【作者】 包永霞；

【导师】 韩烈保；

【作者基本信息】 北京林业大学 ， 草业科学， 2012， 博士

【摘要】 多年生黑麦草(Lolium perenne L.)是我国南北方广泛应用且适应性很强的草坪草种,成坪速度快,抗病虫和分蘖能力强,常作为建植草坪的重要草种。但是,多年生黑麦草不能忍受冷、热、干旱这些气候的极端值。为了改良多年生黑麦草的抗旱遗传性,实验室建立了多年生黑麦草高频再生体系,应用基因枪转化法和农杆菌介导法将DREB1A和BADH-CMO基因分别导入胚性愈伤组织,获得了一批抗性植株。在后续研究中发现,转DREB1A基因的多年生黑麦草和转BADH-CMO基因的多年生黑麦草的抗旱性和耐盐性明显提高的同时出现了明显的矮化现象,这对于草坪草来说是非常有意义的现象。本研究通过研究转基因草坪草的矮化机理和抗逆性,为最终培育抗性增强且矮化的优良草坪草新品种提供理论依据。从营养生长指标、生殖生长指标、日增生长高度、光合效率、细胞观察等方面研究了矮化表型；从赤霉素合成途径和信号传导途径为切入点,研究了转基因植物矮化的机理；分别测定高温、干旱、盐胁迫下的转基因植株的日增生长高度研究转基因植株的抗逆性,并测定了子代苗期的抗盐性以及对干旱+盐双重胁迫的抗性,研究了转基因植株子代苗期的抗逆性；最后主要从环境释放和生产性试验的角度,对转基因多年生黑麦草进行了安全性评价。得到以下研究结果：1.转DREB1A基因的多年生黑麦草(DREB)和转BADH-CMO基因的多年生黑麦草(BC)出现了不同程度的矮化现象。从细胞水平上观察转基因植株和非转化植株的叶片纵切细胞后发现,转基因的叶片细胞明显短于非转化植株。转基因植株的抽穗率也明显降低,尤其BC表现出抽穗率非常低,穗短,穗数少等现象。BC的种子发芽率只有80%,DREB与CK的种子发芽率均能达到100%；BC种子简化活力指数呈最低。光合特性的研究结果表明,只有净光合速率在转基因植株和非转化之间有显著差异,其他各指标均未达显著差异。GA的含量测定结果表明活性GA1和GA4的含量明显降低,成为可能引起转基因多年生黑麦草矮化的因素。喷施外源赤霉素后矮化表型可以恢复至对照的正常表型,细胞大小和日增生长高度对外源赤霉素均有积极响应,表明转基因植株体内的赤霉素受体可以正常发挥功能,排除了赤霉素不敏感型这一可能性。转DREB1A基因的多年生黑麦草GA20ox基因的相对表达量比非转化植株的GA20ox基因的相对表达量低,存在显著差异(P<0.05)。然而,GA20ox基因在转BADH-CMO基因的多年生黑麦草中过量表达,比非转化植株的GA20ox基因的相对表达量高,存在极显著差异(P<0.01)。2.转DREB1A基因多年生黑麦草对高温有一定的抗性；转BADH-CMO基因多年生黑麦草的生长受到高温的严重抑制,BADH-CMO基因并没有提高多年生黑麦草的抗高温能力。DREB1A基因和BADH-CMO基因均提高了多年生黑麦草的抗干旱能力。转基因植株与对照植株相比,具有较强的抗旱性。三种供试材料对0.5%盐胁迫的抗性从大到小的排序是BC>DREB>CK;对1%和1.5%盐胁迫的抗性排序均是DREB>BC> CK。在喷施赤霉素之后,转基因植株的抗旱性受抑制程度较为严重,抗旱性明显减弱。在恢复浇水后,外源赤霉素对转基因植株抗旱性的影响减弱。3.转基因多年生黑麦草T1代种子相对发芽势、相对发芽率均随着盐浓度的不断增加而下降,盐胁迫对对照组的胚芽长有严重抑制作用；转基因组和对照组的胚根长在各浓度盐胁迫下无明显差异和变化规律。随盐浓度递增,叶绿素含量呈降低趋势。转基因组在高浓度盐胁迫下仍具有较高含量的叶绿素,呈良好抗性。转基因组在盐胁迫下膜的结构相对稳定,透性变化较小,受到伤害较轻,因而抗盐性比对照组强。转基因组对NaCl、PEG单独胁迫和NaCl+PEG双重胁迫均具有一定程度抗性,其中对单独胁迫的抗性强于双重胁迫的抗性。胚根长在三种胁迫下的响应规律不明显,所以不适宜作转基因抗性植株筛选指标。转基因多年生黑麦草T1代同样出现矮化表型,所以亲本的矮化性状稳定遗传到子代。4.经环境释放和生产性试验研究发现,转基因植株出现矮化、叶片变窄、穗长变短等表型；对干旱和盐胁迫的抗性明显增强；矮化表型和抗逆性在T1代中能够稳定遗传；对土壤中微生物数量有减少的作用。更多还原

【Abstract】 Perennial Ryegrass(Lolium perenne L.) is a widely planted cool-season turfgrass for utility turf, home lawns, sport fields, and golf fairways. Based on the construction of high frequency regeneration system of Perennial Ryegrass, DREBIA and BADH-CMO gene were transformed into embryogenic calli via biolostic bombardment by Xue Li. The transcription factor dehydration-responsive element binding proteins (DREB1A)/C repeat (CRT) binding factors (CBF) play an important role in regulating gene expression and growth involved in drought and salt stress tolerance. Choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) catalyze the first and second steps in the biosynthesis of glycine betaine in betaine-accumulating plants. Not only enhanced drought and salt stress tolerance but also significant dwarfism had been observed from DREBIA and BADH-CMO transgenic Lolium perenne plants. The dwarf phenotype, dwarf mechanism and stress tolerance of transgenic Perennial Ryegrass was dicussed in this paper. The future vision of turgrass breeding and safety evaluation was also discussed. Conclusions of this reseach showed as followings:1. Height of transgenic plants was decreased obviously compared with the control. Measurement of endogenous GA content demonstrated that both GA1 and GA4 decreased in BADH-CMO transgenic plants and decreased GA1 content was observed in DREB1A/CBF3 transgenic plants, respectively. Dwarf trait of transgenic plants can be recovered by application of exogenous GAs. The result of relative expression level of GA20ox indicates that, relative expression level of GA20ox was decreased in transgenic plants with DREBIA gene and increased in transgenic plants with BADH-CMO gene, compared to control. The further study about function of GA20ox in mechanism of dwarfism was wanted. These results demonstrated that the DREB1A/CBF3 gene and BADH-CMO gene enhanced drought and salt tolerance and induced dwarfism in transgenic Lolium perenne. Dwarfism induced by DREB1A/CBF3 and BADH-CMO was associated with synthesis of endogenous GAs and it could be recovered by application of exogenous GAs.2. Enhanced drought, salt and high temperature stress tolerance had been observed in transgenic plants.In order to further understand the influence of exogenous GA on drought tolerance of transgenic plants with DREB1A/CBF3 and BADH-CMO gene, the research is focused on the analysis of the physiological properties of transgenic plants after drought and restoration treatment. The result of chlorophyll content、relative conductivity、relative water content、water content and sugar content showed that, drought tolerance of all materials was decreased by exogenous GA and the difference between transgenic plants and non-transgenic ones was significant. It suggested that exogenous GA had significant influence on drought stress tolerance of transgenic plants. The further study regarding on the mechanism of this phenomenon is wanted.3. The salt tolerance of transgenic plant seedling with DREB1A gene under different NaCl treatments was identified by determining the physiological indices. The results showed that the seed relative germination potential、relative germination rate、seedling height、chlorophyll Content、Water Content and RWC decreased with NaCl concentration increasing, and the difference was significant between transgenic group and control group. The rule of root length was not obvious. The contents of relative conductivity increased with the increasing of salt concentration and the difference between transgenic plants and CK was significant. This suggested that transgenic group was salt-tolerant. In order to study the double stress tolerance of T1 generation of transgenic plants with DREB1A gene, the research is focused on the analysis of the physiological properties of transgenic plants after PEG、NaCl and PEG+ NaCl treatment at germination stage, with the control group of water treatment. The result of relative germination potential、relative germination rate、seedling height、relative conductivity、wilting degree and chlorophyll content showed that, all materials with DREB1A gene showed the highest tolerance under NaCl stress. The result of relative water content and survival range indicated that materials with DREB1A gene had the highest tolerance under PEG stress. All materials with DREB1A gene had significant tolerance under PEG+ NaCl stress and the difference between transgenic plants and CK was significant. This suggested that T1 generation of transgenic Perennial Ryegrass was stress-tolerant and could be extended in drought and salinity soil to improve drought and salinity land.4. As to safety evaluation, transgenic Perennial Ryegrass with DREB1A/CBF3 and BADH-CMO gene was safe to environment.更多还原