The Study Of Pollen Transgenic Technology And RNA Mediated Broad-Spectrum-PRSV Resistance Papaya

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.