Preliminary Functional Analysis Of BrMYV2/3/4 Involved In Plant Growth And Development,and Indole Glucosinolate Metabolism In Brassica Rapa Ssp.pekinensis

Glucosinolates(GSs)are a class of secondary metabolites with?-D-thioglucose and sulfonated oxime groups unique to Cruciferous plants.GSs and their hydrolysates play an important role in plant morphogenesis and antifungal resistance.Chinese cabbage(Brassicarapa)belongs to Cruciferae Brassica leafy vegetable,shares the same ancestor with Arabidopsis thaliana.MYC2/3/4 belong to the basic helix-loop-helix(b HLH)family of the IIIe subgroup,were the key activation transcription factors in the jasmonic acid response pathway,mediating GS biosynthesis.Functional variation between different copies of BrMYC2/3/4 transcription factors remains unclear.In order to understand the biological function of BrMYC2/3/4 in depth,the full-length coding sequence and amino acid of BrMYC2/3/4 from Chinese cabbage genome database was obtained.Bioinformatics analysis of MYC family genes from the main Brassica vegetables was carried out to determine the key MYC genes of BrMYC2/3/4.BrMYC2/3-1/3-2/4-1/4-2 genes were then isolated and clonedfrom Chinese cabbage.Overexpression and knockout expression vector were constructed to transform Arabidopsis ecotype Columbia and flowering cabbage.Differences of morphogenesis,GS profileand Sclerotinia sclerotiorum resistance among the different genotypes'transgenic plants were analyzed.The main findings are as follows:(1)Analysis of amino acids physicochemical properties showed that BrMYC2/3-1/3-2/4-2/4-3 are hydrophobic and unstable acidic protein,respectively;while BrMYC4-1 is a hydrophobic and unstable basic protein.Domain prediction proved BrMYC2/3/4 belongs to b HLH superfamily and has high homology with Ath MYC2/3/4.(2)The full-length coding sequence of BrMYC2/3-1/3-2/4-1/4-2 gene were isolated and cloned from Chinese cabbage.Expression vectors of p2302MYC and CRISPR-Cas9-MYC were then constructed to transform Arabidopsis and flowering cabbage.Transgenic lines of T₃ generation overexpression Arabidopsis,T₀ generation overexpression and knockout overexpression flowering cabbage were obtained.Root tip cells of T₃overexpressing Arabidopsis lines were observed for subcellular localization,and BrMYC2/3/4 were found expressed in nucleus.(3)Arabidopsis plants expressing each of the five MYC paralogous genes of Chinese cabbage exhibited a wide range of differing phenotypes with respect to the root and shoot elongation,vegetative phase change,flowering time,plant height and tiller number right after flowering,and seed production.Despite the wide variation of phenotypes between the transgenic lines,all of the lines except for BrMYC4-2 exhibited shorter seed length,less seed weight,higher accumulation of GSs and resistance to S.sclerotiorumthan control.Notably the highest GSs level accumulated in BrMYC2 line was correlated with the highest extent of resistance to the necrotic fungal pathogen S.sclerotiorum.Unlike BrMYC3-1/3-2/4-/4-2,BrMYC2expression stimulated the growth of plant height after fluorescence with faster bolting time.(4)Obtained T₀ generation overexpression and knockout expression Chinese cabbage transgenic lines laid the foundation for subsequent analysis of the functional differentiation mechanism of BrMYC2/3/4 and its molecular mechanism in regulating GS biosynthesis in B.rapa.