【摘要】：棉花是一種重要的經(jīng)濟(jì)作物,在我國(guó)國(guó)民經(jīng)濟(jì)發(fā)展等方面有著至關(guān)重要的作用。草害是影響棉花生產(chǎn)的主要因素之一,對(duì)棉花的產(chǎn)量及品質(zhì)有較大的影響。運(yùn)用基因工程創(chuàng)新棉花種質(zhì)資源,培育應(yīng)用轉(zhuǎn)基因抗除草劑的棉花品種對(duì)于棉田化學(xué)除草,降低植棉投入,提高植棉效率具有重要的意義。本研究以具有自主知識(shí)產(chǎn)權(quán)的抗草甘膦除草劑基因(EPSPS-G6)為外源基因,通過(guò)優(yōu)化多種轉(zhuǎn)化條件,完善了基于抗草甘膦基因的棉花莖尖農(nóng)桿菌遺傳轉(zhuǎn)化技術(shù),并將EPSPS-G6基因?qū)腌孀置?01、YZ-1及中棉所49等棉花品種中,創(chuàng)制出抗草甘膦轉(zhuǎn)基因棉花種質(zhì)系。研究結(jié)果如下： 1.以中棉所49、珂字棉201及YZ-1為受體材料,通過(guò)對(duì)培養(yǎng)基草甘膦濃度的篩選、農(nóng)桿菌侵染濃度、共培養(yǎng)時(shí)間以及恢復(fù)培養(yǎng)條件等因素的優(yōu)化,完善了基于抗草甘膦基因的棉花莖尖農(nóng)桿菌遺傳轉(zhuǎn)化技術(shù)。研究結(jié)果表明,適合于棉花莖尖農(nóng)桿菌遺傳轉(zhuǎn)化法的草甘膦篩選濃度為10mg/L；莖尖轉(zhuǎn)化體系：以20min為農(nóng)桿菌侵染時(shí)間,農(nóng)桿菌菌液OD600為0.9-1.0,共培養(yǎng)時(shí)間為48h,選用SH培養(yǎng)基并加入適量活性炭(0.5g/L)作為恢復(fù)培養(yǎng)基。以莖尖外植體為基數(shù),本轉(zhuǎn)化技術(shù)的轉(zhuǎn)化成功率達(dá)6.4%。從轉(zhuǎn)化時(shí)間來(lái)看,該方法從無(wú)菌苗培養(yǎng)開始計(jì)算,獲得再生試管苗的時(shí)間僅為2個(gè)月時(shí)間,4個(gè)半月即能開花,6個(gè)月即能獲得T1代種子,說(shuō)明本轉(zhuǎn)化技術(shù)具有較好的實(shí)用價(jià)值。 2.以本研究建立的棉花莖尖農(nóng)桿菌遺傳轉(zhuǎn)化技術(shù),將具有我校自主知識(shí)產(chǎn)權(quán)的抗草甘膦基因EPSPs-G6導(dǎo)入中棉所49、珂字棉201及YZ-1等3個(gè)陸地棉品種(系)的莖尖。共轉(zhuǎn)化360個(gè)莖尖,共獲得60株抗性再生植株,經(jīng)分子檢測(cè)和草甘膦篩選后移栽成活23株。 3.對(duì)再生植株(T0)的基因組DNA進(jìn)行PCR擴(kuò)增,26株再生植株中能擴(kuò)增出與質(zhì)粒DNA相同大小條帶,初步說(shuō)明外源基因已成功地整合到再生植株的染色體DNA中。Southern雜交鑒定結(jié)果表明,轉(zhuǎn)基因植株的外源基因均為單拷貝。轉(zhuǎn)基因T0植株主要田間農(nóng)藝性狀調(diào)查結(jié)果表明,轉(zhuǎn)EPSPS-G6基因T0當(dāng)代生長(zhǎng)正常,全部能開花結(jié)實(shí),并獲得T1種子。此外,23株轉(zhuǎn)基因植株整體性狀與受體對(duì)照無(wú)明顯差異,不同轉(zhuǎn)化體之間無(wú)明顯的變異。說(shuō)明本研究創(chuàng)造的抗草甘膦棉花種質(zhì)具有較好的育種利用價(jià)值。
[Abstract]:Cotton is an important cash crop, which plays an important role in the development of our national economy. Grass damage is one of the main factors affecting cotton production, and has a great impact on cotton yield and quality. Using genetic engineering to innovate cotton germplasm resources and cultivate cotton varieties with transgenic herbicide resistance are of great significance for chemical weeding in cotton fields, reduction of cotton planting input and improvement of cotton planting efficiency. In this study, glyphosate resistant herbicide gene (EPSPS-G6) with independent intellectual property rights was used as foreign gene, and the genetic transformation technology of Agrobacterium tumefaciens based on glyphosate resistance gene was improved by optimizing various transformation conditions. The transgenic cotton germplasm lines resistant to glyphosate were established by introducing EPSPS-G6 gene into cotton varieties such as Ke cotton 201,YZ 1 and Zhongmiansuo 49. The results showed that the transgenic cotton germplasm was resistant to glyphosate. The results are as follows: 1. The glyphosate concentration, Agrobacterium tumefaciens infection concentration, co-culture time and recovery culture conditions were optimized by screening glyphosate concentration, co-culture time and recovery culture conditions. The genetic transformation technology of Agrobacterium tumefaciens based on glyphosate resistance gene was improved. The results showed that the screening concentration of glyphosate suitable for Agrobacterium tumefaciens genetic transformation was 10 mg 路L ~ (- 1). Stem tip transformation system: using 20min as Agrobacterium tumefaciens infection time, Agrobacterium tumefaciens solution OD600 0.9 鈮，

本文編號(hào)：2474313