FTIR技术在烟草基因编辑素材病害初级筛选中的应用
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摘要
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.
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.
引文
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[15]李旭华,肖明礼,杨庆,等.一种通过紫外光谱评价烟叶醇化质量的方法[P].中国专利:103674874B,2017-10-10.
[16]张鑫,郭佳,倪力军,等.基于红外与近红外光谱的烟叶部位识别[J].光谱学与光谱分析,2007,27(12):2437-2440.
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[21]李淑兰.烟火剂中硝酸盐的红外光谱测定[J].爆破器材,1989(6):5-6.
[22]周晚来,刘文科,杨其长.光对蔬菜硝酸盐累积的影响及其机理[J].华北农学报,2011,26(12):125-130.
[23] Pil Joon Seo, Jaeyong Ryu, Seok Ki Kang, et al. Modulation of sugar metabolism by an INDETERMINATE DOMAIN transcription factor contributes to photoperiodic flowering in Arabidopsis[J]. The Plant Journal,2011,65:418-429.
[24] Yu Sha, Cao Li, Zhou Chuan-Miao, et al. Sugar is an endogenous cue for juvenile-to-adult phase transition in plants[J]. eLife,2013(2):e00269.
[25] Yang Li, Xu Mingli, Koo Yeonjong, et al. Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C[J]. eLife,2013,2(2):e00260.
[26]刘春燕,罗洁,周龙,等.黄化病对葡萄生长发育的影响[J].中国农学通报,2018,11:103-107.
[27]朱本明.类菌原体和烟草类菌原体病害[J].中国烟草,1981,4:37-40.
[28]卢卡斯.第五部份菌原质病害[J].烟草科技通讯,1977,1:55-62,79-80.
[2]肖炳光.烟草基因组计划进展篇:3.烟草分子标记遗传连锁图构建和重要抗病基因定位[J].中国烟草科学,2013,34(3):118-119.
[3]王彦亭.我国烟草育种工作发展思路[J].中国烟草科学,2001(4):1-5.
[4]朱惠琴,戴文新.烟草加倍单倍体群体的构建及其在遗传育种中的应用[J].青海大学学报:自然科学版,2003(6):20-23.
[5]杨金广,樊韦华,杨柳青,等.烟草抗番茄斑萎病毒基因筛选与生物信息学分析[J].中国烟草科学,2016,37(4):60-67.
[6]林国平,王毅,肖宗友.我国白肋烟育种工作现状与发展对策[A].湖北省烟草学会2006年学术年会论文集[C].2006:109-112.
[7]何川生,何兴金,李天飞,等.烤烟品种资源的聚类分析[J].中国农业科学,2000(3):14-18.
[8]王志德,牟建民,王卫锋,等.部分烟草核心种质过氧化物同工酶标记分析[J].中国烟草科学,2003(2):9-11.
[9]林涛,于海燕,应义斌.可见/近红外光谱技术在液态食品检测中的应用研究进展[J].光谱学与光谱分析,2008(2):285-290.
[10]张宁.近红外光谱定性分析技术在食品安全中的应用研究进展[J].食品科技,2008(8):218-221.
[11]陈兰珍,孙谦,叶志华,等.基于神经网络的近红外光谱鉴别蜂蜜品种研究[J].食品科技,2009,34(8):287-289.
[12]王多加,周向阳,金同铭,等.近红外光谱检测技术在农业和食品分析上的应用[J].光谱学与光谱分析,2004(4):447-450.
[13]孙通,徐惠荣,应义斌.近红外光谱分析技术在农产品/食品品质在线无损检测中的应用研究进展[J].光谱学与光谱分析,2009(1):122-126.
[14]阮春生,张强,丁波洋,等.不同部位烟叶紫外-可见光谱特征的研究[J].云南民族大学学报:自然科学版,2009,18(3):237-241.
[15]李旭华,肖明礼,杨庆,等.一种通过紫外光谱评价烟叶醇化质量的方法[P].中国专利:103674874B,2017-10-10.
[16]张鑫,郭佳,倪力军,等.基于红外与近红外光谱的烟叶部位识别[J].光谱学与光谱分析,2007,27(12):2437-2440.
[17]傅永福,孟繁静.植物的成花生理信号[J].中国农业大学学报,1998,3(3):1-11.
[18] Bodson M, Outlaw W H. Elevation in the contents of the shoots apical meristem of sinapis alba at floral evocation[J]. Plant Physiol,1985,79:420-424.
[19] Kinet J M. Environmental, Chemical, and genetic control of flowering[J]. HotRev,1993,7:279-334.
[20] Tran Thanh Van K M. Control of morphogenesis in vitro cultures[J]. Ann Rev Plant Physiol,1981,32:291-311.
[21]李淑兰.烟火剂中硝酸盐的红外光谱测定[J].爆破器材,1989(6):5-6.
[22]周晚来,刘文科,杨其长.光对蔬菜硝酸盐累积的影响及其机理[J].华北农学报,2011,26(12):125-130.
[23] Pil Joon Seo, Jaeyong Ryu, Seok Ki Kang, et al. Modulation of sugar metabolism by an INDETERMINATE DOMAIN transcription factor contributes to photoperiodic flowering in Arabidopsis[J]. The Plant Journal,2011,65:418-429.
[24] Yu Sha, Cao Li, Zhou Chuan-Miao, et al. Sugar is an endogenous cue for juvenile-to-adult phase transition in plants[J]. eLife,2013(2):e00269.
[25] Yang Li, Xu Mingli, Koo Yeonjong, et al. Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C[J]. eLife,2013,2(2):e00260.
[26]刘春燕,罗洁,周龙,等.黄化病对葡萄生长发育的影响[J].中国农学通报,2018,11:103-107.
[27]朱本明.类菌原体和烟草类菌原体病害[J].中国烟草,1981,4:37-40.
[28]卢卡斯.第五部份菌原质病害[J].烟草科技通讯,1977,1:55-62,79-80.