Cloning And Functional Studies Of The BocGTR1s Related To The Glucosinolate Transporter Of Chinese Kale

Chinese kale(Brassica oleracea var.chinensis Lei)is one of the most important cruciferous vegetables.Its unique flavor and high nutritional value are popular with consumers.The Chinese kale is rich in glucosinolates.The glucosinolates are an important plant secondary metabolite.The glucosinolates and their degradation products have active biochemical properties and can impart speciality to food products.Flavors are resistant to diseases and pests on the plants themselves.Among them,the degradation products of Radish glucosinolates(aliphatic glucosides)can prevent the occurrence of multiple cancers.They are widely used in the fields of botany,medicine,animal husbandry and food science,etc.As a result,researchers were extremely interested in glucosinolates.The glucosinolates are synthesized at any tissue parts except seeds,and then form new levels of accumulation at any tissue parts including seeds.The GTR protein is involved in the transport of glucosinolates,but it is a glucoside in different parts of the tissues of the Brassica.However,the function of GTR involved in glucosinlate accumulation in different parts of brassica pants remains unknown.In this study,three glucosinase transport related genes(BocGTR1a,BocGTR1 b and BocGTR1c)were cloned in Chinese kale,and BocGTR1 a may be one of the key genes through expression analysis.In this study,RNAi interference technology was used to reduce the expression of BocGTR1 a in Chinese kale.We explored the function of the gene in the accumulation and distribution of glucosinolates,which provide a basis for regulation of glucosinolate accumulation levels in different tissue sites by genetic engineering.The main results obtained are as follows: 1.Cloning of three BocGTR1 s and analysis for gene sequence characteristics and protein subcellular localization(1)There genes,BocGTR1 a,BocGTR1b and BocGTR1 c,were amplified from Chinese kale.BLAST analysis showed that the sequence of BocGTR1 have a similarity of 99% with the Brassica oleracea BoGTR1 s amino acid sequence,and a which was similar ity of 81% with AtGTR1,which indicated the sequence of glucosinolate transport related genes obtained in Chinese kale were correct.(2)Three genes,BocGTR1 a,BocGTR1b and BocGTR1 c,were analyzed for their gene structure,physicochemical properties,signal peptides,phosphorylation sites,transmembrane structures,functional domains,second and three-dimensional protein structures and genetic relationships.The transmembrane and functional domains of the three genes were found to be identical to that of Arabidopsis AtGTR1,indicating that these three genes may have similar functions to the Arabidopsis glucosinolate transporter gene AtGTR1.(3)Subcellular localization analysis showed that BocGTR1 a and BocGTR1 b proteins were localized in the nucleus and plasma membrane and the BocGTR1 c protein is localized on the plasma membrane.2.Analysis the expression patterns of BocGTR1s(1)The expression patterns of BocGTR1 s in different tissues,at different developmental stages and under different treatment conditions were analyzed.BocGTR1 a and BocGTR1 c have higher expression levels in tissues,especially in leaves and buds.The expression of BocGTR1 b was low in all tissue sites and the expression levels of the three BocGTR1 s gradually increased with the growth and development of the plants.The expression levels were highest in the pods and seeds,and the expression of BocGTR1 a was the highest.Magnesium chloride treatment,damage and the heat stress had a significant effect on the expression of three BocGTR1 s.In particular,BocGTR1 a was significantly up-regulated after being treated with magnesium chloride,noxious stress and heat stress.The above results suggested that BocGTR1 a might be a key glucoside transporter gene.3.Analysis of the function of BocGTR1 a in the accumulation of glucosinolates(1)The best disinfection time for obtaining high-quality sterile seedlings was 9 min,and the lethal concentration of Chinese kale hypocotyl for Phosphinothricin(PPT)screening culture was 14 mg/L.(2)RNAi-BocGTR1 a interference vector was constructed and was transformed into Chinese kale.RNAi-transformed plants with positive PCR were analyzed for BocGTR1 s expression level and glucosinolate content.The expression of BocGTR1 a in the leaf was significantly silenced,the total glucosinolate content in the leaves was significantly reduced,and the total glucosinolate content in the roots increased significantly.The increase in the content of BocGTR1 a indicates that the accumulation of glucosinolates in different tissues has changed and BocGTR1 a is involved in regulating the accumulation of glucosinolates in different tissues.