Lewis Ramsey S, Lopez Harry O, Bowen Steve W, Andres Karen R, Steede William T, Dewey Ralph E
Department of Crop Science, North Carolina State University, Raleigh, North Carolina, United States of America.
PLoS One. 2015 Feb 17;10(2):e0117273. doi: 10.1371/journal.pone.0117273. eCollection 2015.
Motivation exists to develop tobacco cultivars with reduced nicotine content for the purpose of facilitating compliance with expected tobacco product regulations that could mandate the lowering of nicotine levels per se, or the reduction of carcinogenic alkaloid-derived tobacco specific nitrosamines (TSNAs). A berberine bridge enzyme-like (BBL) gene family was recently characterized for N. tabacum and found to catalyze one of the final steps in pyridine alkaloid synthesis for this species. Because this gene family acts downstream in the nicotine biosynthetic pathway, it may represent an attractive target for genetic strategies with the objective of reducing alkaloid content in field-grown tobacco. In this research, we produced transgenic doubled haploid lines of tobacco cultivar K326 carrying an RNAi construct designed to reduce expression of the BBL gene family. Field-grown transgenic lines carrying functional RNAi constructs exhibited average cured leaf nicotine levels of 0.684%, in comparison to 2.454% for the untransformed control. Since numerous barriers would need to be overcome to commercialize transgenic tobacco cultivars, we subsequently pursued a mutation breeding approach to identify EMS-induced mutations in the three most highly expressed isoforms of the BBL gene family. Field evaluation of individuals possessing different homozygous combinations of truncation mutations in BBLa, BBLb, and BBLc indicated that a range of alkaloid phenotypes could be produced, with the triple homozygous knockout genotype exhibiting greater than a 13-fold reduction in percent total alkaloids. The novel source of genetic variability described here may be useful in future tobacco breeding for varied alkaloid levels.
开发尼古丁含量降低的烟草品种具有重要意义，这有助于符合预期的烟草产品法规，这些法规可能会强制降低尼古丁本身的含量，或减少致癌生物碱衍生的烟草特有亚硝胺（TSNA）。最近对烟草中的一个小檗碱桥酶样（BBL）基因家族进行了表征，发现它催化该物种吡啶生物碱合成的最后步骤之一。由于这个基因家族在尼古丁生物合成途径中处于下游，它可能是旨在降低田间种植烟草中生物碱含量的遗传策略的一个有吸引力的靶点。在本研究中，我们培育了烟草品种K326的转基因双单倍体系，该体系携带一个旨在降低BBL基因家族表达的RNAi构建体。携带功能性RNAi构建体的田间种植转基因系的平均烤后叶尼古丁水平为0.684%，而未转化对照为2.454%。由于将转基因烟草品种商业化需要克服众多障碍，我们随后采用了诱变育种方法，以鉴定BBL基因家族三个表达量最高的同工型中的EMS诱导突变。对在BBLa、BBLb和BBLc中具有不同纯合截短突变组合的个体进行田间评估表明，可以产生一系列生物碱表型，三重纯合敲除基因型的总生物碱百分比降低了13倍以上。这里描述的新的遗传变异来源可能在未来烟草育种中用于获得不同的生物碱水平。
