Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (5): 48-55.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1319
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LIN Yan-li1(), QIN Jian-bing1, WU Xiang1, WANG Yan-yan1, PAN You-zhao2, LIU Zhong-yu1()
Received:
2020-10-25
Online:
2021-05-26
Published:
2021-06-11
Contact:
LIU Zhong-yu
E-mail:1329137579@qq.com;zyliu2004@126.com
LIN Yan-li, QIN Jian-bing, WU Xiang, WANG Yan-yan, PAN You-zhao, LIU Zhong-yu. Cloning and Activity Analysis of PcMYB1 Promoter from Polygonum cuspidatum[J]. Biotechnology Bulletin, 2021, 37(5): 48-55.
引物名称Primer name | 引物序列Prime sequence(5'-3') | 酶切位点Enzymatic cleavage site |
---|---|---|
LAD1-1 | ACGATGGACTCCAGAGCGGCCGC(G/C/A)N(G/C/A)NNNGGAA | |
LAD1-2 | ACGATGGACTCCAGAGCGGCCGC(G/C/T)N(G/C/T)NNNGGTT | |
LAD1-3 | ACGATGGACTCCAGAGCGGCCGC(G/C/A)(G/C/A)N(G/C/A)NNNCCAA | |
LAD1-4 | ACGATGGACTCCAGAGCGGCCGC(G/C/T)(G/A/T)N(G/C/T)NNNCGGT | |
AC1 | ACGATGGACTCCAGAG | |
SP0-1 | AAATTTCCACGCTTGAGATCAGGGC | |
SP1-1 | ACGATGGACTCCAGTCCGGCCCCCACATCGAAGAAGTCCAGCAGCT | |
SP2-1 | GCTTTCTCACAACATGGAGACCTACCC | |
P0-F | GATGGACTCCAGAGCGGCC | |
P0-R | TTCTCGAAACTTACCAATC | |
P1-F | AAAACTGCAGGATGGACTCCAGAGCGGCC | PstⅠ |
P2-F | AAAACTGCAGGAGTCTCATACCGTCCAA | PstⅠ |
P3-F | AAAACTGCAGACGATTAGACCCGTTTGG | PstⅠ |
P4-F | AAAACTGCAGTACCTCCATCCTCCTTGT | PstⅠ |
pro-R | CGCGGATCCTTCTCGAAACTTACCAATC | BamHⅠ |
Hyg-F | GAAGTGCTTGACATTGGGGAGT | |
Hyg-R | AGATGTTGGCGACCTCGTATT | |
GUS-F | GTCGCGCAAGACTGTAACCA | |
GUS-R | CGGCGAAATTCCATACCTG |
Table 1 Primer sequences
引物名称Primer name | 引物序列Prime sequence(5'-3') | 酶切位点Enzymatic cleavage site |
---|---|---|
LAD1-1 | ACGATGGACTCCAGAGCGGCCGC(G/C/A)N(G/C/A)NNNGGAA | |
LAD1-2 | ACGATGGACTCCAGAGCGGCCGC(G/C/T)N(G/C/T)NNNGGTT | |
LAD1-3 | ACGATGGACTCCAGAGCGGCCGC(G/C/A)(G/C/A)N(G/C/A)NNNCCAA | |
LAD1-4 | ACGATGGACTCCAGAGCGGCCGC(G/C/T)(G/A/T)N(G/C/T)NNNCGGT | |
AC1 | ACGATGGACTCCAGAG | |
SP0-1 | AAATTTCCACGCTTGAGATCAGGGC | |
SP1-1 | ACGATGGACTCCAGTCCGGCCCCCACATCGAAGAAGTCCAGCAGCT | |
SP2-1 | GCTTTCTCACAACATGGAGACCTACCC | |
P0-F | GATGGACTCCAGAGCGGCC | |
P0-R | TTCTCGAAACTTACCAATC | |
P1-F | AAAACTGCAGGATGGACTCCAGAGCGGCC | PstⅠ |
P2-F | AAAACTGCAGGAGTCTCATACCGTCCAA | PstⅠ |
P3-F | AAAACTGCAGACGATTAGACCCGTTTGG | PstⅠ |
P4-F | AAAACTGCAGTACCTCCATCCTCCTTGT | PstⅠ |
pro-R | CGCGGATCCTTCTCGAAACTTACCAATC | BamHⅠ |
Hyg-F | GAAGTGCTTGACATTGGGGAGT | |
Hyg-R | AGATGTTGGCGACCTCGTATT | |
GUS-F | GTCGCGCAAGACTGTAACCA | |
GUS-R | CGGCGAAATTCCATACCTG |
Fig.2 Analysis of PcMYB1 by electrophoresis of hiTAIL-PCR amplification products M :Marker. A: Preamplification products. B: First round hiTAIL-PCR product.C: Round 2 hiTAIL-PCR products
名称 Name | 识别序列 Identification of sequence | 起始端位点 Start site | 功能 Function |
---|---|---|---|
AE-box | AGAAACAA | -2100 | 部分光响应元件 |
AT1-motif | AATTATTTCTTATT | -640 | 部分光响应元件 |
I-box | GTATAAGGCC | -1410 | 部分光响应元件 |
TCT-motif | TCTTAC | -281;-1296 | 部分光响应元件 |
G-Box | CACGTC | -425-;-683 | 光响应元件 |
GT1-motif | GGTTAA | -1600;-1 984 | 光响应元件 |
ARE | AAACCA | -500;-522;-1 924;-2 479 | 厌氧诱导顺式作用元件 |
CGTCA-motif | CGTCA | -1 021;-1 147;-1 808 | 茉莉酸甲酯响应元件 |
TGACG-motif | TGACG | -1 072;-2 713 | 茉莉酸甲酯响应元件 |
LTR | CCGAAA | -503;-531;-2 812 | 低温诱导顺式作用元件 |
CAAT-box | CCAAT/CAAAT | -23;-710;-1 452;-1 783;-2 098;2 745 | 启动子和增强子常见顺式调控元件 |
TATA-box | TATA | -214;-599;-1 302;1 465;-1 735;-1 882;-1 948;-1 993;-2 202;-2 271;-2 484;-2 781 | 转录起始元件 |
Table 2 PcMYB1 promoter cis-acting element prediction analysis
名称 Name | 识别序列 Identification of sequence | 起始端位点 Start site | 功能 Function |
---|---|---|---|
AE-box | AGAAACAA | -2100 | 部分光响应元件 |
AT1-motif | AATTATTTCTTATT | -640 | 部分光响应元件 |
I-box | GTATAAGGCC | -1410 | 部分光响应元件 |
TCT-motif | TCTTAC | -281;-1296 | 部分光响应元件 |
G-Box | CACGTC | -425-;-683 | 光响应元件 |
GT1-motif | GGTTAA | -1600;-1 984 | 光响应元件 |
ARE | AAACCA | -500;-522;-1 924;-2 479 | 厌氧诱导顺式作用元件 |
CGTCA-motif | CGTCA | -1 021;-1 147;-1 808 | 茉莉酸甲酯响应元件 |
TGACG-motif | TGACG | -1 072;-2 713 | 茉莉酸甲酯响应元件 |
LTR | CCGAAA | -503;-531;-2 812 | 低温诱导顺式作用元件 |
CAAT-box | CCAAT/CAAAT | -23;-710;-1 452;-1 783;-2 098;2 745 | 启动子和增强子常见顺式调控元件 |
TATA-box | TATA | -214;-599;-1 302;1 465;-1 735;-1 882;-1 948;-1 993;-2 202;-2 271;-2 484;-2 781 | 转录起始元件 |
Fig.4 PCR identification of recombinant vectors M :Marker. P1: P1∷GUS amplification product. P2: P2∷GUS amplification product. P3 :P3∷GUS amplification product. P4: P4∷GUS amplification product
Fig.5 Hyg (A) and GUS (B) detection of transgenic hairy roots M :Marker. WT: Wild-type hairy roots. 35S: 35S∷GUS transgenic hairy roots. P1 :P1∷GUS transgenic hairy roots. P2: P2∷GUS transgenic hairy roots. P3: P3∷GUS transposition. P4: P4∷GUS transgenic hairy roots
Fig.6 GUS histochemical staining of transgenic hairy roots M :Marker. WT: Wild-type hairy roots. 35S: 35S∷GUS transgenic hairy roots. P1 :P1∷GUS transgenic hairy roots. P2: P2∷GUS transgenic hairy roots. P3: P3∷GUS transposition. P4: P4∷GUS transgenic hairy roots
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