根癌农杆菌介导的Barnase和iaaM基因转化柑橘的研究
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摘要
柑橘是世界上最重要的水果之一。近30年来,世界鲜食柑橘品种的改良主要围绕品质和成熟期等育种目标进行,而果实无核、易剥皮、风味浓和有香味又是决定品质优良的标准。运用传统的育种手段进行柑橘果实品质的改良,具有周期长、受珠心胚干扰、自交不亲和及基因间的紧密连锁等因素的影响,因而进程缓慢。植物基因工程的出现,成为实现目标性状改良的一种快速、有效的途径。通过基因工程诱导单性结实或特异表达种子切除的细胞毒素基因(Barnase)策略成为柑橘无核分子育种的主要手段。本实验利用根癌农杆菌介导的遗传转化体系,将目的基因TA29:Barnase及AGL5:iaaM转入特色多核柑橘品种,以期获得高品质的无核柑橘新种质。主要研究结果如下:
1.对卡那霉素及头孢霉素进行不同浓度梯度的敏感性试验分析,发现卡那霉素筛选浓度为50 mg/L,头孢霉素的浓度为400 mg/L时,有利于抗性植株的筛选再生,并可有效地抑制“逃逸体”的产生。
2.优化选择早实枳实生苗茎段和椪柑上胚轴切段不定芽的再生体系。结果表明早实枳实生苗茎段和椪柑上胚轴切段不定芽再生的最佳培养基分别为:MT+BA 0.5 mg/L+KT 0.5 mg/L+NAA 0.1 mg/L和MT+BA 0.5 mg/L+NAA 0.1mg/L、MT+BA 0.5 mg/L+IAA 0.2mg/L。
3.利用早实枳实生苗茎段和椪柑上胚轴切段进行遗传转化,结果表明早实枳不定芽再生率明显高于椪柑。同时利用椪柑不同的外植体材料,如:子叶、上胚轴切段、根轴连接体等进行农杆菌介导的遗传转化分析,结果表明根轴连接体不定芽再生率最高,上胚轴切段次之,而子叶再生率最低。
4.对获得的不同品种的抗性植株及抗性愈伤组织进行PCR检测。结果表明:(1)8株椪柑抗性植株及9株早实枳抗性植株含有GUS基因;(2)4株椪柑抗性植株及1株早实枳抗性植株含有iaaM基因;(3)暗柳橙抗性愈伤组织检测含有iaaM基因。
Citrus is one of the most important fruit crops in the world. Over the past 30 years, both quality and maturity are the major breeding objectives in the fresh market of the world, such as seedlessness, easy-peeling, well-flavor and aroma. The traditional breeding program has been hampered by the long juvenility, polyembryony, self-incompatibility, linkage between genes etc. The genetic engineering provided a fast and effective way to improve the traits of plants. Inducing parthenocarpy by genetic engineering and seed-ablated strategy by expressing the cytotoxin gene (Barnase) are the major methods of molecular breeding of seedless citrus. In this study, the chimeric gene TA29: Barnase and AGL5: iaaM were introduced into the seedy varieties to creat seedless citrus varieties by the Agrobacterium-mediated transformation. The main results are as follows:
1. Sensitivity analysis to Kanamycin and Cefotaxime. The results showed that the best growth quality of adventitious shoots were obtained when the explants placed in medium supplemented with 50 mg/L kanamycin and 400 mg/L Cefotaxime. Moveover, the 'escapes' were also effectively prevented under this condition.
2. Optimization of regeneration of stem segments in precocious trifoliate orange and epicotyl segments in Ponkan. The results showed that the most appropriate media for adventitious shoots of precocious trifoliate orange and Ponkan were MT medium supplemented with 0.5 mg/L BA, 0.1 mg/L NAA and 0.5 mg/L KT and MT supplemented with 0.5 mg/L BA and 0.2 mg/L IAA, respectively.
3. Analysis of the regeneration and transformation of precocious trifoliate orange and Ponkan revealed that the regeneration efficiency of Precocious trifoliate orange is much higher than Ponkan. Meanwhile, different explants of the same variety have the different regeneration efficiency, epicotyl segments and cytoledon of Ponkan were also used as explants in this study. The results showed that the regeneration efficiency of the trimmed etiolated shoot/root region was higher than the epicotyl segments, and the regeneration efficiency of cotyledon is the lowest.
4. PCR analysis of different varieties of resistant plants and callus. The results are as follows: 1) GUS gene was integrated into eight transgenic plants of Pokan and nine transgenic precocious trifoliate orange plants; 2) iaaM gene was integrated into four transgenic plants of Pokan and one transgenic precocious trifoliate orange plants; 3) iaaM gene was integrated into resistant callus of Anliucheng.
1.对卡那霉素及头孢霉素进行不同浓度梯度的敏感性试验分析,发现卡那霉素筛选浓度为50 mg/L,头孢霉素的浓度为400 mg/L时,有利于抗性植株的筛选再生,并可有效地抑制“逃逸体”的产生。
2.优化选择早实枳实生苗茎段和椪柑上胚轴切段不定芽的再生体系。结果表明早实枳实生苗茎段和椪柑上胚轴切段不定芽再生的最佳培养基分别为:MT+BA 0.5 mg/L+KT 0.5 mg/L+NAA 0.1 mg/L和MT+BA 0.5 mg/L+NAA 0.1mg/L、MT+BA 0.5 mg/L+IAA 0.2mg/L。
3.利用早实枳实生苗茎段和椪柑上胚轴切段进行遗传转化,结果表明早实枳不定芽再生率明显高于椪柑。同时利用椪柑不同的外植体材料,如:子叶、上胚轴切段、根轴连接体等进行农杆菌介导的遗传转化分析,结果表明根轴连接体不定芽再生率最高,上胚轴切段次之,而子叶再生率最低。
4.对获得的不同品种的抗性植株及抗性愈伤组织进行PCR检测。结果表明:(1)8株椪柑抗性植株及9株早实枳抗性植株含有GUS基因;(2)4株椪柑抗性植株及1株早实枳抗性植株含有iaaM基因;(3)暗柳橙抗性愈伤组织检测含有iaaM基因。
Citrus is one of the most important fruit crops in the world. Over the past 30 years, both quality and maturity are the major breeding objectives in the fresh market of the world, such as seedlessness, easy-peeling, well-flavor and aroma. The traditional breeding program has been hampered by the long juvenility, polyembryony, self-incompatibility, linkage between genes etc. The genetic engineering provided a fast and effective way to improve the traits of plants. Inducing parthenocarpy by genetic engineering and seed-ablated strategy by expressing the cytotoxin gene (Barnase) are the major methods of molecular breeding of seedless citrus. In this study, the chimeric gene TA29: Barnase and AGL5: iaaM were introduced into the seedy varieties to creat seedless citrus varieties by the Agrobacterium-mediated transformation. The main results are as follows:
1. Sensitivity analysis to Kanamycin and Cefotaxime. The results showed that the best growth quality of adventitious shoots were obtained when the explants placed in medium supplemented with 50 mg/L kanamycin and 400 mg/L Cefotaxime. Moveover, the 'escapes' were also effectively prevented under this condition.
2. Optimization of regeneration of stem segments in precocious trifoliate orange and epicotyl segments in Ponkan. The results showed that the most appropriate media for adventitious shoots of precocious trifoliate orange and Ponkan were MT medium supplemented with 0.5 mg/L BA, 0.1 mg/L NAA and 0.5 mg/L KT and MT supplemented with 0.5 mg/L BA and 0.2 mg/L IAA, respectively.
3. Analysis of the regeneration and transformation of precocious trifoliate orange and Ponkan revealed that the regeneration efficiency of Precocious trifoliate orange is much higher than Ponkan. Meanwhile, different explants of the same variety have the different regeneration efficiency, epicotyl segments and cytoledon of Ponkan were also used as explants in this study. The results showed that the regeneration efficiency of the trimmed etiolated shoot/root region was higher than the epicotyl segments, and the regeneration efficiency of cotyledon is the lowest.
4. PCR analysis of different varieties of resistant plants and callus. The results are as follows: 1) GUS gene was integrated into eight transgenic plants of Pokan and nine transgenic precocious trifoliate orange plants; 2) iaaM gene was integrated into four transgenic plants of Pokan and one transgenic precocious trifoliate orange plants; 3) iaaM gene was integrated into resistant callus of Anliucheng.
引文
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