根癌农杆菌介导的GR79Mt基因转化苜蓿和拟南芥的研究

发布时间：2018-05-26 20:25

  本文选题：紫花苜蓿 + 拟南芥　； 参考：《中国农业科学院》2013年硕士论文

【摘要】：草甘膦（Glyphosate）商品名称为农达，是一种广谱高效的灭生性除草剂，在农业生产中被广泛应用。紫花苜蓿（Medicago sativa L.）是一种多年生豆科饲草作物，有“牧草之王”的美誉，在我国种植广泛。农田杂草的滋生，不仅增加了成本投入，而且影响了牧草的产量和品质。本研究将具有自主知识产权的抗草甘膦基因GR79Mt转入拟南芥和苜蓿中，以期获得具有草甘膦抗性的植株。 本实验得出如下结果： 1.建立了中苜6号组织培养再生体系。以下胚轴为外植体愈伤组织诱导率要高于以子叶为外植体的诱导率。在含2mg/L2,4-D和0.2mg/L KT的SH培养基上诱导愈伤组织效率最高，而分化培养基中仅含0.2mg/L的KT即可高效地诱导出胚状体。同时，1g/L水解酪蛋白的加入，对胚状体的产生和成熟有重要作用。胚状体或小植株在不含植物激素的1/2MS培养基中诱导生根。 2.根据已知的抗草甘膦GR79Mt基因序列，在其前端加入叶绿体转运肽基因(CTP2)序列，由增强型启动子CAMV35S启动转录，豌豆rbcS E9基因的多聚腺嘌呤序列终止转录。遗传元件构建完成后人工合成序列，两端加入PvuⅠ酶切位点。植物表达载体pCAMBIA2300用PvuⅠ酶切后留下原核表达框，与合成序列连接，成功的构建了表达载体pCA-GM。 3.通过农杆菌介导法转化拟南芥花序，将收获的种子在含60mg/L草甘膦的MS培养基筛选，获得T1代植株。提取其基因组DNA进行PCR、RT-PCR验证，目的基因可在转录水平上表达，说明目的基因已整合到拟南芥基因组上。 4.通过农杆菌介导的双边界双元载体pCA-GM，，转化中苜6号愈伤组织。诱导培养基中含2mg/L2,4-D、0.2mg/L KT和60mg/L的草甘膦。诱导胚状体时，KT浓度调整为0.4mg/L，草甘膦浓度增加至80mg/L。在无植物生长调节剂的1/2MS培养基中诱导生根。最后获得12株苜蓿再生植株，经PCR验证两株呈阳性，初步表明目的基因已整合到苜蓿基因组中。
[Abstract]:Glyphosate (Glyphosate) is a broad-spectrum and high-efficient herbicide, which is widely used in agricultural production. Medicago sativa L. Is a perennial legume forage crop, with the "King of forage" reputation, widely planted in China. The breeding of weeds in farmland not only increases the cost input, but also affects the yield and quality of forage grass. In this study, glyphosate resistant gene GR79Mt with independent intellectual property rights was transferred into Arabidopsis thaliana and alfalfa in order to obtain glyphosate resistant plants. The results of this experiment are as follows: 1. The regeneration system of tissue culture of Zhongmu No.6 was established. The callus induction rate of cotyledons was higher than that of cotyledon explants. The callus induction efficiency was the highest on SH medium containing 2mg / L _ 2N _ 4-D and 0.2mg/L KT, but the embryoid was induced efficiently by KT containing only 0.2mg/L in the differentiation medium. At the same time, the addition of 1 g / L hydrolyzed casein plays an important role in the formation and maturation of embryoid. Embryoids or small plants were induced to take root in 1/2MS medium without plant hormones. 2. According to the known glyphosate-resistant GR79Mt gene sequence, the chloroplast transporter peptide gene (CTP2) sequence was added to the front of the gene, and the transcription was initiated by the enhanced promoter CAMV35S, and the polyadenine sequence of the pea rbcS E9 gene was terminated. After the construction of the genetic element, the synthetic sequence was constructed and the Pvu 鈪

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