Analysis of Salt Tolerance Function of Soybean Protein Phosphatase Gene GmPP2C72

doi:10.7668/hbnxb.20196133

Abstract

Abstract:

In order to clarify the function of the protein phosphatase 2C gene GmPP2C72 in salt stress response and its salt-tolerance molecular mechanism,bioinformatics analysis of GmPP2C72 protein was conducted, GmPP2C72 gene was cloned to construct the overexpression vector,and then was transferred into Lotus japonicus using the cotyledon node genetic transformation method mediated by Agrobacterium tumefaciens.Finally,the phenotype,physiological indicators and the expression levels of salt tolerance related genes of transgenic plants were analyzed under salt stress condition.The results showed that GmPP2C72 gene contained an open reading frame of 1 206 bp,encoding 401 amino acids,and the encoded protein belonged to the member of the class A subfamily of PP2C family.GmPP2C72 gene did not exhibit tissue expression specificity,it was expressed in various tissues of soybean,and the expression of this gene was induced by salt stress.The overexpression vector of GmPP2C72 gene was successfully constructed.GmPP2C72 gene was successfully transferred into Lotus japonicus,and three transgenic lines with GmPP2C72 gene were successfully obtained.Compared with wild type,the three transgenic lines maintained better growth state,with significantly higher contents of chlorophyll and proline and lower relative plasma membrane permeability and malondialdehyde content in leaves of Lotus japonicus under salt stress.In addition,the expression levels of four key salt tolerance related genes,LjLEA, LjCOR,LjRD29B,and LjP5CS were significantly higher compared with wild type.Overall,the overexpression of GmPP2C72 gene enhances the salt tolerance of Lotus japonicus by reducing the degree of membrane lipid peroxidation and the permeability of the plasma membrane,increasing the contents of chlorophyll and proline in the leaves,and enhancing the expression levels of salt tolerance related genes.

Key words: Soybean, Protein phosphatase 2C(PP2C), Lotus japonicus, Salt tolerance

CLC Number:

KE Danxia, ZHOU Zhaoyuan, HOU Shibo, ZHANG Kexin, SONG Xiaoli, LIN Jianuo. Analysis of Salt Tolerance Function of Soybean Protein Phosphatase Gene GmPP2C72[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 12-20. doi: 10.7668/hbnxb.20196133.

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Figures/Tables 9

Tab.1 The primers and primer sequences used in this study

引物名称 Primer name	引物序列(5'—3') Sequence of primer(5'—3')
GmPP2C72-F	ATGGCTGGAATTTGCTGTGG
GmPP2C72-R	TTAATTGGAGTTTGATGATTG
GmPP2C72-OX-F	GGATCCATGGCTGGAATTTGCTG
GmPP2C72-OX-R	GGTACCTTAATTGGAGTTTGATG
Q-GmPP2C72-F	GCCAAAGTGGAAATCCACGAC
Q-GmPP2C72-R	CCATGTCTCTTCTCCTGCCG
Q-LjUBI-F	CAAGGAAGGTATCCCACCG
Q-LjUBI-R	TTAGAATCCACCACGAAGACG
GUS-F	GTCGCGCAAGACTGTAACCA
GUS-R	CGGCGAAATTCCATACCTG
Q-LjLEA-F	CAAAACTCGTTATGTTGCGG
Q-LjLEA-R	CACCTCAAAGGAAGAAGAACA
Q-LjCOR-F	TTGCAACGGTCGTGTATTTGCG
Q-LjCOR-R	TGGGTAACCACACTCTCTGGATG
Q-LjRD29B-F	TTCACCTTGTGCTCCGTCTTC
Q-LjRD29B-R	AACAACAGCACACACAGACAATC
Q-LjP5CS-F	CTGTTAGGCGTGTTATCGTC
Q-LjP5CS-R	CTTCATACCCCTGAGTGTTTAG

Fig.1 Analysis of conserved domains(A)and phosphorylation sites(B)of GmPP2C72 protein

Fig.2 Expression analysis of GmPP2C72 in different tissues of soybean 1.Young leaves;2.Flowers;3.1 cm pods;4.Pod shells at 10 days after flowering;5.Pod shells at 14 days after flowering;6.Seeds at 10 days after flowering;7.Seeds at 14 days after flowering;8.Seeds at 21 days after flowering;9.Seeds at 28 days after flowering;10.Seeds at 42 days after flowering;11.Roots;12.Nodules.Different lowercase letters indicate significant difference (P<0.05) in gene expression levels among different tissues.

Fig.3 Expression analysis of GmPP2C72 in soybean under salt tress Different lowercase letters indicate significant difference (P<0.05) in gene expression levels among different treatment times.

Fig.4 PCR amplification of GmPP2C72 gene and enzyme digestion identification of recombinant plasmid p1301U-GmPP2C72 A.PCR amplification of GmPP2C72 gene:M.1 kb DNA ladder;1.PCR amplification product of GmPP2C72 gene.B.Enzyme digestion identification of recombinant plasmid p1301U-GmPP2C72:M.1 kb DNA ladder;1—2.The recombinant p1301U-GmPP2C72 plasmid;3—4.Double enzyme digestion product of empty vector p1301U;5—6.Double enzyme digestion product of the recombinant p1301U-GmPP2C72.

Fig.5 Molecular detection of transgenic Lotus japonicas overexpressing GmPP2C72 A.Detection of GUS gene expression in transgenic L.japonicus by PCR;B.Detection of GmPP2C72 gene expression in transgenic L.japonicus by RT-PCR.M.100 bp DNA ladder Marker;nCK.Negative control;pCK.Plasmid positive control;OE1—OE11.Transgenic lines.The same as Fig.6—8.

Fig.6 Phenotypic analysis of transgenic Lotus japonicas overexpressing GmPP2C72 under salt stress A.Phenotype analysis under salt stress;B.Changes of shoot height of transgenic L.japonicus under salt stress;C.Changes of dry shoot weight of transgenic L.japonicus under salt stress.Different lowercase letters indicate significant differences among different lines(P<0.05)under same NaCl concentration.The same as Fig.7.

Fig.7 Physiological indicators of transgenic Lotus japonicas overexpressing GmPP2C72 under salt stress

Fig.8 Expression levels of salt stress related genes in transgenic Lotus japonicas overexpressing GmPP2C72 under salt stress Different lowercase letters indicate significant difference(P<0.05)among different lines.

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