Cloning And Characterization Of Boron Transporter Gene From Rapeseed (Brassica Napus L.)

Rapeseed (Brassica napus L.) is the second largest oil crop in the world, and provides important contribution to food oil consumption in China. China is the largest rapeseed producer in the world. In the Yangtze River region, rapeseed yield accounts for more than 80% in China. However this area is generally lack of Boron nutrient in the soil. Boron nutrient is one of the crucial microelement for rapeseed growing. It is very sensitive when boron is too little or too much. Boron deficiency may lead plant to "flowering but no seeds". Some time it results in nothing yield. One of the conventional ways to deal with this problem is to select and improve the rapeseed cultivar with more efficiency and higher ability to utilize trace of boron nutrient in the soil. However the progress is very limited. Here we cloned and identified boron transporter gene from rapeseed and the provided a new way to improve rapeseed boron utilization efficiency by genetic engineering technology.In this study, gene specific primers were designed according to Arabidopsis boron transporter gene AtBOR1 and homologous ESTs from B. napus. Bioinformatics analysis and gene copy number analysis and expression analysis in various organs had been done after gene cloning. We constructed the plant over-expression vector with the full length cDNA of BnBOR sequence fusioned with the constitutive strong promoter CaMV35S to transform the hypocotyls. And the transplants were obtained after the molecular identification. The main results were as follows:1. This gene, designated BnBOR(GenBank ID:GU362414.1), is 2112 bp in the complete coding sequences, encoding 703 amino acids and with eleven introns in gDNA.2. Bioinformatic analysis showed that, BnBOR is a high molecular weight Rapeseed(Brassica napus L.) is the second largest oilseed crop in the world, and also is the most important oilseed crop in China. China is a big rapeseed producer in the world, and the Yangtze River valley is the main production region with the planting area and total yield accounting for more than 80% of the country. However, soils in this region are generally boron deficient. Rapeseed is sensitive to boron supply and requires a large quantity of boron for its normal growth and development. Boron deficiency may severely affect fertilization and seed setting, resulting in significant decreases in yield and quality. Breeding and extension of low boron-tolerant rapeseed varieties is the most economic and effective countermeasure to overcome the adverse effects of boron deficiency on yield and quality. However, little progress in improvement of low boron tolerance of rapeseed by conventional methods has been made. Development of plant genetic engineering technology and identification of boron transporter gene have provided a new way for improvement of tolerance of rapeseed to boron deficiency.In order to provide evidences and lay a foundation for improvement of low boron tolerance of rapeseed by genetic engineering, a boron transporter gene was cloned from rapeseed (Brassica napus L.) for the first time, analyses of bioinformatics, copy number in genomes and expression profile in different organs for the cloned gene were carried out, and over-expression vector of the cloned gene was constructed and used for rapeseed transformation in this study. The main results were as follows:1. The mRNA sequence of the boron transporter gene BnBOR (GenBank accession number GU362414.1) was 2112 bp in length, coding for 703 amino acids. The genomic DNA sequence of the gene consisted of 11 introns and 12 exons.2. Bioinformatics analysis showed that BnBOR was a high molecular weight transmembrane protein and contained 11 putative transmembrane domains. Homology and phylogenetic analyses indicated that BnBOR was clustered into the same broad category with AtBOR1, AtBOR2 and AtBOR3 from Arabidopsis thalina, OsBOR1 and OsBOR3 from rice, and boron transporter genes from lettuce, grape, citrus, maize, potato, tobacco and castor. The identities of nucleotide and deduced amino acid sequences between BnBOR and these BOR genes were all higher than 70%. Among them, the identity of deduced amino acid sequence between BnBOR and AtBOR1 and AtBOR2 was as high as 97% and 91% respectively.3. Southern blotting analysis indicated that 7 hybridization bands could be detected with a conserved sequence of boron transporter genes as a probe. It was speculated that, therefore, multiple different boron transporter genes might exist in genomes of rapeseed, just as in Arabidopsis thalina and rice.4. Expression profile of boron transporter genes in different organs of rapeseed was analyzed by RT-PCR. It was found that expression level of boron transporter genes in roots and flowers was higher than that in stems and leaves, and developing seeds showed the weakest expression of boron transporter genes.5. The over-expression vector of BnBOR gene was constructed with plasmid pFGC-5941 by fusioning it to constitutive expression promoter CaMV 35S, and used for Agrobacterium tumefaciens-mediated transformation of rapeseed hypocotyls. A batch of Basta-resistant plants were regenerated,36 positive transformed plants of which were confirmed by PCR detection.In summary:In this study, rapeseed boron transporter gene BnBOR was cloned for the first time; biological information, copy number in genomes and expression profile in different organs for BnBOR were basically understood; and 36 transgenic rapeseed plants with constitutive over-expression of BnBOR gene were produced. These results have laid a good foundation for subsequent studies.