6种不结球白菜材料叶片抗氧化活性的比较及花青苷合成相关基因的表达分析
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摘要
[目的]本文旨在比较不结球白菜不同材料间叶片总花青苷含量及其抗氧化活性,探究花青苷合成相关基因的表达特性。[方法]以6种不结球白菜材料为试材,采用DPPH(1,1-二苯基-2-三硝基苯肼)法和铁离子还原能力(FRAP)法检测了叶片提取液抗氧化活性,并测定了花青苷合成相关结构基因(BrcF3H、BrcDFR、BrcANS、BrcCHI、BrcCHS、BrcUFGT)和调节基因(BrcTT8、BrcTTG1、BrcMYB75、BrcMYB90、BrcGL3、BrcEGL3.1、BrcEGL3.2)的表达水平。[结果]在6种不结球白菜材料中,深紫色材料NJZX1-4的总花青苷含量最高,为(801.5±57.4)μg·g~(-1)。6种材料叶片提取液的DPPH自由基清除率为(49.31±3.91)%~(84.94±6.20)%,铁离子还原能力为(256.8±136.3)~(1 279.5±202.0)μg·g~(-1),其中紫色材料NJZX1~(-1)叶片的DPPH自由基清除率和铁离子还原能力最强,且总花青苷含量与DPPH自由基清除率和铁离子还原能力均存在极显著正相关。结构基因BrcF3H、BrcDFR、BrcANS和调节基因BrcTT8在6种材料中的表达量与总花青苷的含量呈正相关。调节基因BrcMYB75和BrcTTG1在紫色材料NJZX1-4叶片中具有较高的表达量,而BrcEGL3.2和BrcMYB90基因在所有材料中均无表达。[结论]不结球白菜不同材料间花青苷含量和抗氧化活性存在显著差异,且紫色材料富含的花青苷在高抗氧化性方面发挥了重要作用。此外,3个结构基因和3个调节基因的表达水平与总花青苷含量密切相关,在调控不结球白菜叶片着色过程中起重要作用。
[Objectives]The aim of this study was to compare the antioxidant activity of total anthocyanins content in the leaves of different non-heading Chinese cabbage materials,and to investigate the expression patterns of genes related to anthocyanins biosynthesis in these materials. [Methods]We used six materials of non-heading Chinese cabbages,and measured the antioxidant activities of leaves by using DPPH(1,1-diphenyl-2-picrylhydrazyl) and FRAP(ferric reducing ability of plasma) methods. In addition,we determined the expression levels of related structural genes(BrcF3H,BrcDFR,BrcANS,BrcCHI,BrcCHS,BrcUFGT) and regulatory genes(BrcTT8,BrcTTG1,BrcMYB75,BrcMYB90,BrcGL3,BrcEGL3.1,BrcEGL3.2) related to anthocyanins biosynthesis.[Results]Among six materials,total anthocyanins content was the highest in the deep purple material NJZX1-4,which was(801.5±57.4) μg·g~(-1). DPPH free radical scavenging rates of six materials were(49.31±3.91) %-(84.94± 6.20) %,and FRAP values were(256.8±136.3)-(1 279.5±202.0) μg·g~(-1),which was higher in purple material NJZX1~(-1) than those of other materials. The correlation among total anthocyanins content,DPPH radical scavenging rate and FRAP antioxidant capacity were extremely significant.Moreover,we found that the expression of anthocyanins structural genes(BrcF3 H,BrcDFR and BrcANS) and regulatory genes(BrcTT8) were positively correlated with the content of total anthocyanins. Regulatory genes BrcEGL3. 2 and BrcMYB90 had no expression in all materials,and BrcMYB75 and BrcTTG1 were highly expressed in the deep purple leaf material NJZX1-4.[Conclusions]There were significant differences in total anthocyanins content and antioxidant activity among different non-headingChinese cabbage materials,and anthocyanins played an important role in antioxidant activities in leaves. The expression levels of three anthocyanins structural genes and three regulatory genes had close relation with the accumulation of anthocyanins in leaves,indicating that these genes might play an important role in regulating the coloring process in leaves of non-heading Chinese cabbage.
[Objectives]The aim of this study was to compare the antioxidant activity of total anthocyanins content in the leaves of different non-heading Chinese cabbage materials,and to investigate the expression patterns of genes related to anthocyanins biosynthesis in these materials. [Methods]We used six materials of non-heading Chinese cabbages,and measured the antioxidant activities of leaves by using DPPH(1,1-diphenyl-2-picrylhydrazyl) and FRAP(ferric reducing ability of plasma) methods. In addition,we determined the expression levels of related structural genes(BrcF3H,BrcDFR,BrcANS,BrcCHI,BrcCHS,BrcUFGT) and regulatory genes(BrcTT8,BrcTTG1,BrcMYB75,BrcMYB90,BrcGL3,BrcEGL3.1,BrcEGL3.2) related to anthocyanins biosynthesis.[Results]Among six materials,total anthocyanins content was the highest in the deep purple material NJZX1-4,which was(801.5±57.4) μg·g~(-1). DPPH free radical scavenging rates of six materials were(49.31±3.91) %-(84.94± 6.20) %,and FRAP values were(256.8±136.3)-(1 279.5±202.0) μg·g~(-1),which was higher in purple material NJZX1~(-1) than those of other materials. The correlation among total anthocyanins content,DPPH radical scavenging rate and FRAP antioxidant capacity were extremely significant.Moreover,we found that the expression of anthocyanins structural genes(BrcF3 H,BrcDFR and BrcANS) and regulatory genes(BrcTT8) were positively correlated with the content of total anthocyanins. Regulatory genes BrcEGL3. 2 and BrcMYB90 had no expression in all materials,and BrcMYB75 and BrcTTG1 were highly expressed in the deep purple leaf material NJZX1-4.[Conclusions]There were significant differences in total anthocyanins content and antioxidant activity among different non-headingChinese cabbage materials,and anthocyanins played an important role in antioxidant activities in leaves. The expression levels of three anthocyanins structural genes and three regulatory genes had close relation with the accumulation of anthocyanins in leaves,indicating that these genes might play an important role in regulating the coloring process in leaves of non-heading Chinese cabbage.
引文
[1]李长新.紫色小白菜花青素理化性质研究[D].杨凌:西北农林科技大学,2011.Li C X.The study of physicochemical property of anthocyanidin in purple pakchoi[D].Yangling:Northwest A&F University,2011(in Chinese with English abstract).
[2]徐学玲,赵岫云,王丹,等.HPLC-ESI-MS分析紫色小白菜中花青苷组成分析[J].食品工业科技,2014,35(11):278-281.Xu X L,Zhao X Y,Wang D,et al.HPLC-ESI-MS analysis of the structure and content of anthocyanins in purple pakchoi[J].Science and Technology of Food Industry,2014,35(11):278-277(in Chinese with English abstract).
[3]Tanaka Y,Sasaki N,Ohmiya A.Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids[J].Plant Journal for Cell and Molecular Biology,2008,54(4):733-749.
[4]Pascual-Teresa S D,Sanchez-Ballesta M T.Anthocyanins:from plant to health[J].Phytochemistry Reviews,2008,7(2):281-299.
[5]Lam T K,Gallicchio L,Boyd K,et al.Cruciferous vegetable consumption and lung cancer risk:a systematic review[J].Cancer Epidemiology Biomarkers&Prevention,2009,18(1):184-195.
[6]Poppel G V,Verhoeven D T H,Verhagen H,et al.Brassica vegetables and cancer prevention[J].Advances in Experimental Medicine&Biology,1999,472(472):159-168.
[7]王荣姣,李昭华,黄红林,等.北陆花青素的抗氧化作用[J].安徽农业科学,2011,39(12):7061-7063.Wang R J,Li Z H,Huang H L,et al.Antioxidative activity of anthocyanins from Northland[J].Journal of Anhui Agricultural Sciences,2011,39(12):7061-7063(in Chinese with English abstract).
[8]倪勤学,霍艳荣,陆国权.花青苷保健功能的研究进展[J].安徽农业科学,2010,38(35):20025-20028.Ni Q X,Huo Y R,Lu G Q.Advances in health functions of anthocyanins[J].Journal of Anhui Agricultural Sciences,2010,38(35):20025-20028(in Chinese with English abstract).
[9]Holton T A,Cornish E C.Genetics and biochemistry of anthocyanin biosynthesis[J].Plant Cell,1995,7(7):1071-1083.
[10]Grotewold E.The genetics and biochemistry of floral pigments[J].Annual Review of Plant Biology,2006,57(1):761-780.
[11]王小青,韩键,文杨,等.呈色机制不同的桃叶片花色素苷积累及合成相关基因表达的季节性差异[J].南京农业大学学报,2016,39(6):924-931.DOI:10.7685/jnau.201602027.Wang X Q,Han J,Wen Y,et al.Seasonal difference in the expression pattern of genes related to anthocyanin accumulation and biosynthesis inleaves of peach with different coloration modes[J].Journal of Nanjing Agricultural University,2016,39(6):924-931(in Chinese with English abstract).
[12]许玉超,侯喜林,徐玮玮,等.紫色不结球白菜花青苷合酶基因BrcA NS的克隆与表达分析[J].作物学报,2016,42(6):850-859.Xu Y C,Hou X L,Xu W W,et al.Cloning and expression analysis of anthocyanidin synthase gene BrcA NS from purple non-heading Chinese cabbage[J].Acta Agronomica Sinica,2016,42(6):850-859(in Chinese with English abstract).
[13]殷丽琴,韦献雅,钟成,等.不同品种彩色马铃薯总花青苷含量与总抗氧化活性[J].食品科学,2014,35(5):96-100.Yin L Q,Wei X Y,Zhong C,et al.Total anthocyanin content and total antioxidant activities in different varieties of pigmented potato(Solanum tuberosum L.)[J].Food Science,2014,35(5):96-100(in Chinese with English abstract).
[14]Benzie I F,Strain J J.The ferric reducing ability of plasma(FRAP)as a measure of‘antioxidant power’:the FRAP assay[J].Analytical Biochemistry,1996,239:70-76.
[15]李妍,王雪花,陈忠文,等.不结球白菜抗坏血酸合成基因BcG ME的同源克隆及胁迫下的表达分析[J].南京农业大学学报,2016,39(2):205-212.DOI:10.7685/jnau.201507026.Li Y,Wang X H,Chen Z W,et al.Homologous cloning and expression analysis of ascorbic acid biosynthesis gene BcG ME under stress from non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2016,39(2):205-212(in Chinese with English abstract).
[16]谭国飞,王枫,贾晓玲,等.芹菜甘露醇脱氢酶基因的分离与表达分析[J].园艺学报,2013,40(11):2189-2198.Tan G F,Wang F,Jia X L,et al.Isolation and expression of mannitol dehydrogenase gene in celery[J].Acta Horticulturae Sinica,2013,40(11):2189-2198(in Chinese with English abstract).
[17]Podsedek A.Natural antioxidants and antioxidant capacity of Brassica vegetables:a review[J].LWT,2007,40(1):1-11.
[18]张淑江,马越,徐学玲,等.芸薹属5种紫红色蔬菜花青素苷含量及组分分析[J].园艺学报,2014,41(7):1451-1460.Zhang S J,Ma Y,Xu X L,et al.Components and amounts of anthocyanins in several Brassica vegetables[J].Acta Horticulturae Sinica,2014,41(7):1451-1460(in Chinese with English abstract).
[19]Gong Z Z,Yamazaki M,Sugiyama M,et al.Cloning and molecular analysis of struetural genes involved in anthocyanin biosynthesis and expressed in a forma-specific manner in Perilla frutescens[J].Plant Molecular Biology,1997,35(6):915-927.
[20]Sparvoli F,Martin C,Scienza A,et al.Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape(Vitis vinifera L.)[J].Plant Molecular Biology,1994,24(5):743.
[21]Xie D Y,Jackson L A,Cooper J D,et al.Molecular and biochemical analysis of two cD NA clones encoding dihydroflavonol-4-reductase from Medicago truncatula[J].Plant Physiology,2004,134(3):979-994.
[22]Huang Y,Gou J Q,Jia Z,et al.Molecular cloning and characterization of two genes encoding dihydroflavonol-4-reductase from Populus trichocarpa[J].PLoS One,2012,7(2):e30364.
[23]Zhang B,Hu Z,Zhang Y,et al.A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale(Brassica oleracea var.acephala f.tricolor)[J].Plant Cell Reports,2012,31(2):281-289.
[24]Albert N W,Davies K M,Lewis D H,et al.A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots[J].The Plant Cell,2014,26(3):962-980.
[25]Baudry A,Caboche M,Lepiniec L.TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and b HLHfactors,allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana[J].The Plant Journal,2006,46(5):768-779.
[26]刘晓芬,李方,殷学仁,等.花青苷生物合成转录调控研究进展[J].园艺学报,2013,40(11):2295-2306.Liu X F,Li F,Yin X R,et al.Recent advances in the transcriptional regulation of anthocyanin biosynthesis[J].Acta Horticulturae Sinica,2013,40(11):2295-2306(in Chinese with English abstract).
[27]Gonzalez A,Zhao M,Leavitt J M,et al.Regulation of the anthocyanin biosynthetic pathway by the TTG1/b HLH/Myb transcriptional complex in Arabidopsis seedlings[J].Plant Journal,2008,53(5):814-827.
[28]Zhang F,Gonzalez A M,Payne C T,et al.A network of redundant b HLH proteins functions in all TTG1-dependent pathways of Arabidopsis[J].Development,2003,130(20):4859-4869.
[2]徐学玲,赵岫云,王丹,等.HPLC-ESI-MS分析紫色小白菜中花青苷组成分析[J].食品工业科技,2014,35(11):278-281.Xu X L,Zhao X Y,Wang D,et al.HPLC-ESI-MS analysis of the structure and content of anthocyanins in purple pakchoi[J].Science and Technology of Food Industry,2014,35(11):278-277(in Chinese with English abstract).
[3]Tanaka Y,Sasaki N,Ohmiya A.Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids[J].Plant Journal for Cell and Molecular Biology,2008,54(4):733-749.
[4]Pascual-Teresa S D,Sanchez-Ballesta M T.Anthocyanins:from plant to health[J].Phytochemistry Reviews,2008,7(2):281-299.
[5]Lam T K,Gallicchio L,Boyd K,et al.Cruciferous vegetable consumption and lung cancer risk:a systematic review[J].Cancer Epidemiology Biomarkers&Prevention,2009,18(1):184-195.
[6]Poppel G V,Verhoeven D T H,Verhagen H,et al.Brassica vegetables and cancer prevention[J].Advances in Experimental Medicine&Biology,1999,472(472):159-168.
[7]王荣姣,李昭华,黄红林,等.北陆花青素的抗氧化作用[J].安徽农业科学,2011,39(12):7061-7063.Wang R J,Li Z H,Huang H L,et al.Antioxidative activity of anthocyanins from Northland[J].Journal of Anhui Agricultural Sciences,2011,39(12):7061-7063(in Chinese with English abstract).
[8]倪勤学,霍艳荣,陆国权.花青苷保健功能的研究进展[J].安徽农业科学,2010,38(35):20025-20028.Ni Q X,Huo Y R,Lu G Q.Advances in health functions of anthocyanins[J].Journal of Anhui Agricultural Sciences,2010,38(35):20025-20028(in Chinese with English abstract).
[9]Holton T A,Cornish E C.Genetics and biochemistry of anthocyanin biosynthesis[J].Plant Cell,1995,7(7):1071-1083.
[10]Grotewold E.The genetics and biochemistry of floral pigments[J].Annual Review of Plant Biology,2006,57(1):761-780.
[11]王小青,韩键,文杨,等.呈色机制不同的桃叶片花色素苷积累及合成相关基因表达的季节性差异[J].南京农业大学学报,2016,39(6):924-931.DOI:10.7685/jnau.201602027.Wang X Q,Han J,Wen Y,et al.Seasonal difference in the expression pattern of genes related to anthocyanin accumulation and biosynthesis inleaves of peach with different coloration modes[J].Journal of Nanjing Agricultural University,2016,39(6):924-931(in Chinese with English abstract).
[12]许玉超,侯喜林,徐玮玮,等.紫色不结球白菜花青苷合酶基因BrcA NS的克隆与表达分析[J].作物学报,2016,42(6):850-859.Xu Y C,Hou X L,Xu W W,et al.Cloning and expression analysis of anthocyanidin synthase gene BrcA NS from purple non-heading Chinese cabbage[J].Acta Agronomica Sinica,2016,42(6):850-859(in Chinese with English abstract).
[13]殷丽琴,韦献雅,钟成,等.不同品种彩色马铃薯总花青苷含量与总抗氧化活性[J].食品科学,2014,35(5):96-100.Yin L Q,Wei X Y,Zhong C,et al.Total anthocyanin content and total antioxidant activities in different varieties of pigmented potato(Solanum tuberosum L.)[J].Food Science,2014,35(5):96-100(in Chinese with English abstract).
[14]Benzie I F,Strain J J.The ferric reducing ability of plasma(FRAP)as a measure of‘antioxidant power’:the FRAP assay[J].Analytical Biochemistry,1996,239:70-76.
[15]李妍,王雪花,陈忠文,等.不结球白菜抗坏血酸合成基因BcG ME的同源克隆及胁迫下的表达分析[J].南京农业大学学报,2016,39(2):205-212.DOI:10.7685/jnau.201507026.Li Y,Wang X H,Chen Z W,et al.Homologous cloning and expression analysis of ascorbic acid biosynthesis gene BcG ME under stress from non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2016,39(2):205-212(in Chinese with English abstract).
[16]谭国飞,王枫,贾晓玲,等.芹菜甘露醇脱氢酶基因的分离与表达分析[J].园艺学报,2013,40(11):2189-2198.Tan G F,Wang F,Jia X L,et al.Isolation and expression of mannitol dehydrogenase gene in celery[J].Acta Horticulturae Sinica,2013,40(11):2189-2198(in Chinese with English abstract).
[17]Podsedek A.Natural antioxidants and antioxidant capacity of Brassica vegetables:a review[J].LWT,2007,40(1):1-11.
[18]张淑江,马越,徐学玲,等.芸薹属5种紫红色蔬菜花青素苷含量及组分分析[J].园艺学报,2014,41(7):1451-1460.Zhang S J,Ma Y,Xu X L,et al.Components and amounts of anthocyanins in several Brassica vegetables[J].Acta Horticulturae Sinica,2014,41(7):1451-1460(in Chinese with English abstract).
[19]Gong Z Z,Yamazaki M,Sugiyama M,et al.Cloning and molecular analysis of struetural genes involved in anthocyanin biosynthesis and expressed in a forma-specific manner in Perilla frutescens[J].Plant Molecular Biology,1997,35(6):915-927.
[20]Sparvoli F,Martin C,Scienza A,et al.Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape(Vitis vinifera L.)[J].Plant Molecular Biology,1994,24(5):743.
[21]Xie D Y,Jackson L A,Cooper J D,et al.Molecular and biochemical analysis of two cD NA clones encoding dihydroflavonol-4-reductase from Medicago truncatula[J].Plant Physiology,2004,134(3):979-994.
[22]Huang Y,Gou J Q,Jia Z,et al.Molecular cloning and characterization of two genes encoding dihydroflavonol-4-reductase from Populus trichocarpa[J].PLoS One,2012,7(2):e30364.
[23]Zhang B,Hu Z,Zhang Y,et al.A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale(Brassica oleracea var.acephala f.tricolor)[J].Plant Cell Reports,2012,31(2):281-289.
[24]Albert N W,Davies K M,Lewis D H,et al.A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots[J].The Plant Cell,2014,26(3):962-980.
[25]Baudry A,Caboche M,Lepiniec L.TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and b HLHfactors,allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana[J].The Plant Journal,2006,46(5):768-779.
[26]刘晓芬,李方,殷学仁,等.花青苷生物合成转录调控研究进展[J].园艺学报,2013,40(11):2295-2306.Liu X F,Li F,Yin X R,et al.Recent advances in the transcriptional regulation of anthocyanin biosynthesis[J].Acta Horticulturae Sinica,2013,40(11):2295-2306(in Chinese with English abstract).
[27]Gonzalez A,Zhao M,Leavitt J M,et al.Regulation of the anthocyanin biosynthetic pathway by the TTG1/b HLH/Myb transcriptional complex in Arabidopsis seedlings[J].Plant Journal,2008,53(5):814-827.
[28]Zhang F,Gonzalez A M,Payne C T,et al.A network of redundant b HLH proteins functions in all TTG1-dependent pathways of Arabidopsis[J].Development,2003,130(20):4859-4869.