拟南芥AtiPGAM2基因参与非生物胁迫的响应

doi:10.13560/j.cnki.biotech.bull.1985.2023-1139

摘要/Abstract

摘要：

【目的】AtiPGAM2 基因是拟南芥（Arabidopsis thaliana）碱性磷酸酶超家族的成员，参与糖酵解过程。研究AtiPGAM2基因在逆境胁迫下的响应机制，为进一步研究AtiPGAM2的抗逆功能奠定基础。【方法】使用PlantCARE在线软件对AtiPGAM2基因启动子顺式作用元件进行分析。采用实时荧光定量PCR，检测AtiPGAM2在NaCl和ABA胁迫下的响应模式。克隆AtiPGAM2基因转入拟南芥，获得AtiPGAM2基因超表达株系。利用三引物法鉴定Atipgam2突变体。对过表达、突变体和野生型在盐胁迫下的萌发率、绿苗率以及各种非生物胁迫（甘露醇、ABA和MeJA）下的表型，进行统计分析。【结果】其启动子上存在多个光、MeJA、低温、ABA、SA、GAs等非生物胁迫和激素响应元件。荧光定量PCR分析显示，AtiPGAM2在NaCl和ABA胁迫下受到不同程度的诱导。成功获得AtiPGAM2基因过表达和突变体株系。在盐胁迫条件下AtiPGAM2过表达株系萌发率以及绿苗率均高于野生型，突变体表型则相反。甘露醇处理下突变体的萌发率低于野生型。ABA处理48 h内突变体和过表达AtiPGAM2株系的萌发率都低于野生型。MeJA处理下过表达的侧根数量高于野生型，突变体则相反。【结论】AtiPGAM2具有耐盐性，该基因响应甘露醇、ABA 和MeJA处理。

关键词: AtiPGAM2, 拟南芥, 盐胁迫, 非生物胁迫, 甘露醇, ABA, MeJA

Abstract:

【Objective】The AtiPGAM2 gene is a member of the alkaline phosphatase superfamily in Arabidopsis and is involved in the glycolysis process. Studying the response mechanism of AtiPGAM2 gene under stress conditions lays the foundation for further studying the stress resistance function of AtiPGAM2.【Method】This study used PlantCARE online software to analyze the cis-acting elements of the AtiPGAM2 gene promoter. Real time fluorescence quantitative PCR was used to detect the response patterns of AtiPGAM2 under NaCl and ABA stress. The AtiPGAM2 gene was cloned and transferred it into Arabidopsis to obtain an overexpressing strain of the AtiPGAM2 gene. The Atipgam2 mutant was identified using the three primer method. Statistical analysis was conducted on the germination rate, green seedling rate, and phenotypes under various abiotic stresses, including mannitol, ABA, and MeJA, for overexpression, mutants, and wild-type under salt stress.【Result】There are multiple abotic stress and hormone responsive elements on its promoter, such as light, MeJA, low temperature, ABA, SA, GAs, etc. Fluorescence quantitative PCR analysis showed that AtiPGAM2 was induced to varying degrees under NaCl and ABA stress. AtiPGAM2 gene overexpression and mutant strains were obtained successfully. Under salt stress, the germination rate and green seedling rate of AtiPGAM2 overexpressing strains were higher than those of the wild type, while the mutant surface type was the opposite. The germination rate of the mutant under mannitol treatment was lower than that of the wild-type. The germination rates of mutant and overexpressing AtiPGAM2 strains treated with ABA within 48 h were lower than those of the wild-type. Under MeJA treatment, the number of overexpressed lateral roots was higher than that of the wild-type, while the mutant was the opposite.【Conclusion】AtiPGAM2 has the tolerance to salt, and this gene responds to mannitol, ABA, and MeJA treatments.

Key words: AtiPGAM2, Arabidopsis, salt stress, abiotic, mannitol, ABA, MeJA

李景艳, 周家婧, 袁媛, 苏晓艺, 乔文慧, 薛岩磊, 李国婧, 王瑞刚. 拟南芥AtiPGAM2基因参与非生物胁迫的响应[J]. 生物技术通报, 2024, 40(5): 215-224.

LI Jing-yan, ZHOU Jia-jing, YUAN Yuan, SU Xiao-yi, QIAO Wen-hui, XUE Yan-lei, LI Guo-jing, WANG Rui-gang. Response of Arabidopsis AtiPGAM2 Gene to Abiotic Stress[J]. Biotechnology Bulletin, 2024, 40(5): 215-224.

图/表 11

表1 本研究中涉及的引物

Table 1 Primers used in this study

引物名称Primer name	序列Sequence（5'-3'）	引物用途Primer usage
AtiPGAM2-HA-F	TACTTCCAATCCAATGCCATGGGTAGCTCCGGCG	基因克隆
AtiPGAM2-HA-R	TTATCCACTTCCAATGCTACTTCTCGACGACTTCGATCAG	Gene clone
AtiPGAM2-qRT-F	TGCGTGTAAACCTGCCAAAT	荧光定量PCR
AtiPGAM2-qRT-R	GTATATCCCTCCGACCTGTTCTAT	RT-qPCR
AtEF1α-F	AGAAGGGTGCCAAATGATGAG	荧光定量PCR
AtEF1α-R	GGAGGGAGAGAGAAAGTCACAGA	RT-qPCR
salk_119825C-LP	TACCGAACCAGATCAATTTGC	突变体鉴定
salk_119825C-RP	TCCTTGATGCCATAGAACAGG	Mutant identification

图1 AtiPGAM2基因ORF的克隆（A）及35S::HA-AtiP-GAM2重组质粒（B）酶切鉴定电泳 A：泳道M为DL 2000 DNA marker；1：AtiPGAM2基因ORF扩增产物；B：M：DL2000 DNA marker；1：Sal I单酶切重组质粒；2：空载体对照

Fig. 1 Gel electrophoresis of the PCR products of AtiP-GAM2 ORF cloned（A）and identification of 35S:: AtiPGAM2-HA recombinant vector（B） A: M: DL2000 DNA marker; 1: PCR products of the AtiPGAM2 ORF; B: M: DL2000 DNA marker; 1: recombinant vector digested with Sal I; 2: vector control

表2 AtiPGAM2启动子中的顺式作用元件

Table 2 Cis-acting elements in AtiPGAM2 promoter

顺式作用元件名称 Name of cis-acting element	顺式作用元件功能 Function of cis-acting element	序列 Sequence（5'-3'）	顺式作用元件数量 Number of cis-acting elements
TGACG-motif	MeJA反应相关	TGACG	4
CGTCA-motif	MeJA反应相关	CGTCA	4
G-box	光反应相关	CACGTC	3
G-box	光反应相关	CACGTT	1
ABRE	脱落酸反应相关	ACGTG	4
TCA-element	水杨酸反应相关	TCAGAAGAGG	1
TCA-element	水杨酸反应相关	CCATCTTTTT	1
TCCC-motif	光反应相关	TCTCCCT	1
P-box	赤霉素相关	CCTTTTG	2
I-box	光反应相关	CCTTATCCT	1
GARE-motif	赤霉素响应元件	TCTGTTG	1
GT1-motif	光响应元件	GGTTAA	1
GATA-motif	光反应相关	GATAGGA	1
GATA-motif	光反应相关	AAGGATAAGG	1
LTR	低温响应元件	CCGAAA	1
TCT-motif	光响应元件	TCTTAC	2

图2 AtiPGAM2的亚细胞定位结果

Fig. 2 Subcellular localization results of AtiPGAM2

图3 转基因株系中AtiPGAM2基因的转录水平检测图中误差线表示标准偏差。下同

Fig. 3 Expressions of AtiPGAM2 in transgenic Arabidopsis lines The error line in the figure refers to the standard deviation. The same below

图4 突变体Atipgam2的T-DNA插入位点以及表达水平鉴定 A：突变体salk_119825C的T-DNA插入位点；B：突变体salk_119825C的PCR鉴定（M：DL 2000 marker；1-4：Atipgam2突变体个体编号；1-4左侧泳道：LP+RP扩增产物；1-4右侧泳道：BP+RP扩增产物）；C：AtiPGAM2基因表达水平的RT-qPCR的检测

Fig. 4 T-DNA insertion site in mutant Atipgam2 and identification of its expression A: T-DNA insertion site of salk_119825C mutant ; B: PCR identification of salk_119825C mutant（M: DL 2000 marker; 1-4: the number of Atipgam2 mutant; 1-4 left swimming lane: PCR products of LP+RP; 1-4 right swimming lane: PCR products of BP+ RP）; C: detection of AtiPGAM2 gene expression levels by RT qPCR

图5 盐和ABA处理下AtiPGAM2转录水平的变化 A：盐胁迫处理下RD29A基因转录水平的变化；B：盐胁迫处理下AtiPGAM2基因转录水平的变化；C：ABA处理下ABI1基因转录水平的变化；D：ABA处理下AtiPGAM2基因转录水平的变化。*P<0.05，** P<0.01，*** P<0.001，ns表示不显著。下同

Fig. 5 Transcriptional changes of AtiPGAM2 under salt stress and ABA A: Transcription level change of RD29A under salt stress. B: Transcription level change of AtiPGAM2 under salt stress. C: Transcription level change of ABI1 under ABA. D: Transcription level change of AtiPGAM2 under ABA. *P<0.05,** P<0.01, *** P<0.001, ns refers to insignificant. The same below

图6 NaCl对野生型、AtiPGAM2过表达纯合体和Atipgam2突变体萌发率和绿苗率的影响 A, C：对照条件下3种基因型拟南芥种子萌发7 d的表型（A）和萌发率统计结果（C）；B, D：200 mmol/L NaCl处理条件下3种基因型拟南芥种子萌发7 d的表型（B）和萌发率统计结果（D）； E：对照条件下3种基因型拟南芥种子萌发10 d的绿苗率表型；F：150 mmol/L NaCl处理下3种基因型拟南芥种子萌发10 d的绿苗率表型；G：对照和150 mmol/L NaCl处理下3种基因型拟南芥种子萌发10 d的绿苗率统计结果

Fig. 6 Effects of NaCl on the germination rate and green seedling rate of wild-type, AtiPGAM2 overexpressed homozygotes and Atipgam2 mutant A, C: Phenotypes（A）and germination rate（C）of Arabidopsis seeds of three genotypes under control conditions after 7 d of germination. B, D: Phenotypes（B）and germination rate（D）of Arabidopsis seeds of three genotypes under 200 mmol/L NaCl treatment after 7 d of germination. E: Phenotypes of green seedling rates of three Arabidopsis genotypes under control after 10 d of germination. F: Phenotypes of green seedling rates of three Arabidopsis genotypes under 150 mmol/L NaCl treatment after 10 d of germination. G: Statistical results of the green seedling rates of Arabidopsis of three genotypes under control and 150 mmol/L NaCl treatment after 10 d of germination

图7 甘露醇对野生型、AtiPGAM2过表达纯合体和Atipgam2突变体萌发率的影响 A, C：对照条件下3种基因型拟南芥种子萌发7 d的表型（A）和萌发率统计结果（C）；B, D：400 mmol/L甘露醇处理条件下3种基因型拟南芥种子萌发7 d的表型（B）和萌发率统计结果（D）

Fig. 7 Effects of mannitol on the germination rate of wild-type, AtiPGAM2 overexpressed homozygotes and Atipgam2 mutant A, C: Phenotypes（A）and germination rate（C）of Arabidopsis seeds of three genotypes under control conditions after 7 d of germination. B, D: Phenotypes（B）and germination rate（D）of Arabidopsis seeds of three genotypes under 400 mmol/L mannitol treatment after 7 d of germination

图8 ABA对野生型、AtiPGAM2过表达纯合体和Atipgam2突变体萌发率的影响 A, D：对照条件下3种基因型拟南芥种子萌发7 d的表型（A）和萌发率统计结果（D）；B, E：1 μmol/L ABA处理条件下3种基因型拟南芥种子萌发7 d的表型（B）和萌发率统计结果（E）；C, F：3 μmol/L ABA处理条件下3种基因型拟南芥种子萌发7 d的表型（C）和萌发率统计结果（F）

Fig. 8 Effects of ABA on the germination rate of wild-type, AtiPGAM2 overexpressed homozygotes and Atipgam2 mutant A, D: Phenotypes（A）and germination rate（D）of Arabidopsis seeds of three genotypes under control conditions after 7 d of germination. B, E: Phenotypes（B）and germination rate（E）of Arabidopsis seeds of three genotypes under 1 μmol /L ABA treatment after 7 d of germination. C, F: Phenotypes（C）and germination rate（F）of Arabidopsis seeds of three genotypes under 3 μmol/L ABA treatment after 7 d of germination

图9 MeJA对野生型、AtiPGAM2过表达纯合体和Atipgam2突变体侧根数量的影响 A：对照条件下3种基因型拟南芥侧根数量的表型；B：75 μmol/L MeJA处理下3种基因型拟南芥侧根数量的表型；C：对照和75 μmol/L MeJA处理3种基因型拟南芥平均侧根数量的统计结果

Fig. 9 Effects of MeJA on the number of lateral roots in wild-type, AtiPGAM2 overexpressed homozygotes, and Atipgam2 mutants A: Phenotypes of lateral root number of three Arabidopsis genotypes under control. B: Phenotypes of lateral root number of three Arabidopsis genotypes under 75 μmol/L MeJA treatment. C: Statistical results of lateral root average number of Arabidopsis of three genotypes under control and 75 μmol/L MeJA treatment

参考文献 25

[1]	Okuda J, Niizuma S, Shioi T, et al. Persistent overexpression of phosphoglycerate mutase, a glycolytic enzyme, modifies energy metabolism and reduces stress resistance of heart in mice[J]. PLoS One, 2013, 8(8): e72173.
[2]	Fothergill-Gilmore LA, Watson HC. The phosphoglycerate mutases[J]. Adv Enzymol Relat Areas Mol Biol, 1989, 62: 227-313. pmid: 2543188
[3]	Jedrzejas MJ. Structure, function, and evolution of phosphoglycerate mutases: comparison with fructose-2, 6-bisphosphatase, acid phosphatase, and alkaline phosphatase[J]. Prog Biophys Mol Biol, 2000, 73(2/3/4): 263-287.
[4]	Poonperm B, Guerra DG, McNae IW, et al. Expression, purification, crystallization and preliminary crystallographic analysis of Leishmania mexicana phosphoglycerate mutase[J]. Acta Crystallogr D Biol Crystallogr, 2003, 59(Pt 7): 1313-1316.
[5]	Wang YL, Wei ZY, Bian Q, et al. Crystal structure of human bisphosphoglycerate mutase[J]. J Biol Chem, 2004, 279(37): 39132-39138. doi: 10.1074/jbc.M405982200 pmid: 15258155
[6]	谢鑫, 刘娜, 魏凤菊. 植物iPGAM的研究进展[J]. 生物技术通报, 2015, 31(9): 1-7. doi: 10.13560/j.cnki.biotech.bull.1985.2015.09.001
	Xie X, Liu N, Wei FJ. Research progress on cofactor-independent phosphoglycerate mutase in plants[J]. Biotechnol Bull, 2015, 31(9): 1-7. doi: 10.13560/j.cnki.biotech.bull.1985.2015.09.001
[7]	Graña X, Pérez de la Ossa P, Broceño C, et al. 2, 3-Bisphosphoglycerate-independent phosphoglycerate mutase is conserved among different phylogenic Kingdoms[J]. Comp Biochem Physiol B Biochem Mol Biol, 1995, 112(2): 287-293.
[8]	Bond CS, Clements PR, Ashby SJ, et al. Structure of a human lysosomal sulfatase[J]. Structure, 1997, 5(2): 277-289. doi: 10.1016/s0969-2126(97)00185-8 pmid: 9032078
[9]	Andriotis VME, Kruger NJ, Pike MJ, et al. Plastidial glycolysis in developing Arabidopsis embryos[J]. New Phytol, 2010, 185(3): 649-662. doi: 10.1111/j.1469-8137.2009.03113.x pmid: 20002588
[10]	Graña X, Ureña J, Ludevid D, et al. Purification, characterization and immunological properties of 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase from maize(Zea mays)seeds[J]. Eur J Biochem, 1989, 186(1/2): 149-153.
[11]	Wang JL, Walling LL, Jauh GY, et al. Lily cofactor-independent phosphoglycerate mutase: purification, partial sequencing, and immunolocalization[J]. Planta, 1996, 200(3): 343-352. pmid: 8931352
[12]	Westram A, Lloyd JR, Roessner U, et al. Increases of 3-phosphoglyceric acid in potato plants through antisense reduction of cytoplasmic phosphoglycerate mutase impairs photosynthesis and growth, but does not increase starch contents[J]. Plant Cell Environ, 2002, 25(9): 1133-1143.
[13]	Lin D, Zhang L, Mei J, et al. Mutation of the rice TCM12 gene encoding 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase affects chlorophyll synthesis, photosynthesis and chloroplast development at seedling stage at low temperatures[J]. Plant Biol, 2019, 21(4): 585-594.
[14]	刘朋虎, 邓优锦, 江玉姬, 等. 草菇PGAM基因克隆、结构及其在同核、异核菌株中的表达量分析[J]. 福建农业学报, 2012, 27(3): 252-256.
	Liu PH, Deng YJ, Jiang YJ, et al. Cloning and structural analysis of phosphoglycerate mutas gene and its expression levels in homokaryon and heterokaryon of Volvariella volvacea[J]. Fujian J Agric Sci, 2012, 27(3): 252-256.
[15]	李春明, 白卉, 于文喜. 低温驯化过程中大青杨叶片差异蛋白质分析[J]. 东北林业大学学报, 2011, 39(10): 45-49.
	Li CM, Bai H, Yu WX. Differential protein in Populus ussuriensis leaves during natural low-temperature acclimation by proteomic analysis[J]. J Northeast For Univ, 2011, 39(10): 45-49.
[16]	Huang Y, Blakeley SD, McAleese SM, et al. Higher-plant cofactor-independent phosphoglyceromutase: purification, molecular characterization and expression[J]. Plant Mol Biol, 1993, 23(5): 1039-1053. doi: 10.1007/BF00021818 pmid: 8260624
[17]	Hajduch M, Casteel JE, Hurrelmeyer KE, et al. Proteomic analysis of seed filling in Brassica napus. Developmental characterization of metabolic isozymes using high-resolution two-dimensional gel electrophoresis[J]. Plant Physiol, 2006, 141(1): 32-46. pmid: 16543413
[18]	龙翔宇, 董绪浓, 等. 橡胶树PGAM基因的克隆及表达特性分析[J]. 热带作物学报, 2013, 34(10): 1895-1901.
	Long XY, Dong XN, et al. Molecular cloning and expression analysis of HbPGAM from Hevea brasiliensis(para rubber tree)[J]. Chin J Trop Crops, 2013, 34(10): 1895-1901.
[19]	Stein M, Gabdoulline RR, Wade RC. Cross-species analysis of the glycolytic pathway by comparison of molecular interaction fields[J]. Mol Biosyst, 2010, 6(1): 152-164. doi: 10.1039/b912398a pmid: 20024078
[20]	Zhao ZX, Assmann SM. The glycolytic enzyme, phosphoglycerate mutase, has critical roles in stomatal movement, vegetative growth, and pollen production in Arabidopsis thaliana[J]. J Exp Bot, 2011, 62(14): 5179-5189.
[21]	Amme S, Matros A, Schlesier B, et al. Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology[J]. J Exp Bot, 2006, 57(7): 1537-1546.
[22]	红格日其其格. 盐胁迫下拟南芥角果的蛋白质组和Khib修饰组学分析及AtATPase-β亚基的功能研究[D]. 呼和浩特: 内蒙古农业大学, 2021.
	Hong G. Proteomic and Khib modification analysis of Arabidopsis thaliana siliques under salt stress and function study on ATATPase-β subunit[D]. Hohhot: Inner Mongolia Agricultural University, 2021.
[23]	Zhang XJ, Yang GY, et al. Arabidopsis cysteine-rich receptor-like kinase 45 functions in the responses to abscisic acid and abiotic stresses[J]. Plant Physiol Biochem, 2013, 67: 189-198.
[24]	Karimi R, Gavili-Kilaneh K, Khadivi A. Methyl jasmonate promotes salinity adaptation responses in two grapevine(Vitis vinifera L.) cultivars differing in salt tolerance[J]. Food Chem, 2022, 375: 131667.
[25]	刘丽婧. 烟草糖酵解途径iPGAM基因的鉴定和功能研究[D]. 重庆: 西南大学, 2020.
	Liu LJ. Identification and functional study of NtiPGAM involved in glycolytic pathway in tobacco[D]. Chongqing: Southwest University, 2020.