Mapping And Cloning Of A Early Bolting Gene Ebm4 In Chinese Cabbage

Chinese cabbage(Brassica rapa ssp.pekinensis)belongs to the genus Brassicaceae and is a traditional Chinese vegetable.Its cultivation area and yield rank first among all kinds of vegetables in China.Bolting is a key developmental event in the transformation of Chinese cabbage from vegetative growth to reproductive growth.Early bolting can lead to severe reduction or even failure of spring cabbage and alpine cabbage.Chinese cabbage has evolved a complex and elaborate regulatory mechanism to strictly control the time of bolting,but the regulatory mechanism is still unclear.Therefore,it is of great theoretical significance and application value to create early and late bolting mutants and to explore important node genes that regulate bolting in Chinese cabbage.1.The germinated seeds of Chinese cabbage DH('FT')were used as mutagenic materials,treated with 0.8% EMS for 12 hours,then treated with running water for 12 hours,and finally528 M0 mutagenized lines were obtained,among which 6 different strains appeared.Early-twitching mutant plants were identified by M2 and M3 generations as early bolting,named ebm3,ebm4,ebm5,ebm6 ebm7 and ebm8.2.Compared with the wild type 'FT',the Chinese cabbage early bolting mutant ebm4 could achieve bolting and flowering without vernalization in autumn,and there was no obvious stage of bulging.Genetic analysis indicated that the mutant trait was controlled by a single recessive nuclear gene(ebm4).3.The improved Mut Map method was used to locate the ebm4 gene.The SNP-index of26 mutation sites was close to 1,among which 10 mutations caused amino acid changes and 1mutation caused the translation to terminate prematurely.Combined with functional prediction,the candidate gene was initially identified as Bra A02g015090.3C,located at7883329 bp on the A02 chromosome.The base of the mutation site changed from C to T,and the corresponding amino acid was mutated to its Arabidopsis homologous gene was AT5G52820.The gene belongs to the WD-40 repeat protein family,which plays an important role in flowering,light transmission and perception,and inflorescence,flower development,chromatin modification,etc.4.Phylogenetic analysis and gene structure analysis of the candidate gene Bra A02g015090.3C revealed that it had a close homology with Brassica napus.There were12 exons,and the amino acid had no transmembrane region,and the signal peptide was cleaved.The locus was located between amino acids 29-30,and the subcellular was localized on the nuclear.5.Transcriptome sequencing of shoot tip meristems(SAM)in wild-type and mutant was performed.After comparison analysis,9221 differentially expressed genes(threshold value0-0.05)were found,and 112 significantly enriched GO terms and 9 significantly enriched metabolic pathways were identified by GO and KEGG enrichment pathway analysis.Among them,the metabolic pathways associated with ticks and flowers are glyoxylic acid anddicarboxylic acid metabolism,biosynthesis of secondary metabolites,phytohormone signal transduction and circadian rhythms.Nine differentially expressed genes were then selected for q PT-PCR to further verify the results of transcriptome sequencing.