生物技术通报 ›› 2022, Vol. 38 ›› Issue (2): 34-43.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1077
收稿日期:
2021-08-21
出版日期:
2022-02-26
发布日期:
2022-03-09
作者简介:
刘萌萌,女,硕士研究生,研究方向:作物遗传育种;E-mail: 基金资助:
LIU Meng-meng(), HAN Li-jun, LIU Bao-ling, XUE Jin-ai(), LI Run-zhi()
Received:
2021-08-21
Published:
2022-02-26
Online:
2022-03-09
摘要:
三酰基甘油脂肪酶(SDP1)是催化三酰甘油降解的关键酶,在植物油脂代谢调控中起着重要作用。克隆棉花SDP1并研究其在3种胁迫下的表达分析,为解析棉花SDP1的生物学功能提供依据。以陆地棉品种冀丰1271为试材,克隆GhSDP1编码序列和上游启动子序列;利用PlantCARE分析GhSDP1启动子区顺式作用元件;qRT-PCR检测逆境胁迫下GhSDP1的表达谱;通过烟草瞬时表达pGhSDP1启动子+GUS载体检测启动子活性。结果表明,GhSDP1的编码序列为2 541 bp,其在盐、低温和干旱胁迫下呈差异表达模式。pGhSDP1除具有启动子所必需的TATA-box和CAAT-box等基本顺式作用元件外,还含有多个与光响应、激素响应及逆境应答等相关的顺式作用元件。棉花pGhSDP1启动子能驱动GUS蛋白高效表达,具有较强的启动子活性。研究揭示了棉花GhSDP1参与胁迫应答的新功能。
刘萌萌, 韩立军, 刘宝玲, 薛金爱, 李润植. 陆地棉GhSDP1及其启动子的克隆与表达分析[J]. 生物技术通报, 2022, 38(2): 34-43.
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 |
表1 本研究所用引物
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 |
图1 构建的pCAMBIA1301-proGhSDP1植物表达载体简图 A:pCAMBIA1301植物表达载体;B:pCAMBIA1301-proGhSDP1载体
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 |
表2 烟草农杆菌侵染液配置
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 |
图3 棉花幼苗在不同胁迫处理下的表型观察 A:NaCl处理(200 mmol/L);B:低温处理(4℃);C:PEG处理(15%)
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%)
图4 GhSDP1在不同胁迫处理下的表达情况 A:NaCl处理;B:低温处理;C:PEG处理。小写字母表示差异达到(P<0.05)显著水平
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)
图5 陆地棉GhSDP1启动子的PCR扩增产物 M:DL2000 DNA marker;1-2:目的片段
Fig.5 PCR amplified product of GhSDP1 promoter in G. hirsutum M:DL2000 DNA marker. 1-2:Target fragment
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 |
表3 棉花GhSDP1基因启动子部分顺式作用元件
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 |
图6 表达载体重组子双酶切电泳图 M:DNA marker DL10 000;1:双酶切结果;2:质粒
Fig.6 Electrophoresis of double-digested expression vector M:DNA marker DL10 000;1:result of enzyme digestion;2:plasmid
图7 携带有pGhSDP1∷GUS载体的农杆菌侵染烟草叶片GUS染色 A:pGhSDP1∷GUS;B:CaMV35S∷GUS(阳性对照);C:GV3101(阴性对照)
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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