Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (9): 42-50.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0509
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ZHANG Xiao-mei1(), ZHOU Nan-ling1, ZHANG Sai-hang2, WANG Chao1, SHEN Yu-long1, GUAN Jun-mei1, MA Ling1()
Received:
2024-05-29
Online:
2024-09-26
Published:
2024-10-12
Contact:
MA Ling
E-mail:1452091644@qq.com;may_ynnu@ynnu.edu.cn
ZHANG Xiao-mei, ZHOU Nan-ling, ZHANG Sai-hang, WANG Chao, SHEN Yu-long, GUAN Jun-mei, MA Ling. Cloning and Expression Analysis of StDREBs Gene in Solanum tuberosum L.[J]. Biotechnology Bulletin, 2024, 40(9): 42-50.
试剂和仪器Reagent and instrument | 用途Fuctinon | 来源Source |
---|---|---|
pRI101-AN-35S-EGFP 载体 | 构建载体 | 实验室提供 |
内切酶 EcoR I | 构建载体 | 南京诺唯赞生物科技股份有限公司 |
内切酶 Kpn I | 构建载体 | 南京诺唯赞生物科技股份有限公司 |
同源重组酶 | 构建载体 | 南京诺唯赞生物科技股份有限公司 |
PCR 高保真酶(PrimeSTAR® GXL DNA Polymerase) | 构建载体 | 宝日医生物技术(北京)有限公司 |
琼脂糖凝胶 DNA 回收试剂盒(TIANgel Purification Kit) | 构建载体 | 天根生化科技(北京)有限公司 |
大肠杆菌 DH5α | 构建载体 | 上海唯地生物技术有限公司 |
农杆菌 GV3101(pSoup-p19) | 构建载体 | 上海唯地生物技术有限公司 |
核定位染料 DAPI 溶液 | 亚细胞定位 | 北京索莱宝科技有限公司 |
TRIzol® reagent | 总RNA提取 | 维百奥(北京)生物科技有限公司 |
氯仿(三氯甲烷) | 总RNA提取 | 实验室提供 |
异丙醇 | 总RNA提取 | 天津市致远化学试剂有限公司 |
无水乙醇 | 总RNA提取 | 天津市致远化学试剂有限公司 |
cDNA 合成试剂盒 | 反转录合成 | 北京全式金生物技术股份有限公司 |
实时荧光定量试剂盒 | 定量分析 | 北京全式金生物技术股份有限公司 |
BIORADPCR 仪 | 构建载体 | 实验室提供 |
化学发光检测仪 | 构建载体 | 实验室提供 |
Zeiss 倒置共聚焦显微镜 | 亚细胞定位 | 实验室提供 |
Applied Biosystems 实时荧光定量PCR 仪 | 定量分析 | 实验室提供 |
Table 1 Reagents and instruments information
试剂和仪器Reagent and instrument | 用途Fuctinon | 来源Source |
---|---|---|
pRI101-AN-35S-EGFP 载体 | 构建载体 | 实验室提供 |
内切酶 EcoR I | 构建载体 | 南京诺唯赞生物科技股份有限公司 |
内切酶 Kpn I | 构建载体 | 南京诺唯赞生物科技股份有限公司 |
同源重组酶 | 构建载体 | 南京诺唯赞生物科技股份有限公司 |
PCR 高保真酶(PrimeSTAR® GXL DNA Polymerase) | 构建载体 | 宝日医生物技术(北京)有限公司 |
琼脂糖凝胶 DNA 回收试剂盒(TIANgel Purification Kit) | 构建载体 | 天根生化科技(北京)有限公司 |
大肠杆菌 DH5α | 构建载体 | 上海唯地生物技术有限公司 |
农杆菌 GV3101(pSoup-p19) | 构建载体 | 上海唯地生物技术有限公司 |
核定位染料 DAPI 溶液 | 亚细胞定位 | 北京索莱宝科技有限公司 |
TRIzol® reagent | 总RNA提取 | 维百奥(北京)生物科技有限公司 |
氯仿(三氯甲烷) | 总RNA提取 | 实验室提供 |
异丙醇 | 总RNA提取 | 天津市致远化学试剂有限公司 |
无水乙醇 | 总RNA提取 | 天津市致远化学试剂有限公司 |
cDNA 合成试剂盒 | 反转录合成 | 北京全式金生物技术股份有限公司 |
实时荧光定量试剂盒 | 定量分析 | 北京全式金生物技术股份有限公司 |
BIORADPCR 仪 | 构建载体 | 实验室提供 |
化学发光检测仪 | 构建载体 | 实验室提供 |
Zeiss 倒置共聚焦显微镜 | 亚细胞定位 | 实验室提供 |
Applied Biosystems 实时荧光定量PCR 仪 | 定量分析 | 实验室提供 |
引物Primer | 序列Sequence(5'-3') |
---|---|
720-CDS-F | ATGATGAAGTTATTCTTAGC |
720-CDS-R | TTATATAGAATAATTCCATA |
730-CDS-F | ATGTTTCCAAGCTATTATTC |
730-CDS-R | TTAGATTGAATAATTCCATA |
740-CDS-F | ATGAATATCTTTGAAACCTA |
740-CDS-R | TTAGATAGAATAATTCCATA |
750-CDS-F | ATGGATATCTTTCGAAGCTA |
750-CDS-R | TTAGATAGAATAGCTCCATA |
pRI101-720-F | agagttgttgattcagaattcATGATGAAGTTATTCTTAGCTTCAAATAAC |
pRI101-720-R | ctgtacaagcccgggggtaccTATAGAATAATTCCATAAAGGCATATAAGC |
pRI101-730-F | agagttgttgattcagaattcATGTTTCCAAGCTATTATTCGGAGC |
pRI101-730-R | ctgtacaagcccgggggtaccTTAGATTGAATAATTCCATAAAGACATGTAA |
pRI101-740-F | agagttgttgattcagaattcATGAATATCTTTGAAACCTATTATTCAGACC |
pRI101-740-R | ctgtacaagcccgggggtaccTTAGATAGAATAATTCCATAAAGTCATGTAAGC |
pRI101-750-F | agagttgttgattcagaattcATGGATATCTTTCGAAGCTATTATTCG |
pRI101-750-R | ctgtacaagcccgggggtaccTTAGATAGAATAGCTCCATAAAGGCATAT |
720qPCR-F | CCAACAGTAGAAATGGCAGCTAGAG |
720qPCR-R | CTTCCGACTGATCTGATGATGATGG |
730qPCR-F | GAACTTTGCTGACTCTGTTTGGAG |
730qPCR-R | CCTCATTCATAGAGAGGGCCTTTTC |
740qPCR-F | TAAAGATATTCAAAAGGCGGCCGC |
740qPCR-R | CTCTGGCGCTTCTTCGTTCATAAA |
750qPCR-F | GCTATAGCATTAAGAGGCCGTTCT |
750qPCR-R | CATCCGGAGTACTAGATTGATCTCC |
Actin-F | GGGATGGAGAAGTTTGGTGGTGG |
Actin-R | CTTCGACCAAGGGATGGTGTAGC |
Table 2 Primer sequence
引物Primer | 序列Sequence(5'-3') |
---|---|
720-CDS-F | ATGATGAAGTTATTCTTAGC |
720-CDS-R | TTATATAGAATAATTCCATA |
730-CDS-F | ATGTTTCCAAGCTATTATTC |
730-CDS-R | TTAGATTGAATAATTCCATA |
740-CDS-F | ATGAATATCTTTGAAACCTA |
740-CDS-R | TTAGATAGAATAATTCCATA |
750-CDS-F | ATGGATATCTTTCGAAGCTA |
750-CDS-R | TTAGATAGAATAGCTCCATA |
pRI101-720-F | agagttgttgattcagaattcATGATGAAGTTATTCTTAGCTTCAAATAAC |
pRI101-720-R | ctgtacaagcccgggggtaccTATAGAATAATTCCATAAAGGCATATAAGC |
pRI101-730-F | agagttgttgattcagaattcATGTTTCCAAGCTATTATTCGGAGC |
pRI101-730-R | ctgtacaagcccgggggtaccTTAGATTGAATAATTCCATAAAGACATGTAA |
pRI101-740-F | agagttgttgattcagaattcATGAATATCTTTGAAACCTATTATTCAGACC |
pRI101-740-R | ctgtacaagcccgggggtaccTTAGATAGAATAATTCCATAAAGTCATGTAAGC |
pRI101-750-F | agagttgttgattcagaattcATGGATATCTTTCGAAGCTATTATTCG |
pRI101-750-R | ctgtacaagcccgggggtaccTTAGATAGAATAGCTCCATAAAGGCATAT |
720qPCR-F | CCAACAGTAGAAATGGCAGCTAGAG |
720qPCR-R | CTTCCGACTGATCTGATGATGATGG |
730qPCR-F | GAACTTTGCTGACTCTGTTTGGAG |
730qPCR-R | CCTCATTCATAGAGAGGGCCTTTTC |
740qPCR-F | TAAAGATATTCAAAAGGCGGCCGC |
740qPCR-R | CTCTGGCGCTTCTTCGTTCATAAA |
750qPCR-F | GCTATAGCATTAAGAGGCCGTTCT |
750qPCR-R | CATCCGGAGTACTAGATTGATCTCC |
Actin-F | GGGATGGAGAAGTTTGGTGGTGG |
Actin-R | CTTCGACCAAGGGATGGTGTAGC |
Fig. 1 Sequence alignment (A) and evolutionary analysis (B) of OsDREB1C homologous protein in potato A: Amino acid sequence alignment of OsDREB1C homologous protein in potato. B: Evolutionary analysis of OsDREB1C homologous protein in potato
蛋白质 Protein | 氨基酸数量Amount of amino acids | 分子质量Molecular mass/Da | 原子总数 Total atoms | 不稳定系数Coefficient of instability | pI | 脂溶性指数Fat solubility index | 亲水指数Hydrophilic index | 亚细胞定位Subcellular localization |
---|---|---|---|---|---|---|---|---|
StDREB720 | 199 | 22 478 | 3 125 | 65.51 | 6.44 | 69.70 | -0.37 | 细胞核 |
StDREB730 | 236 | 26 319 | 3 628 | 61.92 | 4.98 | 67.50 | -0.48 | 细胞核、细胞质 |
StDREB740 | 258 | 28 601 | 3 909 | 70.35 | 4.55 | 57.67 | -0.56 | 细胞核、细胞质 |
StDREB750 | 217 | 24 264 | 3 334 | 55.26 | 5.20 | 57.65 | -0.60 | 细胞核、细胞质 |
Table 3 Physicochemical analysis of StDREBs protein
蛋白质 Protein | 氨基酸数量Amount of amino acids | 分子质量Molecular mass/Da | 原子总数 Total atoms | 不稳定系数Coefficient of instability | pI | 脂溶性指数Fat solubility index | 亲水指数Hydrophilic index | 亚细胞定位Subcellular localization |
---|---|---|---|---|---|---|---|---|
StDREB720 | 199 | 22 478 | 3 125 | 65.51 | 6.44 | 69.70 | -0.37 | 细胞核 |
StDREB730 | 236 | 26 319 | 3 628 | 61.92 | 4.98 | 67.50 | -0.48 | 细胞核、细胞质 |
StDREB740 | 258 | 28 601 | 3 909 | 70.35 | 4.55 | 57.67 | -0.56 | 细胞核、细胞质 |
StDREB750 | 217 | 24 264 | 3 334 | 55.26 | 5.20 | 57.65 | -0.60 | 细胞核、细胞质 |
蛋白质Protein | 无规则卷曲Random coil/% | α-螺旋 α-helix/% | 延伸链 Extended strand/% | β-转角 β-turn/% | 保守结构域Conserved domain(super family) | 保守结构域位置 Conservative domain location/aa |
---|---|---|---|---|---|---|
StDREB720 | 49.25 | 34.17 | 14.57 | 2.01 | AP2 | 28-86 |
StDREB730 | 53.39 | 31.36 | 11.86 | 3.39 | AP2 | 64-122 |
StDREB740 | 49.22 | 35.27 | 11.63 | 3.88 | AP2 | 51-111 |
StDREB750 | 58.99 | 26.73 | 11.06 | 3.23 | AP2 | 62-120 |
Table 4 Structure and secondary structure prediction of StDREBs protein
蛋白质Protein | 无规则卷曲Random coil/% | α-螺旋 α-helix/% | 延伸链 Extended strand/% | β-转角 β-turn/% | 保守结构域Conserved domain(super family) | 保守结构域位置 Conservative domain location/aa |
---|---|---|---|---|---|---|
StDREB720 | 49.25 | 34.17 | 14.57 | 2.01 | AP2 | 28-86 |
StDREB730 | 53.39 | 31.36 | 11.86 | 3.39 | AP2 | 64-122 |
StDREB740 | 49.22 | 35.27 | 11.63 | 3.88 | AP2 | 51-111 |
StDREB750 | 58.99 | 26.73 | 11.06 | 3.23 | AP2 | 62-120 |
名称Name | 序列Sequence | 数量Account | 功能Function |
---|---|---|---|
Circadian | CAAAGATATC | 720/1 740/3 750/1 | 参与昼夜节律控制的顺式作用调节元件Cis-acting regulatory element involved in circadian control |
WUN-motif | AAATTTCCT | 720/1 | 伤口敏感元件Wound-sensitive element |
CGTCA-motif | CGTCA | 720/2 730/1 | 参与水杨酸反应的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness |
Box 4 | ATTAAT | 720/4 730/4 740/5 750/9 | 参与光响应的部分保守 DNA 模块 Part of a conserved DNA module involved in light-responsiveness |
MBS | CAACTG | 720/1 730/2 750/1 | 与干旱诱导有关的 MYB 结合位点MYB binding site involved in drought-inducibility |
TCT-motif | TCTTAC | 720/1 730/1 750/2 | 部分光响应元件Part of a light-responsive element |
G-box | CACGTG | 720/1 730/2 740/2 750/2 | 参与光反应的顺式作用调节元件Cis-acting regulatory element involved in light-responsiveness |
TATC-box | TATCCCA | 730/1 740/1 750/1 | 参与赤霉素反应的顺式作用元件Cis-acting element involved in gibberellin-responsiveness |
GT1-motif | GGTTAA | 720/1 730/5 740/1 | 光响应元件Light-responsive element |
LTR | CCGAAA | 730/4 | 参与低温响应的顺式作用元件Cis-acting element involved in low-temperature responsiveness |
ARE | AAACCA | 730/7 750/1 | 厌氧诱导必不可少的顺式作用的调节元件 Cis-acting regulatory element essential for the anaerobic induction |
AuxRR-core | GGTCCAT | 740/1 | 参与生长素反应的顺式作用调节元件 Cis-acting regulatory element involved in auxin-responsiveness |
TGA-element | AACGAC | 740/1 750/1 | 生长素响应元件Auxin-responsive element |
ABRE | CACGTG | 720/1 740/1 750/2 | 参与脱落酸反应的顺式作用元件Cis-acting element involved in the abscisic acid-responsiveness |
Table 5 Cis-acting elements in promoters of StDREBs gene
名称Name | 序列Sequence | 数量Account | 功能Function |
---|---|---|---|
Circadian | CAAAGATATC | 720/1 740/3 750/1 | 参与昼夜节律控制的顺式作用调节元件Cis-acting regulatory element involved in circadian control |
WUN-motif | AAATTTCCT | 720/1 | 伤口敏感元件Wound-sensitive element |
CGTCA-motif | CGTCA | 720/2 730/1 | 参与水杨酸反应的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness |
Box 4 | ATTAAT | 720/4 730/4 740/5 750/9 | 参与光响应的部分保守 DNA 模块 Part of a conserved DNA module involved in light-responsiveness |
MBS | CAACTG | 720/1 730/2 750/1 | 与干旱诱导有关的 MYB 结合位点MYB binding site involved in drought-inducibility |
TCT-motif | TCTTAC | 720/1 730/1 750/2 | 部分光响应元件Part of a light-responsive element |
G-box | CACGTG | 720/1 730/2 740/2 750/2 | 参与光反应的顺式作用调节元件Cis-acting regulatory element involved in light-responsiveness |
TATC-box | TATCCCA | 730/1 740/1 750/1 | 参与赤霉素反应的顺式作用元件Cis-acting element involved in gibberellin-responsiveness |
GT1-motif | GGTTAA | 720/1 730/5 740/1 | 光响应元件Light-responsive element |
LTR | CCGAAA | 730/4 | 参与低温响应的顺式作用元件Cis-acting element involved in low-temperature responsiveness |
ARE | AAACCA | 730/7 750/1 | 厌氧诱导必不可少的顺式作用的调节元件 Cis-acting regulatory element essential for the anaerobic induction |
AuxRR-core | GGTCCAT | 740/1 | 参与生长素反应的顺式作用调节元件 Cis-acting regulatory element involved in auxin-responsiveness |
TGA-element | AACGAC | 740/1 750/1 | 生长素响应元件Auxin-responsive element |
ABRE | CACGTG | 720/1 740/1 750/2 | 参与脱落酸反应的顺式作用元件Cis-acting element involved in the abscisic acid-responsiveness |
Fig. 3 Expression pattern of StDREBs genes in potato The error bar indicates the standard deviation(n=3); significance analysis uses t test, * 0.05>P≥0.01; ** 0.01>P≥0.001; *** P<0.001; **** P≤0.0001, the same below
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