大豆蛋白磷酸酶基因<i>GmPP2C72</i>的耐盐功能分析

doi:10.7668/hbnxb.20196133

摘要/Abstract

摘要：

为了明确GmPP2C72基因在盐胁迫应答中的功能和耐盐分子机制,对GmPP2C72蛋白进行生物信息学分析,克隆GmPP2C72基因构建植物过表达载体,并利用根癌农杆菌介导的子叶节遗传转化法转化百脉根,分析转基因植株在盐胁迫条件下的表型及生理指标和耐盐相关基因的表达情况。结果表明,GmPP2C72基因含有1个1 206 bp的开放阅读框,编码401 aa,编码蛋白质属于PP2C家族A亚族成员。GmPP2C72基因不具有组织表达特异性,在大豆的各个组织中均有表达,且该基因受盐胁迫诱导表达。成功构建了GmPP2C72基因的过表达载体,并转入百脉根,获得3个转GmPP2C72基因株系。在盐胁迫条件下,与野生型对照相比,3 个转基因株系维持着较好的生长状态,株高和地上部干质量均显著提高。进一步分析发现,3 个转基因株系叶片中叶绿素和脯氨酸含量均显著增加,而相对质膜透性和丙二醛含量均显著降低,且耐盐相关基因LjLEA、LjCOR、LjRD29B和LjP5CS的表达量均显著提升。综上,过表达GmPP2C72基因通过降低膜脂过氧化程度和质膜透性、提高叶片叶绿素和脯氨酸含量及耐盐相关基因的表达量来提高百脉根的耐盐性。

关键词: 大豆, 2C类蛋白磷酸酶(PP2C), 百脉根, 耐盐性

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

中图分类号:

柯丹霞, 周兆源, 侯仕博, 张可馨, 宋晓莉, 林佳诺. 大豆蛋白磷酸酶基因GmPP2C72的耐盐功能分析[J]. 华北农学报, 2026, 41(2): 12-20. doi: 10.7668/hbnxb.20196133.

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.

http://www.hbnxb.net/CN/Y2026/V41/I2/12

图/表 9

表1 本研究中所使用引物及其序列

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

图1 GmPP2C72蛋白的保守结构域(A)和磷酸化位点(B)分析

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

图2 大豆GmPP2C72基因在大豆不同组织中的表达分析 1.幼叶;2.花;3.1 cm豆荚;4.开花后10 d荚壳;5.开花后14 d荚壳;6.开花后10 d种子;7.开花后14 d种子;8.开花后21 d种子;9.开花后28 d种子;10.开花后42 d种子;11.根;12.根瘤。不同小写字母表示不同组织中基因表达水平具有显著性差异(P<0.05)。

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.

图3 大豆GmPP2C72基因在盐胁迫下的表达分析不同小写字母表示不同处理时间基因表达水平具有显著差异(P<0.05)。

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.

图4 GmPP2C72基因的PCR扩增和重组质粒p1301U-GmPP2C72的酶切鉴定 A.GmPP2C72基因的PCR扩增:M.1 kb DNA ladder;1.GmPP2C72基因的PCR扩增产物。B.重组质粒p1301U-GmPP2C72的酶切鉴定:M.1 kb DNA ladder;1—2.重组质粒p1301U-GmPP2C72;3—4.p1301U空载体的双酶切产物;5—6.重组质粒p1301U-GmPP2C72的双酶切产物。

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.

图5 转GmPP2C72基因百脉根的分子检测 A.PCR检测转基因百脉根中GUS基因的表达;B.RT-PCR检测转基因百脉根中GmPP2C72基因的表达。M.100 bp DNA ladder Marker;nCK.阴性对照;pCK.质粒阳性对照;OE1—OE11.转基因株系。图6—8同。

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.

图6 盐胁迫下转GmPP2C72基因百脉根的表型分析 A.盐胁迫下表型分析;B.盐胁迫下转基因百脉根株高变化;C.盐胁迫下转基因百脉根地上部干质量变化。不同小写字母表示相同NaCl浓度不同株系间差异显著(P<0.05)。图7同。

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.

图7 盐胁迫下转GmPP2C72基因百脉根植株中生理指标的变化

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

图8 盐胁迫下转GmPP2C72基因百脉根中耐盐相关基因的表达水平不同小写字母表示不同株系间差异显著(P<0.05)。

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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大豆蛋白磷酸酶基因GmPP2C72的耐盐功能分析

Analysis of Salt Tolerance Function of Soybean Protein Phosphatase Gene GmPP2C72

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