七彩虹竹4-香豆酸辅酶A连接酶基因(Ih4CL)的克隆及功能分析
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摘要
4-香豆酸辅酶A连接酶(4CL)是苯丙烷类代谢途径的关键酶,在上游调控木质素代谢、黄酮代谢。本研究根据转录组数据设计的1对特异引物,采用逆转录PCR(RT-PCR)方法从七彩红竹1年生红秆中克隆到4-香豆酸辅酶A连接酶基因Ih4CL,该基因包含1 677bp完整的c DNA开放阅读框,编码558个氨基酸;生物信息学方法对Ih4CL编码的氨基酸蛋白序列进行的分析表明,Ih4CL与禾本科植物4CL蛋白具有较近的亲缘关系,其中与短花药野生稻的同源性达88%;荧光定量PCR检测结果显示Ih4CL基因在七彩红竹的嫩叶中的表达量明显低于茎秆中的表达量。
4-Coumarate coenzyme A ligase(4CL) is one of the key enzymes in the phenylpropanoid synthesis pathway,which produces several important secondary metabolites,such as flavonoids and lignins. In this study,a4 CL gene(designated as Ih4CL) was obtained from the red culm of Indosasa hispida cv.‘Rainbow'by PCR with a pair of special primers designed based on the transcriptome date. The Ih4 CL gene has a 1677 bp open reading frame(ORF) that is predicted to encode a protein with 558 amino acids. Phylogenetic analysis showed that the Indosasa hispida cv.‘Rainbow'have more closer relative relationship with Poaceae plants and Ih4 CL sequence shared high sequence identity(88%) with Oryza brachyantha. Real time PCR analysis showed that Ih4 CL was most strongly expressed in the young white culm.
4-Coumarate coenzyme A ligase(4CL) is one of the key enzymes in the phenylpropanoid synthesis pathway,which produces several important secondary metabolites,such as flavonoids and lignins. In this study,a4 CL gene(designated as Ih4CL) was obtained from the red culm of Indosasa hispida cv.‘Rainbow'by PCR with a pair of special primers designed based on the transcriptome date. The Ih4 CL gene has a 1677 bp open reading frame(ORF) that is predicted to encode a protein with 558 amino acids. Phylogenetic analysis showed that the Indosasa hispida cv.‘Rainbow'have more closer relative relationship with Poaceae plants and Ih4 CL sequence shared high sequence identity(88%) with Oryza brachyantha. Real time PCR analysis showed that Ih4 CL was most strongly expressed in the young white culm.
引文
[1]郭光艳,柏峰,刘伟,等.转录因子对木质素生物合成调控的研究进展[J].中国农业科学,2015,48(7):1277-1287.
[2]张士花.不同4CL基因家族成员对植物苯丙烷衍生物代谢调控的研究[D].济南:山东师范大学,2011.
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[4]黄胜雄,胡尚连,孙霞,等.木质素生物合成酶4CL基因的遗传进化分析[J].西北农林科技大学学报,2008,36(10):199-206.
[5]倪志勇,王娟,吕萌,等.棉花4-香豆酸辅酶A连接酶基因克隆及原核表达[J].西北植物学报,2010,30(3):0429-0436.
[6]魏建华,宋艳茹.木质素生物合成途径及调控的研究进展[J].植物学报,2001,43(8):771-779.
[7]Xu W J,Dubos C,Lepiniec L.Transcriptional control of flavonoid biosynthesis by MYB-b HLH-WDR complexes[J].Trends in Plant Science,2015,20(3):176-185.
[8]延玺,刘会青,邹永青,等.黄酮类化合物生理活性及合成研究进展[J].有机化学,2008,28(9):1534-1544.
[9]程龙军,童再康,黄华宏,等.光皮桦中4-香豆酸辅酶A连接酶基因Bl4CL的克隆和表达分析[J].植物生理学通讯,2010,46(1):5-10.
[10]刘希华,张丽,邢健宏,等.马尾松4CL基因同源序列克隆与进化分析[J].贵州农业科学,2013,41(12):28-31.
[11]杨晓云.青稞4-香豆酸辅酶A连接酶基因4CL的克隆及表达分析[D].雅安:四川农业大学,2013.
[12]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(3):9-19.
[13]凌瑶,高飞,王安虎,等.苦荞4-香豆酸辅酶A接酶基因(Ft4CL)的克隆及序列分析[J].广西植物,2015,35(5):728-732.
[14]王娟,孙浩,杨宇明,等.浦竹仔红色秆变异个体的r DNA ITS序列及系统发育研究[J].西部林业科学,2012,41(1):1-6.
[15]李悦丰.不同种类竹子中竹叶黄酮含量及Vc含量的比较[J].湖南农业科学,2009(2):37-38.
[16]胡尚连,曹颖,黄胜雄,等.慈竹4CL基因的克隆及其生物信息学分析[J].西北农林科技大学学报,2009,37(8):204-210.
[17]Gao S,Yu HN,Xu RX,et al.Cloning and functional characterization of a 4-coumarate Co A ligase from liverwort Plagiochasma appendiculatum[J].Phytochemistry,2015,111:48-58.
[18]Ata N,Yusuf N A,Tan B C,et al.Expression profiles of flavonoid-related gene,4 coumarate:coenzyme A ligase,and optimization of culturing conditions for the selected flavonoid production in Boesenbergia rotunda[J].Plant Cell,Tissue and Organ Culture,2015,123(1):47-55.
[19]周旭,王晨晨,毕玮,等.七彩红竹查尔酮合成酶Ih CHS1基因的克隆与分析[J].竹子研究汇刊,2014,33(3):11-16.
[20]王晨晨,毕玮,王娟,等.七彩红竹查尔酮异构酶基因Ih CHI1的克隆与表达分析[J].西部林业科学,2014,43(2):85-90.
[21]孔华,郭安平,郭运玲,等.木质素生物合成及转基因调控研究进展[J].热带农业工程,2009,33(5):47-53.
[22]贾彩红,赵华燕,王宏芝,等.抑制4CL基因表达获得低木质素含量的转基因毛白杨[J].科学通报,2004,49(7):662-666.
[23]梁海泳,夏秀英,冯雪松.UGPase和反义4CL基因对转基因烟草纤维素和木质素合成的调控[J].植物生理学通讯,2006,42(6):1067-1072.
[24]陈红年.木质素对剧烯烃塑料的改性研究[D].广州:华南理工大学,2014.
[2]张士花.不同4CL基因家族成员对植物苯丙烷衍生物代谢调控的研究[D].济南:山东师范大学,2011.
[3]Sun H Y,Li Y,Feng S Q,et al.Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice[J].Biochemical and Biophysical Research Communications,2013,430(3):1151-1156.
[4]黄胜雄,胡尚连,孙霞,等.木质素生物合成酶4CL基因的遗传进化分析[J].西北农林科技大学学报,2008,36(10):199-206.
[5]倪志勇,王娟,吕萌,等.棉花4-香豆酸辅酶A连接酶基因克隆及原核表达[J].西北植物学报,2010,30(3):0429-0436.
[6]魏建华,宋艳茹.木质素生物合成途径及调控的研究进展[J].植物学报,2001,43(8):771-779.
[7]Xu W J,Dubos C,Lepiniec L.Transcriptional control of flavonoid biosynthesis by MYB-b HLH-WDR complexes[J].Trends in Plant Science,2015,20(3):176-185.
[8]延玺,刘会青,邹永青,等.黄酮类化合物生理活性及合成研究进展[J].有机化学,2008,28(9):1534-1544.
[9]程龙军,童再康,黄华宏,等.光皮桦中4-香豆酸辅酶A连接酶基因Bl4CL的克隆和表达分析[J].植物生理学通讯,2010,46(1):5-10.
[10]刘希华,张丽,邢健宏,等.马尾松4CL基因同源序列克隆与进化分析[J].贵州农业科学,2013,41(12):28-31.
[11]杨晓云.青稞4-香豆酸辅酶A连接酶基因4CL的克隆及表达分析[D].雅安:四川农业大学,2013.
[12]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(3):9-19.
[13]凌瑶,高飞,王安虎,等.苦荞4-香豆酸辅酶A接酶基因(Ft4CL)的克隆及序列分析[J].广西植物,2015,35(5):728-732.
[14]王娟,孙浩,杨宇明,等.浦竹仔红色秆变异个体的r DNA ITS序列及系统发育研究[J].西部林业科学,2012,41(1):1-6.
[15]李悦丰.不同种类竹子中竹叶黄酮含量及Vc含量的比较[J].湖南农业科学,2009(2):37-38.
[16]胡尚连,曹颖,黄胜雄,等.慈竹4CL基因的克隆及其生物信息学分析[J].西北农林科技大学学报,2009,37(8):204-210.
[17]Gao S,Yu HN,Xu RX,et al.Cloning and functional characterization of a 4-coumarate Co A ligase from liverwort Plagiochasma appendiculatum[J].Phytochemistry,2015,111:48-58.
[18]Ata N,Yusuf N A,Tan B C,et al.Expression profiles of flavonoid-related gene,4 coumarate:coenzyme A ligase,and optimization of culturing conditions for the selected flavonoid production in Boesenbergia rotunda[J].Plant Cell,Tissue and Organ Culture,2015,123(1):47-55.
[19]周旭,王晨晨,毕玮,等.七彩红竹查尔酮合成酶Ih CHS1基因的克隆与分析[J].竹子研究汇刊,2014,33(3):11-16.
[20]王晨晨,毕玮,王娟,等.七彩红竹查尔酮异构酶基因Ih CHI1的克隆与表达分析[J].西部林业科学,2014,43(2):85-90.
[21]孔华,郭安平,郭运玲,等.木质素生物合成及转基因调控研究进展[J].热带农业工程,2009,33(5):47-53.
[22]贾彩红,赵华燕,王宏芝,等.抑制4CL基因表达获得低木质素含量的转基因毛白杨[J].科学通报,2004,49(7):662-666.
[23]梁海泳,夏秀英,冯雪松.UGPase和反义4CL基因对转基因烟草纤维素和木质素合成的调控[J].植物生理学通讯,2006,42(6):1067-1072.
[24]陈红年.木质素对剧烯烃塑料的改性研究[D].广州:华南理工大学,2014.