Preliminary Analysis On Transgeneic Hairy Root Induced From Cucumber By Agrobacterium Rhizogenes K599and Cloning Of Orf14Gene

Agrobacterium rhizogenes, which infected explants and induced hairy roots at the site of plant wound. Hairy roots derived from single-clones and can propagate rapidly. Plant diseases and insects, such as root-knot nematode can host in hairy roots for propagation. Hairy roots could be useful in photyremedation. Hairy roots also can be used as a bioreactor for the production of both exogenous proteins and active ingredients of medicinal plants. T-DNA region is integrated into the plant genome to cause hairy root disease. The utilizing value of the transgenic hairy roots depends on the stability and the high-efficiency expression of the exogenous gene.In this study, we analyze the integrality of T-DNA in hairy roots which were induced from different explants by Agrobacterium rhizogenes K599. Identification the exogenous genes were stably and highly expressed in long-term (3-year-old) cucumber transgenic hairy roots. The orfl4in T-DNA was cloned and a plant expression vector pRI101-AN-orfl4-gfp was constructed which can express the fusion protein of ORF14and GFP. The results as follows:1. Comparison of the frequency of hairy roots formation in the cucumber different explants inoculated with wild-type Agrobacterium rhizogenes K599harboring pRI101-AN-gfp. Hairy roots induced from hypocotyls and epicotyls with a high frequency of100%. Two types of hairy roots were obtained. That is Type-I:highly branched and fast-growing fine roots and type-II:low branched and slow-growing thick roots. A convenient and high-efficiency method for hairy roots induction was established. The results lay foundation for studying T-DNA integration related with hairy roots morphology and developing mechanism of hairy roots.2. Thirty independented culture hairy roots individual were analysed by PCR. Both the T-DNA inner gene included orf2,orf3,orf4,orf8,rolA,rolB,rolC,rolD,rolE, orfl4,cus and the exogenous gfp were integrated into the plant genome. The result showed that all genes in the T-DNA can integrated into hairy roots.3. We investigated the genetic stability of gfp-transgene in the hairy roots that had been cultured on solid medium for3years. The expression of gfp that was driven by the constitutive promoter35S was relatively normal at both mRNA and protein levels. Furthermore, the hairy roots after3-year culturing were still able to express the morphology-related rol genes. Overall, this study provided important laboratory supports for using hairy roots in the long-term industrial production of proteins and active ingredients of medical plants.4. The orfl4gene was successfully cloned from Agrobacterium rhizogenes K599by PCR method. A plant expression vector of pRI101-AN-orfl4-gfp was constructed, which can express GFP fusion protein. It is helpful to ananlyz the function of orfl4.