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摘要：circRNA 是一种新兴的内源性非编码RNA，是目前RNA 研究的热点。circRNA 通过外显子环化或内含子环化将 3'和 5'末端连接起来形成完整的环形结构，因而比线性RNA 更稳定，更具有保守性，在基因表达调控中具有重要作用,但功能不清楚。本实验通过在拟南芥中有效的农杆菌介导转化途径，构建circRNA4235和circRNA4595的突变体和过表达植株，来研究circRNA在拟南芥中的生理作用。结果显示circRNA4235和circRNA4595的突变型和过表达基因成功转入拟南芥中，但是转化成功率很低，在表型上和对照拟南芥一样，目前只获得T0 的种子即T1 代植物，所以circRNA4235和circRNA4595在拟南芥的功能还是不清楚。通过本课题研究，我掌握了基因工程相关领域的基础理论与技术方法，例如基因克隆技术、载体构建技术、植物转基因技术等等，让我受益匪浅。29019
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毕业论文关键词：拟南芥；circRNA；农杆菌；遗传转化
Construct circRNA4235 and circRNA4595 transgenic plants
Abstract:CircRNA is a new kind of endogenous non encoding RNA,.CircRNA is currently a hot topic in RNA research by exon intron ring or ring 3'and 5' terminal connected to form a ring structure, which is more stable than the linear RNA, is more conservative, in gene expression plays an important role in regulation, but the function not clear. Through this experiment effectively in Arabidopsis thaliana by Agrobacterium mediated transformation method to construct circRNA4235 and circRNA4595 mutants and overexpression of circRNA in Arabidopsis plants, to study the students The role of science. The results showed that circRNA4235 mutant and circRNA4595 overexpression and gene transformed into Arabidopsis thaliana successfully, but the conversion success rate is very low, and the control on the phenotype of Arabidopsis thaliana, currently only get the seeds of T0 generation T1 plant, so circRNA4235 and circRNA4595 in Arabidopsis function is not clear. Through this research project I have mastered the basic theory, methods and techniques of gene engineering related fields, such as gene cloning, vector construction technology, transgenic technology and so on, let me benefit.
Key words：Arabidopsis thaliana; circRNA; Agrobacterium tumefaciens; genetic transformation
目录
摘要 1
关键词 1
Abstract 1
Key words 1
引言 1
1材料 2
1.1植物材料 2
1.2实验仪器 2
1.3载体与菌株 2
1.4主要试剂 2
1.5实验所用培养基 3
2方法 3
2.1植物材料培养 3
2.2构建含有目的基因载体 3
2.3酶切 6
2.4 T4连接 6
2.5大肠杆菌JM109感受态细胞的制备及转化 6
2.6circRNA4235:pEG202和circRNA4595:pEG202的突变体和过表达植株的构建 7
2.6.1提质粒 7
2.6.2菌落PCR验证 7
2.6.3测序 7
2.6.4LR反应 7
2.6.5大肠杆菌JM109转化、菌落PCR验证 8
2.6.6农杆菌菌株GV3101感受态的制备和转化 8
2.6.7菌落PCR验证 8
2.6.8农杆菌蘸花转化法 8
2.6.9得到T1植株 8
3结果与分析 9
3.1circRNA4235和circRNA4595转基因拟南芥的分子鉴定 9
3.2转基因拟南芥的正常生长 9
4讨论 11
致谢 11
参考文献: 12
构建 circRNA4235和circRNA4595转基因植物