农杆菌法介导反义咖啡碱合成酶基因转入茶树成熟胚的研究
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摘要
利用基因工程进行茶树遗传改良的研究一直被受人们的关注。本研究以龙井43#、槠叶种、农抗早优良品种为材料,通过建立合适的再生体系,利用农杆菌法转化,将含有反义TCS基因的工程菌,导入龙井43#、槠叶种、农抗早优良品种茶树成熟胚,并通过GUS瞬时表达情况对农杆菌法转化参数进行了优化,用Kan对转化后的材料进行筛选,获得以下结果:
1.茶树成熟胚的培养
本研究在前人基础上首次以成熟胚作为外植体培养,建立了愈伤组织再生体系和胚萌发体系,为茶树的遗传转化奠定了基础。研究证明:在愈伤组织再生体系中,培养基和基因型是影响成熟胚愈伤组织诱导和再生频率的主要因素。龙井43#和农抗早在附加2 mg/L 2,4-D、0.5mg/L KT的MS培养基上出愈率最高,而槠叶种则在附加2 mg/L 2,4-D、1.0mg/L KT的MS培养基上诱导效果较好。基因型主要影响愈伤组织的再生频率,龙井43#分化率为14.2%,而农抗早分化率为8.5%,槠叶种分化率为3.3%,根的分化率达到40%,获得完整植株大概需要一年半的时间;在胚萌发体系中,成熟胚直接培养,快速获得再生苗,2 mg/L 6-BA的MS培养基适合成熟胚直接培养获得幼苗,添加2 mg/L IBA,根的分化率达到86%,大概需要半年时间可以培养出生长健壮的幼苗。
2.农杆菌转化体系的建立
通过GUS瞬时表达对农杆菌法转化条件进行了优化。结果表明:预培养3天,农杆菌浓度OD600为0.6,乙酰丁香酮浓度为500μmol/L,共培养5天时,GUS基因具有较好的表达效果。
3.转化体的筛选结果
对成熟胚进行Kan敏感性实验,确定了不同筛选阶段的Kan选择压浓度:成熟胚生长、生根时Kan分别为50 mg/L、75 mg/L。为了减少Carb对成熟胚生长的抑制作用,以400 mg/L Carb作为抑菌剂的浓度。用建立的转化体系和筛选方案对三个品种进行转化,得到10株抗性茶树幼苗,利用RT-PCR法对转基因植株进行了检测。
Tea (Camellia sinensis (L) O. kuntze) is primary target for the application of genetic engineering to improving agronomic characteristic. In this paper, some of the excellent regeneration system in tea plant were established . The mature embryos used as receptor to be transformed by Agrobacterium tumefaciens with antisense TCS gene and the parameter of transformation were optimized by GUS transient expression. The mature embryos were cultured on selection media with Kan. The main results had been obtained as follows:
1、Cultivation of mature embryos
By establishment of regeneration systems, mature embryos induced young plant firstly, which is the base of heredity transformation on tea plant. In the cultivation, tea plant genotype, concentration of hormone in the medium are the key factors that influence of callus and plant regeneration. The optimize concentration of 2,4-D and KT is defferent, as for as Long jing 43# and Nong kang zao, optimal medium was MS supplemented with 2 mg/L 2,4-D and 0.5mg/L KT, but as for Chu ye zhong, optimal medium was MS supplemented with 2mg/L 2,4-D and 1.0mg/L KT. Genotype mainly influences the regeneration frequency of callus formation, 14.2% buds regeneration frequency can form by Long jing 43#, 8.5% buds regeneration frequency can form by Nong kang zao, 3.3% buds regeneration frequency can form by Chu ye hang , and there is 40% root formation frequency. By induced callus track, mature embryos needed more one year cultivate integrate tea plant. The MS medium with 2 mg/L 6-BA was suitable for mature embryo to culture young plant directly. Root formation frequency is 86%. Mature embryos cultivated stuggy tea plant directly which need more half one year.
2、Establishment of Agrobacterium-mediated transformation systems
The conditions of infection were optimized by GUS transient expression. The results indicated that concentration of Agrobacterium is 0.6 , the efficient density of AS is 500μmol/L and the time of co-cultivation is 5 days.
3、Selection and Result
Selection system of high efficiency was determined by different concentration of Kan. Mature embryos growth and rooting concentration were 50 mg/L and 75 mg/L respectively. Effective concentration of Carb which restrains Agrobacterium tumefaciens is 400mg/L. Under the selection of Kan, there are 10 young tea plant, which were checked by RT-PCR technology.
1.茶树成熟胚的培养
本研究在前人基础上首次以成熟胚作为外植体培养,建立了愈伤组织再生体系和胚萌发体系,为茶树的遗传转化奠定了基础。研究证明:在愈伤组织再生体系中,培养基和基因型是影响成熟胚愈伤组织诱导和再生频率的主要因素。龙井43#和农抗早在附加2 mg/L 2,4-D、0.5mg/L KT的MS培养基上出愈率最高,而槠叶种则在附加2 mg/L 2,4-D、1.0mg/L KT的MS培养基上诱导效果较好。基因型主要影响愈伤组织的再生频率,龙井43#分化率为14.2%,而农抗早分化率为8.5%,槠叶种分化率为3.3%,根的分化率达到40%,获得完整植株大概需要一年半的时间;在胚萌发体系中,成熟胚直接培养,快速获得再生苗,2 mg/L 6-BA的MS培养基适合成熟胚直接培养获得幼苗,添加2 mg/L IBA,根的分化率达到86%,大概需要半年时间可以培养出生长健壮的幼苗。
2.农杆菌转化体系的建立
通过GUS瞬时表达对农杆菌法转化条件进行了优化。结果表明:预培养3天,农杆菌浓度OD600为0.6,乙酰丁香酮浓度为500μmol/L,共培养5天时,GUS基因具有较好的表达效果。
3.转化体的筛选结果
对成熟胚进行Kan敏感性实验,确定了不同筛选阶段的Kan选择压浓度:成熟胚生长、生根时Kan分别为50 mg/L、75 mg/L。为了减少Carb对成熟胚生长的抑制作用,以400 mg/L Carb作为抑菌剂的浓度。用建立的转化体系和筛选方案对三个品种进行转化,得到10株抗性茶树幼苗,利用RT-PCR法对转基因植株进行了检测。
Tea (Camellia sinensis (L) O. kuntze) is primary target for the application of genetic engineering to improving agronomic characteristic. In this paper, some of the excellent regeneration system in tea plant were established . The mature embryos used as receptor to be transformed by Agrobacterium tumefaciens with antisense TCS gene and the parameter of transformation were optimized by GUS transient expression. The mature embryos were cultured on selection media with Kan. The main results had been obtained as follows:
1、Cultivation of mature embryos
By establishment of regeneration systems, mature embryos induced young plant firstly, which is the base of heredity transformation on tea plant. In the cultivation, tea plant genotype, concentration of hormone in the medium are the key factors that influence of callus and plant regeneration. The optimize concentration of 2,4-D and KT is defferent, as for as Long jing 43# and Nong kang zao, optimal medium was MS supplemented with 2 mg/L 2,4-D and 0.5mg/L KT, but as for Chu ye zhong, optimal medium was MS supplemented with 2mg/L 2,4-D and 1.0mg/L KT. Genotype mainly influences the regeneration frequency of callus formation, 14.2% buds regeneration frequency can form by Long jing 43#, 8.5% buds regeneration frequency can form by Nong kang zao, 3.3% buds regeneration frequency can form by Chu ye hang , and there is 40% root formation frequency. By induced callus track, mature embryos needed more one year cultivate integrate tea plant. The MS medium with 2 mg/L 6-BA was suitable for mature embryo to culture young plant directly. Root formation frequency is 86%. Mature embryos cultivated stuggy tea plant directly which need more half one year.
2、Establishment of Agrobacterium-mediated transformation systems
The conditions of infection were optimized by GUS transient expression. The results indicated that concentration of Agrobacterium is 0.6 , the efficient density of AS is 500μmol/L and the time of co-cultivation is 5 days.
3、Selection and Result
Selection system of high efficiency was determined by different concentration of Kan. Mature embryos growth and rooting concentration were 50 mg/L and 75 mg/L respectively. Effective concentration of Carb which restrains Agrobacterium tumefaciens is 400mg/L. Under the selection of Kan, there are 10 young tea plant, which were checked by RT-PCR technology.
引文
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