茉莉花香气相关基因JsGDS启动子的克隆及功能分析

大根香叶烯合成酶在植物萜类化合物合成中具有重要作用。前期从茉莉花cDNA文库中克隆获得香气相关基因Js GDS基因,但是其表达调控机制还不是很清楚,本研究拟采用染色体步移法从茉莉花基因组中克隆了Js GDS上游1 646 bp的启动子序列。生物信息学分析表明,该启动子片段中包含启动子的基本元件TATA-box和CAAT-box以及光调控元件、赤霉素应答元件、ABA应答元件、MeJA应答元件等多个与生长发育或者逆境胁迫相关的顺式作用元件。利用Gateway技术构建Js GDS启动子的GUS融合载体及其系列缺失体,并获得拟南芥遗传转化植株。对转基因拟南芥进行GUS组织化学染色。结果表明,Js GDS启动子及缺失体均具有驱动下游基因转录的活性。本研究为进一步探究Js GDS基因的调控和表达机制提供理论依据。     

MaAQP1过表达拟南芥及对转基因植株抗旱性的分析

MaAQP1 属于水通道蛋白家族的基因,其能响应干旱胁迫。构建植物表达载体pCAMBIA1304-MaAQP1获得转基因拟南芥植株,GUS 染色分析表明,其主要分布于转基因植株的根部。通过Southern blot 检测,选取双拷贝和单拷贝的两个转基因植株进行后续试验。与野生型植株相比,经过干旱胁迫处理后,转基因植株具有较高的存活率,脯氨酸、超氧化物歧化酶和过氧化氢酶含量增加,同时其丙二醛含量降低,提高了对干旱胁迫的抵御能力。     

内含子数量改变GUS基因的瞬时表达调控

内含子是基因的重要组成部分,它与功能基因表达之间的关系越来越被重视。本研究以pCAM-BIA3301为载体,利用玉米泛素启动子Ubi1和水稻肌动蛋白启动子Actin1构建两个GUS基因表达载体p33U1和p33A1,同时设置3个对照载体。通过基因枪轰击法将上述载体转入水稻胚性愈伤组织,探讨内含子对外源目的基因表达的调控作用。组织化学检测结果表明：CaMV35S启动子调控下的iGUS（带内含子）基因能够顺利表达;同样,Actin1启动子（带内含子）调控下的不带内含子的GUS基因也可以正常表达,而当Actin1启动子（带内含子）驱动iGUS基因时,则导致GUS染色反应不能发生。Ubi1启动子（带内含子）调控GUS基因的瞬时表达也得出类似结果,证明表达框中内含子的数量为1个或两个时,对GUS基因的表达起到了不同的调控作用。本研究结果对植物表达载体构建及功能基因表达都具有指导意义。     

根癌农杆菌介导Bt杀虫基因对花椰菜的转化初报

采用 Ti质粒 p KSB带有 Bt杀虫蛋白基因、NPT 选择标记基因及 GUS基因 ,通过根癌农杆菌 L BA40 44菌株的介导 ,将 Bt杀虫基因导入花椰菜品种中。试验以花椰菜下胚轴为外植体 ,将下胚轴切段置于分化培养基 (MS+ 6 - BA1 .0～ 2 .0mg/L + NAA 0～ 0 .5 mg/L)上 ,对影响转化的种种因素进行了试验。得到了转化的愈伤组织及再生植株 ,用组织化学法检测 GUS活性 ,观察到愈伤组织内的转化细胞呈阳性蓝色反应     

一个新的植物转化用质粒pBG1100的构建和检验

在一个10kb双元载体pMOG402的多克隆酶切位点插入一个3.2kb的CaMV35S启动子/GUS编码区/NOS终止区融合基因,得到了一个13.2kb的新植物转化载体pBG1100.pBG1100经三亲交配法或电激法转入农杆菌菌系EHA105,再用农杆菌介导法转化番茄。转基因番茄组成型的强表达GUS基因,在GUS活性测定时表现出强荧光反应。该质粒可用于：1）建立植物转化系统（如检验新的转化方法或测试新的可转化植物种类）;2）用某一基因的启动子取代CaMV35S启动子,可研究该基因在植物体内的表达;3）将目的基因的编码区取代GUS区,可将目的基因转入植物并使其强表达。     

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv. Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110 mM, BAP 10 μM and NAA 0.5 μM. A low light regime of 17 μmol m⁻² s⁻¹ improved regeneration frequency up to 86%. For transformation, leaf discs were inoculated and co-cultivated with two disarmed A. tumefaciens strains, EHA 101 and LBA 4404, both carrying the binary vector pFAJ3000 which contained the nptII selectable gene and the GUS reporter gene. A pre-culture period of 3 days and a short co-cultivation duration (1 day) improved the efficiency of transformation. Inoculation of only 10 min with agitation including (or not) vacuum infiltration was sufficient. If selection on kanamycin containing medium was applied after a 2 weeks culture period on shoot regeneration medium, the percentage of explants forming kanamycin-resistant shoots increased from 3.3 to 13.3%. Integration and expression of the introduced transgene were confirmed by histochemical GUS assay, PCR and Southern blot analysis. Flowering of transgenic plants in glasshouse occurred 10 months after acclimatization.     

莴苣高效瞬时表达体系的建立

 以GUS基因作为报告基因, 利用农杆菌真空渗透法, 采取正交试验设计对莴苣(Lactuca sativa L. sp) 的瞬时表达条件进行研究, 建立了高表达水平的瞬时转化体系(乙酰丁香酮200 μmol /L、菌液密度OD₆₀₀值0.8、真空处理30 min、共培养6 d) 。应用结果表明: 优势组合的表达水平比对照的表达水平提高16.3倍。     

Transient expression of an active human interferon-beta in lettuce

Using Agrobacterium mediated transient expression method, plant bivalent expression vector pBI121 containing GUS as a report gene was transformed into lettuce (Lactuca sativa). Through designed orthogonal analysis, intact lettuce leaves infiltrated with 200 μM acetosyringone and 0.8 OD₆₀₀ bacterial suspensions under vacuum for 30 min, then co-cultured at 24 °C for 6 ds produced a maximum GUS protein of 2.5% TSP with 21.39 nmol mg⁻¹ min⁻¹ MU activity, which was 19 times of the control (1.31 nmol mg⁻¹ min⁻¹ MU). Employed these optimized conditions HuIFN-beta was expressed in lettuce leaves. Western blot and antivirus bioactivity analyses confirmed the HuIFN-beta achieved by agrobacterium infiltration had a high biological activity (3.1 × 10⁴ IU/mL). To our knowledge, it is the first detailed orthogonal optimizing study of Agrobacterium mediated transient expression and the first report on the production of the biologically active therapeutic proteins produced by Agrobacterium mediated transient expression in lettuce. In summary, transient expression by Agrobacterium vacuum infiltration can be adopted as an efficient, inexpensive and small-scaled plant expression system for therapeutic protein production.     

自交系BU-21/3——甜瓜(Cucumis meloL.)转基因育种的有效工具

甜瓜转化率低的问题仍具有挑战性。用1个新特质甜瓜自交系BU-21/3作材料研究其再生和转化能力,表明这个自交系的再生能力优于以往研究过的基因型。用含有报告基因GUS或GFP的根癌农杆菌浸染子叶外植体,研究瞬时表达和稳定表达效率,观察到了大量瞬时表达,稳定表达时每个外植体可产生0.4～1.5个转基因芽,在转化后的8～10周,将转基因植株移入可控温室,发现表型和育性正常。再生能力的遗传分析表明它受单个显性位点控制,因此,这种新特质的基因型有望成为甜瓜转基因育种的有效工具。     

Identification of Resistance to Barley Leaf Stripe Using a Pyrenophora graminea Transformant Expressing β-glucuronidase

A Pyrenophora graminea strain expressing the -glucuronidase gene (GUS) was obtained via genetic transformation, and used to follow the penetration of the pathogen inside barley germinating seeds and the colonization of host tissues. Significant differences between resistant and susceptible barley cultivars were observed in the colonization of artificially-infected embryos by the fungus. These results suggest that the GUS transgenic strain of P. graminea will be useful for the early screening of barley cultivars for resistance to leaf stripe disease.