Stresses induced by the deficiency or excess of trace mineral elements, such as manganese (Mn), represent a common limiting factor for the production of crops like Brassica napus. To identify key genes involved in Mn allocation in B. napus and elucidate the underlying mechanisms, a member of the metal tolerance protein (MTP) family obtained in the previous screening of cDNA library of B. napus under Mn stress was selected as the research subject. Based on the sequence information and phylogenetic analysis, it was named as BnMTP10. It belongs to the Mn-cation diffusion facilitator (CDF) subfamily. Expression of BnMTP10 in yeast significantly improved the tolerance of transformants to excessive Mn and iron (Fe) and reduced the accumulation of Mn and Fe. However, the yeast transformants exhibited no significant changes in tolerance to excess cadmium, boron, aluminum, zinc, or copper. The qRT-PCR results demonstrated that the flowers of B. napus had the highest expression of BnMTP10, followed by roots and leaves. Subcellular localization studies revealed that BnMTP10 was localized in the endoplasmic reticulum (ER). Compared with wild-type plants, transgenic Arabidopsis overexpressing BnMTP10 exhibited enhanced tolerance to excessive Mn stress but showed no significant difference under Fe stress. Correspondingly, under excessive Mn stress, the Mn content in the roots of transgenic Arabidopsis increased significantly. However, under excessive Fe stress, the Fe content in transgenic Arabidopsis did not alter significantly. According to the results, we hypothesize that BnMTP10 may alleviate excessive Mn stress in plants by mediating Mn transport to the ER. This study facilitated our understanding of efficient mineral nutrients, and provided theoretical foundations and gene resources for breeding B. napus.
Brassica napus/genetics* ; Manganese/metabolism* ; Plants, Genetically Modified/genetics* ; Plant Proteins/physiology* ; Arabidopsis/metabolism* ; Gene Expression Regulation, Plant ; Phylogeny ; Cation Transport Proteins/metabolism* ; Stress, Physiological

Improving the nitrogen use efficiency (NUE) of Brassica napus is of significant importance for achieving the national goal of zero growth in chemical fertilizer application and ensuring the green development of the rapeseed industry. This study aims to explore the effects of the nitrate transporter gene BnaNRT1.5s on the nitrogen transport and NUE of B. napus, providing excellent genetic resources for the development of nitrogen-efficient B. napus varieties. The spatiotemporal expression of BnaA05.NRT1.5 as a key nitrogen responsive gene was profiled by qRT-PCR at different growth stages and for different tissue samples of B. napus 'Westar'. Subcellular localization was employed to examine its expression pattern in the cells. Additionally, CRISPR/Cas9 was used to create BnaNRT1.5s knockout lines, which were subjected to hydroponic experiments under high nitrogen (12.0 mmol/L) and low nitrogen (0.3 mmol/L) conditions. After the seedlings were cultivated for 21 days, root and shoot samples were collected for weighing, nitrogen content determination, xylem sap nitrate content assessment, and calculation of total nitrogen and NUE. The B. napus nitrate transporter BnaA05.NRT1.5 was localized to the cell membrane. During the seedling and early bolting stages, BnaA05.NRT1.5 was predominantly expressed in roots, while it was highly expressed in old leaves and mature silique skin during the reproductive stage. Compared with the wild type, the mutant BnaNRT1.5s showed significant increases in the dry weight and total nitrogen of seedlings under both high and low nitrogen conditions. Under low nitrogen conditions, NUE in the roots of BnaNRT1.5s significantly improved. Notably, under both high and low nitrogen conditions, the nitrate content in the shoots of BnaNRT1.5s decreased significantly, while that in the roots increased significantly, resulting in a significantly decreased shoot-to-root nitrate content ratio. BnaNRT1.5s is involved in regulating the transport of nitrate from the roots to the shoots, and its mutation enhances nitrogen absorption and utilization in B. napus seedlings, promoting seedling growth. This study not only provides references for understanding the physiological and molecular mechanisms by which BnaNRT1.5s regulates NUE but also offers valuable genetic resources for improving NUE in B. napus.
Brassica napus/genetics* ; Anion Transport Proteins/metabolism* ; Nitrogen/metabolism* ; Nitrate Transporters ; Plant Proteins/metabolism* ; Nitrates/metabolism* ; Gene Expression Regulation, Plant ; Biological Transport

Under different red (R):blue (B) photon flux ratios, the growth performance of rapeseed (Brassica napus L.) is significantly different. Rapeseed under high R ratios shows shade response, while under high B ratios it shows sun-type morphology. Rapeseed under monochromatic red or blue light is seriously stressed. Transcriptomic and proteomic methods were used to analyze the metabolic pathway change of rapeseed (cv. "Zhongshuang 11") leaves under different R:B photon flux ratios (including 100R:0B%, 75R:25B%, 25R:75B%, and 0R:100B%), based on digital gene expression (DGE) and two-dimensional gel electrophoresis (2-DE). For DGE analysis, 2054 differentially expressed transcripts (|log₂(fold change)|≥1, q<0.005) were detected among the treatments. High R ratios (100R:0B% and 75R:25B%) enhanced the expression of cellular structural components, mainly the cell wall and cell membrane. These components participated in plant epidermis development and anatomical structure morphogenesis. This might be related to the shade response induced by red light. High B ratios (25R:75B% and 0R:100B%) promoted the expression of chloroplast-related components, which might be involved in the formation of sun-type chloroplast induced by blue light. For 2-DE analysis, 37 protein spots showed more than a 2-fold difference in expression among the treatments. Monochromatic light (ML; 100R:0B% and 0R:100B%) stimulated accumulation of proteins associated with antioxidation, photosystem II (PSII), DNA and ribosome repairs, while compound light (CL; 75R:25B% and 25R:75B%) accelerated accumulation of proteins associated with carbohydrate, nucleic acid, amino acid, vitamin, and xanthophyll metabolisms. These findings can be useful in understanding the response mechanisms of rapeseed leaves to different R:B photon flux ratios.
Brassica napus/radiation effects* ; Brassica rapa/radiation effects* ; Carbon/chemistry* ; Chloroplasts/radiation effects* ; Computational Biology ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation, Plant/radiation effects* ; Image Processing, Computer-Assisted ; Light ; Mass Spectrometry ; Metabolic Networks and Pathways ; Nitrogen/chemistry* ; Photons ; Photosystem II Protein Complex/genetics* ; Plant Leaves/radiation effects* ; Plant Proteins/genetics* ; Proteome ; Ribosomes ; Transcription, Genetic ; Transcriptome

In order to broaden Chinese cabbage gene pool, we conducted interspecific somatic hybridization between Chinese cabbage (Brassica campestris, 2n=20, AA) and Cabbage (B. oleracea, 2n=18, CC). Protoplasts were isolated from 10-day-old cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with some plant growth regulators. Fusion products were characterized by their morphological, cytological and molecular biological traits. The results showed that, a total of 35 regenerated green plants were obtained from 320 calli, the plant regeneration frequency was 10.94%, and eleven of which were survived in greenhouse. All regenerants were true hybrids as confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis. Ploidy levels of hybrid plants were determined by chromosome counting and flow cytometry. The sum of the chromosome number (2n = 38) from the two fusion patents were found in 36.4% of regeneratns; another 36.4% had chromosomes range to 58-60; 27.2% had more chromosomes ranges to 70-76. All regenerated plants produced normal flowers. We investigated the pollen fertility and seed set after self-pollination and backcrossing with the parental species. For hybrids with chromosomes more than 38 it was possible to obtain some seeds when they after self-pollination. Within the group of hybrids with 38 chromosomes, seed set were very variable, only 0.11 seeds per pod by self-pollination, 0.23-0.76 by open-pollination, 0.02-0.04 by backcrossing with Chinese cabbage. Progeny lines obtained by self-pollination had larger leaves and leaf shapes intermediate of the parental species. Pollen fertility was gradually recovered in the first and second progenies. The backcrossing progeny lines, as a whole, exhibited morphologies were similar to Chinese cabbage. Morphological variations were observed among the somatic hybrids and their progenies.
Brassica napus ; genetics ; growth & development ; Breeding ; Chromosomes, Plant ; Hybridization, Genetic ; genetics ; Mustard Plant ; genetics ; growth & development ; Plant Somatic Embryogenesis Techniques ; Ploidies ; Pollen ; physiology ; Protoplasts ; cytology ; Random Amplified Polymorphic DNA Technique ; Recombination, Genetic

Recently, more research about the plant bioreactor expressing genes encoding human proteins was reported. In the present study, the cDNA of the human gene keratinocyte growth factor 2 (KGF2) was replaced with plant preferred codons by PCR, and the modified full-length cDNA was cloned into the plant expression vector pCAMBIA-YO containing the oil-body promoter. The fusion construct pCAMBIA-YO-KGF2 was transformed into Brassica napus by Agrobacterium tumefacien-mediated cotyledon transformation method. The transgenic seedlings were identified by PCR, Southern and western blot analysis all showed that KGF2 gene was successfully expressed in in transgenic Brassica napus.
Brassica napus ; genetics ; metabolism ; Cloning, Molecular ; DNA, Complementary ; genetics ; Fibroblast Growth Factor 7 ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Humans ; Plants, Genetically Modified ; genetics ; Rhizobium ; genetics ; Transformation, Genetic