Study On The Transformation Of Eucommia Ulmoides Oliver Gene(EuREF1) Into Dandelion(Taraxacum Mongolicum Hand.-Mazz)

Eucommia ulmoides belonging to Eucommia,Eucommiaceae is a famous medicinal woody species as well as a collagen plant with a wide range of adaptation areas.Eucommia gum is a unique resource in China.Due to its excellent insulation,corrosion resistance and acid and alkali resistance,eucommia gum has great development prospects.At present,it is generally believed that the important components of rubber ion membrane proteins are rubber elongation factor,small rubber granule protein and rubber transferase,which are involved in rubber biosynthesis.The rubber elongation factor plays an important role in the extension of isoprene chains and the polymerization of rubber molecules.In this study,genetic engineering technology was used to successfully establish a regeneration system and a genetic transformation system using Taraxacum mongolicum Hand.-Mazz and Taraxacum kok-saghyz Rodin hybrid varieties,and the Eu REF1 gene was introduced into dandelion,and the effect of Eu REF1 on the phenotype and rubber content of transgenic plants was studied.The main results are as follows:1.In this study,dandelion leaves were used as explants to successfully establish a dandelion regeneration system.The best medium for inducing adventitious bud regeneration from leaf explants is MS+1.5 mg·L^-1 6-BA+0.2 mg·L^-1 NAA,with the maximum induction rate being 91.11±3.85%;screening and culture through rooting induction of regenerated adventitious buds,The best rooting induction medium was 1/2MS+0.4 mg·L^-1 NAA,and the maximum rooting induction rate was 92.41±0.23%.2.This study established an Agrobacterium-mediated leaf disc transformation method for genetic transformation:the optimal selection pressure of kanamycin for bud regeneration is 50 mg·L^-1,and the optimal selection pressure for regenerating bud rooting is 25 mg·L^-1;When the pre-cultivation time of leaf explants was 24 hours,the OD₆₀₀ concentration of Agrobacterium tumefaciens LBA 4404 was about 0.6,and the explants were infected for 15 minutes,the acetosyringone concentration was 100?mol·L^-1,and the co-culture time was 48 hours,resistant bud induction rate is the highest;during genetic transformation,timentin was selected as a bacteriostatic agent at a concentration of 150 mg/L.3.In this study,GUS histochemical staining and genomic PCR were used to test resistant plants.It was proved that Eu REF1 gene was successfully integrated into the dandelion genome,and 7 transgenic plants containing Eu REF1 gene were obtained.Real-time PCR was used to detect the Eu REF1 gene expression in transgenic plants.The expression level of target genes in transgenic plants was up-regulated relative to wild-type plants.The alkaline extraction method was used to extract the rubber of the transgenic plants and the wild-type plants.The results showed that the rubber content of transgenic plants was increased compared with wild-type plants,the average content of 7 transgenic dandelion rubber was 5.03%higher than that of wild type dandelion rubber.In summary,Eu REF1 gene plays a key role in the process of rubber synthesis,and its genetic transformation into dandelion can increase the rubber content.