| Harpin was produced by Gram-negative plant-pathogenic bacteria and elicited hypersensitive cell death (HCD) in non-host plants (tobacco et al). The elicitors of harpin have similar features:glycine-rich, lack of cysteine, protease-sensitive and heat-stable, andincreases plant resistance to diseases and pests and promotes plant growth。study found that:the plants treated with harpin were induced resistance to bacteria, fungus, virus and nematodes and promote plant growth. The genes encoding Harpin were transformed to plants, the resistances and yields of transgenic plants were increased.The hrf2 gene, which encodes harpinxooc, is derived from the rice-pathogenic bacterium Xanthomonas oryzae pv. oryzicola strain RS105, the fragment size of the gene is 414 bp, the molecular weight of harpinxooc is 15.3 Kda and has 138 amino acids and has similar characters and functions with other Harpin. In our study, the hrf2 gene was transformed to soybean by an efficient Agrobacterium tumefaciens-mediated transformation method, the aim of our is to obtain soybean plants will contain better traits and stronger disease resistance and will serve as valuable tools for producing new soybean varieties.In our study, a recombinant plant expression vector pCAMBIA3300::hrf2 containing hrf2 gene was transformed to soybean by an efficient Agrobacterium tumefaciens-mediated transformation method that involves wounding cotyledonary node explants from the soybean (Glycine max [L.] Merrill) cultivar Tianlong 1, we obtained 115 resistant regeneration soybean plants. The expression of hrf2 gene in 41 To plants and T1 plants were analyzed by phenotypic screening and detection using QuickStix (PAT/bar protein detection), polymerase chain reaction (PCR) and reverse transcription polymerase chain reactions (RT-PCR) and Western-blot, results indicated that the hr/2 gene integrated stably in soybean genes and it can be expressed normally in transcription levels and the Harpinxooc can be expressed normally in T1 transgenic soybean plants.The herbicide resistance gene (bar) of 41 healthy T1 plants from the six transgenic lines (T0-1, T0-2, T0-3, T0-4, T0-5 and T0-6) was analyzed by phenotypic and a series of molecular analysis, and datas were validated by χ2-analysis. Result showed that the genetic separation of bar gene showed a close to 3:1 segregation ratio at the 0.05 level and was accorded with the Mendelian inheritance, and suggesting the presence of a single functional locus in the T1 progeny.The resistance of PCR-positive T1 generation transgenic plants to Phytophthora sojae was evaluated by detached leaf inoculation and hypocotyl inoculation methods. results showed that lesions from T17-1, T17-3, T112-1, T119-1 and T119-2 plants were significantly smaller (P< 0.05) than those from the non-transgenic, negative control plants, the lesion diameter of the control plants was approximately 2.0 cm, while the lesion diameter of the transgenic plants ranged from 0.21 to 1.69 cm; in the hypocotyl inoculation methods, lodging were observed in the control plants due to necrosis of the hypocotyl, while hypocotyl necrosis was less pronounced in the transgenic plants than in the control plants, and the necrosis had no significant influence to the growth of the transgenic plants. The two results indicate that the hrf2 gene can increase resistance to Phytophthora sojae in transgenic plants.The resistance of T1 generation transgenic plants to soybean aphids was evaluated by detached leaf inoculation method, results showed that the numbers of soybean aphids were significantly decreased in leaves of transgenic plants when 6 d than control plants, but there were no significant difference between some transgenic plants with control plants, it confirmed that the resistance to soybean aphids was greatly enhanced in the transgenic plants.We investigated agronomic traits in six T1 transgenic lines (T1-1, T1-2, T1-3, T1-4, T1-5 and T1-6) under field conditions and subjected the data to statistical analysis. Results showed that the growth and development of T1 transgenic lines is normal, compared with the control plants, the stem of transgenic plant was significantly wider and seed yield per plant of transgenic plants was higher. It confirmed that the hrf2 gene had beneficial influence to the agronomic traits of transgenic plants... |
PDF Full Text Request