在叶绿体中过量表达AOD1和HPS-PHI创建光合甲醇同化途径增加烟草同化甲醇能力

中国生物工程杂志

2013, Vol. 33

Issue (12): 69-78

技术与方法

在叶绿体中过量表达AOD1和HPS-PHI创建光合甲醇同化途径增加烟草同化甲醇能力

刘蕾, 孙振, 宋中邦, 肖素勤, 陈丽梅

昆明理工大学生命科学与技术学院生物工程技术研究中心昆明 650500

Simultaneous Over-expressions of AOD1 and HPS-PSI in Chloroplasts Creates a Novel Photosynthetic CH₃OH-assimilation Pathway and Enhances Its Ability to Assimilate CH₃OH

LIU Lei, SUN Zhen, SONG Zhong-bang, XIAO Su-qin, CHEN Li-mei

Faculty of Live Science and Engineering, Chenggong Campus, Kunming University of Science and Technology, Kunming 650500, China

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摘要： 最近的研究证明在叶绿体内过量表达甲基营养细菌RuMP中固定HCHO的关键酶6-磷酸己酮糖合成酶（HPS）和6-磷酸果糖异构酶（PHI）的融合蛋白HPS-PHI可以在转基因天竺葵中构建一条甲醛光合同化途径从而提高植物对甲醛的同化和脱毒能力。甲醇氧化酶（AOD1）是甲基营养型酵母甲醇代谢途径的第一个关键酶，可催化甲醇氧化为甲醛。在烟草叶绿体中过量表达AOD1或/和HPS-PHI产生过量表达AOD1（AO）、HPS-PHI（AB）、同时过量表达AOD1和HPS-PHI（AA）的转基因烟草。用2和6mmol/L ¹³CH₃OH处理3种转基因烟草和野生型烟草（WT），¹³C-NMR分析表明AO植株中积累的H¹³COOH最多，[U-¹³C] Gluc和[U-¹³C] Fruc的生成量在AO和WT间无显著差异，在AA和AB中的产生量显著高于AO和WT，且在AA中的生成量又高于AB，这种差异在6mmol/L ¹³CH₃OH处理植株中比2 mmol/L更为明显。这些结果证实在叶绿体中过表达AOD1增加烟草氧化甲醇为甲酸的能力，同时过量表达AOD1和HPS-PHI在烟草中成功地构建一个甲醇光合同化途径，提高烟草同化甲醇为糖类物质的能力。

关键词： HPS-PHI融合蛋白; 甲醇同化作用; 转基因烟草; 甲醇光合同化途径; 甲醇氧化酶

Abstract: 3-hexulose-6-phosphate synthase (HPS) and 6-phosphate-3-hexuloisomerase (PHI) are two key enzymes in a HCHO-assimilation pathway in methylotroph. It has demonstrated that overexpression of HPS/PHI fusion protein in chloroplasts of geranium installs a photosynthetic HCHO-assimilation pathway and thereby enhanced the ability of transgenic plants to assimilate and detoxify formaldehyde. Alcohol oxidase (AOD1) is the first key enzyme in the methanol metabolic pathway in methylotrophic yeasts. AOD catalyzes oxidation of methanol to formaldehyde. AOD1 or/and HPS-PHI were over-expressed in chloroplasts of tobacco to generate AOD1 (AO), HPS-PHI (AB) over-expression line as well as AOD1 and HPS-PHI (AA) simultaneous over-expression line. The transgenic and wild-type tobacco (WT) was treated with 2 mmol/L and 6 mmol/L ¹³CH₃OH. ¹³C-NMR analysis showed that the accumulation of H¹³COOH was maximum in AO trangenic plants. There is no significant difference in the generation of [U-¹³C] glucose (Gluc) and [U-¹³C] fructose (Fruc) between AO and WT lines. However, the production of [U-¹³C] glucose (Gluc) and [U-¹³C] fructose (Fruc) in AA and AB lines was significantly higher than that in AO and WT lines. Moreover, the formation of the two metabolites in AA line was higher than in AB line. These differences were more significant in 6mmol/L¹³CH₃OH treated-plants than in 2 mmol/L ¹³CH₃OH-treated plants. These results suggested that overexpression of AOD1 in chloroplasts enhanced the ability of tobacco to oxidize methanol to formaldehyde, while simultaneous overexpression of AOD1 and HPS-PHI could successfully created a photosynthetic CH₃OH-assimilation pathway, thus enhanced ability of tobacco to assimilate CH₃OH as sugars.

Key words: Transgenic tobacco Photosynthetic HCHO-assimilation pathway Alcohol oxidase HPS-PHI fusion protein CH₃OH-assimilation pathway

收稿日期: 2013-10-12 出版日期: 2013-12-25

ZTFLH:

Q812

基金资助: 国家自然科学基金资助项目（30970263）

通讯作者: 陈丽梅，E-mail：chenlimeikm@yahoo.com.cn E-mail: chenlimeikm@yahoo.com.cn

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引用本文:

刘蕾, 孙振, 宋中邦, 肖素勤, 陈丽梅. 在叶绿体中过量表达AOD1和HPS-PHI创建光合甲醇同化途径增加烟草同化甲醇能力[J]. 中国生物工程杂志, 2013, 33(12): 69-78.

LIU Lei, SUN Zhen, SONG Zhong-bang, XIAO Su-qin, CHEN Li-mei. Simultaneous Over-expressions of AOD1 and HPS-PSI in Chloroplasts Creates a Novel Photosynthetic CH₃OH-assimilation Pathway and Enhances Its Ability to Assimilate CH₃OH. China Biotechnology, 2013, 33(12): 69-78.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I12/69

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