农杆菌介导的抗寒基因转化羽衣甘蓝的初步研究
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摘要
羽衣甘蓝(Brassica oleracea var.acephala),属于十字花科,是芸薹属食用甘蓝的一个园艺变种。羽衣甘蓝营养价值高,热量低,是非常受欢迎的保健蔬菜,同时又具有良好的观赏价值。其色彩丰富绚丽,叶形多样,观赏期长,耐寒性好,可耐多次-5℃～-10℃短暂霜冻,在园林绿化中具有重要的意义。我国北方冬季持续时间长,温度低,羽衣甘蓝在室外观赏的应用中抗冻性还有待于进一步的提高,使其在长期霜冻下保持良好的观赏性。植物的抗寒性是多基因控制的性状,以传统育种手段改良有一定的困难,利用转基因技术将抗寒基因导入栽培品种可能成为提高植物抗寒性的一种比较快速有效的手段。本实验以提高羽衣甘蓝抗冻能力为目的,采用农杆菌介导的方法转入甜菜碱合成基因BADH和抗寒转录调控因子基因TCF,期望得到可以在冬季露天观赏的羽衣甘蓝新品种。主要研究结果如下：
1.优化羽衣甘蓝的再生体系,筛选出再生能力强的3个羽衣甘蓝品种
通过对不同激素配比的培养基上,9种基因型羽衣甘蓝的带柄子叶、下胚轴、真叶的再生情况进行比较分析,从中筛选出皱白1,06-56和05-25这3种高效再生基因型,其最适培养基分别为MS+6-BA1.0mg/L+NAA0.1mg/L, MS+6-BA1.4mg/L, MS+6-BA1.0mg/L,获得的最高不定芽诱导率均可达到100%；芽诱导率差的有玫红、05-20和皱白2,不足70%。说明不同基因型羽衣甘蓝的不定芽诱导率不同,对培养基激素配比的要求也不同。最终优化的羽衣甘蓝离体再生体系为：切取发芽3-4d的带柄子叶,置于与品种对应的最适培养基中诱导芽萌发,待芽伸长到1cm左右时转到生根培养基MS+0.1mg/LNAA上,根长1cm左右,移栽到营养钵中。
2.建立了农杆菌介导的遗传转化体系
确定了羽衣甘蓝最终的转化条件：取发芽3-4d的无菌苗,切取带柄子叶预培养2d,将二次活化,离心,用pH值5.2的MS液体培养基重悬至OD_600值为0.3的菌液侵染,侵染时间5mmin,灭菌滤纸吸干,共培养2d,延迟筛选3d,筛选培养后切去存活不定芽进行生根。预培养培养基：MS+BA 1.0mg/L+NAA 0.1mg/L;共培养培养基：MS+BA 1.0mg/L+NAA 0.1mg/L;延迟培养基：MS+BA 1.0mg/L+NAA 0.1 mg/L+Carb500mg/L；芽筛选培养基：MS+BA1.0mg/L+NAA 0.1 mg/L+Carb500mg/L+0.05‰Gly；生根培养基：MS+NAA0.1mg/L+Carb500mg/L+0.05‰Gly。
3.抗寒调控基因的导入及转基因植株的鉴定
在优化的羽衣甘蓝带柄子叶转化体系上,将抗寒调控因子TCF基因导入羽衣甘蓝,得到阳性抗性芽,诱导率为4.8%,共获得28株伸长的抗性芽。PCR检测显示,其中4株显示PCR阳性。
Ornamental kale(Brassica oleracea var.acephala) is an excellent landscape plant, belonging to cabbge's horticulture varieties of Cruciferae Brassiea. Low temperature is the key factor that limits the growth, colour and appreciatability in winter in the northern of China. Introducing foreign gene into kale through genetic engineering may create novel variety with cold tolerance. In this study, BADH(encoding betaine aldehyde dehydrogenase) and TCH(encoding transcription regulation factors) were used to be transformed into kale with Agrobacterium-mediated transformation. The results are as follows:
1. Get 3 high efficient regeneration genotypes and optimize the regeneration system of kale cotyledon
Trough the analysis of 9 pairs of genotypes, choose 3 high efficient regeneration genotypes and the highest induction rate is 100%. The best medium are MS+6-BA1.0mg/L+NAA0.1mg/L, MS+6-BA1.4mg/L, MS+6-BA1.0mg/L. Cut out for kale cotyledons when germinated 3 days, put them on the culture medium which suit in. When the buds are about 1 cm, transfer them to the culture medium MS+0.1 mg/LNAA to induce roots. After roots being 1cm, the seedling is cultivated on the sterilized soil.
2. The agrobacterium transformation system has been established
Through the comparative test of the factors that influence conversion, the optimum transformation conditions are cutting out for transformation materials and cultivating on MS+BA 1.0mg/L+NAA 0.1mg/L for 2 days'pre-culture. Centrifugation of bacterial liquid with OD_(600)=0.3 which get the second activation, then adding the same volume of MS(pH=5.2)and pre-culture leaves. After 5 minutes, the leaves which remove liquid cultivate in MS+BA1.0mg/L+NAA 0.1mg/L for 2 days. Then take the leaves in MS+BA 1.0mg/L+NAA 0.1 mg/L+Carb500mg/L delay cultured for 3 days. And transfer leaves into differentiation culture which is MS+BA 1.0mg/L+NAA 0.1mg/L+Carb500mg/L+0.05%oGly. Rooting medium is MS+NAA 0.1mg/L+Carb500mg/L+0.05%。 Gly
3. The identification of kale transferred with encoding transcription regulation factors gene
On the transformation systems of optimization kale's cotyledon, transferred with encoding transcription regulation factors TCF. After transferring, we got 4.8% induction rate of positive-resistant bud. We obtained 28 green buds by selection of glyphosate and 4 buds are positive by PCR.
1.优化羽衣甘蓝的再生体系,筛选出再生能力强的3个羽衣甘蓝品种
通过对不同激素配比的培养基上,9种基因型羽衣甘蓝的带柄子叶、下胚轴、真叶的再生情况进行比较分析,从中筛选出皱白1,06-56和05-25这3种高效再生基因型,其最适培养基分别为MS+6-BA1.0mg/L+NAA0.1mg/L, MS+6-BA1.4mg/L, MS+6-BA1.0mg/L,获得的最高不定芽诱导率均可达到100%；芽诱导率差的有玫红、05-20和皱白2,不足70%。说明不同基因型羽衣甘蓝的不定芽诱导率不同,对培养基激素配比的要求也不同。最终优化的羽衣甘蓝离体再生体系为：切取发芽3-4d的带柄子叶,置于与品种对应的最适培养基中诱导芽萌发,待芽伸长到1cm左右时转到生根培养基MS+0.1mg/LNAA上,根长1cm左右,移栽到营养钵中。
2.建立了农杆菌介导的遗传转化体系
确定了羽衣甘蓝最终的转化条件：取发芽3-4d的无菌苗,切取带柄子叶预培养2d,将二次活化,离心,用pH值5.2的MS液体培养基重悬至OD_600值为0.3的菌液侵染,侵染时间5mmin,灭菌滤纸吸干,共培养2d,延迟筛选3d,筛选培养后切去存活不定芽进行生根。预培养培养基：MS+BA 1.0mg/L+NAA 0.1mg/L;共培养培养基：MS+BA 1.0mg/L+NAA 0.1mg/L;延迟培养基：MS+BA 1.0mg/L+NAA 0.1 mg/L+Carb500mg/L；芽筛选培养基：MS+BA1.0mg/L+NAA 0.1 mg/L+Carb500mg/L+0.05‰Gly；生根培养基：MS+NAA0.1mg/L+Carb500mg/L+0.05‰Gly。
3.抗寒调控基因的导入及转基因植株的鉴定
在优化的羽衣甘蓝带柄子叶转化体系上,将抗寒调控因子TCF基因导入羽衣甘蓝,得到阳性抗性芽,诱导率为4.8%,共获得28株伸长的抗性芽。PCR检测显示,其中4株显示PCR阳性。
Ornamental kale(Brassica oleracea var.acephala) is an excellent landscape plant, belonging to cabbge's horticulture varieties of Cruciferae Brassiea. Low temperature is the key factor that limits the growth, colour and appreciatability in winter in the northern of China. Introducing foreign gene into kale through genetic engineering may create novel variety with cold tolerance. In this study, BADH(encoding betaine aldehyde dehydrogenase) and TCH(encoding transcription regulation factors) were used to be transformed into kale with Agrobacterium-mediated transformation. The results are as follows:
1. Get 3 high efficient regeneration genotypes and optimize the regeneration system of kale cotyledon
Trough the analysis of 9 pairs of genotypes, choose 3 high efficient regeneration genotypes and the highest induction rate is 100%. The best medium are MS+6-BA1.0mg/L+NAA0.1mg/L, MS+6-BA1.4mg/L, MS+6-BA1.0mg/L. Cut out for kale cotyledons when germinated 3 days, put them on the culture medium which suit in. When the buds are about 1 cm, transfer them to the culture medium MS+0.1 mg/LNAA to induce roots. After roots being 1cm, the seedling is cultivated on the sterilized soil.
2. The agrobacterium transformation system has been established
Through the comparative test of the factors that influence conversion, the optimum transformation conditions are cutting out for transformation materials and cultivating on MS+BA 1.0mg/L+NAA 0.1mg/L for 2 days'pre-culture. Centrifugation of bacterial liquid with OD_(600)=0.3 which get the second activation, then adding the same volume of MS(pH=5.2)and pre-culture leaves. After 5 minutes, the leaves which remove liquid cultivate in MS+BA1.0mg/L+NAA 0.1mg/L for 2 days. Then take the leaves in MS+BA 1.0mg/L+NAA 0.1 mg/L+Carb500mg/L delay cultured for 3 days. And transfer leaves into differentiation culture which is MS+BA 1.0mg/L+NAA 0.1mg/L+Carb500mg/L+0.05%oGly. Rooting medium is MS+NAA 0.1mg/L+Carb500mg/L+0.05%。 Gly
3. The identification of kale transferred with encoding transcription regulation factors gene
On the transformation systems of optimization kale's cotyledon, transferred with encoding transcription regulation factors TCF. After transferring, we got 4.8% induction rate of positive-resistant bud. We obtained 28 green buds by selection of glyphosate and 4 buds are positive by PCR.
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