论文标题:番木瓜花粉管转基因技术及RNA介导广谱抗病性的初步研究
The Study of Pollen Transgenic Technology and RNA Mediated Broad-Spectrum-PRSV Resistance Papaya
论文作者
论文导师 周鹏,论文学位 博士后,论文专业 作物遗传育种
论文单位 华南热带农业大学,点击次数 103,论文页数 74页File Size6717K
2006-12-01论文网 http://www.lw23.com/lunwen_13083147/
papaya ringspot virus(PRSV);; homologous DNA segments;; papaya ringspot virus coat protein(PRSV-CP);; pollen tube channel technology;; RNA-mediated virus resistance
番木瓜原产于南美洲，是著名的热带亚热带作物之一。番木瓜环斑病毒(Papaya ringspot virus,PRSV)是番木瓜生产上重要的世界性病害，极大地影响世界各地的番木瓜生产及其相关产业发展。为研究番木瓜高抗谱抗病毒的机制以及培育出适合在我国栽培的高抗谱抗环斑病毒且安全性好的番木瓜新品系。本研究采用RNA介导-花粉管转基因技术体系对抗病毒番木瓜品系的培育进行了研究，主要结果如下： 1运用RT-PCR技术及相关分子生物学技术对我国海南、云南、广西和广东等7个不同番木瓜生产区的番木瓜环斑病毒外壳蛋白基因(PRSV-CP)进行了克隆和鉴定分析。结果表明PRSV-CP编码区范围在864-873核苷酸之间，编码蛋白是288-291氨基酸。对分离的7个PRSV-CP的核苷酸及氨基酸序列的比较结果表明大部分序列差异都处于N端，而各产区番木瓜PRSV-CP基因的3′端586—864 bp区段，即278 bp DNA片断的同源率最高，达到98.5％。 2采用RT-PCR技术对番木瓜环斑病毒CP基因的同源区段进行了克隆和鉴定分析。序列分析结果表明获得PRSV-CP基因3′端同源率最高的278 bp DNA片段，并分别构建了六种RNA介导的植物表达载体，分别为：p2301CP+、p2301CP-、p2301CPu、p2301CPuL、pBCP+、pBCP-，并通过电激法导入农杆菌中。 3采用花粉管通道技术将含有CP基因同源DNA片段的植物表达载体导入番木瓜穗中红植株，通过对花粉管导入时的花朵大小、导入液类别、环境因子等参数进行了较为系统的研究，建立了利用质粒DNA和农杆菌为导入液的花粉管通道技术平台。获得了番木瓜花粉管转基因技术中比较理想的导入条件：受体花一般长3.0-4.5cm；一般供体的花采用3.5cm左右的两性花：而对受体来说，质粒DNA为导入液的一般采用2.0-4.0cm的雌花，农杆菌为导入液的一般采用3.0-4.5cm左右的雌花；转化时间一般选择在晴朗天气，气温在20-30℃时比较适宜。 4通过花粉管直接转化技术对6种RNA介导的植物表达载体进行基因转化，获得相应的转基因植株，并对转基因植株进行了分子鉴定，为筛选、培育出高抗谱抗环斑病毒且安全性好的番木瓜株系奠定基础。
Papaya (Carica papaya L.), which originated in tropical America, is one of themost widely grown crops in tropical and subtropical areas. The potyvirus Papayaringspot virus (PRSV) is an important pathogen of papaya which causing severelosses in economic crops papaya production globally. In order to designing anddeveloping durable virus resistance-PRSV transgenic papaya in china, meanwhilebroad-spectrum-virus resistance, strongly resistance-PRSV and good safe papayalines used RNA mediated-pollen tube channel technology. The the main results wereas follows: 1 The coat protein gene of Papaya ringspot virus (PRSV) isolates originating fromseven different china plantation (four from Hainan, one from Guangdong, one fromGuangxi and one from Yunnan) were cloned and sequenced using RT-PCR. TheCP-coding region varied in size from 864 to 873 nucleotides, encoding proteins of288-291 amino acids. Considerable heterogeneity in the CP length was observed andall differences in length were confined to the N terminus of the CP-coding region. Thehigher sequence identity regions were found in 586-864 nt of the seven PRSVCP-coding region of C terminus as 98.5％. 2 The coat protein gene homologous segment of Papaya ringspot virus (PRSV)was cloned and sequenced using RT-PCR. The 278 bp highest homologous segmentssequence identity regions at C terminus of the PRSV-CP-coding region was gained.The six kinds of RNA-mediated plant expression vectors are gained respectively byusing a series of cloning methods.: p2301CP+、p2301CP-、p2301CPu、p2301CPuL、pBCP+、pBCP-. The recombinant plasmid was transferred into competent cells ofAgrobacterium tumefaciens EHA105, 3 The RNA-mediated plant expression vectors with the CP gene homology DNAfragment were introduced into papaya suizhonghong adult plant using the pollen tubechannel technology. The pollen tube channel technology platform using materialparticle DNA and the Agrobacterium tumefaciens was established through more systematic research the flowers size, inducts parameter and environment factor and soon parameters. The ideal condition of the papaya pollen tube channel technology werethe follows: The general donor flower were about 3.5 centimeters hermaphroditeflowers; But when said to the acceptor flower, material particle DNA for inductorgenerally to pick 2.0-4.0 centimeter female flower, the Agrobacterium tumefaciensfor inductor generally to pick 3.0-4.5 centimeter female flower; The transformed timegenerally chooses when the sunny weather, the temperature 20-30℃compares issuitable. 4 The six kinds of RNA-mediated plant expression vectors were introduced intopapaya soloⅡadult plant through the pollen tube direct transformation technologyand obtains the corresponding transgenic plant, which of them had been recognizedusing molecular appraisal. It would provide theory and research bases for gainingbroad-spectrum-virus resistance of papaya line.
|
