转蜡样芽孢杆菌acdS基因改善烟草耐盐性研究

发布时间：2018-05-05 02:14

本文选题：烟草 + acdS基因　；参考：《郑州大学》2017年硕士论文

【摘要】：烟草是我国重要的经济作物,土地盐碱化引起的土壤板结、利用率低等问题一直制约着烟草在盐碱地上的种植。植物受逆境胁迫时,乙稀含量升高且过量的乙稀会抑制植物生长并对植物造成损害。ACC氧化酶(ACO)能氧化ACC(1-aminocyclopropane-1-carboxylate)从而生成乙稀,而acdS基因编码的ACC脱氨酶(ACD)能通过水解ACC,降低植物体内乙稀的合成,有效缓解高浓度乙稀对植物生长的抑制作用,促进植物生长。实验室前期研究结果显示海滨锦葵内生细菌蜡样芽孢杆菌HK012(Bacillus cereus HK012),具有较高ACC脱氨酶活性,接种于小麦等作物根系后,能显著增强这些植物在高盐胁迫下的抗性。基于上述研究结果,本文采用叶盘法将蜡样芽孢杆菌HK012的acdS基因转入烟草,获得基因工程耐盐烟草新品种,为提高烟草对盐碱地的利用率提供理论基础和技术支持。论文从蜡样芽孢杆菌HK012中克隆acdS基因,利用Gateway技术构建植物pMDC 45-acdS(2×35S-GFP-acdS-Nos)表达载体,并通过叶盘法将其转入烟草中,根据重组质粒携带的筛选标记基因Hyg,用潮霉素浓度为15 mg/L的愈伤培养基(MS+1 mg/L 6-BA+0.1 mg/L NAA+500 mg/L Cefotaxime)进行烟草不定芽的筛选,潮霉素浓度为10 mg/L的生根培养基(MS+250 mg/L Cefotaxime)进一步筛选转基因苗,获得转基因株。通过DNA水平、mRAN水平以及蛋白质表达水平(Western blot)检验,表明acdS融合基因在烟草中获得稳定表达,荧光显微镜观测显示融合蛋白定位于烟草根系的细胞膜(或细胞壁)上。对转基因烟草T2代幼苗进行耐盐性检测,实验结果表明转基因烟草株高、根长、干重、鲜重、叶绿素含量均大于未转化烟草。脯氨酸含量测定结果显示,150 mM NaCl、300 mM NaCl浓度下转基因烟草叶片中脯氨酸含量相对于未转化烟草分别提高55.15%、42.70%。保护酶(SOD、POD、CAT)活性测定结果显示,当NaCl浓度为150 mM时,转基因烟草SOD、POD、CAT活性相对于未转化烟草分别提高28.87%、21.57%、21.84%;当NaCl浓度为300 mM时,转基因烟草SOD、POD、CAT活性相对于未转化烟草分别提高31.83%、32.24%、26.30%。ACC脱氨酶活性测定结果显示,转基因烟草ACC脱氨酶活性均大于未转化烟草,在150 mM NaCl、300 mM NaCl浓度下转基因烟草叶片中ACC脱氨酶活性相对于未转化烟草分别提高15.3%、58.1%。综合以上实验结果表明蜡样芽孢杆菌acdS基因成功转入烟草并获得稳定表达,从而提高了烟草在高盐胁迫下的抗性。另一方面表明,acdS基因在植物耐盐基因工程中具有一定应用价值,通过基因工程方法开发新基因改良烟草品种有望提高烟草对盐碱土地的利用率。
[Abstract]:Tobacco is an important cash crop in China. The problems of soil consolidation caused by soil salinization and low utilization rate have been restricting the cultivation of tobacco on saline soil. When plants are stressed by stress, ethylene content increases and excess ethylene inhibits plant growth and causes damage to plants. ACC oxidase ACO) can oxidize ACC-1-aminocyclopropane-1-carboxylate) to produce ethylene. ACC deaminase (ACD) encoded by acdS gene can reduce the synthesis of ethylene in plants by hydrolyzing ACCs, alleviate the inhibitory effect of high concentration ethylenes on plant growth and promote plant growth. The experimental results showed that the endophytic Bacillus cereus HK012(Bacillus cereus HK012 had high ACC deaminase activity. Inoculation with wheat roots could significantly enhance the resistance of these plants under high salt stress. Based on the above results, the acdS gene of Bacillus cereus HK012 was transferred to tobacco by leaf disk method, and a new genetic engineering salt-tolerant tobacco variety was obtained, which provided the theoretical basis and technical support for improving the utilization rate of tobacco to saline-alkali soil. In this paper, acdS gene was cloned from Bacillus cereus HK012, and the expression vector of plant pMDC 45-acdS(2 脳 35S-GFP-acdS-Nosin was constructed by Gateway technique and transferred into tobacco by leaf disk method. According to the screening marker gene Hyg carried by the recombinant plasmid, the adventitious buds of tobacco were screened on the callus medium MS 1 mg/L 6-BA 0.1 mg/L NAA 500 mg/L Cefotaxime with hygromycin concentration of 15 mg/L. The transgenic seedlings were further screened on the rooting medium MS 250 mg/L Cefotaxime with the concentration of hygromycin for 10 mg/L, and the transgenic plants were obtained. AcdS fusion gene was stably expressed in tobacco by DNA level and protein expression level. Fluorescence microscopy showed that the fusion protein was located on the cell membrane (or cell wall) of tobacco root system. The salt tolerance of transgenic tobacco seedlings of generation T2 was tested. The results showed that the height, root length, dry weight, fresh weight and chlorophyll content of transgenic tobacco were higher than those of untransformed tobacco. The results of proline content measurement showed that the proline content in transgenic tobacco leaves increased 55.15% and 42.70%, respectively, compared with untransformed tobacco at the concentration of 150mm NaCl-300mm NaCl. When the concentration of NaCl was 150 mm, the cat activity of transgenic tobacco was increased by 28.87% and 21.57 mm than that of untransformed tobacco, respectively, and when the concentration of NaCl was 300mm, the activity of cat in transgenic tobacco was increased by 28.87 mm and 21.57 mm, respectively, when the concentration of NaCl was 300mm. Compared with untransformed tobacco, the activity of cat in transgenic tobacco was increased by 31.833,32.24 and 26.30.ACC deaminase activity. The results showed that the activity of ACC deaminase in transgenic tobacco was higher than that in untransformed tobacco. The activity of ACC deaminase in transgenic tobacco leaves was increased by 15.3% compared with that of untransformed tobacco at the concentration of 150mm NaCl-300mm NaCl, respectively. The results showed that the acdS gene of Bacillus cereus was successfully transformed into tobacco and expressed stably, which improved the resistance of tobacco under high salt stress. On the other hand, it is suggested that the acdS gene has certain application value in plant salt tolerance gene engineering, and it is expected to improve the utilization rate of saline-alkali land by developing new genetic modified tobacco varieties by genetic engineering method.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S572

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