农杆菌介导的Bt、CBF1基因对马铃薯的遗传转化
摘要
本研究以马铃薯品种东农303的脱毒微型薯为外植体，利用农杆菌介导法分别将Bt-Cry V基因和CBF1基因转入马铃薯中，经PCR和PCR-Southern检测证明Bt-CryV基因和CBF1基因已转入马铃薯中。同时对影响遗传转化因素激素种类与浓度、蔗糖和琼脂浓度、脱菌抗生素种类与浓度、共培养时间、筛选方式等进行了研究，优化了马铃薯再生体系和遗传转化体系。主要研究结果如下：
1薯块再生培养基确定为MS+ZT 3.5mg／L+IAA 1.0mg／L，出芽率为89.85％。以经过三个月休眠期的薯块为最佳外植体生理状态，基本培养基中蔗糖和琼脂浓度分别为30g／L，0.7％。
2利用农杆菌介导法进行遗传转化时的最适条件受所转入的目的基因影响不大，两种基因最适的农杆菌侵染时间均为5min、菌液浓度均为OD_(600)=0.5，此条件下的出芽率分别为61.90％和68.33％，污染率较低。
3共培养时间受目的基因活性影响较大，东农303薯块与CryV基因与薯块的最佳共培养时间为60h，CBF1基因与薯块的最佳共培养时间为36h。
4对不同产地脱菌抗生素的浓度进行了比较试验，国产头孢噻肟钠(哈药)对CryV基因和CBF1基因的有效抑菌浓度分别为400mg／L、500mg／L。选用进口头孢噻肟钠(invitrogen)抑菌效果较好，且在有效的抑菌浓度下对外值体无不良影响，它对CryV基因和CBF1基因的有效抑菌浓度均为150mg／L。
5试验采用的筛选方式为延迟7d筛选，抗性选择剂为卡那霉素(Kan)，结果表明薯块芽诱导阶段Kan浓度为100mg／L，生根阶段的Kan浓度为100mg／L。
6目的基因CryV试验共获得68株kan抗性植株，其中21株通过了生根筛选(Kan100mg／L)，PCR检测结果表明有10株呈阳性，进一步的PCR-Southern杂交检测结果呈阳性。目的基因CBF1试验总共获得96株kan抗性植株，其中31株通过了生根筛选(Kan100mg／L)，PCR检测结果表明有15株呈阳性，进一步的PCR-Southern杂交检测结果呈阳性。证明了目的基因已整合到了马铃薯基因组中。
A regeneration system available for potato transformation was established considering byseverial factors, such as the kind and concentration of different phytohormones and bactericides,the concentration of sucrose and agar, the time of co-culture, the effect of selection. In this study,Bt-CryV and CBF1 genes were transferred into Dongnong303, a main local cultivars, withAgrobacterium-mediated method. The explants were microtuber discs. The transgenic plantswere tested by PCR and PCR-Southern, and they were confirmed to be integrated in the genomeof potato. The main results were showed as following:
1 The oplimal regeneration medium for tuber disc was MS+3.5 mg/L ZT+1.0 mg/L IAA.The highest rate of inducing shoot is 89.85%. The best physiological estate of tuber explant fortransformation is three month ofresting stage. The concentration of sucrose and agar in basic culture medium is 30 g/L and0.7%.
2 The optimal conditions of Agrobacterium-mediated method had no difference in theintention of gene. Both of CryV gene and CBF1 gene, the optimal concentration ofAgrobacterium was OD_(600) 0.5, the optimal time of infection was 5 minutes. In this condition,the rate of tuber disc with CryV gene was 61.90%, the rate of tuber disc with CBF1 gene was68.33%, both of them, the rate of pollution were low.
3 Co-culture time had great influence on activity of gene. The optimal co-culture time ofDongnong303 with CryV gene was 60 hours and that of CBF1 gene was 36 hours.
4 In the comparative experiment of several bactericides, the effective concentration withCefotaxime sodium made in China: for CryV gene was 400mg/L, for CBF1 gene was 500mg/L.The best one was Cefotaxime sodium made in USA, and its effective concentration was150mg/L for both CryV and CBF1 genes, which didn't have negative effect to the growth ofexplants.
5 The manner of selection was confirmed seven days later. Kanamycin was used as theselective agent. The concentration was 100mg/L for the tuber discs at the stage of shoot and rootformation.
6 Transformants were attained for CryV gene in this experiment, in which 21 plants wetepassed rootage (Kan100mg/L), and in which 10 transformants were confirmed by the test ofPCR and PCR-Southern blotting to be transgenic plants. 96 transformants were attained forCBF1 gene in this experiment, in which 31 plants wete passed rootage (Kan100mg/L), and inwhich 15 transformants were confirmed by the test of PCR and PCR-Southern blotting to betransgenic plants. Both CryV gene and CBF1 genes were integrated into potato genomes.
1薯块再生培养基确定为MS+ZT 3.5mg／L+IAA 1.0mg／L，出芽率为89.85％。以经过三个月休眠期的薯块为最佳外植体生理状态，基本培养基中蔗糖和琼脂浓度分别为30g／L，0.7％。
2利用农杆菌介导法进行遗传转化时的最适条件受所转入的目的基因影响不大，两种基因最适的农杆菌侵染时间均为5min、菌液浓度均为OD_(600)=0.5，此条件下的出芽率分别为61.90％和68.33％，污染率较低。
3共培养时间受目的基因活性影响较大，东农303薯块与CryV基因与薯块的最佳共培养时间为60h，CBF1基因与薯块的最佳共培养时间为36h。
4对不同产地脱菌抗生素的浓度进行了比较试验，国产头孢噻肟钠(哈药)对CryV基因和CBF1基因的有效抑菌浓度分别为400mg／L、500mg／L。选用进口头孢噻肟钠(invitrogen)抑菌效果较好，且在有效的抑菌浓度下对外值体无不良影响，它对CryV基因和CBF1基因的有效抑菌浓度均为150mg／L。
5试验采用的筛选方式为延迟7d筛选，抗性选择剂为卡那霉素(Kan)，结果表明薯块芽诱导阶段Kan浓度为100mg／L，生根阶段的Kan浓度为100mg／L。
6目的基因CryV试验共获得68株kan抗性植株，其中21株通过了生根筛选(Kan100mg／L)，PCR检测结果表明有10株呈阳性，进一步的PCR-Southern杂交检测结果呈阳性。目的基因CBF1试验总共获得96株kan抗性植株，其中31株通过了生根筛选(Kan100mg／L)，PCR检测结果表明有15株呈阳性，进一步的PCR-Southern杂交检测结果呈阳性。证明了目的基因已整合到了马铃薯基因组中。
A regeneration system available for potato transformation was established considering byseverial factors, such as the kind and concentration of different phytohormones and bactericides,the concentration of sucrose and agar, the time of co-culture, the effect of selection. In this study,Bt-CryV and CBF1 genes were transferred into Dongnong303, a main local cultivars, withAgrobacterium-mediated method. The explants were microtuber discs. The transgenic plantswere tested by PCR and PCR-Southern, and they were confirmed to be integrated in the genomeof potato. The main results were showed as following:
1 The oplimal regeneration medium for tuber disc was MS+3.5 mg/L ZT+1.0 mg/L IAA.The highest rate of inducing shoot is 89.85%. The best physiological estate of tuber explant fortransformation is three month ofresting stage. The concentration of sucrose and agar in basic culture medium is 30 g/L and0.7%.
2 The optimal conditions of Agrobacterium-mediated method had no difference in theintention of gene. Both of CryV gene and CBF1 gene, the optimal concentration ofAgrobacterium was OD_(600) 0.5, the optimal time of infection was 5 minutes. In this condition,the rate of tuber disc with CryV gene was 61.90%, the rate of tuber disc with CBF1 gene was68.33%, both of them, the rate of pollution were low.
3 Co-culture time had great influence on activity of gene. The optimal co-culture time ofDongnong303 with CryV gene was 60 hours and that of CBF1 gene was 36 hours.
4 In the comparative experiment of several bactericides, the effective concentration withCefotaxime sodium made in China: for CryV gene was 400mg/L, for CBF1 gene was 500mg/L.The best one was Cefotaxime sodium made in USA, and its effective concentration was150mg/L for both CryV and CBF1 genes, which didn't have negative effect to the growth ofexplants.
5 The manner of selection was confirmed seven days later. Kanamycin was used as theselective agent. The concentration was 100mg/L for the tuber discs at the stage of shoot and rootformation.
6 Transformants were attained for CryV gene in this experiment, in which 21 plants wetepassed rootage (Kan100mg/L), and in which 10 transformants were confirmed by the test ofPCR and PCR-Southern blotting to be transgenic plants. 96 transformants were attained forCBF1 gene in this experiment, in which 31 plants wete passed rootage (Kan100mg/L), and inwhich 15 transformants were confirmed by the test of PCR and PCR-Southern blotting to betransgenic plants. Both CryV gene and CBF1 genes were integrated into potato genomes.
引文
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