苹果蛋白磷酸酶基因MdPP2C24的克隆及功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2025-1231

摘要/Abstract

摘要：

目的 2C型蛋白磷酸酶（2C type protein phosphatase, PP2C）是脱落酸（ABA）信号转导途径的核心组分，在植物逆境胁迫响应中起重要作用，克隆苹果PP2C家族基因，并解析其在ABA信号及干旱胁迫响应中的作用，为苹果抗逆育种提供理论依据和基因资源。方法以‘嘎啦’苹果为材料克隆MdPP2C24，通过进化树和蛋白质结构分析其保守域及同源性，采用实时荧光定量PCR（RT-qPCR）检测表达模式，利用洋葱内表皮瞬时表达观察蛋白亚细胞定位情况，通过花序侵染法获得MdPP2C24过表达拟南芥，并分析其对ABA敏感性及干旱胁迫耐受性的影响。结果苹果MdPP2C24的开放阅读框长度为1 218 bp，编码405个氨基酸，蛋白质分子量为44.29 kD，等电点为6.63。进化树和蛋白质结构分析发现，MdPP2C24与梨PbPP2C24亲缘关系较近，且含有1个保守的PP2C结构域。RT-qPCR表明MdPP2C24在苹果的各组织器官中均有表达，其中在根系中的表达水平最高，且其表达水平受ABA和甘露醇处理的诱导显著上调。亚细胞定位分析显示，MdPP2C24为核质共定位蛋白。此外，在拟南芥中异源过表达MdPP2C24显著降低了植株对ABA的敏感性及干旱胁迫耐受性，同时抑制了胁迫响应标记基因的表达。结论苹果蛋白磷酸酶基因MdPP2C24通过负调控ABA敏感性和抑制胁迫响应标记基因的表达来降低植株的耐旱性。

关键词: 苹果, 蛋白磷酸酶, MdPP2C24, 表达分析, 脱落酸, 抗旱性

Abstract:

Objective Type 2C protein phosphatases (PP2Cs) are core components in the abscisic acid (ABA) signal transduction pathway and play crucial roles in plant responses to abiotic stresses. This study aimed to clone PP2C family genes from apple and investigate their roles in ABA signaling and drought stress responses, providing a theoretical basis and genetic resources for apple stress-resistant breeding. Method The MdPP2C24 gene was cloned from ‘Gala’ apple. Phylogenetic tree and protein structure analyses were performed to identify conserved domains and homology. Quantitative real-time PCR (RT-qPCR) was used to detect expression patterns, transient expression in onion inner epidermis was employed to observe protein localization, and MdPP2C24-overexpressing Arabidopsis thaliana plants were generated via floral dip method to analyze ABA sensitivity and drought tolerance. Result The open reading frame of MdPP2C24 was 1 218 bp, encoding 405 amino acids with a molecular weight of 44.29 kD and the pI was 6.63. Phylogenetic and protein structure analyses revealed that MdPP2C24 was closely related to PbPP2C24 from pear (Pyrus × bretschneideri) and contained one conserved PP2C domain. RT-qPCR showed that MdPP2C24 was expressed in all apple tissues, with the highest expression in the roots, and its expression was significantly upregulated by ABA and mannitol treatments. Subcellular localization indicated that MdPP2C24 was a nuclear-cytoplasmic co-localized protein. Furthermore, heterologous overexpression of MdPP2C24 in Arabidopsis significantly reduced ABA sensitivity and drought tolerance, while inhibiting the expression of stress-responsive marker genes. Conclusion The apple protein phosphatase gene MdPP2C24 reduces plant drought tolerance by negatively regulating ABA sensitivity and suppressing the expression of stress-responsive marker genes.

Key words: apple, protein phosphatase, MdPP2C24, expression analysis, abscisic acid, drought resistance

陈萃, 兰丽霞, 苏梦雨, 翁年东, 张震. 苹果蛋白磷酸酶基因MdPP2C24的克隆及功能鉴定[J]. 生物技术通报, 2026, 42(8): 153-161.

CHEN Cui, LAN Li-xia, SU Meng-yu, WENG Nian-dong, ZHANG Zhen. Cloning and Functional Identification of Apple Protein Phosphatase Gene MdPP2C24[J]. Biotechnology Bulletin, 2026, 42(8): 153-161.

图/表 7

图1 MdPP2C24的克隆及其蛋白进化树分析A：MdPP2C24的扩增；B：不同物种间PP2C蛋白的系统进化树分析。Os：水稻；Zm：玉米；Pb：梨；Ta：小麦；At：拟南芥；Md：苹果；Pe：胡杨；Nt：烟草；Zj：枣；Vv：葡萄。下同

Fig. 1 Cloning of the MdPP2C24 gene and phylogenetic analysis of its proteinA: RT-PCR of MdPP2C24. B: Phylogenetic tree analysis of PP2C proteins from different species. Os: Oryza sativa; Zm: Zea mays; Pb: Pyrus × bretschneideri; Ta: Triticum aestivum; At: Arabidopsis thaliana; Md: Malus × domestica; Pe: Populus euphratica; Nt: Nicotiana tomentosiformis; Zj: Ziziphus jujuba; Vv: Vitis vinifera.The same below

图2 MdPP2C24蛋白结构域的预测（A）与氨基酸序列比对（B）

Fig. 2 Prediction of the conserved domains (A) and amino acid sequence alignment (B) of protein MdPP2C24

图3 MdPP2C24的表达模式分析A：MdPP2C24的组织表达分析；B：MdPP2C24在ABA处理下的表达水平分析；C：MdPP2C24在甘露醇处理下的表达水平分析；D：MdPP2C24亚细胞定位分析。*P<0.05，**P<0.01，***P<0.001。下同

Fig. 3 Expression pattern of MdPP2C24A: Expression of MdPP2C24 in different tissues. B: Expression of MdPP2C24 under ABA treatment. C: Expression of MdPP2C24 under mannitol treatment. D: Subcellular localization of MdPP2C24. *P<0.05, **P<0.01, ***P<0.001. The same below

图4 MdPP2C24转基因拟南芥株系的DNA（A）和RNA（B）水平的检测

Fig. 4 Detection of DNA (A) and RNA (B) levels of MdPP2C24 in transgenic Arabidopsis lines

图5 ABA处理下Col-0和MdPP2C24转基因拟南芥株系的种子萌发情况A：种子萌发表型；B：种子萌发率的统计

Fig. 5 Seed germination of Col-0 and MdPP2C24 transgenic Arabidopsis lines under ABA stressA: Phenotype of seed germination. B: Statistics of seed germination rate

图6 过表达MdPP2C24拟南芥植株干旱处理后的表型及相关生理指标A：干旱处理表型；比例尺=2 cm；B：叶绿素含量；C：净光合速率；D：气孔开张度观察，比例尺=10 μm；E：气孔开张度的统计

Fig. 6 Phenotype and related physiological indexes of MdPP2CA transgenic Arabidopsis plants under drought stressA: Drought treatment phenotype. Bar=2 cm. B: Chlorophyll content. C: Net photosynthetic rate. D: Stomatal apertures, bar=10 μm. E: Statistical results of stomatal aperture

图7 干旱处理下MdPP2C24过表达拟南芥植株中胁迫响应标记基因的表达分析

Fig. 7 Relative expressions of stress-responsive marker genes in MdPP2C24-overexpressing Arabidopsis plants under drought treatment

参考文献 34

[1]	Li SH, Liu S, Zhang Q, et al. The interaction of ABA and ROS in plant growth and stress resistances [J]. Front Plant Sci, 2022, 13: 1050132.
[2]	Yang WQ, Zhang W, Wang XX. Post-translational control of ABA signalling: The roles of protein phosphorylation and ubiquitination [J]. Plant Biotechnol J, 2017, 15(1): 4-14.
[3]	Chen XX, Ding YL, Yang YQ, et al. Protein kinases in plant responses to drought, salt, and cold stress [J]. J Integr Plant Biol, 2021, 63(1): 53-78.
[4]	Ben-Ari G. The ABA signal transduction mechanism in commercial crops: Learning from Arabidopsis [J]. Plant Cell Rep, 2012, 31(8): 1357-1369.
[5]	Fujii H, Chinnusamy V, Rodrigues A, et al. In vitro reconstitution of an abscisic acid signalling pathway [J]. Nature, 2009, 462(7273): 660-664.
[6]	Soon FF, Ng LM, Zhou XE, et al. Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases [J]. Science, 2012, 335(6064): 85-88.
[7]	Ruan YX, Wang M, Xiao M, et al. Identification and function analysis of PP2C gene family shows that BnaA2.PP2C73 negatively regulates drought and salt stress responses in Brassica napus L. [J]. Ind Crops Prod, 2024, 214: 118568.
[8]	MacKintosh C, Cohen P. Identification of high levels of type 1 and type 2A protein phosphatases in higher plants [J]. Biochem J, 1989, 262(1): 335-339.
[9]	Yu LP, Miller AK, Clark SE. POLTERGEIST encodes a protein phosphatase 2C that regulates CLAVATA pathways controlling stem cell identity at Arabidopsis shoot and flower meristems [J]. Curr Biol, 2003, 13(3): 179-188.
[10]	张春玲, 周李杰, 王桂栾, 等. 苹果蛋白磷酸酶基因MdPP2C-Like的克隆及其对ABA的响应分析 [J]. 园艺学报, 2018, 45(7): 1225-1236.
	Zhang CL, Zhou LJ, Wang GL, et al. Molecular cloning of protein phosphatase gene MdPP2C-Like in apple and the response analysis of ABA [J]. Acta Hortic Sin, 2018, 45(7): 1225-1236.
[11]	Sheen J. Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants [J]. Proc Natl Acad Sci USA, 1998, 95(3): 975-980.
[12]	Gosti F, Beaudoin N, Serizet C, et al. ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling [J]. Plant Cell, 1999, 11(10): 1897-1910.
[13]	Schweighofer A, Hirt H, Meskiene I. Plant PP2C phosphatases: emerging functions in stress signaling [J]. Trends Plant Sci, 2004, 9(5): 236-243.
[14]	Kuhn JM, Boisson-Dernier A, Dizon MB, et al. The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA [J]. Plant Physiol, 2006, 140(1): 127-139.
[15]	Hao Q, Yin P, Li WQ, et al. The molecular basis of ABA-independent inhibition of PP2Cs by a subclass of PYL proteins [J]. Mol Cell, 2011, 42(5): 662-672.
[16]	Zhao Y, Chan ZL, Xing L, et al. The unique mode of action of a divergent member of the ABA-receptor protein family in ABA and stress signaling [J]. Cell Res, 2013, 23(12): 1380-1395.
[17]	Ghanizadeh H, Qamer Z, Zhang Y, et al. The multifaceted roles of PP2C phosphatases in plant growth, signaling, and responses to abiotic and biotic stresses [J]. Plant Commun, 2025, 6(9): 101457.
[18]	Wang JY, Li CN, Li L, et al. DIW1 encoding a clade I PP2C phosphatase negatively regulates drought tolerance by de-phosphorylating TaSnRK1.1 in wheat [J]. J Integr Plant Biol, 2023, 65(8): 1918-1936.
[19]	Wang Y, Chen MX, Wu D, et al. OsPP2C03 positively regulates rice drought stress resistance through a non-canonical ABA pathway [J]. Plant Sci, 2026, 362: 112772.
[20]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-△△Ct method [J]. Methods, 2001, 25(4): 402-408.
[21]	Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [J]. Plant J, 1998, 16(6): 735-743.
[22]	Chen C, Zhang Z, Lei YY, et al. MdMYB44-like positively regulates salt and drought tolerance via the MdPYL8-MdPP2CA module in apple [J]. Plant J, 2024, 118(1): 24-41.
[23]	王宏, 蔺经, 李晓刚, 等. 耐盐杜梨蛋白磷酸酶基因PbPP2C的克隆及表达分析 [J]. 江苏农业学报, 2014, 30(6): 1464-1471.
	Wang H, Lin J, Li XG, et al. Molecular cloning and expression analysis of PbPP2C gene from salt-tolerant Pyrus betulaefolia Bunge [J]. Jiangsu J Agric Sci, 2014, 30(6): 1464-1471.
[24]	张婷婷, 李雨欣, 张德遥, 等. 铁皮石斛蛋白磷酸酶PP2C家族基因鉴定及其表达分析 [J]. 园艺学报, 2021, 48(12): 2458-2470.
	Zhang TT, Li YX, Zhang DY, et al. Genome-wide identification and expression analyses of PP2C gene family in Dendrobium catenatum [J]. Acta Hortic Sin, 2021, 48(12): 2458-2470.
[25]	Sobol G, Chakraborty J, Martin GB, et al. The emerging role of PP2C phosphatases in tomato immunity [J]. Mol Plant Microbe Interact, 2022, 35(9): 737-747.
[26]	Li L, Li B, Zhu SR, et al. TMK4 receptor kinase negatively modulates ABA signaling by phosphorylating ABI2 and enhancing its activity [J]. J Integr Plant Biol, 2021, 63(6): 1161-1178.
[27]	Komatsu K, Nishikawa Y, Ohtsuka T, et al. Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss Physcomitrella patens [J]. Plant Mol Biol, 2009, 70(3): 327-340.
[28]	Tougane K, Komatsu K, Bhyan SB, et al. Evolutionarily conserved regulatory mechanisms of abscisic acid signaling in land plants: characterization of abscisic acid insensitive1-like type 2C protein phosphatase in the liverwort Marchantia polymorpha [J]. Plant Physiol, 2010, 152(3): 1529-1543.
[29]	Reyes D, Rodríguez D, González-García MP, et al. Overexpression of a protein phosphatase 2C from beech seeds in Arabidopsis shows phenotypes related to abscisic acid responses and gibberellin biosynthesis [J]. Plant Physiol, 2006, 141(4): 1414-1424.
[30]	倪飞. 鸢尾蛋白磷酸酶2C(PP2C)基因的克隆和功能分析及拟南芥PP2C基因家族的鉴定和基因组学分析 [D]. 泰安: 山东农业大学, 2009.
	Ni F. Cloning and functional analysis of a protein phosphatase 2C gene (IrisPP2C1) in the flower of Iris tectorum and genome-wide analysis of PP2C family in Arabidopsis [D]. Tai’an: Shandong Agricultural University, 2009.
[31]	杨进, 周俊琴, 卢梦琪, 等. 油茶ABI5基因的克隆及其表达分析 [J]. 植物生理学报, 2020, 56(7): 1583-1592.
	Yang J, Zhou JQ, Lu MQ, et al. Cloning and expression analysis of ABI5 from Camellia oleifera [J]. Plant Physiol J, 2020, 56(7): 1583-1592.
[32]	Chen KQ, Song MR, Guo YN, et al. MdMYB46 could enhance salt and osmotic stress tolerance in apple by directly activating stress-responsive signals [J]. Plant Biotechnol J, 2019, 17(12): 2341-2355.
[33]	许璇, 何钰贤, 姚允聪, 等. 苹果属PP2C A亚家族基因的克隆及生信分析 [J]. 北京农学院学报, 2022, 37(2): 33-38.
	Xu X, He YX, Yao YC, et al. Cloning and bioinformatics analysis of group A-PP2C genes in ‘Malus hupehensis Rehd.’ [J]. J Beijing Univ Agric, 2022, 37(2): 33-38.
[34]	Liu YY, Shi WS, Liu Y, et al. MdPP2C24/37, protein phosphatase type 2Cs from apple, interact with MdPYL2/12 to negatively regulate ABA signaling in transgenic Arabidopsis [J]. Int J Mol Sci, 2022, 23(22): 14375.