Identification Of Key Genes Of Swelling Gall Formation Zizania Latifolia Induced By Ustilago Esculenta Based On RNA-seq

Zizania latifolia（Zizania latifolia（Griseb.）Stapf）was a perennial aquatic vegetable due to the establishment of symbiosis between Z.latifolia and U.esculenta results in the swelling gall formation.In the early stage,the study group applied exogenous hormone IAA to Z.latifolia,which could significantly increase the photosynthesis,carbohydrate and yield of Z.latifolia.It was speculated that the swelling gall of Z.latifolia might be related to the changes of endogenous hormones.Relevant theoretical speculations would help to explore the mechanism of swelling gall formation.In this study,the single-season Z.latifolia"Dabieshan NO.1"was used as the experimental material.We analyzed Z.latifolia and U.esculenta symbiotic circumstances using Triadimefon（TDF）treatment and RNA-seq technique.17541DEGs were identified by reference genome analysis.By KEGG pathway analysis,most genes are dominated by phytohormone signal transduction pathways,phenylpropane biosynthesis,starch and sucrose metabolism,and the contents of related indicators were determined.Furthermore,the related information of the ZlARF family was analyzed by genomic information.The main results are as follows:1.Screening of TDF concentrationA novel method TDF was analyzed Z.latifolia and U.esculenta symbiotic circumstances using TDF treatment and RNA-seq technique.The samples were taken before and after gall formation.It was found that the TDF concentration of 80 mg/L was the best during the period from the growth of Zizania to theⅢstage,which could significantly inhibit the production of gall formation.U.esculenta was not observed in the gall tip samples of swelling gall no formation.Further,through the identification of ITS1 and ITS4 fungus-specific to identify U.esculenta reached 98%,and the results were positive.Meanwhile,the Actin of Z.latifolia and U.esculenta was further used as the specific primers and further verified by q RT-PCR.Through the above verification,it was found that the U.esculenta was not found before TDF treatment and gall no formation.It was further judged that U.esculenta did not infect Z.latifolia after TDF treatment,while the swelling gall was formed in the normal group through the infection of U.esculenta.2.Observation of the whole period and microstructure of Z.latifoliaThe whole growth period of Z.latifolia was tracked,and the growth cycle was determined.Aniline blue staining was used to observe the U.esculenta,and U.esculenta were not observed in the treatment group.Furthermore,it was found that the U.esculenta were enriched around the cell wall and in the intercellular space by ultrastructure.Then,it was found that the U.esculenta were obviously attached to the cell wall of plant,carrying out certain signal transmission by scanning electron microscopy.3.Transcriptome identification and analysisTranscriptome sequencing was used to explain the symbiosis mechanism of Z.latifolia and to further understand the molecular mechanism of formation of Z.latifolia and the expression of DEGs.Most of the genes were in cell wall softening factor（XTH,EXPA,EXPB,FLA and so on）and plant hormone signal transduction（AUX1,ARF,AUX/IAA and so on）.U.esculenta infection may regulate the expression of these genes for swelling gall formation.4.Measurement of relevant indexesAs revealed by UHPLC analysis,the IAA,GA₃,Z and ZR content were increase while U.esculenta infected and ratio of these hormones to ABA also increased.The up-regulated expression of most genes was also found by q RT-PCR.Meanwhile,physiological indexes were identified after gall formation,among which sugar metabolism:sucrose,fructose,starch,SS and SPS increased while AI content decreased;Cell wall:β-1,3 glucanase,H₂O₂ and CAT content increased,while chitin content decreased;Phenylpropane metabolism:cellulose and lignin content increased.5.Identification and analysis of ZlARF familyBased on the genome database of Z.latifolia.The results showed that 33 candidate ZlARF genes were found in the genome of Z.latifolia.More comprehensive information was obtained on the conserved domain analysis,evolutionary relationship,intron-exon,protein secondary structure and subcellular localization of the ZlARF family of Z.latifolia.Therefore,the biological functions of Z.latifolia can be identified and predicted,which provides an important theoretical basis for regulating various growth and development processes.The results of transcriptome analysis were used to verify the relevant functions and expression patterns of ARF genes.The Z1ARF gene family has a highly conserved structure and was involved in various aspects of cell development and gall enlargement after Z.latifolia infection by U.esculenta.Based on the above information,we come up with the hormone-cell wall loosening model to study symbiotic mechanism of gall formation after U.esculenta infected Z.latifolia.Meanwhile,our study provides a new perspective for studying the physiological and molecular mechanisms of U.esculenta infection Z.latifolia that causes swelling gall formation and theoretical basis for further increasing the future yield of Z.latifolia.