烤烟烟叶类黄酮合成关键酶活性及其调控基因的表达分析
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摘要
为系统探究类黄酮在烟叶成熟期合成积累的关键点、合成相关酶及调控基因的变化规律,测定了豫烟6号、K326和ND202三个烤烟品种中部叶成熟期叶片的类黄酮含量、类黄酮合成上游的苯丙氨酸解氨酶(PAL)、肉桂酸4-羟化酶(C4H)、4-香豆酰CoA连接酶(4CL)、查尔酮合成酶(CHS)和查尔酮异构酶(CHI)酶活性及其基因表达模式的变化。结果表明,三个烤烟品种烟叶类黄酮含量变化呈现先增加后降低的规律,打顶后0～30d是烟叶类黄酮合成积累的关键时期,在叶片成熟期含量达最大值;在同一生态环境及栽培管理条件下,成熟期各阶段均以豫烟6号中部叶类黄酮含量最高;测定类黄酮合成酶活性发现,PAL、CHS和CHI酶活性与烟叶类黄酮合成的相关性较大,其中CHS合成酶最大(p<0.01,r=0.88);基因表达模式研究发现,PAL、C4H、4CL2、CHS和CHI基因与烟叶类黄酮含量的积累相关性较大,其中CHS基因最大(p<0.01,r=0.96),是烟叶类黄酮合成过程中的关键基因。
In order to systematically explore the key points for ?avonoids synthesis and accumulation during tobacco leaf maturation, the changes of related synthases and regulatory genes, matured leaves of three ?ue-cured tobacco varieties i.e., Yuyan6, K326 and ND202, were tested and ?avonoid concentration, Phenylalanine ammonia lyase(PAL)activity, Cinnamate 4-hydroxylase(C4H)activity, 4-coumaroyl Co A ligase(4CL)activity, Chalcone synthase(CHS)activity, Chalcone isomerase(CHI)activity were determined and hence changes in gene expression pattern. Results showed that the ?avonoids content of three varieties of ?ue-cured tobacco increased ?rst and then decreased.30 days after topping is the key period for synthesis and accumulation of ?avonoids in tobacco leaves, and the content reaches maximum during maturity period. Under same ecological environment and cultivation and management conditions, the content of flavonoids in the middle leaves of Yuyan6 at maturity stage was the highest. By determining the activity of ?avonoid synthase, ?avonoid synthase activities of PAL, CHS and CHI can be determined and they were correlated more with synthesis of ?avonoids in tobacco leaves, and the correlation degree for CHS synthetase was the largest(p<0.01, r=0.88). The PAL, C4H, 4 CL2, CHS and CHI genes were correlated more with accumulation of?avonoids in tobacco leaves, especially for CHS gene(p<0.01, r=0.96), which is a key gene in the synthesis of ?avonoids in tobacco leaf.
In order to systematically explore the key points for ?avonoids synthesis and accumulation during tobacco leaf maturation, the changes of related synthases and regulatory genes, matured leaves of three ?ue-cured tobacco varieties i.e., Yuyan6, K326 and ND202, were tested and ?avonoid concentration, Phenylalanine ammonia lyase(PAL)activity, Cinnamate 4-hydroxylase(C4H)activity, 4-coumaroyl Co A ligase(4CL)activity, Chalcone synthase(CHS)activity, Chalcone isomerase(CHI)activity were determined and hence changes in gene expression pattern. Results showed that the ?avonoids content of three varieties of ?ue-cured tobacco increased ?rst and then decreased.30 days after topping is the key period for synthesis and accumulation of ?avonoids in tobacco leaves, and the content reaches maximum during maturity period. Under same ecological environment and cultivation and management conditions, the content of flavonoids in the middle leaves of Yuyan6 at maturity stage was the highest. By determining the activity of ?avonoid synthase, ?avonoid synthase activities of PAL, CHS and CHI can be determined and they were correlated more with synthesis of ?avonoids in tobacco leaves, and the correlation degree for CHS synthetase was the largest(p<0.01, r=0.88). The PAL, C4H, 4 CL2, CHS and CHI genes were correlated more with accumulation of?avonoids in tobacco leaves, especially for CHS gene(p<0.01, r=0.96), which is a key gene in the synthesis of ?avonoids in tobacco leaf.
引文
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[18]李佶芸.不同抗性品种烤烟感染黑胫病后生理指标的变化[J].中国农业信息,2013(09):142-143.LI Jiyun.Changes of physiological indexes of flue-cured tobacco with different resistance varieties after infection with black shank[J].China Agriculture Information,2013(09):142-143.
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[21]钟楚,陈宗瑜,毛自朝,等.滤减UV-B辐射对烟叶可溶性蛋白、光合色素和类黄酮的影响[J].广西植物,2010,30(04):501-506+457.ZHONG Chun,CHEN Zongyu,MAO Zichao,et al.Effect of UV-BRadiation on Soluble Protein,Photosynthetic Pigment and Flavonoids in Tobacco Leaves[J].Guihaia,2010,30(04):501-506+457.
[22]董卓娅.云南不同纬度烟区烤烟K326类黄酮和丙二醛含量比较研究[J].安徽农业科学,2012,40(26):12805-12807.DONG Zhuoya.Comparative Study on the Contents of K326Flavonoids and Malondialdehyde in Flue-cured Tobacco in Different Latitude Areas of Yunnan Province[J].Journal of Anhui Agricultural Sciences,2012,40(26):12805-12807.
[23]陈帅,杨爱国.烟草重要基因篇14:烟草类黄酮合成相关基因[J].中国烟草科学,2016,37(02):95-98.CHEN Shuai,YANG Aiguo.Important Genes of Tobacco Part 14:Tobacco Flavonoid Synthesis Related Genes[J].Chinese Tobacco Science,2016,37(02):95-98.
[24]彭东,陈爱国,梁洪波,等.不同种质材料烟草各时期多酚含量和组成差异分析[C]//山东植物生理学会第七次代表大会暨植物生物学与现代农业研讨会论文集.2012.PENG Dong,CHEN Aiguo,LIANG Hongbo,et al.Analysis of polyphenol content and composition difference in tobacco of different germplasm materials in different periods[C]//Proceedings of the Seventh Congress of the Plant Physiology Society of Shandong and the Symposium on Plant Biology and Modern Agriculture.2012.
[25]王爱华.烤烟生长和调制过程中主要多酚类物质代谢动态的研究[D].郑州:河南农业大学,2005.WANG Aihua.Study on the metabolism of major polyphenols in the growth and regulation of flue-cured tobacco[D].Zhengzhou:Henan Agricultural University,2005.
[26]连文力.施氮量对烟叶成熟期重要物质代谢及基因表达的影响研究[D].郑州:河南农业大学,2016.LIAN Wenli.Effects of nitrogen application rate on metabolism and gene expression of important substances in tobacco leaf maturity[D].Zhengzhou:Henan Agricultural University,2016.
[27]杨慧芹.温度和水分对烟草多酚代谢的影响及种子引发对提高烟草种子和幼苗抗逆性的效应[D].昆明:云南师范大学,2015.Yang Huiqin,2015.Effects of Temperature and Water Status on Polyphenols Metabolism in Tobacco Plants and Effects of Seed Priming on Stress Resistance in Tobacco Seeds and Seedlings[D].Kunming:Yunnan Normal University.
[28]Hu W J,Kawaoka A,Tsai C J,et al.Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoAligase genes in aspen(Populus tremuloides)[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(9):5407-5412.
[2]李富强,宫长荣,宋朝鹏,等.河南浓香型烤烟与进口烟叶的内在品质比较[J].浙江农业科学,2008(06):780-783.LI Fuqiang,GONG Changrong,SONG Zhaopeng,et al.Comparison of intrinsic quality between Henan Luzhou flue-cured tobacco and imported tobacco leaves[J].Journal of Zhejiang Agricultural Sciences,2008(06):780-783.
[3]Layten Davis D,Nielsen M T.Tobacco:production,chemistry and technology.[J].Tobacco Production Chemistry&Technology,1999.
[4]Winkel-Shirley B.Flavonoid biosynthesis.A colorful model for genetics,biochemistry,cell biology,and biotechnology.[J].Plant Physiology,2001,126(2):485-493.
[5]Xie D Y,Dixon R A.Role of anthocyanidin reductase,encoded by BANYULS in plant flavonoid biosynthesis[J].Science,2003,299(5605):396-9.
[6]郭磊,蔡志翔,张斌斌,等.叶面喷施脱落酸对桃果实着色及相关基因表达的影响[J].植物营养与肥料学报,2013,19(06):1464-1470.GUO Lei,CAI Zhixiang,ZHANG Binbin,et al.Effects of foliar abscisic acid on peach fruit coloring and expression of related genes[J].Journal of Plant Nutrition and Fertilizer,2013,19(06):1464-1470.
[7]许海峰,王楠,姜生辉,等.新疆红肉苹果杂种一代4个株系类黄酮含量及其合成相关基因表达分析[J].中国农业科学,2016,49(16):3174-3187.XU Haifeng,WANG Nan,JIANG Shenghui,et al.Content and Analysis of Biosynthesis-Related Genes of Flavonoid Among Four Strains of Malus sieversii f.neidzwetzkyana F1 Population.[J].Scientia Agricultura Sinica,2016,49(16):3174-3187.
[8]李春芳.茶树类黄酮等次生代谢产物的合成及基因的表达分析[D].北京:中国农业科学院,2016.LI Chunfang.Synthesis of secondary metabolites such as tea flavonoids and gene expression analysis[D].Beijing:Chinese Academy of Agricultural Sciences,2016.
[9]周,王玉锋,娄来清,等.甜高粱4-香豆酸辅酶A连接酶基因(4CL)的克隆与鉴定及时空表达分析[J].南京农业大学学报,2014,37(03):9-19.ZHOU Sha,WANG Yufeng,LOU Laiqing,et al.Cloning and Identification of Sweet Sorghum 4-Coumaric Acid Coenzyme A Ligase Gene(4CL)in Time and Space Analysis[J].Journal of Nanjing Agricultural University,2014,37(03):9-19.
[10]张刚,逄涛,王莎莎,等.不同生长温度对烟草叶片黄酮类化合物含量及其代谢途径的调控[J].基因组学与应用生物学,2016,35(12):3527-3537.ZHANG Gang,PANG Tao,WANG Shasha,et al.Regulation of Flavonoids Content and Metabolic Pathway in Tobacco Leaves under Different Growth Temperatures[J].Genomics and Applied Biology,2016,35(12):3527-3537.
[11]张刚.生长温度和施氮水平对烟草叶片黄酮类化合物含量及其代谢途径的调控[D].昆明:云南师范大学,2016.ZHANG Gang.Regulation of Growth Hormone and Nitrogen Levels on Flavonoids Content and Metabolic Pathway in Tobacco Leaves[D].Kunming:Yunnan Normal University,2016.
[12]贾宏昉,张洪映,张松涛,等.不同生态区烤烟黄酮类物质代谢途径关键基因表达分析[J].河南农业科学,2014,43(05):52-55.JIA Hongfang,ZHANG Hongying,ZHANG Songtao,et al.Analysis of key genes expression of flavonoids in flue-cured tobacco in different ecological regions[J].Journal of Henan Agricultural Sciences,2014,43(05):52-55.
[13]Jia H,Wang J,Yang Y,et al.Changes in flavonol content and transcript levels of genes in the flavonoid pathway in tobacco under phosphorus deficiency[J].Plant Growth Regulation,2015,76(2):225-231.
[14]丁福章,李继新,袁有波,等.烟草不同组织总RNA的提取方法初探[J].中国农学通报,2007,23(12):98-101.DING Fuzhang,LI Jixin,YUAN Youbo,et al.Preliminary study on extraction methods of total RNA from different tissues of tobacco[J].Chinese Agricultural Science Bulletin,2007,23(12):98-101.
[15]蒋博文,魏硕,王涛,等.采后烟叶软化与淀粉、糖代谢及相关基因表达的研究[J].中国农业科技导报,2018(03):30-38.JIANG Bowen,WEI Shuo,WANG Tao,et al.Study on postharvest tobacco leaf softening and starch,sugar metabolism and related gene expression[J].Journal of Agricultural Science and Technology,2018(03):30-38.
[16]Yang Y,He F,Yu L,et al.Influence of Drought on Oxidative Stress and Flavonoid Production in Cell Suspension Culture of Glycyrrhiza inflata Batal[J].Zeitschrift Fur Naturforschung C AJournal of Biosciences,2007,62(5-6):410-416.
[17]杨新周,金诚,刘汗青.热裂解-冷阱聚焦-气相色谱/质谱联用法研究烟草中芸香苷的热裂解行为[J].化学试剂,2014,36(01):61-64.YANG Xinzhou,JIN Cheng,LIU Hanqing.Thermal cracking-cold trap focusing-gas chromatography/mass spectrometry for the pyrolysis behavior of rutin in tobacco[J].Chemical Reagents,2014,36(01):61-64.
[18]李佶芸.不同抗性品种烤烟感染黑胫病后生理指标的变化[J].中国农业信息,2013(09):142-143.LI Jiyun.Changes of physiological indexes of flue-cured tobacco with different resistance varieties after infection with black shank[J].China Agriculture Information,2013(09):142-143.
[19]颜侃,陈宗瑜.不同生态条件对烤烟形态及相关生理指标的影响[J].生态学报,2012,32(10):3087-3097.YAN Kan,CHEN Zongyu.Effects of Different Ecological Conditions on Morphology and Related Physiological Indexes of Flue-cured Tobacco[J].Acta Ecologica Sinica,2012,32(10):3087-3097.
[20]金霞,赵正雄,李忠环,等.不同施氮量烤烟赤星病发生与发病初期氮营养、生理状况关系研究[J].植物营养与肥料学报,2008(05):940-946.JIN Xia,ZHAO Zhengxiong,LI Zhonghuan,et al.Study on the relationship between nitrogen nutrition and physiological status of flue-cured tobacco with different nitrogen application rates[J].Journal of Plant Nutrition and Fertilizers,2008(05):940-946.
[21]钟楚,陈宗瑜,毛自朝,等.滤减UV-B辐射对烟叶可溶性蛋白、光合色素和类黄酮的影响[J].广西植物,2010,30(04):501-506+457.ZHONG Chun,CHEN Zongyu,MAO Zichao,et al.Effect of UV-BRadiation on Soluble Protein,Photosynthetic Pigment and Flavonoids in Tobacco Leaves[J].Guihaia,2010,30(04):501-506+457.
[22]董卓娅.云南不同纬度烟区烤烟K326类黄酮和丙二醛含量比较研究[J].安徽农业科学,2012,40(26):12805-12807.DONG Zhuoya.Comparative Study on the Contents of K326Flavonoids and Malondialdehyde in Flue-cured Tobacco in Different Latitude Areas of Yunnan Province[J].Journal of Anhui Agricultural Sciences,2012,40(26):12805-12807.
[23]陈帅,杨爱国.烟草重要基因篇14:烟草类黄酮合成相关基因[J].中国烟草科学,2016,37(02):95-98.CHEN Shuai,YANG Aiguo.Important Genes of Tobacco Part 14:Tobacco Flavonoid Synthesis Related Genes[J].Chinese Tobacco Science,2016,37(02):95-98.
[24]彭东,陈爱国,梁洪波,等.不同种质材料烟草各时期多酚含量和组成差异分析[C]//山东植物生理学会第七次代表大会暨植物生物学与现代农业研讨会论文集.2012.PENG Dong,CHEN Aiguo,LIANG Hongbo,et al.Analysis of polyphenol content and composition difference in tobacco of different germplasm materials in different periods[C]//Proceedings of the Seventh Congress of the Plant Physiology Society of Shandong and the Symposium on Plant Biology and Modern Agriculture.2012.
[25]王爱华.烤烟生长和调制过程中主要多酚类物质代谢动态的研究[D].郑州:河南农业大学,2005.WANG Aihua.Study on the metabolism of major polyphenols in the growth and regulation of flue-cured tobacco[D].Zhengzhou:Henan Agricultural University,2005.
[26]连文力.施氮量对烟叶成熟期重要物质代谢及基因表达的影响研究[D].郑州:河南农业大学,2016.LIAN Wenli.Effects of nitrogen application rate on metabolism and gene expression of important substances in tobacco leaf maturity[D].Zhengzhou:Henan Agricultural University,2016.
[27]杨慧芹.温度和水分对烟草多酚代谢的影响及种子引发对提高烟草种子和幼苗抗逆性的效应[D].昆明:云南师范大学,2015.Yang Huiqin,2015.Effects of Temperature and Water Status on Polyphenols Metabolism in Tobacco Plants and Effects of Seed Priming on Stress Resistance in Tobacco Seeds and Seedlings[D].Kunming:Yunnan Normal University.
[28]Hu W J,Kawaoka A,Tsai C J,et al.Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoAligase genes in aspen(Populus tremuloides)[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(9):5407-5412.