大白菜BrCYP83B1基因的克隆及表达分析

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

摘要/Abstract

摘要：

【目的】 细胞色素P450家族是十字花科植物硫苷合成重要的酶系，其中CYP83亚家族主要参与核心结构的合成，旨在探究大白菜（Brassica rapa ssp. pekinensis）CYP83B1基因的功能。【方法】 利用RT-PCR技术克隆BrCYP83B1基因，通过生物信息学软件分析其编码蛋白理化性质、同源性及启动子顺式作用元件，利用RT-qPCR技术分析BrCYP83B1的表达模式，并构建其植物超表达载体。【结果】 BrCYP83B1 cDNA序列全长为1 500 bp，编码499个氨基酸，编码蛋白属于细胞色素P450超家族，主要定位于细胞质，二级结构主要由α-螺旋和无规则卷曲构成，与甘蓝型油菜、青花菜的CYP83B1蛋白具有较高的同源性。启动子分析表明，该基因启动子区域包含水杨酸、脱落酸及茉莉酸甲酯等激素响应的顺式作用元件，说明BrCYP83B1基因表达可能受激素调控。RT-qPCR分析结果表明，BrCYP83B1基因在大白菜的根、茎、叶、花和果中均有表达，且以叶中的表达量最高；茉莉酸甲酯够显著促进该基因的表达，而水杨酸处理对其表达具有一定的抑制作用，脱落酸处理下基因先上调后又下调。【结论】 BrCYP83B1可能参与大白菜对激素的响应调控。

关键词: 大白菜, BrCYP83B1, 基因克隆, 植物激素, 表达分析, 超表达载体

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

王玉书, 赵琳琳, 赵爽, 胡琦, 白慧霞, 王欢, 曹业萍, 范震宇. 大白菜BrCYP83B1基因的克隆及表达分析[J]. 生物技术通报, 2024, 40(6): 152-160.

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.

图/表 10

表1 本研究所用的引物

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

图1 BrCYP83B1基因PCR扩增 M：DNA marker；P：PCR产物

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

图2 BrCYP83B1蛋白保守结构域

Fig. 2 Conservative domain of BrCYP83B1 protein

图3 BrCYP83B1蛋白的二级结构（A）和三级结构（B）预测 A图中大写字母表示BrCYP83B1蛋白氨基酸序列；小写字母代表不同的二级结构，h 代表α-螺旋，c 代表无规则卷曲，e 代表延伸链，t 代表β 转角

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

图4 BrCYP83B1与其他植物同源蛋白序列比对 BnCYP83B1：甘蓝型油菜，CDY35791.1；RsCYP83B1：萝卜，CDY35791.1；AtCYP83B1：拟南芥，NP_194878.1；BoCYP83B1 ：青花菜，APZ76511.1；ItCYP83B1：菘蓝，APY26702.1；EsCYP83B1：盐芥，XP_006396006.1；EvCYP83B1：芝麻菜，AGS49168.1；CrCYP83B1：荠菜，XP_006285988.1；ThCYP83B1：醉蝶花，XP_010530341.1；BcCYP83B1：小白菜，HM347235.1；图中黑色、粉色及蓝色分别代表氨基酸完全保守、部分保守及相似

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

图5 BrCYP83B1和其他植物同源蛋白系统发育分析

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

表2 BrCYP83B1启动子上游元件

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

图6 BrCYP83B1基因表达模式 A：BrCYP83B1在不同组织中表达；B：BrCYP83B1在MeJA、SA、ABA处理下表达。不同小写字母表示表达量在 P<0.05水平上差异显著

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

图7 植物表达载体双酶切及农杆菌菌液PCR电泳检测 M：DNA marker；1：双酶切产物；2：农杆菌菌液PCR产物

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

图8 BrCYP83B1 基因超表达载体模式图

Fig. 8 Model of overexpression vector for BrCYP83B1 gene

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