Plant Genetic Transformation And Antifungal Activity Analysis Of EuAFP1 Gene

Diseases caused by fungi often lead to a decline in the yield and quality of cash crops,which is extremely harmful.Plant antibacterial peptides can effectively inhibit the pathogenic fungi of tomatoes,potatoes,tobacco,wheat,alfalfa and other crops and pastures.They are a major type of biological pesticides.Eucommia ulmoides has antibacterial,anti-disease,anti-virus and other disease resistance,which is related to the antibacterial protein it contains.In this thesis,cloning and plant genetic transformation of EuAFP1 gene encoding Eucommia ulmoides antibacterial peptide,heatherin,as well as antifungal activity research,provide a basis for improvement of fungal disease resistance of crops and pastures.The main research contents and results are as follows:1.Cloning of EuAFP1 gene of Eucommia ulmoides.The EuAFP1 gene(>GWHPAAAL014598)was screened from Eucommia ulmoides genome(hmmer3.0)according to the conserved domains of plant antimicrobial peptides,and full-length amplification primers were designed.EuAFP1 sequence was obtained by homologous amplification using Eucommia ulmoides young leaf c DNA as template.The EuAFP1 gene is 1002 bp in length and encodes a hydrophilic protein of 329 amino acids.It is clustered with disease resistance genes such as Eucommia Chitinase gene EuCHIT2,Petunia Pn-AMP and Portulaca Ac-AMP.The plant expression vector p SH-35S-EuAFP1 was constructed with GUS::NPTII as the reporter gene and resistance screening gene,and 35 S promoter driving the target gene.2.Model plant transformation and antibacterial activity of EuAFP1 gene.The fifth to sixth leaves of K326 tobacco at the 6?7 leaf stage were injected and transformed,and the high expression period of the GUS reporter gene was 4?6 d after transformation;the EuAFP1 gene was genetically transformed into Arabidopsis thaliana by the flower method to obtain 4 transgenes(TP)Plant.The antibacterial activity of the crude protein extracts of the recipient tobacco and T1 generation TP Arabidopsis leaves transformed from 4 to 6 days were analyzed by perforation method and spectrophotometry,and the crude protein(rEuAFP1)of the two plants was found to be crude.The extract has obvious inhibitory activity against powdery mildew,Botrytis cinerea and Fusarium oxysporum(p<0.05).3.Disease resistance of EuAFP1 genetically transformed tomato and transgenic tomato.The EuAFP1 gene was transformed into tomato leaves by injection,and the 3 d transformed leaves that began to express GUS protein were inoculated with Botrytis cinerea.Compared with the wild-type parent(WT),TP leaves had a later onset,smaller lesions,and slower spread during the injection period.It is better to keep green.Transgenic EuAFP1 tomato has certain resistance to gray mold.4.The disease resistance analysis of EuAFP1 genetically transformed Lotus japonicus and transgenic Lotus japonicus.A non-tissue culture genetic transformation system of Lotus japonicus was established,and 18 TP Lotus japonicus strains were obtained through this transformation system.The powdery mildew fungus inoculation experiment found that compared with WT leaves in the same period of infection,TP can keep green better,with small lesion area and slow spreading.Inoculation experiments of Fusarium oxysporum showed that the root tissue of TP Lotus japonicus had a light degree of browning during the same period,and the integrity was good,the lesions were transferred slowly,and the growth characteristics were maintained.Transgenic EuAFP1 gene Lotus japonicus has good resistance to powdery mildew and root rot.Studies have shown that Eucommia EuAFP1 has three fungi inhibitory activities:powdery mildew,gray mold and Fusarium oxysporum.It can effectively improve the resistance of plants to gray mold,powdery mildew and root rot.The research results can provide a reference for molecular breeding of disease-resistant crops and diseaseresistant pastures using EuAFP1 gene.