The Study On Agrobacterium Tumefaciens-medited Cecropin Foreign Gene Transfer In Houttuynia Cordata Thunb

Plant Genetic Engineering achieved remarkable results after more than 20 years of development,so far,the world has isolated more than 100 used target gene for the purpose of genetic engineering,transgenic plants were nearly 200.Nearly 1,000 cases of transgenic plants were approved to enter field trials,involving more than 50 plant species,has approved 48 transgenic plants for commercial production.In the aspect of transgenic medicinal plants,China has realized the foreign gene expression in the Chinese wolfberry, pinellia tuberifera tenore,and other Chinese herbal medicine.Cecropin is a small molecule antibacterial activity of the cationic peptides,on a strong anti-bacterial effect,some Cecropin have anti-viral,anti-fungal activity,and kill cancer cells,which are,or even can enhance immunity,speed up wound healing process.Its unique mechanism of broad-spectrum anti-microbial activity,known as natural antibiotics. At present,the Cecropin has been transferred to tobacco,rice,vegetables,fruit trees,herbs and other plants successfully.Houttuyniae is a perennial plant which comes from the entire plants of Houttuynia cordata.It can clear heat and detoxicate,diminish carbuncle,eliminate purulence,induce diuresis for treating strangurtia.Useing for asthma,heat dysentery,heat stranguria,and carbuncle.Because of the artificial cultivation,Houttuynia growth environment of change, reduce the capacity of plant diseases and insect pests,plant diseases and insect pests in general and more serious,directly affecting the quality and yield of Houttuynia.Foreign antibacterial peptide gene transfer into Houttuynia can improve its anti-pest′s ability to enhance its antibacterial activity.Because Agrobacterium(Agrobacterium tumefaciens)Ti plasmid(Tumor inducing plasmid)transformation system is one of gene conversion systems which studies most,the theory mechanism to be clearest,the technique maturest at present.Therefore this experiment application Agrobacterium-medited method,transforms foreign antibacterial peptide gene Cecropin B and NP-1 to the cordate houttuynia.We explored the two kinds of different Agrobacterium EHA105 and LBA4404 the conditions which transforms to the cordate houttuynia.1 Experimental research1.1 Construction Cecropin plant expression vector pCAMBIA1305.2-CNCut off the target gene CN and the cauliflower mosaic virus promoter which on the expression vector pBI121-CN that have constructed the laboratory,inserted them into the multiple cloning sites of plasmid pCAMBIA1305.2,constructing the plant expression vector pCAMBIA1305.2 - CN.Identified by double- digestion,PCR,and DNA sequencing show that recombinant pCAMBIA1305.2-CN on the Cecropin B and NP-1 fusion gene have the same gene sequence with the original design gene.Integration of plant gene expression vector pCAMBIA 1305.2-CN has successfully constructed.1.2 Construction Agrobacterium EHA 105/pCAMBIA1305.2-CNPlant expression vector pCAMBIA1305.2-CN was transformed into Agrobacterium EHA105 for the infected leaves.Extraction pCAMBIA1305.2-CN boiled from the EHA105/pCAMBIA1305.2-CN. Identified by PCR,that successful construction of EHA105/pCAMBIA1305.2-CN.1.3 Agrobacterium EHA105/pCAMBIA1305.2-CN transfer into Houttuynia and conditions of the optimizationAt different temperatures,different pre-culture time,different co-culture time, different acetosyringone concentration use Agrobacterium EHA105/pCAMBIA1305.2-CN transfer into Houttuynia,through GUS detection,and optimize the conditions for transformation.In 25～26℃,pre-culture 5d,co-culture 3d,addding 200μmol/L acetosyringone in co-culture medium conditions,the application of EHA105/pCAMBIA1305.2-CN transfer into Houttuynia,the initial access Houttuynia resistance buds.1.4 Construction Agrobacterium LBA4404/pBI121-CN and LBA4404/pBI121Plant expression vector pBI121-CN and pBI121 were transformed into Agrobacterium LRA4404 for the infected leaves. Extraction LBA4404/pBI121-CN and LBA4404/pBI121 boiled from the plasmid pBI121-CN and pBI121,identified by PCR,that successful construction of Agrobacterium LBA4404/pBI121-CN and LBA4404/pBI121.1.5 Agrobacterium LBA4404/pBI121 transfer into Houttuynia and conditions of the optimizationAt different temperatures,different pre-culture time,different co-culture time, different acetosyringone concentration use Agrobacterium LBA4404/pBI121 transfer into Houttuynia,through GUS detection,and optimize the conditions for transformation.In 23℃,pre-culture 6d,co-culture 7d,addding 200μmol/L acetosyringone in co-culture medium conditions,the application of Agrobacterium LBA4404/pBI121-CN transfer into Houttuynia,the initial access Houttuynia resistance plants.2 ConclusionThrough exploration the different Agrobacterium transformed condition of houttuynia, initially established the agrobacterium-mediated transformation cordate houttuynia system of foreign Cecropin gene.For the next step research Cecropin gene′s in in the expression of cordate houttuynia and the transgene cordate houttuynia′s antibacterial performance and so on has laid the foundation.And provide a theory and the application basis for the antibacterial peptide expression in the Chinese medicine plants.