【作者】 徐春波；

【导师】 米福贵；

【作者基本信息】 内蒙古农业大学 ， 草畜种质资源学， 2016， 博士

【摘要】 紫花苜蓿(Medicago sativa L.)为豆科苜蓿属多年生草本植物,是世界上最重要的牧草资源,具有极高的饲用及生态价值。近些年,随着紫花苜蓿栽培面积的不断扩大,抗寒性弱成为限制紫花苜蓿向北方地区推广的主要因素,转入抗寒基因提高紫花苜蓿抗寒性是解决该问题的高效育种方法之一。本研究从拟南芥中克隆得到冷诱导转录因子AtCBFl基因,采用农杆菌介导法转入到优良紫花苜蓿品种中,通过转化植株分子检测、抗寒性及田间鉴定形成株系,筛选出了转AtCBFl基因抗寒高产的转基因紫花苜蓿新材料。主要研究结果如下：1.研究了紫花苜蓿品种、外植体以及激素组合配比等影响组培的再生因子,结果表明,组织培养最佳基因型为猎人河苜蓿；最佳外植体为下胚轴；最佳愈伤组织诱导培养基为SH+2.0mg · L-12,4-D+0.5 mg · L11 6-BA,愈伤组织继代培养基为MSO+0.5mg · L-1 NAA+1.0mg · L-1 6-BA+1.0mg · L-1 AgNO3和MS+0.3mg·L-1TDZ分化培养基为MS+0.2mg · L-1 KT。2.克隆了拟南芥冷诱导转录因子AtCBF1基因并构建了适于紫花苜蓿农杆菌遗传转化的植物表达载体PBI121-CBF1。3.紫花苜蓿遗传转化因子研究确定出最佳农杆菌抑制剂为350mg·L-1 Carb:最佳Kan筛选浓度为60mg·L-1；最佳受体基因型为猎人河苜蓿；最佳外植体为下胚轴；最适宜农杆菌菌液浓度OD600为0.4～0.6,侵染时间10min；AS最适浓度为10mg·L-1。4.利用农杆菌介导法将AtCBF1基因转入紫花苜蓿,获得了抗Kan的转基因紫花苜蓿植株。经PCR检测,部分转化植株扩增出了大小为650bp左右特异带,初步表明目的基因AtCBF1已经整合到紫花苜蓿基因组,进一步通过RT-PCR检测,结果显示外源基因AtCBF1在部分转化植株的转录水平上表达。5.抗寒性生理研究测定了转基因苜蓿相对电导率、脯氨酸含量、可溶性糖含量及丙二醛含量,结果显示转AtCBF1基因紫花苜蓿植株较对照抗寒性有所提高,转基因株系以T9、T11和T3的抗寒性相对较高,且T9株系抗寒性最强。6.研究了转基因苜蓿人工模拟冷冻天气抗寒性,叶片受害及恢复生长结果表明转基因紫花苜蓿植株表现出较强的耐低温胁迫能力,即过量表达AtCBF1基因的转基因紫花苜蓿的抗寒性获得了提高。7.转基因紫花苜蓿形态、产量、再生速度及品质方面研究显示,转基因株系与对照相比在形态上没有明显生长延缓、植株矮小等不良变化,只是叶片长度较对照植株稍短;在产量和再生速度上,转基因各株系有的高于对照,有的低于对照,T11和T9株系在产量及再生速度上均显著高于对照和其他株系；营养成分转基因株系均表现出高于对照的粗蛋白含量,营养品质有所提高。综合研究结果,T9和T11株系为表现最优的转基因株系。更多还原

【Abstract】 Alfalfa (Medicago sativa L.) as one of perennial herb of legume, is the world’s most important forage resource and has a very high forage and ecological value. In recent years, with the expansion of alfalfa cultivation area, limit of cold resistance of alfalfa became major factor in areas in North China. Transfer the cold resistance genes into Alfalfa to improve cold resistance became an efficient breeding method to solve the problem.In this research, AtCBF1 gene, one cold-induced transcription factor cloned from Arabidopsis, were transfered into diverse varieties of Alfalfa through mediated by Agrobacterium. the genetically engineered alfalfa with cold resistance and high forage yield were selected according to molecular detection of the transformation plants, identification of cold tolerance and field forming strains with AtCBFl gene. The main results are as follows:1. Research on affection of tissues regeneration factor include alfalfa varieties, explants and hormones ratio. Results showed that the best genotype is Hunter-River alfalfa in tissue culture. The best explant is hypocotyl. The best medium for callus induction is SH+2.0mg·L-12,4-D+0.5 mg·L-1 6-BA. The best Medium for the callus subculture is MSO+0.5mg·L-1 NAA+1.0mg·L-1 6-BA+1.0mg·L-1 AgNO3 and MS+ 0.3mg·L-1 TDZ. The best Differentiation medium is MS+0.2mg·L-1 KT.2. Arabidopsis thaliana transcription factors AtCBFl gene induced by cold were Cloned.The Agrobacterium transformation of alfalfa plant expression vector PBI121-CB Fl were constructed.3. Research on genetic transformation factors showed that, the best inhibitors of Agrobacterium is 350 mg-L^Carb, best Kan concentration is 60mg·L-1, best receptor genotype is the Hunter-River alfalfa, best explant is hypocotyl, most suitable concentration OD600 Agrobacterium bacteria is between 0.4-0.6, best time of infection is lOmin, AS optimal concentration is 10 mg · L-1.4. AtCBFl gene were transfered to alfalfa by Agrobacterium-mediated. Kan-resistant transgenic alfalfa plants were obtained. PCR Detection amplified around 650bp specific bands, indicated that AtCBFl gene is already integrated into the alfalfa genome. RT-PCR showed AtCBFl foreign gene transcriptional expression in part of transgenic plants.5. Cold resistance were evaluated by determination of relative conductivity, contents of PROLINE, soluble sugar content and MDA content in transgenic alfalfa plants. Plants with AtCBF1 gene showed higher cold resistance than control plants. Among transgenic plants, T9, T11, and T3 showed relatively higher cold resistance and T9 of the strongest cold resistance.6. Cold resistance of genetically engineered alfalfa were evaluated under artificial freezing temperature, leaves victims and restore growth showed that transgenic alfalfa plants have strong resistance to low temperature stress. Over expression of AtCBFl improved cold tolerance of alfalfa.7. The production, regeneration speed and the quality research of transgenic plants showed that, Transgenic alfalfa strains have no obviously growth delay, plant short, and severe changes compared to control plants. The only obviously change is leaves length were slightly shorter. Compare to control plant in production and regeneration speed, transgenic plants and strains show some above and some below control. Strains T11 and T9 are significantly better than control and other strains in production and the regeneration speed. Nutrition quality were also improved in transgenic strains on some nutrition components like rough protein content. Based on research results, T9 and T11 are best two transgenic alfalfa strains.更多还原