Screening And Preliminary Functional Verification Of Genes Related To Flower Bud Differentiation Based On Transcriptome Sequencing In Brassica Napus

As one of the five major oil crops in the world,Oilseed rape(Brassica napus L.)has a high oil content of 30～50%,and its economic value mainly includes food value,industrial value,as well as other added values.So increasing yield of rape is of great significance for the growth of the national economy.Fower bud differentiation implies a transition from vegetative to reproductive phase and is one of the most important physiological process in plants,"flowering determination" affects the length of growth period and is closely related to crop yield anf quality,which is of great significance for production and application.while the molecular mechanisms of the floral process(from flower bud differentiation to floral organ formation and flowering)in higher plant are more complex.The more intensively studied species are mainly in model plants,such as Arabidopsis thaliana,goldfish grass,etc.At present,the studies about floral development of oilseed rape have focused on the processes of morphological development and physiological changes.The study of its molecular mechanisms is still in its infancy.In this study,three main rape cultivars(Zheyou 50,Zheyou 51 and Zheyouza 108)in Zhejiang Province were used as materials.With the buds from tree phase(Pre-,mid-,and post flower bud differentiation),we obtained gene expression profiles by RNA-seq.Finally,we selected and cloned two genes(KAO2 and RGA1)in oilseed rape,then constructed the overexpression vectors and GUS fusion protein expression vectors,which were transformed into Arabidopsis for preliminary functional verification with a view to further study the molecular mechanism of floral bud differentiation in rapeseed,as well as to provide a basis for the breeding of excellent cultivars in rape.The main conclusions are as follows:(1)In this study,a mass of Unigene information was obtained.RNA-seq technique was applied to analyze the expression profiles of from three oilseed rape cultivars and three differentiation periods.The result revealed that more than 31 M raw reads were obtained for each sample.Analysis of pairwise comparisons between the three periods in each cultivar,abtained a total of nine comparative pools,The down-regulated genes were approximately 2-3 times the number of up-regulated genes.The number of up and down regulated genes in the comparison pool of the pre and post differentiation stage were higher.(2)By comparing the reference genome of Arabidopsis in this study,we screened and analyzed expression patterns of 24 key floral bud differentiation-related genes,most of which were up-regulated in the later stages of differentiation.Through the identification of genes involved in phytohormones biosynthesis and signaling,we found that some genes were significantly up regulated,such as ET signaling pathway related genes EBF1/2,GA signaling pathway related genes RGA1.Whereas some genes were significantly downregulated,such as ABA related genes NCED1/3/5、ABI5、HAI1 and PYL5,IAA related genes CYP79B2、IAA19/7/28、SAUR30/34/48/9,SA related genes PAL1/2、NPR1/3、PR1、TGA1/3/4,GA related genes GA2OX1/2、KAO2、GIDIA/B、PIF4.Through the recognition and classification of transcription factors,a number of transcription factor families with differential expression were identified,among which ERF,WRKY,NAC,MYB,bHLH,C2H2,bZIP,Trihelix,HSF,GRAS,C3H and other transcription factor families were significantly up-regulated,and were estimated to be potentially involved in the rape flower bud differentiation transition process.(3)In this study,the homologous genes KAO2 and RGA1 in oilseed rape were cloned,and multiple sequence alignment and phylogenetic tree analysis showed that they had a close relative to Brassica species.Subcellular localization experiments showed that both proteins were localized to the nucleus and the cell membrane.By constructing an overexpression vector and transforming into Arabidopsis,the results of transgenic line phenotyping showed that the overexpression of KAO2 significantly delayed flowering time in Arabidopsis,whereas histochemical staining results showed that BnRGAl was specifically expressed in mature inflorescence tissues,flower organs,pod fruits and leaf veins at seedling stage.And the overexpression of RGA1 increased flower number with higher inflorescence meristem activity.