FTIR技术在烟草基因编辑素材病害初级筛选中的应用

Application of FTIR Technology in the Primary Screening of Gene Editing Tobacco Material Diseases

针对基因编辑素材出现的病害,以差异性分析为基础,围绕素材病害特征为研究核心,开发高通量的FTIR初级筛选方法。对早花成苗期与正常成苗期烟叶研究表明,早花后烟叶糖类化合物明显增加,而蛋白质含量变化不明显,糖/蛋白质比值明显增加,糖类化合物可能是早花烟草植株的成花信号因子。黄化烟叶与正常烟叶的研究表明,黄化烟叶糖类化合物明显减少,而蛋白质含量基本不变,同时黄化烟叶中出现硝酸盐的富集,这可能是由于在黄化烟叶中,光合作用的降低使植物硝酸盐的吸收和同化降低,同化作用降低更明显,出现硝酸盐的富集。也有可能是由于编辑基因位点阻断或降低了植物硝酸盐的同化作用。烟叶中硝酸盐的富集可以作为该类型黄化病初步筛选标准,1384 cm^-1和829 cm^-1为其烟叶黄化标志特征峰。此外,通过行业标准测试方法对上述化学成分变化进行了验证且分析结果一致。主成分分析研究表明,可以明显地将上述病害烟叶与对照烟叶进行区分,并建立相关病害的初步判定模型。研究结果说明,FTIR技术的分析结果具有较高的准确度,且不同样品间区别明显,可以用于基因编辑素材的早期病害诊断和病害分析等的高通量初级筛选。

Aiming at the diseases that appear in gene editing tobacco materials, based on differential analysis,we developed a high-throughput FTIR primary screening method focused on the characteristics of tobacco material diseases as the research core. Study on the premature flowering and normal seedling formation period showed that tobacco leaf carbohydrates increased significantly after premature flowering, while the protein content did not change significantly, ratio of sugar to protein increased significantly. The carbohydrates could be flowering signal factor of premature flowering tobacco plants. Study on the chlorisis leaves and normal leaves showed that the carbohydrate in chlorisis leaves decreased significantly, and the protein content was basically unchanged. The nitrate was enriched in chlorisis leaves, since the decrease of photosynthesis in chlorisis leaves might cause the decrease of the absorption and assimilation of plant nitrate, and the decrease of assimilation was more obvious. It was also possible that the nitrate assimilation was blocked or reduced by the editing of gene loci. The enrichment of nitrate in tobacco leaves could be used as a preliminary screening criterion for this type of chlorisis disease, and the characteristic peaks of the chlorisis tobacco were in 1384 cm^-1 and 829 cm^-1. In addition, the changes in the chemical composition were verified by other analytical methods and the results were consistent. The principal component analysis showed that the disease tobacco could be distinctly distinguished from the control tobacco, and a preliminary determination model of the related diseases was established. In general, the analysis results of FTIR technology has high accuracy, and the difference between the samples was obvious, it could be applied to high-throughput primary screening for early disease diagnosis and disease analysis of gene editing tobacco materials.