青花菜<i>BolMYC2</i>基因克隆、亚细胞定位及表达分析

doi:10.7668/hbnxb.20196159

摘要/Abstract

摘要：

为系统地分析青花菜BolMYC2的序列特征、表达模式,探究该基因在青花菜应对软腐病致病菌侵染时的作用,以青花菜cDNA为模板进行PCR扩增,克隆BolMYC2基因的编码序列,利用生物信息学软件分析BolMYC2蛋白的基本性质,构建BolMYC2-EGFP融合表达载体瞬时转化本氏烟草,分析BolMYC2蛋白亚细胞定位情况,利用qRT-PCR技术分析BolMYC2在青花菜不同组织部位转录表达特征以及软腐病致病菌胁迫条件下BolMYC2转录表达特征。结果表明:BolMYC2基因CDS序列长度为1 833 bp,编码蛋白含有610个氨基酸,理论分子质量为66.297 ku,理论等电点为5.21。BolMYC2为不稳定亲水蛋白,无跨膜结构和信号肽。保守结构域分析表明,BolMYC2蛋白含有MYC转录因子家族保守结构域bHLH-MYC_N和bHLH_SF。系统进化树分析表明,BolMYC2蛋白与抱子甘蓝亲缘关系较近。对BolMYC2启动子顺式作用元件分析表明,其含有参与光响应、激素响应、胁迫应答的顺式作用元件和多个MYB、MYC、WRKY转录因子识别元件。亚细胞定位结果表明,BolMYC2定位于细胞核。BolMYC2在叶片和种子中呈现高表达,并且在青花菜受到软腐病菌侵染胁迫后其表达水平呈现先下降后上升趋势,暗示BolMYC2在青花菜响应生物胁迫中发挥重要作用。

关键词: 青花菜, 软腐病, BolMYC2基因, 表达分析, 亚细胞定位

Abstract:

In order to systematically analyze the sequence characteristics and expression pattern of BolMYC2 in broccoli,and to explore the role of BolMYC2 in response to the infection of soft rot pathogens in broccoli,the coding sequence of BolMYC2 gene was cloned by PCR amplification using broccoli cDNA as a template.The basic properties of BolMYC2 protein were analyzed by bioinformatics software.BolMYC2-EGFP fusion expression vector was constructed and transiently transformed into Nicotiana benthamiana to analyze the subcellular localization of BolMYC2 protein.The transcriptional expression characteristics of BolMYC2 in different tissues of broccoli and the transcriptional expression characteristics of BolMYC2 under the stress of soft rot pathogens were analyzed by qRT-PCR.The results showed that the coding sequence of BolMYC2 was 1 833 bp,encoding 610 amino acids.The theoretical molecular weight was 66.297 ku and the theoretical isoelectric point was 5.21.BolMYC2 was an unstable hydrophilic protein without transmembrane structure and signal peptide.Conserved domain analysis showed that BolMYC2 protein contained the conserved domains bHLH-MYC_N and bHLH_SF of MYC transcription factor family.Phylogenetic tree analysis showed that BolMYC2 protein was closely related to brussels sprouts.Analysis of the cis-acting elements of the BolMYC2 promoter showed that it contained cis-acting elements involved in light response,hormone response,stress response,and multiple MYB,MYC,and WRKY transcription factor recognition elements.Subcellular localization results showed that BolMYC2 was localized in the nucleus.BolMYC2 was highly expressed in leaves and seeds.In addition,the expression level of BolMYC2 decreased first and then increased after broccoli was infected by soft rot pathogens,suggesting that BolMYC2 plays an important role in broccoli response to biotic stress.

Key words: Broccoli, Soft rot disease, BolMYC2 gene, Expression analysis, Subcellular localization

中图分类号:

S635.3

马超超, 罗兰, 孔垚, 陶婧婷, 周显涛, 李慧. 青花菜BolMYC2基因克隆、亚细胞定位及表达分析[J]. 华北农学报, 2026, 41(3): 32-39. doi: 10.7668/hbnxb.20196159.

MA Chaochao, LUO Lan, KONG Yao, TAO Jingting, ZHOU Xiantao, LI Hui. Cloning,Subcellular Localization and Expression Analysis of BolMYC2 Gene in Broccoli[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(3): 32-39. doi: 10.7668/hbnxb.20196159.

http://www.hbnxb.net/CN/Y2026/V41/I3/32

图/表 10

参考文献 27

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引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	用途 Usage
qBolMYC2-F	AGACGGAATCTGAGAAAACT	实时荧光定量PCR
qBolMYC2-R	CACCCTTATCATCGCATC
β-actin-F	GCTCCTCTTAACCCAAAGGC	实时荧光定量PCR
β-actin-R	CACACCATCACCAGAATCCAGC
GFP-BolMYC2-F	GAGAACACGGGGGACTCTAGAATGACGGAGCCGACGATGA	GFP融合载体构建
GFP-BolMYC2-R	GCTCACCATGGTACCCCCGGGACCAATCTTTGAGATTAAACTCGCC
BolMYC2-F	ATGACGGAGCCGACGATGA	CDS序列扩增
BolMYC2-R	TTAACCAATCTTTGAGATTAAACTCGCC

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	用途 Usage
qBolMYC2-F	AGACGGAATCTGAGAAAACT	实时荧光定量PCR
qBolMYC2-R	CACCCTTATCATCGCATC
β-actin-F	GCTCCTCTTAACCCAAAGGC	实时荧光定量PCR
β-actin-R	CACACCATCACCAGAATCCAGC
GFP-BolMYC2-F	GAGAACACGGGGGACTCTAGAATGACGGAGCCGACGATGA	GFP融合载体构建
GFP-BolMYC2-R	GCTCACCATGGTACCCCCGGGACCAATCTTTGAGATTAAACTCGCC
BolMYC2-F	ATGACGGAGCCGACGATGA	CDS序列扩增
BolMYC2-R	TTAACCAATCTTTGAGATTAAACTCGCC

功能 Function	软件 Software	网址 Website
信号肽预测 Signal peptides prediction	SignalP4.1	https://services.healthtech.dtu.dk/services/SignalP-4.1/
跨膜结构预测 Transmembrane structure prediction	TMHMM Server v.2.0	https://services.healthtech.dtu.dk/services/TMHMM 2.0/
磷酸化位点预测 Phosphorylation site prediction	NetPhos-3.1	https://services.healthtech.dtu.dk/services/NetPhos-3.1/
保守功能结构域预测 Conserved functional domain prediction	NCBI网站CDD工具模块	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
二级结构预测 Secondary structure prediction	SOPMA	https://npsa.lyon.inserm.fr/cgi-bin/secpred_sopma.pl
三级结构预测 Tertiary structure prediction	SWISS-MODEL	https://swissmodel.expasy.org/
启动子顺式作用元件预测 Promoter cis-acting element prediction	PlantCARE	http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

功能 Function	软件 Software	网址 Website
信号肽预测 Signal peptides prediction	SignalP4.1	https://services.healthtech.dtu.dk/services/SignalP-4.1/
跨膜结构预测 Transmembrane structure prediction	TMHMM Server v.2.0	https://services.healthtech.dtu.dk/services/TMHMM 2.0/
磷酸化位点预测 Phosphorylation site prediction	NetPhos-3.1	https://services.healthtech.dtu.dk/services/NetPhos-3.1/
保守功能结构域预测 Conserved functional domain prediction	NCBI网站CDD工具模块	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
二级结构预测 Secondary structure prediction	SOPMA	https://npsa.lyon.inserm.fr/cgi-bin/secpred_sopma.pl
三级结构预测 Tertiary structure prediction	SWISS-MODEL	https://swissmodel.expasy.org/
启动子顺式作用元件预测 Promoter cis-acting element prediction	PlantCARE	http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

名称 Name	序列 Sequence	功能 Function	数量 Number
MRE	AACCTAA	参与光响应的MYB结合位点	1
TCCC-motif	TCTCCCT	光响应元件的一部分	1
ARE	AAACCA	厌氧诱导调节元件	2
I-box	GTATAAGGCC/GGATAAGGTG	光响应元件的一部分	2
DRE core	GCCGAC	脱水反应元件	2
circadian	CAAAGATATC	昼夜节律调控元件	1
MYC	CATGTG	MYC 响应元件	4
MYB	CAACCA/TAACCA	MYB 响应元件	4
AAGAA-motif	GGTAAAGAAA/GAAAGAA	脱落酸响应元件	5
W box	TTGACC	WRKY转录因子家族结合位点	1
GT1-motif	GGTTAAT	光响应元件	3
TCA-element	CCATCTTTTT	水杨酸响应元件	1
Myc	TCTCTTA	MYC 响应元件	1
G-Box	CACGTT	光响应元件	1
TGA-element	AACGAC	生长素响应元件	1
G-box	CACGTC	光响应元件	1
MYB-like sequence	TAACCA	MYB响应元件	3
3-AF1 binding site	TAAGAGAGGAA	光响应元件	1
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件	2
P-box	CCTTTTG	赤霉素响应元件	1
ATCT-motif	AATCTAATCC	参与光响应的保守DNA模块的一部分	1
GATA-motif	GATAGGA	光响应元件的一部分	1
ABRE	ACGTG	脱落酸响应元件	2
as-1	TGACG	氧化应激反应元件	2
STRE	AGGGG	胁迫响应元件	2
Box 4	ATTAAT	参与光响应的保守DNA模块的一部分	1

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青花菜BolMYC2基因克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization and Expression Analysis of BolMYC2 Gene in Broccoli

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