Screening and Functional Analysis of UGT Genes Involved in the Flavonoid Biosynthesis of Brassica oleracea var. acephala

doi:10.13560/j.cnki.biotech.bull.1985.2022-0434

Abstract

Abstract:

In order to explore the function of UDP glycosyltransferase（UGT）gene in the flavonoids synthesis in kale，the UGT gene was screened and analyzed based on transcriptome data of kale with different leaf colors. The correlation between the expression of candidate UGT gene and the content of anthocyanin and flavonoids was verified by RT-qPCR. As results，a total of 32 UGT genes were screened. Among them，the C-terminal of 32 BoUGTs generally contained Motif 1 and Motif 2，and the N-terminal of 29 BoUGTs contained Motif 3. The number of amino acids ranged from 222 to 501，and the molecular weight was in 24.69-56.53 kD. Moreover，the isoelectric point was 4.85-7.19. The prediction of subcellular location showed that most BoUGTs were located in chloroplast. Further phylogenetic analysis uncovered that the protein encoded by 21 BoUGTs might be involved in the glycosylation of plant hormones，while 11 BoUGTs might function in the flavonoid glycosylation. The results revealed that expressions of Bol018968，Bol011466 and Bol028297 were positively correlated with the contents of anthocyanin. Furthermore expressions of Bol021317，Bol027055 and Bol026392 were positively correlated with the contents of flavonoid，while that of Bol018968 was negatively correlated with the flavonoid content. This study provides candidate genes for the follow-up study on the biosynthesis pathway of flavonoids in kale.

Key words: Brassica oleracea var. acephala, transcriptome, UGT gene, flavonoids, anthocyanin

LUO Ya-fang, ZHU Chun-hua, XIAO Yu-ting, LI Fang-quan, ZHANG Jiang, WANG Yu-shu. Screening and Functional Analysis of UGT Genes Involved in the Flavonoid Biosynthesis of Brassica oleracea var. acephala[J]. Biotechnology Bulletin, 2022, 38(11): 194-201.

Figures/Tables 9

References 22

[1]	Hou BK, Lim EK, Higgins GS, et al. N-glucosylation of cytokinins by glycosyltransferases of Arabidopsis thaliana[J]. J Biol Chem, 2004, 279(46):47822-47832. doi: 10.1074/jbc.M409569200 URL
[2]	吴一凡, 张睿琪, 董泽安, 等. 植物糖基转移酶的分类、分布及在果实发育中作用研究进展[J]. 食品科技, 2021, 46(7):1-6.
	Wu YF, Zhang RQ, Dong ZA, et al. Classification and distribution of glycosyltransferases in plant and its roles in the fruit development[J]. Food Sci Technol, 2021, 46(7):1-6. doi: 10.1016/j.tifs.2015.07.006 URL
[3]	解林峰, 任传宏, 张波, 等. 植物类黄酮生物合成相关UDP-糖基转移酶研究进展[J]. 园艺学报, 2019, 46(9):1655-1669.
	Xie LF, Ren CH, Zhang B, et al. Plant UDP-glycosyltransferases in flavonoids biosynthesis[J]. Acta Hortic Sin, 2019, 46(9):1655-1669.
[4]	Wu BP, Gao LX, Gao J, et al. Genome-wide identification, expression patterns, and functional analysis of UDP glycosyltransferase family in peach(Prunus persica L. batsch)[J]. Front Plant Sci, 2017, 8:389.
[5]	王玉书, 赵爽, 张琳, 等. 羽衣甘蓝花青素的定位及含量成分测定[J]. 中国农业大学学报, 2020, 25(11):45-53.
	Wang YS, Zhao S, Zhang L, et al. Location, content and composition determination of anthocyanin in kale[J]. J China Agric Univ, 2020, 25(11):45-53.
[6]	王玉书, 王欢, 范震宇, 等. 基于转录组测序的羽衣甘蓝叶色相关基因分析[J]. 基因组学与应用生物学, 2020, 39(1):200-206.
	Wang YS, Wang H, Fan ZY, et al. Identifying genes associated with leaf color in kale(Brassica oleracea l. var. acephala DC. )based on transcriptome analysis[J]. Genom Appl Biol, 2020, 39(1):200-206.
[7]	Tian L, Blount JW, Dixon RA. Phenylpropanoid glycosyltransferases from osage orange(Maclura pomifera)fruit[J]. FEBS Lett, 2006, 580(30):6915-6920. pmid: 17157841
[8]	Masada S, Terasaka K, Oguchi Y, et al. Functional and structural characterization of a flavonoid glucoside 1, 6-glucosyltransferase from Catharanthus roseus[J]. Plant Cell Physiol, 2009, 50(8):1401-1415. doi: 10.1093/pcp/pcp088 URL
[9]	Priest DM, Ambrose SJ, Vaistij FE, et al. Use of the glucosyltransferase UGT71B6 to disturb abscisic acid homeostasis in Arabidopsis thaliana[J]. Plant J, 2006, 46(3):492-502. pmid: 16623908
[10]	Husar S, Berthiller F, Fujioka S, et al. Overexpression of the UGT73C6 alters brassinosteroid glucoside formation in Arabidopsis thaliana[J]. BMC Plant Biol, 2011, 11:51. doi: 10.1186/1471-2229-11-51 URL
[11]	Noutoshi Y, Okazaki M, Kida T, et al. Novel plant immune-priming compounds identified via high-throughput chemical screening target salicylic acid glucosyltransferases in Arabidopsis[J]. Plant Cell, 2012, 24(9):3795-3804. doi: 10.1105/tpc.112.098343 URL
[12]	Tognetti VB, van Aken O, Morreel K, et al. Perturbation of indole-3-butyric acid homeostasis by the UDP-glucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance[J]. Plant Cell, 2010, 22(8):2660-2679. doi: 10.1105/tpc.109.071316 URL
[13]	Wang J, Ma XM, Kojima M, et al. N-glucosyltransferase UGT76C2 is involved in cytokinin homeostasis and cytokinin response in Arabidopsis thaliana[J]. Plant Cell Physiol, 2011, 52(12):2200-2213. doi: 10.1093/pcp/pcr152 pmid: 22051886
[14]	张桂芝, 林继山, 李燕洁, 等. 植物激素糖基化修饰研究进展[J]. 植物学报, 2014, 49(5):515-523. doi: 10.3724/SP.J.1259.2014.00515
	Zhang GZ, Lin JS, Li YJ, et al. Research advances in the glycosylation of plant hormones[J]. Chin Bull Bot, 2014, 49(5):515-523.
[15]	安建平, 宋来庆, 赵玲玲, 等. 苹果细胞分裂素O-糖基转移酶基因MdZOG1的分离与功能鉴定[J]. 园艺学报, 2016, 43(8):1429-1436.
	An JP, Song LQ, Zhao LL, et al. Molecular cloning and functional characterization of a cytokinin O-glycosyltransferase gene MdZOG1 in apple[J]. Acta Hortic Sin, 2016, 43(8):1429-1436.
[16]	Lim EK, Doucet CJ, Hou BK, et al. Resolution of(+)-abscisic acid using an Arabidopsis glycosyltransferase[J]. Tetrahedron Asymmetry, 2005, 16(1):143-147. doi: 10.1016/j.tetasy.2004.11.062 URL
[17]	Ma YL, Cao J, Chen QQ, et al. Abscisic acid receptors maintain abscisic acid homeostasis by modulating UGT71C5 glycosylation activity[J]. J Integr Plant Biol, 2021, 63(3):543-552. doi: 10.1111/jipb.13030
[18]	Bowles D, Lim EK, Poppenberger B, et al. Glycosyltransferases of lipophilic small molecules[J]. Annu Rev Plant Biol, 2006, 57:567-597. pmid: 16669774
[19]	Pollastri S, Tattini M. Flavonols:old compounds for old roles[J]. Ann Bot, 2011, 108(7):1225-1233. doi: 10.1093/aob/mcr234 URL
[20]	Liu XG, Lin CL, Ma XD, et al. Functional characterization of a flavonoid glycosyltransferase in sweet orange(Citrus sinensis)[J]. Front Plant Sci, 2018, 9:166. doi: 10.3389/fpls.2018.00166 URL
[21]	Devaiah SP, Tolliver BM, Zhang C, et al. Mutational analysis of substrate specificity in a Citrus paradisi flavonol 3-O-glucosyltransferase[J]. J Plant Biochem Biotechnol, 2018, 27(1):13-27. doi: 10.1007/s13562-017-0411-0 URL
[22]	赵倩倩, 宋艳红, 宋盼, 等. 森林草莓糖基转移酶基因家族生物信息学及其表达分析[J]. 南方农业学报, 2021, 52(6):1615-1624.
	Zhao QQ, Song YH, Song P, et al. Analysis of bioinformatics and gene expression of glycosyltransferase gene family in Fragaria vesca[J]. J South Agric, 2021, 52(6):1615-1624.

基因Gene	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
Bol021317	GGGTGTGTTTGTGACGCATTGTG	CTTCCACCGCTCTTCCGTTCAG
Bol018968	CCGTTCTTCGCCGATCAGTTGAC	CCTAACCACCGCCTCCACTCTC
Bol027055	ATCACGGAGCAACCATCACATACG	GGAGAACCAAGCATAGGACAAGCC
Bol026392	GGGAATCGAGAGAGGGAGTTAGGG	AACCTTAGCCACAAGTCCGTCAAC
Bol011466	GGAACTGTGGCAACGAGGGTAAC	GCCGACGACGATGAAGGTTACG
Bol028297	GCCCGTAACTGTCTTCTCCATCTTC	GGAACTGTGGCAACGAGGGTAAC
18S RNA	CCAGGTCCAGACATAGTAAG	GTACAAAGGGCAGGGACGTA

基因Gene	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
Bol021317	GGGTGTGTTTGTGACGCATTGTG	CTTCCACCGCTCTTCCGTTCAG
Bol018968	CCGTTCTTCGCCGATCAGTTGAC	CCTAACCACCGCCTCCACTCTC
Bol027055	ATCACGGAGCAACCATCACATACG	GGAGAACCAAGCATAGGACAAGCC
Bol026392	GGGAATCGAGAGAGGGAGTTAGGG	AACCTTAGCCACAAGTCCGTCAAC
Bol011466	GGAACTGTGGCAACGAGGGTAAC	GCCGACGACGATGAAGGTTACG
Bol028297	GCCCGTAACTGTCTTCTCCATCTTC	GGAACTGTGGCAACGAGGGTAAC
18S RNA	CCAGGTCCAGACATAGTAAG	GTACAAAGGGCAGGGACGTA

基因登录号Gene ID	氨基酸数No. of amino acids	分子量Molecular weight/kD	等电点pI	亚细胞定位Subcellular localization
Bol005901	448	50.49	6.38	叶绿体Chloroplast
Bol005786	436	47.69	5.24	叶绿体Chloroplast
Bol010781	478	52.64	5.77	细胞核Nucleus
Bol011466	481	52.61	6.05	内质网Endoplasmic reticulum
Bol013632	495	55.74	5.36	细胞质Cytoplasm
Bol014127	456	51.22	5.63	叶绿体Chloroplast
Bol016956	486	53.90	5.14	液泡Vacuoles
Bol017633	464	52.12	5.32	叶绿体Chloroplast
Bol018968	476	53.75	5.27	叶绿体Chloroplast
Bol021317	460	50.11	5.41	叶绿体Chloroplast
Bol022050	468	52.98	5.46	细胞质Cytoplasm
Bol023225	483	54.07	4.88	细胞核Nucleus
Bol025091	501	56.53	7.19	叶绿体Chloroplast
Bol026363	483	53.91	4.94	叶绿体Chloroplast
Bol026392	480	53.18	6.04	过氧化物酶体Peroxisome
Bol026589	466	52.31	6.01	内质网Endoplasmic reticulum
Bol026592	477	53.16	5.14	细胞质Cytoplasm
Bol026593	260	29.15	4.99	细胞质Cytoplasm
Bol027055	457	50.14	5.20	叶绿体Chloroplast
Bol028297	476	54.45	5.55	叶绿体Chloroplast
Bol029484	423	47.38	5.81	叶绿体Chloroplast
Bol030030	450	50.57	5.76	叶绿体Chloroplast
Bol031911	484	53.95	5.72	细胞质Cytoplasm
Bol036924	464	51.48	5.57	内质网Endoplasmic reticulum
Bol037703	496	55.69	5.34	叶绿体Chloroplast
Bol038332	260	28.89	4.85	细胞核Nucleus
Bol038351	222	24.69	5.58	叶绿体Chloroplast
Bol039726	496	56.04	5.52	叶绿体Chloroplast
Bol040697	483	52.83	5.50	叶绿体Chloroplast
Bol041114	477	52.89	5.06	细胞质Cytoplasm
Bol041115	475	53.02	5.85	细胞质Cytoplasm
Bol043480	484	54.48	5.80	叶绿体Chloroplast

基因登录号Gene ID	氨基酸数No. of amino acids	分子量Molecular weight/kD	等电点pI	亚细胞定位Subcellular localization
Bol005901	448	50.49	6.38	叶绿体Chloroplast
Bol005786	436	47.69	5.24	叶绿体Chloroplast
Bol010781	478	52.64	5.77	细胞核Nucleus
Bol011466	481	52.61	6.05	内质网Endoplasmic reticulum
Bol013632	495	55.74	5.36	细胞质Cytoplasm
Bol014127	456	51.22	5.63	叶绿体Chloroplast
Bol016956	486	53.90	5.14	液泡Vacuoles
Bol017633	464	52.12	5.32	叶绿体Chloroplast
Bol018968	476	53.75	5.27	叶绿体Chloroplast
Bol021317	460	50.11	5.41	叶绿体Chloroplast
Bol022050	468	52.98	5.46	细胞质Cytoplasm
Bol023225	483	54.07	4.88	细胞核Nucleus
Bol025091	501	56.53	7.19	叶绿体Chloroplast
Bol026363	483	53.91	4.94	叶绿体Chloroplast
Bol026392	480	53.18	6.04	过氧化物酶体Peroxisome
Bol026589	466	52.31	6.01	内质网Endoplasmic reticulum
Bol026592	477	53.16	5.14	细胞质Cytoplasm
Bol026593	260	29.15	4.99	细胞质Cytoplasm
Bol027055	457	50.14	5.20	叶绿体Chloroplast
Bol028297	476	54.45	5.55	叶绿体Chloroplast
Bol029484	423	47.38	5.81	叶绿体Chloroplast
Bol030030	450	50.57	5.76	叶绿体Chloroplast
Bol031911	484	53.95	5.72	细胞质Cytoplasm
Bol036924	464	51.48	5.57	内质网Endoplasmic reticulum
Bol037703	496	55.69	5.34	叶绿体Chloroplast
Bol038332	260	28.89	4.85	细胞核Nucleus
Bol038351	222	24.69	5.58	叶绿体Chloroplast
Bol039726	496	56.04	5.52	叶绿体Chloroplast
Bol040697	483	52.83	5.50	叶绿体Chloroplast
Bol041114	477	52.89	5.06	细胞质Cytoplasm
Bol041115	475	53.02	5.85	细胞质Cytoplasm
Bol043480	484	54.48	5.80	叶绿体Chloroplast

试材 Test materials	花青素含量Anthocyanin content/（mg·g^-1）	类黄酮含量Flavonoid content/（mg·g^-1）
W05	1.429±0.413a	0.125±0.048c
W09	0.979±0.287ab	0.149±0.037c
D07	0.595±0.169bc	0.268±0.059c
W08	-	15.083±1.958a
D06	-	12.216±2.065b