Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

Brassica rapa orphan genes largely affect soluble sugar metabolism

Orphan genes(OGs),which are genes unique to a specific taxon,play a vital role in primary metabolism.However,little is known about the functional significance of Brassica rapa OGs(BrOGs)that were identified in our previous study.To study their biological functions,we developed a BrOG overexpression(BrOGOE)mutant library of 43 genes in Arabidopsis thaliana and assessed the phenotypic variation of the plants.We found that 19 of the 43 BrOGOE mutants displayed a mutant phenotype and 42 showed a variable soluble sugar content.One mutant,BrOG1OE,with significantly elevated fructose,glucose,and total sugar contents but a reduced sucrose content,was selected for indepth analysis.BrOG1OE showed reduced expression and activity of the Arabidopsis sucrose synthase gene(AtSUS);however,the activity of invertase was unchanged.In contrast,silencing of two copies of BrOG1 in B.rapa,BraA08002322(BrOG1A)and BraSca000221(BrOG1B),by the use of an efficient CRISPR/Cas9 system of Chinese cabbage(B.rapa ssp.campestris)resulted in decreased fructose,glucose,and total soluble sugar contents because of the upregulation of BrSUS1b,BrSUS3,and,specifically,the BrSUS5 gene in the edited BrOG1 transgenic line.In addition,we observed increased sucrose content and SUS activity in the BrOG1 mutants,with the activity of invertase remaining unchanged.Thus,BrOG1 probably affected soluble sugar metabolism in a SUS-dependent manner.This is the first report investigating the function of BrOGs with respect to soluble sugar metabolism and reinforced the idea that OGs are a valuable resource for nutrient metabolism.