Cloning Of Tomato Flowering Gene BOSS And Functional Research Of Its RNA Editing

Tomato is an important plant food resource,which can be eaten fresh or processed,and its annual supply is limited by flowering time.It is of great significance to cultivate diversified flowering varieties by biotechnology for regulating the market supply of tomato.However,flowering time of tomato is easily affected by environmental factors,so it is difficult to clone related regulatory genes quickly and efficiently by using traditional map based cloning technology.As a new biotechnology,genome wide association study(GWAS)has been used to clone functional genes comprehensively and rapidly at the whole genome level.Therefore,this project revealed the genetic structure of tomato flowering regulation at the whole genome level by GWAS,and the key gene BOSS was cloned.Furthermore,the eukaryotic RNA editing phenomenon was found in BOSS.The biological functions of the two transcripts formed by RNA editing of BOSS were studied from plant phenotype level,cell level and gene expression level by using transgenic function verification,cell section technology and transcriptomics.The biological regulation network of BOSS RNA editing was preliminarily analyzed by using yeast one hybrid technology,Luc technology,yeast two hybrid technology and Bi FC technology.It provides a certain theoretical guidance for the cultivation of new tomato varieties with different florescence.The main work of this paper is as follows:1.Genetic basis of flowering period and cloning of a key flowering gene BOSS.Firstly,GWAS was used to locate four control locus q FIN3,q FIN3,q FIN9,q FIN12,and then solyc03g097830.2 in q FIN3,solyc06g074350.2 in q FIN3,and solyc09g010650.1(BOSS)were selected in q FIN9.2.Discovery and verification of BOSS RNA editing.Bioinformatics found RING-H2 in BOSS gene,and an EL5?Like domain and transmembrane helix were found only in some dicotyledons.BOSS mainly expressed in tomato root,stem,leaf,flower,bud,fruit and seed.And the expression level in young leaves was not related to the flowering time of tomato.Sequencing analysis showed that there was nuclear gene RNA editing phenomenon in BOSS gene.The amino acid of BOSS gene was changed by RNA editing,which was found in most organs of tomato,with the highest in stem tip and the lowest in fruit;it was also induced by continuous light.Subcellular localization showed that BOSS gene transcripts were located in nuclear membrane and cell membrane before and after RNA editing.Further studies showed that the RNA editing of BOSS was common in dicotyledons,which indicated that RNA editing of nuclear gene had universal biological significance.More abundant RNA editing types were found in melon and potato.Analysis revealed that RNA editing was significantly correlated with flowering time.The significance of this study is that it's confirmed that RNA editing of nuclear genes regulates plant traits,which has a certain supplementary significance to the "biological center principle" of plants.3.Biological function analysis of BOSS gene RNA editing.The transgenic materials with two transcripts related to RNA editing of BOSS gene(? is the original transcript and ? is edited by ?)are obtained by using transgenic function verification technology.From the phenotypic level,it was found that RNA editing version ? decreased the node position of the first inflorescence(a measure of flowering time in production)and the interval node of the first and second inflorescence,which accelerated the flowering,affected the self pruning of tomato,and improved the fruit yield per plant.However,the original transcript ? had no significant effect on Tomato flowering.RNA editing version ? promoted the transformation of shoot tip meristem to inflorescence meristem and flower meristem in advance,but ? had no such function.The overexpression of RNA editor ? resulted in the up regulation of three MADS genes and the expression changes of known important flowering genes;however,the original transcript ? did not affect differential expression of of flowering genes,although it could increase one Wo gene by 7000 times.4.Analysis of RNA editing pathway of BOSS gene.Based on the analysis of phenotype of transgenic materials,we found that the transgenic lines with overexpression of ? presented the type of inflorescence interval(2L-2L-1L-1L-1L),which was similar to the penotype(2L-2L-1L-1L-1L-0L)drived by super dominant heterosis combination of a single gene(sp/sp sft/+).Yeast one hybrid and LUC analysis showed that BOSS gene could bind to the promoter of SFT(promoting flowering gene)and SP(inhibiting flowering gene)to enhance its expression and balance the flowering time.Using yeast two hybrid and Bi FC,it was found that ?-protein or ?-protein could interact with SP protein or SFT protein respectively,but ? and ? could not form trimer with SP and SFT respectively.In addition,yeast two hybrid test was used to verify that ? and ?,? and ?,? and ? could not form dimers.These results provide a theoretical basis for further elucidating the regulation of BOSS gene on tomato flowering.