Marker gene excision in transgenic Brassica napus via Agrobacterium-mediated Cre/lox transient expression system

Oilseed rape(Brassica napus L.)is one of the most important oil crops in the world.However,study on marker-free transgene of B.napus for bio-safety purpose is limited in this allotetraploid crop.In order to advance marker gene excision research,a newly designed Cre/lox system combining crossing and auto-excision strategy was introduced into B.napus.The system consists of 2 sets of independent vectors including pC35 Spro::T7 RP carrying T7 RNA polymerase and pCT7 pro::Cre carrying T7 promoter respectively.After hybridization of 2 according types of transgenic B.napus,marker gene would be removed as T7 RNA polymerase facilitate T7 promoter to promote Cre gene expression.Totally 52 and 46 positive To transgenic lines of these 2 vectors were obtained after identification by PCR and test trip.T1 plants from 3 T0 positive pC35 Spro::T7 RP lines and 2 T0 positive pC35 Spro::T7 RP lines were identified as single copy according to segregation ratio and were chosen for crossing.However,expression of CP4 EPSPS(glyphosate resistance gene)and OXY(bromoxynil resistance gene)were not found in F1 progeny,which proved that the excision was not complete.The possible reasons for our limited success were investigated and detailed analyses were performed.Although this system is not applicable for generating transgenic B.napus free from selectable marker gene,it provided valuable experience and clue for further improvement of this technique.Many other advantages and further improvement will be progressed in future work.

NMR-based metabolomics pro!le analysis in understanding metabolic variations of Bra LTP1-overexpressing Brassica napus

BraLTP1 is an important member of lipid transfer protein family in Brassica napus.The aim of current study was to detect, classify and follow variations in metabolite profiling of B. napus with overexpression of BraLTP1. In this study, metabolic change in leaves of BraLTP1-overexpressing B. napus plants (BraLTP1-22) and negative control (BraLTP1-22N) was investigated using nuclear magnetic resonance (NMR). Statistical strategy of principle component analysis (PCA) was performed to identify related difference metabolites. PCA score plots indicated not only high reproducibility of various treatments, but also significant difference of metabolite levels between different treatments. PCA loading plots indicated main responsible metabolites and a total of 50 metabolites were quantitatively determined. A wide range of metabolites were detected due to BraLTP1 overexpression,including biosynthesis and metabolism of sugars, amino acids, ammoniums compounds and organic acids. Furthermore, concentrations of 17 amino acids were determined for other set of samples by amino acids component analysis assay. The link between metabolite variations and phenotype were also discussed in BraLTP1-overexpressing B. napus.This work will help to gain insight into BraLTP1 function in B. napus in metabolism.

Effects of non-procedural factors in Brassica napus genetic transformation

Rapeseed (Brassica napus) is the second largest oil crop in the world. However, transformation efficiency of rapeseed still needs to be improved. To evaluate non-procedural factors (e.g. explants, section of explant, marker genes and number of exogenous genes) effects on transformation efficiency, 6-day-old hypocotyl explants from in vitro grown seedlings were co-cultivated with Agrobacterium strain GV3101 harboring a binary vector using optimized transformation procedure. Results showed that normal maturing variety ‘Zhongshuang 6 (ZS6)’ had the highest overall capacity to produce rooted shoots among 5 common varieties and 6 early maturing varieties, with green callus induction rate 81.45% and shoot regeneration rate 21.66%. Early maturing variety 14M645 has relatively high regeneration rate (4.69%) and one of the shortest growth periods (107.54 d). Data showed that choosing neomycin phosphotransferase II gene (NPTII) as selectable marker led to the best transformation rate (17.38%). Selecting upper hypocotyl segments near cotyledon as explant provided the higest transformation efficiency, with regeneration rate of 25.59% when using NPTII as selectable marker and 22.19% for Bar. B. napus transformed with single gene showed higher transformation frequency than vectors with multiple genes,highlighting difficulty of multiple gene transformation. This work helped to further improve genetic transformation of B. napus by optimizing factors that impact transformation efficiency,and it would ultimately improve research in transgenic B. napus varieties with commercial potential.

LC-MS based secondary metabolic profile of BraLTP2 overexpressing Brassica napus

BraLTP2 is an important member of lipid transfer protein family, and its molecular biology function in Brassica napus （B. napus） had been explored by prerious study. How-ever, affection of BraLTP2 on secondary metabolites is still not clear. In this study, we inves-tigated difference of leaf secondary metabolite profling between BraLTP2 overexpressing B. napus and wild type. Liquid chromatography tandem mass spectrometry （LC-MS） was utilized. Wide range of secondary metabolites was found in BraLTP2 overexpressing plants. A total of 100 secondary metabolites were determined, 42 of which had signifcant differ-ences, including favonoids, phenylpropanoids and phenolamides. These results were in accordance with signifcant increasing trichomes of overexpressing BraLTP2 plants, which might produce and store secondary metabolites. Partial least squares discriminant anal-ysis （PLS-DA） was performed to identify difference of secondary metabolites. PLS-DA score plots showed high reproducibility of each treatment. Signifcant changes were found between transformed and wild type. Permutation test validates the reliability rigorously. Fur-thermore, overexpressing of BraLTP2 led to seed germination improvement during the frst 48 h under oxidation stress. Increased oxidation resistance of transgenic B. napus was in accordance with the signifcant variations of phenylpropanoids, phenylpropanoids and phe-nolamides.This work was supported by Central Public-interest Scientifc Institution Basal Research Fund, Major Research Project of CAAS Science and National Genetically Modifed Organisms Breeding Major Projects China （2018ZX0801023B）.