| Stock raising plays a very important role in Heilongjiang Province. Meliorating grazing and improving absorption rate of feedstuff are urgent problems at present in stock raising. Maize as an important feedstuff is of vital concernment in stock raising. Because of its large quantity of cellulose, the digestion rate and absorption rate are seriously affected. Using cellulase as an additive in feedstuff has resolved the problem in certain way but there are still some questions such as low production, instability and high cost of the production.In addition, drought, high salt and low temperature stress also lead to reduction of crop production. There is about 6,000,000 hectares salt alkaline soil in Heilongjiang Province. Cultivating the feedstuff plants with drought, salt and temperature resistance can make good use of tilth of our province, and will play an important role in developing our stockbreeding. Therefore, fostering transgenic feedstuff crops with high digestion rate and resistance to low temperature, drought, salt can make tremendous economical and social benefit.In this research, the regeneration system of Maize has been established. The main factors affecting maize regeneration are analyzed. Endo-l,4-beta-glucanase V(EGV) gene is cloned and had been constructed into the plant expression vector which contain a transcription factor DREB1A. DREB1A is a transcription factor that can regulate several genes involved in stress reaction. Both of the two genes were transformed into Maize mediated by Agrobacterium. The research work will help to breed the new maize with high drought, salt and low temperature resistance and high maize feedstuff absorption rate.The main results were summarized as follows.1. Gene cloneAccording to the EGV gene sequence in GenBank in 1994, a pair of specific primers was designed, and the EGV gene was cloned from genome DNA of Trichoderma reesei by PCR method. The result of sequence analysis indicated that this fragment showed 100% identity in nucleotide and 100% in amino acid to the previously reported one.2. Vector constructionBivalent expression vector pAEG29A was constructed, on which EGV gene was regulated by the constitutive promoter Ubi of maize. DREB1A gene was regulated by the rd29A and bar gene as selectable marker.3. Establishment of Maize regeneration system(1) The order of four main factors on affecting callus tissue type II is: 2, 4-D > BA > L-Pro > CH. The best combination is: MS medium adding 4mg/L 2.4-D, 750mg/L L-Pro and 250mg/LCH.(2) Establishing the optimization induction medium of callus tissue II: MS+4mg/L 2.4-D+750mg/L L-Pro+250mg/LCH+250mg/L L-Glu+1 Omg/L AgNO3,30g/L sucrose, 0.8%agar. pH5.8(3) Establishing Callus subculture medium : N6 + 2mg/L 2,4-D+ 0.25mg/L BA+ 750mg/L L-Pro+ 250mg/L CH,30g/L sucrose, 0.8% agar, pH5.8. Properly decreasing concentration of 2,4-D to keep the embryonic character of callus tissue and at the same time differentiation of adventitious buds is not affected.(4) Differentiation rate of adventitious buds and average number of buds from each explants are different among different genotypes. For the three genotypes Tianl, Longkang and Dong8112, differentiation rate of adventitious buds are 89.97%, 74.92% and 38.53% respectively, average number of buds from each explant are 1.13, 1.02 and 0.69 respectively.(5) Establishing adventitious buds differentiation medium of every genotypes. Composition of differentiation medium of Tianl is MS+2mg/L BA+0.5mg/L NAA, 30g/L sucrose., 0.8% agar, pH5.8. That of Longkangll is MS+0.2mg/L BA+500mg/L CH, 30g/L sucrose., 0.8% agar, pH5.8. That of Dong 8112 is MS+2mg/L BA+0.2mg/L 2,4-D,30g/L sucrose, 0.8% agar, pH5.8.(6) Establishing the optimization medium of root generation: MS+1mg/L NAA+ 0.25mg/L BA+0.1% active carbon+30g/L sucrose, 0.8% agar, pH5.8.4. Transformation into Maize by Agrobacterium-medium methodEstablishing Bialaphos selection pressure in the stage of callus differentiation: Tianl is 1.25 mg/L...
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