细辛甲基丁香酚合成相关酶基因的克隆与序列分析
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摘要
目的基于细辛转录组测序结果,克隆甲基丁香酚合成相关酶基因,了解相应的序列信息。方法以细辛叶片为材料,通过RT-PCR扩增得到苯丙氨酸裂解酶(PAL)、肉桂酸-4-羟化酶(C4H)、4-羟基肉桂酰辅酶A连接酶(4CL)、肉桂醇脱氢酶(CAD)等基因的c DNA全长,并进行生物信息学分析。结果 AhPAL、AhC4H、Ah4CL和AhCAD基因的ORF长度分别为2 157、1 278、1 623、1 071 bp,编码718、425、540、356个氨基酸。4种蛋白均具有各自的保守结构域。除Ah C4H外,AhPAL、Ah4CL和AhCAD与已报道的其它物种相应酶的氨基酸序列相似性都较高。结论对细辛甲基丁香酚合成相关酶基因进行克隆和生物信息学分析,为后续基因功能分析及甲基丁香酚生物合成调控机制的解析奠定基础。
Objective To clone the enzyme genes related with methyl eugenol synthesis and characterize the corresponding sequence information based on the transcriptome sequencing of Asarum heterotropoides. Methods RT-PCR was performed to obtain the full length c DNA of the phenylalanine lyase(PAL) gene, cinnamic acid-4-hydroxylase(C4 H) gene, 4-hydroxycinnamoyl-CoA ligase(4 CL), and cinnamyl alcohol dehydrogenase(CAD) genes by using young leaves as materials. The acquired enzyme genes were analyzed by bioinformatics. Results The ORF lengths of AhPAL, AhC4 H, Ah4 CL, and AhCAD were 2 157, 1 278, 1 623 and 1 071 bp, which respectively encoded 718, 425, 540, and 356 amino acids. Four proteins had respective conserved domains. The amino acid sequences of AhPAL, Ah4 CL, and AhCAD were similar to those of other reported species except for AhC4 H. Conclusion Four enzyme genes related with methyl-eugenol synthesis in A. heterotropoides were separated and analyzed using bioinformatics method. These results would lay the important foundation for functional analysis of corresponding genes and for elucidating the regulation mechanism of methyl-eugenol biosynthesis.
Objective To clone the enzyme genes related with methyl eugenol synthesis and characterize the corresponding sequence information based on the transcriptome sequencing of Asarum heterotropoides. Methods RT-PCR was performed to obtain the full length c DNA of the phenylalanine lyase(PAL) gene, cinnamic acid-4-hydroxylase(C4 H) gene, 4-hydroxycinnamoyl-CoA ligase(4 CL), and cinnamyl alcohol dehydrogenase(CAD) genes by using young leaves as materials. The acquired enzyme genes were analyzed by bioinformatics. Results The ORF lengths of AhPAL, AhC4 H, Ah4 CL, and AhCAD were 2 157, 1 278, 1 623 and 1 071 bp, which respectively encoded 718, 425, 540, and 356 amino acids. Four proteins had respective conserved domains. The amino acid sequences of AhPAL, Ah4 CL, and AhCAD were similar to those of other reported species except for AhC4 H. Conclusion Four enzyme genes related with methyl-eugenol synthesis in A. heterotropoides were separated and analyzed using bioinformatics method. These results would lay the important foundation for functional analysis of corresponding genes and for elucidating the regulation mechanism of methyl-eugenol biosynthesis.
引文
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[22]宋修鹏,黄杏,莫凤连,等.甘蔗苯丙氨酸解氨酶基因(PAL)的克隆和表达分析[J].中国农业科学,2013, 46(14):2856-2868.
[23] Fukasawa-Akada T, Kung S D, Watson J C.Phenylalanine ammonia-lyase gene structure, expression,and evolution in Nicotiana[J]. Plant Mol Biol, 1996,30(4):711-722.
[24] Sykes R W, Gjersing E L, Foutz K, et al. Downregulation of p-coumaroyl quinate/shikimate3′-hydroxylase(C3'H)and cinnamate 4-hydroxylase(C4H)genes in the lignin biosynthetic pathway of Eucalyptus urophylla×E. grandis leads to improved sugar release[J]. Biotechnol Biof, 2015, 8:128-138.
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[28]丁霄,曹彩荣,李朋波,等.植物木质素的合成与调控研究进展[J].山西农业科学, 2016, 44(9):1406-1411.
[29] Cukovic D, Ehlting J, Vanaiffle J A. Structure and evolution of 4-coumarate:Coenzyme a ligase(4CL)gene families[J]. Biol Chem, 2001, 382(4):645-654.
[30]史经昂,张兵,肖晓琳,等.结缕草肉桂醇脱氢酶基因家族全基因组序列鉴定和表达分析[J].草业学报,2017, 26(6):111-119.
[31]朱金鑫,孙金金,原晓龙,等.巴拉圭瓜多竹CAD基因的克隆与分析[J].西部林业科学, 2017, 46(1):6-11.
[32]潘磊,林懋怡,赵晓冰,等.细辛苯丙素类化合物生物合成相关酶基因的挖掘及其表达分析[J].天然产物研究与开发, 2017, 29(5):735-740.
[2]中国药典[S].一部. 2015.
[3] Ramalingam M, Kim S J. Phytochemical, toxicological and pharmacological studies of Asiasari Radix et Rhizoma[J].Trop J Pharm Res, 2015, 14(3):545-554.
[4]赵晓冰.华细辛肉桂醇脱氢酶基因CAD的克隆、表达分析和功能验证[D].上海:华东师范大学, 2017.
[5]董艳珍.植物苯丙氨酸解氨酶基因的研究进展[J].生物技术通报, 2006(S1):31-33.
[6]林懋怡,郑柳,刘晋杰,等.华细辛苯丙氨酸解氨酶基因的克隆与生物信息学分析[J].中国实验方剂学杂志, 2018, 24(1):38-43.
[7] Yuan Y, Wang Z Y, Jiang C, et al. Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes[J]. Gene, 2014, 534(2):408-416.
[8]许锋,曹腾,宁迎晶,等.夏枯草苯丙氨酸解氨酶基因的克隆与表达分析[J].华北农学报, 2012, 27(1):39-44.
[9]刘建福,钟书淳,王明元,等.姜黄苯丙氨酸解氨酶基因的克隆与序列分析[J].中草药, 2014, 45(21):3141-3148.
[10]曾祥玲,郑日如,罗靖,等.桂花C4H基因的克隆与表达特性分析[J].园艺学报, 2016, 43(3):525-537.
[11] Chen Z, Tang N, You Y, et al. Transcriptome analysis reveals the mechanism underlying the production of a high quantity of chlorogenic acid in young leaves of Lonicera macranthoides Hand.-Mazz.[J]. PLoS One,2015, 10(9):1-18.
[12]赵乐,马利刚,杨泽岸,等.独行菜C4H基因克隆与表达分析[J].药学学报, 2017, 52(5):821-831.
[13]王丽华,李杰勤,詹秋文,等.高粱SbCAD4基因的克隆与序列分析及原核表达分析[J].湖南农业大学学报:自然科学版, 2015, 41(6):595-601.
[14] Wyrambik D, Grisebach H. Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures[J]. Eur J Biochem,1975, 59(1):9-15.
[15]曹佳强,李波,杨洋,等.木质素生物合成中肉桂醇脱氢酶基因(CAD)的研究进展[J].分子植物育种, 2014, 12(5):1034-1043.
[16]何朋,刘思妤,王宏宇,等.北细辛叶柄愈伤组织的增殖培养及器官分化研究[J].中草药, 2016, 47(13):2341-2345.
[17]刘思妤,杨悦,王鹰,等.北细辛悬浮培养体系的建立及优化[J].草业科学, 2017, 34(11):2254-2260.
[18] Jing Y, Zhang Y F, Shang M Y. Chemical constituents from the roots and rhizomes of Asarum heterotropoides var. mandshuricum and the in vitro anti-inflammatory activity[J]. Molecules, 2017, 22(1):125-144.
[19] Wong J H, Namasivayam P, Abdullah M P. The PAL2promoter activities in relation to structural development and adaptation in Arabidopsis thaliana[J]. Planta, 2012,235(2):267-277.
[20] Wanner L A, Li G Q, Ware D, et al. The phenylalaninc ammonixlyase gene family in Arabidopsis thaliana[J].Plant Mol Biol, 1995, 27(2):327-338.
[21]孙海燕,全雪丽,付爽,等.拟南芥苯丙氨酸解氨酶(PAL)基因的研究进展[J].延边大学农学学报, 2016,38(1):88-92.
[22]宋修鹏,黄杏,莫凤连,等.甘蔗苯丙氨酸解氨酶基因(PAL)的克隆和表达分析[J].中国农业科学,2013, 46(14):2856-2868.
[23] Fukasawa-Akada T, Kung S D, Watson J C.Phenylalanine ammonia-lyase gene structure, expression,and evolution in Nicotiana[J]. Plant Mol Biol, 1996,30(4):711-722.
[24] Sykes R W, Gjersing E L, Foutz K, et al. Downregulation of p-coumaroyl quinate/shikimate3′-hydroxylase(C3'H)and cinnamate 4-hydroxylase(C4H)genes in the lignin biosynthetic pathway of Eucalyptus urophylla×E. grandis leads to improved sugar release[J]. Biotechnol Biof, 2015, 8:128-138.
[25] Baek M H, Chung B Y, Kim J H, et al. cDNA cloning and expression pattern of cinnamate-4-hydroxylase in the Korean black raspberry[J]. BMB Rep, 2008, 41(7):529-536.
[26] Boerjan W, Ralph J, Baucher M. Lignin biosynthesis[J].Annual Review Plant Biol, 2003, 54(1):519-546.
[27] Whetten R W, Mackay J J, Sederoff R R. Recent advances in understanding lignin biosynthesis[J]. Annual Review Plant Physiol Plant Mol Biol, 1998, 49(49):585-609.
[28]丁霄,曹彩荣,李朋波,等.植物木质素的合成与调控研究进展[J].山西农业科学, 2016, 44(9):1406-1411.
[29] Cukovic D, Ehlting J, Vanaiffle J A. Structure and evolution of 4-coumarate:Coenzyme a ligase(4CL)gene families[J]. Biol Chem, 2001, 382(4):645-654.
[30]史经昂,张兵,肖晓琳,等.结缕草肉桂醇脱氢酶基因家族全基因组序列鉴定和表达分析[J].草业学报,2017, 26(6):111-119.
[31]朱金鑫,孙金金,原晓龙,等.巴拉圭瓜多竹CAD基因的克隆与分析[J].西部林业科学, 2017, 46(1):6-11.
[32]潘磊,林懋怡,赵晓冰,等.细辛苯丙素类化合物生物合成相关酶基因的挖掘及其表达分析[J].天然产物研究与开发, 2017, 29(5):735-740.