Cloning and Functional Analysis of MbbZIP43 Gene in ‘Hongmantang’ Red-flesh Apple

doi:10.13560/j.cnki.biotech.bull.1985.2023-0676

Abstract

Abstract:

【Objective】 To investigate the regulatory role of basic leucine zipper（bZIP）transcription factors in anthocyanin metabolism in ‘Hongmantang’ apple.【Method】 We cloned a bZIP gene from ‘Hongmantang’ red-flesh apple, analyzed it and its sequence characteristics, studied the subcellular localization of its encoded protein using onion epidermis cell transient overexpression, and investigated its expression pattern in ‘Hongmantang’ red-flesh apple fruits at different ripening stages using quantitative real time PCR（RT-qPCR）. Furthermore, we explored its regulatory roles in the expressions of genes related to anthocyanin accumulation and biosynthesis based on the transient overexpression experiments in tobacco leaves, apple leaves and fruits.【Result】 This bZIP gene was intron-less and shared the highest similarity with MdbZIP43（XP_008393381.1）, thus was named as MbbZIP43. Its coding sequence was 522 bp, which encoded a 173 aa hydrophilic nucleus-localized protein with conserved bZIP_plant_GBF1 domain. RT-qPCR analysis revealed that MbbZIP43 showed a ‘rise-fall’ expression change pattern in ‘Hongmantang’ fruits during ripening, with the highest expression in the fruits at 11 weeks post flowering. Correlation analysis results showed that MbbZIP43 expression was positively correlated with the total flavonoids and anthocyanin contents in ‘Hongmantang’ fruits, indicating that it might play regulatory roles in flavonoid biosynthesis. Consistently, transient overexpression of MbbZIP43 improved significantly the anthocyanin accumulations by 17.42%, 25.66% and 48.99% in tobacco leaves, apple leaves and apple fruits, respectively. Moreover, RT-qPCR analysis results showed that, compared to the empty vector control, the expressions of anthocyanin biosynthesis related structural genes（CHI, F3'H, DFR and UFGT）in the apple leaves overexpressing MbbZIP43 were up-regulated by 2.27-, 1.84-, 2.39- and 2.89-fold, respectively. And their expressions in the peels of apple fruits overexpressing MbbZIP43 were up-regulated by 1.79-, 1.70-, 1.35- and 1.51-fold, respectively. These results indicated that the gene's transient overexpression may improve anthocyanin accumulation by activating the expressions of the anthocyanin biosynthesis related structural genes.【Conclusion】 MbbZIP43 can promote anthocyanins accumulation by activating the expression of anthocyanin biosynthesis related structural genes, including CHI, UFGT and so on.

Key words: bZIP transcription factor, apple, regulation to anthocyanin biosynthesis, gene cloning, transient overexpression, expression analysis, subcellular localization

YANG Yan, HU Yang, LIU Ni-ru, YIN Lu, YANG Rui, WANG Peng-fei, MU Xiao-peng, ZHANG Shuai, CHENG Chun-zhen, ZHANG Jian-cheng. Cloning and Functional Analysis of MbbZIP43 Gene in ‘Hongmantang’ Red-flesh Apple[J]. Biotechnology Bulletin, 2024, 40(2): 146-159.

Figures/Tables 10

References 56

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引物名称 Primer name	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	引物用途 Application
MbbZIP43-F	ATGGAGCCAAATGAATCAAAGG	53	基因克隆 Gene cloning
MbbZIP43-R	TGGCGTACTTGAGTCTTCG	53	基因克隆 Gene cloning
MbbZIP43-OE-F	ACGGGGGACTCTAGAGGATCCATGGAGCCAAATGAATCAAAGG	65	载体构建 Vector construction
MbbZIP43-OE-R	GCTCACCATCGCTGCACTAGTTGGCGTACTTGAGTCTTCG	65	载体构建 Vector construction
ACTIN-F	TGACCGAATGAGCAAGGAAATTACT	60	RT-qPCR
ACTIN-R	TACTCAGCTTTGGCAATCCACATC
Q-MbbZIP43-F	GGAGCCAAATGAATCAAAGG
Q-MbbZIP43-R	CGCATTCTTCTTCGTCTAACT
CHS-F	GGAGACAACTGGAGAAGGACTGGAA
CHS-R	CGACATTGATACTGGTGTCTTC
CHI-F	GGGATAACCTCGCGGCCAAA
CHI-R	GCATCCATGCCGGAAGCTACAA
F3'H-F	TGGAAGCTTGTGAGGACTGGGGT
F3'H-R	CTCCTCCGATGGCAAATCAAAGA
DFR-F	CCAAGTGAAGCGGGTTGTGCT
DFR-R	CAAAGCAGGCGGACAGGAGTAGC
ANS-F	GATAGGGTTTGAGTTCAAGTA
ANS-R	TCTCCTCAGCAGCCTCAGTTTTCT
UFGT-F	CCACCGCCCTTCCAAACACTCT
UFGT-R	CACCCTTATGTTACGCGGCATGT

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	引物用途 Application
MbbZIP43-F	ATGGAGCCAAATGAATCAAAGG	53	基因克隆 Gene cloning
MbbZIP43-R	TGGCGTACTTGAGTCTTCG	53	基因克隆 Gene cloning
MbbZIP43-OE-F	ACGGGGGACTCTAGAGGATCCATGGAGCCAAATGAATCAAAGG	65	载体构建 Vector construction
MbbZIP43-OE-R	GCTCACCATCGCTGCACTAGTTGGCGTACTTGAGTCTTCG	65	载体构建 Vector construction
ACTIN-F	TGACCGAATGAGCAAGGAAATTACT	60	RT-qPCR
ACTIN-R	TACTCAGCTTTGGCAATCCACATC
Q-MbbZIP43-F	GGAGCCAAATGAATCAAAGG
Q-MbbZIP43-R	CGCATTCTTCTTCGTCTAACT
CHS-F	GGAGACAACTGGAGAAGGACTGGAA
CHS-R	CGACATTGATACTGGTGTCTTC
CHI-F	GGGATAACCTCGCGGCCAAA
CHI-R	GCATCCATGCCGGAAGCTACAA
F3'H-F	TGGAAGCTTGTGAGGACTGGGGT
F3'H-R	CTCCTCCGATGGCAAATCAAAGA
DFR-F	CCAAGTGAAGCGGGTTGTGCT
DFR-R	CAAAGCAGGCGGACAGGAGTAGC
ANS-F	GATAGGGTTTGAGTTCAAGTA
ANS-R	TCTCCTCAGCAGCCTCAGTTTTCT
UFGT-F	CCACCGCCCTTCCAAACACTCT
UFGT-R	CACCCTTATGTTACGCGGCATGT

软件 Software	用途 Application	网址 Website
GDR	序列下载	https://www.rosaceae.org/
CELLO v.2.5	亚细胞定位预测	http://cello.life.nctu.edu.tw/
TMHMM-2.0	跨膜结构预测	https://services.healthtech.dtu.dk/services/TMHMM-2.0/
SignalP-5.0	信号肽预测	https://services.healthtech.dtu.dk/services/SignalP-5.0/
NetPhos 3.1	蛋白磷酸化位点预测	https://services.healthtech.dtu.dk/services/NetPhos-3.1/
SOPMA	蛋白二级结构预测	https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl
SWISS-MODEL	蛋白三级结构预测	https://swissmodel.expasy.org/
MEME	蛋白保守基序预测	http://meme-suite.org/tools/meme
CDD	结构域验证	https://www.ncbi.nlm.nih.gov/cdd
NCBI BLASTP	同源蛋白搜索	https://blast.ncbi.nlm.nih.gov/Blast.cgi
PlantCARE	启动子顺式作用元件预测	http://bioinformatics.psb.ugent.be/
TFBS	启动子转录因子结合位点预测	http://plantregmap.gao-lab.org/binding_site_prediction.php

软件 Software	用途 Application	网址 Website
GDR	序列下载	https://www.rosaceae.org/
CELLO v.2.5	亚细胞定位预测	http://cello.life.nctu.edu.tw/
TMHMM-2.0	跨膜结构预测	https://services.healthtech.dtu.dk/services/TMHMM-2.0/
SignalP-5.0	信号肽预测	https://services.healthtech.dtu.dk/services/SignalP-5.0/
NetPhos 3.1	蛋白磷酸化位点预测	https://services.healthtech.dtu.dk/services/NetPhos-3.1/
SOPMA	蛋白二级结构预测	https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl
SWISS-MODEL	蛋白三级结构预测	https://swissmodel.expasy.org/
MEME	蛋白保守基序预测	http://meme-suite.org/tools/meme
CDD	结构域验证	https://www.ncbi.nlm.nih.gov/cdd
NCBI BLASTP	同源蛋白搜索	https://blast.ncbi.nlm.nih.gov/Blast.cgi
PlantCARE	启动子顺式作用元件预测	http://bioinformatics.psb.ugent.be/
TFBS	启动子转录因子结合位点预测	http://plantregmap.gao-lab.org/binding_site_prediction.php

种类 Type	功能 Function	元件 Element	数目 Number
光响应元件 Light responsive elements	光响应Light responsiveness	GATA-motif	1
	光响应Light responsiveness	MRE	1
	光响应Light responsiveness	Box 4	2
	光响应Light responsiveness	ATCT-motif	1
	光响应Light responsiveness	G-box	1
	光响应Light responsiveness	Gap-box	1
	光响应Light responsiveness	3-AF1 binding site	3
	光响应Light responsiveness	GT1-motif	6
生长发育相关元件 Plant growth and development related elements	分生组织表达Meristem expression	CAT-box	1
生长发育相关元件 Plant growth and development related elements	胚乳表达Endosperm expression	GCN4_motif	1
激素响应元件 Phytohormone responsive elements	水杨酸响应Salicylic acid responsiveness	TCA-element	1
	脱落酸响应Abscisic acid responsiveness	ABRE	1
	茉莉酸甲酯响应MeJA-responsiveness	CGTCA-motif	2
	茉莉酸甲酯响应MeJA-responsiveness	TGACG-motif	2
	赤霉素响应Gibberellin-responsiveness	P-box	2
	赤霉素响应Gibberellin-responsiveness	TATC-box	1
逆境响应元件 Stress responsive elements	厌氧诱导Anaerobic induction	ARE	1
	低温Low-temperature	LTR	1
	伤害反应元件Wound-responsive element	WUN-motif	2
	高温High-temperature	STRE	4
	干旱响应Drought-inducibility	MYC	4
	防御和胁迫Defense and stress	DRE core	1
	防御和胁迫Defense and stress	TC-rich repeats	1
	防御和胁迫Defense and stress	W box	2
	防御和胁迫Defense and stress	MYB	4