摘要
杨树属杨柳科(Salicaceae)杨属(Populus),在我国分布广泛,是主要的造林和经济树种,并且在生态环境治理和解决木材短缺等方面占有重要位置。杨树具有生长快、材质好、适应性强等特点。本实验以三倍体毛白杨、741杨、107杨、108杨和烟草无菌苗为试验材料,建立了叶片再生体系并对农杆菌介导的遗传转化体系进行了优化。通过农杆菌介导法将杨树维管束特异表达载体pProNAC068::GUS转入烟草,将pProNAC157::GUS转入杨树和烟草。对转化植株进行DNA和GUS活性检测,研究该启动子在杨树和烟草中的特异表达情况。主要结果如下:
确定烟草转化的卡那霉素临界筛选浓度为50 mg·L-l,头孢噻肟钠的抑菌浓度为300mg·L-l。利用农杆菌介导叶盘转化法,将植物表达载体pProNAC068::GUS和pProNAC157::GUS转入烟草中,分别获得17个株系和3个株系。经PCR检测,初步证明目的基因已整合到烟草的基因组中。GUS组织化学分析表明:植物表达载体pProNAC068::GUS在根、茎和叶中检测到GUS活性;植物表达载体pProNAC157::GUS在根、茎和叶中没有检测到GUS活性。
对农杆菌介导的三倍体毛白杨和741杨遗传转化体系进行了优化,确立了最佳分化培养基MS+6-BA 1.0 mg·L-1+NAA 0.1 mg·L-1和MS+TDZ 0.1 mg·L-1+NAA 0.1 mg·L-1,生根培养基1/2MS+IBA 0.3~0.5 mg·L-1。适宜菌液浓OD600=0.4~0.6,侵菌时间为8~10 min,共培养时间以2 d为宜。卡那霉素临界筛选浓度为50 mg·L-1,抗生素头孢噻肟钠的抑菌浓度为400 mg·L-1。
用农杆菌介导法将植物表达载体pProNAC157::GUS转入三倍体毛白杨和741杨。经过卡那霉素筛选,分别得到16个和2个抗性株系。经过PCR检测显示目的基因已整合到各基因组中。GUS组织化学分析显示:三倍体毛白杨仅在少量茎中检测到了GUS活性,在根和叶中没有检测到GUS活性;在741杨根、茎和叶中没有检测到GUS活性。
采用无菌苗108杨叶片为外植体,筛选出MS+6-BA 0.6 mg·L-1+NAA 0.2 mg·L-1以诱导不定芽的分化,再生芽生根培养基为1/2 MS+IBA0.3 mg·L-1。107杨和108杨卡那霉素临界筛选浓度为30~35 mg·L-1,抗生素头孢噻肟钠的抑菌浓度为400 mg·L-1。遗传转化的适宜菌液浓OD600=0.4,侵菌时间为8~10 min,共培养时间以2 d为宜。用农杆菌介导法将植物表达载体pProNAC157::GUS转入107杨和108杨。筛选转化植株,107杨共获得2个卡那霉素抗性系号,经PCR分子检测,目的基因已整合到107杨基因组中。GUS组织化学分析显示:在根、茎和叶中没有检测到GUS活性。
Poplars belong to Salicaceae Populus, which is very popular throughout in the china. It not only is an important sylvicultural and commercial tree, but also has important use in maintenance balance of ecosystem and the resolvent of wood shortage.Popla has many advantages,such as fast growth, good adaptability, wide adaptation. Selecting Triploid of PopulusTomentosa,741poplar,Populus×euramericana‘Neva’,Populus×euramericana‘Guariento’and tobacco the experiment materials,the leaf regeneration systems were established and optimized the Agrobacterium tumefaciens mediated transformation system. Mediated by Agrobacterium tumefaciens, transfer pProNAC068::GUS were integrated into tobacco,pProNAC157::GUS were integrated into Poplars and tobacco genome.Some transgenic plants were tested on DNA levels and GUS gene activity.The main results were as follows:
Kanamycin (Km) critical concentration was 50 mg·L-1 and Cefotaxime was 300 mg·L-1for the tobacco transformation. pProNAC068::GUS and pProNAC157::GUS were transformed into mode plant with leaf disc transformation respectively. At last we obtained 17 and 3 plants of Km resistant.PCR detections indicated that genes were integrated into the tobacco genome.A GUS histochemical assay showed that pProNAC068::GUS of GUS activity was found in root, stem and leave, but the pProNAC157::GUS of GUS activity was not found in root, stem and leave.
The leaf high frequency transformation system of Triploid of PopulusTomentosa and 741 poplar was established.First, a high frequency regeneration system of Triploid of PopulusTomentosa has been established.The best shoot regeneration mediumwas MS+6-BA 1.0 mg·L-1+NAA 0.1 mg·L-1 and MS+TDZ 0.1 mg·L-1+NAA 0.1 mg·L-1, the root induction medium was 1/2 MS+6-BA 0.3~0.5 mg·L-1.Infected liquid concentration OD600 was 0.4~0.6, infection of leaf explants for 8~10min with Agrobacteria and cocultivation for 2 days after infection would befavorable for the transformation. Kanamycin critical concentration was 50 mg·L-1and Cefotaxime 400 mg·L-1 was a suitable concentration to control the propagation of Agrobacteria.
16 and 2 plants of Km resistant with pProNAC157::GUS were gained via strictly selection by Km. The results of PCR showed that the foreign gene has been integrated into genome of Triploid of Populus Tomentosa and 741 poplars. A GUS histochemical assay showed that GUS activity of Triploid Populus Tomentosa was only found in some small amount of the stem and was not found in root and leave; the GUS activity of 741 poplars was not found.
The regeneration system of Populus×euramericana‘Guariento’was established. The shoots regeneration medium were MS+ 6-BA 0.6 mg·L-1+NAA 0.2 mg·L-1 .Then the regenerated plants were rooted on 1/2 MS+IBA0.3 mg·L-1.Kanamycin (Km) critical concentration was 30~35mg·L-1and Cefotaxime 400 mg·L-1 was a suitable concentration to control the propagation of Agrobacteria. Several crucial factors influencing the transformation efficiency were studied.It was found that OD600=0.4, infection of leaf explants for 8~10 min with Agrobacteria and cocultivation for 2 days after infection would befavorable for the transformation.
Mediated by Agrobacterium tumefaciens pProNAC157 genes were transformed into Populus×euramericana‘Neva’and Populus×euramericana‘Guariento’,there were 2 Kanamycin resistant buds of Populus×euramericana‘Neva’.PCR detections indicated that genes were integrated into the Populus×euramericana‘Neva’genome. A GUS histochemical assay showed that the GUS activity was not found in root, stem and leave.
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