目录
摘要Ⅰ
关键词Ⅱ
引言
引言1
1 材料与方法2
1.1 供试材料2
1.2 主要试剂2
1.3 培养基2
1.4 预实验3
1.4.1 大豆萌发时间3
1.4.2 大豆萌发培养环境3
1.4.3 伤口处理高度3
1.5 农杆菌转化4
1.6 菌液和侵染液制备4
1.7 大豆种子的消毒和外植体的获得4
1.7.1 大豆种子的消毒4
1.7.2 大豆种子的萌发5
1.7.3 大豆外植体的获得5
1.8 大豆毛状根的诱导6
1.9 大豆毛状根的检测6
2 结果与分析7
2.1 大豆毛状根的形态7
2.2 不同萌发时间的大豆转化率7
2.3 不同浓度的筛选剂条件下毛状根发生效率8
2.4 客观分析8
3 讨论与结论 8
致谢9
参考文献9
大豆下胚轴毛状根发生体系的建立
摘 要
本实验利用热激法将pFGC5941GUS质粒转入发根农杆菌(Agrobacterium rhizogenes)K599感受态中，该质粒含有35S启动子驱动的GUS基因表达框。分别将萌发3d、5d和7d的大豆（Williams 82）下胚轴自身生长出的根部除去，并进行伤口处理。通过浸泡法让含有菌株的侵染液侵染大豆下胚轴上的伤口，发根农杆菌中的Ri质粒上的TDNA片段进入植物细胞，在植物细胞中随机整合，利用植物体内的酶系统进行转录和表达，诱导大豆下胚轴产生毛状根。改变诱导毛状根发生的培养基筛选剂浓度，从而探索不同萌发时间和不同浓度筛选剂下大豆毛状根的转化效率。通过GUS染液染色法对诱导产生的毛状根进行染色，观察染色后毛状根的阳性根数量计算转化率。其中萌发时间3d、5d、7d在浓度为3mg/ml的筛选剂条件下的转化率分别为41.15％、87.50％和51.16％。根据转化率确定适合大豆下胚轴毛状根发生的条件，建立高效的毛状根发生体系。
 Es *51今日免费论文网|www.51jrft.com +Q: ^351916072* 
tablishment of Hairy Root Generation System from Soybean Hypocotyl
Abstract
The pFGC5941GUSK599 strain was synthesized by transferring pFGC5941GUS plasmid into Agrobacterium rhizogenes K599. The plasmid contained GUS gene expression frame driven by 35S promoter. The roots of 3d, 5d and 7d germinated soybean (Williams 82) hypocotyls were removed and the wounds were treated. TDNA fragments from Ri Plasmid of agrobacterium Rhizogenes were inserted into the plant cells, and were integrated randomly in the plant cells, soybean hypocotyls were induced to produce hairy roots by transcription and expression of enzymes in plants. To explore the transformation efficiency of soybean hairy roots under different germination time and concentration, the concentration of culture medium for hairy root induction was changed. The hairy roots were stained by GUS staining method, and the number of positive roots was observed to calculate the conversion rate. Among them, the conversion rates were 41.15% , 87.50% and 51.16% at the concentration of 3 mg / ml sieving agent on 3d, 5d and 7d respectively.The suitable conditions for hypocotyl hairy root formation in soybean were determined according to the transformation rate, and a high efficient hairy root formation system was established.
KEY WORDS： Agrobacterium rhizogenesK599；soybean hypocotyl；hairy root；Agrobacterium transformation; GUS staining
引言：大豆是一种双子叶作物，在全球众多转基因植物中，大豆虽然种植面积最广但是转化效率却最低。研究表明，大豆中富含的大量蛋白质和油脂是人体所需的必要营养物质，但是目前我国的大豆很大程度上依赖于进口[1]。为摆脱这种现状，发展我国的大豆转基因技术，通过建立利用农杆菌介导大豆下胚轴毛状根的发生体系，可以为研究基因功能提供技术支持，同时为以后的大豆转基因技术提供基础。

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