Cloning,Expression And Preliminary Functional Studies Of The BocGTR2s Glucosinolate Transporter In Chinese Kale

Chinese kale(Brassica oleracea var.chinensis Lei)is an important cruciferous vegetable with high nutritional value and unique flavor,which is favored by consumers.Chinese kale is rich in glucosinolate(Glucosinolate),and its glucosinolates and its degradation products have active biochemical characteristics.Glucosinolates are famous for their anti-cancer and anti-aging properties.The researchers’ high attention has been widely used in the fields of botany,medicine,food science and animal science.Sulfate is synthesized in various tissue parts except seeds,and a new accumulation level is formed in storage sites such as seeds,roots,and leaves by transport of GTR proteins.However,the effect of GTR protein on the accumulation of glucosinolates in different tissues of Brassica vegetables is not clear.In this study,three glucosinolate-related genes(BocGTR2 a,BocGTR2 b and BocGTR2c)were cloned in Chinese kale,and BocGTR2 c was found to be a key gene by expression analysis.We analyzed three BocGTR2 s and tried to identify the function of BocGTR2 c in the glucosinolates transport by RNAi and CRISPRCas9 technology,regulating the accumulation of glucosinolates in different tissues of Brassica vegetables.The study provided a basis for regulation of glucosinolate accumulation levels in different tissue sites by genetic engineering.The main results obtained are as follows:1.Three BocGTR1 s were cloned and analyzed for gene sequence characteristics.(1)The DNA and c DNA sequences of BocGTR2 a,BocGTR2 b and BocGTR2 c were cloned.The similarity between the three genes and the Arabidopsis thaliana transport gene At GTR2 was over 86%,indicating BocGTR2 s may be involved in glucosinolate transport in Chinese kale;(2)Bioinformatics analysis of Bo GTR2 s revealed that the three genes have great similarity with At GTR2 in gene structure,conserved domain and protein structure,indicating that Bo GTR2 s may have similar functions to At GTR2.2.Analysis of the accumulation of glucosinolates in different tissues of Chinese kaleThe determination of glucosinolates in different tissues of Chinese kale showed that the total glucosinolate content of roots was higher than that of other tissues.The total glucosinolate content in florescence inflorescences and pods was also significantly higher than that in vegetative organs;In terms of content,the glucosinolate component of each tissue part is mainly aliphatic glucoside,the inflorescence has the most aliphatic glucoside,and the root contains the most steroidal glucoside.3.The expression patterns of three BocGTR2 s were analyzed.The expression pattern of BocGTR2 s was analyzed.It was found that the three genes of BocGTR2 s may play an important role in the transport of glucosinolates.The accumulation of glucosinolates at the tissue sites may be positively correlated with the expression of BocGTR2 s.The expression of roots is much higher than that of stems and leaves.BocGTR2 c has the highest expression in roots and may play a major role in root glucosamine transport.4.The function of BocGTR2 c in the accumulation of kale glucosinolate was analyzed.The RNAi vector of BocGTR2 c and the CRISPR/Cas9 vector were constructed and the kale was successfully transformed.The expression level of BocGTR2 s was analyzed in PCR positive RNAi transgenic plants.RNAi plants T01,T02,T03,T04,T05 and T06 were selected as materials for subsequent experiments;the target mutation rate of CRISPR-BocGTR2 c was 0.067% and 3 Mutations(T03,T018,and T019)were selected as materials for subsequent experiments.