Response of Arabidopsis AtiPGAM2 Gene to Abiotic Stress

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

Abstract

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

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.

Figures/Tables 11

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

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

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

Fig. 2 Subcellular localization results of AtiPGAM2

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

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

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

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

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

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

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

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