【作者】 吴关庭；

【导师】 夏英武； 陈锦清；

【作者基本信息】 浙江大学 ， 生物物理学， 2004， 博士

【摘要】 高羊茅是目前正在国内外广泛应用的主要冷季型草坪草之一，利用基因工程技术改良其耐逆性对保持草坪四季常绿，节约水资源，扩大建植区域，尤其是对改善我国西部地区的生态环境具有十分重要的意义。本论文在对高羊茅胚性愈伤组织植株再生与农杆菌介导遗传转化的多种影响因素进行系统研究的基础上，将组成型表达启动子CaMV 35S引导的耐逆相关CBF1基因导入该草种的基因组，获得耐逆性增强的转基因植株。主要研究结果如下： （1）通过对成熟种子愈伤组织诱导、胚性愈伤组织继代发生和分化的多种影响因素的研究，建立了高羊茅胚性愈伤组织植株再生体系。研究表明，高羊茅成熟种子愈伤组织诱导需要较高浓度的2，4-D，以8mg／L 2，4-D与2mg／L ABA配合能获得最佳的诱导效果；种子灭菌后纵切或切胚，可使出愈率成倍提高；采用MS基本培养基和在培养基中添加0.5g／L的水解酪蛋白与谷氨酰胺也有助于提高出愈率；低剂量（10Gy）γ射线辐照处理对成熟种子愈伤组织尤其是胚性愈伤组织形成有一定的刺激效应。在继代培养基中添加0.1～0.2mg／L BAP或2.5mg／L硫酸铜，或将继代培养基中蔗糖浓度提高到60g／L能促进胚性愈伤组织发生，提高胚性愈伤组织频率。高羊茅胚性愈伤组织分化时，BAP的作用要比KT大，但2mg／L BAP与0.5mg／L KT配合可获得更佳的效果；在该细胞分裂素水平下，生长素NAA用0.5mg／L，愈伤组织再生率最高；胚性愈伤组织先在含60g／L蔗糖的分化培养基上高渗预分化，以及经高渗预分化后的致密愈伤组织在琼脂浓度为10g／L的分化培养基上分化，能明显促进愈伤组织的植株再生；在分化培养基中添加脯氨酸导致愈伤组织再生率下降，但同时有减少白化苗再生的趋势。试验还发现，高羊茅成熟种子愈伤组织诱导特别是胚性愈伤组织发生及植株再生存在明显的品种间差异，说明对品种进行选择是必要的。 （2）以gus基因瞬时表达频率为指标，研究确定农杆菌介导高羊茅胚性愈伤组织转化的适宜条件为：预培养培养基中添加较高浓度（0.5mg／L）的BAP和NAA，预培养时间3～5d；农杆菌悬浮液OD₆₀₀值0.5～0.7，感染时间10～20min；共培养基pH值5.2～5.6，共培养温度23～25℃，共培养时间3d；农杆菌预培养基和悬浮培养基以及共培养基中均添加100μmol／L的乙酰丁香酮。试验表明，在农杆菌介导高羊茅遗传转化中，抑菌剂宜选用梭节青霉素;以潮霉素作为选择剂时，采用低浓度（50一15om留L）连续筛选的方式比较合适，在该方式下，获得的转基因植株较多。通过以上研究，建立起了农杆菌介导高羊茅胚性愈伤组织遗传转化体系。 （3）采用所建立的农杆菌介导转化体系在世界上首次将耐逆相关CBFI基因导入4个供试高羊茅品种的基因组，经PCR检测、GUS组织化学染色、离体叶片潮霉素抗性鉴定和Sotlthem杂交分析，共获得112株独立来源的转基因植株，平均转化频率为1.7%（按GUS+愈伤组织数计算）和1.9%（按再生转基因植株数计算），但不同品种间存在明显差异。 （4）整株存活试验表明，在相同的高盐与高渗胁迫下，转基因植株具有明显的生长优势，植株存活率分别达65.7%和71.4%，高于非转化对照植株的28.6%和42.9%。经低温、高温、干旱和高盐等逆境胁迫处理后的叶片相对电导率测定结果显示，转基因植株的相对电导率平均较非转化对照植株低20%一30%。这些事实说明，CBFI基因的组成型表达使高羊茅转基因植株的耐逆性得到了增强。考察表明，在正常环境条件下，转基因植株的生长受到了一定抑制。对1个Tl代转基因株系进行PCR检测后发现，外源基因已传递至后代，但发生了严重的偏分离。 （5）作为本论文的相关研究内容，从高羊茅基因组中克隆出1个608bP的C刀尸同源基因片段，该片段与3个拟南芥 CBF基因的核昔酸及其推导氨基酸序列分别具有81%一84%和75%一79%的同源性，而且推导氨基酸序列中包含有同源性更高的APZ DNA结合域以及为CBF蛋白独有的两段特征序列PKK/RRAGRxKFxETRHP和DSAWR。更多还原

【Abstract】 Tall fescue (Festuca arundinacea Schreb.) is one of the major cool-season turfgrasses widely utilized at home and abroad, and improvement of its stress tolerances by genetic engineering is of momentous significance to maintaining of perennial evergreen turf, saving of water resource, enlargement of establishment area, and especially, to melioration of ecological environments in western regions of China. In this dissertation, influences of various factors on plant regeneration and Agrobacterium-mediated transformation of tall fescue embryogenic calli were systematically studied, and thereafter, stress tolerance-related CBF1 gene guided by constituent promoter CaMV 35S was incorporated into genome of this grass to obtain transgenic plants with increased stress tolerances. The main results are as follows:(1) Plant regeneration system of tall fescue embryogenic calli was established according to studies on impacts of manifold factors on callus induction from mature seeds and subculture production and differentiation of embryogenic calli. The results showed that high concentration of 2,4-D was required for callus induction from mature seeds of tall fescue, and combination of 8mg/L 2,4-D with 2mg/L ABA gave best induction effects. By slicing sterilized seeds longitudinally or cutting embryos, callus induction frequency was profoundly increased over intact seeds from one and half to eight times. Adoption of MS basal medium and supplementation of 0.5g/L casamino acids and 0.5g/L glutamine in medium were found to help to facilitate callus induction. It was also noted that irradiation treatment with low dose (10Gy) of y rays exerted slight stimulating effects on callus induction and formation of embryogenic calli in particular. Production of embryogenic calli was obviously promoted by addition of 0.1~0.2mg/L BAP or 2.5mg/L CuSO4 5H2O, or enhancement of sucrose concentration to 60g/L in subculture medium. BAP performed more important function than KT in differentiation of tall fescue embryogenic calli, but better results could be achieved with combination of 2mg/L BAP and 0.5mg/L KT. At this cytokinin level, 0.5mg/L NAA was recommended to obtain the highest callus regeneration frequency. Plant regeneration could be evidently boosted when embryogenic calli were pre-differentiated on high-osmoticum medium with 60g/L sucrose, and/or when the pre-differentiated compact calli were differentiated on differentiation medium solidified with l0g/L agar. Callus regeneration frequency was decreased but numbers of regenerated albino plantlets slightly reduced on differentiation medium supplemented with L-proline. In addition, it was observed that distinct differences in culture response existed among the tested cultivars, indicating the necessity of cultivar selection.(2) Based on transient expression of gus gene, the optimal conditions for Agrobacterium-mediated transformation of tall fescue embryogenic calli were determined as follows: addition of 0.5mg/L BAP and 0.5mg/L NAA to callus pre-culture medium, pre-culture time of 3~5d, bacterial suspension OD600 of 0.5-0.7, infection time of 10~20min, co-culture medium pH of 5.2-5.6, co-culture temperatureof 23-25 , co-culture time of 3d, and supplementation of 100 mol/L acetosyringone to Agrobacterium pre-cultivation and suspension media and co-culture medium. The results also revealed that it was a reasonable choice to use carbenicillin as the antibiotics of inhibiting growth of remnant Agrobacterium after co-culture, and when hygromycin was used as the selective agent, continuous selection at low concentrations (50~150mg/L) produced the highest numbers of transgenic plants without escapes. On the basis of the above studies, Agrobacterium-mediated transformation system of tall fescue embryogenic calli has been set up.(3) By adopting the established Agrobacterium-mediated transformation system, stress tolerance-related CBF1 gene was incorporated into genomes of four tall fescue cultivars for the first time in the world and 112 independent transgenic plants were obtaine更多还原