羽衣甘蓝类黄酮糖基转移酶基因的筛选及分析

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

摘要/Abstract

摘要：

筛选羽衣甘蓝类黄酮糖基转移酶（UDP glycosyltransferase，UGT）基因，研究其在不同材料中的表达模式，为后续的功能研究提供依据。以不同叶色羽衣甘蓝转录组数据为材料，筛选UGT基因并对其进行分析，通过RT-qPCR验证候选UGT基因的表达与花青苷和类黄酮含量的相关性。结果共筛选获得32个UGT基因，32个BoUGT蛋白的C端均含有Motif 1和Motif 2，29个BoUGT蛋白的N端含有Motif 3。其氨基酸数目为222-501个，分子量为24.69-56.53 kD，等电点为4.85-7.19。亚细胞定位预测结果显示，BoUGT以叶绿体定位居多。蛋白聚类分析发现，有21个BoUGT基因编码的蛋白可能参与激素的糖基化修饰过程，11个可能参与类黄酮的糖基化修饰过程。花青素含量与Bol018968、Bol011466和Bol028297表达呈正相关；类黄酮含量与Bol021317、Bol027055和Bol026392表达呈正相关，而与Bol018968负相关。为后续羽衣甘蓝类黄酮物质生物合成途径研究提供了候选基因。

关键词: 羽衣甘蓝, 转录组, UGT, 类黄酮, 花青素

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

罗雅方, 朱春花, 肖钰婷, 李方全, 张江, 王玉书. 羽衣甘蓝类黄酮糖基转移酶基因的筛选及分析[J]. 生物技术通报, 2022, 38(11): 194-201.

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.

图/表 9

参考文献 22

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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