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Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes

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posted on 2020-01-04, 03:20 authored by Zhongbang Song, Xueyi Sui, Meiyun Li, Yulong Gao, Wenzheng Li, Lu Zhao, Feng Li, Xuefeng Yao, Chunming Liu, Bingwu Wang

Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the CYP82E2 family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized population. In this study, we developed dCAPS markers for each nonsense mutation. Using marker-assisted selection, NND mutants were crossed with each other to generate a triple mutant GP449. In line with previous reports, the triple knockout caused significantly decreased levels of nornicotine and NNN in flue-cured tobacco. With the decreased nornicotine, the nicotine level was expected to accumulate. However, the nicotine level in GP449 was significantly decreased to 72.80% of wild type. Realtime RT-PCR analysis showed that the nicotine reduction was correlated with inhibited expression of nicotine biosynthetic pathway genes. The triple mutant and dCAPS markers can be utilized to develop new flue-cured tobacco varieties with lower levels of nornicotine and NNN.

Funding

This study was supported by the National Natural Science Foundation of China [31560080]; the grants from China Tobacco Company and Yunnan Tobacco Company [110201801027 (JY-04), 2016YN25, 2017YN03 and 2018530000241001].

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