Stability And Resistance Analysis Of Houttuynia Cordata Thunb. Transformed With Foreign Antimicrobial Peptides

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. Using for curing 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 much more serious, directly affecting the quality and yield of Houttuynia. Foreign antibacterial peptide gene transfered into Houttuynia can improve its anti-pest's ability and enhance its antibacterial activity.Cecropin is a small molecule antibacterial activity of 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. The antimicrobial peptide genes were used in genetic engineering for breeding, disease resistance.Our laboratory designed and synthesized the insect antibacterial peptide cecropinB and rabbit defensin fusi, because both the structure and mechanism of different characteristics, may play a synergistic role in the performance of a stronger antibacterial ability.In this paper, we optimize the method of Agrobacterium-mediated transformation on Houttuynia, and analyse the stability of exogenous genes initially in transgenic Houttuynia. Western blot analysis is employed to detect expression of exogenous antimicrobial peptide in transgenic plant. Both wild and transgenic plants are inoculated with E.coil.K12 and Rhizoctonia solani to detect the activity of foreign antimicrobial peptide. The results are list blow:1. Obtained of Agrobacterium-mediated transgenic HouttuyniaThe Agrobacterium tumefaciens EHA105 has powerful virulence. The transformation plants can express stably, but the effect is not very well when use it mediate the transformed on Houttuynia. In order to solve this issue, we optimize the method of transformation based on the system our laboratory has already built. In this study, we find the best selection concentration of hygromycin is 1.5mg/L,we obtain 6 resistance buds under this selection concentration. Rapid PCR detection results show that 6 resistance buds are positive.2. Screening and stability analysis of transgenic HouttuyniaThe laboratory has constructed expression vectors LBA4404/pBI121-CN that harbor kanamycin resistance selection marker gene and the exogenous peptide genes. Kananmycin is smeared to the surface of the leaves to screen the transgenic plants containing the nptâ…¡gene.The plants with resistance to Kananmycin is further validated by PCR detection of the npt II gene and target gene CN.The study show the optimum for screening to smear Kanamycin 5000mg/L and then observe the result after 4 days. The PCR analysis shows that the equivalent ratio of resistant gene nptâ…¡was 96% in the resistant plants, and the equivalent ratio of target gene CN was 37%. A method of quick screening transgenic Houttuynia cor data Thunb.has been established. The stability of target gene is less than the selecting maker gene.3. Expression of foreign antimicrobial peptide and resistance disease of transgenic HouttuyniaSDS-PAG electrophresis and Western blot analysis are used to detect the exogenous antimicrobial peptide expression.Extracing Houttuynia total protein to do the zone of inhibition experiments. Wild type Houttuynia and transgenic Houttuynia are inoculated with different concentrations of spores of Rhizoctonia solani to detect and the infected situation. In comparison with non-transgenic plant, transgenic Houttuynia produced a new band larger than 7 kDa, similar with expected 8 kDa of target protein, indicating that this band is probably the product of foreign gene CN. Western blot analysis show a specific band with size larger than 7 kDa appeared in the lane for transgenic samples, while did not for non-transformed. The experiment of zone of inhibition show the exogenous antimicrobial peptide expression can enhance transgenic Houttuynia resistance to E.coil. K12. The exogenous antimicrobial peptide gene in the transgenic plant can be expressed. It is optimum to inoculate spores 3 X 105/mL concentration and observe the result after 3 days. The resistance of transgenic plant has been enhanced. The exogenous antimicrobial peptide expression can enhance transgenic Houttuynia resistance to stem rot disease.4. Comparative morphology of transgenic and non-transgenic HouttuyniaIn this study, we find that transgenic and non-transgenic plants have some apparent differences in morphplogy.Compared with non-transgenic plants,we find that the leaf of transgenic plants turned curl, shrinkage. In addition, the leaves become thinner and the petioles become thicking. The results of temporary installation pieces show epidermal cells of transgenic Houttuynia is smaller than that of non-transgenic plant, epidermal cells are more closely arranged.However, in further study of plant tissue paraffin sections, we do not find apparent difference between transgenic and non-trasngenic plants.Conclusions:Through the study of smearing Kanamycin on the resisitant transgenic plants which grow well in the medium of Kan, a method of quick screening transgenic Houttuynia cordata Thunb.has been established,and find the stability of target gene is less than the selecting maker gene.The study of Houttuynia total protein show that the exogenous antimicrobial peptide expression can enhance transgenic Houttuynia resistance.Then, we find that ransgenic and non-transgenic plants have some apparent differences in morphplogy,for further study of foreign genes into plants genome and the production of unintended effects provide theoretical and application basis.