Screening Of Root Hair Mutants By TILLING And Isolation Of The Genes Involved In Root Hair Development In Brassica Napus

Rapeseed is an important oil crop. Phosphorus (P) deficiency leads to yield and quality reduction in rapeseed. Molecular biology technique is available to unveil the characteristics in uptake, utilization and distribution of P in rapeseed, which can provide basic information for us to understand the mechanism on P nutrient, and improve P nutrition trait in rapeseed. In the present study, it bases on the Ningyou 7 EMS mutants' library, which was built by our laboratory. We cloned the genes BnWER,Bn-AT5G49270 and BnCPC that control the development of root hair in Brassica napus, according to the gene information on Arabidopsis Thaliana by homologue-sequence method and PCR walking. According to the difference between the two homologous genes of BnWER that isolated by sequence analysis, we designed gene-specific primers and screened NingYou7 EMS mutant library, to find the root hair mutants by TILLING (Targeting Induced Local Lesions IN Genomes) technology, and identified the phosphate uptake efficiency of the mutants. The main results were listed as follows.1. The isolation of the genes BnWER, Bn-A T5G492 70, BnCPC in Brassica napusHomologue-sequence method and PCR walking were used to isolate the genes in B. napus. We obtained two full-length homologous genes of BnWER in B.napus, which were named as BnWER-1 and BnWER-2. The sequence homology of them was 98%, the main difference of the two homologous genes is 26 bp fragment deletion on the 1355th bp of the intron 2 of BnWER-1 compared with BnWER-2. Using the same methods, we cloned the gene of Bn-AT5G49270 that control the root hair development in Arabidopsis, the sequence homology of them was 86% compared with the corresponding Arabidopsis genes. Bn-AT5G49270 has at least two homologous genes by sequence analysis, and there has 9 bp (CTGTATATA) fragment deletion in one homologous gene fragment. For BnCPC, 682 bp fragment was gained in B. napus, the homology of them was from 85% to 93% compared with the corresponding Arabidopsis gene.2. Screening BnWER mutantsAccording to the difference between BnWER-1 and BnWER-2, we designed two pairs of gene specific primers, they were BnWER-1F/1R and BnWER-2F/2R. With these two pairs of primers, we detected 16 mutations by screening 2980 M₂ plants. The PCR products of the mutants were sequenced using the unlabeled primers, found that these 16 BnWER mutations, 14 represented G/C to A/T transitions and two represented T to G transversions. Of the 16 BnWER mutations, 7 mutations were located in the intron of the gene, they will be cut in the proceeding of transcription, so they have no effect on the function of the gene. And there have 9 mutations located on the exon of the gene, they were predicted that they will alter the encoding of amino acid by PARSESNP programme.3. Identified the phenotype of the mutant and evaluated the relationship between the mutant and the phosphorus efficiencyThe number and length of the root hair of the mutant M273,M024 and K413 haven't obvious increased compared with the wild type by the solution culture, and the advantage of these three mutants on improving the efficiency of phosphorus uptake and utilization has not been manifested.4. The expression analysis and gene mapping of BnWER-1 and BnWER-2The expression of the BnWER-1 has no difference under both two phosphorus treatments. The BnWER-1 has no polymorphism either in the parent or in the population; And BnWER-2 has polymorphism in the parent, but there has no polymorphism in the population. Considering both gene expression and gene mapping didn't acquire ideal results, so we need design primers to do gene expression and gene mapping over again.