【作者】 TCHOUOPOU LONTCHI Josine（思妮）；

【导师】 季静；

【作者基本信息】 天津大学 ， 生物分子工程， 2012， 博士

【摘要】 月季(Rosa chinensis Jacq.),是中国最主要的花卉之一,已在中国栽培了上千年,于1793年传入西欧。它是一种城市花卉,主要种植于北京等其他37个城市。同时,月季花种植受土壤盐碱条件的影响,这是对其生长和产量最有害的因素之一。盐胁迫在世界范围内制约着农作物的生长和产量,已成为中国灌溉地区主要和日趋严重的问题之一,影响着7%的土地面积和5%的耕地。虽然植物盐胁迫调控的研究已经取得了成果,但月季仍然没有取得进展。改进作物的耐盐性仍需要获得新的方法(无论是通过自然繁育还是转基因手段)和鉴定耐盐性的高效技术。本研究为了提高月季的耐盐性,我们首先在NaCl压力培养基上通过组织培养再生技术进行自然繁殖。其次,脱水应答元件结合因子(DREB)主要与植物对非生物压力如寒冷、干旱和高盐的反应有关。此外,月季花大部分DREB基因的功能和调控这些反应的潜在机制依然需要进一步研究。所以,我们先以拟南芥作为模式植物对AtDREB2A-CA基因(拟南芥转录因子DREB2A脱水应答顺式作用元件相互作用序列)进行了功能分析。最后,在月季花中表达了AtDREB2A-CA基因。在适宜的压力选择培养基上胚胎发育之后,对NaCl压力的缓解使小植株有了更好的适应性和易恢复性。与盐胁迫植株(SSIV)(434.75±0.44ug/g)相比,盐胁迫恢复植株(RIV)中脯氨酸含量(371.42±0.88ug/g)减少,但此值仍然显著高于对照组(368.79±0.58 ug/g)。盐胁迫恢复植株含水量(69.24±0.65%)也显著高于对照组(54.67±1.31%)。经过温室栽培再转移至试验田中的选育植株在生理和超微结构上表现出对NaCl压力的适应性。表达AtDREB2A-CA基因的转基因拟南芥增加了水、脯氨酸、N、P和K含量,通过激活它们的合成提供应答和阻止植物免受NaCl伤害。而且,AtDREB2A-CA基因降低了叶绿素和总类胡萝卜素含量,这并没有影响盐胁迫条件下24小时之后的植物生长。本研究所观察到的转基因植株叶片上表皮的形态学改变仅发生在胁迫的初始阶段,并且栅栏薄壁组织体积的增加诱导了对NaCl胁迫的抗性。AtDREB2A-CA基因诱导了转基因植株无NaCl时类囊体膨胀和NaCl胁迫下轻微的类囊体聚积。同时,也发现对了淀粉粒合成的抑制作用。在NaCl压力条件下,当质体小球持续增加时白色体增加并最终保持恒定。这表明,NaCl胁迫能引起野生型植株叶绿体的损伤。有趣的是,AtDREB2A-CA基因表达引起的(这些)变化引起了植物叶片内膜对NaCl胁迫的耐受性。此外,一定浓度的H2O2(特别是0.3, 1.2 and 1.5 mMol/l)能够激活种子萌发和根的伸长,而LiCl只能激活种子萌发。本研究提出了一种由农杆菌介导的侵染月季芽点的新方法。利用半芽点作为外植体进行转基因是其中最有效的方法,最佳条件为侵染时间6min,AS浓度100μM。转AtDREB2A-CA基因月季植株积累了较高的含水量(91.56±3.15%),脯氨酸含量(555.11±7.8μg/g),以及N(2.93±0.09%)、P (2.59±0.26%)、K (1.44±0.21%)含量,较低的叶绿素a、b(0.50±0.01 (X=m/M)和总类胡萝卜素(0.21±0.01 (X=m/M)含量,这些在野生型(植株)中的含量分别为78.87±1.25%; 350.76±13.93μg/g; 0.83±0.01%; 0.633±0.03%; 1.11±0.14%; 0.88±0.09 (X=m/M); 0.50±0.05 (X=m/M)。拟南芥研究结果表明,这种生理活性可用来参照其对NaCl的耐受力。蛋白质印迹分析表明AtDREB2A-CA介导了Cu/Zn-SOD蛋白的活性,并且高浓度NaCl条件下(200和300mM),Cu/Zn-SOD蛋白调节机制受AtDREB2A-CA激活。叶片上表皮超微结构分析表明,在野生型植株中叶片分泌细胞膨胀更为明显,转基因植株细胞间隙变小。我们还发现除了只有当NaCl浓度达200mM的时候,外褶皱的合成会受抑制之外,AtDREB2A-CA基因的表达也抑制压力和非压力性转基因植株中叶片角质层的合成。在NaCl浓度为200和300 mM条件下,转基因植株叶绿体呈纺锤形,并且显示出类囊体排列成特定的基粒,褶皱和小型的淀粉粒清晰可见。我们首先可以推断,转基因繁殖的小植株具有水分保持的能力。脯氨酸的合成和可用性可以解释它在新环境中的调节机制。生理和超微结构的适应性说明,可能在养分获取和利用过程中存在一种遗传变异性,在植物育种过程中,利用养分将是一种可行的技术手段。其次,在模式植物拟南芥中AtDREB2-CA激活与盐胁迫耐受性相关下游基因,表明在遗传改良培育压力耐受性植物中,它可能是一个良好的候选基因。本研究提出的一种新的农杆菌介导的月季侵染方法,可以用于盐压力环境中大规模快速繁殖盐胁迫耐受性植物。在月季中AtDREB2-CA基因的表达调节了植物在适应NaCl压力时的生理活性。此外,该基因与月季通过激活Zn/Cu-SOD蛋白表达的盐胁迫应答途径有关。表达该基因的月季植株所提供的叶片细胞超微结构的构造(淀粉粒的缺失或微小化和角质层的薄化)与NaCl压力耐受性植物的特征相似。最后,表达AtDREB2-CA基因并具有NaCl压力耐受性的月季植株已被移栽至温室中正常生长三个月。更多还原

【Abstract】 Rosa chinensis Jacq., commonly called Chinese rose has been one of the most grown in China for thousands of years and reached Western Europe in 1793. It is a city flower principally cultivated in Beijing as well as other 37 cities. Meanwhile, China rose is highly influenced by soil saline conditions, one of the most harmful factors concerning its growth and productivity. In China, 7% of the land’s surface and 5% of cultivated lands are affected by salinity stress being one of the major and increasing problems in irrigated land, limiting crop plants development and productivity worldwide. Although studies in plants salinity stress control have brought successful results, Chinese rose is still without positive salt stress response. Improving the salt tolerance of crop and pasture species requires access to new diversity (either natural breeding or transgenic) and efficient techniques for identifying salt-tolerance.In our research to improved Chinese rose salt tolerance, we first naturally bred by tissue culture and regenaration in NaCl stress culture medium. Secondly, the Dehydration Response Element Binding factors is known to be involved mainly in plant responses to abiotic stresses such as cold, drought or high salinity. Moreover, the function of most DREB genes in Rosa chinensis Jacq. and the underlying mechanisms controlling these responses remains elusive. We therefore functionally analysed an AtDREB2A-CA gene (Arabidopsis thaliana transcription factor DREB2A interacting with a cis-acting dehydration-responsive element (DRE) sequence) in A.thaliana as a model plant. Thirdly, the AtDREB2A-CA gene was then applied by expressing to Chinese rose.The alleviation of NaCl stress after embryogenesis on convenient stress media could provide better adaptation and recovery to plantlets. Proline content was reduced in Recovered In Vitro (RIV) (371.42±0.88ug/g) compare to Salt Stress In Vitro (SSIV) (434.75±0.44ug/g), but this value still remained significantly higher compare to control (368.79±0.58 ug/g). Water retention was significantly higher in RIV (69.24±0.65%) plantlets compare to control (54.67±1.31%). This bred veriaties after cultivation in greenhouse and transplantation to our experimental garden presented physiological and ultrastructural adaptation to NaCl stress.Transgenic arabidopsis expressing AtDREB2A-CA increased water, proline, N, P and K content by activating their synthesis and providing potentialities to response and prevent plant from NaCl damage. Furthermore, AtDREB2A-CA decreased chlorophyll and total carotenoids content and this does not defect plant growth after 24h in salinity stress conditions. Morphological changes were observed in the leaf upper epidermis of transgenic plants only at the initial step of the stress and the increase in the volume of the palissade parenchyma induced resistance during NaCl stress. AtDREB2A-CA gene induced swollen thylacoids in NaCl-free and slightly stacked thylacoids in NaCl stress in the chloroplast of transgenic plant. In the meantime, an inhibition of starch grains synthesis was observed. Also, etioplasts increased and remained constant while plastoglobules remained incremental when submitted to NaCl stress. These induced that NaCl stress induce damage in the chloroplast of WT plant. Interrestingly, AtDREB2A-CA expression induced changes that caused plants leaf inner membrane NaCl stress tolerant. Furthermore, H2O2 could activate at a certain concentration (especially 0.3, 1.2 and 1.5 mMol/l) seeds germination and roots elongation, while LiCl could only activate seeds germination along the treatment in an AtDREB2A-CA dependent manner.A novel Agrobacterium tumefaciens mediated transformation method was described using the BBPs (Branch Burgeon Points) of Rosa chinensis Jacq. This method was the most efficient when choosing Half-BBPs as explants for a timely productive transgenic with 6 min infection time and 100μM AS concentration. AtDREB2A-CA transgenic Rosa chinensis Jacq. plant accumulated higher water (91.56±3.15%), proline (555.11±7.8μg/g), N (2.93±0.09%), P (2.59±0.26%) and K (1.44±0.21%), then lower Chla+b (0.50±0.01 (X=m/M)) and total carotenoids (0.21±0.01 (X=m/M)) compare to WT respectively of 78.87±1.25%; 350.76±13.93μg/g; 0.83±0.01%; 0.633±0.03%; 1.11±0.14%; 0.88±0.09 (X=m/M); 0.50±0.05 (X=m/M). Conclusions drawn from studies on A. thaliana indicated that, this physiological activity conferred NaCl stress tolerance ability. Western blot analysis demonstrated that AtDREB2A-CA mediated the activation of Cu/Zn-SOD protein. Moreover, at high NaCl concentration (200 and 300mM), the Cu/Zn-SOD regulation mechanism is activated by AtDREB2A-CA. Furthermore, Leaf upper epidermis ultrastructural analysis revealed that the secretory cells were swelling and more accentuated in wild type plants. Also, intercellular spaces were reduced in transgenic plants. It was also found that AtDREB2A-CA expression inhibit the leaf cuticule synthesis in stressed and unstressed transgenic plants, except that the synthesis of the external lamellae was inhibited only when NaCl reached 200mM. Chloroplasts of transgenic plants were fusiform and showed thylacoids arranged into defined grana, lamella and little size starch grains visible at 200 and 300 mM NaCl.At first, it could be concluded that, bred plantlet had the ability of water retention. Proline synthesis and availability could unravel the regulation of metabolism in its new environment. Physiological and ultrastructural adaptation emerged a possibility that, there might be genetic variability for the processes of nutrient acquisition and utilization, and that nutritional efficiency is a viable technique in plant breeding programs. Secondly, AtDREB2-CA activates expression of downstream genes involved in salt-stress tolerance in the model plant Arabidopsis thaliana, indicating that it might be a good candidate gene for genetic improvement to produce stress-tolerant plants. The novel Agrobacterium mediated transformation of Rosa chinensis jacq. described here can be used for rapid production in large scale of salinity stress tolerant plants for application in salt stress environments. The expression of AtDREB2A-CA in Rosa chinensis Jacq. regulates plant physiological activity in response to NaCl stress. Furthermore, AtDREB2A-CA is involved in Chinese rose NaCl stress response pathways by activating Zn/Cu-SOD protein expression. The expression of AtDREB2A-CA gene in Chinese rose plants provide the formation of the leaf cell ultrastructure (absence or small sized of the starch grains and the thiness of the cuticle) similar to a characteristic of NaCl-stress-tolerant plants. Finally, Rosa chinensis Jacq. expressing AtDREB2A-CA and NaCl stress tolerant was transplanted and grown normally in greenhouse for already three months.更多还原