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作物学报 ›› 2025, Vol. 51 ›› Issue (10): 2713-2726.doi: 10.3724/SP.J.1006.2025.55028

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

花生抗旱基因AhCPK8及其互作蛋白GAPDH的克隆及分子鉴定

何梅1,2(), 张佳蕾1, 范士凯1, 孟静静1, 王建国1, 郭峰1, 李新国1, 万书波1,*(), 杨莎1,*()   

  1. 1山东省农业科学院农作物种质资源研究所, 山东济南 250100
    2山东师范大学生命科学学院, 山东济南 250399
  • 收稿日期:2025-04-17 接受日期:2025-07-09 出版日期:2025-10-12 网络出版日期:2025-07-22
  • 通讯作者: *万书波, E-mail: wanshubo2016@163.com;杨莎, E-mail: yangsha0904@126.com
  • 作者简介:E-mail: hemei920@163.com
  • 基金资助:
    国家自然科学基金项目(32272020);山东省重点研发计划项目(ZFJH202310);泰山学者工程项目(tspd20221107);泰山学者工程项目(tsqn202408305)

Cloning and molecular identification of the peanut drought-resistance gene AhCPK8 and its interacting protein GAPDH

HE Mei1,2(), ZHANG Jia-Lei1, FAN Shi-Kai1, MENG Jing-Jing1, WANG Jian-Guo1, GUO Feng1, LI Xin-Guo1, WAN Shu-Bo1,*(), YANG Sha1,*()   

  1. 1Crop Germplasm Resources Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
    2College of Life Sciences, Shandong Normal University, Jinan 250399, Shandong, China
  • Received:2025-04-17 Accepted:2025-07-09 Published:2025-10-12 Published online:2025-07-22
  • Contact: *E-mail: wanshubo2016@163.com;E-mail: yangsha0904@126.com
  • Supported by:
    National Natural Science Foundation of China(32272020);Shandong Key R&D Program(ZFJH202310);Taishan Scholars Program(tspd20221107);Taishan Scholars Program(tsqn202408305)

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摘要：

花生(Arachis hypogaea L.)是重要的油料和经济作物, 干旱是限制花生产量的主要因素之一。探明花生响应干旱胁迫的关键基因, 对后续花生品种改良, 提高产量具有重要意义。本研究以花生品种花育22号(HY22)为研究对象, 足钙(SC)、缺钙(NC) 2种营养液水培培养, 终浓度为20% PEG-6000溶液模拟干旱胁迫处理(DSC、DNC)。通过干旱处理后转录组数据结合花生钙传感蛋白编码基因CPKs的差异表达筛选出花生抗旱基因AhCPK8。以干旱处理后的花生叶片为材料, 利用酵母双杂交筛选到其互作蛋白甘油醛-3-磷酸脱氢酶(GAPDH)并得到初步验证。GAPDH家族基因在调节植物对非生物胁迫的反应中发挥着重要作用。基于AhGAPDH基因家族的生物信息学分析, 在花生中共鉴定出21个GAPDH基因, 再根据大豆和拟南芥系统发育树关系以及基因在12条染色体上的先后顺序进行命名。基本理化特征结果表明, AhGAPDH家族成员大多数为稳定、疏水性蛋白; 同一聚类的AhGAPDH家族成员大多数表现出相似的motif结构、保守结构域和基因结构; 启动子区域中有丰富的光响应、植物激素响应及非生物胁迫响应相关的顺式作用元件。AhCPK8与AhGAPDH存在相互作用, 共同调控花生的抗旱性。

关键词: 花育22号, 抗旱性, AhCPK8, 互作蛋白, GAPDH

Abstract:

Peanut is an important oilseed and economic crop, and drought is one of the major factors limiting its yield. Identifying key genes involved in the peanut drought response is of great significance for improving drought tolerance and enhancing yield. In this study, the peanut variety Huayu 22 (HY22) was grown hydroponically using two nutrient solutions: one with sufficient calcium (SC) and one without calcium (NC). Drought stress treatments (DSC and DNC) were simulated using PEG-6000 at a final concentration of 20%. Based on transcriptome analysis and differential expression of calcium-dependent protein kinases (CPKs), we identified the drought-responsive gene AhCPK8, which encodes a calcium-sensing protein in peanut. Using yeast two-hybrid assays and drought-treated peanut leaves, we screened for and validated its interacting protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The GAPDH gene family plays a crucial role in regulating plant responses to abiotic stress. Through bioinformatics analysis, a total of 21 AhGAPDH genes were identified in peanut. These genes were named according to their phylogenetic relationships with soybean and Arabidopsis thaliana, as well as their chromosomal positions across the 12 peanut chromosomes. Physicochemical characterization revealed that most AhGAPDH proteins are stable and hydrophobic. Members within the same phylogenetic cluster shared similar motif structures, conserved domains, and gene architectures. Moreover, their promoter regions contain numerous cis-acting elements related to light responsiveness, phytohormone signaling, and abiotic stress responses. The interaction between AhCPK8 and AhGAPDH suggests a coordinated regulatory mechanism that contributes to drought resistance in peanut.

Key words: Huayu 22, drought resistance, AhCPK8, interacting protein, GAPDH

表1

基因名称及qRT-PCR引物"

基因名称
Gene name		 正向引物序列
Forword primer sequence (5′-3′)		 反向引物序列
Reverse primer sequence (5′-3′)
TUA5 CTGATGTCGCTGTGCTCTTGG CTGTTGAGGTTGGTGTAGGTAGG
AhCPK2 TGGGCAGAGACCGAACAAGGG CCGCTGTCAACCGCTTCTTAGG
AhCPK8 CCTGCAAGTCCATCCCAAAGCG CGAGATGAACGGCAAGGCTGTC
AhCPK36 TTCCTCCTCATCCGCCACCATC CCCAGCTCCCTACCGAAGACG
AhCPK40 TGGAGGCGGCTGATGTGGAC CCTCGCGCTCTAGCTTGTTGAG

图1

FH18和NH5中CaM/CML、CPKs和CBL家族基因对干旱胁迫的响应 A~C图分别是CaM/CML、CPKs和CBL三大类钙离子识别蛋白家族基因的表达谱。18/63: 在干旱胁迫下表现上调的18个CaM/CML家族基因成员占全部63个家族基因成员的比例; 24/45: 在干旱胁迫下表现上调的24个CPKs家族基因成员占全部45个家族基因成员的比例; 7/23: 在干旱胁迫下表现上调的7个CBL家族基因成员占全部23个家族基因成员的比例。FH18: 干旱敏感型花生品种; NH5: 耐旱型花生品种。"

图2

AhCPK2、AhCPK8、AhCPK36、AhCPK40在不同处理下的表达模式 *、**、***、****分别表示在0.05、0.01、0.001、0.0001水平差异显著。DSC: 干旱处理足钙培养条件下的花育22号幼苗; DNC: 干旱处理缺钙培养条件下的花育22号幼苗。"

图3

诱饵毒性及自激活检测"

图4

AhCPK8互作蛋白的筛选 A: Y2Hgold [pGBKT7-AhCPK8] +10 μL文库质粒的转化效率。B: QDO/X-α-gal平板上筛选阳性克隆。"

图5

蛋白互作验证 a: 阳性对照pGBKT7-53+pGADT7-T; b: 阴性对照pGBKT7- Lam+pGADT7-T; c: 试验组pGBKT7-AhCPK8+pGADT7; d: 试验组pGBKT7-AhCPK8+pGADT7-AhGAPDH。"

表2

AhGAPDH基因家族成员命名表"

名称
Symbol		 序列ID
Sequence ID		 名称
Symbol		 序列ID
Sequence ID
AhGAPA1 arahy.Tifrunner.gnm2.ann1.D2DMXG.1 AhGAPC3 arahy.Tifrunner.gnm2.ann1.8UAX1T.1
AhGAPA2 arahy.Tifrunner.gnm2.ann1.YN8IJ2.1 AhGAPC4 arahy.Tifrunner.gnm2.ann1.YH7XEC.1
AhGAPA3 arahy.Tifrunner.gnm2.ann1.8EQN3M.1 AhGAPC5 arahy.Tifrunner.gnm2.ann1.YH7XEC.3
AhGAPA4 arahy.Tifrunner.gnm2.ann1.HFT5ZK.1 AhGAPC6 arahy.Tifrunner.gnm2.ann1.SIHD8Y.1
AhGAPA5 arahy.Tifrunner.gnm2.ann1.FN6RIW.1 AhGAPC7 arahy.Tifrunner.gnm2.ann1.VRBE94.1
AhGAPA6 arahy.Tifrunner.gnm2.ann1.BA5EUQ.1 AhGAPC8 arahy.Tifrunner.gnm2.ann1.QHP0ED.1
AhGAPA7 arahy.Tifrunner.gnm2.ann1.BFW5UL.1 AhGAPCp1 arahy.Tifrunner.gnm2.ann1.5PGU74.1
AhGAPA8 arahy.Tifrunner.gnm2.ann1.6W2CKZ.1 AhGAPCp2 arahy.Tifrunner.gnm2.ann1.5PGU74.2
AhGAPA9 arahy.Tifrunner.gnm2.ann1.ZX8DBB.1 AhGAPCp3 arahy.Tifrunner.gnm2.ann1.F06IQB.2
AhGAPC1 arahy.Tifrunner.gnm2.ann1.YL3Z6V.1 AhGAPCp4 arahy.Tifrunner.gnm2.ann1.F06IQB.3
AhGAPC2 arahy.Tifrunner.gnm2.ann1.PV44JT.1

表3

AhGAPDH基因家族成员基本信息表"

名称
Symbol		 氨基酸数目
Number of amino acid		 分子量
Molecular weight (Da)		 等电点
pI		 不稳定指数
Instability index		 脂溶指数
Aliphatic index		 总平均亲水性值
Grand average of
hydropathicity
AhGAPA1 388 41,339.06 8.92 24.19 89.95 -0.079
AhGAPA2 509 54,458.07 5.59 29.04 89.80 -0.101
AhGAPA3 359 38,725.28 5.48 24.14 93.31 -0.040
AhGAPA4 428 45,980.96 9.40 41.61 92.69 -0.130
AhGAPA5 405 43,179.17 8.44 24.73 91.68 -0.056
AhGAPA6 478 51,430.76 6.80 27.71 89.08 -0.114
AhGAPA7 381 40,951.94 5.50 25.43 94.88 -0.005
AhGAPA8 469 50,071.35 5.32 32.03 99.10 0.082
AhGAPA9 586 63,306.33 7.93 41.38 91.96 -0.102
AhGAPC1 336 36,546.89 7.06 23.08 91.34 -0.108
AhGAPC2 347 37,480.97 8.23 33.58 90.49 -0.065
AhGAPC3 278 31,231.71 6.07 30.71 90.43 -0.282
AhGAPC4 339 36,841.07 7.02 28.17 90.27 -0.087
AhGAPC5 223 24,220.73 8.68 26.63 83.50 -0.122
AhGAPC6 336 36,250.48 8.24 31.23 89.67 -0.080
AhGAPC7 336 36,532.86 7.06 23.33 91.04 -0.109
AhGAPC8 339 36,855.10 7.02 27.16 90.27 -0.087
AhGAPCp1 416 44,435.84 9.00 32.74 89.30 -0.039
AhGAPCp2 345 37,141.40 6.61 29.29 86.75 -0.051
AhGAPCp3 440 47,250.28 9.06 33.15 89.52 -0.027
AhGAPCp4 416 44,479.96 9.04 31.08 89.06 -0.044

图6

AhGAPDH基因在染色体上的分布"

图7

AhGAPDH基因家族的聚类分析和保守基序、保守结构域及基因结构分析 A: 聚类分析; B: 保守基序分析; C: 保守结构域分析; D: 基因结构分析。Motif: 基序; UTR: 非编码区; CDS: 编码区序列。"

图8

AhGAPDH基因启动子区顺式作用元件预测分析"

图9

花生、大豆、油菜、拟南芥和烟草的GAPDH基因家族成员系统进化树"

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