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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2012, Vol. 32 Issue (08): 56-61    
研究报告     
甲基营养菌 Methylobacterium sp MB200 中crtI基因的克隆及表达
林琼珊1,2,3, 唐咸来1,4, 李献1,2,3, 蒋承建1,2,3, 李俊芳1,2,3, 申佩弘1,2,3
1. 广西大学生命科学与技术学院 南宁 530005;
2. 微生物及植物遗传工程教育部重点实验室 南宁 530005;
3. 亚热带农业生物资源保护与利用国家重点实验室 南宁 530005;
4. 广西壮族自治区科学技术厅 南宁 530005
Cloning and Expression crtI Gene in Methylobacterium sp MB200
LIN Qiong-shan1,2,3, TANG Xian-lai1,4, LI Xian1,2,3, JIANG Cheng-jian1,2,3, LI Jun-fang1,2,3, SHEN Pei-hong 1,2,3
1. College of Life Science and Technology of Guangxi University, Nanning 530005, China;
2. The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, Nanning 530005, China;
3. State Key Laboratory for Conservation and Utilization of Agricultural Bioresources in the Subtropics, Nanning 530005, China;
4. The Science and Techenology Department of Guangxi, Nanning 530005, China
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摘要: 八氢番茄红素脱氢酶(CrtI)催化八氢番茄红素经过4次脱氢合成番茄红素,或者经过3次脱氢合成链孢红素,在类胡萝卜素的生物合成中发挥重要的作用。以甲基营养菌Methylobacterium sp MB200为原始菌株,首先采用转座子突变技术构建部分突变体库共11552株,筛选得到33株颜色发生变化的目的突变体,随后利用分子克隆技术从目的突变体中获得crtI基因的完整ORF,长为1539 bp,编码512个氨基酸。与来自M. populi BJ001、M. chloromethanicum CM4和M. extorquens AM1的crtI一致性均为93%。将crtI与载体pCM80连接得到重组质粒pCM80-crtI,导入原始菌株中得到重组菌MB200/pCM80-crtI。测定原始菌株与重组菌株的CrtI酶活,结果发现,重组菌株CrtI的酶活与原始菌株相比约提高了40%。实验结果为完善甲基营养菌中类胡萝卜素的生物合成代谢途径提供了理论参考。
关键词: 甲基营养菌crtI八氢番茄红素克隆表达    
Abstract: The partial mutants library of M. sp MB200 was constructed by transposon mutagenesis and 11552 mutants were obtained which could survived in medium with methanol as the sole carbon source. Rescreening results showed that 33 strains were colorless mutants. The crtI gene was got from a colorless mutant by molecular cloning techniques. It shared 93% identity with the crtI gene of M. populi BJ001, M. chloromethanicum CM4 and M. extorquens AM1 at the nucleotide level reported in Genbank. The gene size is 1539 bp, encoding the phytoene desaturase (CrtI) that catalyzes phytoene to form lycopene with four desaturations, or catalyzes phytoene to form neurosporene with three desaturations, therefore it plays an important role in the biosynthetic pathway of carotenoid. Here, a recombinant plasmid pCM80-crtI was constructed by ligating the crtI to the vector pCM80, then imported into M. sp MB200 to obtain recombinant strain MB200/pCM80-crtI. The activity of enzyme from MB200/pCM80-crtI was more by 40% than M. sp MB200. The experimental results provided a theoretical reference to improve the biosynthetic and metabolic pathways of carotenoid in Methylobacterium.
Key words: M. sp    crtI    Phytoene    Cloning    Expression
收稿日期: 2012-03-20 出版日期: 2012-08-25
ZTFLH:  Q786  
基金资助: 国家自然科学基金(31160022)、第四十九批博士后基金(20110490866)资助项目
通讯作者: 申佩弘     E-mail: sos89@126.com
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引用本文:

林琼珊, 唐咸来, 李献, 蒋承建, 李俊芳, 申佩弘. 甲基营养菌 Methylobacterium sp MB200 中crtI基因的克隆及表达[J]. 中国生物工程杂志, 2012, 32(08): 56-61.

LIN Qiong-shan, TANG Xian-lai, LI Xian, JIANG Cheng-jian, LI Jun-fang, SHEN Pei-hong. Cloning and Expression crtI Gene in Methylobacterium sp MB200. China Biotechnology, 2012, 32(08): 56-61.

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https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I08/56

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