Agriobacterium-mediated Transformation Of Antibacterial Peptide Gene For Anthurium Andraeanum

Anthurium andraeanum,a kind of perennial herb which belongs to Anthurium genus of araceae family,is gradually favored by consumers because of its beautiful patterns,unique pattern and the long flowering period.Now it has become the second largest tropical flowers goods.However,anthurium is highly vulnerable to bacterial blight in production process,even a devastating loss.So it has positive significance to develop anthurium antibacterial transgenic research.Agrobacterium mediated method is currently the most widespread transgenic method,callus also commonly used for genetic transformation receptor material.Therefore,the establishment of the high frequency regeneration system and genetic transformation system for anthurium transgenic breeding research has important value.Based on previous researchers,this study was mainly armed to the establishment of the high frequency regeneration system in anthurium,prokaryotic expression of foreign genes and genetic transformation system for agrobacterium mediated method.The main research results were as follows:1.Used the anthurium andraeanum plantlets as test materials and the 1/2 MS medium as basic medium,the cytokinin of TDZ,processing methods and the type of explants were researched the influence of the callus induction of anthurium andraeanum.The results showed that when the concentration of TDZ was 2.0 mg/L,appropriate treatment of the blade could increase the callus induction rate and shorten the induction time.The callus could be induced after 20 d.Therefore,the best way to induce callus of Anthurium were that Anthurium andraeanum explants were moderately cut with the culture medium of 1/2 MS+TDZ 2.0 mg/L+2,4-D 0.1 mg/L.2.Used anthurium callus as test materials,the kanamycin,hygromycin and cefotaxime were discussed the effects on callus growth and differentiation.The results showed that anthurium callus were more sensitive to hygromycin than kanamycin,and no sensitive to cefotaxime.So in the cultivation of Anthurium callus screening,using 30mg/L of hygromycin for resistance screening,500 mg/L of cefotaxime for bacteriostatic culture was more appropriate.3.In order to improve the antibacterial effect of agrobacterium tumefaciens,nano-antibacterial materials were used to process bacteriostatic test.The four different kinds of nano-antibacterial materials were been discussed the antibacterial effect on agrobacterium tumefaciens and the influence on anthurium callus growth and differentiation.The results showed that the reagent silver nanoparticles can inhibit the growth of agrobacterium tumefaciens,and the minimum inhibitory concentration was 25mg/L.With 25mg/L reagent silver nanoparticles treated anthurium material what were contaminated by agrobacterium tumefaciens,the agrobacterium pollution could be suppressed,the antibacterial rate was 100%,and the growth of the anthurium callus and adventitious buds were normal.4.The antibacterial peptide gene AaAMP is connected with the vector pET-28a,the prokaryotic expression vector pET-28a-AaAMP was constructed and transferred to E.coli BL21.After the IPTG inducing expression 1 h,in the Tricine-SDS-PAGE chromatogram of the total protein of the bacteria,the differential proteins were observed.And its molecular weight was consistent with the theoretical value of the target protein.The resulte showed that he antibacterial peptide gene can induce expression of antibacterial peptide protein.5.In order to optimize the Anthurium genetic transformation system,the pre-culture time and co-culture time of the Anthurium callus were studied.The results showed that under the condition of pre-culture 3d and co-culture 1d,the contamination rate of callus was lower,the survival rate of callus was the highest,more resistant materials can be screened.So,the genetic transformation system was that:the callus were cut into 1 cm×1 cm×0.5 cm and pre-cultured for 3 days,then infection 20 minutes in Agrobacterium tumefacien suspension,co-culture for 1 day,then put in the screening culture medium for screening culture until the resistant callus and adventitious buds were obtained.Further identification of the resistant callus showed that the antibacterial peptide gene AaAMP had been integrated into the genome of partial transgenic callus.Therefore,the andreanum callus and adventitious bud of transgenic AaAMP gene were obtained.