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园艺学报 ›› 2020, Vol. 47 ›› Issue (3): 445-454.doi: 10.16420/j.issn.0513-353x.2019-0773

• 研究论文 • 上一篇    下一篇

响应黄龙病侵染的柑橘乙醇脱氢酶基因CsADH1的克隆与表达分析

谢 竹,赵 珂,郑 林,龙俊宏,杜美霞,何永睿,陈善春*,邹修平*   

  1. 西南大学/中国农业科学院柑桔研究所,国家柑桔品种改良中心,重庆 400712
  • 出版日期:2020-03-25 发布日期:2020-03-25
  • 基金资助:
    国家自然科学基金项目(31972393);科技部国家重点研发计划专项(2018YFD0201500);国家现代农业产业技术体系建设专项资金项目(CARS-26);广东省科技计划项目(2018B020202009);广西创新驱动发展专项资金课题(桂科 AA18118046)

Cloning and Expression Analysis of Alcohol Dehydrogenase Gene CsADH1Responding to Huanglongbing Infection in Citrus

XIE Zhu,ZHAO Ke,ZHENG Lin,LONG Junhong,DU Meixia,HE Yongrui,CHEN Shanchun*,and ZOU Xiuping*   

  1. Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences;National Center for Citrus Varieties Improvement,Chongqing 400712,China
  • Online:2020-03-25 Published:2020-03-25

摘要: 对前期在感染黄龙病的‘晚锦橙’中发现的明显差异表达的柑橘乙醇脱氢酶(CsADH1)基因进行克隆测序分析,结果表明,CsADH1 的 CDS 序列长为 1 143 bp,编码 380 个氨基酸。蛋白质结构预测显示,CsADH1 含有乙醇脱氢酶 GroES(ADH_N)和 ADH_zinc_N 保守结构域。进化树分析显示,CsADH1 蛋白与拟南芥、蓖麻和中国莲的 ADH 蛋白亲缘关系最近。亚细胞定位结果显示,CsADH1 蛋白定位在细胞质中。以感病‘晚锦橙’的根和叶脉为材料,qPCR 分析显示 CsADH1 在感病根和叶脉中均上调表达,且根中的表达水平是叶脉中的 10 倍。离子胁迫试验和外源植物生长调节剂诱导试验表明,Zn、水杨酸(SA)、生长素(IAA)和脱落酸(ABA)均显著诱导 CsADH1 上调表达,并且根中的表达水平明显高于叶中。本结果表明,CsADH1 可能在柑橘根响应黄龙病菌侵染中起着重要作用。

关键词: 柑橘, 黄龙病, 基因表达, 亚细胞定位, qPCR, Zn 离子, CsADH1

Abstract: Huanglongbing(HLB)often causes the loss and abnormal distribution of zinc(Zn)element in citrus plant. Zn,which is required for activity of alcohol dehydrogenases(ADH),plays important roles in regulation of ADH responding to biotic- and abiotic-stress. Our proteome sequencing showed that a citrus alcohol dehydrogenase 1(CsADH1)had significantly different expression in HLB-affected citrus compared with healthy control. Here,the 1 143 bp-long CDS sequence of CsADH1,which encodes a protein of 380 amino acid residues,was cloned from‘Wanjincheng’orange(Citrus sinensis Osbeck). Informatic analysis showed that CsADH1 had the GroES and ADH_zinc_N functional domains. Sequence alignment showed that CsADH1 shared high amino acid identities with alcohol dehydrogenase(ADH)members from Arabidopsis,castor and lotus. Subcellular localization analysis showed that CsADH1 protein located in the cytoplasm of onion epidermic cells. Real-time quantitative analysis revealed that CsADH1 had up-regulated expression in root and midrib of‘Wanjincheng’orange after HLB pathogen infection and the expression level in root was 10 times higher than that in midrib. CsADH1 expression was also upregulated by Zn ion,salicylic acid(SA),auxin(IAA)and abscisic acid(ABA)induction and its expression levels in roots were obviously higher than that in leaves. The results suggested that CsADH1 play a more important role in the responses of citrus roots to HLB infection compared with midrib.

Key words: citrus, Huanglongbing(HLB), gene expression, subcellular localization, qPCR, Zn ion, CsADH1

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