Screening Of Effector Protein FonSIX6 And Non-coding RNA In Fusarium Oxysporum F.sp.niveum In Watermelon

Watermelon[Citrullus lanatus(Thunb.)Matsum&Nadai]belongs to the cucurbitaceae family,the watermelon genus,which is widely cultivated around the world as one of the highest yielding cash crops.However,it is vulnerable to a variety of pests and diseases in the production,especially Fusarium wilt,on the yield and quality of watermelon has a very serious impact.Breeding varieties resistant to blight is one of the most rapid and effective solutions.Therefore,it is very important to explore the mechanism of watermelon resistance to Fusarium wilt and find the corresponding genes of resistance to Fusarium wilt.Effector proteins are a class of small molecule pathogen proteins,whose role is to change the host cell structure and function.It generally activates downstream immune regulation by interacting with disease-fighting proteins in the host.It was found that the effector protein FonSIX6 was a non-toxic factor of fusarium oxysporum and a key factor affecting pathogenicity.Bio informatics analysis showed that the FonSIX6 protein has a RN A-binding motif,suggesting that it is likely to interact with RNA in the host.A large number of studies have confirmed that non-coding RNAs in plants are involved in disease resistance regulation and play an important role.However,the regulatory mechanism of FonSIX6 interaction with host non-coding RNA in disease response has not been reported.Study of protein and RNA interaction technology is few,and most are not mature,among them,the GoldCLIP technology(Gel-omitted and ligation-dependent CLIP),it can be efficient and rapid screening of RNA and protein interactions,but the technology is currently only in animal reports,this research probes into the technology improved,with FonSIX6 protein interactions of watermelon and filtered the noncoding RNA oftobacco.It is hoped that a relatively mature GoldCLIP system can be established to provide technical support for future studies on plant protein-RNA interaction.It is also hoped that the non-coding RNA interacting with FonSIX6 can be screened to have a preliminary understanding of its disease-mediated resistance mechanism in watermelon.The main results of this study are as follows:(1)the P3 5S:Halotag-FonSIX6-PBI121 vector was constructed,which was transformed into watermelon ’JJZ’ and tobacco ’bens’ varieties by agrobacterium-mediated genetic transformation.PCR detection was used to select 2 watermelons and 12 tobacco P35S:Halotag-FonSIX6-PBI 121 To generation transgenic plants.(2)The purple diplomacy was conducted on the positive P35S:Halotag-FonSIX6PBI121 watermelon transgenic leaves.A total of 84 clones were obtained through GoldCLIP technology,and multiple sequence alignment was performed.After removing the joint and repeated sequence,a total of 4 specific RNA sequences were obtained.Found that they can’t locate to the existing watermelon genome database,but part of the RNA sequences in melon(DHL92)mRNAv 4.0 repository location on the two genes(MELO3c027805.2.1 and MELO3c031322.2.1),and inferred the possible reasons are that the material used in the existing watermelon gene database is different f om the watermelon material used in this experiment,which may cause the obtained RNA sequence cannot be located.(3)The positive P35:Halotag-FonSIX6-PBI 121 transgenic tobacco leaves were subjected to purple diplomacy,a total of 75 clones were screened by GoldCLIP technology,and multiple sequence alignment was performed.After removing the joint and repeated sequence,a total of 7 specific RNA sequences were obtained.The 7 sequences were blasted on the website(https://solgenomics.net/).It was found that nitab4.5-0660344,0359768,0309180,0251996,0376295,074600,0110193,0052963,0233874,0747497 could be localized on 11 genes in the N.t-abacum gene bank(Current version),respectively.Through the GoldCLIP technology,the above data results are obtained,indicating the preliminary establishment of the technical system.The technology compared with other traditional methods,the success of the Halotag label simplified after being mixed with FonSIX6 protein in vitro purified protein and nucleic acid complex operation steps,and magnetic beads form covalent fixed together,no matter in any detergent or degeneration conditions will not wash the interactions of RNA,the compound can be purified,greatly improved the efficiency,reduces the isotope labeling as well as some cumbersome steps turn run rubber membrane.It provides a new technique for screening plant protein and RNA interaction in the future.