本文關鍵詞： 花青素 直觀標記 載體構建 瞬時表達 轉基因　出處：《山西大學》2014年碩士論文　論文類型：學位論文

【摘要】：目前轉基因中常用的選擇標記基因一般為抗除草劑基因、抗生素耐受基因及一些對細胞有毒害作用的代謝產物基因,由于這類基因本身對植物有毒害作用,且容易引起一些安全性方面的擔憂,因而在很大程度上限制了轉基因作物在生產及食品上的廣泛應用。本研究用花青素調控因子C1/Bperu分別與玉米胚特異性啟動子Glb1和組成型啟動子CaMV35S構建成植物轉化載體pGlb1CB和p35SCB,并利用基因槍轉化方法,將重組表達載體轉入玉米幼胚。顯微觀察結果證實,這兩個載體均能在玉米幼胚細胞中瞬時表達。用花青素作為標記基因不僅可以在一定程度上減少公眾對轉基因生物安全性方面的擔憂,而且可以幫助直觀地從轉化當代和后代種子中通過顏色標記篩選到轉化籽粒,從而可以大大簡化篩選程序,提高效率,節(jié)約檢測成本,對轉基因研究具有十分重要的意義。此外,花青素作為一種天然存在的水溶性色素,具有極強的抗氧化及清除機體自由基的能力,在預防衰老,心血管病,癌癥等方面具有重要作用,花青素作為一種具有雙重功能的安全標記基因,既有標記基因的作用,其本身又可以作為目的基因,用其做標記基因具有廣闊的應用前景。超聲波輔助的花粉介導法是我國第一例具有完全自主知識產權和廣泛應用前景的非組培轉基因方法,對許多開花植物都適用。該方法利用花粉作為載體介導外源基因轉化,既能避免傳統組培轉化方法所要求的基因型依賴性,以及長期組培所帶來的再生困難,幼苗早期夭亡,后代育性不正常,易產生體細胞變異等一系列問題,又能避免轉化體細胞所帶來的易形成嵌合體以及基因槍法的耗資大、轉化率低等問題。本研究擬將花青素作為標記基因構建的載體pGlb1CB和p35SCB首次與超聲波輔助的花粉介導法相結合對玉米進行轉化。轉化后代中花青素在玉米籽粒中特異性表達或者在玉米植株中的表達,使我們能通過肉眼直接觀察,對玉米籽粒是否為轉化種子作初步的篩選,即轉化體的籽粒或胚部呈深(紅、藍或紫)色,而非轉化體的胚仍為原色。二者的結合將會極大地簡化植物轉基因操作過程,避免了傳統方法需要經過組培再生植株過程的繁瑣、轉化率低、耗時長、易突變等缺點,也可大大簡化初篩過程,縮短篩選時間。
[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姳綺変綔涓鴻澆浣撲粙瀵煎婧愬熀鍥犺漿鍖，

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