Cloning And Expression Characterization Of Early Bolting Mutant Gene Ebm3 In Chinese Cabbage

Chinese cabbage(Brassica rapa ssp.Pekinensis)is a kind of vegetable crop originating in China,which has high economic and nutritional value.Timely bolting is very important to the production and breeding of Chinese cabbage,but premature bolting will have a serious impact on the production of spring-heading Chinese cabbage,so it is particularly important to explore the molecular mechanism that affects the bolting and flowering.In this study,the seeds of Chinese cabbage DH line 'FT' were treated by EMS mutagenesis,and a stable early bolting mutant(ebm3)was obtained.Based on the modified Mut Map technique,the mutant gene was mapped.The main research results included the following four aspects:1.Compared with the wild type 'FT',the mutant ebm3 which was not subjected to vernalization showed obvious early bolting characteristics in the spring and autumn cultivation,accompanied by the upward curling of the leaves.Genetic analysis showed that the early bolting phenotype of mutant ebm3 was controlled by a single recessive nuclear gene.2.The wild type 'FT' and the mutant ebm3 were used as parents to construct the F2 generation segregating population,and the modified Mut Map method was used to locate the mutant gene.The result showed that due to the single base mutation of the Bra A04001706gene(which encoded a CURLY LEAF transcription factor and was an important H3K27methyltransferase)on the A04 chromosome(C?T),the codon was changed from TCT to TTT,and then the non-synonymous mutation of S?F(serine?proline)resulted in the occurrence of early bolting.3.q RT-PCR analysis showed that the expression level of the mutant gene on flower and bud was very significant,while the expression level in the stem was extremely low.The promoter fusion GUS vector was constructed and transformed into Arabidopsis thaliana by dipping flower method.GUS histochemical staining showed that the promoter of the gene could activate the expression of the gene in flowers,buds,leaves and pods.The fusion expression vector of p BWA(V)HS-LZY-GLosgfp was constructed and transfected into Arabidopsis protoplasts by PEG-mediated method.Subcellular localization results showed that the protein products of the gene were mainly distributed in the nucleus.4.A total of 1906 differentially expressed genes were obtained by transcriptome sequencing of shoot tip growth point.Compared with wild type,1079 DEGs were up-regulated and 827 DEGs were down-regulated in mutants.The results of GO enrichment analysis and KEGG pathway analysis showed that there were 338 GO-enriched terms and 20 significant metabolic pathways,respectively.Among them,the first three items with the mostdifferential gene enrichment were the plasma membrane(GO: 0005886),chloroplast(GO:0009507),and extracellular region(GO: 0005576).Finally,12 genes were randomly selected and q RT-PCR was used to verify the reliability of the transcriptome data.