Screening Of Novel Antimicrobial Peptides And Design Of A New Strategy To Control Sclerotinia Sclerotiorum Disease In Brassica Napus

Sclerotinia sclerotiorum is an important plant pathogen which can infect many crops worldwide,including Brassica napus oilseed rape,one of major oil crops in the world,and causes rot of leaves,stems and pods,resulting in a huge yield loss.Although a control strategy against this disease has been in place which integrates resistant varieties as a primary measure with chemical application as a reinforcement and other control measures as a supplement," to control S.sclerotiorum measures,until now there has not yet found immune or highly resistant cultivars and thus disease resistance levels are far from requirement by crop production while chemical fungicide application is cost and not eco-friendly,and more importantly pose difficulty in spraying fungicides at the flowering stage.In order to achieve more effective control of Sclerotium disease,we need to open up new ways or develop new technology.In the infecting cycle of Sclerotinia disease,90% of infection on oilseed rape leaf and stem was mediated by infected petals while infection on pods comes from their touch with diseased leaves or/and stems.Thus if petals are conferred resistance or with a ablity to kill the pathogen,the infection cycle may be able to be stopped and the diesae may be prevented from spread onto stems and leaves.To this aim,our lab has developed an idea that is to use petals as a bioreactor to specifically and sustainably express antifungal peptides/compounds at an effective concentration that can kill the pathogen in situ in petals and thereby block the infection cycle,to achieve the purpose of controlling the S.sclerotiorum disease of B.napus.For this disease cycle,the laboratory has designed a kind of new technical route,using the petals as bioreactor,in which specific,high capacity,sustained and stable expression of antimicrobial substances,in situ kill germs,reach the purpose of blocking disease infection cycle and preventing,treatmenting of disease.The design of technical route for this petal bioreactor include 3 key elements: 1)Specific,highly efficient,sustained expression of an oilseed rape petal promoter;2)genes introduced can highly effective synthesize antifungal peptides/compound(s);3)Synthesized product(peptides/compound(s))will be able to be efficiently transported to extracellular regions and and resist degradation.This study focused on screening of antimicrobial peptides and assembling of gene elements.The main research results are as follows: 1.Screening of effective antimicrobial peptides from Brassica napus oilseed rape.Antimicrobial peptides(AMPs)are a group of diverse and endogenous antibiotics and innate immune components that protect hosts against microbial infection and are produced ubiquitously in the natural environment.In this study,we first predicted and analysed the antibacterial peptide genes from B.napus whole genome through bioinformatics approaches.We preliminarily selected 40 candidate genes which contain similar sequences to the known antimicrobial peptides.Then through the overlap polymerase chain reaction(PCR),we artificially synthesized the complete genes and cloned the synthetic gene into pET30a-EDDIE-GFP expression vector to express in Escherichia coli.After an in vitro protein renaturation process of the expressed inclusion body-fused protein and through the self-cleavage of the fusion protein EDDIE,we obtained antimicrobial peptide product without any unwanted amino acids.Inhibitory activity detection results showed that there are 32 antimicrobial peptides with strong antibacterial activity and 24 with strong activity against S.sclerotiorum.2.Identification of a novel proline-rich peptide with strong antimicrobial activity from Brassica napus.Proline-rich antimicrobial peptides(PR-AMPs)are a group of cationic host defense peptides that are characterized by a high content of proline residues.Up to now,they have been reported in some insects and animals,but are not found in plants.In this study,BnPRP1 as a new type of proline-rich antimicrobial peptide was first found in plants through the above screening.This gene designated BnPRP1 encodes a 35-amino acid peptide with 13 proline residues(> 35% of the total number of amino acids).The best homology(40.5% identity)of BnPRP1 is a known proline-rich antimicrobial peptide SP-B from pig.There are four homologous sequences in B.napus(AACC,2n=38),and two homologous sequences in B.rapa(AA,2n=20)and in B.oleracea(CC,2n=18),respectively.Experimental results of circular dichroism(CD)revealed that the main secondary structure of BnPRP1 was the random coil.BnPRP1 exhibits strong inhibitory activity against strains of some bacterial and fungal species including S.sclerotiorum.BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation.Fourty eight hours after S.sclerotiorum inoculation,the expression of BnPRP1 increased significantly in the susceptible lines while expression in resistant lines was slightly decreased.These suggested that BnPRP1 might play a role in the plant defense response against S.sclertiotum.3.Preliminary assembly of petal bioreactor.In order to verify the feasibility of this new strategy,we first used 35 S promoter,radish antimicrobial peptide gene Rs and ?-amylase signal peptide ?Amy3SP to transform Arabidopsis.To do this,we cloned Rs and ?Amy3SP.Experiments with GFP indicated that ?Amy3SP correctly directed GFP into intercellular space.Transgenic plants with the 35S-Rs-?Amy3SP construct showed that 4 homozygous transgenic lines had significant increases in Rs expression and exhibited significant decreases in lesion sizes after inoculation with S.sclertiotum mycelial plugs on leaves.Next we replaced 35 S of 35S-Rs-?Amy3SP with the petal-specific promoter P76247(P7)which was screened,cloned and verified by RNA-seq and GUS detection methods.The results from transgenic Arabidopsis indicated that Rs expression in flowers was significantly higher than that in stems and leaves of transgenic plants(relative to Actin expression quantity),and that in stems,leaves and flowers of transgenic plants with the 35S-Rs-?Amy3SP construct.Athough inhibitory effects of transgenic plants with the P7-Rs-?Amy3SP construct have not measured,we can expect,from its high expression in flowers and inhibitory effect of transgenic plants with the 35S-Rs-?Amy3SP construct,that transgenic petals with the P7-Rs-?Amy3SP construct have reasonable effect.These studies provide a new route by genetic engineering technology to improve crops resistance to Sclerotinia disease.