陆地棉GhSDP1及其启动子的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1077

摘要/Abstract

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。

关键词: 陆地棉, SDP1, 启动子, 载体构建, GUS染色

Abstract:

Triacylglycerol lipase（SDP1）is a key enzyme in the degradation of triacylglycerol and plays an important role in the regulation of vegetable oils. We focused on the cloning of the cotton SDP1 gene and tits expression analysis under three stresses,which provides basis for analyzing the biological function of the cotton SDP1 gene. The coding sequence and upstream promoter sequence of GhSDP1 were cloned in cotton variety Jifeng 1271. PlantCARE was used to analyze the cis-acting elements in the promoter region of GhSDP1,and qRT-PCR was applied to detect the expression profile of GhSDP1 under stress. The promoter activity analysis of pGhSDP1+GUS fusion vector was carried out by tobacco transient expression. The results showed that the coding sequence of GhSDP1 was 2 541 bp,and the gene presented a differential expression pattern under salt,low temperature and drought stress. pGhSDP1 not only had the basic cis-acting elements such as TATA-box and CAAT-box,but also contained a number of cis-acting elements related to light response,hormone response and adversity response. The pGhSDP1 promoter in cotton can drive the high-efficiency expression of GUS protein and has strong promoter activity. The study revealed that the cotton GhSDP1 gene participates in the corresponding new functions of the stress.

Key words: Gossypium hirsutum, SDP1, promoter, vector construction, GUS staining

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.

图/表 10

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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

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

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

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

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