發(fā)布時間：2018-02-23 19:26

  本文關(guān)鍵詞： 花青素 直觀標(biāo)記 載體構(gòu)建 瞬時表達(dá) 轉(zhuǎn)基因　出處：《山西大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目前轉(zhuǎn)基因中常用的選擇標(biāo)記基因一般為抗除草劑基因、抗生素耐受基因及一些對細(xì)胞有毒害作用的代謝產(chǎn)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(dān)憂,因而在很大程度上限制了轉(zhuǎn)基因作物在生產(chǎn)及食品上的廣泛應(yīng)用。本研究用花青素調(diào)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(gòu)建成植物轉(zhuǎn)化載體pGlb1CB和p35SCB,并利用基因槍轉(zhuǎn)化方法,將重組表達(dá)載體轉(zhuǎn)入玉米幼胚。顯微觀察結(jié)果證實(shí),這兩個載體均能在玉米幼胚細(xì)胞中瞬時表達(dá)。用花青素作為標(biāo)記基因不僅可以在一定程度上減少公眾對轉(zhuǎn)基因生物安全性方面的擔(dān)憂,而且可以幫助直觀地從轉(zhuǎn)化當(dāng)代和后代種子中通過顏色標(biāo)記篩選到轉(zhuǎn)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(zhuǎn)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(qiáng)的抗氧化及清除機(jī)體自由基的能力,在預(yù)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(biāo)記基因,既有標(biāo)記基因的作用,其本身又可以作為目的基因,用其做標(biāo)記基因具有廣闊的應(yīng)用前景。超聲波輔助的花粉介導(dǎo)法是我國第一例具有完全自主知識產(chǎn)權(quán)和廣泛應(yīng)用前景的非組培轉(zhuǎn)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(dǎo)外源基因轉(zhuǎn)化,既能避免傳統(tǒng)組培轉(zhuǎn)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(chǎn)生體細(xì)胞變異等一系列問題,又能避免轉(zhuǎn)化體細(xì)胞所帶來的易形成嵌合體以及基因槍法的耗資大、轉(zhuǎn)化率低等問題。本研究擬將花青素作為標(biāo)記基因構(gòu)建的載體pGlb1CB和p35SCB首次與超聲波輔助的花粉介導(dǎo)法相結(jié)合對玉米進(jìn)行轉(zhuǎn)化。轉(zhuǎn)化后代中花青素在玉米籽粒中特異性表達(dá)或者在玉米植株中的表達(dá),使我們能通過肉眼直接觀察,對玉米籽粒是否為轉(zhuǎn)化種子作初步的篩選,即轉(zhuǎn)化體的籽粒或胚部呈深(紅、藍(lán)或紫)色,而非轉(zhuǎn)化體的胚仍為原色。二者的結(jié)合將會極大地簡化植物轉(zhuǎn)基因操作過程,避免了傳統(tǒng)方法需要經(jīng)過組培再生植株過程的繁瑣、轉(zhuǎn)化率低、耗時長、易突變等缺點(diǎn),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，

本文編號：1527324