The Construction Of VIGS System In Peach Leaf And Strawberry Fruit

Virus-introduced gene silencing is a technique that exploits the RNA-mediated antiviral mechanism. The modified virus vector carrying the fragment of a target gene were constructed and then inserted to silence the target gene within host plants by Agrobacterium-meditated transformation. The study chose tobacco rattle virus (TRV) and peach leaf or strawberry fruit to construct the gene-silencing vector carrying the fragment of CHLH gene and the infection of the vector indicated that the silencing of the target gene CHLH resulted in photo bleached young peach leaf or delay the strawberry fruit development. semiquantiative RT-PCR and quantiative RT-PCR analysis was employed to test the effect of silencing the target gene CHLH which show that the gene CHLH was significantly degraded.This gene-silencing system will benefit the high-flux analysis of plant genes in the future.Compared with advance in model plants or crops, the studies of molecular biology in fruit trees have been very backward due to the difficulties in gene transformation and manipulation. Therefore, exploring an efficient system for gene manipulation is of particular significance in pomology science.In this papaer, we report using virus-induced gene silencing (VIGS) technique for gene function analysis in peach and strawberry fruit. A 4,445-bp cDNA, encoding a putative H subunit of magnesium chelatase(CHLH) that loss function coupling with yellow or while color, was cloned from peach and in strawberry it is 4,312bp. To establish VIGS,A 650-bp cDNA fragment of CHLH gene in peach and strawberry were selected, and cloned into virus vector pTRV2. The pTRVl, pTRV2 and its recombinant plasmids pTRV2-PpCHLH(650bp) were transformed into Agrobacterium strain GV3101 via freeze-thaw method respectively.The recombinant Agrobacterium mixture was infiltrated into leaves of 4-5 week-old peach seedlings and strawberry fruit with a needleless 50ml syringe. About 15 days after transformation inpeach leaf,it was observed that the infected areas of the leaves faded, and finally tuned into yellow or white color, which is just identical with the loss of PpCHLH function.while in strawberry,the fruit did not start color after 7 days infection. Semiquantitative PCR, real-time PCR and siRNA northern blot analysis all showed that the expression of CHLH was completely silenced, demonstrating that the VIGS in peach and strawberry was successfully developed. This would contribute to promote the studies of molecular biology in fruit trees.