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园艺学报 ›› 2014, Vol. 41 ›› Issue (3): 417-428.

• 果树 • 上一篇    下一篇

分泌型Cecropin B 抗菌肽基因转化血橙提高其抗溃疡病水平

邹修平*,彭爱红,刘琦琦,何永睿,王军政,许兰珍,雷天刚,姚利晓,陈善春*   

  1. 中国农业科学院柑橘研究所/西南大学柑橘研究所,国家柑橘品种改良中心,柑橘学重庆市级重点实验室,重庆
    400712
  • 出版日期:2014-03-25 发布日期:2014-03-25
  • 基金资助:

    重庆市自然科学基金项目(cstc2011jjA80010);科技部农村领域国家科技计划课题(2011AA100205);国家现代农业产业技
    术体系建设专项资金项目(CARS-27);农业部公益性行业(农业)科研专项(2010 03067-02-4);柑橘学重庆市市级重点实验室开放基金计
    划项目(CKLC201105);中央高校基本科研业务费专项(XDJK2012B023)

Secreted Expression of Cecropin B Gene Enhances Resistance to Xanthomonas
axonopodis pv. citri in Transgenic Citrus sinensis‘Tarocco’

ZOU Xiu-ping*,PENG Ai-hong,LIU Qi-qi,HE Yong-rui,WANG Jun-zheng,XU Lan-zhen,LEI Tian-gang,
YAO Li-xiao,and CHEN Shan-chun*   

  1. Citrus Research Institute,Southwest University;Citrus Research Institute,Chinese Academy of Agricultural Sciences;
    National Citrus Engineering Research Center;National Center for Citrus Varieties Improvement,Chongqing 400712,
    China
  • Online:2014-03-25 Published:2014-03-25

摘要: 为了利用Cecropin B(CB)抗菌肽基因提高柑橘对溃疡病的抗性,人工合成了两个含有信
号肽的Cecropin B 抗菌肽基因PR1aCB 和AATCB。洋葱表皮瞬时表达分析表明,与非分泌型CB 抗菌肽
相比,PR1aCB 和AATCB 抗菌肽在细胞间隙中优势积累。进一步构建了CaMV 35S 调控 PR1aCB 和AATCB
基因的植物表达载体,转化塔罗科血橙(Citrus sinensis Osbeck)上胚轴,获得转基因植株。GUS 组织化
学染色、PCR 和Southern blot 分析表明外源基因已成功整合入柑橘基因组。Real-time PCR 定量分析表明,
抗菌肽基因在转基因植株中成功表达。柑橘叶片离体抗性分析表明,转PR1aCB 和AATCB 基因植株的抗
性显著强于非转基因植株,其抗性水平与高抗品种‘南丰蜜橘’(C. succosa Hort. Ex Tan)和‘四季橘’
(C. madurensis)相当。

关键词: 柑橘, 信号肽, 抗菌肽, 柑橘溃疡病, 遗传转化, 抗性

Abstract: A Citrus canker caused by Xanthomonas axonopodis pv. citri(Xac)is a very destructive
disease,which affects the citrus industry in most citrus-producing areas of the world. Here,we reported the
production of transgenic Tarocco blood orange(C. sinensis Osbeck)containing the synthetic antibacterial
peptide genes and the evaluation of transgenic plants for resistance to Xac. Two new Cecropin B(CB)
antibacterial peptide genes PR1aCB and AATCB with signal peptide(SP)sequence were synthesized by PCR. Transient RFP expression showed that fluorescence accumulation was observed predominantly in
intercellular space surrounding the onion epidermal cells transformed with PR1aCB︰rfp and AATCB︰rfp
genes compared with CB︰rfp without SP,indicating the SPs could direct protein secretion to the apoplast.
PR1aCB and AATCB gene cassettes driven by CaMV 35S were introduced into Tarocco blood orange by
Agrobacterium-mediated transformation. Integration of transgenes into citrus genome was confirmed by
GUS histochemical staining,PCR and Southern blot. Transcriptions of PR1aCB and AATCB genes were
detected by Real-time quantitative PCR in transgenic plants. Transgenic leaves were in vitro inoculated
with suspension of Xac. Compared to control(nontrangenic plants and pGN transgenic plants without
antibacterial gene),ten transgenic lines showed significant increase in resistance to citrus canker. The
levels of the resistance of the transgenic lines were equivalent to that of the highly resistant varieties:
Nanfeng Miju(C. succosa Hort. Ex Tan)and Calamondin(C. madurensis).

Key words: Citrus, signal peptide, antibacterial peptide, citrus canker, genetic transformation, resistance

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