Study On Transformation Of Papaya Ringspot Vir (PRSV) Ci And Nib Gene Inverted Repeats Experssion Vector

Papaya ringspot virus(PRSV) is the most destructive virus disease of papaya, and cause great damage in the warldwide. Up to now, there was still no efficient chemical preparation to completely control this disease. The traditional method were based mainly on the measures of cultivation. The development of plant genetic engineering provided a new effective way to the study of plant virus resistance, expecially the big progress of RNAi, the study on plant virus resistance using inverted repeats expression vector became a hot spot in the study of plant virus resistance.In our study, Agrobacterium mediated gene transformation method were used to harboured pHellsgate-CIIR and pHellsgate-NIbIR, which structured the ci gene and the nib gene inverted repeats. we also employed protoplast transient expression system to study repeats expression vector's silence effect to the target gene. The main results of this study is as follows:(1) Papaya hypocotyledonary axis, leafstalk, zygotic embryo and callus were used to induce embryoid. We only gained embryoid using zygotic embryo and callus. Among the two materials, zygotic embryo was better for induce embryoid. Embryoid could generated in 40 days.(2) We established regeneration system of cucumber using cotyledonary node. Using the agrobacterium carrying pHellsgate-CIIR and pHellsgate-NibIR transformed cotyledonary node of cucumber. Transformed bud were obtained through antibiotics screening.(3) PRSV virion and repeats expression vector pHellsgate-CIIR were used to cotransform papaya protoplasts. The cotransformed protoplasts incubated at room temperature for 12 to 48 hours until RNA extraction and RT-PCR. From 24 hours to 48 hours, the expression of CI mRNA could be detected in the protoplasts only infecting with PRSV. As time increasing, the amount of CI mRNA also increased. But in protoplasts which cotransformed both PRSV and pHellsgate-CIIR, the expression of CI mRNA was not detected. It illustration that repeats expression vector pHellsgate-CIIR's product could inhibit the expression of ci gene.