Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (9): 191-197.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0570
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SHI Guang-zhen(), WANG Zhao-ye, SUN Qi, ZHU Xin-xia()
Received:
2022-05-09
Online:
2022-09-26
Published:
2022-10-11
Contact:
ZHU Xin-xia
E-mail:1106517620@qq.com;302641316@qq.com
SHI Guang-zhen, WANG Zhao-ye, SUN Qi, ZHU Xin-xia. Cloning and Activity Analysis of SikCDPK1 Promoter from Saussurea involucrata[J]. Biotechnology Bulletin, 2022, 38(9): 191-197.
引物Primer name | 引物序列Primer sequence(5'-3') | 用途Purpose |
---|---|---|
AD1 | NTCGASTWTSGWGTT | TAIL-PCR |
AD2 | NGTCGASWGANAWGAAAA | TAIL-PCR |
AD3 | WGTCNACWANCANACA | TAIL-PCR |
AD4 | TGWGNAGWANCANAGA | TAIL-PCR |
AD5 | AGWGNAGWANCAWAGG | TAIL-PCR |
SP1 | CGTTATCCCAAACTGCCCTTGTCCTA | TAIL-PCR |
SP2 | TCCGTTAGGGGTAGGTGGGGTATCTT | TAIL-PCR |
SP3 | TTCGGTCCAACACAAGTATTCCCCAT | TAIL-PCR |
pSikCDPK1-F1 | CCCAAGCTTCCTTAGCATCTATGAGGGTCG | 启动子克隆 Promoter cloning |
pSikCDPK1-R1 | GACTAGTTCCAACACAAGTATTCCCCATG | 启动子克隆 Promoter cloning |
pSikCDPK1-F2 | CCCAAGCTTCTCTTCTTATGGGTTCAAAGGGTCA | 5'端缺失分析 5'-end deletion analysis |
pSikCDPK1-F3 | CCCAAGCTTAAGCGTCATGCCAGTCAAGC | 5'端缺失分析 5'-end deletion analysis |
pSikCDPK1-F4 | CCCAAGCTTTTTCAATGGAGAAGCGACGAGC | 5'端缺失分析 5'-end deletion analysis |
Table 1 Primers used in this study
引物Primer name | 引物序列Primer sequence(5'-3') | 用途Purpose |
---|---|---|
AD1 | NTCGASTWTSGWGTT | TAIL-PCR |
AD2 | NGTCGASWGANAWGAAAA | TAIL-PCR |
AD3 | WGTCNACWANCANACA | TAIL-PCR |
AD4 | TGWGNAGWANCANAGA | TAIL-PCR |
AD5 | AGWGNAGWANCAWAGG | TAIL-PCR |
SP1 | CGTTATCCCAAACTGCCCTTGTCCTA | TAIL-PCR |
SP2 | TCCGTTAGGGGTAGGTGGGGTATCTT | TAIL-PCR |
SP3 | TTCGGTCCAACACAAGTATTCCCCAT | TAIL-PCR |
pSikCDPK1-F1 | CCCAAGCTTCCTTAGCATCTATGAGGGTCG | 启动子克隆 Promoter cloning |
pSikCDPK1-R1 | GACTAGTTCCAACACAAGTATTCCCCATG | 启动子克隆 Promoter cloning |
pSikCDPK1-F2 | CCCAAGCTTCTCTTCTTATGGGTTCAAAGGGTCA | 5'端缺失分析 5'-end deletion analysis |
pSikCDPK1-F3 | CCCAAGCTTAAGCGTCATGCCAGTCAAGC | 5'端缺失分析 5'-end deletion analysis |
pSikCDPK1-F4 | CCCAAGCTTTTTCAATGGAGAAGCGACGAGC | 5'端缺失分析 5'-end deletion analysis |
Fig. 2 Cloning of SikCDPK1 promoter A:The first round of PCR amplification. B:The second round of PCR amplification. C:The third round of PCR amplification. M:DNA2000 marker. 1:AD1. 2:AD2. 3:AD3. 4:AD4
顺式作用元件名称 Name of cis-acting element | 序列 Sequence(5'-3') | 功能 Function | 位置 Location/ nt |
---|---|---|---|
O2-site | GATGA(C/T)(A/G)TG(A/G)或GATGATGTGG | 玉米醇溶蛋白代谢调节 Zein metabolism regulation | -56 to -48,-596 to -587 |
CGTCA-motif | CGTCA | 茉莉酸甲酯响应 MeJA-responsiveness | -94 to -89,-562 to -557,-479 to -474 |
CBFHV | RYCGAC | 低温反应元件 Cis-acting element for cold | -103 to -97,-379 to -373,-672 to -666 |
GT1GMSCAM4 | GAAAAA | 盐诱导响应相关元件 NaCl-induced | -159 to -153 |
LTRECOREATCOR15 | CCGAC | 冷诱导低温反应元件 Cis-acting element for cold induction | -215 to -210,-459 to -454 |
GC-motif | GCCCCC | 参与缺氧特异性诱导的类增强子Enhancer-like element involved in anoxic specific inducibility | -287 to -281 |
TGACG-motif | TGACG | 茉莉酸甲酯响应 MeJA-responsiveness | -310 to -305,-499 to -494 |
ABRE | TACGGTC | ABA响应Abscisic acid responsiveness | -327 to -320 |
W-box | TTGACC | WRKY转录因子的结合位点WRKY transcription factor binding site | -387 to -381 |
box-S | AGCCACC | 无功能No function | -571 to -564 |
MYC | CAATTG | 无功能No function | -533 to -539 |
G-box | CACGTC | 光响应 Light responsiveness | -689 to -683 |
MYB | CAACCA | MYB 顺式作用元件MYB Cis-acting element | -759 to -753 |
OSE2ROOTNODULE | CTCTT | 在根瘤的感染细胞中被激活 Activated in infected cells of root nodules | -821 to -816 |
MYB-Core | CGTTAG | 脱水胁迫反应元件Responsive to dehydration | -958 to -952 |
DPBFCOREDCDC3 | ACACNNG | 脱落酸响应元件ABA-responsive elements | -975 to -967 |
ACGTATERD1 | ACGT | 脱水诱导反应元件 Early responsive to dehydration | -1 038 to -1 034 |
Table 2 Cis-elements and functions in the promoter pSikCDPK1 sequence
顺式作用元件名称 Name of cis-acting element | 序列 Sequence(5'-3') | 功能 Function | 位置 Location/ nt |
---|---|---|---|
O2-site | GATGA(C/T)(A/G)TG(A/G)或GATGATGTGG | 玉米醇溶蛋白代谢调节 Zein metabolism regulation | -56 to -48,-596 to -587 |
CGTCA-motif | CGTCA | 茉莉酸甲酯响应 MeJA-responsiveness | -94 to -89,-562 to -557,-479 to -474 |
CBFHV | RYCGAC | 低温反应元件 Cis-acting element for cold | -103 to -97,-379 to -373,-672 to -666 |
GT1GMSCAM4 | GAAAAA | 盐诱导响应相关元件 NaCl-induced | -159 to -153 |
LTRECOREATCOR15 | CCGAC | 冷诱导低温反应元件 Cis-acting element for cold induction | -215 to -210,-459 to -454 |
GC-motif | GCCCCC | 参与缺氧特异性诱导的类增强子Enhancer-like element involved in anoxic specific inducibility | -287 to -281 |
TGACG-motif | TGACG | 茉莉酸甲酯响应 MeJA-responsiveness | -310 to -305,-499 to -494 |
ABRE | TACGGTC | ABA响应Abscisic acid responsiveness | -327 to -320 |
W-box | TTGACC | WRKY转录因子的结合位点WRKY transcription factor binding site | -387 to -381 |
box-S | AGCCACC | 无功能No function | -571 to -564 |
MYC | CAATTG | 无功能No function | -533 to -539 |
G-box | CACGTC | 光响应 Light responsiveness | -689 to -683 |
MYB | CAACCA | MYB 顺式作用元件MYB Cis-acting element | -759 to -753 |
OSE2ROOTNODULE | CTCTT | 在根瘤的感染细胞中被激活 Activated in infected cells of root nodules | -821 to -816 |
MYB-Core | CGTTAG | 脱水胁迫反应元件Responsive to dehydration | -958 to -952 |
DPBFCOREDCDC3 | ACACNNG | 脱落酸响应元件ABA-responsive elements | -975 to -967 |
ACGTATERD1 | ACGT | 脱水诱导反应元件 Early responsive to dehydration | -1 038 to -1 034 |
Fig. 3 PCR amplification of promoter SikCDPK1 and double restriction identification of expression vector A:PCR amplification SikCDPK1 promoter(M:DNA2000 marker. 1,2:PCR product of P0. 3,4:PCR product of P1. 5,6:PCR product of P2. 7,8:PCR product of P3). B,C:Double digestion identification of expression vector(B1:P0 double digestion. B2:P0 plasmid. B3:P1 double digestion. B4:P1 plasmid. C1:P2 double digestion. C2:P2 plasmid. C3:P3 double digestion. C4:P3 plasmid. M:DNA marker III)
Fig. 4 GUS staining analysis of transient transformed tobacco with SikCDPK1 promoter A:Negative control(Non-transgenic). B:Positive control(35S∷GUS). C:P0. D:P1. E:P2. F:P3
Fig. 5 Gus staining analysis and enzyme activity determin-ation under stress treatment A:Gus staining and enzyme activity after low temperature stress. B:Gus staining and enzyme activity after PEG stress. a:Positive control p35S-GUS-1304. b:Experimental group pSikCDPK1-GUS-1304. c:Experimental group control at room temperature
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