Functional Identification and Expression Analysis of Cinnamonyl Alcohol Dehydrogenase AsCAD in Angelica sinensis

doi:10.13560/j.cnki.biotech.bull.1985.2024-0760

Abstract

Abstract:

Objective The study is to explore the catalytic activity and expression characteristics of cinnamyl alcohol dehydrogenase, which is a key enzyme of lignin synthesis in Angelica sinensis, and to provide a new research idea and theoretical basis for solving the issue of lignification in the roots of A. sinensis. Method Unbolted roots (UBP) and bolted roots (BP) were used as experimental materials to identify candidate cinnamyl alcohol dehydrogenase genes based on transcriptome data. The full-length CDS of AsCADs gene was cloned, the prokaryotic expression vector pET-28a-AsCADs was constructed, transferred into E. coli BL21 (DE3), and the recombinant protein expression was induced and enzyme activity was measured in vitro. The active CAD protein sequences were analyzed by bioinformatics. Real-time quantitative PCR was used to analyze the gene expression patterns of UBP and BP. Result A total of 30 AsCADs genes were identified, among which 3 cinnamyl alcohol dehydrogenase genes, AsCAD1, AsCAD4 and AsCAD24, were reported and cloned, all containing two Zn²⁺ binding sites and 1 NAD(H) coenzyme binding site. In vitro enzymatic assay revealed that AsCAD1 catalyzed the reduction of coniferaldehyde and cafferaldehyde to the corresponding alcohols, and AsCAD4 and AsCAD24 catalyzed the reduction of p-coumaraldehyde, coniferaldehyde, cafferaldehyde and sinapaldehyde to the corresponding alcohols. AsCAD1, AsCAD4 and AsCAD24 contained an open reading frame of 1 083 bp, encoded 360 amino acids, and theoretical isoelectric points (pI) of 6.1 and 5.52, of which AsCAD1 was a hydrophobic protein, AsCAD4 and AsCAD24 were hydrophilic proteins, none of which contained a transmembrane structure and signal peptide, and the subcellular localization were in cytoplasm. RT-qPCR results showed that the expression of AsCAD1 was high in UBP, while AsCAD4 and AsCAD24 was high in BP. Conclusion The gene of AsCAD1, AsCAD4 and AsCAD24 are successfully cloned. The proteins all have catalytic activity after prokaryotic expression, and their relative expression levels are different in UBP and BP.

Key words: Angelica sinensis, lignification, cinnamyl alcohol dehydrogenase, prokaryotic expression, enzyme activity in vitro, expression analysis

XIANG Chun-fan, LI Le-song, WANG Juan, LIANG Yan-li, YANG Sheng-chao, LI Meng-fei, ZHAO Yan. Functional Identification and Expression Analysis of Cinnamonyl Alcohol Dehydrogenase AsCAD in Angelica sinensis[J]. Biotechnology Bulletin, 2025, 41(2): 295-308.

Figures/Tables 12

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence （5'-3'）	备注 Note
AsCAD1-F	agcaaatgggtcgcggatccATGGAAAAATCAGCAGAAACCCAAC	基因克隆 Gene cloning
AsCAD1-R	ctcagtggtggtggtggtggtgGGCAGCCTGCAATGTATTTCCA
AsCAD4-F	acagcaaatgggtcgcggatccATGGGCAGCTTGGAAGTG
AsCAD4-R	tcagtggtggtggtggtggtgGGTTTCTTGATCAAGTTTGCTGC
AsCAD24-F	acagcaaatgggtcgcggatccATGGGCAGCTTGGAAGTG
AsCAD24-R	ggtggtggtggtggtgTGTTTCTCGATCAAGTTTGCTACCTG
环P-pET-28a-F	tggacagcaaatgggtcgcggatcc	环状载体克隆 Circular vector cloning
环P-pET-28a-R	ggatccgcgacccatttgctgtccac	环状载体克隆 Circular vector cloning
pET-28a-F-质粒	atctcgatcccgcgaaattaatac	原核表达Prokaryotic expression
pET-28a-R-质粒	tcctttcagcaaaaaacccct	原核表达Prokaryotic expression
q-AsCAD1-F	GGGAGGAAAATAGTAGCCGGTAG	实时荧光定量PCR RT-qPCR
q-AsCAD1-R	CAAGACGTTCCATTGCAGTGTTC
q-AsCAD4-F	GAAAGACGAGGCAATGGATCATC
q-AsCAD4-R	CAACAAGGAGAGGTAAGGTTCGA
q-AsCAD24-F	CACTCGAACCTTACCTCTCCTTG
q-AsCAD24-R	CCCTATGAAGCTCCCTGTTATGG
q-AsEEF1G-F	GTCCCAGCAGCCAAAAAGTC	内参基因 Reference gene
q-AsEEF1G-R	TCTGCCTTGGGCAATTCCTT	内参基因 Reference gene

Table 2 Website links for bioinformatics analysis

生物信息学分析 Bioinformatics analysis	网址 Website links
开放阅读框（ORF）	https：//www.ncbi.nlm.nih.gov/orffinder/ORF
蛋白理化性质分析	https：//web.expasy.org/protparam/
蛋白质二级结构	https：//npsa-prabi.ibcp.fr/cgi-bin/secpredsopma.pl
蛋白质三级结构	https：//swissmodel.expasy.org/interactive
蛋白信号肽预测	https：//services.healthtech.dtu.dk/services/SignalP-6.0/
蛋白跨膜结构域预测	https：//services.healthtech.dtu.dk/services/TMHMM-2.0/
蛋白亚细胞定位	https：//www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2

Fig. 1 Safranin-Fast Green staining sections of roots from unbolted plant (UBP) and bolted plant (BP) in Angelica sinensis

Fig. 2 Phylogenetic tree and expression pattern analysis of cinnamyl alcohol dehydrogenase genes in A. sinensisA: Analysis of the CAD family evolutionary tree. B: Heatmap of the expressions of CAD genes in UBP and BP of A. sinensis

Fig. 3 Multiple alignment of amino acid sequences from AsCAD1, AsCAD4andAsCAD24

Fig. 4 Cloning of the AsCAD1, AsCAD4 and AsCAD24 genes and SDS-PAGE analysis of the recombinant proteinsM: Marker. A: Electrophoresis of full-length cloning PCR products of AsCAD genes (1, 2, and 3 are the full-length cloning products of AsCAD1, AsCAD4, and AsCAD24 genes, respectively). B-D: SDS-PAGE analysis of pET-28a-His-AsCAD recombinant proteins (B: Renatured supernatant of pET-28a-His-AsCAD1. C: Renatured supernatant of pET-28a-His-AsCAD4. D: Renatured supernatant of pET-28a-His-AsCAD24)

Fig. 5 Enzymatic reaction and product analysis of coniferaldehyde catalyzed by AsCAD1, AsCAD4 and AsCAD24A: AsCAD1, AsCAD4 and AsCAD24 catalyze the reduction reaction of coniferaldehyde. B: HPLC is used to detect the products of enzymatic reaction mixtures and blank controls. C: LC-MS is used to detect the molecular weights of standards and enzymatic reaction mixtures (1, 2, 3, and 4 are the LC-MS results of standards, AsCAD1, AsCAD4 and AsCAD24, respectively)

Fig. 6 Enzymatic reaction and product analysis of caffealdehyde catalyzed by AsCAD1, AsCAD4and AsCAD24A: Reduction reaction of caffealdehyde catalyzed by AsCAD1, AsCAD4 and AsCAD24. B: Detection of enzymatic reaction mixture and blank control product by HPLC. C: Detection of molecular weights of standard and enzymatic reaction mixture by LC-MS (1,2,3,4 are the LC-MS results of standard, AsCAD1, AsCAD4 and AsCAD24, respectively)

Fig. 7 Enzymatic reaction and product analysis of p-coumaraldehyde catalyzed by AsCAD1, AsCAD4 and AsCAD24A: Reduction reaction of p-coumaraldehyde catalysed by AsCAD1, AsCAD4 and AsCAD24. B: Detection of enzymatic reaction mixture and blank control product by HPLC. C: Detection of molecular weights of standard and enzymatic reaction mixture by LC-MS (1,2,3 are the LC-MS results of standard, AsCAD4 and AsCAD24, respectively)

Fig. 8 Enzymatic reaction and product analysis of sinapaldehyde catalyzed by AsCAD1, AsCAD4 and AsCAD24A: Reduction reaction of sinapaldehyde catalysed by AsCAD1, AsCAD4 and AsCAD24. B: Detection of enzymatic reaction mixture and blank control product by HPLC. C: Detection of molecular weights of standard and enzymatic reaction mixture by LC-MS (1,2,3 are the LC-MS results of standard, AsCAD4 and AsCAD24, respectively)

Fig. 9 Secondary and tertiary structure prediction of three AsCAD proteinsA-C: Predicted secondary structures of AsCAD1, AsCAD4 and AsCAD24. D-F: Predicted tertiary structure of AsCAD1, AsCAD4 and AsCAD24 (Purple: Spiral; green: extended chain; silver white: irregular curl)

Fig. 10 Relative expressions of AsCAD1, AsCAD4 and AsCAD24 genes in the UBP and BP of A. sinensis

References 48

1	栗孟飞, 刘学周, 魏建和, 等. 基于生物量、活性物质积累和抗氧化能力的当归高海拔种植区域选择 [J]. 中草药, 2020, 51(2): 474-481.
	Li MF, Liu XZ, Wei JH, et al. Selection of high altitude planting area of Angelica sinensis based on biomass, bioactive compounds accumulation and antioxidant capacity [J]. Chin Tradit Herb Drugs, 2020, 51(2): 474-481.
2	刘方舟,李园白,王静,等. 当归药材道地性系统评价与分析 [J]. 世界科学技术中医药现代化,2018, 20 (9):1531-1539.
	Liu FZ, Li YB, Wang J, et al. Systematic evaluation and analysis on Dao-di Herbs Angelica Sinensis [J]. World Science and Technology/Modemization of Traditional Chinese Medicine and Materia Medica J. 2018, 20, (9):1531-1539.
3	马依林, 张虹. 《中华人民共和国药典》中含当归方剂的组方规律 [J]. 中医学报, 2021, 36(12): 2708-2712.
	Ma YL, Zhang H. Prescription composition principles of Danggui (Radix angelicae sinensis) contained ones in Chinese pharmacopoeia [J]. Acta Chin Med, 2021, 36(12): 2708-2712.
4	Yao WL, Zhang L, Hua YL, et al. The investigation of anti-inflammatory activity of volatile oil of Angelica sinensis by plasma metabolomics approach [J]. Int Immunopharmacol, 2015, 29(2): 269-277.
5	Zhou WJ, Wang S, Hu Z, et al. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation [J]. Biochem Biophys Res Commun, 2015, 467(3): 562-569.
6	Ma JP, Guo ZB, Jin L, et al. Phytochemical progress made in investigations of Angelica sinensis (Oliv.) Diels [J]. Chin J Nat Med, 2015, 13(4): 241-249.
7	Wei WL, Zeng R, Gu CM, et al. Angelica sinensis in China-a review of botanical profile, ethnopharmacology, phytochemistry and chemical analysis [J]. J Ethnopharmacol, 2016, 190: 116-141.
8	栗孟飞, 康天兰, 晋玲, 等. 当归抽薹开花及其调控途径研究进展 [J]. 中草药, 2020, 51(22): 5894-5899.
	Li MF, Kang TL, Jin L, et al. Research progress on bolting and flowering of Angelica sinensis and regulation pathways [J]. Chin Tradit Herb Drugs, 2020, 51(22): 5894-5899.
9	梁婕, 王辉, 睢宁. 伞形科根茎类药用植物早期抽薹的研究进展 [J]. 中国农学通报, 2022, 38(13): 90-95.
	Liang J, Wang H, Sui N. Early bolting of rhizome medicinal plants in Umbelliferae: research progress [J]. Chin Agric Sci Bull, 2022, 38(13): 90-95.
10	马艳春, 吴文轩, 胡建辉, 等. 当归的化学成分及药理作用研究进展 [J]. 中医药学报, 2022, 50(1): 111-114.
	Ma YC, Wu WX, Hu JH, et al. Research progress on chemical constituents and pharmacological effects of Angelica sinensis [J]. Acta Chin Med Pharmacol, 2022, 50(1): 111-114.
11	管西芹, 毛近隆, 闫滨, 等. 当归不同提取液中阿魏酸、咖啡酸含量及抗氧化作用的比较研究 [J]. 天然产物研究与开发, 2018, 30(12): 2033-2038.
	Guan XQ, Mao JL, Yan B, et al. A comparative study among ferulic acid, caffeic acid content and antioxidation in different extracts of Angelica sinensis [J]. Nat Prod Res Dev, 2018, 30(12): 2033-2038.
12	Ono K, Hirohata M, Yamada M. Ferulic acid destabilizes preformed beta-amyloid fibrils in vitro [J]. Biochem Biophys Res Commun, 2005, 336(2): 444-449.
13	Li ML, Cui XW, Jin L, et al. Bolting reduces ferulic acid and flavonoid biosynthesis and induces root lignification in Angelica sinensis [J]. Plant Physiol Biochem, 2022, 170: 171-179.
14	Yuan CX, Li LS, Zhou PH, et al. Decoding the root lignification mechanism of Angelica sinensis through genome-wide methylation analysis [J]. J Exp Bot, 2024: erae392.
15	Huang LQ, Jin L. Suitable technology for production and processing of Angelica Sinensis [J]. Pharmaceutical Science and Technology Press: Beijing, China, 2018: 1-14.
16	张真, 刘学周, 包亚军, 等. 基于生物量、活性物质积累和抗氧化能力的当归种植茬口选择 [J]. 甘肃农业大学学报, 2018, 53(6): 82-89.
	Zhang Z, Liu XZ, Bao YJ, et al. Selection of cropping rotations of Angelicasinensisbased on biomass, bioactive compounds accumulation and antioxidant capacity [J]. J Gansu Agric Univ, 2018, 53(6): 82-89.
17	Miedes E, Vanholme R, Boerjan W, et al. The role of the secondary cell wall in plant resistance to pathogens [J]. Front Plant Sci, 2014, 5: 358.
18	Boerjan W, Ralph J, Baucher M. Lignin biosynthesis [J]. Annu Rev Plant Biol, 2003, 54: 519-546.
19	Bonawitz ND, Chapple C. The genetics of lignin biosynthesis: connecting genotype to phenotype [J]. Annu Rev Genet, 2010, 44: 337-363.
20	Baucher M, Chabbert B, Pilate G, et al. Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar [J]. Plant Physiol, 1996, 112(4): 1479-1490.
21	Halpin C, Knight ME, Grima-Pettenati J, et al. Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems [J]. Plant Physiol, 1992, 98(1): 12-16.
22	Ma QH. Functional analysis of a cinnamyl alcohol dehydrogenase involved in lignin biosynthesis in wheat [J]. J Exp Bot, 2010, 61(10): 2735-2744.
23	Sibout R, Eudes A, Pollet B, et al. Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis. Isolation and characterization of the corresponding mutants [J]. Plant Physiol, 2003, 132(2): 848-860.
24	Zhang KW, Qian Q, Huang ZJ, et al. GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice [J]. Plant Physiol, 2006, 140(3): 972-983.
25	Chao N, Liu SX, Liu BM, et al. Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa [J]. Planta, 2014, 240(5): 1097-1112.
26	Porter S, Sederoff RR. Purification, characterization, and cloning of cinnamyl alcohol dehydrogenase in loblolly pine (Pinus taeda L.) [J]. Plant Physiol, 1992, 98(4): 1364-1371.
27	Lee CJ, Kim SE, Park SU, et al. Tuberous roots of transgenic sweetpotato overexpressing IbCAD1 have enhanced low-temperature storage phenotypes [J]. Plant Physiol Biochem, 2021, 166: 549-557.
28	Park HL, Kim TL, Bhoo SH, et al. Biochemical characterization of the rice cinnamyl alcohol dehydrogenase gene family [J]. Molecules, 2018, 23(10): 2659.
29	Pan HY, Zhou R, Louie GV, et al. Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis [J]. Plant Cell, 2014, 26(9): 3709-3727.
30	Vasupalli N, Hou D, Singh RM, et al. Homo- and hetero-dimers of CAD enzymes regulate lignification and abiotic stress response in moso bamboo [J]. Int J Mol Sci, 2021, 22(23): 12917.
31	Baghdady A, Blervacq AS, Jouanin L, et al. Eucalyptus gunnii CCR and CAD2 promoters are active in lignifying cells during primary and secondary xylem formation in Arabidopsis thaliana [J]. Plant Physiol Biochem, 2006, 44(11-12): 674-683.
32	Kim SJ, Kim MR, Bedgar DL, et al. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis [J]. Proc Natl Acad Sci USA, 2004, 101(6): 1455-1460.
33	朋冬琴, 罗蜜蜜, 郭欣慰, 等. 当归实时荧光定量PCR内参基因筛选 [J]. 中草药, 2024, 55(1): 269-278.
	Peng DQ, Luo MM, Guo XW, et al. Selection of reference genes for quantitative real-time PCR analysis in Angelica sinensis [J]. Chin Tradit Herb Drugs, 2024, 55(1): 269-278.
34	Rong W, Luo MY, Shan TL, et al. A wheat cinnamyl alcohol dehydrogenase TaCAD12 contributes to host resistance to the sharp eyespot disease [J]. Front Plant Sci, 2016, 7: 1723.
35	Tobias CM, Chow EK. Structure of the cinnamyl-alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification [J]. Planta, 2005, 220(5): 678-688.
36	Li X, Ma DM, Chen JL, et al. Biochemical characterization and identification of a cinnamyl alcohol dehydrogenase from Artemisia annua [J]. Plant Sci, 2012, 193/194: 85-95.
37	Barakat A, Bagniewska-Zadworna A, Choi A, et al. The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression [J]. BMC Plant Biol, 2009, 9: 26.
38	Kim SJ, Kim KW, Cho MH, et al. Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: lessons for database annotations? [J]. Phytochemistry, 2007, 68(14): 1957-1974.
39	Chen F, Tobimatsu Y, Havkin-Frenkel D, et al. A polymer of caffeyl alcohol in plant seeds [J]. Proc Natl Acad Sci USA, 2012, 109(5): 1772-1777.
40	Tobimatsu Y, Chen F, Nakashima J, et al. Coexistence but independent biosynthesis of catechyl and guaiacyl/syringyl lignin polymers in seed Coats [J]. Plant Cell, 2013, 25(7): 2587-2600.
41	Chen F, Tobimatsu Y, Jackson L, et al. Novel seed coat lignins in the Cactaceae: structure, distribution and implications for the evolution of lignin diversity [J]. Plant J, 2013, 73(2): 201-211.
42	Youn B, Camacho R, Moinuddin SGA, et al. Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4 [J]. Org Biomol Chem, 2006, 4(9): 1687-1697.
43	王贺萍, 孙震, 刘雨辰, 等. 蒙古冰草肉桂醇脱氢酶基因序列鉴定及功能分析 [J]. 植物学报, 2024, 59(2): 204-216.
	Wang HP, Sun Z, Liu YC, et al. Sequence identification and functional analysis of cinnamyl alcohol dehydrogenase gene from Agropyron mongolicum [J]. Chin Bull Bot, 2024, 59(2): 204-216.
44	Bomati EK, Noel JP. Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase [J]. Plant Cell, 2005, 17(5): 1598-1611.
45	Yang M, Fehl C, Lees KV, et al. Functional and informatics analysis enables glycosyltransferase activity prediction [J]. Nat Chem Biol, 2018, 14(12): 1109-1117.
46	宋希茜. 超积累型东南景天SaCAD基因的克隆及其功能分析 [D]. 北京: 中国林业科学研究院, 2016.
	Song XQ. Cloning and functional analysis of SaCAD gene of hyperaccumulator Sedum alfredii [D]. Beijing: Chinese Academy of Forestry, 2016.
47	Ponniah SK, Shang ZH, Akbudak MA, et al. Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase, cinnamoyl CoA reductase, and cinnamyl alcohol dehydrogenase leads to lignin reduction in rice (Oryza sativa L. ssp. Japonica cv. nipponbare) [J]. Plant Biotechnol Rep, 2017, 11(1): 17-27.
48	Xun HW, Qian XY, Wang M, et al. Overexpression of a cinnamyl alcohol dehydrogenase-coding gene, GsCAD1, from wild soybean enhances resistance to soybean mosaic virus [J]. Int J Mol Sci, 2022, 23(23): 15206.

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