本文選題：柑橘 + cor8基因��； 參考：《湖南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：本課題組已經(jīng)從耐寒性柑橘枳中克隆獲得一個(gè)與低溫鍛煉有關(guān)的基因,并將其命名為Ptcor8。同時(shí)發(fā)現(xiàn)該基因在金柑、檸檬等柑橘種類中具有高度同源性,而且該同源基因表達(dá)與柑橘抗寒能力大小呈現(xiàn)相同趨勢,枳最耐寒,能耐受-26℃低溫,檸檬最不耐寒,枳中的表達(dá)量是檸檬的25倍。另外研究比對(duì)不同柑橘種類中同源基因啟動(dòng)子的序列發(fā)現(xiàn),枳啟動(dòng)子與檸檬啟動(dòng)子的同源性為88.17%,并且枳啟動(dòng)子含有3個(gè)與逆境響應(yīng)相關(guān)的特有調(diào)控元件。于是構(gòu)建了含有啟動(dòng)子和cor8基因的融合植物表達(dá)載體,用農(nóng)桿菌介導(dǎo)的瞬時(shí)表達(dá)法,將含有不同載體的菌液注射到墨西哥萊檬葉片中,進(jìn)行瞬時(shí)表達(dá),發(fā)現(xiàn)枳啟動(dòng)子對(duì)低溫響應(yīng)的敏感度更高。在此條件下,將pPtcor8::Ptc-or8::YFP, pPtcor8::Clcor8::YFP, pClcor8::Ptcor8::YFP3個(gè)植物表達(dá)載體轉(zhuǎn)入到不耐寒的檸檬,并對(duì)檸檬遺傳轉(zhuǎn)化條件進(jìn)行優(yōu)化,篩選出最佳組合的再生條件和篩選壓,最終得到了轉(zhuǎn)基因植株本研究取得了以下主要結(jié)果：1、將構(gòu)建好的4個(gè)植物表達(dá)載體(35S::Ptcor8::YFP, pPtcor8::Ptcor8::YFP, pClcor8::Ptcor8::YFP,pPtcor8::Clcor8::YFP)采用農(nóng)桿菌介導(dǎo)的瞬時(shí)表達(dá)法注射墨西哥萊檬葉片,YFP報(bào)告基因的熒光檢測說明枳的啟動(dòng)子比檸檬啟動(dòng)子在低溫下更敏感。2、對(duì)檸檬遺傳轉(zhuǎn)化條件進(jìn)行優(yōu)化,在再生培養(yǎng)基中不同濃度的6-BA,卡那霉素以及除草劑進(jìn)行篩選,得出了再生培養(yǎng)基為MT+0.75 mg/L 6-BA,添加75 mg/LKan或者10mg/LBastao3、將pPtcor8::Ptcor8::YFP, pPtcor8::Clcor8::YFP, pClcor8::Ptcor8::YFP3個(gè)植物表達(dá)載體轉(zhuǎn)入到不耐寒的檸檬,得到含有ppPtcor8::Ptcor8::YFP抗性芽34個(gè),pClcor8::Ptcor8::YFP抗性芽48個(gè),pPtcor8::Clcor8::YFP抗性芽29個(gè),通過報(bào)告基因的熒光檢測得到成功轉(zhuǎn)入pPtcor8::Ptcor8::YFP, pPtcor8::Ptcor8::YFP, pClcor8:: Ptcor8::YFP,pPtcor8::Clcor8::YFP的檸檬植株分別是3株,4株,2株；并移栽于溫室大棚。
[Abstract]:Our team has cloned a gene related to low temperature exercise from cold tolerant citrus trifoliate orange and named it Ptcor8. At the same time, it was found that the gene had high homology in citrus species such as orange and lemon, and the expression of the homologous gene showed the same trend as the cold resistance of citrus. The trifoliate orange was the most cold tolerant, able to tolerate -26 鈩，

本文編號(hào)：2101666