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作物学报 ›› 2023, Vol. 49 ›› Issue (1): 97-104.doi: 10.3724/SP.J.1006.2023.14239

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗ScPR10基因的克隆及其响应赤条病菌侵染的表达特征分析

李娟, 周敬如, 储娜, 孙会东, 黄美婷, 傅华英, 高三基()   

  1. 福建农林大学国家甘蔗工程技术研究中心, 福建福州350002
  • 收稿日期:2021-12-17 接受日期:2022-03-25 出版日期:2023-01-12 网络出版日期:2022-04-20
  • 通讯作者: 高三基
  • 基金资助:
    本研究由财政部和农业农村部国家现代农业产业技术体系建设专项(糖料, CARS-170302(CARS-170302);国家甘蔗工程技术研究中心主任基金项目(KJG16005M)

Gene cloning and expression analysis of ScPR10 in sugarcane under Acidovorax avenae subsp. avenae infection

LI Juan, ZHOU Jing-Ru, CHU Na, SUN Hui-Dong, HUANG Mei-Ting, FU Hua-Ying, GAO San-Ji()   

  1. National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2021-12-17 Accepted:2022-03-25 Published:2023-01-12 Published online:2022-04-20
  • Contact: GAO San-Ji
  • Supported by:
    Earmarked Fund of the China Agriculture Research System of MOF and MARA(CARS-170302);Director’s Innovation Fund in National Engineering Research Center for Sugarcane, FAFU, China(KJG16005M)

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摘要：

PR10是一种病程相关蛋白(pathogenesis-related protein, PR), 在植物生长发育、应激生物和非生物胁迫时发挥重要作用。本研究利用RT-PCR技术从甘蔗赤条病抗病品种新台糖22号和感病品种闽糖11-610叶片克隆获得ScPR10基因, 并利用实时荧光定量PCR (RT-qPCR)、转录组和蛋白质组分析, 研究在接种燕麦食酸菌燕麦亚种(Acidovorax avenae subsp. avenae, Aaa)之后这2个品种ScPR10基因的转录和蛋白表达模式。序列分析显示, 本研究克隆获得2个ScPR10基因, 编码187个氨基酸, 比其他植物PR10蛋白多了27个氨基酸, 含有保守结构域P-loop和Bet v 1; 与已报道的甘蔗、高粱和斑茅的PR10亲缘关系较近。在Aaa胁迫下, RT-qPCR分析表明抗病、感病品种ScPR10基因的表达量均显著上调, 尤其在接种24 h时, 表达量最高, 分别为对照的27.2倍和39.7倍; 转录组数据分析结果显示, 该基因表达水平在抗病、感病品种上均显著提高, 尤其在接种72 h时, 表达量最高, log2 FC值为5.3~5.4。蛋白互作预测发现, ScPR10与植物PDR型ABCG转运蛋白(Cluster-13677.282407)、蛋白激酶(Cluster- 13677.166559)之间存在互作关系。蛋白质组数据分析显示, 在Aaa接种24 h时, 抗病、感病品种ScPR10均为上调表达, log2FC值为1.65~1.69; 与ScPR10互作的PDR型ABCG转运蛋白成员的表达量在抗病、感病品种上有不同程度提高; 蛋白激酶表达量在感病品种上有显著提高, 但是, 在抗病品种上表达量没有显著变化。本研究结果表明, ScPR10可能与PDR型ABCG转运蛋白正向协同参与甘蔗寄主应答Aaa病菌侵染的防御响应。

关键词: PR10蛋白, 燕麦食酸菌燕麦亚种, 生物胁迫, 基因表达, 甘蔗

Abstract:

PR10 is a pathogenesis-related protein (PR), which plays critical roles in the growth and development and various environmental stress responses in plants. This study demonstrated that two ScPR10 genes were cloned from the leaf samples of ROC22 (resistant to red stripe) and Mintang 11-610 (susceptible to red stripe) by RT-PCR, and then RT-qPCR, transcriptome, and proteomics assays were carried out to explore the expression patterns of two genes in varieties ROC22 and Mintang 11-610 post inoculation with red stripe pathogen Acidovorax avenae subsp. avenae (Aaa). Sequence analysis showed that the two ScPR10 genes encoding 187 amino acids had more 27 amino acids than PR10 proteins from other plants, which contained the conserved domains P-loop and Bet v 1. ScPR10 obtained in this study shared high homology with these published PR10 proteins encoded by Saccharum spp., Sorghum bicolor, and Erianthus arundinaceus. The RT-qPCR analysis showed that the relative expression levels of ScPR10 genes were significantly increased by 27.2 times and 39.7 times in the resistant and susceptible varieties under Aaa infection, especially at 24 hours post inoculation (24 hpi), compared with the controls (0 hpi), respectively. Meanwhile, the transcriptome data revealed that the relative expression levels of ScPR10 genes were significantly increased by 5.3-5.4 folds [log2(Fold Change)] in the two varieties under Aaa infection, particular at 72 hours post inoculation (72 hpi). Prediction of protein interaction revealed that ScPR10 would interact with the pleiotropic drug resistance (PDR) protein of ABCG transporter subfamily (Cluster-13677.282407) and protein kinase (Cluster-13677.166559). Proteomics analysis referred that the relative expression of ScPR10 proteins were increased by 1.65-1.69 folds (log2FC) evaluated by 24 hpi versus 0 hpi in resistant and susceptible varieties. Similarly, the relative expression levels of the ABC transporter (Cluster-13677.282407) were upregulated to a certain extent in two varieties, but the relative expression levels of protein kinase (Cluster-13677.166559) were only enhanced in Mintang 11-610, not in ROC22. In conclusion, the ScPR10 may synergize with the ABC transporter (Cluster-13677.282407) involving in the defense response in sugarcane in response to Aaa infection.

Key words: PR10 protein, Acidovorax avenae subsp. avenae, biotic stress, gene expression, Saccharum spp.

表1

甘蔗ScPR10蛋白的理化性质"

NCBI登录号
NCBI
accession ID.		 基因长度
Gene length (bp)		 CDS长度
CDS length
(bp)		 氨基酸
No. of amino acid (aa)		 分子式
Molecular
formula		 分子量
Molecular weight (kD)		 等电点
pI		 不稳定系数
Instability index		 亲水性
Hydrophilicity
OK290572 697 564 187 C883H1401N231O274S5 19,797.52 4.98 32.02 0.042
OK290571 695 564 187 C885H1406N230O275S5 19,828.58 4.98 31.93 0.067

图1

甘蔗ScPR10蛋白结构分析 A和B分别是来自新台糖22号和闽糖11-610的ScPR10蛋白二级结构; C为甘蔗ScPR10蛋白三级结构; 蓝色、粉红色、红色和绿色分别表示α-螺旋、无规则卷曲、延伸链和β-折叠; α (1~3)、β (1~7)和L (1~9)分别表示α-螺旋、β-折叠和环肽链。"

图2

几种禾本科植物PR10蛋白序列多重比对 P-loop保守结构域用红色方框表示, Bet v 1用下画线标注, 疏水性蛋白结合位点用黄色三角符号表示。"

图3

几种禾本科植物PR10蛋白的系统进化树分析(A)和一致性热图(B)"

图4

甘蔗ScPR10基因应答Aaa胁迫的转录表达模式 A: RT-qPCR验证; B: 转录组数据(log2FC)。新台糖22号(ROC22)为抗病品种, 闽糖11-610 (MT11-610)为感病品种。柱上不同小写字母表示显著差异(P < 0.05)。"

图5

ScPR10及其互作蛋白的转录后表达水平 蓝色、红色方框数值表示抗、感病品种在Aaa胁迫24 h (R24或S24)与对照组处理0 h (CK0)的蛋白表达量的比值, 用log2 FC值表示。"

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