Cloning and Expression of the Anoectochilus roxburghii C4H Gene and Its Role in the Flavonoid Synthesis Pathway

doi:10.7668/hbnxb.20194639

Abstract

Abstract:

In order to investigate the effect of cinnamic acid 4-hydroxylase(C4H)on the accumulation of flavonoids in Anoectochilus roxburghii,the total flavonoid content of different tissues of A.roxburghii was determined by using the aluminium nitrate assay.Based on homologous cloning and RACE techniques,the C4H gene(ArC4H,GenBank accession number ON455229)was isolated and cloned from A.roxburghii;the expression of the C4H gene in different tissues of A.roxburghii was analysed by Real-time Quantitative PCR(qRT-PCR).The results showed that the flavonoid contents of different tissues of A.roxburghii differed significantly(leaf>stem>root),with the total flavonoid content of leaf being more than three times that of root and stem;the phylogenetic tree analysis revealed that A.roxburghii was close to most orchids and the open reading frame of the ArC4H gene was 1 518 bp,encoding 505 amino acids,and belonged to the cytochrome P450 superfamily,with a theoretical molecular weight of 65.17 ku and an isoelectric point of 9.21,which belonged to hydrophilic proteins;ArFLS had sequence conservatism,and was expressed in different tissues.qRT-PCR analysis showed that the gene had the highest expression in stems,followed by roots,and the expression in leaves was extremely low,and the total flavonoid content also showed a similar trend.Accordingly,the gene was initially identified as ArC4H gene.

Key words: Anoectochilus roxburghii, C4H gene, Gene cloning, Expression analysis, Flavonoid

LIN Hongjun, LÜ Ke, PAN Pingping, DONG Lili, XU Zhihao, WANG Zhonghua. Cloning and Expression of the Anoectochilus roxburghii C4H Gene and Its Role in the Flavonoid Synthesis Pathway[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 31-38. doi: 10.7668/hbnxb.20194639.

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Figures/Tables 13

Fig.1 Conserved region sequence

Fig.2 3'RACE and 5'RACE sequence 1.Negative control;2.Middle fragment sequence;3.5'end sequence;4.3'end sequence.

Fig.3 Homology alignment of amino acid sequences Shaded portion:in each nucleic acid sequence,the portion is consistent with the rest of the sequence.

Fig.4 Phylogenetic tree of ArC4H

Fig.5 Analysis of protein functional structural domains

Tab.1 Basic physicochemical properties of the proteins encoded by the ArC4H gene of Anoectochilus roxburghii

类别 Category	数量 Amount	类别 Category	数量 Amount
氨基酸数	572	相对分子量	65.167 23 ku
Number of amino acids		Molecular weight
理论等电点Theoretical pI	9.21	分子式Formula	C₂₉₈₈H₄₇₀₉N₇₉₃O₈₀₁S₁₉
脂肪指数Aliphatic index	102.40	原子总数Total number of atoms	9 310
亲水性的平均值Grand average of hydropathicity	-0.044	不稳定指数The instability index	44.07
带正电荷的残基总数(g+Lys)	74	带负电荷的残基总数(Asp+Glu)	64
Positively charged residues		Negatively charged residues

Fig.6 Protein hydrophobicity analysis

Fig.7 Protein transmembrane region Purple lines on the Y-axis indicate that they are not on the structural cell membrane;blue lines indicate that they are on the structural cell membrane;red lines on the X-axis are transmembrane helical structures.

Fig.8 Phosphorylation sites Red.Serine;Green.Threonine;Blue.Tyrosine;Purple.Threshold assessment.Only those above the threshold are considered loci.

Fig.9 Protein signal peptide analysis

Fig.10 Protein secondary structure Blue.Alpha helix;Green.Beta turn;Yellow.Random coil;Red.Extended strand.

Fig.11 Protein tertiary structure

Fig.12 Relative expression and total flavonoid content of C4H gene Different letters indicate significant differences,P<0.05.

References 29

[1]	刘英孟, 张海燕, 汪镇朝, 杨明, 钟萍. 金线莲的研究进展[J]. 中成药, 2022, 44(1):186—192.doi:10.3969/j.issn.1001-1528.2022.01.035.
	Liu Y M, Zhang H Y, Wang Z C, Yang M, Zhong P. Research progress of Anoectochilus roxburghii[J]. Chinese Traditional Patent Medicine, 2022, 44(1):186—192.
[2]	陈育青, 林艺华, 邹毅辉, 田惠桥, 林美珍. 金线莲生药鉴定、活性成分影响因素及药理作用研究进展[J]. 中成药, 2020, 42(8):2141—2144.doi:10.3969/j.issn.1001-1528.2020.08.033.
	Chen Y Q, Lin Y H, Zou Y H, Tian H Q, Lin M Z. Research progress on pharmacognosy identification,influencing factors of active components and pharmacological effects of Anoectochilus roxburghii[J]. Chinese Traditional Patent Medicine, 2020, 42(8):2141—2144.
[3]	李莉, 赵越, 马君兰. 苯丙氨酸代谢途径关键酶:PAL、C4H、4CL研究新进展[J]. 生物信息学, 2007, 5(4):187—189.doi:10.3969/j.issn.1672-5565.2007.04.015.
	Li L, Zhao Y, Ma J L. Recent progress on key enzymes:PAL,C4H,4CL of phenylalanine metabolism pathway[J]. China Journal of Bioinformatics, 2007, 5(4):187—189.
[4]	卢学琴, 周美娟, 胡尚连, 曹颖, 任鹏. 慈竹木质素合成关键基因C4H克隆及组织表达分析[J]. 竹子研究汇刊, 2014, 33(2):11—15.doi:10.3969/j.issn.1000-6567.2014.02.003.
	Lu X Q, Zhou M J, Hu S L, Cao Y, Ren P. Cloning and expression of the C4H gene,the key gene of lignin biosynthesis in Neosinocalamus affinis[J]. Journal of Bamboo Research, 2014, 33(2):11—15.
[5]	李广柱, 朱成磊, 杨克彬, 王新悦, 高志民. 毛竹C4H基因的鉴定及其表达模式分析[J]. 热带亚热带植物学报, 2022, 30(2):151—160.doi:10.11926/jtsb.4455.
	Li G Z, Zhu C L, Yang K B, Wang X Y, Gao Z M. Identification and expression pattern analysis of C4H genes in Phyllostachys edulis[J]. Journal of Tropical and Subtropical Botany, 2022, 30(2):151—160.
[6]	车玉红, 杨波, 郭春苗, 刘晨曦, 木巴热克·阿尤普, 吴津蓉, 杜鹃. 榅桲C4H基因的克隆、序列分析及表达[J]. 经济林研究, 2020, 38(2):1—8.doi:10.14067/j.cnki.1003-8981.2020.02.001.
	Che Y H, Yang B, Guo C M, Liu C X, Mubazek A, Wu J R, Du J. Cloning,sequence analysis and expression of C4H gene in Cydonia oblonga[J]. Non-wood Forest Research, 2020, 38(2):1—8.
[7]	黄利娜, 吴光斌, 匡凤元, 张珅, 陈发河. 莲雾果实C4H基因的克隆及在NO处理下的表达分析[J]. 集美大学学报(自然科学版), 2020, 25(2):105—112.doi:10.19715/j.jmuzr.2020.02.04.
	Huang L N, Wu G B, Kuang F Y, Zhang S, Chen F H. Cloning and expression analysis of cinnamic acid-4-hydroxylase from wax apple fruit under NO treatment[J]. Journal of Jimei University (Natural Science), 2020, 25(2):105—112.
[8]	刘荣华, 王丽, 孙朝霞, 侯思宇, 李红英. 苦荞C4H基因的cDNA克隆及生物信息学分析[J]. 山西农业大学学报(自然科学版), 2017, 37(11):767—773.doi:10.13842/j.cnki.issn1671—8151.2017.11.002.
	Liu R H, Wang L, Sun Z X, Hou S Y, Li H Y. Cloning and bioinformatics analysis of C4H gene in Tatary Buckwheat[J]. Journal of Shanxi Agricultural University (Natural Science Edition), 2017, 37(11):767—773.
[9]	Mizutani M, Ohta D, Sato R. Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta[J]. Plant Physiology, 1997, 113(3):755—763.doi:10.1104/pp.113.3.755. pmid: 9085571
[10]	Chen F, Dixon R A. Lignin modification improves fermentable sugar yields for biofuel production[J]. Nature Biotechnology, 2007, 25(7):759—761.doi:10.1038/nbt1316. pmid: 17572667
[11]	嵇元烨, 吴梅, 吴秋丽, 江晴儿, 谢晓鸿, 孔向军, 王忠华. 金线莲UGPase基因克隆与表达及在多糖合成中的作用[J]. 中草药, 2021, 52(12):3671—3678.doi:10.7501/j.issn.0253-2670.2021.12.025.
	Ji Y Y, Wu M, Wu Q L, Jiang Q E, Xie X H, Kong X J, Wang Z H. Cloning and expression of UGPase gene and its role in polysaccharide synthesis of Anoectochilus roxburghii[J]. Chinese Traditional and Herbal Drugs, 2021, 52(12):3671—3678.
[12]	杨宁宁. 金线莲花青素DFR基因筛选与克隆及功能分析[D]. 杭州: 浙江农林大学, 2021.doi:10.27756/d.cnki.gzjlx.2021.000263.
	Yang N N. Screening,cloning and functional of analysis of the DFR gene Anoectochilus roxburghii[D]. Hangzhou: Zhejiang Agriculture and Forestry University, 2021.
[13]	林江波, 王伟英, 邹晖, 戴艺民. 金线莲呋甾皂苷26-O-β-葡萄糖苷酶基因克隆与表达分析[J]. 福建农业学报, 2020, 35(4):422—428.doi:10.19303/j.issn.1008-0384.2020.04.009.
	Lin J B, Wang W Y, Zou H, Dai Y M. Cloning and expression analysis of furostanol glycoside 26-O-β-glucosidase gene in Anoectochilus roxburhii[J]. Fujian Journal of Agricultural Sciences, 2020, 35(4):422—428.
[14]	拓昊苑. 金线莲苯丙氨酸解氨酶基因(PAL)和辣椒红素合成酶基因(CCS)的克隆与功能验证[D]. 成都: 四川农业大学, 2019.doi:10.27345/d.cnki.gsnyu.2019.000898.
	Tuo H Y. Cloning and functional validation of phenylalanine ammonia-lyase gene(PAL)and capsaicin synthase gene(CCS)in Anoectochilus roxburghii[D]. Chengdu: Sichuan Agricultural University, 2019.
[15]	黄哲, 高云, 党友超, 杨烨, 李蒙禹, 王世清. 黔产金线莲总黄酮含量测定研究[J]. 贵州中医药大学学报, 2020, 42(6):23—27.doi:10.16588/j.cnki.issn2096-8426.2020.06.005.
	Huang Z, Gao Y, Dang Y C, Yang Y, Li M Y, Wang S Q. Determsination of total flavonoids in Anoectochilus of Guizhou[J]. Journal of Guizhou University of Traditional Chinese Medicine, 2020, 42(6):23—27.
[16]	Zhang B X, Lewis K M, Abril A, Davydov D R, Vermerris W, Sattler S E, Kang C. Structure and function of the cytochrome P450 monooxygenase cinnamate 4-hydroxylase from Sorghum bicolor[J]. Plant Physiology, 2020, 183(3):957—973.doi:10.1104/pp.20.00406.
[17]	张晓辉. 金线莲多糖结构分析及抗糖尿病活性研究[D]. 汕头: 汕头大学, 2011.
	Zhang X H. Structural analysis and anti-diabetic activity of Anoectochilus roxburghii polysaccharide[D]. Shantou: Shantou University, 2011.
[18]	沈廷明, 吴仲玉, 黄春情, 刘知远. 金线莲化学成分、药理、组培及栽培研究进展[J]. 海峡药学, 2016, 28(12):26—30.doi:10.3969/j.issn.1006-3765.2016.12.010.
	Shen T M, Wu Z Y, Huang C Q, Liu Z Y. Study on Anoectochilus roxburghii's chemical constituents,pharmacodynamics,tissue culture and cultivation[J]. Strait Pharmaceutical Journal, 2016, 28(12):26—30.
[19]	王勇, 陈硕, 卢端萍, 张丽蓉. 金线莲化学成分的研究[J]. 中草药, 2017, 48(13):2619—2624.doi:10.7501/j.issn.0253-2670.2017.13.006.
	Wang Y, Chen S, Lu D P, Zhang L R. Chemical constituents of Anoectochilus roxburghii[J]. Chinese Traditional and Herbal Drugs, 2017, 48(13):2619—2624.
[20]	尹泽楠, 徐柯心, 樊娇娇, 马志强, 林瑞超. 中国兰科开唇兰属植物化学成分研究进展[J]. 环球中医药, 2016, 9(9):1153—1160.doi:10.3969/j.issn.1674-1749.2016.09.041.
	Yin Z N, Xu K X, Fan J J, Ma Z Q, Lin R C. Research progress on chemical constituents of Anoectochilus and pharmacological activities[J]. Global Traditional Chinese Medicine, 2016, 9(9):1153—1160.
[21]	徐静. 金线莲化学成分及脱氧鬼臼衍生物活性研究[D]. 兰州: 兰州大学, 2011.
	Xu J. Research on chemical composition and activity of deoxynivalenol derivatives of Anoectochilus roxburghii[D]. Lanzhou: Lanzhou University, 2011.
[22]	朱善岚. 金线莲活性成分的分析[D]. 福州: 福建医科大学, 2010.doi:10.7666/d.Y1711658.
	Zhu S L. Analysis on active constituents of Anoectochilu roxburghii[D]. Fuzhou: Fujian Medical University, 2010.
[23]	李轶奕, 刘顺治, 谢婷婷, 范世明, 丘鹰昆. 金线莲HPLC指纹图谱研究[J]. 亚热带植物科学, 2021, 50(4):267—271.doi:10.3969/j.issn.1009-7791.2021.04.005.
	Li Y Y, Liu S Z, Xie T T, Fan S M, Qiu Y K. HPLC fingerprinting of Anoectochilus roxburgii[J]. Subtropical Plant Science, 2021, 50(4):267—271.
[24]	李帅玲. 金线莲多糖的含量变化、结构表征及药理活性研究[D]. 杭州: 浙江农林大学, 2017.
	Li S L. Content change,structural characterization and pharmacological activity of Anoectochilus roxburghii polysaccharide[D]. Hangzhou: Zhejiang A&F University, 2017.
[25]	刘婷婷. 金线莲苷靶向树突状细胞与CD8⁺T细胞的相互作用抗自身免疫性肝炎作用机制研究[D]. 武汉: 华中科技大学, 2017.doi:10.7666/d.D01313065.
	Liu T T. Study on the anti-autoimmune hepatitis mechanism of the interaction between anoectoside targeted dendritic cells and CD8⁺T cells[D]. Wuhan: Huazhong University of Science and Technology, 2017.
[26]	朱亮, 徐菲拉, 王群星, 徐斌. 浙江省金华市防治新型冠状病毒感染肺炎中药处方分析[J]. 浙江中西医结合杂志, 2020, 30(3):179—181.doi:10.3969/j.issn.1005-4561.2020.03.003.
	Zhu L, Xu F L, Wang Q X, Xu B. Analysis of traditional Chinese medicine prescription for the prevention and treatment of new coronavirus infection pneumonia in Jinhua City,Zhejiang Province[J]. Zhejiang Journal of Integrated Traditional Chinese and Western Medicine, 2020, 30(3):179—181.
[27]	叶旭星, 王晓波, 马双双, 何钦, 徐斌. 中西医结合治疗新型冠状病毒肺炎合并肾功能异常一例[J]. 浙江中西医结合杂志, 2020, 30(11):920—923,957.doi:10.3969/j.issn.1005-4561.2020.11.016.
	Ye X X, Wang X B, Ma S S, He Q, Xu B. A case of novel coronavirus complicated with renal dysfunction treated by integrated traditional Chinese and Western medicine[J]. Zhejiang Journal of Integrated Traditional Chinese and Western Medicine, 2020, 30(11):920—923,957.
[28]	Wohl J, Petersen M. Functional expression and characterization of cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis in Physcomitrella patens[J]. Plant Cell Reports, 2020, 39(5):597—607.doi:10.1007/s00299-020-02517-z.
[29]	Dong N Q, Lin H X. Contribution of phenylpropanoid metabolism to plant development and plant-environment interactions[J]. Journal of Integrative Plant Biology, 2021, 63(1):180—209.doi:10.1111/jipb.13054.

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