Functional Analysis Of Glucosinolate Metabolites And Screening Of Proteins Interacting With BoaMYB28 In Chinese Kale Sprouts

Chinese kale(Brassica oleracea var.alboglabra)is an important Brassica vegetable.As a native Chinese vegetable,Chinese kale sprouts are rich in nutrients,especially abundant in glucosinolates(GLSs).Together with their short growth period and easy cultivation,cultivation of Chinese kale sprouts becomes a new high-quality and efficient production mode.GLSs are a class of important secondary metabolite widely distributed in cruciferous plants.GLSs and their breakdown products(GBPs)have multiple biological functions,such as affecting the flavor of Brassica vegetables and exerting anticancer activity.MYB28 is a key regulator of aliphatic GLS.Till now,the relationship between the flavor quality and anticancer activity of Chinese kale and the components of GLSs and GBPs is still unclear.GLSs metabolic patterns vary in different varieties of Brassica vegetables,but most relevant research focused on the GLSs themselves,and little is known about the genetic variation in the composition and content of GBPs in Chinese kale.In this study,different varieties of Chinese kale sprouts were used to investigate the metabolic patterns of GLSs and GBPs,and the relationships between the flavor formation together with anticancer activity of Chinese kale sprouts and GLS metabolites were also analyzed.The interacting proteins of Boa MYB28 were also screened by yeast two-hybrid assay.Our results help to provide a theoretical basis and technical support for the elucidation of GLS regulatory network and function,as well as the quality improvement of Chinese kale and other Brassica vegetables in the future.The main results are as follows:1.The compositions and contents of GLSs and GBPs vary greatly among different varieties of Chinese kale sprouts.Thirteen GLSs and 16 GBPs were identified in 22 different varieties of Chinese kale sprouts.It was found that the compositions contents of GLSs and GBPs varied greatly among different varieties,of which the variation range were 5.51-104.57 μmol/g DW and 1.13-16.73 μmol/g FW,respectively.Aliphatic GLSs and epithionitriles were the main GLSs and GBPs,respectively,accounting for 85% of the total GLSs content and more than 76% of the total GBPs content,respectively.3-butenyl GLS was the most abundant GLS,and the GBP of 3-butenyl GLS,1-cyano-3,4-epithiobutane,was the most abundant GBP.In addition,the total aliphatic GLS content in different Chinese kale varieties showed greater differences,while the variance in the total indolic GLS content was smaller,which indicates the influence of genetic factors on aliphatic GLSs is greater,while the influence on the metabolism of indolic GLSs is relatively small.Moreover,BOA 3 and BOA 10 maybe good candidates for future breeding programs since the two varieties contain high levels of glucosinolate breakdown products such as isothiocyanates and epithionitriles in Chinese kale sprouts.2.3-Butenyl GLS and indole-3-methyl GLS affect the bitterness of Chinese kale sprouts.According to the content of GLSs and GBPs,13 different varieties of kale sprouts were selected for tasting experiments.We also conducted the correlation analysis between the flavor quality and GLSs/GBPs contents,and found that 3-butenyl GLS and indole-3-methyl GLS correlated with the bitterness of Chinese kale sprouts significantly,respectively,indicating that 3-butenyl GLS and indole-3-methyl GLS could affect the bitterness of Chinese kale sprouts.Furthermore,BOA 1 and BOA 13 maybe good candidates for future breeding programs since the two varieties of Chinese kale sprouts have the lightest bitterness and pungency.3.The anticancer activity of Chinese kale sprouts correlates with 1-cyano-3,4-epithiobutane.The inhibition rates of the crude extracts of different varieties of Chinese kale sprouts on the proliferation of two kinds of cancer cell lines(human cervical cancer cell Hela and human colon cancer cell HT-29)was determined by the anticancer cell proliferation experiment,and it was found that 200 mg/m L crude extract of Chinese kale sprouts could significantly inhibited the proliferation of cancer cells,especially HT-29 cells,with inhibition rates of 70% and 80%,respectively,indicating that Chinese kale sprouts have high anticancer activity.Correlation analysis showed that there was a significant positive correlation between the content of 1-cyano-3,4-epithiobutane in the crude extract of Chinese kale sprouts and the inhibition rate of human colon cancer cell(HT-29)proliferation,indicating that 1-cyano-3,4-epithiobutane in Chinese kale sprouts might function in its anticancer activity against human colon cancer.4.Screening the interacting proteins of the GLS regulator Boa MYB28.We cloned the Boa MYB28 gene in Chinese kale,constructed the bait vector,and screened the interacting proteins of Boa MYB28 in the Chinese kale c DNA library using yeast twohybrid assay,and obtained 19 candidate interacting proteins.The identification of these interacting proteins will help to further elucidate the regulation network of aliphatic GLS metabolism,and then provide a theoretical basis for improving the flavor and nutritional quality of Chinese kale through metabolic engineering in the future.