Cloning And Functional Identification Of The Petal Morphogenesis Gene BrACX1 In Chinese Cabbage

Chinese cabbage(Brassica campestris ssp.pekinensis)belongs to the Brassica of Cruciferae family,and is an important vegetable crop.Petal color,size,and morphology play important roles in protecting other floral organs,attracting pollinators,and facilitating sexual reproduction in plants.Elucidating the molecular mechanism of petal morphogenesis is not only one of the tasks of plant developmental biology research,but also the attention of horticulturalists.In a previous study,using isolated microspore culture and 60 Co ?-ray mutagenesis,we obtained a petal degeneration mutant(pdm)of the ‘FT' line of Chinese cabbage and found that the candidate gene for pdm,Bra040093(Huang et al.,2014).In the present study,we further identified and analyzed the pdm candidate gene Bra040093 in order to characterize the gene networks regulating petal development in pdm plants.The main results are summarized below.1.Cloning and sequencing of candidate genes confirmed the existence of two SNP mutations.Cloning and sequence alignment found that the length of the c DNA sequence of wild type ‘FT' and mutant pdm candidate gene,Bra040093,were 1995 bp.SNP mutations occurred at 1696 and 1701 sites of the mutant pdm,respectively,from A to T and C to T.The results of SNP analysis obtained by DNA cloning and sequencing were consistent with those of the c DNA,indicating that the two SNPs were located on the exon.The candidate gene Bra040093 encodes 664 amino acids in wild type ‘FT'.In pdm,the codon AAA mutates to TAA due to base substitution at position 1696,resulting in the early termination of the amino acid sequence,encoding only 565 amino acids.The phylogenetic tree was constructed by using the amino acid sequence of ‘FT'.Bra040093 was highly homologous to the ACX1 gene of Arabidopsis thaliana.It was named Br ACX1.It was predicted that Br ACX1 was an alkaline hydrophilic protein of ACOX family on peroxisome.2.The down-regulation of Br ACX1 gene expression in mutant pdm proves that it plays an important role in petal development.The activity of ACX in tissues of‘FT' and pdm was consistent,and the activity of ACX in tissues of ‘FT' was significantly higher than that of pdm.The activity of ACX was the strongest in the flowers blooming on the day.The differential expression of Br ACX1 in tissues of ‘FT' was higher than that in pdm by Western Blot.There was almost no expression in all tissues;q RT-PCR was used to analyze the expression of ‘FT' in different tissues,buds and organs.The results showed that the expression of ‘FT' in all tissues,buds and organs was significantly higher than that of pdm.3.Jasmonic acid(JA)spraying test verified the function of Br ACX1 gene in regulating petal morphogenesis.The JA contents in buds and petals of ‘FT' and pdm were measured.The results showed that there were significant differences between them,and ‘FT' was significantly higher than that of pdm.The inflorescence of mutant pdm was sprayed with exogenous JA and the petals of wild-type ‘FT' were sprayed with DIECA.The petals of subsequent blooming flowers were shrunk and the phenotype was similar to that of pdm.These results indirectly validate the effect of candidate gene Br ACX1 on petal morphogenesis.In addition,we also analyzed the duration and transport direction of JA,and found that JA began to play a role on the second day(48h)after exogenous spraying treatment,and lasted until the fifth day;JA played a role in the bottom-up transport of plants.4.RNA-Seq analysis preliminarily constructed JA gene network regulating petal development.Comparative analyses of the transcriptomes of ‘FT',pdm and pdm + JA(p JA)plants revealed 10,160 differentially expressed genes(DEGs)with consistent expression tendencies in ‘FT' vs.pdm and p JA vs.pdm comparisons.Among these DEGs,we identified 69 DEGs related to floral organ development,11 of which are involved in petal development regulated by JA.They involved in flower petal development,involving 7 JA-responsive regulators,including MYB21,AP1,SPK1,SPT,TCP4,GIF1 and JAG,in which MYB21 and AP1 directly regulate petal development,SPK1 and SPT regulate petal morphology through cell expansion,and TCP4,GIF1 and JAG regulate petal morphology through cell proliferation.Ultimately,the combined effects of these genes affect the development of petals in the mutant pdm.