Study On Antimicrobial Analysis And Effect Of Antimicrobial Peptide From Elaeis Guineensis

Oil palm stem base rot disease,which occurs in almost all palm growing areas.The pathogen of oil palm stem base rot is Thielaviopsis paradoxa,which has the character of Ceratocystis paradoxa.It mainly infects the fruit,leaf and stem of oil palm and causes fruit drop and death of oil palm,which has a certain impact on the global oil palm industry.At present,chemical and physical control methods are mainly used to control the disease.Chemical control was harmful to environment.Physical control is cutting down infected plants for disinfection,which is more likely to trigger fires.Because oil palm belongs to tropical crops,it is not conducive to the promotion and application of biological control technologies such as microbial agents and botanical preparations.Therefore,the prevention and control of stem base rot of oil palm need to find new ways,develop new technologies.Antimicrobial peptides(AMPs)are small molecular polypeptides with broad-spectrum antimicrobial activity.They are widely distributed in various organisms in nature.They are widely used in agriculture.In this paper,the gene prediction,screening and antimicrobial activity analysis of AMPs in oil palm genome were carried out by bioinformatics technology and genetic engineering technology,and a broad-spectrum and highly effective AMPs of oil palm was obtained.According to the pathogenic characteristics of oil palm stem base rot,the broad-spectrum and high-efficiency oil palm AMPs were transformed into Arabidopsis thaliana and expressed stably,which provided a new way for the control of oil palm stem base rot.The main results are as follows:(1)89 aseptic oil palm seedlings were successfully cultured by embryo induction method,and the induced survival rate was 4.57%,which was close to the current international standard of the induced survival rate of oil palm tissue culture seedlings.(2)The pathogens of oil palm stem base rot and oil palm leaf anthracnose were systematically identified by morphological and molecular biological methods.The pathogens of oil palm stem base rot disease were identified as T.paradoxa,and the pathogen of leaf anthracnose was Colotrichum siamese.(3)Using genomics and bioinformatics methods to mine AMPs genes and identify and apply their antimicrobial and antipathogenic fungal activities.Based on 22 candidate genes of antimicrobial peptide,13 AMPs were further screened to show antifungal and bacteriological activities.The antimicrobial activities against T.paradoxa and C.siamese.were further verified,and 13 AMPs were found.All showed bacteriostatic activity.(4)The expression vectors p ET30a(+)and p GE-4T-2 were used to express the highly pathogenic AMPs gene 8-1,respectively.It was found that the two vectors were expressed in the form of inclusion bodies and water-soluble.The results of bacteriostasis showed that the antimicrobial activity of the three antimicrobial peptides 8-1 was: chemical synthesis > p ET30a(+)expression product >8-1-GST.(5)A novel LTPs antimicrobial peptide 8-1 gene against T.paradoxa was screened and synthesized,purified and tested for its antimicrobial activity.The protein was composed of 119 amino acids without tryptophan and phenylalanine.The active region of 8-1 protein was estimated to be around 28-115 amino acids at the N-terminal.The results of protein three-dimensional structure model showed that the structure of LTPs AMPs 8-1 contained six alpha-helixes and no beta-folding,and nine Loop angles formed four disulfide bonds with eight conservative angles.Antimicrobial activity test showed that LTPs antimicrobial peptide 8-1 not only had strong antifungal activity,but also had strong antimicrobial activity,indicating that the antimicrobial peptide gene played a certain role in the defense of T.paradoxa.(6)In this study,the fusion plant expression vectors p CAMBIA2301-AP8.1 and p CAMBIA2301-AP3.8 containing LTPs AMPs 8-1 gene and Defensins a AMPs 3-8 gene were constructed respectively,and the transgenic plants of pure line T3 of Arabidopsis thaliana were successfully transformed.The results showed that the LTPs AMPs 8-1 gene transformed plants showed antimicrobial activity,while the Defensins AMPs 3-8 transformed plants did not show antimicrobial activity.This indicated that it was feasible to design a method to control the stembased rot of oil palm by using the disease-resistant gene in this study.