花青素合成调控基因作为直观标记的转化载体构建及其在玉米中表达的初步研究

发布时间：2018-02-23 19:26

  本文关键词： 花青素 直观标记 载体构建 瞬时表达 转基因　出处：《山西大学》2014年硕士论文　论文类型：学位论文

【摘要】：目前转基因中常用的选择标记基因一般为抗除草剂基因、抗生素耐受基因及一些对细胞有毒害作用的代谢产物基因,由于这类基因本身对植物有毒害作用,且容易引起一些安全性方面的担忧,因而在很大程度上限制了转基因作物在生产及食品上的广泛应用。本研究用花青素调控因子C1/Bperu分别与玉米胚特异性启动子Glb1和组成型启动子CaMV35S构建成植物转化载体pGlb1CB和p35SCB,并利用基因枪转化方法,将重组表达载体转入玉米幼胚。显微观察结果证实,这两个载体均能在玉米幼胚细胞中瞬时表达。用花青素作为标记基因不仅可以在一定程度上减少公众对转基因生物安全性方面的担忧,而且可以帮助直观地从转化当代和后代种子中通过颜色标记筛选到转化籽粒,从而可以大大简化筛选程序,提高效率,节约检测成本,对转基因研究具有十分重要的意义。此外,花青素作为一种天然存在的水溶性色素,具有极强的抗氧化及清除机体自由基的能力,在预防衰老,心血管病,癌症等方面具有重要作用,花青素作为一种具有双重功能的安全标记基因,既有标记基因的作用,其本身又可以作为目的基因,用其做标记基因具有广阔的应用前景。超声波辅助的花粉介导法是我国第一例具有完全自主知识产权和广泛应用前景的非组培转基因方法,对许多开花植物都适用。该方法利用花粉作为载体介导外源基因转化,既能避免传统组培转化方法所要求的基因型依赖性,以及长期组培所带来的再生困难,幼苗早期夭亡,后代育性不正常,易产生体细胞变异等一系列问题,又能避免转化体细胞所带来的易形成嵌合体以及基因枪法的耗资大、转化率低等问题。本研究拟将花青素作为标记基因构建的载体pGlb1CB和p35SCB首次与超声波辅助的花粉介导法相结合对玉米进行转化。转化后代中花青素在玉米籽粒中特异性表达或者在玉米植株中的表达,使我们能通过肉眼直接观察,对玉米籽粒是否为转化种子作初步的筛选,即转化体的籽粒或胚部呈深(红、蓝或紫)色,而非转化体的胚仍为原色。二者的结合将会极大地简化植物转基因操作过程,避免了传统方法需要经过组培再生植株过程的繁琐、转化率低、耗时长、易突变等缺点,也可大大简化初筛过程,缩短筛选时间。
[Abstract]:The current selection marker used in transgenic gene for herbicide resistance gene, antibiotic resistant genes and some toxic effects on cell metabolism genes, because the gene itself is toxic to plants, and easily lead to some security concerns, because of the extensive application in the largely restricted in the production of genetically modified crops and food. This study used C1/Bperu anthocyanin regulatory factor respectively with maize embryo specific promoter Glb1 and constitutive promoter CaMV35S to construct plant transformation vector pGlb1CB and p35SCB, and using the biolistic transformation method, the recombinant expression vector into maize immature embryos. Microscopic observation results show that the two vectors could express instantaneous in maize immature embryo cells. With anthocyanin as a marker gene not only can to some extent reduce the public on GMO safety concerns Sorrow, but also can help directly from the conversion of present and future generations of seeds through the color marker into the kernel, which can greatly simplify the screening procedures, improve efficiency, save the cost of testing, has very important significance for transgenic research. In addition, the anthocyanin as a natural water soluble pigment, has strong antioxidant and the scavenging capacity of free radicals, in the prevention of aging, cardiovascular disease, cancer and other aspects play an important role in anthocyanin, as a safe marker gene with dual function, both the marker gene for itself and can be used as the target gene, the marker gene has a wide application prospect. Pollen mediated ultrasonic the auxiliary guide method is the first example of a completely independent intellectual property rights and broad application prospects of the transgenic non tissue culture method for the number of flowering plants. The method 鐢ㄨ姳绮変綔涓鸿浇浣撲粙瀵煎�栨簮鍩哄洜杞�鍖，

本文编号：1527324