The Establishment Of CRISPR/Cas9 Technology And The Function Study Of BjFT Gene In Brassica Juncea Var.Tumida

Tumorous stem mustard(Brassica juncea var.tumida)is belonging to Brassica of cruciferous.It has high economic value due to its mainly used as a raw material for Fuling mustard.The stem mustard often shows abnormal growth phenomena,such as flowering and bolting early,hollow heart,and non-swelling of the tuber in production.The previous studies of tumorous stem mustard were mainly focused on breeding,industrial cultivation,and antioxidant capacity,but rarely reports on the molecular function.The Phosphatidyl Ethanolamine-Binding Protein(PEBP)gene family,mainly regulates flowering development in plants.Systematic research these genes of stem mustard can provide a basis for studying the phenomenon of early bolting and flowering in stem mustard.In this study,21 Bj PEBP genes were identified in stem mustard.The length of the encoded protein was about 175 aa and the conservative domain was round 135 aa.Phylogenetic tree revealed that these genes were clustered in four subfamilies,FT-Like,TFL1-Like,MFT-Like,and ybh B-like.They were mainly located at chromosomes A06,A07,A10,B08,and B02,and some of them may have been subject to purifying selection.RNA-seq data showed that the expression level of Bj TFL1 and Bj TFL3 genes gradually increase at YA3 and YA4,that suggest they were involved in the reproductive growth of stem mustard.The q PCR results showed that the expression of Bj PEBP family genes was significantly different in roots,stems,leaves,flowers and fruit pods.Bj CLA and Bj PDS were used as marker genes in the VIGS technology system.By using Agrobacterium-mediated methods,the stem mustard cotyledons were injected,and showed the whitening of the leaves.The q PCR result showed that the expression of the two genes were reduced.Further investigates the function of the stem mustard gene FT.The subcellular localization results showed that the fluorescence of Bj FT-GFP fusion protein was observed at the cytoplasmic membrane.The results of yeast two-hybrid experiments showed that Bj FT and Bj14-3-3 proteins could interact in vitro.After silence the Bj FT gene by VIGS method,the expression of Bj FT gene was significantly decreased.The expression of downstream genes of the FT flowering pathway was examined,AP1,FUL,LFY and SOC1 genes were significantly decreased.This result suggested that silencing of FT genes may lead to decreased expression of their downstream genes.Since the effect usually of VIGS technology lasts 4 weeks,but more than 6 months from injection to flowering,no significant effect has been observed in Bj FT gene silencing experiment.These results suggest that Bj FT,like Arabidopsis FT,may have a function in regulating flowering,possibly by interacting with Bj14-3-3 and participating in the expression of downstream flowering genes.The CRISPR/Cas9 technology system was constructed also using the Bj CLA and Bj PDS genes as marker genes.No editing products were detected after Agrobacterium-mediated transient transformation.