刺葡萄查尔酮合成酶基因CHS对不同光质的响应及转录因子调控分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0077

摘要/Abstract

摘要：

查尔酮合成酶（chalcone synthase，CHS）是花青素合成途径的第一个关键酶，为探明CHS在不同光质下的表达模式及转录因子调控特征，以刺葡萄愈伤组织为材料，克隆了2个CHS基因（VdCHS2和VdCHS3），进行生物信息学分析和8种不同光质培养处理下的表达分析，并对CHS启动子顺式作用元件和转录因子结合位点进行预测。结果显示，VdCHS2和VdCHS3基因长度分别为1 382 bp和1 398 bp，均由2个外显子和1个内含子组成，编码蛋白均为无信号肽、定位于细胞质的亲水性蛋白。光照促进VdCHS的表达，VdCHS在短波光诱导下，先呈上调表达，当表达量达到一定水平后则呈现下调作用，而长波光抑制VdCHS的表达。靶向CHS的转录因子预测得到13个MYB成员，2个CHS启动子上存在23个MYB转录因子识别和结合元件。研究结果表明，不同光质的波长对VdCHS表达影响显著，13个MYB转录因子可能在刺葡萄花青素合成中参与CHS的转录调控。

关键词: 刺葡萄, 查尔酮合成酶, CHS, 光质, 转录因子, MYB, 花青素

Abstract:

Chalcone synthase（CHS）is the first key enzyme in anthocyanidin synthesis pathway. To explore the CHS expression pattern under different light quality and transcription factor regulation，2 CHS genes（VdCHS2 and VdCHS3）were cloned from Vitis davidii callus，and VdCHS bioinformatics and relative expression by 8 different light quality treatments were conducted. Moreover，CHS promoter cis-acting elements and transcription factor binding sites were predicted. The results showed that VdCHS2 and VdCHS3 were cloned with 1 382 bp and 1 398 bp length，respectively. Both CHS were composed of 2 exons and 1 intron，and they encoded hydrophilic proteins without signal peptide，and were predicted to be cytoplasm-located. Light promoted the expression of VdCHS，which was up-regulated under short wavelength light induction，then down-regulated when its expression level reached a certain level. But long wavelength light inhibited the expression of VdCHS. Total 13 MYB transcription factors targeted at CHS were predicted，and there were 23 MYB recognition and binding elements on the 2 CHS promoters. The above results revealed that light quality affected greatly the expression of CHS. Moreover，13 MYB transcription factors may be involved in CHS transcription regulation in anthocyanidin synthesis of V. davidii.

Key words: Vitis davidii, chalcone synthase, CHS, light quality, transcription factor, MYB, anthocyanidin

赖恭梯, 阙秋霞, 潘若, 刘雨轩, 王琦, 赖谱富, 高慧颖, 赖呈纯. 刺葡萄查尔酮合成酶基因CHS对不同光质的响应及转录因子调控分析[J]. 生物技术通报, 2022, 38(11): 129-139.

LAI Gong-ti, QUE Qiu-xia, PAN Ruo, LIU Yu-xuan, WANG Qi, LAI Pu-fu, GAO Hui-ying, LAI Cheng-chun. Response of Chalcone Synthase Gene（CHS）to Different Light Quality and Transcription Factor Regulation in Vitis davidii[J]. Biotechnology Bulletin, 2022, 38(11): 129-139.

图/表 10

参考文献 52

[1]	Hatier JHB, Clearwater MJ, Gould KS. The functional significance of black-pigmented leaves:photosynthesis, photoprotection and productivity in Ophiopogon planiscapus ‘Nigrescens’[J]. PLoS One, 2013, 8(6):e67850.
[2]	Ju YL, Yue XF, Cao XY, et al. Targeted metabolomic and transcript level analysis reveals quality characteristic of Chinese wild grapes(Vitis davidii Foex)[J]. Foods, 2020, 9(10):1387. doi: 10.3390/foods9101387 URL
[3]	Fu YF, Chen M, Ye XL, et al. Variation laws of anthocyanin content in roots and their relationships with major economic traits in purple-fleshed sweetpotato[Ipomoea batatas(L.)Lam][J]. Agric Sci China, 2008, 7(1):32-40. doi: 10.1016/S1671-2927(08)60019-X URL
[4]	刘恺媛, 王茂良, 辛海波, 等. 植物花青素合成与调控研究进展[J]. 中国农学通报, 2021, 37(14):41-51.
	Liu KY, Wang ML, Xin HB, et al. Anthocyanin biosynthesis and regulate mechanisms in plants:a review[J]. Chin Agric Sci Bull, 2021, 37(14):41-51.
[5]	Wang XC, Wu J, Guan ML, et al. Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis[J]. Plant J, 2020, 101(3):637-652. doi: 10.1111/tpj.14570 URL
[6]	Yonekura-Sakakibara K, Higashi Y, Nakabayashi R. The origin and evolution of plant flavonoid metabolism[J]. Front Plant Sci, 2019, 10:943. doi: 10.3389/fpls.2019.00943 pmid: 31428108
[7]	周小理, 王青, 等. 植物中查尔酮酶合成黄酮类化合物的研究进展[J]. 食品工业, 2009, 30(6):72-74.
	Zhou XL, Wang Q, et al. Advances of plant chalcone synthase in synthesis of flavonoids[J]. Food Ind, 2009, 30(6):72-74.
[8]	苏全胜, 王爽, 孙玉强, 等. 植物原花青素生物合成及调控研究进展[J]. 中国细胞生物学学报, 2021, 43(1):219-229.
	Su QS, Wang S, Sun YQ, et al. Advances in biosynthesis and regulation of plant proanthocyanidins[J]. Chin J Cell Biol, 2021, 43(1):219-229.
[9]	胡可, 韩科厅, 戴思兰. 环境因子调控植物花青素苷合成及呈色的机理[J]. 植物学报, 2010, 45(3):307-317.
	Hu K, Han KT, Dai SL. Regulation of plant anthocyanin synthesis and pigmentation by environmental factors[J]. Chin Bull Bot, 2010, 45(3):307-317.
[10]	潘红, 赖呈纯, 黄贤贵, 等. 不同处理对刺葡萄愈伤组织花青素和原花青素生物合成的影响[J]. 热带作物学报, 2018, 39(12):2404-2409.
	Pan H, Lai CC, Huang XG, et al. Effects of different treatments on anthocyanins and procyanidins biosynthesis in spine grape callus[J]. Chin J Trop Crops, 2018, 39(12):2404-2409.
[11]	卢雯瑩, 赵磊, 李天奇, 等. 蔷薇科植物果实花青苷积累研究进展[J]. 生物技术通报, 2021, 37(1):234-245. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0717
	Lu WY, Zhao L, Li TQ, et al. Research advances of fruit anthocyanin accumulation in Rosaceae plants[J]. Biotechnol Bull, 2021, 37(1):234-245.
[12]	崔虎亮, 贺霞, 张前. 不同牡丹品种开花期间花瓣花青素和类黄酮组成的动态变化[J]. 中国农业科学, 2021, 54(13):2858-2869.
	Cui HL, He X, Zhang Q. Anthocyanins and flavonoids accumulation forms of five different color tree peony cultivars at blooming stages[J]. Sci Agric Sin, 2021, 54(13):2858-2869.
[13]	Ramsay NA, Glover BJ. MYB-bHLH-WD40 protein complex and the evolution of cellular diversity[J]. Trends Plant Sci, 2005, 10(2):63-70. pmid: 15708343
[14]	Mishra AK, Puranik S, Prasad M. Structure and regulatory networks of WD40 protein in plants[J]. J Plant Biochem Biotechnol, 2012, 21(1):32-39. doi: 10.1007/s13562-012-0134-1 URL
[15]	Kui LW, Bolitho K, Grafton K, et al. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae[J]. BMC Plant Biol, 2010, 10:50. doi: 10.1186/1471-2229-10-50 URL
[16]	Bogs J, Jaffé FW, et al. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development[J]. Plant Physiol, 2007, 143(3):1347-1361. pmid: 17208963
[17]	Walker AR, Lee E, Bogs J, et al. White grapes arose through the mutation of two similar and adjacent regulatory genes[J]. Plant J, 2007, 49(5):772-785. pmid: 17316172
[18]	Spelt C, Quattrocchio F, Mol JN, et al. anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes[J]. Plant Cell, 2000, 12(9):1619-1632. pmid: 11006336
[19]	Jin HL, Cominelli E, Bailey P, et al. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis[J]. EMBO J, 2000, 19(22):6150-6161. pmid: 11080161
[20]	张静, 潘红, 赖呈纯, 等. ALA对刺葡萄愈伤组织生长及主要抗氧化物质积累的影响[J]. 热带作物学报, 2021, 42(8):2305-2312.
	Zhang J, Pan H, Lai CC, et al. Effect of 5-aminolevulinic acid (ALA)on callus growth of spine grape callus and accumulation of its main antioxidants[J]. Chin J Trop Crops, 2021, 42(8):2305-2312.
[21]	Ju YL, Yang L, Yue XF, et al. Anthocyanin profiles and color properties of red wines made from Vitis davidii and Vitis vinifera grapes[J]. Food Sci Hum Wellness, 2021, 10(3):335-344. doi: 10.1016/j.fshw.2021.02.025 URL
[22]	王静, 周广胜. 中国毛葡萄和刺葡萄分布的气候适宜性[J]. 应用生态学报, 2020, 31(1):97-103. doi: 10.13287/j.1001-9332.202001.019
	Wang J, Zhou GS. Climatic suitability for the distribution of Vitis heyneana and V. davidii in China[J]. Chin J Appl Ecol, 2020, 31(1):97-103.
[23]	洪艳, 武宇薇, 宋想, 等. 光照调控园艺作物花青素苷生物合成的分子机制[J]. 园艺学报, 2021, 48(10):1983-2000.
	Hong Y, Wu YW, Song X, et al. Molecular mechanism of light-induced anthocyanin biosynthesis in horticultural crops[J]. Acta Hortic Sin, 2021, 48(10):1983-2000.
[24]	刘帅, 张亚红, 徐伟荣, 等. 基于转录组研究光质对转色期红地球葡萄果实着色及品质的影响[J]. 果树学报, 2021, 38(12):2045-2058.
	Liu S, Zhang YH, Xu WR, et al. Effects of light quality on the berry coloration and quality of Red Globe grape during veraison based on transcriptome sequencing[J]. J Fruit Sci, 2021, 38(12):2045-2058.
[25]	袁华招, 赵密珍, 吴伟民, 等. 葡萄CHS和STS基因家族生物信息学鉴定和表达分析[J]. 植物遗传资源学报, 2016, 17(4):756-765.
	Yuan HZ, Zhao MZ, Wu WM, et al. Genome-wide identification and expression analysis of CHS and STS gene families in grape(Vitis vinifera L.)[J]. J Plant Genet Resour, 2016, 17(4):756-765.
[26]	潘红, 赖呈纯, 张静, 等. 不同光质条件下刺葡萄红色愈伤组织的RT-qPCR内参基因筛选[J]. 应用与环境生物学报, 2019, 25(6):1407-1413.
	Pan H, Lai CC, Zhang J, et al. Selection of reference genes for RT-qPCR from the red callus of Vitis davidii(Rom. CailL.)Foëx under different light qualities[J]. Chin J Appl Environ Biol, 2019, 25(6):1407-1413.
[27]	葛翠莲, 黄春辉, 徐小彪. 果实花青素生物合成研究进展[J]. 园艺学报, 2012, 39(9):1655-1664.
	Ge CL, Huang CH, Xu XB. Research on anthocyanins biosynthesis in fruit[J]. Acta Hortic Sin, 2012, 39(9):1655-1664.
[28]	时晓芳, 林玲, 白先进, 等. 阳光玫瑰葡萄果实生长发育及品质对不同光质的响应[J]. 南方农业学报, 2021, 52(6):1641-1647.
	Shi XF, Lin L, Bai XJ, et al. Response of berries development and quality of Shine Muscat grape to different light qualities[J]. J South Agric, 2021, 52(6):1641-1647.
[29]	张克坤, 刘凤之, 王孝娣, 等. 不同光质补光对促早栽培‘瑞都香玉’葡萄果实品质的影响[J]. 应用生态学报, 2017, 28(1):115-126. doi: 10.13287/j.1001-9332.201701.003
	Zhang KK, Liu FZ, Wang XD, et al. Effects of supplementary light with different wavelengths on fruit quality of ‘Ruidu Xiangyu’ grape under promoted cultivation[J]. Chin J Appl Ecol, 2017, 28(1):115-126.
[30]	Li Q, Kubota C. Effects of supplemental light quality on growth and phytochemicals of baby leaf lettuce[J]. Environ Exp Bot, 2009, 67(1):59-64. doi: 10.1016/j.envexpbot.2009.06.011 URL
[31]	Ren J, Guo SS, Xu CL, et al. Effects of different carbon dioxide and LED lighting levels on the anti-oxidative capabilities of Gynura bicolor DC[J]. Adv Space Res, 2014, 53(2):353-361. doi: 10.1016/j.asr.2013.11.019 URL
[32]	Kadomura-Ishikawa Y, Miyawaki K, Noji S, et al. Phototropin 2 is involved in blue light-induced anthocyanin accumulation in Fragaria x ananassa fruits[J]. J Plant Res, 2013, 126(6):847-857. doi: 10.1007/s10265-013-0582-2 pmid: 23982948
[33]	解潇冬, 刘晓莹, 汪文杰, 等. 光质对红阳猕猴桃愈伤组织生长速度和花青素合成的影响[J]. 山西农业科学, 2021, 49(10):1166-1172.
	Xie XD, Liu XY, Wang WJ, et al. Effects of light quality on callus growth rate and anthocyanin synthesis of Hongyang kiwifruit[J]. J Shanxi Agric Sci, 2021, 49(10):1166-1172.
[34]	Liu CC, Chi C, Jin LJ, et al. The bZip transcription factor HY5 mediates CRY1a-induced anthocyanin biosynthesis in tomato[J]. Plant Cell Environ, 2018, 41(8):1762-1775. doi: 10.1111/pce.13171 URL
[35]	王峰, 王秀杰, 赵胜男, 等. 光对园艺植物花青素生物合成的调控作用[J]. 中国农业科学, 2020, 53(23):4904-4917.
	Wang F, Wang XJ, Zhao SN, et al. Light regulation of anthocyanin biosynthesis in horticultural crops[J]. Sci Agric Sin, 2020, 53(23):4904-4917.
[36]	Borevitz JO, Xia Y, Blount J, et al. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis[J]. Plant Cell, 2000, 12(12):2383-2394. pmid: 11148285
[37]	Tohge T, Nishiyama Y, Hirai MY, et al. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor[J]. Plant J, 2005, 42(2):218-235. doi: 10.1111/j.1365-313X.2005.02371.x URL
[38]	Zhang YZ, Xu SZ, Ma HP, et al. The R2R3-MYB gene PsMYB58 positively regulates anthocyanin biosynthesis in tree peony flowers[J]. Plant Physiol Biochem, 2021, 164:279-288. doi: 10.1016/j.plaphy.2021.04.034 URL
[39]	Lijavetzky D, Ruiz-García L, Cabezas JA, et al. Molecular genetics of berry colour variation in table grape[J]. Mol Genet Genomics, 2006, 276(5):427-435. pmid: 16924546
[40]	Azuma A, Kobayashi S, Goto-Yamamoto N, et al. Color recovery in berries of grape(Vitis vinifera L.)‘Benitaka’, a bud sport of ‘Italia’, is caused by a novel allele at the VvmybA1 locus[J]. Plant Sci, 2009, 176(4):470-478. doi: 10.1016/j.plantsci.2008.12.015 URL
[41]	Azuma A, et al. Haplotype composition at the color locus is a major genetic determinant of skin color variation in Vitis × labruscana grapes[J]. Theor Appl Genet, 2011, 122(7):1427-1438. doi: 10.1007/s00122-011-1542-7 URL
[42]	Albert NW, Davies KM, et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots[J]. Plant Cell, 2014, 26(3):962-980. doi: 10.1105/tpc.113.122069 URL
[43]	Zhang H, Gong JX, Chen KL, et al. A novel R3 MYB transcriptional repressor, MaMYBx, finely regulates anthocyanin biosynthesis in grape hyacinth[J]. Plant Sci, 2020, 298:110588.
[44]	Fu ZZ, Wang LM, Shang HQ, et al. An R3-MYB gene of Phalaenopsis, MYBx1, represses anthocyanin accumulation[J]. Plant Growth Regul, 2019, 88(2):129-138. doi: 10.1007/s10725-019-00493-3 URL
[45]	Wan SZ, Li CF, Ma XD, et al. PtrMYB57 contributes to the negative regulation of anthocyanin and proanthocyanidin biosynthesis in poplar[J]. Plant Cell Rep, 2017, 36(8):1263-1276. doi: 10.1007/s00299-017-2151-y pmid: 28523445
[46]	Xu HF, Yang GX, Zhang J, et al. Overexpression of a repressor MdMYB15L negatively regulates anthocyanin and cold tolerance in red-fleshed callus[J]. Biochem Biophys Res Commun, 2018, 500(2):405-410. doi: 10.1016/j.bbrc.2018.04.088 URL
[47]	Deng GM, Zhang S, et al. MaMYB4, an R2R3-MYB repressor transcription factor, negatively regulates the biosynthesis of anthocyanin in banana[J]. Front Plant Sci, 2021, 11:600704.
[48]	Pérez-Díaz JR, Pérez-Díaz J, Madrid-Espinoza J, et al. New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco[J]. Plant Mol Biol, 2016, 90(1/2):63-76. doi: 10.1007/s11103-015-0394-y URL
[49]	Zhu ZG, Li GR, Liu L, et al. A R2R3-MYB transcription factor, VvMYBC2L2, functions as a transcriptional repressor of anthocyanin biosynthesis in grapevine(Vitis vinifera L.)[J]. Molecules, 2018, 24(1):92. doi: 10.3390/molecules24010092 URL
[50]	Tirumalai V, Swetha C, Nair A, et al. miR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes[J]. J Exp Bot, 2019, 70(18):4775-4792. doi: 10.1093/jxb/erz264 pmid: 31145783
[51]	Chagné D, Kui LW, Espley RV, et al. An ancient duplication of apple MYB transcription factors is responsible for novel red fruit-flesh phenotypes[J]. Plant Physiol, 2013, 161(1):225-239. doi: 10.1104/pp.112.206771 pmid: 23096157
[52]	Cui DL, Zhao SX, Xu HN, et al. The interaction of MYB, bHLH and WD40 transcription factors in red pear(Pyrus pyrifolia)peel[J]. Plant Mol Biol, 2021, 106(4/5):407-417. doi: 10.1007/s11103-021-01160-w URL

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Application
CHS2-F	CACTGATTCAAGCAGCCAAAAATG	CHS2 克隆
CHS2-R	TTACTTAAGTGTGAAAGGAGAGAAG	CHS2 克隆
CHS3-F	CTCCAACCACCACAACCCTCATC	CHS3 克隆
CHS3-R	ATGGGGCTAGAAAAAGCATATCATC	CHS3 克隆
CHS2-qPCR-F	TGGGTCTGAAGGAAGAGAAAC	CHS2 qPCR
CHS2-qPCR-R	TTGTGTAGCAAGGCTGTGC	CHS2 qPCR
CHS3-qPCR-F	AGTTACGCTCCACACGACAC	CHS3 qPCR
CHS3-qPCR-R	AGCACCACGGTCTCAACAGT	CHS3 qPCR

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Application
CHS2-F	CACTGATTCAAGCAGCCAAAAATG	CHS2 克隆
CHS2-R	TTACTTAAGTGTGAAAGGAGAGAAG	CHS2 克隆
CHS3-F	CTCCAACCACCACAACCCTCATC	CHS3 克隆
CHS3-R	ATGGGGCTAGAAAAAGCATATCATC	CHS3 克隆
CHS2-qPCR-F	TGGGTCTGAAGGAAGAGAAAC	CHS2 qPCR
CHS2-qPCR-R	TTGTGTAGCAAGGCTGTGC	CHS2 qPCR
CHS3-qPCR-F	AGTTACGCTCCACACGACAC	CHS3 qPCR
CHS3-qPCR-R	AGCACCACGGTCTCAACAGT	CHS3 qPCR

基因名称 Gene name	GenBank 登陆号 GenBank accession	片段长度 Fragment length/bp	ORF长度 ORF length/bp	氨基酸数目 Amino acid number	分子量 Molecular weight/kD	理论等电点pI	亲水性 GRAVY	信号肽 Signal peptide	亚细胞定位 Subcellular localization
VdCHS2	OL906400	1 382	1 182	393	42.90	6.1	-0.057	无 No	细胞质Cytoplasmic
VdCHS3	OL906401	1 398	1 170	389	42.60	6.18	-0.132	无 No	细胞质Cytoplasmic

基因名称 Gene name	GenBank 登陆号 GenBank accession	片段长度 Fragment length/bp	ORF长度 ORF length/bp	氨基酸数目 Amino acid number	分子量 Molecular weight/kD	理论等电点pI	亲水性 GRAVY	信号肽 Signal peptide	亚细胞定位 Subcellular localization
VdCHS2	OL906400	1 382	1 182	393	42.90	6.1	-0.057	无 No	细胞质Cytoplasmic
VdCHS3	OL906401	1 398	1 170	389	42.60	6.18	-0.132	无 No	细胞质Cytoplasmic

光质类型 Light quality type	光质名称 Light quality	光波长 Light wavelength/nm		表达模式 Expression pattern
光质类型 Light quality type	光质名称 Light quality	主峰 Main peak	次峰 Secondary peak	VdCHS2	VdCHS3
短波光	紫光	448	/	先升后降	先升后降
	蓝光	456	/	先升后降	先升后降
	绿光	514	/	先升后降	先升后降
长波光	黄光	590	/	下调	下调
长波光	红光	637	/	下调	下调
混合光质	白光	453	550	先升后降	下调
	暖白光	588	450	先升后降	上调
	暖黄光	595	450	先升后降	先升后降