【作者】 徐凤萍；

【导师】 安利佳； 杨君；

【作者基本信息】 大连理工大学 ， 作物遗传育种， 2008， 硕士

【摘要】 线性DNA直接转化方法因其易操作、转化效率高而且转化受体不受限制等诸多优势在植物基因转化中已被广泛使用,所转化外源基因能够整合、表达并且稳定遗传给后代。线性DNA直接转化系统主要用于稳定转化,而在转化过程中,却存在着转化动力不足的问题。为了提高转化效率,本研究以报告基因smGFP和GUS的线性DNA片段为转化表达框,研究不同物理化学因素包括表面活性剂、渗透处理、钙等处理对线性DNA转化表达框进入细胞核及其在细胞中瞬时表达效率的影响,以期建立一种以线性DNA片段为转化表达框、低成本高效率的瞬时表达直接转化体系,并进一步用于稳定转化体系。通过构建植物smGFP和GUS基因双元表达载体,以洋葱下表皮为实验材料,借助农杆菌瞬时表达系统,研究不同浓度的CaCl₂、6-BA、2,4-D、Silwet L-77、DowCorningQ2-5211和渗透处理对瞬时表达效率的影响。SmGFP和GUS基因的瞬时表达结果表明各种物理化学因素在不同程度上促进了农杆菌瞬时表达,且各因素的最佳处理浓度依次为CaCl₂,0.5mol/L;6-BA,0.2mg/L;2,4-D,0.2 mg/L;Silwet L-77,0.1‰;DowCorning Q2-5211,0.1‰。通过荧光表达强度和GUS染色分析发现,农杆菌高渗处理能较好的提高转化效率,0.1‰的Dow Corning Q2-5211和Silwet L-77诱导也能提高农杆菌转化效率,但效果较渗透稍差。将农杆菌瞬时表达系统筛选出的各种物理化学因素的最佳浓度用于线性基因表达框的转化。以洋葱下表皮为转化材料,对不同处理的线性smGFP和GUS基因表达框的瞬时表达分时段检测;同时检测FITC和SYBR Green I标记的GUS线性基因表达框转化后在细胞中的定位和荧光强度,并利用流式细胞仪对荧光标记基因进入细胞核的效率进行定量分析。结果表明,洋葱组织块经过高渗处理15～20min后,再在smGFP/GUS线性表达框终浓度为100ng/mL的MS液体培养基中悬浮共培养1.5～2h即能有效的将smGFP/GUS线性基因表达框转化入细胞核内,并在转换后36h左右得到高效的瞬时表达,能快速建立瞬时表达系统。通过荧光标记基因的定位和定量分析,高渗处理是促进线性表达框直接转化的最佳方法。借助洋葱下表皮中优化的瞬时表达体系,将smGFP基因的线性片段转化烟草叶片诱导的愈伤组织。烟草愈伤组织转化后与对照相比有很强的荧光差异。通过高渗转化法处理的转化组愈伤荧光强度最强,且生长状态几乎不受影响,成活率高。通过对转化T₀代植株的PCR,RT-PCR,点杂交等分子检测发现,几种直接转化处理均能获得转基因植株,其中高渗转化法获得的转化植株成活率和转化阳性率更高。本文建立的方法适用于外源基因的直接转化,且不受转化受体的影响,在单子叶、双子叶植株、以及组织和细胞中均能应用。通过瞬时和稳定表达优化出高渗直接转化线性基因表达框是国内的首篇报道。更多还原

【Abstract】 Direct transformation of minimal linear gene cassette has been widely used in plant gene transformation,with the advantages of easily operation,high transformation efficiency and wide range host transformants.Exogenous gene could be integrated into the host DNA, expressed and stably inherited to progenies.Direct transformation of linear gene cassette has ever been used in stable transformation,however,it remains unconvinced because of its insufficiency and unrepeatability.Using genes smGFP and GUS as two linear gene cassettes, the present work aims at evaluating different physicochemical co-factors,including surfactants,osmotic and calcium ion,and imaging the delivery and localization of the linear gene cassettes through transient expression in vivo.It was expected to establish a direct transformation system for transient expression with minimal linear gene cassette,and may also contribute to the potent application in stable gene transformation.Binary expression vector of smGFP and GUS were constructed to transform onion epidermal cells mediated via Agrobacterium-mediated transient transformation system.The transformation efficiency of gradient concentration of transformation co-factors,including CaCl₂,6-BA,2,4-D,Silwet L-77,Dow CorningQ2-5211,as well as osmotic treatement were evaluated.The results showed that each physicochemical factor had active effects on Agrobacterium-mediated transformation and transient expression,and the optimum concentration of was 0.5mol/L of CaCl₂,0.2mg/L of 6-BA,0.2 mg/L of 2,4-D,0.1‰Silwet-77, and 0.1‰Dow Coming Q2-5211,respectively.Compared with the expression intensity of Green Fluorescent Protein and quantity of punctate blue stain,Agrobacterium infiltration was the optimal treatment in improving transformation efficiency.The transient expression of liner smGFP/GUS was evaluated through observation of fluorescent/stain intensity at 30min.2h.6h,12h,36h,48h post transformation.Compared with the expression intensity of GFP and quantity of punctate blue stains,Agrobacterium infiltration obtained the higher transformation efficiency compared with the co-factor treatements.Meanwhile,the delivery and orientation of linear transformation cassettes were traced through fluorescent indicators of FITC or SYBR Green I.The entry rate of the linear transformation cassette into nuclei was quantitatively measured by flow cytometry.The results showed that the efficiency of transient expression in lower onion epidermal was dramatically increased after 15-20min hypertonic treatment followed by 100ng/mL linear DNA co-culturvatied in MS liquid medium for 1.5-2h,suggesting its feasibility in direct transformation of linear gene cassette.The linear smGFP gene cassette was then transformed into tobacco leaf callus and To trangenic seedlings were further identified by the PCR,RT-PCR and dot blotting.High regeneration rate as well as higher transformation efficiency was achieved.Our study first reports the direct hypertonic transformation of liner gene cassette in transient and stable expression.更多还原