⁺ showed no obvious change before prolonging stage but declined at the 7^th day,and their extractability showed no rule change.The ethylene inhibitors inhibit the ethylene emission;promote the axis prolonging and Zn²⁺ concentration and HCl-extractability. The ACC showed the contrary effect.These findings suggested that,the triple response in soybean affected by ethylene may be regulated the concentration of Zn²⁺ into axis tissues from cotyledon or HCl-extractability in axis tissues.（3） Different concentrations of ethylene inhibitor AVG and promoter ACC were sprayed to soybean,and their effects on soyben flower traits,pollen morphology and yield were studied.Results showed that total number of flower,flower length,petal length, stamina column length,pollen quantity,pollen tube length,straw weight,pod number per plant and yield increased along with higher AVG concentration,while abortion ratio of pollen decreased.ACC,the precursor of ethylene showed an opposite effect.Both AVG and ACC treatments had no significant effects on pollen germination ratio.Scanning electron microscope（SEM） observation results showed that AVG treatments promoted pollen length,while distorted deformation occurred after ACC treatments.（4） A linear gene cassettes direct transformation method was developed and used in transient expression of a GUS reporter gene in onion epidermal cells and soybean cotyledon nod system,respectively.Linear gene cassette was labeled by 5’-fluorescent dye（fluorescein isothiocyanate,FITC） and its delivery pathway was traced.The basic transformation solution used was Murashige and Skoog basal salt mixture（MS） liquid medium.Hypertonic pretreatment of explants and transformation cofactors,including Ca²⁺,surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated.Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h,the onion low epidermal explants were pre-cultured in darkness at 27℃for 48h.After transformation for 3h,the explants were transferred to MS solid media for 72h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei.By GUS staining and flow-cytometry-mediated fluorescent detection,a significant increase of the ratios of fluorescent nuclei as well as expression of the GUS reporter gene was observed by each designed transformation solution.This potent and feasible method showed prospective applications in plant transgenic research.（5） RNAi vector of conservative region from ethylene biosynthesis limiting enzyme ACS gene was constructed.The liner DNA cassettes of RNAi-ACS acquired by PCR action was used as the transformation elements and the soybean cotyledon nod was transformed as the explants to study the effect of ethylene on flower bud differentiation of soybean.The results of PCR and RT-PCR showed that the linear RNAi-ACS gene cassettes have entered into the cotyledons node of soybean.Ethylene emission and ACS gene expression abundance was significantly inhibited after the cotyledon node was transformed.Compared with the control,more flower bud was differentiated at the cotyledon section.The above results showed that the ethylene affects soybean growth and development through its lifecycle.The typical ethylene triple response was relative with the Zn²⁺ concentration and extractability in axis tissues regulated by ethylene.And inhibition of ethylene in soybean increased soybean yields by improving photosynthesis characteristics, promoting the vegetable growth,and enhancing the forage quality and quantity.The results presented above provide a general theoretical basis for the soybean cultivation and breeding.更多还原