山杨转双价抗虫基因的研究
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摘要
本文以山杨(Populus davidiana Dodetumefaciens)为受体材料,采用根癌农杆菌介导的叶盘转化法进行外源双价抗虫基因(Bt+蜘蛛杀虫肽)的遗传转化研究,探讨了影响山杨遗传转化的若干因子,初步建立了山杨遗传转化体系;获得了转化再生的植株,经PCR检测和Southern杂交检测,证明了目的基因已经整合到山杨的基因组中,为山杨的遗传转化及抗虫育种提供了理论基础和研究材料。
本试验的研究结果如下:
1、优化了山杨组培再生体系,结果表明,山杨不同组织的适宜培养基分别为:分化培养基:MS+BA 0.5 mg/L+NAA 0.01 mg/L;生根培养基:WPM+IBA 0.4 mg/L。
2、筛选了山杨转化体及选择生根培养的卡那霉素和头孢噻肟钠浓度。卡那霉素抑制叶片分化的浓度下限为20 mg/L,芽诱导生根的卡那霉素临界浓度为20 mg/L;700mg/L的头孢噻肟钠能有效的除菌,且对叶片分化及生根培养没有不利影响。
3、探讨了影响山杨遗传转化的主要因子,初步确立了山杨遗传转化体系。将带切口的健壮叶片预培养2 d后,用无菌蒸馏水稀释成OD_(600)=0.3～0.4、培养至对数生长期的农杆菌(OD_(600)=0.5)侵染叶片10～15min;共培养2 d后,转入到筛选培养基(附加700mg/L头孢噻肟钠+20 mg/L卡那霉素的MS培养基)中进行诱导抗性不定芽生长,当抗性不定芽长到1.5～2 cm时转入选择生根培养基(附加800 mg/L头孢噻肟钠+20 mg/L卡那霉素的WPM培养基)中诱导其生根。
4、试验共侵染山杨叶片1862个,经过多次筛选和选择获得56个抗性芽,抗性芽率为3.0%。由抗性芽在经过选择生根培养后得到13株抗性植株,抗性芽生根率为23.2%
5、对13个抗性植株的叶片进行PCR检测,其中有3株呈阳性,阳性率为23.1%。将呈阳性的叶片进行Southern杂交检测,3株均呈阳性,阳性率为100%,转化率为1.6‰。
In this study, Populus davidiana Dodetumefaciens was selected as target material for genetic transformation by Agrobacterium. Some factors that affected the efficiency of genetic transformation were studied. Agrobacterium mediated genetic transformation system of P. davidiana Dodetumefaciens was established. And the transformation regeneration plants were obtained. Stable integration of the goal gene (Spider insecticidal peptide and Bt) in the plant genome was confirmed by PCR detection and Southern blot analysis. The theory base was given for P. davidiana Dodetumefaciens transformation and fastness breeding. The conclusions were drawn as follows:
1. A high frequency regeneration system of P. davidiana Dodetumefaciens has been established. The results shown that different Phytohormone proportion can greatly effect Poplar's growing. The best leaf regeneration medium is MS supplemented with 0.5 mg/L 6-BA, 0.01 mg/L NAA; the best root regeneration medium is WPM supplemented with 0.4 mg/L IBA.
2. The appropriate concentration of Kanamycin and Cefotaxime, Sodium Salt for sensitivity experiment of the leaves and rooting was determined. The concentration of Kanamycin was 20 mg/L when screening transformed hybrid buds, while 20 mg/L inducing roots. The concentration of Cefotaxime, Sodium Salt was 700 mg/L when disposing Agrobacterium, and the concentration isn't any effect on growing.
3. The main factors that affected the efficiency of genetic transformation were studied, the transformation procedure Agrobacterium -mediated of P. davidiana Dodetumefaciens was established successfully. Take the vigorous leaves that were cut to pre-culture for 3 days; centrifuge Agrobacterium when being developped to the logarithm growth time(OD_(600)=0.5), then dilute Agrobacterium to the concentration of OD_(600)=0.3～0.4 with asepsis distilled water for infection; infection time was 10～15 minute; after being co-cultured 2 days, inoculate on the selecting medium(containing Kanamycin 20 mg/L and Cefotaxime, Sodium Salt 700 mg/L) to be filtrated in succession during inducing unsureness buds. When the unsureness buds grow about 1.5～2 cm, cut them off and take them into the selecting medium (containing Kanamycin 30 mg/L and Cefotaxime, Sodium Salt 800 mg/L) for rooting.
4. 1862 leaves were transformated, 56 Km-resistant buds were obtained after many selections, the rate of km-resistant buds is 3 %.13 Km-resistant plant were obtained after selection rooting, the rate of rooting is 23.2 %.
5. After PCR analysis, three positive transformants were achieved, the rate of positive is 23.1 %. Then by the Southern blotting analysis, the differential hybridizing strips appeared and the positive frequentcy was 100 percent, the rate of transformation was 1.6‰.
本试验的研究结果如下:
1、优化了山杨组培再生体系,结果表明,山杨不同组织的适宜培养基分别为:分化培养基:MS+BA 0.5 mg/L+NAA 0.01 mg/L;生根培养基:WPM+IBA 0.4 mg/L。
2、筛选了山杨转化体及选择生根培养的卡那霉素和头孢噻肟钠浓度。卡那霉素抑制叶片分化的浓度下限为20 mg/L,芽诱导生根的卡那霉素临界浓度为20 mg/L;700mg/L的头孢噻肟钠能有效的除菌,且对叶片分化及生根培养没有不利影响。
3、探讨了影响山杨遗传转化的主要因子,初步确立了山杨遗传转化体系。将带切口的健壮叶片预培养2 d后,用无菌蒸馏水稀释成OD_(600)=0.3～0.4、培养至对数生长期的农杆菌(OD_(600)=0.5)侵染叶片10～15min;共培养2 d后,转入到筛选培养基(附加700mg/L头孢噻肟钠+20 mg/L卡那霉素的MS培养基)中进行诱导抗性不定芽生长,当抗性不定芽长到1.5～2 cm时转入选择生根培养基(附加800 mg/L头孢噻肟钠+20 mg/L卡那霉素的WPM培养基)中诱导其生根。
4、试验共侵染山杨叶片1862个,经过多次筛选和选择获得56个抗性芽,抗性芽率为3.0%。由抗性芽在经过选择生根培养后得到13株抗性植株,抗性芽生根率为23.2%
5、对13个抗性植株的叶片进行PCR检测,其中有3株呈阳性,阳性率为23.1%。将呈阳性的叶片进行Southern杂交检测,3株均呈阳性,阳性率为100%,转化率为1.6‰。
In this study, Populus davidiana Dodetumefaciens was selected as target material for genetic transformation by Agrobacterium. Some factors that affected the efficiency of genetic transformation were studied. Agrobacterium mediated genetic transformation system of P. davidiana Dodetumefaciens was established. And the transformation regeneration plants were obtained. Stable integration of the goal gene (Spider insecticidal peptide and Bt) in the plant genome was confirmed by PCR detection and Southern blot analysis. The theory base was given for P. davidiana Dodetumefaciens transformation and fastness breeding. The conclusions were drawn as follows:
1. A high frequency regeneration system of P. davidiana Dodetumefaciens has been established. The results shown that different Phytohormone proportion can greatly effect Poplar's growing. The best leaf regeneration medium is MS supplemented with 0.5 mg/L 6-BA, 0.01 mg/L NAA; the best root regeneration medium is WPM supplemented with 0.4 mg/L IBA.
2. The appropriate concentration of Kanamycin and Cefotaxime, Sodium Salt for sensitivity experiment of the leaves and rooting was determined. The concentration of Kanamycin was 20 mg/L when screening transformed hybrid buds, while 20 mg/L inducing roots. The concentration of Cefotaxime, Sodium Salt was 700 mg/L when disposing Agrobacterium, and the concentration isn't any effect on growing.
3. The main factors that affected the efficiency of genetic transformation were studied, the transformation procedure Agrobacterium -mediated of P. davidiana Dodetumefaciens was established successfully. Take the vigorous leaves that were cut to pre-culture for 3 days; centrifuge Agrobacterium when being developped to the logarithm growth time(OD_(600)=0.5), then dilute Agrobacterium to the concentration of OD_(600)=0.3～0.4 with asepsis distilled water for infection; infection time was 10～15 minute; after being co-cultured 2 days, inoculate on the selecting medium(containing Kanamycin 20 mg/L and Cefotaxime, Sodium Salt 700 mg/L) to be filtrated in succession during inducing unsureness buds. When the unsureness buds grow about 1.5～2 cm, cut them off and take them into the selecting medium (containing Kanamycin 30 mg/L and Cefotaxime, Sodium Salt 800 mg/L) for rooting.
4. 1862 leaves were transformated, 56 Km-resistant buds were obtained after many selections, the rate of km-resistant buds is 3 %.13 Km-resistant plant were obtained after selection rooting, the rate of rooting is 23.2 %.
5. After PCR analysis, three positive transformants were achieved, the rate of positive is 23.1 %. Then by the Southern blotting analysis, the differential hybridizing strips appeared and the positive frequentcy was 100 percent, the rate of transformation was 1.6‰.
引文
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