Chloroplast-targeted expression of the codon-optimized truncated cry1Ah gene in transgenic tobacco confers a high level of protection against insects

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• 作者：Xiuying Li (1) (2)
  Shengyan Li (2) (4)
  Zhihong Lang (2)
  Jie Zhang (3)
  Li Zhu (2)
  Dafang Huang (1) (2)
  
• 关键词：Truncated cry1Ah gene ; Codon optimization ; Chloroplast targeting ; Transgenic tobacco ; Insect resistance
• 刊名：Plant Cell Reports
• 出版年：2013
• 出版时间：August 2013
• 年：2013
• 卷：32
• 期：8
• 页码：1299-1308
• 全文大小：1203KB
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• 作者单位：Xiuying Li (1) (2)
  Shengyan Li (2) (4)
  Zhihong Lang (2)
  Jie Zhang (3)
  Li Zhu (2)
  Dafang Huang (1) (2)
  
  1. College of Life Science, Northeast Agricultural University, 150030, Harbin, China
  2. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China
  4. College of Biological Sciences, China Agricultural University, 100193, Beijing, China
  3. Plant Protection Institute, Chinese Academy of Agricultural Sciences, 100193, Beijing, China
  

文摘

Key message The study of insect-resistant transgenic tobacco provides a good foundation for the further application of the cry1Ah gene in other important crops. Abstract To improve transgene expression levels and insect resistance, the coding sequence of the novel Bacillus thuringiensis insecticidal gene cry1Ah (truncated cry1Ah) was modified according to the codon bias of the plant by increasing its GC content from the original 37?% to 48, 55, and 63?% (designated m1-cry1Ah, m2-cry1Ah, and m3-cry1Ah, respectively). In addition, the m3-cry1Ah gene was linked with a transit peptide sequence for chloroplast-targeted expression (designated ctp-m3-cry1Ah). Four plant expression vectors were constructed harboring m1-cry1Ah, m2-cry1Ah, m3-cry1Ah, or ctp-m3-cry1Ah. A total of 23 transgenic tobacco lines were produced with the four constructs by Agrobacterium tumefaciens-mediated transformation. PCR, Southern hybridization, quantitative RT-PCR and ELISA indicated that the cry1Ah gene was not only integrated into the tobacco genome, but was also successfully expressed at the mRNA and protein levels. The Cry1Ah protein level in ctp-m3-cry1Ah plants reached 4.42?μg/g fresh weight, which was a 2- to 10-fold increase over the levels observed in m1-cry1Ah, m2-cry1Ah, and m3-cry1Ah plants and resulted in the highest resistance to Helicoverpa armigera based on bioassays. Our results demonstrated that combining the codon optimization of cry1Ah gene with the targeting of Cry1Ah protein to the chloroplasts conferred a high level of protection against insects. The results of our experiments in tobacco, an important model system, provide a good foundation for enhancing the insecticidal efficacy of staple crops.