| Stock raising plays a very important role in Heilongjiang Province. Improving feedstuff and its absorption rate are urgent problems at present in stock raising. Maize as an important feedstuff is of vital concernment in Stock raising. Because it contains a large quantity of cellulose, the digestion rate and absorption rate is seriously affected. Using cellulase as an additive in feedstuff industry has made obvious good effect to the problem although 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, salina had reached 90,000,000 units in Heilongjiang Province. Cultivating the feedstuff plants with drought, salt and temperature resistance can make good use of tilth of our province, which will play an important role to 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, bgll gene and cbhII gene are cloned and had been constructed into the plant expression vector which contain a transcription factor DREB1A. DREBIA is a transcription factor that can regulate several genes involved in stress reaction, which is prepared for transferring the cfeW/gene and bgll gene with DREB1A gene respectively to 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 was summarized as follows.1. Gene clone(1) According to the bgll gene sequence in Genbank, a pair of specific primer was designed, and the bgll gene was cloned from genome DNA of Trichoderma reesei by PCR method. The result of sequence analysis shows the DNA fragment we got shares 99.9% in nucleotide and 99.9% in amino acid with that of in Genbank.(2) According to the Trichoderma reesei gene sequence in Genbank, a pair of specific primer was designed, and the cbhll gene was cloned by PCR method. The sequence analysis is making now.2. Vector constructionThe intermediate expression vector (pAHG) was constructed which harbored bgll gene regulated under the constitutive promoter Ubi of maize and the selective marker Amp gene.3. Maize regeneration system was established.(1) The best combination of normal experiments of four main factors that affect callus tissue II inductions are N6 medium adding 4mg/L 2, 4-D, 0.25mg/L BA, 750mg/L L-Pro, 250mg/L CH. The order of affecting factors is: 2, 4-D > BA > L-Pro > CH.(2) The best induction medium of callus tissue II is: N6+4mg/L 2,4-D+0.25mg/L BA+750mg/L L-Pro+250mg/LCH+250mg/L L-Glu+10 mg/L AgNO3+30g/L sucrose, 0.8% agar, pH5.8(3) Callus subculture medium is N6 + 2mg/L 2.4-D+ 0.25mg/L BA+ 750mg/L L-Pro+ 250mg/L CH+250mg/L L-Glu+ 10mg/L AgNO3+ 30g/L sucrose, 0.8%agar, pH5.8. Probably the decreased concentration of 2,4-D to keep the embryonic character of callus tissue can 't affect the differentiation of adventitious buds.(4) Different genotypes showed different bud differentiation rate and differentiation efficiency. Adventitious buds differentiation rate of TianK Longkang and Dong 8112 are 89.97%, 74.92% and 38.53% respectively. Differentiation efficiency are 113.37%, 102.93% and 69.12% respectively.(5) Differentiation medium for adventitious shoots of different genotypes were decided. The composition of differentiation medium of Tianlis MS+2mg/L BA+0.5mg/L NAA, 30g/Lsuc. , 0.8% agar, pH5.8. The composition of differentiation medium of Longkangll is MS+0.2mg/L BA+500mg/L CH, 30g/Lsuc., 0.8% agar, pH5.8. The composition of differentiation medium of Dong 8112 is MS+2mg/L BA+0.2mg/L 2,4-D, 30g/Lsuc., 0.8% agar, pH5.8.(6) The best medium for root generation is MS+2 mg/LNAA+ 0.25mg/L BA+0.1%a... |
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