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园艺学报 ›› 2017, Vol. 44 ›› Issue (10): 1881-1893.doi: 10.16420/j.issn.0513-353x.2017-0216

• 研究论文 • 上一篇    下一篇

柑橘响应溃疡病菌转录因子基因CsAP2-09的克隆与功能分析

贾瑞瑞,胡安华,陈善春,邹修平,彭爱红,许兰珍,雷天刚,姚利晓,白晓晶,何永睿*,李 强*   

  1. (西南大学/中国农业科学院柑桔研究所,国家柑桔工程技术研究中心,重庆 400712)
  • 出版日期:2017-10-25 发布日期:2017-10-25

Cloning and Expression Analysis of CsAP-09:a Transcription Factor Related to Citrus Canker Disease

JIA Ruirui,HU Anhua,CHEN Shanchun,ZOU Xiuping,PENG Aihong,XU Lanzhen,LEI Tiangang,YAO Lixiao,BAI Xiaojing,HE Yongrui*,and LI Qiang*   

  1. (Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences,National Citrus Engineering Research Center,Chongqing 400712,China)
  • Online:2017-10-25 Published:2017-10-25

摘要: 对柑橘中AP2转录因子进行注释,并克隆柑橘溃疡病相关的CsAP2-09,研究外源水杨酸、茉莉酸甲酯、乙烯利、机械损伤以及溃疡病菌对该基因的诱导表达,确定其表达模式。从柑橘全基因组公共数据库一共注释出12个AP2成员,据系统发育和结构可以将其分为4个亚类;可能与抗、感溃疡病相关的CsAP2-09基因全长4 159 bp,开放阅读框1 467 bp,编码488个氨基酸,具有典型的AP2结构,同时也具有一些结构特殊性;该基因在细胞核中优势表达,与定位信号预测结果吻合;上游启动子元件含多个与植物逆境或激素应答相关的顺式作用元件,如BOX-W1、CGTCA-motif、TCA-element、WUN-motif等;诱导结果表明CsAP2-09对外源水杨酸、茉莉酸甲酯、乙烯利及机械损伤均有响应;柑橘溃疡病菌侵染可诱导抗病品种四季橘中此基因明显上调表达,而在感病品种纽荷尔中变化趋势不显著。上述研究表明CsAP2-09是一个响应溃疡病菌侵染的,可作为研究柑橘抗溃疡病的候选基因。目前已将其在锦橙中超表达,共筛选出8个转基因植株,后期将对其进行抗病性评价以确定其分子育种价值。

关键词: 柑橘, 柑橘溃疡病, AP2, 转录因子, 亚细胞定位, 基因表达

Abstract: The AP2 family of Citrus sinensis will be annotated and analyzed while the citrus canker related transcription factor CsAP2-09 will be cloned and analyzed in this study. It is also aimed to confirm the subcellular localization and the expression profiles induced by exogenous hormones,Xanthomonas citri subsp. citri(Xcc)and mechanical wounding. Twelve AP2 genes are extracted and annotated from the public genomic databases of C. sinensis. These 12 AP2s can be divided into 4 categories based on the phylogeny and the motifs. Amino acid sequence and structure analysis indicates that the full-length of CsAP2-09 is 4 159 bp with a 1 467 bp open reading frame which codes a protein containing 488 amino acids. CsAP2-09 contains classic AP2 domains and some specific motifs which give essential and special functions. Subcellular localization results confirm the prediction of the protein localization in nucleus. The promoter contains multiple cis-acting elements involved in plant adversity or hormone responses,such as BOX-W1,CGTCA-motif,TCA-element,WUN-motif and so on. Based on the qPCR data,the CsAP2-09 responds to the exogenous salicylic acid,jasmonic acid methyl ester,mechanical damage and ethylene. Xcc attack can significantly increase the expression of CsAP2-09 in Calamondin but no significant change in Newhall navel orange. All the experiments show CsAP2-09 would be an important transcription factor which is closely associated with the resistance of citrus canker. This gene should be a potential candidate in the molecular breeding to improve the canker resistance of citrus. We have over expressed CsAP2-09 in C. sinensis and obtain 8 transgenic seedlings. Then the evaluation of the resistance will be performed to determine the value of molecular breeding.

Key words: Citrus, citrus canker disease, AP2, transcription factors, subcellular localization, gene expression

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