基于转录组测序的金柑类黄酮糖基转移酶基因的初步分析
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摘要
植物体内类黄酮通常以糖基化的形式存在,其糖基化由UDP–糖基转移酶(UDPglycosyltransferase,UGT)催化完成。以‘浏阳金柑’果实为研究对象,利用UPLCG2-SQTOF技术对其类黄酮种类进行鉴定,共检测出11种类黄酮,包括C–葡萄糖苷类黄酮、C–新橙皮糖苷类黄酮和O–新橙皮糖苷类黄酮。基于转录组测序,筛选出49个金柑果实发育过程中高表达的UGT基因。进化分析显示,13个高表达的FcUGT可能参与了类黄酮的糖基化修饰过程。其中,1个FcUGT编码C–葡萄糖基转移酶,参与了C–葡萄糖苷类黄酮的修饰过程;9个FcUGT编码7–O–葡萄糖基转移酶,可能参与了O–新橙皮糖苷类黄酮的修饰过程;3个FcUGT编码1,2–鼠李糖基转移酶,可能参与了O–或C–新橙皮糖苷类黄酮的修饰过程。
O-and C-glycoside flavonoids are important metabolites in Fortunella crassifolia plants.Glycosylation of flavonoids is catalyzed by UDP-glycosyltransferase(UGT). In this study,we determined the flavonoid glycosides in kumquat fruits by using UPLC G2-S QTOF. A total of 11 flavonoid glycosides were detected,including C-glucoside,O-neohesperidoside,and C-neohesperidoside flavonoids.Subsequently,based on transcriptomes,we isolated 49 UGT genes with high expression levels in kumquat fruits. Further phylogenetic analysis showed that 13 UGTs of them might be involved in the glycosylation process of flavonoids. One UGT encoding C-glucosyltransferase catalyzed the C-glucosylation,9 UGTs encoding 7-O-glucosyltransferase were involved in the 7-O-glucosylation,and 3 UGTs encoding1,2-rhamnosyltransferase,can perform rhamnosylation of flavonoids.
O-and C-glycoside flavonoids are important metabolites in Fortunella crassifolia plants.Glycosylation of flavonoids is catalyzed by UDP-glycosyltransferase(UGT). In this study,we determined the flavonoid glycosides in kumquat fruits by using UPLC G2-S QTOF. A total of 11 flavonoid glycosides were detected,including C-glucoside,O-neohesperidoside,and C-neohesperidoside flavonoids.Subsequently,based on transcriptomes,we isolated 49 UGT genes with high expression levels in kumquat fruits. Further phylogenetic analysis showed that 13 UGTs of them might be involved in the glycosylation process of flavonoids. One UGT encoding C-glucosyltransferase catalyzed the C-glucosylation,9 UGTs encoding 7-O-glucosyltransferase were involved in the 7-O-glucosylation,and 3 UGTs encoding1,2-rhamnosyltransferase,can perform rhamnosylation of flavonoids.
引文
Abad-García B,Garmón-Lobato S,Sánchez-Ilárduya M B,Berrueta L A,Gallo B,Vicente F,Alonso-Salces R M.2014.Polyphenolic contents in citrus fruit juices:authenticity assessment.European Food Research and Technology,238(5):803-818.
Barreca D,Bellocco E,Caristi C,Leuzzi U,Gattuso G.2011.Kumquat(Fortunella japonica Swingle)juice:flavonoid distribution and antioxidant properties.Food Research International,44:2190-2197.
Bowles D,Isayenkova J,Lim E K,Poppenberger B.2005.Glycosyltransferases:managers of small molecules.Current Opinion in Plant Biology,8(3):254-263.
Brazier-Hicks M,Evans K M,Gershater M C,Puschmann H,Steel P G,Edwards R.2009.The C-glycosylation of flavonoids in cereals.Journal of Biological Chemistry,284(27):17926-17934.
Brugliera F,Holton T A,Stevenson T W,Farcy E,Lu C Y,Cornish E C.1994.Isolation and characterization of a cDNA clone corresponding to the Rt locus of Petunia hybrida.The Plant Journal,5(1):81-92.
Cao Yunlin,Xing Mengyun,Xu Changjie,Li Xian.2018.Biosynthesis of flavonol and its regulation in plants.Acta Horticulturae Sinica,45(1):177-192.(in Chinese)曹运琳,邢梦云,徐昌杰,李鲜.2018.植物黄酮醇生物合成及其调控研究进展.园艺学报,45(1):177-192.
Caputi L,Malnoy M,Goremykin V,Nikiforova S,Martens S.2012.A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyl transferases revealed the expansion of the family during the adaptation of plants to life on land.The Plant Journal,69(6):1030-1042.
Chen Jia-jing,Peng Zhao-xin,Shi Mei-yan,Xu Juan.2016.Advances in on flavonoid composition and metabolism in Citrus.Acta Horticulturae Sinica,43(2):384-400.(in Chinese)陈嘉景,彭昭欣,石梅艳,徐娟.2016.柑橘中类黄酮的组成与代谢研究进展.园艺学报,43(2):384-400.
Frydman A,Weisshaus O,Bar-Peled M,Huhman D V,Sumner L W,Marin F R,Lewinsohn E,Fluhr R,Gressel J,Eyal Y.2004.Citrus fruit bitter flavors:isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2-rhamnosyltransferase,a key enzyme in the biosynthesis of the bitter flavonoids of citrus.The Plant Journal,40(1):88-100.
Gachon C M,Langlois-Meurinne M,Saindrenan P.2005.Plant secondary metabolism glycosyltransferases:the emerging functional analysis.Trends in Plant Science,10(11):542-549.
Gattuso G,Barreca D,Gargiulli C,Leuzzi U,Caristi C.2007.Flavonoid composition of citrus juices.Molecules,12(8):1641-1673.
Hirade Y,Kotoku N,Terasaka K,Saijo-Hamano Y,Fukumoto A,Mizukami H.2015.Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean(Glycine max).FEBS Lett,589(15):1778-1786.
Ito T,Fujimoto S,Suito F,Shimosaka M,Taguchi G.2017.C-glycosyltransferases catalyzing the formation of di-C-glucosyl flavonoids in citrus plants.The Plant Journal,91(2):187-198.
Joe E J,Kim B G,An B C,Chong Y,Ahn J H.2010.Engineering of flavonoid O-methyltransferase for a novel regioselectivity.Molecules&Cells,30(2):137-141.
Jones P,Messner B,Nakajima J,Schaffner A R,Saito K.2003.UGT73C6 and UGT78D1,glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana.Journal of Biological Chemistry,278(45):43910-43918.
Kubo H,Nawa N,Lupsea S A.2007.Anthocyanin less1 gene of Arabidopsis thaliana encodes a UDP-glucose:flavonoid-3-O-glucosyltransferase.Journal of Plant Research,120(3):445-449.
Lewinsohn E,Berman E,Mazur Y,Gressel J.1989.(7)-Glucosylation and(1-6)-rhamnosylation of exogenous flavanones by undifferentiated Citrus cell cultures.Plant Science,61:23-28.
Lim E K,Bowles D J.2004.A class of plant glycosyltransferases involved in cellular homeostasis.EMBO Journal,23(15):2915-2922.
Lou S N,Lai Y C,Huang J D,Ho C T,Ferng L H,Chang Y C.2015.Drying effect on flavonoid composition and antioxidant activity of immature kumquat.Food Chemistry,171:356-363.
Martin C,Prescott A,Mackay S,Bartlett J,Vrijlandt E.1991.Control of anthocyanin biosynthesis in flowers of Antirrhinum majus.The Plant Journal,1(1):37-49.
Masada S,Terasaka K,Oguchi Y,Okazaki S,Mizushima T,Mizukami H.2009.Functional and structural characterization of a flavonoid glucoside1,6-glucosyltransferase from Catharanthus roseus.Plant Cell Physiology,50(8):1401-1415.
Moehs C P,Allen P V,Friedman M,Belknap W R.1997.Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato.The Plant Journal,11(2):227-236.
Nagatomo Y,Usui S,Ito T,Kato A,Shimosaka M and Taguchi G.2014.Purification,molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M.(buckwheat)cotyledon.The Plant Journal,80(3):437-448.
Ogawa K,Kawasaki A,Omura M,Yoshida T,Ikoma Y,Yano M.2001.3′,5′-Di-C-β-glucopyranosylphloretin,a flavonoid characteristic of the genus Fortunella.Phytochemistry,57:737-742.
Paquette S,M?ller B L,Bak S.2003.On the origin of family 1 plant glycosyltransferases.Phytochemistry,62(3):399-413.
Ralston E J,English J J,Dooner H K.1988.Sequence of three bronze alleles of maize and correlation with the genetic fine structure.Genetics,119(1):185-197.
Shu Qingyan,Zhu Jin,Men Siqi,Hao Qing,Wang Qianyu,Liu Zheng’an,Zeng Xiuli,Wang Liangsheng.2018.Establishing virus induced gene silencing(VIGS)system in tree peony using PsUFGT genes.Acta Horticulturae Sinica,45(1):168-176.(in Chinese)舒庆艳,朱瑾,门思琦,郝青,王倩玉,刘政安,曾秀丽,王亮生.2018.基于牡丹类黄酮糖基转移酶基因建立VIGS技术体系.园艺学报,2018,45(1):168-176.
Tanaka Y,Yonekura K,Fukuchi-Mizutani M,Fukui Y,Fujiwara H,Ashikari T,Kusumi T.1996.Molecular and biochemical characterization of
three anthocyanin synthetic enzymes from Gentiana triflora.Plant Cell Physiology,37(5):711-716.
Tohge T,Nishiyama Y,Hirai M Y,Yano M,Nakajima J,Awazuhara M,Inoue E,Takahashi H,Goodenowe D B,Kitayama M,Noji M,Yamazaki M,Saito K.2005.Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor.The Plant Journal,42(2):218-235.
Wise R P,Rohde W,Salamini F.1990.Nucleotide sequence of the Bronze-1 homologous gene from Hordeum vulgare.Plant Molecular Biology,14(2):277-279.
Yamazaki M,Gong Z,Fukuchi-Mizutani M,Fukui Y,Tanaka Y,Kusumi T,Saito K.1999.Molecular cloning and biochemical characterzation of a novel anthocyanin 5-O-glucosyltransferase by m RNA differential display for plant forms regarding anthocyanin.The Journal of Biology Chemistry,274(11):7405-7411.
Yonekura-Sakakibara K,Fukushima A,Nakabayashi R,Hanada K,Matsuda F,Sugawara S,Inoue E,Kuromori T,Ito T,Shinozaki K,Wangwattana B,Yamazaki M,Saito K.2012.Two glycosyltransferases involved in anthocyanin modification delineated by transcriptome independent component analysis in Arabidopsis thaliana.The Plant Journal,69(1):154-167.
Yonekura-Sakakibara K,Nakabayashi R,Sugawara S,Tohge T,Ito T,Koyanagi M,Kitajima M,Takayama H,Saito K.2014.A flavonoid3-O-glucoside:2″-O-glucosyltransferase responsible for terminal modification of pollen-specific flavonols in Arabidopsis thaliana.The Plant Journal,79(5):769-782.
Yonekura-Sakakibara K,Tohge T,Matsuda F,Nakabayashi R,Takayama H,Niida R,Watanabe-Takahashi A,Inoue E,Saito K.2008.Comprehensive flavonol profiling and transcriptome coexpression analysis leading to decoding gene metabolite correlations in Arabidopsis.Plant Cell,20(8):2160-2176.
Yonekura-Sakakibara K,Tohge T,Niida R,Saito K.2007.Identification of a flavonol 7-O-rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis by transcriptome coexpression analysis and reverse genetics.Journal of Biological Chemistry,282(20):14932-14941.
Barreca D,Bellocco E,Caristi C,Leuzzi U,Gattuso G.2011.Kumquat(Fortunella japonica Swingle)juice:flavonoid distribution and antioxidant properties.Food Research International,44:2190-2197.
Bowles D,Isayenkova J,Lim E K,Poppenberger B.2005.Glycosyltransferases:managers of small molecules.Current Opinion in Plant Biology,8(3):254-263.
Brazier-Hicks M,Evans K M,Gershater M C,Puschmann H,Steel P G,Edwards R.2009.The C-glycosylation of flavonoids in cereals.Journal of Biological Chemistry,284(27):17926-17934.
Brugliera F,Holton T A,Stevenson T W,Farcy E,Lu C Y,Cornish E C.1994.Isolation and characterization of a cDNA clone corresponding to the Rt locus of Petunia hybrida.The Plant Journal,5(1):81-92.
Cao Yunlin,Xing Mengyun,Xu Changjie,Li Xian.2018.Biosynthesis of flavonol and its regulation in plants.Acta Horticulturae Sinica,45(1):177-192.(in Chinese)曹运琳,邢梦云,徐昌杰,李鲜.2018.植物黄酮醇生物合成及其调控研究进展.园艺学报,45(1):177-192.
Caputi L,Malnoy M,Goremykin V,Nikiforova S,Martens S.2012.A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyl transferases revealed the expansion of the family during the adaptation of plants to life on land.The Plant Journal,69(6):1030-1042.
Chen Jia-jing,Peng Zhao-xin,Shi Mei-yan,Xu Juan.2016.Advances in on flavonoid composition and metabolism in Citrus.Acta Horticulturae Sinica,43(2):384-400.(in Chinese)陈嘉景,彭昭欣,石梅艳,徐娟.2016.柑橘中类黄酮的组成与代谢研究进展.园艺学报,43(2):384-400.
Frydman A,Weisshaus O,Bar-Peled M,Huhman D V,Sumner L W,Marin F R,Lewinsohn E,Fluhr R,Gressel J,Eyal Y.2004.Citrus fruit bitter flavors:isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2-rhamnosyltransferase,a key enzyme in the biosynthesis of the bitter flavonoids of citrus.The Plant Journal,40(1):88-100.
Gachon C M,Langlois-Meurinne M,Saindrenan P.2005.Plant secondary metabolism glycosyltransferases:the emerging functional analysis.Trends in Plant Science,10(11):542-549.
Gattuso G,Barreca D,Gargiulli C,Leuzzi U,Caristi C.2007.Flavonoid composition of citrus juices.Molecules,12(8):1641-1673.
Hirade Y,Kotoku N,Terasaka K,Saijo-Hamano Y,Fukumoto A,Mizukami H.2015.Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean(Glycine max).FEBS Lett,589(15):1778-1786.
Ito T,Fujimoto S,Suito F,Shimosaka M,Taguchi G.2017.C-glycosyltransferases catalyzing the formation of di-C-glucosyl flavonoids in citrus plants.The Plant Journal,91(2):187-198.
Joe E J,Kim B G,An B C,Chong Y,Ahn J H.2010.Engineering of flavonoid O-methyltransferase for a novel regioselectivity.Molecules&Cells,30(2):137-141.
Jones P,Messner B,Nakajima J,Schaffner A R,Saito K.2003.UGT73C6 and UGT78D1,glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana.Journal of Biological Chemistry,278(45):43910-43918.
Kubo H,Nawa N,Lupsea S A.2007.Anthocyanin less1 gene of Arabidopsis thaliana encodes a UDP-glucose:flavonoid-3-O-glucosyltransferase.Journal of Plant Research,120(3):445-449.
Lewinsohn E,Berman E,Mazur Y,Gressel J.1989.(7)-Glucosylation and(1-6)-rhamnosylation of exogenous flavanones by undifferentiated Citrus cell cultures.Plant Science,61:23-28.
Lim E K,Bowles D J.2004.A class of plant glycosyltransferases involved in cellular homeostasis.EMBO Journal,23(15):2915-2922.
Lou S N,Lai Y C,Huang J D,Ho C T,Ferng L H,Chang Y C.2015.Drying effect on flavonoid composition and antioxidant activity of immature kumquat.Food Chemistry,171:356-363.
Martin C,Prescott A,Mackay S,Bartlett J,Vrijlandt E.1991.Control of anthocyanin biosynthesis in flowers of Antirrhinum majus.The Plant Journal,1(1):37-49.
Masada S,Terasaka K,Oguchi Y,Okazaki S,Mizushima T,Mizukami H.2009.Functional and structural characterization of a flavonoid glucoside1,6-glucosyltransferase from Catharanthus roseus.Plant Cell Physiology,50(8):1401-1415.
Moehs C P,Allen P V,Friedman M,Belknap W R.1997.Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato.The Plant Journal,11(2):227-236.
Nagatomo Y,Usui S,Ito T,Kato A,Shimosaka M and Taguchi G.2014.Purification,molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M.(buckwheat)cotyledon.The Plant Journal,80(3):437-448.
Ogawa K,Kawasaki A,Omura M,Yoshida T,Ikoma Y,Yano M.2001.3′,5′-Di-C-β-glucopyranosylphloretin,a flavonoid characteristic of the genus Fortunella.Phytochemistry,57:737-742.
Paquette S,M?ller B L,Bak S.2003.On the origin of family 1 plant glycosyltransferases.Phytochemistry,62(3):399-413.
Ralston E J,English J J,Dooner H K.1988.Sequence of three bronze alleles of maize and correlation with the genetic fine structure.Genetics,119(1):185-197.
Shu Qingyan,Zhu Jin,Men Siqi,Hao Qing,Wang Qianyu,Liu Zheng’an,Zeng Xiuli,Wang Liangsheng.2018.Establishing virus induced gene silencing(VIGS)system in tree peony using PsUFGT genes.Acta Horticulturae Sinica,45(1):168-176.(in Chinese)舒庆艳,朱瑾,门思琦,郝青,王倩玉,刘政安,曾秀丽,王亮生.2018.基于牡丹类黄酮糖基转移酶基因建立VIGS技术体系.园艺学报,2018,45(1):168-176.
Tanaka Y,Yonekura K,Fukuchi-Mizutani M,Fukui Y,Fujiwara H,Ashikari T,Kusumi T.1996.Molecular and biochemical characterization of
three anthocyanin synthetic enzymes from Gentiana triflora.Plant Cell Physiology,37(5):711-716.
Tohge T,Nishiyama Y,Hirai M Y,Yano M,Nakajima J,Awazuhara M,Inoue E,Takahashi H,Goodenowe D B,Kitayama M,Noji M,Yamazaki M,Saito K.2005.Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor.The Plant Journal,42(2):218-235.
Wise R P,Rohde W,Salamini F.1990.Nucleotide sequence of the Bronze-1 homologous gene from Hordeum vulgare.Plant Molecular Biology,14(2):277-279.
Yamazaki M,Gong Z,Fukuchi-Mizutani M,Fukui Y,Tanaka Y,Kusumi T,Saito K.1999.Molecular cloning and biochemical characterzation of a novel anthocyanin 5-O-glucosyltransferase by m RNA differential display for plant forms regarding anthocyanin.The Journal of Biology Chemistry,274(11):7405-7411.
Yonekura-Sakakibara K,Fukushima A,Nakabayashi R,Hanada K,Matsuda F,Sugawara S,Inoue E,Kuromori T,Ito T,Shinozaki K,Wangwattana B,Yamazaki M,Saito K.2012.Two glycosyltransferases involved in anthocyanin modification delineated by transcriptome independent component analysis in Arabidopsis thaliana.The Plant Journal,69(1):154-167.
Yonekura-Sakakibara K,Nakabayashi R,Sugawara S,Tohge T,Ito T,Koyanagi M,Kitajima M,Takayama H,Saito K.2014.A flavonoid3-O-glucoside:2″-O-glucosyltransferase responsible for terminal modification of pollen-specific flavonols in Arabidopsis thaliana.The Plant Journal,79(5):769-782.
Yonekura-Sakakibara K,Tohge T,Matsuda F,Nakabayashi R,Takayama H,Niida R,Watanabe-Takahashi A,Inoue E,Saito K.2008.Comprehensive flavonol profiling and transcriptome coexpression analysis leading to decoding gene metabolite correlations in Arabidopsis.Plant Cell,20(8):2160-2176.
Yonekura-Sakakibara K,Tohge T,Niida R,Saito K.2007.Identification of a flavonol 7-O-rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis by transcriptome coexpression analysis and reverse genetics.Journal of Biological Chemistry,282(20):14932-14941.