Functional Analysis of Soybean GmPP2C89 Gene under Salt Stress

doi:10.7668/hbnxb.20193021

Abstract

Abstract:

To investigate the function of soybean GmPP2C89 gene in plant abiotic stress response and adaptation. The expression patterns of GmPP2C89 under NaCl,PEG and mannitol treatments were detected by transcriptome data and Real-time quantitative PCR. Then,the cis-acting elements on the promoter of GmPP2C89 in response to abiotic stress were analyzed,and promoters of different lengths were cloned according to the distribution of cis-elements to construct fusion GUS vectors to obtain the corresponding transgenic Arabidopsis. The response of the promoters to NaCl,PEG and mannitol was analyzed by GUS staining. Transgenic Arabidopsis overexpressing the GmPP2C89 was constructed,and the root length,leaf MDA content and electrolytic leakage,and the expression of salt stress-related genes(SOD,POD,CAT,RD26,RD29A,and RD29B)were measured under normal and NaCl treatment conditions. The results showed that NaCl,PEG and mannitol treatments all led to a significant increase in the expression level of soybean GmPP2C89;the promoter region contained many cis-acting elements such as ABRE,DRE,G-box,MBS,MYB,MYC and TC-rich repeats which were involved in abiotic stress response,and this promoter was more responsive to NaCl treatment. In addition,under the salt treatment,the root length of transgenic Arabidopsis GmPP2C89-OX was significantly greater than that of WT,while the MDA content and electrolytic leakage were significantly lower than those of WT,and the salt tolerance was significantly enhanced;the expression of antioxidant enzyme genes(SOD and POD)and ABA pathway key gene RD29B in GmPP2C89-OX was significantly higher than that in WT. These results indicated that soybean GmPP2C89 was induced by NaCl,PEG and mannitol,and GmPP2C89 overexpression could enhance the salt tolerance of transgenic Arabidopsis by activating antioxidant and ABA pathways.

Key words: Soybean, GmPP2C89 gene, Promoter, cis-acting element, Salt tolerance

ZHANG Bin. Functional Analysis of Soybean GmPP2C89 Gene under Salt Stress[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 20-27. doi: 10.7668/hbnxb.20193021.

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Figures/Tables 7

Tab.1 Primers used in this study

名称 Name	序列(5'-3') Sequences (5'-3')	目的 Purpose
q-GmPP2C89F	GATAGGGTTTGCTGCGAAGTG	基因表达检测
q-GmPP2C89R	CTCCGCCACCAGCCTGTA
q-AtSODF	TGGAACTGCCACCTTCACAA
q-AtSODR	TTCCGAGGTCATCAGGGTCT
q-AtPODF	TAACCAACCAGACGAGACGC
q-AtPODR	TCGAATCTTGCAGGAGAGGC
q-AtCATF	CACCCTGAAAGTTTGAACATGTTCAC
q-AtCATR	TGAGCTTTGCCAGCTTTGTTG
q-AtRD26F	AGGTCTTAATCCAATTCCAG
q-AtRD26R	GAGTTCTGCTGCCGATTC
q-AtRD29AF	CACAATCACTTGGCTCCACTGTTG
q-AtRD29AR	ACCTAGTAGCTGGTATGGAGGAACT
q-AtRD29BF	GTTGAAGAGTCTCCACAATCACTTG
q-AtRD29BR	ATACAAAAACACAAACATTCAAAAG
q-AtACTINF	CAACTATGTTCTCAGGTATTGCAGA	内参基因
q-AtACTINR	GTCATGGAAACGATGTCTCTTTAGT
q-GmUBI3F	GTGTAATGTTGGATGTGTTCCC
q-GmUBI3R	ACACAATTGAGTTCAACACAAACCG
P1-2000F	TGCCTGCAGGTCGACTCTAGATACTCTCTGGTAATCGAATACA	不同长度启动子克隆
P2-1370F	TGCCTGCAGGTCGACTCTAGAAGGGTGTGGCCGAACCAGT
P3-980F	TGCCTGCAGGTCGACTCTAGATATGTCTTTTTGATGGAAACTC
P4-240F	TGCCTGCAGGTCGACTCTAGACCCTCAGTAGAATCCAACTCA
ProR	ACCACCCGGGGATCCTCTAGAAAACACAACAGAACCACAGAAA

Fig.1 The expression level of GmPP2C89 gene under abiotic stress A.FPKM value of GmPP2C89 gene in soybean transcriptome data treated with salt;B.Expression level of GmPP2C89 gene in soybean treated with 20% PEG; C.Expression level of GmPP2C89 gene in soybean treated with 300 mmol/L mannitol.;Different letters indicate significant differences(P<0.05).The same as Fig.5,6.

Fig.2 Distribution of abiotic stress response cis-elements in the promoter of GmPP2C89 gene

Fig.3 Cloning of GmPP2C89 gene promoters with different lengths and GUS vector construction A.Promoters of different lengths and cis-element positions;B.Promoter fragment cloning electrophoresis diagram; C.Vector construction successful digestion electrophoresis diagram.

Fig.4 Screening and identification of transgenic Arabidopsis and GUS staining analysis under different stress treatments A-D. Screening and identification of Arabidopsis transformed with promoters with different lengths fused with GUS vector;E. GUS staining treated with 150 mmol/L NaCl,20% PEG and 300 mmol/L mannitol.

Fig.5 Overexpression of soybean GmPP2C89 gene enhanced salt tolerance of transgenic Arabidopsis A-B.Root length phenotype and statistics on MS medium and MS medium with 100 mmol/L NaCl;C-E.Phenotype and MDA content, electrolytic leakage of Arabidopsis thaliana treated with 150 mmol/L NaCl for 10 d.

Fig.6 Expression pattern of stress-related genes in WT and GmPP2C89-OX seedlings

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