Regulatory Mechanisms And Function Of Indolic Glucosinolate Metabolism By BoaMYB51 And BoaBIM1 In Chinese Kale

Glucosinolates(GS)are diverse kinds of sulfur-and nitrogen-containing plant secondary metabolites that exist mainly in crucifers.Glucosinolates have extensive biological functions,which play an important role in plant resistance to pathogen invasion,insect defense,and autoimmunity.The breakdown products of glucosinolates can prevent multiple cancers and repress the initiation and progression of carcinogenesis.Brassica vegetables belong to one of the most important kinds of vegetables in our country.Glucosinolates are abundant in Brassica vegetables,which not only affect the flavor and nutrition of Brassica vegetables,but also play an important role in resisting important vegetable diseases such as black spot.The regulatory network of glucosinolate biosynthetic pathway has been elucidated in Arabidopsis thaliana,but there are few studies and applications in crops.Researches on glucosinolates of Brassica vegetables by using metabolic engineering and other biotechnology approaches to improve the resistance and quality of vegetables is of significance in development of the vegetable industry and environmental protection.Chinese kale is an original Chinese vegetable from the Brassicaceae family which is rich in glucosinolates.It is a good system for studying the metabolic regulation network of glucosinolates in Brassica vegetables.The present study used Chinese kale as the research material,molecular biology,physiology,genetics,genomics,and other methods are explored to elucidate the indole glucosinolates transcriptional regulatory mechanism of indole glucosinolate metabolic pathways.Following are the main results:1.The gene silencing of Boa MYB51 by VIGS systerm reduced the resistance of Chinese kale to black spot disease.Virus-induced gene silencing(VIGS)system was explored to obtain Boa MYB51 gene silencing lines which were then inoculated with Alternaria brassicicola spore suspension.Disease assay showed that lesion in Boa MYB51-VIGS line was larger than that in control and the spore concentration was higher when the plaque was dissolved in water.The results showed that Boa MYB51 positively regulated the resistance of Chinese kale to black spot disease.2.Overexpression of Boa MYB51 promoted the biosynthesis of IGS in Chinese kale.To verify the function of Boa MYB51 in IGS pathway,we obtained Boa MYB51 genesilencing lines and over expression transgenic plants.The results showed that the contents of indole glucosinolates profiles in Boa MYB51-VIGS lines were significantly decreased compared with those of the control.However,IGS contents in Boa MYB51 overexpression Chinese kale lines were increased signicantly.Correspondingly,the expression of genes related to IGS biosynthesis such as BoaCYP79B2,BoaCYP79B3 and BoaCYP83B1 was also modulated by Boa MYB51.The results showed that Boa MYB51 positively regulated the biosynthesis of IGS in Chinese kale.3.The bHLH transcription factor BoaBIM1 interacted with Boa MYB51.A yeast two-hybrid system was used to screen the Chinese kale cDNA library,30 proteins that may interact with Boa MYB51 were identified.We performed the yeast two-hybrid point-to-point and pull-down tests on the bHLH transcription factors BoaBIM1 and Boa MYB51 for further protein interaction verification.Two BoaBIM1 genes were identified in Chinese kale.Amino acid comparison and phylogenetic tree analysis showed that BoaBIM1 and At BIM1 shared high homology.We found that BoaBIM1 were localized in the nucleus by tobacco transient expression analysis.The expression of BoaBIM1 in different tissues and organs of Chinese kale and different growth stages of Chinese kale sprouts was investigated.The results showed that the expression levels of BoaBIM1 in young leaves,flowers,stems,and roots is relatively high while in siliques is very low.In addition,the expression level of BoaBIM1.1 was significantly higher than that of BoaBIM1.2.4.BoaBIM1 transcriptionally regulated the IGS biosynthesis.To elucidate the function of BoaBIM1 in Chinese kale,we generated the BoaBIM1-VIGS line in Chinese kale.The results showed the contents of indole glucosinolate profiles in BoaBIM1-VIGS lines were decreased significantly.When compared with the control,the expression level of Boa MYB51.1 was decreased significantly while there was no significant difference in the gene expression of Boa MYB51.2 in BoaBIM1-VIGS lines.The expression level of genes in IGS biosynthetic pathway,notably BoaCYP79B2-2and BoaSUR1.1 was significantly reduced.We also generated BoaBIM1 overexpression lines in Arabidopsis by Agrobacterium infection method and further determined the contents of IGS profiles and expression levels of IGS biosynthetic gene in transgenic lines.Consistent with the results in Chinese kale,the contents of indole glucosinolates in bim123 were significantly reduced compared with the wile type,while Col-0 35S:BoaBIM1.1-1 and Col-0 35S: BoaBIM1.2-3 had higher expression level of BoaBIM1.1and BoaBIM1.2,respectively,showed significant increase in IGS contents.We further analyzed the BIM1 binding cis origins in the promoter regions of IGS biosynthetic genes,such as CYP79B2,CYP79B3,CYP83B1,and CYP81F2 and chromosome immunoprecipitation tests were conducted to verify that BIM1 can directly bind to the promoters of IGS biosynthetic genes.These results further indicated that BoaBIM1 might regulate IGS-related genes at the level of transcription.In summary,we verified the function of the transcription factor Boa MYB51,positively regulating the biosynthesis of indolic glucosinolates and resisting black spot diseases in Chinese kale.We also identified a novel IGS regulatory protein,BoaBIM1 which is a bHLH transcription factor interaction with Boa MYB51.Boa MYB51 and BoaBIM1 could form a complex which regulates the expression of downstream genes.Our study enriches the research in metabolic regulation network and biological functions of indolic glucosinolates in Brassica vegetables,and provides a new strategy for the control of the severe fungal disease such as black spot disease in Brassica vegetables.