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Production of Transgenic Anliucheng Sweet Orange (Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance |
LI Ding-li, XIAO Xuan, GUO Wen-wu |
1、Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture & Forestry Sciences, Huazhong Agricultural
University, Wuhan 430070, P.R.China
2、College of Life and Environmental Sciences, Gannan Normal University, Ganzhou 341000, P.R.China |
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摘要 Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR (pattern recognition receptor) gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene (HPT) was introduced into Anliucheng sweet orange (Citrus sinensis Osbeck) via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin (HYG) and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identified by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was briefly discussed.
Abstract Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR (pattern recognition receptor) gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene (HPT) was introduced into Anliucheng sweet orange (Citrus sinensis Osbeck) via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin (HYG) and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identified by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was briefly discussed.
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Received: 28 October 2013
Accepted: 14 November 2014
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Fund: This research was financially supported by the National High- Tech R&D Program of China (863, 2011AA100205), and the National Natural Science Foundation of China (31125024). The authors thank Prof. Zhu Lihuang (the Institute of Genetics & Developmental Biology, Chinese Academy of Sciences, Beijing) for providing the plasmid pCXK1301. |
Corresponding Authors:
GUO Wen-wu, Tel: 86-27-87281543, Fax: 86-27-87280622,E-mail: guoww@mail.hzau.edu.cn
E-mail: guoww@mail.hzau.edu.cn
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About author: LI Ding-li, E-mail: qauldl@163.com; XIAO Xuan, E-mail: xiaoxuanxx@163.com;* These authors contributed equally to this study. |
Cite this article:
LI Ding-li, XIAO Xuan, GUO Wen-wu.
2014.
Production of Transgenic Anliucheng Sweet Orange (Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance. Journal of Integrative Agriculture, 13(11): 2370-2377.
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Afroz A, Chaudhry Z, Rashid U, Ali G M, Nazir F, Iqbal J,Khan M R. 2011. Enhanced resistance against bacterialwilt in transgenic tomato (Lycopersicon esculentum) linesexpressing the Xa21 gene. Plant Cell, Tissue and OrganCulture, 104, 227-237Alva A K, Graham J H, Anderson C A. 1995. Soil pH andcopper effects on young ‘Hamlin’ orange trees. Soil ScienceSociety of America Journal, 59, 481-487Barbosa-Mendes J M, Mourao Filho F A A, Filho A B,Harakava R, Beer S V, Mendes B M J. 2009. Genetictransformation of Citrus sinensis cv. Hamlin with hrpNgene from Erwinia amylovora and evaluation of thetransgenic lines for resistance to citrus canker. ScientiaHorticulturae, 122, 109-115Behlau F, Belasque J, Bergamin Filho A, Graham J H, Leite RP, Gottwald T R. 2008. Copper sprays and windbreaks forcontrol of citrus canker on young orange trees in southernBrazil. Crop Protection, 27, 807-813Brunings A M, Gabriel D W. 2003. Xanthomonas citri:Breaking the surface. Molecular Plant Pathology, 4,141-157Cardoso S C, Barbosa-Mendes J M, Boscariol-Camargo RL, Christiano R S C, Bergamin Filho A, Vieira M L C,Mendes B M J, Mourão Filho F A A. 2010. Transgenicsweet orange (Citrus sinensis L. Osbeck) expressing theattacin A gene for resistance to Xanthomonas citri subsp.citri. Plant Molecular Biology Reporter, 28, 185-192Cheng Y J, Guo W W, Yi H L, Pang X M, Deng X X. 2003. Anefficient protocol for genomic DNA extraction from citrusspecies. Plant Molecular Biology Reporter, 21, 177-178Collinge D B, Lund O S, Thordal-Christensen H. 2008.What are the prospects of genetically engineered, diseaseresistant plants? European Journal of Plant Pathology,121, 217-231Duan Y X, Guo W W, Meng H J, Tao N G, Li D D, DengX X. 2007. High efficient transgenic plant regenerationfrom embryogenic calluses of Citrus sinensis. BiologiaPlantarum, 51, 212-216Fu X Z, Chen C W, Wang Y, Liu J H, Moriguchi T. 2011.Ectopic expression of MdSPDS1 in sweet orange(Citrus sinensis Osbeck) reduces canker susceptibility:Involvement of H2O2 production and transcriptionalalteration. BMC Plant Biology, 11, 55.He Y R, Chen S C, Peng A H, Zou X P, Xu L Z, Lei T G, Liu XF, Yao L X. 2011. Production and evaluation of transgenicsweet orange (Citrus sinensis Osbeck) containing bivalentantibacterial peptide genes (Shiva A and Cecropin B) via anovel Agrobacterium-mediated transformation of mature axillary buds. Scientia Horticulturae, 128, 99-107Jefferson R A. 1987. Assaying chimeric genes in plants:The GUS gene fusion system. Plant Molecular BiologyReporter, 5, 387-405Lee S W, Han S W, Sririyanum M, Park C J, Seo Y S, RonaldP C. 2009. A type I-secreted, sulfated peptide triggersXA21-mediated innate immunity. Science, 326, 850-853Li D D, Shi W, Deng X X. 2002. Agrobacterium-mediatedtransformation of embryogenic calluses of Ponkanmandarin and the regeneration of plants containing thechimeric ribonuclease gene. Plant Cell Reports, 21, 153-156Liu Q, Xu J, Liu Y Z, Zhao X L, Deng X X, Guo L L, Gu J Q.2007. A novel bud mutation that confers abnormal patternsof lycopene accumulation in sweet orange fruit (Citrussinensis L. Osbeck). Journal of Experimental Botany, 58,4161-4171Liu Y Z, Liu Q, Tao N G, Deng X X. 2006. Efficient isolationof RNA from fruit peel and pulp of ripening navelorange (Citrus sinensis Osbeck). Journal of HuazhongAgricultural University, 25, 300-304(in Chinese)Livak K J, Schmittgen T D. 2001. Analysis of relative geneexpression data using real-time quantitative PCR and the2−ΔΔCT method. Methods, 25, 402-408Mendes B M J, Cardoso S C, Boscariol-Camargo R L, CruzR B, Mourão Filho F A A, Bergamin Filho A. 2010.Reduction in susceptibility to Xanthomonas axonopodispv. citri in transgenic Citrus sinensis expressing the riceXa21 gene. Plant Pathology, 59, 68-75Miao L, Shou S, Zhu Z, Jiang F, Zai W, Yang Y. 2008. Isolationof a novel tomato caffeoyl CoA 3-O-methyltransferasegene following infection with bacterium Ralsotoniasolanacearum. Journal of Phytopathology, 156, 588-596Murashige T, Tucker D P H. 1969. Growth factor requirementsof citrus tissue culture. Proceedings of the First InternaionalCitrus Symposium, 3, 1155-1161Omar A A, Song W Y, Grosser J W. 2007. Introduction ofXa21, a Xanthomonas-resistance gene from rice, into‘Hamlin’ sweet orange [Citrus sinensis (L.) Osbeck]using protoplast-GFP cotransformation or single plasmidtransformation. Journal of Horticultural Science andBiotechnology, 82, 914-923Rinaldi D A M F, Leite Jr R P. 2000. Adaptation ofXanthomonas axonopodis pv. citri population to thepresence of copper compounds in nature. Proceedings ofInternational Society of Citriculture, 2, 1064.Schubert T S, Rizvi S A, Sun X, Gottwald T R, Graham J H,Dixon W N. 2001. Meeting the challenge of eradicatingcitrus canker in Florida-again. Plant Disease, 85, 340-356Sendín, L N, Filippone M P, Orce I G, Rigano L, Enrique R,Peña L, Vojnov A A, Marano M R, Castagnaro A P. 2012.Transient expression of pepper Bs2 gene in Citrus limon asan approach to evaluate its utility for management of citruscanker disease. Plant Pathology, 61, 648-657Song W Y, Wang G L, Chen L L, Kim H S, Pi L Y, Holsten T,Gardner J, Wang B, Zhai W X, Zhu L H, Fauquet C, RonaldP. 1995. A receptor kinase-like protein encoded by the ricedisease resistance gene, Xa21. Science, 270, 1804-1806Wang G L, Song W Y, Ruan D L, Sideris S, Ronald P C.1996. The cloned gene, Xa21, confers resistance to multipleXanthomonas oryzae pv. oryzae isolates in transgenicplants. Molecular Plant-Microbe Interacttions, 9, 850-855Zhai W X, Li X B, Tian W Z, Zhou Y L, Pan X B, Cao S Y,Zhao X F, Zhao B, Zhang Q, Zhu L H. 2000. Introductionof a rice blight resistance gene, Xa21, into five Chineserice varieties through an Agrobacterium-mediated system.Science in China (Series C, Life Sciences), 43, 361-368 (in Chinese) |
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