Genetic Transformation Via Microprojectile Bombardment, Cloning And Identification Of NBS-LRR Gene In Wheat And Agrobacterium Rhizogenes-mediated Genetic Transformation In Tartary Buckwheat

The major research contents of this dissertation included: (â… ) Regeneration of transgenic wheats containing rice RC24 gene via microprojectile bombardment, and study of rust resistance of T₀ and T₁ generations. (â…¡) Establishment of appropriate regeneration and transformation protocol for tartary buckwheat, and analysis of rutin content in regenerants and hairy root lines. (â…¢) Cloning and identification of NBS-LRR gene analogs of wheat with primers designed on the basis of cloned resistance genes, and expression analysis of obtained sequences by Northern blotting.â…  .Immature embryos (0.8-1.5mm) of wheat Xinong88, Baofeng7228, Heixiaomai, Yan 2, 80101T, Ganmai, Xinongl376 and Xinong8727 were used as initial materials. After being precultured for 3-4 days, rice chitinase gene RC24 was transferred into scutellum by microprojectile bombardment with PDS1000/He DNA delivery system. Three plasmid combinations were applied in this experiment, Immature embryos bombarded with plasmid pYAO24 (containing rice chitinase gene RC24 and selective maker gene npt â…¡) were selected on the medium supplemented with 150 mg/L kanamycin, while immature embryos bombarded with plasmid combination pARN6 + pDBl (pARN6 contains rice chitinase gene RC24, pDBl contains gus gene and selective maker gene bar), and plasmid combination pBAB3(containing rice chitinase gene RC24) + pDBl were selected on the medium supplemented with 2mg/L phosphinothricin. After bombardment, selection and regeneration of immature embryos, 5 green plants were regenerated, and 4 transgenic plants were confirmed by PCR and Southern blotting analysis. The average transformation frequency was 0.30%. 8 cultivars of wheat exhibited differenttransformation frequencies. Transformation frequency of Xinong 88 was 1.38%. Transformation frequency of Baofeng 7228 reached 0.59%. Transformation frequency of Ganmai got to 0.49%. No transgenic plant was obtained from Xinong 1376> Heixiaomah Yan 2^ 80101T and Xinong 8727. In comparison to with control plants, plant height, ear length, spikelet number, effective tillers and grain number per plant of To transgenic plants had no obvious changes. To and Ti transgenic plants of Xinong 88 had strong resistance to rust, they were almost immune. To and Ti transgenic plants of Baofeng 7228 and Ganmai also showed resistance to rust.II. It is an effective way to clone gene using PCR with the primers designed on the basis of conserved motifs of cloned resistance genes. In this work, specific primers sequences were designed on the basis of NBS> LRR conserved motifs of cloned resistance genes and subsequently were used to amplify the resistance gene analog (RGA) from wheat (Triticum aestivum L.) Xinong 88 with rust resistance characteristic. The amplified products of WRGA1, WRGA2 and WRGA14 were cloned and sequenced. They wers submitted to NCBI, and the accession number were AY520908, AY555270 and AY550176 respectively. Phylogenetic analysis of deduced amino acid sequences showed that WRGA1, WRGA2 and WRGA14 were homologous toL6 gene (U27081) ,Lr21 gene (AAQ01784) ,Lrl0gene (AAO12455) and N gene (U15605) . WRGA1, WRGA2 and WRGA14 maybe belong to the resistance gene family of wheat L21 gene. They contain the same conserved motifs such as Kinase-2a, Kinase-3a and HD presented in known NBS-LRR resistance genes from other plants and share 46.0%-9.9% amino acid sequence identity with them. The amino acid sequence identity among three RGAs was 80.7%-56.8%. The sequence distance analysis of deduced amino acid sequences showed WRGA1, WRGA2 and WRGA14 were resistance gene analog (RGA). The WRGA1 expression which was positively regulated by salicylic acid was detected by Northern blotting.III. The regeneration system of tartary buckwheat was established and hairy roots were induced from hypocotyl and cotyledon explants of tartary buckwheat by Agrobacterium rhizogenes-mediated transformation.The hypocotyl and cotyledon explants of sterile seedings of tartary buckwheatwere cut and transferred onto different media containing different phytohormones for calli induction. It was found that the calli could be induced efficiently on MS basal medium supplemented with 2.0mg/L 2,4-D, 0.5mg/L 6-BA, lOmg/L AgNCb and 3% sucrose. Shoots were regenerated on MS medium containing 2.0mg/L 6-BA, 0.5mg/L NAA and 3%sucrose, and the differentiation frequency was about 30.6%. The regenerated shoots or shoot cuttings produced roots on 1/2 MS phytohormone-free medium, and over 80% transplanted plants were survived. Rutin content (dry weight) of different organs of the regenerants were determined by HPLC. Rutin content of leaf, root, stem and flower was 0.3242mg/g, 0.0040mg/g, 0.0344mg/g and 0.4460mg/g respectively. Rutin content in leaf of the regenerated plants was 1.01 times higher than the rutin content (dry weight) in leaf of wild tartary buckwheat. The rutin content (dry weight) in root, stem and flower of the regenerated plant was lower than the rutin content in root, stem and flower of wild tartary buckwheat, and they were 0.61, 0.34 and 0.63 times respectively.Hairy roots could be induced by Agrobacterium rhizogenes strain 15834 from the hypocotyl explants of tartary buckwhat at a frequency of 28.33%, and they grew well on phytohormone-free solid or liquid 1/2 MS medium (30g/L sucrose). After a culture period of 20d, the fresh weight of solid-cultured hairy roots increased 13.50 + 0.60 times, while the fresh weight of liquid-cultured hairy roots increased almost one thousand times. The growth curve of hairy roots on phytohormone-free solid 1/2 MS medium was similar to hairy roots grew in phytohormone-free liquid 1/2 MS medium, and the growth rate became faster after 4⁵d. At the range of sucrose concentrations from 15 to 75 g/L, 30g/L was favorable for the growth of hairy roots. The stable integration of rol genes into genomic DNA of tartary buckwheat was confirmed by opine detection and PCR analysis. The rutin contents of six lines of hairy roots positive in PCR were determined by HPLC, two hairy roots lines could produce rutin. The rutin content (dry weight) of NO.l hairy root lines was 0.848 lmg/'g, 21.2 times higher than roots of regenerated plants, 3.35 times higher than wild roots. The rutin content (dry weight) of NO.2 hairy root lines was 0. 1344mg/g, 12.85 times higher than roots of regenerated plants, 2.03 times higher than wild roots.