Cloning,Analysis And Genetic Transformation Of Purple Character Gene In Leaf Mustard

Brassica juncea（Brassica juncea Czern.et Coss.）belongs to Brassica Crops of Cruciferae.It is an important cultivated vegetable,oil crop and seasoning crop in China,which has high economic value.As a kind of leaf mustard,the anthocyanin content of red leaf mustard is significantly higher than that of common leaf mustard.Based on the six-generation genetic population constructed by Zhang Tian,we cloned and transformed the candidate gene Bj.Pur（transcription factor MYB90）on chromosome B02,using purple mustard inbred line（L17-P）and green mustard inbred line（L17-G）as the main research materials,and based on the results of Liu Liyan（2015）and Zhang Tian（2017）.The results are as follows:1.Analysis of protein structure encoded by Bj.Pur gene.The amino acid sequence information encoded by Bj.Pur gene was submitted to NCBI Conserved domains for protein domain and functional analysis.The results showed that the N-terminal highly conserved MYB domain was located in 8-105 amino acids and belonged to R2R3-MYB transcription factor.The protein had a high similarity with the transcription factor MYB114-like of radish（92%）.2.Comparative analysis of anthocyanin content and chlorophyll content.We have analyzed the anthocyanin content of purple mustard inbred line（L17-P）and green mustard inbred line（L17-G）and hybrid progeny F₁.The results showed that the anthocyanin content of purple mustard was significantly higher than that of green mustard,while the anthocyanin content of hybrid progeny F₁ was between parents.We also analyzed the chlorophyll content of purple leaf mustard,green leaf mustard and their hybrid progeny F₁.The results showed that the chlorophyll content of purple leaf mustard was slightly higher than that of green leaf mustard,and the chlorophyll content of hybrid progeny F₁ was between parents.3.Screening of linkage markers.Referring to the mustard genome（https://www.ncbi.nlm.nih.gov/nuccore/LFQT00000000）,132 pairs of primers were developed and screened on the other side of Bj.Pur gene located by SSR,In Del and SNP molecular marker technology,using purple and green plants from F₂ generation isolation population as locating populations,and no stable linkage markers were found.4.Analysis of gene expression related to anthocyanin synthesis pathway.The results of gene expression analysis of anthocyanin synthesis in purple leaf mustard,green leaf mustard and F₁ generation showed that the relative expression of candidate gene Bj.Pur（transcription factor MYB90）was significantly different between the two parents,and the expression of F₁ was between the two parents.The downstream genes of anthocyanin synthesis were significantly up-regulated in the purple leaf mustard,followed by F₁.The expression of anthocyanin synthesis related genes increased with the prolongation of treatment time under low temperature treatment,but did not change significantly under shading treatment.5.Genetic transformation and functional verification of candidate gene Bj.Pur.The p35s:P-MYB90 superexpression vector was constructed and the genetic transformation of Arabidopsis thaliana and Greenery was carried out.The results showed that there were obvious purple phenotypes in the T₁ generation of transgenic Arabidopsis thaliana.The phenotype of leaf color of T₁ generation of transgenic green mustard was purple,and the content of anthocyanin was significantly increased compared with that of green mustard.The expression of genes related to anthocyanin synthesis was also significantly increased in T₁ generation of transgenic green mustard.6.Cloning and analysis of promoter sequence of Bj.Pur gene.Referring to the mustard genome,we amplified the 1980 bp upstream fragments of the Bj.Pur gene and the purple leaf mustard.The amplified results showed that the promoter fragments of the green leaf mustard had a total of 36 bases inserted into the purple leaf mustard.The two fragments were identical in the 959 bp upstream of the Bj.Pur gene,and the promoter-related elements of the two fragments were almost identical.Two groups of GUS reporter gene vectors with 959 bp promoter truncation and full promoter length were constructed and injected into Ben’s tobacco for transient expression.The results showed that 959 bp promoter fragments with the same length upstream of Bj.Pur genes in purple leaf mustard and green leaf mustard had basic promoter activity,and there was no significant difference in promoter activity between them.7.Preliminary study on the mechanism of leaf color difference among different mustard germplasm resources.A 500 bp molecular marker was designed at the first intron insertion and the second exon of the green leaf mustard,and 52 different mustard varieties were screened.Only the green leaf phenotype of the Japanese round leaf mustard amplified the expected band.Sequence alignment results showed that they were identical,which preliminarily indicated that there was a close relationship between P.japonica and P.greenery.The results of amplification and screening of promoter region of Bj.Pur gene showed that no band was amplified in spring vegetable.The preliminary results showed that the promoter structure of 52 mustard materials was basically the same.