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

doi:10.3969/j.issn.1004-1524.20221285

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (9): 2090-2096.DOI: 10.3969/j.issn.1004-1524.20221285

• Horticultural Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S634.1

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.

Figures/Tables 6

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

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
•	•			•			•			•			•			•			•

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)

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

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.

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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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
•	•			•			•			•			•			•			•