Research Of CeRNA Related To The Occurrence Of Paulownia Fortunei Witches’ Broom

Paulownia is a fast-growing tree species which is widely used for landscape engineering and making wood product.It plays an important role in windbrake and sand fixation,improving the ecological environment,and ensuring food security in China.The Paulownia witches’ broom(Pa WB)caused by phytoplasma may lead to slow growth of large trees and death of young trees,causing huge economic losses to the Paulownia industry.In the past,although scientists carried out a series of researches on Pa WB,and have made certain progress.However,the pathogenesis of Pa WB is not very clear.Competitive endogenous RNA(ceRNA)has important biological significance in regulating gene expression,resisting environmental stress,and plant growth and development.In this study,healthy Paulownia fortunei(PF),Pa WB-infected P.fortunei(PFI),methyl methane sulfonate(MMS)and rifampicin(Rif)treated PFI were used as materials.The ceRNA expression profile,Pa WB-related ceRNA regulatory network,and molecular functions of Pa WB-related miR156 f molecular functions have been studied,a series of results have been achieved.The main research results are as follows:1.Established a simulation system for Pa WB.In this study,different concentrations of MMS and Rif were used to treat PFI to simulate the Pa WB infection and recovery processes,in the 60 mg·L-1 MMS or60 mg·L-1 Rif treatment,with the extension of the treatment time,the seedlings returned to a healthy state,and phytoplasma could not be detected in the seeding.In the 20 mg·L-1 MMS or 30 mg·L-1 Rif treatment,the seedlings also recovered,but the seedlings still contained phytoplasma.Substituting the recovered seedlings into a medium without MMS and Rif,the symptoms of witches’ broom seedlings appeared again,and the phytoplasma content gradually increased.The results showed that MMS and Rif treatment could control the level of phytoplasma in Paulownia.The system could simulate the dynamic change process of Pa WB,overcome the difficulty of phytoplasma cultured in vitro,and laid the foundation for subsequent research on Pa WB.2.The ceRNA expression profile of Pa WB in Paulownia fortunei was conducted.In this study,we used PFI treated with two reagents at different points and healthy seedlings,through high-throughput sequencing,28,514 transcripts,668 miRNA and its 4,395 target genes,5,118 lnc RNAs and its 7,242 target genes,9,927 circ RNA and its 5,045 hosting genes were identified.GO functional analysis showedthat these genes are mainly involved in biological processes,transcriptional regulation and transcription;KEGG analysis showed that these genes were mainly involved in metabolic pathways such as plant pathogen interaction,plant hormone signal transduction,starch and sucrose metabolism,indicated that the genes in these metabolic processes were closely related to the occurrence of Paulownia witches’ broom disease,and provided data support for the study of the occurrence of Paulownia witches’ broom disease.3.Constructed the ceRNA regulatory network related to the occurrence of Paulownia witches’ broom disease,verified their function by transgenosis.In this study,based on the established ceRNA expression profile associated with the occurrence of Pa WB,and using trend analysis,a total of 17 transcripts,4miRNAs,4 lnc RNAs and 6 circ RNAs related to the occurrence of Pa WB were identified.Through ceRNA analysis,it was found that circ RNA7714-miR156f-SPL3 is the ceRNA specifically related to the occurrence of Pa WB,and its function was verified in Populus trichocarpa.The results showed that miR156 f over-expression plants increased branching,while Pf SPL3 over-expression plants did not appear.These results provided a theoretical basis for the study of the pathogenesis of Pa WB.4.Clarified the molecular function of miR156 f and its target Pf SPL3,which were specifically related to the occurrence of Pa WB.The targeting relationship between miR156 f and Pf SPL3 were used to verified using degradome sequencing,RLM-5′-RACE and luciferase.In addition,yeast two-hybrid,co-immunoprecipitation,bimolecular fluorescence complementation and Glutathione S transferase-pull down were used to discover and confirm the interaction between SPL3 and RPN3.It was found that Pf SPL3 is ubiquitinated,and its regulated target genes were mainly involved in plant hormone signal transduction,phenylpropane biosynthesis,carbon metabolism and brassinosteroid biosynthesis.The results laid the foundation for elucidating the pathogenesis of Pa WB.