The Acquisition Of The High Efficient And Broad-spectrum Papaya Ringspot Virus Resistant Transgenic Papaya By Exploiting The RNAi Technology

Papaya(Carica papaya)is one of the most important consumer and export fruit in tropical regions of the world.It is named as the first value of the top ten most nutritious fruit by World Health Organization and loved by the majority of consumers.papaya ringspot virus(papaya ringspot virus;PSPV)is the biggest limiting factor in the papaya planting,which can lead to serious damage or even without harvest.Hainan is the largest papaya planting areas in China.Currently,the papaya planting is seriously threatened by PRSV without high resistant papaya species.Due to the lack of resistance germplasm,conventional breeding methods can not breed resistant varieties;and no effective pesticide can prevent.From a global perspective,the most effective method of prevention and control of PRSV are to cultivate virus-resistant transgenic varieties.PRSV with geographical strains,the virus strains are different in different regions.Previous studies find that the PSV coat protein gene transformed varieties from Hawaii or Taiwan have varying defence against PRSV isolates from other geographical regions.And those transgenic cultivars fail to show resistance to Hainan isolates.That is the reason for the world's first commercialized transgenic papaya"Rainbow" from 1998 restricted on the use in HawaiiTo date,the practical application of the anti-viral genes are derived from papaya viral gene itself.Such transgenic resistant behavior is caused by RNA-mediated post-transcriptional gene silencing(PTGS)that is RNA interference.RNAi(inverted repeat double stranded RNA molecule)is considered as the most appropriate RNA silencing initiator.Generally viral single-stranded sense or antisense gene confers resistance to the plant is only about 20%.But an inverted repeat sequence of a IR plant the dsRNA contribute to the virus resistance up to 90%.On the basis of diversity analysis and conserved sequence analysis for Hainan PRSV strains,we perform 3 vectors by hairpin RNAi antiviral efficient design.More than 3,000 transformed seedlings are obtained by optimized transformation system.A number of highly efficient and broad-spectrum resistant lines have entered the GMO safety assessment procedures.It has laid a good foundation for high resistant,high quality and high yield papaya breeding.The specific results are represented as follows:1.By survey,sampling,PCR identification,sequencing,cluster analysis,etc.,species and distribution of Hainan PRSV virus strains are studied in physiological and molecular level.Present work has provided many new insights into the diversity of PRSV in Hainan.(1)We confirmed and expanded the two previously published Hainan populations(Hainan I,and II).(2)We also reported a novel PRSV population,Hainan III.(3)Three Hainan types of PRSV have varied distribution among the cities.Hainan I group is mainly in Sanya,Hainan ? group is mainly in Chengmai,Hainan III group is mainly in Wenchang and Changjiang.Ledong is located in the intersection of three types of strains,virms strains are most complex and diverse.(4)By surveying the diversity of PRSV in Hainan,we verified our previous hypothesis that the higher divergence and distinct differences from other sources of PRSV suggest that transgenic papaya from Taiwan and Hawaii would not be successful in Hainan.(5)Since greatly influenced by Non-Darwin and Darwinian selection,Hainan PRSV has a high mutation rate,that need to be considered in breeding.2.According to previous transgenic virus-resistant breeding experience of papaya,we select the important functional genes CP,Nib,Hc-Pro of Hainan PRSV for sequencing.After bioinformatics comparison,the conserved functional regions of these genes are cloned from Hainan typical PRSV strain to construct efficient RNAi hairpin vectors.We use the RNAi vectors to transform papaya and obtain batch transgenic lines.The design incorporates the successes and shortcomings of papaya breeding for virus resistance and introduces the high efficient vector idea of RNAi.3.The transgenic papaya lines are identified at the molecular level.Then they are propagated and evaluated in virus resistance on the physiological level,in the laboratory and greenhouse conditions.4.The next step is to focus on the further resistance and stability evaluation of papaya resistant transgenic lines and their third generation plants and to complete the GMO safety assessment experiments for genetically modified plant.The paper comprehensively applies Virology,Bioinformatics,Disease,physiology,Molecular biology,Transgenic breeding etc.to cultivate high efficient and broad-spectrum anti-PRSV papaya new germplasm for Hainan papaya commercial planting.The success of the project will push the headed papaya varieties in Hainan and South China with our own intellectual property and can even greatly promote China's papaya industry.