發(fā)布時(shí)間：2018-03-28 05:13

  本文選題：煙草　切入點(diǎn)：orf基因　出處：《分子植物育種》2017年05期

【摘要】：雜種優(yōu)勢(shì)的利用可以有效地實(shí)現(xiàn)煙草高產(chǎn)、高抗、優(yōu)質(zhì)的育種目標(biāo),目前,培育雄性不育系是獲取雜種煙草最有效的途徑。研究發(fā)現(xiàn)orf25基因位于線(xiàn)粒體基因組中,與煙草雄性不育密切相關(guān)。本研究以煙草雄性不育相關(guān)基因orf25為研究對(duì)象,根據(jù)RNA干擾載體構(gòu)建原則,將長(zhǎng)度為606 bp的orf25基因干擾片段分別以正向、反向連接到大小為190 bp的linker連接片段的兩側(cè),構(gòu)成發(fā)夾結(jié)構(gòu)的RNA干擾載體片段。將RNA干擾載體片段插入到中間載體pET30a上,最后克隆到植物表達(dá)載體pCAMBIA1301中,成功構(gòu)建orf25基因的RNAi表達(dá)載體。利用農(nóng)桿菌介導(dǎo)法將pCAMBIA1301-orf25-RNAi轉(zhuǎn)入到煙草保持系中煙90中,經(jīng)抗性篩選以及分子檢測(cè)最終獲得35株轉(zhuǎn)pCAMBIA1301-orf25-RNAi煙草植株,其中陽(yáng)性煙草有18株,轉(zhuǎn)化率為51.4%。結(jié)果表明pCAMBIA1301-orf25-RNAi干擾載體已成功轉(zhuǎn)入到煙草中煙90中。
[Abstract]:The utilization of heterosis can effectively achieve the goal of tobacco breeding with high yield, high resistance and good quality. At present, breeding male sterile lines is the most effective way to obtain hybrid tobacco. It is found that orf25 gene is located in the mitochondrial genome. In this study, the male sterility related gene (orf25) of tobacco was studied. According to the principle of RNA interference vector construction, the interference fragments of 606 BP orf25 gene were divided into positive direction. The RNA interference vector fragment with hairpin structure was constructed. The RNA interference vector fragment was inserted into the intermediate vector pET30a and cloned into the plant expression vector pCAMBIA1301. The RNAi expression vector of orf25 gene was successfully constructed. The pCAMBIA1301-orf25-RNAi was transferred into tobacco maintainer line Yan90 by Agrobacterium tumefaciens. 35 transgenic pCAMBIA1301-orf25-RNAi tobacco plants were obtained by resistance screening and molecular analysis, among which 18 were positive. The results showed that the pCAMBIA1301-orf25-RNAi interference vector had been successfully transferred into tobacco smoke 90.
【作者單位】： 江西農(nóng)業(yè)大學(xué)農(nóng)學(xué)院作物生理生態(tài)與遺傳育種教育部重點(diǎn)實(shí)驗(yàn)室江西省作物生理生態(tài)與遺傳育種重點(diǎn)實(shí)驗(yàn)室;南昌職業(yè)學(xué)院;湖南農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院植物病蟲(chóng)害生物學(xué)與防控湖南省重點(diǎn)實(shí)驗(yàn)室;江西農(nóng)業(yè)大學(xué)園林與藝術(shù)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(31260350;31301388) 中國(guó)博士后科學(xué)基金(2015T8087;2014M562109) 江西省教育廳科技計(jì)劃項(xiàng)目(GJJ13275)共同資助
【分類(lèi)號(hào)】：Q943.2
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本文編號(hào)：1674900