Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (2): 34-43.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1077
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LIU Meng-meng(), HAN Li-jun, LIU Bao-ling, XUE Jin-ai(), LI Run-zhi()
Received:
2021-08-21
Online:
2022-02-26
Published:
2022-03-09
Contact:
XUE Jin-ai,LI Run-zhi
E-mail:2574227154@qq.com;306214803@qq.com;rli2001@126.com
LIU Meng-meng, HAN Li-jun, LIU Bao-ling, XUE Jin-ai, LI Run-zhi. Cloning and Expression Analysis of GhSDP1 and Its Promoter in Gossypium hirsutum[J]. Biotechnology Bulletin, 2022, 38(2): 34-43.
引物名称Primer name | 引物序列Primer sequence(5'-3') | 用途Application |
---|---|---|
GhSDP1-F | ATGGATATAACTAATGAGGCCAGTGT | GhSDP1引物 |
GhSDP1-R | TTAGGCATCTACAACACCACCCTG | Primer pairs for GhSDP1 gene |
pGhSDP1-F | TTGGAGTTAGATTGTAAATAAT | GhSDP1启动子引物 |
pGhSDP1-R | AGCTAGTTTCTAATAGGGAATG | Primer pairs for the promoter of GhSDP1 |
1301-pGhSDP1-F | tcctctagagtcgacctgcagTTGGAGTTAGATTGTAAATAAT | GUS染色载体引物 |
1301-pGhSDP1-R | ttaccctcagatctaccatggAGCTAGTTTCTAATAGGGAATG | Primer pairs for GUS staining vector |
qGhSDP1-F | CTTCGGCAACCAGATTCTTCC | GhSDP1荧光定量引物 |
qGhSDP1-R | AGCGACTCCACCACCTACT | Primer pairs for qRT-PCR of GhSDP1 |
histone-F | AAGCCTCATCGATACCGTCC | 内参基因引物 |
histone-R | GGCTCTGGAAACGCAAATCA | Primer pairs for internal reference gene |
Table 1 Primers used in this study
引物名称Primer name | 引物序列Primer sequence(5'-3') | 用途Application |
---|---|---|
GhSDP1-F | ATGGATATAACTAATGAGGCCAGTGT | GhSDP1引物 |
GhSDP1-R | TTAGGCATCTACAACACCACCCTG | Primer pairs for GhSDP1 gene |
pGhSDP1-F | TTGGAGTTAGATTGTAAATAAT | GhSDP1启动子引物 |
pGhSDP1-R | AGCTAGTTTCTAATAGGGAATG | Primer pairs for the promoter of GhSDP1 |
1301-pGhSDP1-F | tcctctagagtcgacctgcagTTGGAGTTAGATTGTAAATAAT | GUS染色载体引物 |
1301-pGhSDP1-R | ttaccctcagatctaccatggAGCTAGTTTCTAATAGGGAATG | Primer pairs for GUS staining vector |
qGhSDP1-F | CTTCGGCAACCAGATTCTTCC | GhSDP1荧光定量引物 |
qGhSDP1-R | AGCGACTCCACCACCTACT | Primer pairs for qRT-PCR of GhSDP1 |
histone-F | AAGCCTCATCGATACCGTCC | 内参基因引物 |
histone-R | GGCTCTGGAAACGCAAATCA | Primer pairs for internal reference gene |
Fig.1 Diagram of constructing pCAMBIA1301-proGhS-DP1 plant expression vector A:pCAMBIA1301 plant expression vector. B:pCAMBIA1301-proGhSDP1 plant expression vector
试剂Reagent | 母液浓度 Mother liquor | 终浓度 Final concentration | 使用量 Volume of use |
---|---|---|---|
乙酰丁香酮(AS) | 100 μmol/mL | 100 μmol/L | 100 μL |
硫酸镁(MgSO4) | 1 mmol/mL | 10 mmol/L | 1 mL |
2-(N-吗啡啉)- 乙磺酸(MES) | 1 mmol/mL | 10 mmol/L | 1 mL |
无菌水(ddH2O) | 定容至100 mL |
Table 2 Preparation of tobacco infecting solution
试剂Reagent | 母液浓度 Mother liquor | 终浓度 Final concentration | 使用量 Volume of use |
---|---|---|---|
乙酰丁香酮(AS) | 100 μmol/mL | 100 μmol/L | 100 μL |
硫酸镁(MgSO4) | 1 mmol/mL | 10 mmol/L | 1 mL |
2-(N-吗啡啉)- 乙磺酸(MES) | 1 mmol/mL | 10 mmol/L | 1 mL |
无菌水(ddH2O) | 定容至100 mL |
Fig.3 Phenotype observation of cotton seedlings under dif-ferent stress treatments A:NaCl treatment(200 mmol/L). B:Cold stress treatment(4℃). C:PEG treatment(15%)
Fig.4 Relative expressions of GhSDP1 gene under different stress treatments A:NaCl treatment. B:Cold stress treatment. C:PEG treatment. The lowercase letters indicate that the difference reached a significant level(P<0.05)
元件名称 Cis- element | 核心序列 Sequence | 功能 Function | 分布 Position | 数量Number |
---|---|---|---|---|
GATABOX | GATA | 光应答元件 | 138(+),680(-),686(-),773(+),870(-),1116(-),1194(-),1304(-),1397(+),1577(+),1626(-),1680(+),1765(-) | 13 |
ARR1AT | NGATT | 应答调控因子ARR1结合元件 | 9(+),55(+),111(-),116(-),132(+),382(-),707(-),715(-),806(-),845(-),1111(-),1150(-),1170(-),1439(+),1509(-),1538(+),1638 | 21 |
ACGTATERD1 | ACGT | 脱水相关顺式作用元件 | (-),1690(-),1801(-),817(-),883(-) 141(+),851(+) | 2 |
MYB1AT | WAACCA | 干旱响应相关元件 | 1308(-),1381(-),1480(-),1566(-) | 4 |
MYBCORE | CNGTTR | 干旱应答 | 947(+),1203(-),1389(-),1828(+) | 4 |
MYCCONSENSUSAT | CANNTG | 干旱及低温响应元件 | 947(+),1673(-) | 2 |
GT1CONSENSUS | GRWAAW | 盐、水杨酸及光响应元件 | 773(+),818(+),819(+),1223(-),1316(-),1326(-),1371(+),1397(+),1450(+),1451(+),1461(+),1649(+),1663(-),1700(-),1718(+),1763(-),1823(-) | 17 |
GAREAT | TAACAAR | 赤霉素应答元件 | 634(-),830(-) | 2 |
WRKY71OS | TGAC | W-box 核心区,参与赤霉素信号途径 | 224(-),734(-),793(-),849(+),1581(-) | 5 |
BIHD1OS | TGTCA | 抗病响应相关元件 | 1351(+) | 1 |
WBOXNTERF3 | TGACY | ERF3结合位点,损伤响应元件 | 792(-),1580(-) | 2 |
POLLEN1LELAT52 | AGAAA | 花粉特异表达相关元件 | 50(-),261(+),415(-),475(-),570(+),704(-),748(+),788(+),933(-),1050(+),1255(+) | 11 |
Table 3 Part of putative cis-acting elements and their positions in the GhSDP1 promoter
元件名称 Cis- element | 核心序列 Sequence | 功能 Function | 分布 Position | 数量Number |
---|---|---|---|---|
GATABOX | GATA | 光应答元件 | 138(+),680(-),686(-),773(+),870(-),1116(-),1194(-),1304(-),1397(+),1577(+),1626(-),1680(+),1765(-) | 13 |
ARR1AT | NGATT | 应答调控因子ARR1结合元件 | 9(+),55(+),111(-),116(-),132(+),382(-),707(-),715(-),806(-),845(-),1111(-),1150(-),1170(-),1439(+),1509(-),1538(+),1638 | 21 |
ACGTATERD1 | ACGT | 脱水相关顺式作用元件 | (-),1690(-),1801(-),817(-),883(-) 141(+),851(+) | 2 |
MYB1AT | WAACCA | 干旱响应相关元件 | 1308(-),1381(-),1480(-),1566(-) | 4 |
MYBCORE | CNGTTR | 干旱应答 | 947(+),1203(-),1389(-),1828(+) | 4 |
MYCCONSENSUSAT | CANNTG | 干旱及低温响应元件 | 947(+),1673(-) | 2 |
GT1CONSENSUS | GRWAAW | 盐、水杨酸及光响应元件 | 773(+),818(+),819(+),1223(-),1316(-),1326(-),1371(+),1397(+),1450(+),1451(+),1461(+),1649(+),1663(-),1700(-),1718(+),1763(-),1823(-) | 17 |
GAREAT | TAACAAR | 赤霉素应答元件 | 634(-),830(-) | 2 |
WRKY71OS | TGAC | W-box 核心区,参与赤霉素信号途径 | 224(-),734(-),793(-),849(+),1581(-) | 5 |
BIHD1OS | TGTCA | 抗病响应相关元件 | 1351(+) | 1 |
WBOXNTERF3 | TGACY | ERF3结合位点,损伤响应元件 | 792(-),1580(-) | 2 |
POLLEN1LELAT52 | AGAAA | 花粉特异表达相关元件 | 50(-),261(+),415(-),475(-),570(+),704(-),748(+),788(+),933(-),1050(+),1255(+) | 11 |
Fig.7 GUS staining of the tobacco leaves infected by agrob-acterium containing the pGhSDP1∷GUS vector A:pGhSDP1∷GUS. B:CaMV35S∷GUS(positive control). C:GV3101(negative control)
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