‘红满堂’苹果MbbZIP43基因的克隆与功能研究

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

摘要/Abstract

摘要：

【目的】为探究碱性亮氨酸拉链（bZIP）转录因子在‘红满堂’苹果花青素代谢中的调控作用。【方法】从‘红满堂’苹果克隆得到一个bZIP基因，分析了其基因及编码蛋白序列特性，利用洋葱表皮细胞瞬时转化确定其编码蛋白亚细胞定位，利用实时荧光定量PCR（RT-qPCR）研究了该基因在不同成熟时期‘红满堂’果实中的表达情况，基于烟草叶片、苹果叶片以及苹果果实瞬时转化研究了其对花青素积累和花青素合成相关结构基因表达的调控作用。【结果】该基因不含内含子，与MdbZIP43（XP_008393381.1）相似度最高，故命名为MbbZIP43。其编码序列长为522 bp，可编码由173 aa组成、含有bZIP_plant_GBF1结构域、定位于细胞核的亲水蛋白。RT-qPCR分析结果显示，随着果实成熟，MbbZIP43在‘红满堂’果实中的表达水平呈“先升后降”的变化趋势，在花后11周的果实中表达量最高。相关性分析显示MbbZIP43基因表达量与总黄酮和花青素含量正相关。瞬时过表达该基因的烟草叶片、苹果叶片和果皮中花青素含量分别提高了17.42%、25.66%和48.99%，过表达MbbZIP43的苹果叶片中花青素合成结构基因CHI、F3'H、DFR和UFGT分别提高了2.27倍、1.84倍、2.39倍和2.89倍；过表达MbbZIP43的苹果果皮中CHI、F3'H、DFR和UFGT分别提高了1.79倍、1.70倍、1.35倍和1.51倍，说明MbbZIP43可以通过上调这些结构基因的表达正调控苹果花青素合成。【结论】MbbZIP43可通过激活花青素合成相关结构基因CHI和UFGT等的表达进而促进花青素的积累。

关键词: bZIP转录因子, 苹果, 花青素合成调控, 基因克隆, 瞬时转化, 表达分析, 亚细胞定位

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

杨艳, 胡洋, 刘霓如, 殷璐, 杨锐, 王鹏飞, 穆霄鹏, 张帅, 程春振, 张建成. ‘红满堂’苹果MbbZIP43基因的克隆与功能研究[J]. 生物技术通报, 2024, 40(2): 146-159.

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.

图/表 10

表1 本研究所用引物信息

Table 1 Information for the primers used in this study

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

表2 本研究所用生物信息学分析网站信息

Table 2 Information for the software used for the bioinformatic analysis in this study

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

图1 MbbZIP43基因PCR产物电泳检测（A）及序列比对（B）结果 M：DL2000 marker；1：cDNA为模板；2：gDNA为模板；MbbZIP43-1为参考基因组CDS序列；MbbZIP43-2为测序序列

Fig. 1 Electrophoresis detection（A）and sequence alignment（B）results of PCR products for the MbbZIP43 M: DL2000 marker; 1: using cDNA as template; 2: using gDNA as template; MbbZIP43-1: reference CDS sequence in the Malus baccata genome; MbbZIP43-2: amplified MbbZIP43 gene sequence

表3 MbbZIP43启动子区顺式作用元件预测结果

Table 3 Predicted cis-regulatory elements in the promoter region of MbbZIP43

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

图2 不同植物bZIP43以及已知花青素调控相关bZIP蛋白多序列比对结果 MdbZIP43：苹果bZIP43（XP_008393381.1）；PbbZIP43：白梨bZIP43（XP_009339530.2）；PybZIP43：沙梨bZIP43（UXW88004.1）；PabZIP43：甜樱桃bZIP43（XP_021809905.1）；PsbZIP43：李子bZIP43（XM_008241838.1）；PdbZIP43：扁桃bZIP43（XP_034216145.1）；PpbZIP43：桃bZIP43（XP_007209676.1）；RcbZIP43：月季bZIP43（XP_024190316.2）；MdbZIP44：苹果bZIP44（XP_008377201.2）；AtHY5：拟南芥HY5（BAA21327.1）；VvHY5：葡萄HY5（AGX85877.1）；MdHY5：苹果HY5（NP_001280752.1）；PaHY5：甜樱桃HY5（XP_021827650.1）；PpHY5：桃HY5（XP_020411091.1）；GbHY5：银杏HY5（Gb_12012）。图中红色框标出的是bZIP保守结构，红色线条表示α螺旋，绿色线条代表β折叠

Fig. 2 Multiple sequence alignment results for bZIP43 proteins and bZIP proteins related to known anthocyanin biosynthesis from different plant species MdbZIP43: Malus domestica bZIP43（XP_008393381.1）; PbbZIP43: Pyrus bretschneideri bZIP43（XP_009339530.2）; PybZIP43: Pyrus pyrifolia bZIP43（UXW88004.1）; PabZIP43: Prunus avium bZIP43（XP_021809905.1）; PsbZIP43: P. salicina bZIP43（XM_008241838.1）; PdbZIP43: P. dulcis bZIP43（XP_034216145.1）; PpbZIP43: P. persica bZIP43（XP_007209676.1）; RcbZIP43: Rosa chinensis bZIP43（XP_024190316.2）; MdbZIP44: M. domestica bZIP44（XP_008377201.2）; AtHY5: Arabidopsis thaliana HY5（BAA21327.1）; VvHY5: Vitis vinifera HY5（AGX85877.1）; MdHY5: M. domestica HY5（NP_001280752.1）; PaHY5: P. avium HY5（XP_021827650.1）; PpHY5: P. persica HY5（XP_020411091.1）; GbHY5: Ginkgo biloba HY5（Gb_12012）. Red box, red line and green line indicates the conserved bZIP domain, α-helix and β folding, respectively

图3 不同植物bZIP蛋白保守基序（A）及motif1序列Logo图（B）

Fig. 3 Conserved motifs in bZIP proteins from different plant species（A）and sequence Logo for the motif1（B）

图4 MbbZIP43蛋白亚细胞定位分析结果

Fig. 4 Subcellular localization analysis results of MbbZIP43 protein

图5 不同发育时期‘红满堂’苹果MbbZIP43相对表达量、总酚含量、总黄酮含量、总黄酮醇含量和花青素含量 PS1-PS5：分别指花后7周、11周、15周、19周和23周的果实；FW：鲜重。不同小写字母表示差异显著（P<0.05）。下同

Fig. 5 Relative expressions of MbbZIP43, total phenol content, total flavonoid content, total flavonol content and anthocyanin content in ‘Hongmantang’ apple fruits at different ripening stages PS1-PS5 represent fruits at 7, 11, 15, 19 and 23 weeks post flowering, respectively. FW: Fresh weight. Different lowercase letters indicate significant differences（P<0.05）. The same below

表4 MbbZIP43基因表达水平与总黄酮、总酚、总黄酮醇和花青素含量相关性分析结果

Table 4 Correlation analysis results among the MbbZIP43 expressions, total flavonoids content, total phenolic content, total flavonol content and anthocyanin content

测定指标 Index	MbbZIP43表达水平 MbbZIP43 expression level	总黄酮含量 Total flavonoids content	总酚含量 Total phenolic content	总黄酮醇含量 Total flavonol content	花青素含量 Anthocyanin content
MbbZIP43表达水平 MbbZIP43 expression level	1.0
总黄酮含量 Total flavonoids content	0.60	1.0
总酚含量 Total phenolic content	0.08	0.69	1.0
总黄酮醇含量 Total flavonol content	-0.60	-0.97*	-0.81	1.0
花青素含量 Anthocyanin content	0.34	-0.02	-0.70	0.21	1.0

图6 MbbZIP43瞬时过表达对花青素积累的影响 A：瞬时过表达MbbZIP43对烟草叶片花青素积累的影响；B：瞬时过表达MbbZIP43的苹果叶片；C：瞬时过表达MbbZIP43的苹果果皮；D：瞬时过表达MbbZIP43对苹果叶片花青素积累的影响；E：瞬时过表达MbbZIP43对苹果叶片中MbbZIP43和花青素合成相关结构基因表达的影响；F：瞬时过表达MbbZIP43对苹果果皮花青素积累的影响；G：瞬时过表达MbbZIP43对苹果果皮中MbbZIP43和花青素合成相关结构基因表达的影响

Fig. 6 Influences of transient overexpression of MbbZIP43 gene on anthocyanin accumulations A: Influences of transient overexpression of MbbZIP43 gene on the anthocyanin accumulation in tobacco. B: Apple leaves overexpressing MbbZIP43. C: Apple peel overexpressing MbbZIP43. D: Influences of transient overexpression of MbbZIP43 gene on anthocyanin accumulation in apple leaves. E: Influences of transient overexpression of MbbZIP43 gene on the expressions of MbbZIP43 and structural genes related to anthocyanin biosynthesis in apple leaves. F: Influences of transient overexpression of MbbZIP43 gene on anthocyanin accumulation in apple. G: Influences of transient overexpression of MbbZIP43 gene on the expressions of MbbZIP43 and structural genes related to anthocyanin biosynthesis in apple peel

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