Construction Of Biocontrol Bacteria For Sugarcane Smut By Genetic Tranformation Of JMT(Jasmonate Methyl Transferase)Gene

Sugarcane smut is caused by Sporisorium scitamineum,which have two opposite mating types MAT-1 and MAT-2.Sexual mating between MAT-1 and MAT-2 sporidia gives rise to dikaryotic hypha/mycelium capable of infecting host cane and causing disease.In plant growth of pathogens is affected by(biotic)environment and signal molecules,including phytohormones.Phytohormones e.g.Jasmonic acid(JA)and Methyl Jasmonate(MeJA),play various physiological roles in plant growth and development,including resistance to pathogen infection.It has been reported that JA participates in resistance response to pest invasion,while both JA and MeJA are involved in inducing resistance genes expression upon microbial infection.In this research we intended to screen and identified small molecules including phytohormones and microbial signal molecules,that is capable of suppressing S.scitamineum mating/filamentation,and to transform symbiotic/endophytic bacteria of sugarcane via genetic modification,for the purpose of disease control.The results are as follows:1.Screening for small molecules suppressing S.scitamineum mating/filamentation: we found that in vitro condition,MeJA of 200 ?M,400 ?M and 800 ?M concentration could effectively suppress S.scitamineum mating/filamentation.2.Identification and validation of enzyme of MeJA synthesis: we found that Jasmonate MethylTransferase(JMT)encodes an enzyme capable of catalyzing MeJA production with JA as precursor and SAM as methyl donor.It has been reported that JA content is relatively high in young tissues of plant,where S.scitamineum mating may occur and systematic infection starts,while MeJA was hardly detected in such parts of plant.Therefor in this study,we plan to introduce JMT gene to the young tissues of plant cane to produce sufficient MeJA and suppress S.scitamineum mating and systematic infection at the early stage.3.Expression of JMT gene in Escherichia coli: JMT gene was cloned and ligated to expression vector pRSFDUET-1,driven by T7 promoter.pRSFDUET-1(JMT)expression plasmid was transformed into E.coli to test its catalyzing activity.Our result showed that by IPTG induction and with presence of JA and SAM,the JMT-carrying E.coli could produce MeJA.4.Screening for symbiotic/endophytic bacteria for genetic transformation: considering the difficulty in genetic transformation of sugarcane,we plan to introduce the JMT gene into symbiotic/endophytic bacteria of sugarcane to achieve smut disease by suppression of S.scitamineum mating and systematic infection.Our initial screening obtained 79 endophytes,one of which was preliminarily identified as belonging to genus of Burkholderia,and was showed to suppress S.scitamineum mating in vitro.We named it as CGB10.In summary,in this study we found that MeJA is able to suppress S.scitamineum sexual matingg/hyphal forming,and it could be produced in vitro by the enzyme encoded by JMT.We identified abundant endophytes from young leaves of sugarcane,which are of potential in biocontrol bacteria construction via genetic modification.One of the endophytes showed inhibitory effect on S.scitamineum mating in vitro and we named it as CGB10.