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假微型海链藻DGAT基因的重组质粒构建
所在学院 专业班级 生物工程 学生姓名 学号 指导教师 职称 完成日期 年 月
目 录
摘 要
引 言............................................................................................................................................................... 1 1 绪论 ........................................................................................................................................................... 1
1.1 生物柴油的研究现状 ....................................................................................................................... 1 1.2 藻类的特点 ....................................................................................................................................... 2 1.3 二脂酰甘油酰基转移酶DGAT的研究现状 ..................................................................................... 2
1.3.1 二脂酰甘油酰基转移酶DGAT的特点 ................................................................................. 2 1.3.2 DGAT基因对肌内脂肪含量的影响 ...................................................................................... 2 1.3.3 拟南芥中DGAT活性变化 ..................................................................................................... 3
2 实验材料 ................................................................................................................................................... 3
2.1 DGAT片段 ....................................................................................................................................... 3 2.2 菌株 ................................................................................................................................................... 3 2.3 质粒 ................................................................................................................................................... 3
2.3.1 表达质粒 ............................................................................................................................... 3 2.3.2 对照质粒 ............................................................................................................................... 4 2.4 引物 ................................................................................................................................................... 4 2.5 药品和常用药剂 ............................................................................................................................... 4
2.5.1 酶和生化试剂 ....................................................................................................................... 4 2.5.2 抗生素 ................................................................................................................................... 4 2.5.3 其他试剂 ............................................................................................................................... 4 2.5.4 培养基的配制 ....................................................................................................................... 5
3 实验方法 ................................................................................................................................................... 5
3.1 质粒抽提（试剂盒） ....................................................................................................................... 5 3.2 电泳检测 ........................................................................................................................................... 6 3.3 质粒双酶切 ....................................................................................................................................... 6 3.4 DNA片段回收（试剂盒） ............................................................................................................. 7 3.5 目的基因的获取 ............................................................................................................................... 7
3.5.1 PCR扩增目的基因 ................................................................................................................ 7 3.5.2 割胶回收目的基因（试剂盒） ........................................................................................... 8 3.6 大肠杆菌DH5α感受态的制备 ...................................................................................................... 8 3.7 连接与转化 ....................................................................................................................................... 9
3.7.1 连接（采用In-Fusion PCR Cloning Kit试剂盒） ....................................................... 9 3.7.2 转化 ....................................................................................................................................... 9 3.7.3 PCR检测重组质粒 ................................................................................................................ 9
4 结果 ........................................................................................................................................................... 9
4.1 目的基因PCR扩增 ....................................................................................................................... 10 4.2 电泳检测割胶回收图 ..................................................................................................................... 10 4.3 PCR检测重组质粒 ........................................................................................................................ 10 4.4 测序结果 ......................................................................................................................................... 11 5 小结 ......................................................................................................................................................... 11 参考文献 ......................................................................................................................................................... 13 附录................................................................................................................................................................. 15
摘要：在高产油微藻假微型海链藻中，二酰甘油酰基转移酶（DGAT）是三酰甘油合成途径的关键酶。本研究试验采用In-Fusion PCR Cloning Kit试剂盒进行重组质粒的构建，从而解决pCAMBIA1300表达质粒的多克隆位点在DGAT基因序列的酶切位点上都存在的问题。用XbaⅠ和KpnⅠ限制性内切酶对pCAMBIA1300质粒进行双酶切得到线性质粒。根据此试剂盒的引物设计原理，利用线性表达质粒的序列和已知DGAT基因序列设计引物，以用于进行DGAT基因的PCR扩增，得到目的片段。使用此试剂盒特有的In-Fusion连接酶连接目的片段和表达质粒，该酶不受酶切位点的限制可高效连接目的片段和表达质粒，从而构建含假微型海链藻DGAT基因的重组质粒。该质粒适用于研究拟南芥三酰甘油含量的变化及该基因在拟南芥中的表达定位。
关键词：假微型海链藻；尼罗红；乙酰辅酶A羧化酶；二酰甘油酰基转移酶
Abstract: Thalassiosira pseudonana is the oleaginous alga,and its diacylglycerol acyltransferase (DGAT) is the key enzyme in synthesis pathway of triglyceride.The experiment constructs recombinantplasmid using In-Fusion PCR Cloning Kit,in order to solve the problem of the multiple cloning sites of pCAMBIA1300 are the same to the restriction sites of DGAT. The linear plasmid obtained by double-enzyme cleavage of pCAMBIA1300 plasmid with XbaⅠand KpnⅠ.Based on the principle of primer designing in the kit,the primer is designed with the sequence of linear plasmid and the sequence of DGAT gene to obtain the fragment .Target DNA fragment and expression plasmid is connected by unique In-Fusion ligase of the kit.The enzyme can connect the DNA fragment and expression plasmid efficiently without the restrictions of restriction site, so that we can construct recombinantplasmid which contains the DGAT gene of Thalassiosira pseudonana.The plasmid can be used for observing the content change of triacylglycerol in Arabidopsis thaliana and the expression of the gene in Arabidopsis thaliana.
Keywords: Thalassiosira pseudonana; Nile red; Acetyl CoA carboxylase; Diacylgycerol Acyltransgerase
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假微型海链藻DGAT基因的重组质粒构建【毕业作品】
