蒙古沙冬青外向钾离子通道AmGORK启动子克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0192

摘要/Abstract

摘要：

克隆蒙古沙冬青钾离子通道AmGORK的启动子区域,探明在干旱胁迫下它对保卫细胞运动的调控机制,为深入研究该基因在水分及养分利用、光合作用、抗旱等过程的功能奠定基础。以蒙古沙冬青保卫细胞外向钾离子通道基因AmGORK编码序列为基础,通过染色体步移技术克隆该基因的启动子序列,并对其进行生物信息学分析,构建pCambia1301-AmGORK∷GUS双元载体转化野生型拟南芥,对T₃转基因拟南芥各组织进行GUS活性染色,转基因植株经PEG或ABA处理后,分析AmGORK启动子的表达情况。结果表明,获得AmGORK上游1 723 bp序列,该区域含有多个响应干旱、ABA、光照等的顺式调控元件,说明AmGORK可能参与干旱响应过程。经GUS活性染色,发现AmGORK在根中柱、叶柄、叶脉、叶片保卫细胞、花萼均有表达,表明AmGORK的表达具有组织特异性。定量分析发现PEG或ABA处理后的转基因植株中GUS分别上调3.2倍和4.1倍。AmGORK表达可能受干旱诱导。

关键词: AmGORK, 启动子, 蒙古沙冬青, GUS, 表达分析

Abstract:

To investigate its regulation mechanism on guard cell movement under drought stress,the promoter region of potassium channel AmGORK in Ammopiptanthus mongolicus was cloned so as to lay a foundation for further studying the functions of AmGORK in water and nutrient utilization,photosynthesis,drought resistance and other processes. Based on the coding sequence of the outward potassium channel gene AmGORK in the guard cells of A. mongolicus,the promoter sequence of the gene was cloned by chromosome walking technique,and its bioinformatics analysis was further conducted. The pCambia1301-AmGORK∷GUS binary vector was constructed and transformed into the wild-type A. thaliana. The tissues of T3 in the transgenic A. thaliana were stained for analyzing GUS activity. After being treated with PEG or ABA,the transgenic plants were analyzed for the expression of AmGORK promoter. The results showed that the 1 723 bp upstream sequence of AmGORK was obtained,and contained several ci regulatory elements in response to drought,ABA and light,indicating that AmGORK may be involved in the drought response process. GUS activity staining showed that AmGORK was expressed in the root stele,petiole,vein,guard cell of a leaf,and calyx,indicating that the expression of AmGORK was tissue-specific. According to quantitative analysis,GUS was up-regulated by 3.2-fold and 4.1-fold in the PEG- or ABA-treated transgenic plants,respectively. Consequently,the expression of AmGORK may be induced by drought.

Key words: AmGORK, promoter, Ammopiptanthus mongolicus, GUS, expression analysis

李俊林, 张焕朝, 聂文婧, 张海洋, 王向誉, 郭洪恩, 韩蕾. 蒙古沙冬青外向钾离子通道AmGORK启动子克隆及表达分析[J]. 生物技术通报, 2021, 37(10): 9-16.

LI Jun-lin, ZHANG Huan-chao, NIE Wen-jing, ZHANG Hai-yang, WANG Xiang-yu, GUO Hong-en, HAN Lei. Cloning and Expression Analysis of Outward Potassium Ion Channel Gene AmGORK Promoter from Ammopiptanthus mongolicus[J]. Biotechnology Bulletin, 2021, 37(10): 9-16.

图/表 7

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序号Sequence No.	引物名称Primer name	引物序列Primer sequence（5'-3'）
1	GP1-1	GCCGAATTCCATTGGGGTGAAG
2	GP1-2	ACGATGGACTCCAGTCCGGCCGTGGTGGATCCTCCAATCCAAC
3	GP1-3	CTTCCTCCTCCGAATCATCGCT
4	GP2-1	CTGGAGTCCATCGTAATCGTCGA
5	GP2-2	ACGATGGACTCCAGTCCGGCCCAACTCCAGCTTGACTCAAGCAAC
6	GPP2-3	CAACTTGAACAGGAGCTTGCTCG
7	LAD1-1	ACGATGGACTCCAGAGCGGCCGC（G/C/A）N（G/C/A）NNNGGAA
8	LAD1-2	ACGATGGACTCCAGAGCGGCCGC（G/C/T）N（G/C/T）NNNGGTT
9	LAD1-3	ACGATGGACTCCAGAGCGGCCGC（G/C/A）（G/C/A）N（G/C/A）NNNCCAA
10	LAD1-4	ACGATGGACTCCAGAGCGGCCGC（G/C/T）（G/A/T）N（G/C/T）NNNCGGT
11	AC1	ACGATGGACTCCAGAG
12	AmGORK-pro-F	GGTACCGGCACTTTTTTTAACAAGGTGAATCAATG
13	AmGORK-pro-R	CCATGGGGCGATGCTCTTTGCCATTCTC
14	AmGORK-pro-F-375	GGTACCCTCCGATCCTATTTTGGTATATCA
15	AmGORK-pro-F-856	GGTACCCTGGGTTGCAGTCAGGATAATAC
16	AmGORK-pro-F-1245	GGTACCGCTGATTCTTGAAGAATGGAG

序号Sequence No.	引物名称Primer name	引物序列Primer sequence（5'-3'）
1	GP1-1	GCCGAATTCCATTGGGGTGAAG
2	GP1-2	ACGATGGACTCCAGTCCGGCCGTGGTGGATCCTCCAATCCAAC
3	GP1-3	CTTCCTCCTCCGAATCATCGCT
4	GP2-1	CTGGAGTCCATCGTAATCGTCGA
5	GP2-2	ACGATGGACTCCAGTCCGGCCCAACTCCAGCTTGACTCAAGCAAC
6	GPP2-3	CAACTTGAACAGGAGCTTGCTCG
7	LAD1-1	ACGATGGACTCCAGAGCGGCCGC（G/C/A）N（G/C/A）NNNGGAA
8	LAD1-2	ACGATGGACTCCAGAGCGGCCGC（G/C/T）N（G/C/T）NNNGGTT
9	LAD1-3	ACGATGGACTCCAGAGCGGCCGC（G/C/A）（G/C/A）N（G/C/A）NNNCCAA
10	LAD1-4	ACGATGGACTCCAGAGCGGCCGC（G/C/T）（G/A/T）N（G/C/T）NNNCGGT
11	AC1	ACGATGGACTCCAGAG
12	AmGORK-pro-F	GGTACCGGCACTTTTTTTAACAAGGTGAATCAATG
13	AmGORK-pro-R	CCATGGGGCGATGCTCTTTGCCATTCTC
14	AmGORK-pro-F-375	GGTACCCTCCGATCCTATTTTGGTATATCA
15	AmGORK-pro-F-856	GGTACCCTGGGTTGCAGTCAGGATAATAC
16	AmGORK-pro-F-1245	GGTACCGCTGATTCTTGAAGAATGGAG

基序名称Site name	物种Species	序列Sequence	功能Function
AAAC-motif	菠菜 Spinacia oleracea	CAATCAAAACCT	光响应元件 Light-responsive element
AACA-motif	水稻 Oryza sativa	TAACAAACTCCA	胚乳特异性表达 Involved in endosperm-specific expression
ABRE	拟南芥 A. thaliana	TACGTG	ABA响应顺式作用元件cis-acting element involved in the abscisic acid responses
CAAT-box	拟南芥 A. thaliana	GGCAAT	启动子和增强子区域共有顺式作用元件Common cis-acting element in promoter and enhancer regions
G-box	金鱼草 Antirrhinum majus	CACGTA	光反应中的顺式调节元件cis-acting regulatory element involved in light responses
GAG-motif	大麦 Hordeum vulgare	GGAGATG	光响应元件的一部分 Part of a light-responsive element
GARE-motif	甘蓝 Brassica oleracea	AAACAGA	赤霉素响应元件 Gibberellin-responsive element
GCN4	水稻 O. sativa	TGAGTCA	胚乳表达顺式调控元件 cis-regulatory element involved in endosperm expression
HSE	甘蓝 B. oleracea	AAAAAATTTC	热响应顺式作用元件 cis-acting element involved in heat stress responses
I-box	豌豆 Pisum sativum	ATGATATGA	光响应元件的一部分 Part of a light-responsive element
LAMP-element	菠菜 S. oleracea	CCTTATCCA	光响应元件的一部分 Part of a light-responsive element
LTR	大麦 H. vulgare	CCGAAA	低温响应顺式作用元件 cis-acting element involved in low-temperature responses
MBS	拟南芥 A. thaliana	TAACTG	干旱诱导MYB结合位点 MYB binding site involved in drought-inducibility
MRE	荷兰芹 Petroselinum crispum	AACCTAA	MYB结合位点参与光响应 MYB binding site involved in light responses
O2-site	玉米 Zea mays	GATGACATGG	玉米蛋白代谢调控顺式作用元件 cis-acting regulatory element involved in zein metabolism regulation
Skn-1 motif	水稻 Oryza sativa	GTCAT	胚乳表达必需的顺式作用元件 cis-acting regulatory element required for endosperm expression
Sp1	玉米 Zea mays	CC（G/A）CCC	光响应元件 Light-responsive element
TATA-box	拟南芥 A. thaliana	TATA	在转录开始前30 bp核心启动子元件 Core promoter element around -30 bp of transcription start
TC-rich repeats	烟草 Nicotiana tabacum	ATTTTCTCCA	防御及胁迫响应顺式作用元件 cis-acting element involved in defense and stress responses
TCT-motif	拟南芥 A.s thaliana	TCTTAC	光响应元件的一部分 Part of a light-responsive element
circadian	番茄 Lycopersicon esculentum	CAANNNNATC	昼夜节律控制顺式作用元件 cis-acting regulatory element involved in circadian control
rbcS-CMA7a	膨胀浮萍 Lemna gibba	GTCGATAAGG	光响应元件的一部分 Part of a light-responsive element

基序名称Site name	物种Species	序列Sequence	功能Function
AAAC-motif	菠菜 Spinacia oleracea	CAATCAAAACCT	光响应元件 Light-responsive element
AACA-motif	水稻 Oryza sativa	TAACAAACTCCA	胚乳特异性表达 Involved in endosperm-specific expression
ABRE	拟南芥 A. thaliana	TACGTG	ABA响应顺式作用元件cis-acting element involved in the abscisic acid responses
CAAT-box	拟南芥 A. thaliana	GGCAAT	启动子和增强子区域共有顺式作用元件Common cis-acting element in promoter and enhancer regions
G-box	金鱼草 Antirrhinum majus	CACGTA	光反应中的顺式调节元件cis-acting regulatory element involved in light responses
GAG-motif	大麦 Hordeum vulgare	GGAGATG	光响应元件的一部分 Part of a light-responsive element
GARE-motif	甘蓝 Brassica oleracea	AAACAGA	赤霉素响应元件 Gibberellin-responsive element
GCN4	水稻 O. sativa	TGAGTCA	胚乳表达顺式调控元件 cis-regulatory element involved in endosperm expression
HSE	甘蓝 B. oleracea	AAAAAATTTC	热响应顺式作用元件 cis-acting element involved in heat stress responses
I-box	豌豆 Pisum sativum	ATGATATGA	光响应元件的一部分 Part of a light-responsive element
LAMP-element	菠菜 S. oleracea	CCTTATCCA	光响应元件的一部分 Part of a light-responsive element
LTR	大麦 H. vulgare	CCGAAA	低温响应顺式作用元件 cis-acting element involved in low-temperature responses
MBS	拟南芥 A. thaliana	TAACTG	干旱诱导MYB结合位点 MYB binding site involved in drought-inducibility
MRE	荷兰芹 Petroselinum crispum	AACCTAA	MYB结合位点参与光响应 MYB binding site involved in light responses
O2-site	玉米 Zea mays	GATGACATGG	玉米蛋白代谢调控顺式作用元件 cis-acting regulatory element involved in zein metabolism regulation
Skn-1 motif	水稻 Oryza sativa	GTCAT	胚乳表达必需的顺式作用元件 cis-acting regulatory element required for endosperm expression
Sp1	玉米 Zea mays	CC（G/A）CCC	光响应元件 Light-responsive element
TATA-box	拟南芥 A. thaliana	TATA	在转录开始前30 bp核心启动子元件 Core promoter element around -30 bp of transcription start
TC-rich repeats	烟草 Nicotiana tabacum	ATTTTCTCCA	防御及胁迫响应顺式作用元件 cis-acting element involved in defense and stress responses
TCT-motif	拟南芥 A.s thaliana	TCTTAC	光响应元件的一部分 Part of a light-responsive element
circadian	番茄 Lycopersicon esculentum	CAANNNNATC	昼夜节律控制顺式作用元件 cis-acting regulatory element involved in circadian control
rbcS-CMA7a	膨胀浮萍 Lemna gibba	GTCGATAAGG	光响应元件的一部分 Part of a light-responsive element