农杆菌介导IP-10基因遗传转化马铃薯的研究
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摘要
本论文利用马铃薯微型薯为试验材料,通过不同激素组合试验,优化了东农303品种的高效再分化体系。采用农杆菌介导法将IP-10基因导入马铃薯基因组中,并通过PCR、RT-PCR、GFP蛋白荧光检测,验证了目的基因已转入马铃薯基因组中。另外,探讨了转基因植株试管薯形成的最佳条件。
1.利用马铃薯脱毒试管苗茎段、叶片和薯块为试验材料,通过IAA、NAA、GA_3、IBA、6-BA、ZT的不同组合,进行了再分化试验。结果表明,诱导茎段愈伤的最适培养基为:MS+IBA 0.1mg/L+6-BA 0.5mg/L,愈伤诱导率为86%;但其出芽率较低;诱导叶片再分化的最适培养基为:MS+ZT 10.0mg/L+NAA 0.1mg/L+GA_3 0.2mg/L,出芽率达150%;薯块再分化最适培养基为:MS+IAA 1.0mg/L+ZT 3.5mg/L,分化率为120%。
2.对农杆菌侵染并通过潮霉素筛选的转基因植株,进行了IP-10基因和潮霉素磷酸转移酶基因(HPT)的PCR扩增,分别获得了276bp和602bp所需目的条带。通过进一步的RT-PCR反应和GFP荧光蛋白的激光共聚焦检测,最终获得3个转基因株系。
3.探讨了KT、B9、CCC等激素和蔗糖浓度对转基因株系形成试管薯的影响。结果表明,当KT浓度为100mg/L,B9的浓度为150mg/L,蔗糖的浓度为8%时试管薯形成量较多,其MT/Plantlet分别为1.4,1.5和1.6,表明KT、B9和蔗糖均为转基因试管薯诱导的可选试剂。
Potato minituber was used as explant in this study.A efficient regeneration system of cultivar Dongnong303 was optimized by testing different hormone combinations.At the same time IP-10 gene was introduced into potato genome with Agrobacterium mediated method. PCR,RT-PCR and GFP fluorescence detection was done to prove that target gene had been conformed into potato genome.In addition,the best conditions of transgene potato microtuber formation was discussed.
1.Virus-free potato plantlets was used to do regeneration testing by different IAA,NAA, GA3,IBA,6-BA and ZT combinations.The results showed that the optimal medium for callus induction from stem was MS+IBA 0.1mg/L+6-BA 0.5mg/L and its inductivity was 86%,but germination rate was low;The best differentiation medium for leaves was MS+ZT 10.00mg/L+NAA 0.1 mg/L+GA_3 0.2mg/L and the shoot induction rate was 150%.On the other hand,the optimal differentiation medium for tuber disc was MS+IAA 1.0mg/L+ZT 3.5 mg/L and the differentiation rate reached 120%.
2.Transgenic plants which were obtained by Agrobacterium infection and hygromycin selection were performed to PCR amplification of IP-10 and hygromycin phosphortransferase genes,respectively.Corresponding bands of 276bp and 602bp were obtained.Three transgenic lines were identified by further RT-PCR reaction and protein GFP fluorescence laser scanning confocal detection.
3.The effect of phytohormone and sucrose content to transgenic lines of tuber formed test-tube tuber was discussed.Result showed that the most formation was obtained when KT concentration was 100mg/L,B9 concentration was 150mg/L and sucrose was 8%,and MT/Plantlet were 1.4,1.5 and 1.6,respectively.We confirmed KT,B9 and sucrose could be used as reagent for induction of transgenic test tube-tuber.
1.利用马铃薯脱毒试管苗茎段、叶片和薯块为试验材料,通过IAA、NAA、GA_3、IBA、6-BA、ZT的不同组合,进行了再分化试验。结果表明,诱导茎段愈伤的最适培养基为:MS+IBA 0.1mg/L+6-BA 0.5mg/L,愈伤诱导率为86%;但其出芽率较低;诱导叶片再分化的最适培养基为:MS+ZT 10.0mg/L+NAA 0.1mg/L+GA_3 0.2mg/L,出芽率达150%;薯块再分化最适培养基为:MS+IAA 1.0mg/L+ZT 3.5mg/L,分化率为120%。
2.对农杆菌侵染并通过潮霉素筛选的转基因植株,进行了IP-10基因和潮霉素磷酸转移酶基因(HPT)的PCR扩增,分别获得了276bp和602bp所需目的条带。通过进一步的RT-PCR反应和GFP荧光蛋白的激光共聚焦检测,最终获得3个转基因株系。
3.探讨了KT、B9、CCC等激素和蔗糖浓度对转基因株系形成试管薯的影响。结果表明,当KT浓度为100mg/L,B9的浓度为150mg/L,蔗糖的浓度为8%时试管薯形成量较多,其MT/Plantlet分别为1.4,1.5和1.6,表明KT、B9和蔗糖均为转基因试管薯诱导的可选试剂。
Potato minituber was used as explant in this study.A efficient regeneration system of cultivar Dongnong303 was optimized by testing different hormone combinations.At the same time IP-10 gene was introduced into potato genome with Agrobacterium mediated method. PCR,RT-PCR and GFP fluorescence detection was done to prove that target gene had been conformed into potato genome.In addition,the best conditions of transgene potato microtuber formation was discussed.
1.Virus-free potato plantlets was used to do regeneration testing by different IAA,NAA, GA3,IBA,6-BA and ZT combinations.The results showed that the optimal medium for callus induction from stem was MS+IBA 0.1mg/L+6-BA 0.5mg/L and its inductivity was 86%,but germination rate was low;The best differentiation medium for leaves was MS+ZT 10.00mg/L+NAA 0.1 mg/L+GA_3 0.2mg/L and the shoot induction rate was 150%.On the other hand,the optimal differentiation medium for tuber disc was MS+IAA 1.0mg/L+ZT 3.5 mg/L and the differentiation rate reached 120%.
2.Transgenic plants which were obtained by Agrobacterium infection and hygromycin selection were performed to PCR amplification of IP-10 and hygromycin phosphortransferase genes,respectively.Corresponding bands of 276bp and 602bp were obtained.Three transgenic lines were identified by further RT-PCR reaction and protein GFP fluorescence laser scanning confocal detection.
3.The effect of phytohormone and sucrose content to transgenic lines of tuber formed test-tube tuber was discussed.Result showed that the most formation was obtained when KT concentration was 100mg/L,B9 concentration was 150mg/L and sucrose was 8%,and MT/Plantlet were 1.4,1.5 and 1.6,respectively.We confirmed KT,B9 and sucrose could be used as reagent for induction of transgenic test tube-tuber.
引文
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[12]张明洲,应华冠.转基因植物牛物反应器的研究进展.中国计量学院学报,2005,16(3):242-246
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