Cloning and Expression Analysis of Outward Potassium Ion Channel Gene AmGORK Promoter from Ammopiptanthus mongolicus

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

Abstract

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

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.

Figures/Tables 7

References 29

[1]	马倩, 马宝月, 穆波, 等. 植物基因启动子的克隆及分析的研究进展[J]. 中国农业文摘-农业工程, 2018, 30(3):23-29.
	Ma Q, Ma BY, Mu B, et al. Research progress on cloning and analysis of plant gene promoter[J]. Agric Sci Eng China, 2018, 30(3):23-29.
[2]	杨鹏芳, 段国琴, 胡晓炜, 等. 高等植物启动子研究概述[J]. 分子植物育种, 2018, 16(5):1482-1493.
	Yang PF, Duan GQ, Hu XW, et al. Overview of higher plant promoters research[J]. Mol Plant Breed, 2018, 16(5):1482-1493.
[3]	Maheshwari P, Assmann SM, Albert R. A guard cell abscisic acid(ABA)network model that captures the stomatal resting state[J]. Front Physiol, 2020, 11:927. doi: 10.3389/fphys.2020.00927 pmid: 32903539
[4]	Kuromori T, Seo M, Shinozaki K. ABA transport and plant water stress responses[J]. Trends Plant Sci, 2018, 23(6):513-522. doi: S1360-1385(18)30085-2 pmid: 29731225
[5]	Hosy E, Vavasseur A, Mouline K, et al. The Arabidopsis outward K⁺channel GORK is involved in regulation of stomatal movements and plant transpiration[J]. PNAS, 2003, 100(9):5549-5554. doi: 10.1073/pnas.0733970100 URL
[6]	Ache P, Becker D, Ivashikina N, et al. GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K +-selective, K⁺-sensing ion channel[J]. FEBS Lett, 2000, 486(2):93-98. pmid: 11113445
[7]	Eisenach C, Papanatsiou M, Hillert EK, et al. Clustering of the K⁺channel GORK of Arabidopsis parallels its gating by extracellular K⁺[J]. Plant J, 2014, 78(2):203-214. doi: 10.1111/tpj.12471 URL
[8]	Becker D, Hoth S, Ache P, et al. Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress[J]. FEBS Lett, 2003, 554(1/2):119-126. doi: 10.1016/S0014-5793(03)01118-9 URL
[9]	Ooi A, Lemtiri-Chlieh F, Wong A, et al. Direct modulation of the guard cell outward-rectifying potassium channel(GORK)by abscisic acid[J]. Mol Plant, 2017, 10(11):1469-1472. doi: 10.1016/j.molp.2017.08.010 URL
[10]	Lim CW, Kim SH, Choi HW, et al. The shaker type potassium channel, GORK, regulates abscisic acid signaling in Arabidopsis[J]. Plant Pathol J, 2019, 35(6):684-691. doi: 10.5423/PPJ.OA.07.2019.0199 URL
[11]	Adem GD, Chen G, Shabala L, et al. GORK channel:a master switch of plant metabolism?[J]. Trends Plant Sci, 2020, 25(5):434-445. doi: 10.1016/j.tplants.2019.12.012 URL
[12]	Li J, Zhang H, Lei H, et al. Functional identification of a GORK potassium channel from the ancient desert shrub Ammopiptanthus mongolicus(Maxim. )Cheng F[J]. Plant Cell Rep, 2016, 35(4):803-815. doi: 10.1007/s00299-015-1922-6 URL
[13]	段义忠, 杜忠毓, 亢福仁. 西北干旱区孑遗濒危植物蒙古沙冬青群落特征及与环境因子的关系[J]. 植物研究, 2018, 38(6):834-842.
	Duan YZ, Du ZY, Kang FR. Community characteristics of endangered plant of Ammopiptanthus mongolicus to environmental factors in northwest arid area of China[J]. Bull Bot Res, 2018, 38(6):834-842.
[14]	马惠成, 李小伟, 杨君珑, 等. 蒙古沙冬青群落区系组成及分类研究[J]. 西北植物学报, 2020, 40(4):706-716.
	Ma HC, Li XW, Yang JL, et al. Study on the community classification and floristic composition of Ammopiptanthus mongolicus[J]. Acta Bot Boreali Occidentalia Sin, 2020, 40(4):706-716.
[15]	麦尔哈巴·阿布拉, 刘博, 李征珍, 等. 蒙古沙冬青群落组成与结构研究[J]. 中央民族大学学报:自然科学版, 2019, 28(2):12-16.
	Merhaba ABLA, Liu B, Li ZZ, et al. Composition and structure of community of Ammopiptanthus mongolicus in alasan left banner, Inner Mongolia[J]. J Minzu Univ China:Nat Sci Ed, 2019, 28(2):12-16.
[16]	Liu YG, Chen Y. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. Biotechniques, 2007, 43(5):649-50, 652, 654 passim. doi: 10.2144/000112601 URL
[17]	杜传慧, 付艳, 王利敏, 等. 苹果异戊烯基转移酶基因启动子调控特性研究[J]. 核农学报, 2019, 33(3):455-463.
	Du CH, Fu Y, Wang LM, et al. Regulatory characteristics of the apple isopentenyl transferase gene promoter[J]. J Nucl Agric Sci, 2019, 33(3):455-463.
[18]	Han L, Li J, Jin M, et al. Functional analysis of a type 2C protein phosphatase gene from Ammopiptanthus mongolicus[J]. Gene, 2018, 653:29-42. doi: 10.1016/j.gene.2018.02.015 URL
[19]	杨瑞娟, 白建荣, 李锐, 等. 诱导型启动子在植物基因工程中的研究进展[J]. 山西农业科学, 2018, 46(2):292-298.
	Yang RJ, Bai JR, Li R, et al. Research progress of the inducible promoters in plant genetic engineering[J]. J Shanxi Agric Sci, 2018, 46(2):292-298.
[20]	田晨菲, 李建华, 王勇. 植物合成生物学调控元件的研究进展[J]. 植物生理学报, 2020, 56(11):2261-2274.
	Tian CF, Li JH, Wang Y. Research advances of regulatory elements in plant synthetic biology[J]. Plant Physiol J, 2020, 56(11):2261-2274.
[21]	王海波, 郭俊云. 小桐子低温诱导型启动子JcDnaJ20p的克隆及烟草转化功能鉴定[J]. 生物技术通报, 2021, 37(2):24-31.
	Wang HB, Guo JY. Molecular cloning of cold-induced JcDnaJ20p promoter from Jatropha curcas and its functional identification in transgenic tobacco[J]. Biotechnol Bull, 2021, 37(2):24-31.
[22]	周涛, 王娟, 胡佳蕙, 等. 番茄转录因子基因SlWRKY6的克隆与原核表达分析[J]. 西北植物学报, 2020, 40(11):1824-1832.
	Zhou T, Wang J, Hu JH, et al. Cloning and prokaryotic expression analysis of a WRKY transcription factor gene SlWRKY6 in Solanum lycopersicum[J]. Acta Bot Boreali Occidentalia Sin, 2020, 40(11):1824-1832.
[23]	Ma YL, Cao J, He JH, et al. Molecular mechanism for the regulation of ABA homeostasis during plant development and stress responses[J]. Int J Mol Sci, 2018, 19(11):E3643.
[24]	Klessig DF, Choi HW, Dempsey DA. Systemic acquired resistance and salicylic acid:past, present, and future[J]. Mol Plant Microbe Interact, 2018, 31(9):871-888. doi: 10.1094/MPMI-03-18-0067-CR URL
[25]	Prodhan MY, Munemasa S, Nahar MN, et al. Guard cell salicylic acid signaling is integrated into abscisic acid signaling via the Ca²⁺/CPK-dependent pathway[J]. Plant Physiol, 2018, 178(1):441-450. doi: 10.1104/pp.18.00321 pmid: 30037808
[26]	Gaymard F, Pilot G, Lacombe B, et al. Identification and disruption of a plant shaker-like outward channel involved in K⁺ release into the xylem Sap[J]. Cell, 1998, 94(5):647-655. pmid: 9741629
[27]	Plesch G, Ehrhardt T, Mueller-Roeber B. Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression[J]. Plant J, 2001, 28(4):455-464. pmid: 11737782
[28]	Corratgé-Faillie C, Ronzier E, Sanchez F, et al. The Arabidopsis guard cell outward potassium channel GORK is regulated by CPK33[J]. FEBS Lett, 2017, 591(13):1982-1992. doi: 10.1002/1873-3468.12687 pmid: 28543075
[29]	Hedrich R, Geiger D. Biology of SLAC1-type anion channels-from nutrient uptake to stomatal closure[J]. New Phytol, 2017, 216(1):46-61. doi: 10.1111/nph.14685 pmid: 28722226

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