异源表达irrE转基因烟草的耐盐耐旱性

发布时间：2018-06-02 02:08

本文选题：密码子优化 + irrE　；参考：《中国农业科学》2017年20期

【摘要】：【目的】IrrE是从耐辐射异常球菌中发现的全局调控蛋白,主要通过修复强辐射等逆境条件下DNA损伤,提高耐辐射异常球菌对极端逆境环境的抗性。研究按植物密码子优化的irrE后导入烟草对转基因烟草耐逆能力的提高,为棉花等作物耐逆育种研究打下基础。【方法】按照植物密码子优化细菌irrE并合成基因;通过酶切连接法构建irrE植物表达载体;通过叶盘法转化烟草并PCR验证获得阳性转基因再生苗;通过实时荧光定量PCR(q RT-PCR)分析转基因株系中irrE的表达量;通过蛋白免疫印迹法(Western blot)检测IrrE编码蛋白;通过NaCl和甘露醇模拟盐处理和干旱处理分析纯和转基因株系的耐盐耐旱性,通过测定抗逆相关生理指标鉴定其对植物耐逆的贡献。【结果】按照植物密码子对irrE进行改造,共优化了241个密码子;构建了高效植物表达载体GBI-IE;利用除草剂草甘膦作为筛选剂获得转基因再生幼苗,并通过PCR验证共获得15个独立的转基因株系;通过q RT-PCR分析从中选取两个表达量最高的株系GO1和GO2进行后续的抗逆性分析;Western blot验证IrrE编码蛋白在GO1和GO2中能正确翻译。转基因烟草耐盐耐旱性分析:种子萌发试验表明,正常1/2 MS培养基上,转基因株系GO1和GO2发芽率和非转基因野生型对照之间没有明显的差异。然而250 mmol·L~(-1)NaCl培养基上GO1和GO2萌发率分别为78.8%和90.0%,野生型仅为10.3%,分别提高了68.5%和79.7%。类似地,300 mmol·L~(-1)甘露醇条件下,野生型的萌发率为39.7%,转基因株系GO1和GO2分别提高了42.9%和50.8%;正常萌发的种子移栽到250 mmol·L~(-1) NaCl和300 mmol·L~(-1)甘露醇条件12 d后,转基因株系根长、侧根数以及鲜重等生理指标显著高于野生型对照;温室中正常生长30 d的苗期烟草在250 mmol·L~(-1) NaCl处理下,转基因烟草SOD、CAT活性比野生型对照分别提高了48.80%和88.55%,而MDA含量比野生型对照降低了61.61%,胁迫响应基因Nt ABF2、Nt LEA5、Ntzfp、Nt CDPK2在GO1和GO2转基因株系中表达量均显著高于非转基因野生型。和盐处理结果类似,300 mmol·L~(-1)甘露醇的处理下,转基因烟草的耐旱生理生化指标均优于非转基因对照。【结论】烟草中异源表达耐辐射异常球菌irrE可以显著提高耐盐耐旱性;其多效性耐非生物胁迫能力的提高表明其可作为植物耐逆基因工程的优良基因源。
[Abstract]:[objective] IrrE is a global regulatory protein found from the actinomycetes radiodurans. It can improve the resistance to extreme stress environment by repairing DNA damage in severe radiation stress conditions. To study the improvement of stress tolerance of transgenic tobacco introduced into tobacco by irrE optimized by plant codon. [methods] to optimize bacterial irrE and synthesize genes according to plant codon, which will lay a foundation for the research of stress tolerance breeding in cotton and other crops. The irrE plant expression vector was constructed by restriction endonuclease ligation method, the positive transgenic regenerated seedlings were obtained by leaf disk method and PCR verification, and the expression of irrE in transgenic lines was analyzed by real-time fluorescence quantitative PCR(q RT-PCR. IrrE coding protein was detected by Western blotting, salt tolerance of pure and transgenic lines was analyzed by NaCl and mannitol simulated salt treatment and drought treatment. The contribution to plant stress tolerance was identified by determining the physiological indexes related to stress resistance. [results] 241 codon were optimized by modifying irrE according to plant codon. The high efficient plant expression vector GBI-IEwas constructed, and 15 independent transgenic lines were obtained by PCR verification by using herbicide glyphosate as screening agent. Two of the most expressed lines, GO1 and GO2, were selected by Q RT-PCR analysis for further analysis of resistance to stress. Western blot was used to verify the correct translation of IrrE encoded proteins in GO1 and GO2. Analysis of salt tolerance of transgenic tobacco: seed germination test showed that there was no significant difference between GO1 and GO2 germination rate of transgenic lines on 1 / 2 MS medium and non-transgenic wild-type control. However, the germination rates of GO1 and GO2 on 250 mmol L~(-1)NaCl medium were 78.8% and 90.0% respectively, and the wild type was only 10.3, which increased 68.5% and 79.7%, respectively. Similarly, under the condition of mannitol, the germination rate of wild type was 39.7 and the GO1 and GO2 of transgenic lines increased by 42.9% and 50.8%, respectively, and the root length of transgenic lines was increased after transplanting the normally germinated seeds to 250 mmol Lng-1) NaCl and 300 mmol Lny-1) mannitol for 12 days. The physiological indexes of lateral root number and fresh weight were significantly higher than that of wild-type control, and the seedlings of tobacco grown normally for 30 days in greenhouse were treated with 250 mmol / L ~ (-1) NaCl. The activity of cat in transgenic tobacco was increased by 48.80% and 88.55% than that of wild type control, while the content of MDA was decreased by 61.61. The expression of stress response gene NtABF2NtLEA5NtLEA5Nt CDPK2 in GO1 and GO2 transgenic lines was significantly higher than that in non-transgenic wild-type lines. The physiological and biochemical indexes of drought tolerance of transgenic tobacco were superior to those of non-transgenic control, and the results of salt treatment were similar to those of C300 mmol / L ~ (-1) mannitol. [conclusion] the heterologous expression of irrE in tobacco could significantly improve the tolerance to salt drought. The improvement of its ability of tolerance to abiotic stress indicates that it can be used as a good gene source for plant stress tolerance gene engineering.
【作者单位】：中国农业科学院生物技术研究所;
【基金】：国家转基因生物新品种培育科技重大专项(2016ZX08005-004) 国家“863”计划(2013AA102601-2)
【分类号】：Q943.2

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