title: A Quantitative Real-Time PCR-Based Strategy for Molecular Evaluation of Nicotine Conversion in Burley Tobacco.
abstract: Nornicotine production in Nicotiana tabacum is undesirable because it is the precursor of the carcinogen N'-nitrosonornicotine. In some individual burley tobacco plants, a large proportion of the nicotine can be converted to nornicotine, and this process of nicotine conversion is mediated primarily by enzymatic N-demethylation of nicotine which is controlled mainly by CYP82E4. Here we report a novel strategy based on quantitative real-time polymerase chain reaction (qPCR) method, which analyzed the ratio of nicotine conversion through examining the transcript level of CYP82E4 in burley leaves and do not need ethylene induction before detected. The assay was linear in a range from 1 × 10¹ to 1 × 10⁵ copies/mL of serially diluted standards, and also showed high specificity and reproducibility (93%-99%). To assess its applicability, 55 plants of burley cultivar Ky8959 at leaf maturing stage were analyzed, and the results were in accordance with those from gas chromatograph-mass spectrometry (GC-MS) method. Moreover, a linear correlation existed between conversion level and CYP82E4 transcript abundance. Taken together, the quantitative real-time PCR assay is standardized, rapid and reproducible for estimation of nicotine conversion level in vivo, which is expected to shed new light on monitoring of burley tobacco converter.
authors.affiliation: College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China. sunadam011@163.com.|National Tobacco Gene Center, Zhengzhou Tobacco Research Institute, Zhengzhou 450001, China. sunadam011@163.com.
authors.forename: Bo
authors.initials: B
authors.lastname: Sun
authors.affiliation: College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China. 18161250603@163.com.
authors.forename: Sheng-Ling
authors.initials: SL
authors.lastname: Xue
authors.affiliation: College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China. zhangf_12@163.com.|National Tobacco Gene Center, Zhengzhou Tobacco Research Institute, Zhengzhou 450001, China. zhangf_12@163.com.
authors.forename: Fen
authors.initials: F
authors.lastname: Zhang
authors.affiliation: National Tobacco Gene Center, Zhengzhou Tobacco Research Institute, Zhengzhou 450001, China. luozhaopeng163@163.com.
authors.forename: Zhao-Peng
authors.initials: ZP
authors.lastname: Luo
authors.affiliation: National Tobacco Gene Center, Zhengzhou Tobacco Research Institute, Zhengzhou 450001, China. mingzhuwus@126.com.
authors.forename: Ming-Zhu
authors.initials: MZ
authors.lastname: Wu
authors.affiliation: College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China. supnovel@gmail.com.
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.affiliation: College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China. htang@sicau.edu.cn.
authors.forename: Hao-Ru
authors.initials: HR
authors.lastname: Tang
authors.affiliation: National Tobacco Gene Center, Zhengzhou Tobacco Research Institute, Zhengzhou 450001, China. fuchenglin@zju.edu.cn.
authors.forename: Fu-Cheng
authors.initials: FC
authors.lastname: Lin
authors.affiliation: National Tobacco Gene Center, Zhengzhou Tobacco Research Institute, Zhengzhou 450001, China. yangjun@ztri.com.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
mesh_terms: D019143:Evolution, Molecular; D008401:Gas Chromatography-Mass Spectrometry; D009538:Nicotine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009538:Nicotine; C005311:nornicotine
keywords: CYP82E4; burley tobacco; nicotine conversion; quantitative real-time PCR (qPCR)