Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (2): 146-159.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0676
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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()
Received:
2023-07-14
Online:
2024-02-26
Published:
2024-03-13
Contact:
CHENG Chun-zhen, ZHANG Jian-cheng
E-mail:18404968975@163.com;ld0532cheng@sxau.edu.cn;zjcnd001@sxau.edu.cn
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.
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 | ||
MbbZIP43-OE-F | ACGGGGGACTCTAGAGGATCCATGGAGCCAAATGAATCAAAGG | 65 | 载体构建 Vector construction |
MbbZIP43-OE-R | GCTCACCATCGCTGCACTAGTTGGCGTACTTGAGTCTTCG | ||
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 |
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 | ||
MbbZIP43-OE-F | ACGGGGGACTCTAGAGGATCCATGGAGCCAAATGAATCAAAGG | 65 | 载体构建 Vector construction |
MbbZIP43-OE-R | GCTCACCATCGCTGCACTAGTTGGCGTACTTGAGTCTTCG | ||
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 | |
CELLO v.2.5 | 亚细胞定位预测 | |
TMHMM-2.0 | 跨膜结构预测 | |
SignalP-5.0 | 信号肽预测 | |
NetPhos 3.1 | 蛋白磷酸化位点预测 | |
SOPMA | 蛋白二级结构预测 | |
SWISS-MODEL | 蛋白三级结构预测 | |
MEME | 蛋白保守基序预测 | |
CDD | 结构域验证 | |
NCBI BLASTP | 同源蛋白搜索 | |
PlantCARE | 启动子顺式作用元件预测 | |
TFBS | 启动子转录因子结合位点预测 | |
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 | |
CELLO v.2.5 | 亚细胞定位预测 | |
TMHMM-2.0 | 跨膜结构预测 | |
SignalP-5.0 | 信号肽预测 | |
NetPhos 3.1 | 蛋白磷酸化位点预测 | |
SOPMA | 蛋白二级结构预测 | |
SWISS-MODEL | 蛋白三级结构预测 | |
MEME | 蛋白保守基序预测 | |
CDD | 结构域验证 | |
NCBI BLASTP | 同源蛋白搜索 | |
PlantCARE | 启动子顺式作用元件预测 | |
TFBS | 启动子转录因子结合位点预测 | |
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
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 |
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 |
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 |
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 |
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
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
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 |
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 |
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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