柑橘4CL基因家族的结构及其功能分析
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摘要
植物中编码类黄酮和木质素生物合成关键酶,4–香豆酸:辅酶A连接酶(4CL)的基因通常是家族基因。通过生物信息分析,从克里曼丁橘(Citrus clementina)基因组数据库中筛选出若干条拟为4CL基因的序列。通过对其蛋白结构进行分析,发现其中仅有3个基因具有典型的4CL家族基因的特性。聚类分析结果表明柑橘Cit4CL1聚到ClassⅠ,Cit4CL2和Cit4CL3聚到ClassⅡ。利用荧光定量PCR(q PCR)对北京柠檬不同组织和发育过程中的果实Cit4CL的表达进行分析,发现各成员的表达存在明显的差异,其中Cit4CL1的表达水平与类黄酮含量呈显著正相关,说明该基因对调控柑橘类黄酮的生物合成具有重要作用。Cit4CL2和Cit CL3的表达与类黄酮的含量相关性不显著,但其表达的组织特异性和木质素生物合成较为活跃的组织明显相关,表明这两个基因是与木质素生物合成相关的基因。
4-coumarate co-enzyme A ligase(4 CL) gene is a gene family in the plants. It is responsible for biosynthesis of flavonoides and lignin. A group of 4 CL-like genes have been discovered from the genomic database of Citrus clementina. Analysis of their protein structures indicated that only three genes possessed typical characters of 4 CL family. Phylogenetic relationship analysis divided those three citrus Cit4 CL genes into two groups:ClassⅠand ClassⅡ. Gene expression profiles in Meyer lemon[C. limon(L.)Burm. f.] demonstrated a tissue-specific and temporal-specific fashion. The expression of Cit4 CL1 was highly positively correlated with the concentration of flavonoids,indicating that this gene is important in the biosynthesis of flavonoids. However,the expression of the Cit4 CL2 and Cit4 CL3 genes did not relate to the flavonoids contents,but showed strong relationship with the tissues where the lignin biosynthesis occur red. This suggested that those two genes could be involved in the lignification. This study provided the basic information on function of 4 CL family genes in citrus.
4-coumarate co-enzyme A ligase(4 CL) gene is a gene family in the plants. It is responsible for biosynthesis of flavonoides and lignin. A group of 4 CL-like genes have been discovered from the genomic database of Citrus clementina. Analysis of their protein structures indicated that only three genes possessed typical characters of 4 CL family. Phylogenetic relationship analysis divided those three citrus Cit4 CL genes into two groups:ClassⅠand ClassⅡ. Gene expression profiles in Meyer lemon[C. limon(L.)Burm. f.] demonstrated a tissue-specific and temporal-specific fashion. The expression of Cit4 CL1 was highly positively correlated with the concentration of flavonoids,indicating that this gene is important in the biosynthesis of flavonoids. However,the expression of the Cit4 CL2 and Cit4 CL3 genes did not relate to the flavonoids contents,but showed strong relationship with the tissues where the lignin biosynthesis occur red. This suggested that those two genes could be involved in the lignification. This study provided the basic information on function of 4 CL family genes in citrus.
引文
Akachi T,Shiina Y,Ohishi Y,Kawaguchi T,Kawagishi H,Morita T,Mori M,Sugiyama K.2010.Hepatoprotective effects of flavonoids from shekwasha(Citrus depressa)against D-galactosamine-induced liver injury in rats.Journal of Nutritional Science and Vitaminology(Tokyo),56(1):60-67.
Asikin Y,Taira I,Inafuku-Teramoto S,Sumi H,Ohta H,Takara K,Wada K.2012.The composition of volatile aroma components,flavanones,and polymethoxylated flavones in Shiikuwasha(Citrus depressa Hayata)peels of different cultivation lines.Journal of Agricultural and Food Chemistry,60(32):7973-7980.
Bailey T L,Williams N,Misleh C,Li W W.2006.MEME:discovering and analyzing DNA and protein sequence motifs.Nucleic Acids Research,34:W369-W373.
Cao Y P,Han Y H,Li D H,Lin Y,Cai Y P.2016.Systematic analysis of the 4-coumarate:coenzyme A ligase(4CL)related genes and expression profiling during fruit development in the Chinese pear.Genes,7(10):doi.org/10.3390/genes7100089.
Costa M A,Bedgar D L,Moinuddin S G A,Kim K W,Cardenas C L,Cochrane F C,Shockey J M,Helms G L,Amakura Y,Takahashi H,Milhollan J K,Davin L B,Browse J,Lewis N G.2005.Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis:syringyl lignin and sinapate/sinapyl alcohol derivative formation.Phytochemistry,66(17):2072-2091.
Douglas C,Hoffmann H,Schulz W,Hahlbrock K.1987.Structure and elicitor or UV-light-stimulated expression of 2,4-coumarate-CoA ligase genes in parsley.Embo Journal,6(5):1189-1195.
Du Q Z,Chen H.2010.The methoxyflavones in Citrus reticulata Blanco cv.Ponkan and their antiproliferative activity against cancer cells.Food Chemistry,119(2):567-572.
Durand-Hulak M,Dugrand A,Duval T,Bidel L P R,Jay-Allemand C,Froelicher Y,Bourgaud F,Fanciullino A L.2015.Mapping the genetic and tissular diversity of 64 phenolic compounds in Citrus species using a UPLC-MS approach.Annals of Botany,115(5):861-877.
Ehlting J,Buttner D,Wang Q,Douglas C J,Somssich I E,Kombrink E.1999.Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms.Plant Journal,19(1):9-20.
Endler A,Martens S,Wellmann F,Matern U.2008.Unusually divergent 4-coumarate:CoA-ligases from Ruta graveolens L.Plant Molecular Biology,67(4):335-346.
Golawska S,Sprawka I,Lukasik I,Golawski A.2014.Are naringenin and quercetin useful chemicals in pest-management strategies?Journal of Pest Science,87(1):173-180.
Goodstein D M,Shu S Q,Howson R,Neupane R,Hayes R D,Fazo J,Mitros T,Dirks W,Hellsten U,Putnam N,Rokhsar D S.2012.Phytozome:a comparative platform for green plant genomics.Nucleic Acids Research,40(D1):D1178-D1186.
Gui J S,Shen J H,Li L G.2011.Functional characterization of evolutionarily divergent 4-coumarate:coenzyme A ligases in rice.Plant Physiology,157(2):574-586.
Hamberger B,Hahlbrock K.2004.The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare,sinapate-activating and three commonly occurring isoenzymes.Proceedings of the National Academy of Sciences of the United States of America,101(7):2209-2214.
Harding S A,Leshkevich J,Chiang V L,Tsai C J.2002.Differential substrate inhibition couples kinetically distinct 4-coumarate:coenzyme a ligases with spatially distinct metabolic roles in quaking aspen.Plant Physiology,128(2):428-438.
Hu B,Jin J,Guo A,Zhang H,Luo J,Gao G.2014.GSDS 2.0:an upgraded gene feature visualization server.Oxford,England:Bioinformatics.
Hu W J,Kawaoka A,Tsai C J,Lung J,Osakabe K,Ebinuma H,Chiang V L.1998.Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen(Populus tremuloides).Proceedings of the National Academy of Sciences of the United States of America,95(9):5407-5412.
Hu Y,Gai Y,Yin L,Wang X,Feng C,Feng L,Li D,Jiang X N,Wang D C.2010.Crystal structures of a Populus tomentosa 4-coumarate:CoA ligase shed light on its enzymatic mechanisms.The Plant Cell,22(9):3093-3104.
Hue H T T,Ha D T T,Hai N V,Hien L T T.2016.Isolation and characterization of the 4-coumarate:coenzyme A ligase(4CL1)promoter from Eucalyptus camaldulensis.Physiology and Molecular Biology of Plants,22(3):399-405.
Lee Y S,Cha B Y,Saito K,Choi S S,Wang X X,Choi B K,Yonezawa T,Teruya T,Nagai K,Woo J T.2011.Effects of a Citrus depressa Hayata(shiikuwasa)extract on obesity in high-fat diet-induced obese mice.Phytomedicine,18(8-9):648-654.
Li Y,Gu H,Qi C.2013.Expression of lignin synthesis key genes of F5H,4CL and COMT on loding resistance of Brassica napus L.Chinese Journal of Oil Crop Sciences,35(1):24-28.
Li Z B,Li C F,Li J,Zhang Y S.2014.Molecular cloning and functional characterization of two divergent 4-coumarate:coenzyme A ligases from Kudzu(Pueraria lobata).Biological&Pharmaceutical Bulletin,37(1):113-122.
Lin Jianzhong.2009.Functional analysis of Arabidopsis 4CL3 gene in flavonoid[Ph.D.Dissertation].Changsha:Hunan University.(in Chinese)林建中.2009.拟南芥4CL3基因在类黄酮合成代谢中的功能分析[博士论文].长沙:湖南大学.
Lindermayr C,M?llers B,Fliegmann J,Uhlmann A,Lottspeich F,Meimberg H,Ebel J.2002.Divergent members of a soybean(Glycine max L.)4-coumarate:coenzyme A ligase gene family.European Journal of Biochemistry,269(4):1304-1315.
Lois R,Hahlbrock K.1992.Differential wound activation of members of the phenylalanine ammonia-lyase and 4-coumarate:CoA ligase gene families in various organs of parsley plants.Zeitschrift für Naturforschung section C,47(1-2):90-94.
Nakatsu T,Ichiyama S,Hiratake J,Saldanha A,Kobashi N,Sakata K,Kato H.2006.Structural basis for the spectral difference in luciferase bioluminescence.Nature,440(7082):372-376.
Osakabe Y,Osakabe K,Chiang V L.2009.Isolation of 4-coumarate CoA ligase gene promoter from loblolly pine(Pinus taeda)and characterization of tissue-specific activity in transgenic tobacco.Plant Physiology and Biochemistry,47(11-12):1031-1036.
Ran Yue,Jiao Bining,Zhao Qiyang,Tian Ling,Su Xuesu,Zeng Chaobo.2013.Simultaneous determination of 11 flavonoids in citrus fruits by ultra performance liquid chromatography.Food Science,34(4):168-172.(in Chinese)冉玥,焦必宁,赵其阳,田玲,苏学素,曾朝波.2013.超高效液相色谱法同时测定柑橘中11种类黄酮物质.食品科学,34(4):168-172.
Schneider K,Hovel K,Witzel K,Hamberger B,Schomburg D,Kombrink E,Stuible H P.2003.The substrate specificity-determining amino acid code of 4-coumarate:coA ligase.Proceedings of the National Academy of Sciences of the United States of America,100(14):8601-8606.
Sergeev I N,Ho C T,Li S M,Colby J,Dushenkov S.2007.Apoptosis-inducing activity of hydroxylated polymethoxyflavones and polymethoxyflavones from orange peel in human breast cancer cells.Molecular Nutrition&Food Research,51(12):1478-1484.
Soltani B M,Ehlting J,Hamberger B,Douglas C J.2006.Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members.Planta,224(5):1226-1238.
Sun H Y,Li Y,Feng S Q,Zou W H,Guo K,Fan C F,Si S L,Peng L C.2013b.Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice.Biochemical and Biophysical Research Communications,430(3):1151-1156.
Sun Y J,Qiao L P,Shen Y,Jiang P,Chen J C,Ye X Q.2013a.Phytochemical profile and antioxidant activity of physiological drop of Citrus fruits.Journal of Food Science,78(1):C37-C42.
Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.2011.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,28(10):2731-2739.
Tong Hongmei.2012.Detection of oxidation resisting activity and content of flavonoid in different Chenpi with HPLC.Western Journal of Traditional Chinese Medicine,25(7):17-19.(in Chinese)童红梅.2012.用HPCL检测不同成药时间陈皮中类黄酮含量及其抗氧化活性.西部中医药,25(7):17-19.
Wang C H,Yu J,Cai Y X,Zhu P P,Liu C Y,Zhao A C,Lu R H,Li M J,Xu F X,Yu M D.2016.Characterization and functional analysis of4-coumarate:CoA ligase genes in mulberry.PLoS ONE,11(6):doi:10.1371/journal.pone.0155814.
Zhou Jianying,Cao Ying,Sun Xia,Chen Ke,Lu Xueqin,Hu Shanglian.2010.Construction of RNAi carrier of 4CL gene of Neosinocalamus affinis and its transformation into tobacco.Journal of Fujian Forestry Science and Techology,37:28-32.(in Chinese)周建英,曹颖,孙霞,陈珂,卢学琴,胡尚连.2010.慈竹木质素合成酶基因4CL RNAi载体构建与烟草转化.福建林业科技,37(2):28-32.
Asikin Y,Taira I,Inafuku-Teramoto S,Sumi H,Ohta H,Takara K,Wada K.2012.The composition of volatile aroma components,flavanones,and polymethoxylated flavones in Shiikuwasha(Citrus depressa Hayata)peels of different cultivation lines.Journal of Agricultural and Food Chemistry,60(32):7973-7980.
Bailey T L,Williams N,Misleh C,Li W W.2006.MEME:discovering and analyzing DNA and protein sequence motifs.Nucleic Acids Research,34:W369-W373.
Cao Y P,Han Y H,Li D H,Lin Y,Cai Y P.2016.Systematic analysis of the 4-coumarate:coenzyme A ligase(4CL)related genes and expression profiling during fruit development in the Chinese pear.Genes,7(10):doi.org/10.3390/genes7100089.
Costa M A,Bedgar D L,Moinuddin S G A,Kim K W,Cardenas C L,Cochrane F C,Shockey J M,Helms G L,Amakura Y,Takahashi H,Milhollan J K,Davin L B,Browse J,Lewis N G.2005.Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis:syringyl lignin and sinapate/sinapyl alcohol derivative formation.Phytochemistry,66(17):2072-2091.
Douglas C,Hoffmann H,Schulz W,Hahlbrock K.1987.Structure and elicitor or UV-light-stimulated expression of 2,4-coumarate-CoA ligase genes in parsley.Embo Journal,6(5):1189-1195.
Du Q Z,Chen H.2010.The methoxyflavones in Citrus reticulata Blanco cv.Ponkan and their antiproliferative activity against cancer cells.Food Chemistry,119(2):567-572.
Durand-Hulak M,Dugrand A,Duval T,Bidel L P R,Jay-Allemand C,Froelicher Y,Bourgaud F,Fanciullino A L.2015.Mapping the genetic and tissular diversity of 64 phenolic compounds in Citrus species using a UPLC-MS approach.Annals of Botany,115(5):861-877.
Ehlting J,Buttner D,Wang Q,Douglas C J,Somssich I E,Kombrink E.1999.Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms.Plant Journal,19(1):9-20.
Endler A,Martens S,Wellmann F,Matern U.2008.Unusually divergent 4-coumarate:CoA-ligases from Ruta graveolens L.Plant Molecular Biology,67(4):335-346.
Golawska S,Sprawka I,Lukasik I,Golawski A.2014.Are naringenin and quercetin useful chemicals in pest-management strategies?Journal of Pest Science,87(1):173-180.
Goodstein D M,Shu S Q,Howson R,Neupane R,Hayes R D,Fazo J,Mitros T,Dirks W,Hellsten U,Putnam N,Rokhsar D S.2012.Phytozome:a comparative platform for green plant genomics.Nucleic Acids Research,40(D1):D1178-D1186.
Gui J S,Shen J H,Li L G.2011.Functional characterization of evolutionarily divergent 4-coumarate:coenzyme A ligases in rice.Plant Physiology,157(2):574-586.
Hamberger B,Hahlbrock K.2004.The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare,sinapate-activating and three commonly occurring isoenzymes.Proceedings of the National Academy of Sciences of the United States of America,101(7):2209-2214.
Harding S A,Leshkevich J,Chiang V L,Tsai C J.2002.Differential substrate inhibition couples kinetically distinct 4-coumarate:coenzyme a ligases with spatially distinct metabolic roles in quaking aspen.Plant Physiology,128(2):428-438.
Hu B,Jin J,Guo A,Zhang H,Luo J,Gao G.2014.GSDS 2.0:an upgraded gene feature visualization server.Oxford,England:Bioinformatics.
Hu W J,Kawaoka A,Tsai C J,Lung J,Osakabe K,Ebinuma H,Chiang V L.1998.Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen(Populus tremuloides).Proceedings of the National Academy of Sciences of the United States of America,95(9):5407-5412.
Hu Y,Gai Y,Yin L,Wang X,Feng C,Feng L,Li D,Jiang X N,Wang D C.2010.Crystal structures of a Populus tomentosa 4-coumarate:CoA ligase shed light on its enzymatic mechanisms.The Plant Cell,22(9):3093-3104.
Hue H T T,Ha D T T,Hai N V,Hien L T T.2016.Isolation and characterization of the 4-coumarate:coenzyme A ligase(4CL1)promoter from Eucalyptus camaldulensis.Physiology and Molecular Biology of Plants,22(3):399-405.
Lee Y S,Cha B Y,Saito K,Choi S S,Wang X X,Choi B K,Yonezawa T,Teruya T,Nagai K,Woo J T.2011.Effects of a Citrus depressa Hayata(shiikuwasa)extract on obesity in high-fat diet-induced obese mice.Phytomedicine,18(8-9):648-654.
Li Y,Gu H,Qi C.2013.Expression of lignin synthesis key genes of F5H,4CL and COMT on loding resistance of Brassica napus L.Chinese Journal of Oil Crop Sciences,35(1):24-28.
Li Z B,Li C F,Li J,Zhang Y S.2014.Molecular cloning and functional characterization of two divergent 4-coumarate:coenzyme A ligases from Kudzu(Pueraria lobata).Biological&Pharmaceutical Bulletin,37(1):113-122.
Lin Jianzhong.2009.Functional analysis of Arabidopsis 4CL3 gene in flavonoid[Ph.D.Dissertation].Changsha:Hunan University.(in Chinese)林建中.2009.拟南芥4CL3基因在类黄酮合成代谢中的功能分析[博士论文].长沙:湖南大学.
Lindermayr C,M?llers B,Fliegmann J,Uhlmann A,Lottspeich F,Meimberg H,Ebel J.2002.Divergent members of a soybean(Glycine max L.)4-coumarate:coenzyme A ligase gene family.European Journal of Biochemistry,269(4):1304-1315.
Lois R,Hahlbrock K.1992.Differential wound activation of members of the phenylalanine ammonia-lyase and 4-coumarate:CoA ligase gene families in various organs of parsley plants.Zeitschrift für Naturforschung section C,47(1-2):90-94.
Nakatsu T,Ichiyama S,Hiratake J,Saldanha A,Kobashi N,Sakata K,Kato H.2006.Structural basis for the spectral difference in luciferase bioluminescence.Nature,440(7082):372-376.
Osakabe Y,Osakabe K,Chiang V L.2009.Isolation of 4-coumarate CoA ligase gene promoter from loblolly pine(Pinus taeda)and characterization of tissue-specific activity in transgenic tobacco.Plant Physiology and Biochemistry,47(11-12):1031-1036.
Ran Yue,Jiao Bining,Zhao Qiyang,Tian Ling,Su Xuesu,Zeng Chaobo.2013.Simultaneous determination of 11 flavonoids in citrus fruits by ultra performance liquid chromatography.Food Science,34(4):168-172.(in Chinese)冉玥,焦必宁,赵其阳,田玲,苏学素,曾朝波.2013.超高效液相色谱法同时测定柑橘中11种类黄酮物质.食品科学,34(4):168-172.
Schneider K,Hovel K,Witzel K,Hamberger B,Schomburg D,Kombrink E,Stuible H P.2003.The substrate specificity-determining amino acid code of 4-coumarate:coA ligase.Proceedings of the National Academy of Sciences of the United States of America,100(14):8601-8606.
Sergeev I N,Ho C T,Li S M,Colby J,Dushenkov S.2007.Apoptosis-inducing activity of hydroxylated polymethoxyflavones and polymethoxyflavones from orange peel in human breast cancer cells.Molecular Nutrition&Food Research,51(12):1478-1484.
Soltani B M,Ehlting J,Hamberger B,Douglas C J.2006.Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members.Planta,224(5):1226-1238.
Sun H Y,Li Y,Feng S Q,Zou W H,Guo K,Fan C F,Si S L,Peng L C.2013b.Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice.Biochemical and Biophysical Research Communications,430(3):1151-1156.
Sun Y J,Qiao L P,Shen Y,Jiang P,Chen J C,Ye X Q.2013a.Phytochemical profile and antioxidant activity of physiological drop of Citrus fruits.Journal of Food Science,78(1):C37-C42.
Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.2011.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,28(10):2731-2739.
Tong Hongmei.2012.Detection of oxidation resisting activity and content of flavonoid in different Chenpi with HPLC.Western Journal of Traditional Chinese Medicine,25(7):17-19.(in Chinese)童红梅.2012.用HPCL检测不同成药时间陈皮中类黄酮含量及其抗氧化活性.西部中医药,25(7):17-19.
Wang C H,Yu J,Cai Y X,Zhu P P,Liu C Y,Zhao A C,Lu R H,Li M J,Xu F X,Yu M D.2016.Characterization and functional analysis of4-coumarate:CoA ligase genes in mulberry.PLoS ONE,11(6):doi:10.1371/journal.pone.0155814.
Zhou Jianying,Cao Ying,Sun Xia,Chen Ke,Lu Xueqin,Hu Shanglian.2010.Construction of RNAi carrier of 4CL gene of Neosinocalamus affinis and its transformation into tobacco.Journal of Fujian Forestry Science and Techology,37:28-32.(in Chinese)周建英,曹颖,孙霞,陈珂,卢学琴,胡尚连.2010.慈竹木质素合成酶基因4CL RNAi载体构建与烟草转化.福建林业科技,37(2):28-32.