大白菜番茄红素β-环化酶基因BrLCYB的鉴定与功能分析

doi:10.3969/j.issn.1004-1524.20221285

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (9): 2090-2096.DOI: 10.3969/j.issn.1004-1524.20221285

大白菜番茄红素β-环化酶基因BrLCYB的鉴定与功能分析

李必元(), 岳智臣, 赵彦婷, 雷娟利, 胡齐赞, 陶鹏()

浙江省农业科学院蔬菜研究所,浙江杭州 310021

收稿日期:2022-09-01 出版日期:2023-09-25 发布日期:2023-10-09
作者简介:*陶鹏,E-mail: taopeng-84@163.com
李必元(1976—),男,湖北仙桃人,学士,副研究员,主要从事十字花科蔬菜育种工作。E-mail: 16061944@qq.com
通讯作者: 陶鹏
基金资助:
浙江省农业新品种选育重大科技专项(2021C02065-5-2);浙江省自然科学基金(LY21C150006);浙江省农业重大技术协同推广计划(2021XTTGSC01-2)

Identification and functional analysis of the BrLCYB gene of lycopene β-cyclase from Chinese cabbage

LI Biyuan(), YUE Zhichen, ZHAO Yanting, LEI Juanli, HU Qizan, TAO Peng()

Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2022-09-01 Online:2023-09-25 Published:2023-10-09
Contact: TAO Peng

摘要/Abstract

摘要：

本研究从大白菜中克隆了唯一的番茄红素β-环化酶基因(BrLCYB),编码序列长度为1 482 bp,BrLCYB只在5 'UTR所对应的基因序列中存在一个内含子,导致其转录后形成2种类型的转录本(BrLCYB-1和BrLCYB-2),其中BrLCYB-1类型占98.26%。BrLCYB广泛表达在多种器官中和整个胚胎发育阶段,其中在花和成熟胚胎中有着较高的表达。系统进化树显示,高等植物的LCYB起源于蓝藻,LCYB基因在进化过程中出现了内含子获得和内含子丢失事件,最终在高等植物的编码序列中丢失全部内含子。BrLCYB蛋白包含番茄红素β-环化酶结构域,为了进一步验证BrLCYB的功能,构建了pET-BrLCYB原核表达载体。pACCRT-EIB和pET-BrLCYB共转化到大肠埃希菌中,可使得大肠埃希菌富集橙黄色的β-胡萝卜素,证明大白菜BrLCYB具有番茄红素β-环化酶的催化功能。

关键词: 大白菜, 番茄红素β-环化酶, 基因, 功能分析

Abstract:

In this study, the only lycopene β-cyclase was cloned from Chinese cabbage (BrLCYB) and had 1 482 bp coding sequence. BrLCYB contains the only one intron in its gene sequence, corresponding to 5' UTR. The intron resulted in BrLCYB producing two types of transcripts (BrLCYB-1 and BrLCYB-2) after transcription. BrLCYB-1 accounted for 98.26% of the total BrLCYB transcripts. BrLCYB was widely expressed in a variety of organs and the whole embryonic development stage, and has a higher expression in flowers and mature embryos. The phylogenetic tree of LCYBs showed that the LCYBs of higher plants originated from cyanobacteria. During the evolution of LCYB genes, intron gain and intron loss, occurred successively in LCYBs, and finally no intron existed in coding sequences of LCYBs in higher plants. BrLCYB contained a lycopene β-cyclase domain. In order to further verify the function of BrLCYB, the pET-BrLCYB prokaryotic expression vector was constructed. The co-transformation of pACCRT-EIB and pET-BrLCYB into E. coli can make E. coli enriching orange-yellow β-carotene, proving that BrLCYB of Chinese cabbage had the function of lycopene β-cyclase.

Key words: Chinese cabbage, lycopene β-cyclase, gene, functional analysis

中图分类号:

S634.1

李必元, 岳智臣, 赵彦婷, 雷娟利, 胡齐赞, 陶鹏. 大白菜番茄红素β-环化酶基因BrLCYB的鉴定与功能分析[J]. 浙江农业学报, 2023, 35(9): 2090-2096.

LI Biyuan, YUE Zhichen, ZHAO Yanting, LEI Juanli, HU Qizan, TAO Peng. Identification and functional analysis of the BrLCYB gene of lycopene β-cyclase from Chinese cabbage[J]. Acta Agriculturae Zhejiangensis, 2023, 35(9): 2090-2096.

图/表 6

表1 本研究中所使用的引物/检索序列

Table 1 The retrieval sequences used in this study

引物/检索序列名称Name	序列Sequence (5 '→3')
LCYBF	TTTTGTTTAACTTTAAGAAGGAGATATACCATGGATACTCTCTT
LCYBR	TCGGGCTTTGTTAGCAGCCGGATCTCAGTGTTAGTCTCTATCT
BrLCYB-1S	CTTCTGGGTGATCTTCAATTATCTGAAAGTTAAGA
BrLCYB-2S	CATGTAGGTGATCTTCAATTATCTGAAAGTTAAGA

表2 LCYB基因在芸薹属物种中的亚基因组定位和共线性关系

Table 2 Subgenomic localization and syntenic relationship of LCYB genes in Brassica species

At	Br			Bni			Bo			Bna_A			Bna_C			Bju_A			Bju_B
At	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2
•	•			•			•			•			•			•			•

图1 大白菜BrLCYB基因在DNA和cDNA模板中的扩增结果(A),大白菜He2 BrLCYB基因结构及转录本类型(B)及BrLCYB-1和BrLCYB-2两种转录本类型在总的BrLCYB转录本中的比例(C)

Fig.1 Amplification of the BrLCYB gene with the template DNA and cDNA of Chinese cabbage (A), gene structure and transcript type of BrLCYB in Chinese cabbage He2 line (B) and the proportion of BrLCYB-1 and BrLCYB-2 in the total BrLCYB transcripts (C)

图2 BrLCYB基因在不同器官和不同胚胎发育阶段的转录表达水平

Fig.2 The transcriptional expression level of BrLCYB gene in different organs and different embryonic development stages

图3 LCYB系统发育进化树方框中的数字表示相应物种的LCYB基因编码序列中内含子的数量。

Fig.3 The LCYB phylogenetic tree The number of the box representing the number of introns in the coding sequence of LCYB gene of the corresponding specie.

图4 大白菜He2的BrLCYB蛋白的功能域及其对应的蛋白序列(A)及BrLCYB蛋白的酶活性分析(B)

Fig.4 Functional domain of BrLCYB of Chinese cabbage He2 line and its corresponding protein sequence (A) and enzyme activity analysis of BrLCYB (B)

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大白菜番茄红素β-环化酶基因BrLCYB的鉴定与功能分析

Identification and functional analysis of the BrLCYB gene of lycopene β-cyclase from Chinese cabbage

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At	Br			Bni			Bo			Bna_A			Bna_C			Bju_A			Bju_B
At	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2
•	•			•			•			•			•			•			•

At	Br			Bni			Bo			Bna_A			Bna_C			Bju_A			Bju_B
At	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2
•	•			•			•			•			•			•			•

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At	Br			Bni			Bo			Bna_A			Bna_C			Bju_A			Bju_B
At	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2
•	•			•			•			•			•			•			•

At	Br			Bni			Bo			Bna_A			Bna_C			Bju_A			Bju_B
At	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2
•	•			•			•			•			•			•			•

At	Br			Bni			Bo			Bna_A			Bna_C			Bju_A			Bju_B
At	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2
•	•			•			•			•			•			•			•

At	Br			Bni			Bo			Bna_A			Bna_C			Bju_A			Bju_B
At	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2	LF	MF1	MF2
•	•			•			•			•			•			•			•