Cloning and Expression Analysis of BrCYP83B1 Gene in Chinese Cabbage

doi:10.13560/j.cnki.biotech.bull.1985.2023-1127

Abstract

Abstract:

【Objective】 The cytochrome P450 family is an important enzyme system involved in glucosinolate synthesis in cruciferous plants, where in the CYP83 subfamily plays a predominant role in core structure formation, this work aims to investigate the Chinese cabbage (Brassica rapa ssp. Pekinensis) CYP83B1 gene functional role.【Method】 RT-PCR was used to clone the CYP83B1 gene. Bioinformatics analysis software was utilized to predict the encoded protein's physicochemical properties, homology of the encoded protein, and promoter cis-acting elements. The expression pattern of BrCYP83B1 was analyzed by RT-qPCR, and a plant overexpression vector was constructed for further experimentation. 【Result】 The cDNA length of BrCYP83B1 was 1 500 bp, encoding a total of 499 amino acids. The protein belonged to cytochrome P450 superfamily and predominantly located in the cytoplasm. Its secondary structure primarily comprised of α-helixes and irregular coil. Homologous comparison illustrated that BrCYP83B1 had close relationship with Brassica napus L and Brassica oleracea L. var. italica. The BrCYP83B1 promoter contained cis-acting elements that were involved in the response to salicylic acid（SA）, abscisic acid（ABA）, and methyl jasmonate（MeJA）, suggesting that the expression of BrCYP83B1 gene may be regulated by hormones. The expression of BrCYP83B1 was detected in various plant organs, including the roots, stems, leaves, flowers and fruits from RT-qPCR results. Notably, the highest expression was observed in the leaves. Moreover, BrCYP83B1 significantly presented induction upon treatment with MeJA, while its expression was repressed by SA. Additionally, ABA treatment initially up-regulated and subsequently down-regulated the gene. 【Conclusion】 BrCYP83B1 may be involved in the response regulation of Chinese cabbage to hormones.

Key words: Brassica rapa ssp. pekinensis, BrCYP83B1, gene cloning, plant hormones, expression analysis, overexpression vector

WANG Yu-shu, ZHAO Lin-lin, ZHAO Shuang, HU Qi, BAI Hui-xia, WANG Huan, CAO Ye-ping, FAN Zhen-yu. Cloning and Expression Analysis of BrCYP83B1 Gene in Chinese Cabbage[J]. Biotechnology Bulletin, 2024, 40(6): 152-160.

Figures/Tables 10

Table 1 Primer used in this study

引物名称Primer name	序列Sequence（5'-3'）	用途 Application
BrCYP83B1-F	GC GTCGACATGGATCTCTTCTTGATTATTGCCG	片段扩增Fragment amplification
BrCYP83B1-R	G GAATTCTCAAATGTGCGTCCTTGGTGC	片段扩增Fragment amplification
RT-CYP83B1-F	CGCAAGTTTCCGACCCGTTAGAG	荧光定量PCR RT-qPCR
RT-CYP83B1-R	ACAGTTGGTGAAGGACAAGAGAAGC	荧光定量PCR RT-qPCR 荧光定量PCR RT-qPCR
RT-BrActin-F	GGAGCTGAGAGATTCCGTTG	荧光定量PCR RT-qPCR 荧光定量PCR RT-qPCR
RT-BrActin-R	GAACCACCACTGAGGACGAT	荧光定量PCR RT-qPCR

Fig. 1 PCR amplification of BrCYP83B1 gene M: DNA marker; P: PCR amplification product

Fig. 2 Conservative domain of BrCYP83B1 protein

Fig. 3 Prediction of secondary（A）and tertiary structure（B）of BrCYP83B1 protein Capital letters indicate amino acid sequence of BrCYP83B1 protein. Lowercase letters indicate different secondary structures, where h refers to α helix, c to random coil, e to extended strand, and t to β turn

Fig. 4 Multiple sequence alignment of BrCYP83B1 with its homologous protein from other plant species+ BnCYP83B1: Brassica napus, CDY35791.1; RsCYP83B1: Raphanus sativus, CDY35791.1; AtCYP83B1: Arabidopsis thaliana, NP_194878.1; BoCYP83B1: Brassica oleracea var. italica, APZ76511.1; ItCYP83B1: Isatis tinctoria, APY26702.1; EsCYP83B1: Eutrema salsugineum, XP_006396006.1; EvCYP83B1: Eruca vesicaria subsp. sativa, AGS49168.1; CrCYP83B1: Capsella rubella, XP_006285988.1; ThCYP83B1: Tarenaya hassleriana, XP_010530341.1; BcCYP83B1: Brassica rapa subsp. chinensis, HM347235.1. Amino acids highlighted in black, pink, and blue respectively represent residues completely conserved, partially conserved and similar

Fig. 5 Phylogenetic analysis of BrCYP83B1 with its homologues from other plant species

Table 2 Upstream elements in BrCYP83B1 promoter

元件名称Name of element	序列Sequence（5'-3'）	元件类型Type of element	数量Amount
TGACG-motif	TGACG	茉莉酸甲酯响应元件Cis-acting regulatory element involved in the MeJA-responsiveness	3
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件Cis-acting regulatory element involved in the MeJA-responsiveness	2
GT1-motif	GGTTAA	光响应元件Light responsive element	2
ABRE	AACCCGG	脱落酸响应元件Cis-acting element involved in the abscisic acid responsiveness	1
Box 4	ATTAAT	光响应元件Light responsive element	1
G-Box	CACGTT	光响应元件Light responsive element	1
GARE-motif	TCTGTTG	赤霉素响应元件Gibberellin-responsive element	1
MRE	AACCTAA	光响应元件Light responsive element	1
TCA-element	CCATCTTTTT	水杨酸响应元件Cis-acting element involved in salicylic acid responsiveness	1

Fig. 6 Expression pattern of BrCYP83B1 A: Expression of BrCYP83B1 in different tissues. B: Expression of BrCYP83B1 treated with MeJA, SA and ABA. Different lowercase letters indicate significant differences in relative expression at the P<0.05 level

Fig. 7 Electrophoretic detection on double enzyme digestion of plant expression vector and PCR of Agro-bacterium tumefaciens liquid M: DNA marker; 1: double digestion products; 2: PCR products of Agrobacterium tumefaciens liquid

Fig. 8 Model of overexpression vector for BrCYP83B1 gene

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