【作者】 张彬彬；

【导师】 朱延明；

【作者基本信息】 东北农业大学 ， 植物学， 2003， 硕士

【摘要】 畜牧业是黑龙江省的半壁江山，进行饲草改良和提高饲料作物的吸收率是当前发展畜牧业亟需解决的问题。玉米是重要的饲用作物，在畜牧业中占有支柱地位。但由于其中含有大量的纤维素，影响了草食性动物对它的消化率和吸收率。迄今，为提高对纤维的消化率和吸收率，将纤维素酶作为添加剂应用于饲料工业，取得了一定成就，但仍存在着诸多问题，如纤维素酶的生产菌株产酶量低、酶制剂在加工过程中易失活、酶制剂的成本较高等。 低温、干旱、盐碱等逆境条件严重制约着黑龙江省的农业生产，仅盐碱地就达9000万亩，培育出耐低温、干旱、盐碱的饲草作物，可充分利用我省的耕地资源，对我省的畜牧业发展将会起到重大作用。因此，培育耐低温、干旱、盐碱以及高消化率的转基因饲料作物，可创造巨大的经济和社会效益。 本研究的目的是系统探讨提高玉米再生效率的主要因子，建立稳定的玉米植株再生体系；克隆β-葡萄糖苷酶基因(bglⅠ)和葡聚糖外切酶基因(cbhⅡ)，并分别将其构建在含有对抗渗透胁迫基因起调控作用的转录因子—DREB1A基因的植物表达载体上，为后期利用农杆菌介导法将编码纤维素酶的基因分别和DREB1A基因联合导入玉米中做准备，并为培育耐低温、干旱、盐碱的高消化率的饲用玉米新品种(系)奠定基础。 主要研究结果如下： 1．基因克隆 (1)利用PCR方法，用GenBank上1993年注册的β-葡萄糖苷酶基因的特异性引物，从里氏木霉的基因组DNA中克隆了β-葡萄糖苷酶基因(bglⅠ)。序列分析结果表明，该序列与GenBank上的核苷酸序列同源性达99.9％，氨基酸序列同源性达99.9％。 (2)利用PCR方法，用GenBank上1987年注册的葡聚糖外切酶基因的特异性引物，从里氏木霉的基因组DNA中克隆了葡聚糖外切酶基因(cbhⅡ)，序列测定正在进行中。 2．载体构建 构建了中间表达载体pAHG。pAHG带有玉米组成型启动子Ubi调控的β-葡萄糖苷酶基因和Amp基因选择标记。 3．玉米再生体系建立 (1) 影响Ⅱ型愈伤组织诱导的4个主要因素的主次顺序是：2，4-D＞BA＞L-脯氨酸＞水解酪蛋白。试验较优组合为：N6培养基添加4mg／L 2，4-D、0.25mg／L BA、750mg／L L-脯氨酸、250mg／L水解酪蛋白。 东北农业大学理学硕士学位论文一 （2）确定了最佳 ＊ 型愈伤组织诱导培养基为：N6＋4mg／L 2，4－Ded．25wt BA＋750mg／L L－脯氨酸＋250mg／L＋水解酪蛋白＋250mg／L谷氨酞胺＋10 mg／L AgNO3，30gb蔗糖，0．8％琼脂，pHS．8 （3）确定了继代培养基为：N6＋2mglL 2，4D－－m．25mgh BA＋750mg／L L－脯氢酸、 ＋250mgh 7K解酪蛋白，30gb蔗糖，0．8％＄脂，pHS．8。适当地降低 2，4－D的 浓度为了保持愈伤组织胚性的同时，不影响不定芽的分化。 （4）不同基因型的不定芽分化率和分化效率不同。在供试的3个基因型中，甜1、 龙抗11和东8］口的不定芽分化率分别为89，97％、74．92％、38．53％，分化效 率分别为门．37％、102．93％和69．12％。 （5）确定了各基因型的不定芽分化培养基。甜1 的分化培养基为MS＋Zm叭 BA＋0．sing／L NAA，30g／L蔗糖，0．8％琼脂，pHS．8。龙抗 11 的分化培养基为 MS＋0．Zmg／LBA＋500mg／L水解酪蛋白，30g／L蔗糖，0．8％琼脂，pHS．8。东sllZ 的分化培养基为 MS＋Zing／L BA＋0．Zing／L 2，4D，30g／L蔗糖，0．8％琼脂，pHS．8。 间 确定了生根培养基为：＊队2＊叭＊AA＋o．25wt＊＊仍．1％活性炭，30g／L 蔗糖，0．8％琼脂，pHS．8。 （7） 不定芽分化阶段双丙氨磷的选择压力确定为：东sllZ为1．om叭，龙抗＊为 1二sing／L，甜 1为 1．5 m叭。更多还原

【Abstract】 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更多还原