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| Prokaryotic Expression and Purification of Bt-Cry5Aa Insecticidal Gene and Its Identification in Gossypium |
| MA Xiao*, ZHAO Shi-Hao*, WANG Feng, HE Wen, CHEN Jin-Xiang, ZHANG Qiu-Ping**, ZHOU Zhong-Hua** |
| College of Agronomy, Hunan Agricultural University, Changsha 410128, China |
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Abstract The single transgenic Bacillus thuringiensis (Bt) gene Cry1Ab/Cry1Ac insect-resistant cotton (Gossypium) is a large-scale planted crop in China. With the increase of planting time and area, the resistance of pests to Bt toxin protein increases, new Bt gene research work is necessary. Cry5Aa is a novel Bt insecticidal gene with resistance to both Lepidoptera and Nematodes, was successfully transferred into cotton by Cotton Research Institute of Hunan Agricultural University and a stable genetic 'JX0010', 'JX0020' strain was obtained. In this study, the Cry5Aa full-length gene prokaryotic expression vector was constructed, the prokaryotic expression induction conditions were optimized, and its expression in the Bt-Cry5Aa gene of transgenic cotton line was identified. The results showed that the protein expression was the highest when induced at 37 ℃ with 220 r/min 1.0 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) for 4 h. Through Western blot experiment, it was found that the molecular weight of the protein was 79 kD, which was consistent with the theoretical value; at the same time, the protein existed in the form of inclusion body and was purified by Glutathione-Sepharose 4B small particle affinity chromatography. The identification experiment of transgenic cotton lines with Bt-Cry5Aa gene showed that Cry5Aa gene could express insecticidal protein in transgenic line 'JX0010', 'JX0020', and the expression of Cry5Aa toxin protein of 'JX0010' was higher than that of 'JX0020' in different periods and different parts of cotton. The expression in bud stage was the highest, and the spatial distribution was the highest in leaves. In this study, the prokaryotic expression vector of Bt-Cry5Aa insecticidal gene was constructed, the purified protein of Cry5Aa was obtained, and its expression in cotton was preliminarily identified, which provides a theoretical basis for the further application of Bt-Cry5Aa insecticidal gene in cotton.
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Received: 08 January 2020
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Corresponding Authors:
** , zhanqiuping@hunau.edu.cn; zhouzhonghua1976@hotmail.com.
* The authors who contribute equally
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