Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

doi:10.1016/S2095-3119(13)60531-6

Journal of Integrative Agriculture ›› 2014, Vol. 13 ›› Issue (5): 937-944.DOI: 10.1016/S2095-3119(13)60531-6

Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

LI Xiu-ying, LANG Zhi-hong, ZHANG Jie, HE Kang-lai, ZHU Li , HUANG Da-fang

1、College of Life Science, Northeast Agricultural University, Harbin 150030, P.R.China
2、Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
3、State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection, Chinese Academy of Agricultural Sciences,
Beijing100193, P.R.China

收稿日期:2013-03-15 出版日期:2014-05-01 发布日期:2014-05-06
通讯作者: HUANG Da-fang, Tel/Fax: +86-10-82109857, E-mail: huangdafang@caas. cn; LANG Zhi-hong, Tel/Fax: +86-10-82109857, E-mail: langzhihong@caas.cn
作者简介:LI Xiu-ying, Mobile: 15811024861, E-mail: lixiuying1987@gmail.com
基金资助:
the Genetically Modified Organisms Breeding Major Project, China (2011ZX08003-001), the National Basic Research Program of China (973 Program, 2009CB118902) and the National Natural Science Foundation of China (30970231). Additionally, the authors thank Dr. Yu Jingjuan at the College of Biological Sciences, China Agricultural University for supplying the maize seed.

Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

LI Xiu-ying, LANG Zhi-hong, ZHANG Jie, HE Kang-lai, ZHU Li , HUANG Da-fang

1、College of Life Science, Northeast Agricultural University, Harbin 150030, P.R.China
2、Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
3、State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection, Chinese Academy of Agricultural Sciences,
Beijing100193, P.R.China

Received:2013-03-15 Online:2014-05-01 Published:2014-05-06
Contact: HUANG Da-fang, Tel/Fax: +86-10-82109857, E-mail: huangdafang@caas. cn; LANG Zhi-hong, Tel/Fax: +86-10-82109857, E-mail: langzhihong@caas.cn
About author:LI Xiu-ying, Mobile: 15811024861, E-mail: lixiuying1987@gmail.com
Supported by:
the Genetically Modified Organisms Breeding Major Project, China (2011ZX08003-001), the National Basic Research Program of China (973 Program, 2009CB118902) and the National Natural Science Foundation of China (30970231). Additionally, the authors thank Dr. Yu Jingjuan at the College of Biological Sciences, China Agricultural University for supplying the maize seed.

摘要/Abstract

摘要： A novel insecticidal gene cry1Ah was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active Cry1Ah toxin has a toxicity level similar to that of the full-length Cry1Ah toxin. In this study, plant expression vector pMhGM harboring truncated cry1Ah gene was transformed into maize (Zea mays L.) immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions (madMARs) were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer (Ostrinia furnacalis). These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the cry1Ah gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene cry1Ah was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T1-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.

关键词: Bacillus thuringiensis , truncated cry1Ah gene , insect resistant maize , stable inheritance

Abstract: A novel insecticidal gene cry1Ah was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active Cry1Ah toxin has a toxicity level similar to that of the full-length Cry1Ah toxin. In this study, plant expression vector pMhGM harboring truncated cry1Ah gene was transformed into maize (Zea mays L.) immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions (madMARs) were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer (Ostrinia furnacalis). These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the cry1Ah gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene cry1Ah was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T1-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.

Key words: Bacillus thuringiensis , truncated cry1Ah gene , insect resistant maize , stable inheritance

LI Xiu-ying, LANG Zhi-hong, ZHANG Jie, HE Kang-lai, ZHU Li , HUANG Da-fang. Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation[J]. Journal of Integrative Agriculture, 2014, 13(5): 937-944.

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Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

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