Study On Transformation Of Papaya Ringspot Virus Coat Protein Gene IR Expression Vector And Virus Resistance

Papaya is widely damaged by virus in production, among which Papaya ringspot virus (PRSV) is a main restriction factor and a world-wide serious disease. For the lack of PRSV resistance in the recently cultivars, the differences between PRSV lines at different places, the incompatible phenomenon of distant hybridization, make it difficult for the breeding of PRSV-resistance papaya.With the development of genetic engineering, a new approach for the research of plant virus resistance came out. With the big progress of RNAi, the study on plant virus resistance using inverted repeats expression vector became a hot spot. People can obtain stably inherited virus-resistance plants by the way of integrating the virus genes into plant genome. This way can avoid the difficulty by the regular method of screening plant strains.In this study, the host plants cucumber and papaya were used as plant materials. The main experiment methods were agrobacterium mediated gene transformation and PEG mediated gene transformation. The agrobacteria with genes which were structured with the CP gene inverted repeats were transformed into plant materials. And then we analyzed the virus-resistance of transgenic plant materials. The main results of this study were as follows:(1) Different parts of papaya were placed on different embryonic callus inducing culture media. It was found that embryonic callus could be induced from stem segments and immature zygotic embryos when they were placed on C3 culture medium. Embryonic callus could then be induced into somatic embryo. What's more, the time of somatic embryos induced form immature zygotic embryos was much shorter than from stem segments, which was about 80 days. When the somatic embryos were separated and put on C3 medium, new somatic embryos could be induced in 27 days. It is a way to get somatic embryos which can save much time and energy.The induced somatic embryos were transformed by agrobacterium mediate gene transformation. However, no transformation seedlings were obtained as a result of agrobacterium pollution. Some of the somatic embryos were not polluted, but they became browning on the screening culture medium with the time. (2) Different parts of cucumber were used as explants, different culture media were used to induce adventitious buds. It was discovered that adventitious buds in good condition could be induced on culture medium MS+1.5 mg/L 6-BA+2.0 mg/L AgNO3 when cotyledon node of cucumber was selected as explant. The adventitious buds could elongate and take root in 1/2MS medium.Different methods were tried to transform the expression vectors pWatergate-CP861IR and pHellsgatel2-CP215IR which contain the inverted repeat sequence of coat protein gene. 11 presumptive transformation seedlings were obtained, one of them was verified to be a pWatergate-CP861IR gene transformation seedling by PCR detection, three of them were pHellsgate12-CP215IR gene transformation seedlings. The positive rate is 36.4%. It was discovered that all of the four transformation seedlings were obtain from the cotyledonary nodes which were treated with supersonic wave for 2 to 3 seconds. There isn't any transformation seedlings obtained from other treatments. It was illustrated that the transformation rate could be increased by ultrasonication. The obtained transformation cucumber seedlings were transplanted after seedling adaptation, but they didn't survive.(3) PRSV particles were extracted from PRSV infected leaves. The concentration of the extracted virus was 1.0875mg/L. Protoplasts were made from papaya leaves of seedlings, and were co-transformed with pHellsgatel2-CP861IR gene and PRSV particles. The RNA of protoplasts was extracted, and the inhibition effect of pHellsgate12-CP861IR gene to PRSV was detected by RT-PCR.It was showed that the papaya we used was CP gene transformation papaya. No CP gene was detected by RT-PCR when the papaya protoplasts were transfected with 0.2μg or 2μg PRSV. And CP gene could be detected when the protoplasts were transfected with 20μg PRSV. CP gene couldn't be detected when the protoplast were co-transfected with 10μg pHellsgatel2-CP861IR gene and 0.2μg,2μg or 20μg PRSV. It demonstrated that the expression of CP gene was suppressed by the RNAi effect launched by pHellsgate12-CP861IR gene.