中国农业科学 ›› 2017, Vol. 50 ›› Issue (20): 3860-3870.doi: 10.3864/j.issn.0578-1752.2017.20.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

异源表达irrE转基因烟草的耐盐耐旱性

赵雷霖,范鑫,聂星,梁成真,张锐,孙国清,孟志刚,林敏,王远,郭三堆   

  1. 中国农业科学院生物技术研究所,北京 100081
  • 收稿日期:2017-03-30 出版日期:2017-10-16 发布日期:2017-10-16
  • 通讯作者: 王远,E-mail:wangyuan07@caas.cn。郭三堆,E-mail:guosandui@caas.cn
  • 作者简介:赵雷霖,Tel:010-82106129;E-mail:happy_lin92@163.com。
  • 基金资助:
    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)

Salt and Drought Tolerance in Heterologous-Expression of irrE Transgenic Tobacco

ZHAO LeiLin, FAN Xin, NIE Xing, LIANG ChengZhen, ZHANG Rui, SUN GuoQing, MENG ZhiGang, LIN Min, WANG Yuan, GUO SanDui   

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2017-03-30 Online:2017-10-16 Published:2017-10-16

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(qRT-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qRT-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NtABF2、NtLEA5、Ntzfp、NtCDPK2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。

关键词: 密码子优化;irrE;转基因烟草;耐旱耐盐 ,

Abstract: 【Objective】 IrrE is a protein found in Deinococcus radiodurans, and it process the transcription regulating function mainly through repairing DNA damage in conditions of strong radiations and other adverse environments. It is of great significance to study the tolerance of optimized irrE against drought and salt in transgenic plants and to lay a theoretical basis of crops resistance breeding.【Method】The bacteria irrE codons were optimized with the plant preferences and the gene was synthesized. Efficient plant expression vector was constructed by using the method of enzyme digestion and connection. Leaf disc method was used for transforming tobacco, and the positive regenerated seedlings were screened with PCR. The expression level of irrE in transgenic strain was analyzed. The IrrE protein was tested using Western blot. Pure transgenic generations were recruited and treated with the mannitol and NaCl treatments, which simulated drought and salt conditions. Physiological tests were conducted to determine drought and salt tolerance.【Result】The irrE was modified according to plant codes, and 241 codes were optimized. The efficient plant expression vector GBI-IE was constructed. The regenerated seedlings were screened by herbicide glyphosate, and 15 independent transgenic strains were obtained using PCR, and two highest expression lines, GO1 and GO2, were used for subsequent analysis of tolerance under adverse environmental conditions. Western blot proved that protein IrrE encoded in GO1 and GO2 could be translated correctly. Salt and drought tolerance of transgenic tobacco was analyzed. Germinating experiments displayed that there was no obvious difference between transgenic lines (GO1 and GO2) and non-GMO wild type (WT) in terms of germination rate in 1/2 MS culture medium, however, in medium containing 250 mmol·L-1 NaCl, germination rate of GO1 and GO2 was 78.8% and 90.0%, respectively, while the one of the WT was 10.3%, increased by 68.5% and 79.7%, respectively. Similarly, germination rate of WT was 39.7% in medium containing 300 mmol·L-1 mannitol, the rate of GO1 and GO2 increased by 42.9% and 50.8%, respectively. Normally germinated seedlings were transplanted into 250 mmol·L-1 NaCl and 300 mmol·L-1 mannitol environment for 12 days, and root length, lateral roots number and fresh weight of those two transgenic lines were markedly higher than the ones of WT. Tobacco seedlings of 30 days normal growth in the greenhouse were treated with 250 mmol·L-1 NaCl of which transgenic tobacco SOD and CAT activity was more 48.80% and 88.55% than WT, respectively, and the MDA content was less 61.61% than WT, In addition, the expression level of drought responsive gene in GO1 and GO2, like NtABF2, NtLEA5, Ntzfp, NtCDPK2, was significantly higher than WT. Similarly, the changes of physiological and biochemical indexes in transgenetic lines in 300 mmol·L-1 mannitol treatment were higher than the ones in WT.【Conclusion】Expression of irrE from D. radiodurans remarkably improved the tolerance of tobacco to drought and salt stresses in which the pleiotropy could be beneficial gene resource of gene engineering about resistance to stress in plant.

Key words: codon optimization; irrE, transgenic tobacco, drought and salt tolerance