【作者】 蔡能；

【导师】 易自力； 蒋建雄；

【作者基本信息】 湖南农业大学 ， 植物学， 2008， 博士

【摘要】 多年生黑麦草(Lolium perenne L.)不仅是一种优质牧草,更是一种常用的观赏型和运动型草坪草种。针对草坪杂草难以防除和冷季型草坪草抗旱性差、绿色期短的不足,该研究以多年生黑麦草常用品种爱神特、卡特等为材料,建立并优化了多年生黑麦草高频离体培养再生体系、农杆菌转化技术体系和拟转化子的筛选方案,通过农杆菌介导法将外源双价抗除草剂基因——草丁膦抗性基因(bar)和抗旱基因——烟碱酰胺合成酶基因(nas)导入多年生黑麦草中,得到了具有抗除草剂和抗旱两种抗性的多年生黑麦草转基因植株。主要研究结果如下:1.建立并优化了多年生黑麦草胚性愈伤组织诱导与分化的培养条件研究结果表明,含胚的半粒种子是诱导愈伤组织较为理想的外植体,其平均愈伤组织诱导率可达86%,其愈伤组织分化率可达85%;MS培养基是绝大多数多年生黑麦草愈伤组织的最适诱导与继代培养基;随着2,4-D浓度的增加,愈伤组织诱导率上升,8.0 mg/L较为适合;为避免高浓度2,4-D对愈伤组织生长及再分化的伤害,愈伤组织继代培养时可适当降低2,4-D浓度;继代培养基中添加25g/L的甘露醇可以有效地改善愈伤组织的生长状态,促进胚性愈伤组织的形成;MS培养基上诱导的丛生不定芽较粗壮,NB培养基上诱导的丛生不定芽较细密。具体操作方式为:胚端半粒种子于MS+2,4-D 8.0 mg/L(或另添加甘露醇25g/L)上诱导愈伤组织;愈伤组织接种于MS+2,4-D 4.0mg/L+甘露醇25 g/L上进行1-2次继代后于NB+6-BA2.0 mg/L上进行分化;不定芽转接至MS+6-BA2.0 mg/L上进行扩繁或于1/2MS+NAA0.5mg/L+IAA0.5mg/L上进行生根。2.将外源bar+nas双价基因导入多年生黑麦草中获得了转基因植株构建了bar+nas双价基因植物表达载体(EHA105—p3301-gus-nas,LBA4404—p3301-gus-nas),并进行了nas基因的原核表达和多克隆抗体制备,为多年生黑麦草的转化和检测提供了材料。优化了多年生黑麦草愈伤组织农杆菌转化方法。实验结果表明,根癌农杆菌EHAl05菌株对多年生黑麦草的转化效果优于LBA 4404菌株的转化效果;在转化之前一直将受体愈伤组织在含甘露醇的培养基上继代培养可提高受体材料的质量;在共培养过程中采用浸泡共培养基的滤纸代替固体共培养基,既能增强抑菌效果,又可维持愈伤组织状态,提高转化频率,还可简化操作程序。选用Basta作为筛选剂对多年生黑麦草抗性愈伤组织和抗性植株进行筛选,其适宜的筛选浓度为7.5-10mg/L,其筛选时间以40-60天为宜。获得了转基因多年生黑麦草植株。应用本研究所建立的农杆菌转化系统,将bar基因和nas基因导入多年生黑麦草愈伤组织,经筛选剂筛选获得20株抗性植株。通过对部分植株进行PCR,RT-PCR和Western blot杂交等分子检测的结果表明,外源目的基因已整合进了多年生黑麦草转化植株基因组。3.进行了除草剂抗性和抗旱性鉴定经叶面喷施除草剂溶液,转基因植株具有不同程度的抗性,抗性最高可达150-200mg/L Basta。通过反复干旱法,发现转基因植株抗旱性得到增强,整体观感较好;而未转化株经过长期的反复干旱,失水严重,最后死亡。在自然干旱条件下,对转基因植株和未转化株生理生化指标进行了测定,结果表明:转化株土壤保水能力、植株增长速度、根生长速度、叶片相对水含量、叶绿素含量均高于未转化株;丙二醛含量、脯氨酸含量、超氧化物歧化酶活性、过氧化物酶活性、过氧化氢酶活性、叶片水势和叶片外渗溶液相对电导率的变化规律表明转基因植株与未转化株相比,抗旱性得到增强。更多还原

【Abstract】 Perennial ryegrass(Lolium perenne L.) is not only a kind of high-quality forage, but also a kind of turfgrass used for ornamental lawn and sport turf.Based on the difficulty of weed control on turf establishment and management,disadvantages of drought sensitivity and short green stage of cool-season turfgrass,bivalent herbicide-resistant gene and drought-resistant gene were transformed into perennial ryegrass to obtain new lines with two resistance.In this thesis,taking some good varieties of perennial ryegrass such as "Accent" and "Kutter" as explants,the initiation and regeneration system at very high frequency for embryogenic callus and the selection scheme for tolerant callus were established and modified,and the transformation method mediated by Agrobacterium for callus of these perennial ryegrass were optimized too.By using the Agrobacterium-mediated transformation system,bivalent herbicide-resistant gene(bar) and nicotianamine synthase gene(nas) were introduced into the callus of perennial ryegrass and 20 tentative transgenic lines were obtained.PCR,RT-PCR and Western blot analysis showed that target genes were integrated into the genome of these transgenic plants. Through identification,the transgenic plants were more resistant to Basta and drought than non-transgenic plants.1.The initiation and regeneration system for embryogenic callus of perennial ryegrass were established and optimized at very high frequencyThe result showed that the half-seeds with embryo were the best explants for callus induction.Compared to other explants such as mature seeds,the frequency of calli induction and buds differentiation from half-seeds with embryo could reach to 86%and 85%.MS medium was the best basic medium for induction and subculture of the calli among all the media tested.Calli induction frequency raised with the rise of 2,4-D concentration,and 8.0 mg/L was suitable.In order to reducing the damage of high 2,4-D concentration on the growth of calli and buds differentiation,2,4-D concentration was decreased on the calli subculture medium.Mannitol could improve the quality of calli and promote the formation of embryonic calli.Buds induced on the MS medium were strong and robust,while those induced on the NB medium were thin and close.The whole regeneration process was:the calli induced on medium of MS+2.4-D8.0 mg/L(with mannitol 25 g/L or not) were subcultured on medium of MS+2.4-D8.0 mg/L+ mannitol 25 g/L for 1 to 2 months,then cultured on medium of NB+6-BA2.0 mg/L to induce adventitious buds.Adventitious buds can be extending propagated on MS+6-BA2.0 mg/L or induced roots on medium of 1/2MS+NAA0.5mg/L+IAA0.5mg/L.2.Transgenic perennial ryegrass strains were obtained by Agrobacteriummediated transformation with bivalent bar and has genesPlant expressing vector with bivalent bar gene and nas gene was constructed.Nas gene was cloned into a prokaryotic expression vector pGEM-KG to express the recombinant protein pKG-NAS,the polyclonal antibody of NAS was prepared.The transformed methods mediated by Agrobacterium tumefaciens were optimized.In the experiment,the Agrobacterium tumefaciens strain EHA105 was more efficient than LBA4404 for perennial ryegrass transformation.The quality of receptor could be improved through subculturing the calli on subculture medium with mannitol.Taking filter papers dipped with liquid co-culture medium as co-culture carrier instead of solid co-culture medium,it was beneficial to inhibit the growth of Agrobacterium,maintain the quality of calli,increase transformation efficiency and simplify the procedure.The selection scheme for tolerant calli and buds of perennial ryegrass was established.The suitable selection scheme was culturing the callui and buds on the medium containing 7.5-10 mg/L Basta for 40-60 days.By using the Agrobacterium-mediated transformation system,bivalent bialaphos resistance gene(bar) and nicotianamine synthase gene(nas) were introduced into the calli of perennial ryegrass and 20 transgenic lines were obtained through selection with Basta.PCR,RT-PCR and Western blot analysis showed that target genes were integrated into the genome of these transgenic plants.3.Resistance identification of transgenic plants to herbicide and droughtThrough identification by spraying Basta solution on the leaves,the transgenic plants were more resistant to Basta than non-transgenic plants,could resist 150-200mg/L Basta.Drought resistance identification with repeated drought showed:The transgenic plants were more resistant to drought than non- transgenic plants,having longer green stage;the non- transgenic plants lost water seriously,died at last.Under natural drought conditions,the carrying-water ability,whole plant growth speed,root growth speed,relative water content of leaves,chlorophyll content of transgenic plants were higher than those of non-transgenic plants.On the process of drought treatment,the malondiadehyde(MDA) content,Proline(Pro) content,superoxide dismutase (SOD) activity,peroxidase(POD) activity,catalase(CAT) activity,leaf water potential(LWP) and relative electrical conductivity(LEC)showed certain variation laws and changing trends.更多还原