甘蔗蔗糖转运蛋白基因SoSUT5的遗传转化

发布时间：2018-05-23 20:16

  本文选题：甘蔗 + 烟草　； 参考：《广西大学》2016年硕士论文

【摘要】：甘蔗是世界上最重要的糖料和能源作物。在我国,甘蔗的产糖量占全国糖总产量的92%-94%。培育高产高糖抗逆性强的甘蔗新品种是确保我国甘蔗糖业可持续发展的重要基础。在甘蔗品种遗传改良中,常规育种仍然是培育甘蔗优良新品种的主要手段,但基因工程育种正表现出明显的优势,其运用也愈加广泛。本研究以甘蔗蔗糖转运蛋白基因SoSUT5基因作为外源基因构建了植物表达载体,进行了烟草和甘蔗的遗传转化。主要研究结果如下：1.烟草的遗传转化。构建了双子叶植物表达载体pRI101-ON-SoSUT5,通过农杆菌介导转化法侵染烟草的叶盘,将载体上的T-DNA区域转入烟草品种K 346中,经过组织培养获得烟草再生植株31株。通过PCR检测,扩增npt Ⅱ基因和目的基因,分别得到转化效率为100%(npt Ⅱ+)和93.55%(SoSUT5+)。测定了野生型、转空载体pRI 101-ON型和转重组载体pRI 101-ON-SoSUT5型烟草的可溶性糖含量，，以转pRI101-ON-SoSUT5型转空载体pRI 101-ON型野生型。2. RNAi载体的构建。利用SiRNA Target Finder预测软件,分析SoSUT5基因的siRNA干扰片段,最终选择一段550 bp的序列作为干扰片段。将该片段分别正向和反向插入干扰载体pTCK 303中,构建了RNAi载体pTCK 303-SoSUT5。同时以甘蔗品种B8的愈伤组织为材料,进行了Hyg的浓度筛选实验。结果表明,甘蔗愈伤诱导阶段和分化阶段的Hyg的最佳筛选浓度为50 mg/L,生根阶段的Hyg的最佳筛选浓度为40 mg/L3.甘蔗的遗传转化。构建了单子叶植物表达载体pUBTC-SoSUT5,通过农杆菌介导转化法侵染甘蔗愈伤组织,将载体的T-DNA区域转入甘蔗品种B8中。经过组织培养,在PPT 2.5 mg/L压力下筛选48 d获得甘蔗再生植株128株。移栽后进行PCR检测标记基因bar,其中阳性植株有91株,转化效率71.09%。
[Abstract]:Sugar cane is the most important sugar and energy crop in the world. In our country, sugar yield of sugarcane accounts for 92-94 percent of total sugar production in China. Cultivating new sugarcane varieties with high yield and high sugar resistance is an important basis to ensure the sustainable development of sugarcane industry in China. In the genetic improvement of sugarcane varieties, conventional breeding is still the main means of breeding sugarcane fine new varieties, but genetic engineering breeding is showing obvious advantages, and its application is becoming more and more extensive. In this study, the sugarcane sucrose transporter gene SoSUT5 gene was used as the exogenous gene to construct the plant expression vector, and the genetic transformation of tobacco and sugarcane was carried out. The main results are as follows: 1. Genetic transformation of tobacco. A dicotyledonous plant expression vector pRI101-ON-SoSUT5 was constructed. The T-DNA region of the vector was transferred into tobacco variety K346 by Agrobacterium tumefaciens-mediated transformation, and 31 tobacco regenerated plants were obtained by tissue culture. Npt 鈪

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