水稻4香豆酸辅酶A连接酶基因缺失突变载体的构建
水稻4香豆酸辅酶A连接酶基因缺失突变载体的构建[20200408204248]
摘 要
4-香豆酸辅酶A连接酶（4CL）是木质素合成途径中的关键酶，处在总途径向分支途径的转折点，控制着苯丙烷类化合物的代谢方向。将水稻Os4CL5的氨基酸序列与拟南芥At4CL4 和大豆Gm4CL1（已证明具有芥子酸活性）的氨基酸序列比对，发现了Os4CL5底物结合区保守缬氨酸的存在，此保守氨基酸的存在可能妨碍了芥子酸与酶的结合。本实验采用重叠延伸PCR的方法对水稻的Os4CL5基因进行定点突变，删除了该基因中编码缬氨酸的密码子，然后构建该突变基因的表达载体，为后续的酶学性质研究提供物质基础。
*查看完整论文请 +Q: 3 5 1 9 1 6 0 7 2
关键字:DNA的定点突变重叠延伸PCRDNA重组技术载体构建
目 录
前言 1
1材料和方法 3
1.1试剂和仪器 3
1.1.1菌株和质粒 3
1.1.2试剂...............................................................................................................3
1.1.3酶制剂...........................................................................................................3
1.1.4仪器...............................................................................................................4
1.1.5溶液配制.......................................................................................................4
1.2实验方法..............................................................................................................5
1.2.1含载体pET22b(+)的大肠杆菌的培养........................................................5
1.2.2 pET22b(+)质粒DNA的提取......................................................................5
1.2.3 r Tag酶对水稻4CL5进行全长扩增和分段扩增.......................................5
1.2.4野生型Os4CL5的PCR扩增（高保真酶）..............................................7
1.2.5重叠延伸PCR对突变型Os4CL5的PCR扩增.................................8
1.2.6表达载体的构建..........................................................................................11
1.2.7大肠杆菌感受态细胞（DH5a）的制备....................................................13
1.2.8连接产物转化DH5α感受态（热激法）..................................................13
1.2.9 LB平板的转板............................................................................................14
1.2.10对连接产物假阳性的菌落PCR检测......................................................14
2结果..........................................................................................................................16
2.1r Tag酶对水稻4CL5进行全长扩增和分段扩增...............................................16
2.2野生型Os4CL5的PCR扩增（高保真酶）....................................................16
2.3重叠延伸PCR对突变型Os4CL5的PCR扩增...............................................17
2.4 PET载体的双酶切及电泳检测..........................................................................19
2.5野生型4CL5与载体的连接、转化及菌落PCR鉴定.............................20
2.5.1野生型4CL5与载体的连接、转化............................................................20
2.5.2野生型单菌落的转板...................................................................................21
2.5.3野生型连接产物的菌落PCR检测.............................................................21
2.6野生型4CL5与载体的连接、转化及菌落PCR鉴定. ...........................22
2.6.1突变型4CL5与载体的连接、转化...........................................................22
2.6.2突变型单菌落的转板...................................................................................22
2.6.3突变型连接产物的菌落PCR检测............................................................23
2.7野生型及突变型构建载体的测序结果..............................................................24
3讨论..........................................................................................................................25
3.1重叠延伸PCR....................................................................................................25
摘 要
4-香豆酸辅酶A连接酶（4CL）是木质素合成途径中的关键酶，处在总途径向分支途径的转折点，控制着苯丙烷类化合物的代谢方向。将水稻Os4CL5的氨基酸序列与拟南芥At4CL4 和大豆Gm4CL1（已证明具有芥子酸活性）的氨基酸序列比对，发现了Os4CL5底物结合区保守缬氨酸的存在，此保守氨基酸的存在可能妨碍了芥子酸与酶的结合。本实验采用重叠延伸PCR的方法对水稻的Os4CL5基因进行定点突变，删除了该基因中编码缬氨酸的密码子，然后构建该突变基因的表达载体，为后续的酶学性质研究提供物质基础。
*查看完整论文请 +Q: 3 5 1 9 1 6 0 7 2
关键字:DNA的定点突变重叠延伸PCRDNA重组技术载体构建
目 录
前言 1
1材料和方法 3
1.1试剂和仪器 3
1.1.1菌株和质粒 3
1.1.2试剂...............................................................................................................3
1.1.3酶制剂...........................................................................................................3
1.1.4仪器...............................................................................................................4
1.1.5溶液配制.......................................................................................................4
1.2实验方法..............................................................................................................5
1.2.1含载体pET22b(+)的大肠杆菌的培养........................................................5
1.2.2 pET22b(+)质粒DNA的提取......................................................................5
1.2.3 r Tag酶对水稻4CL5进行全长扩增和分段扩增.......................................5
1.2.4野生型Os4CL5的PCR扩增（高保真酶）..............................................7
1.2.5重叠延伸PCR对突变型Os4CL5的PCR扩增.................................8
1.2.6表达载体的构建..........................................................................................11
1.2.7大肠杆菌感受态细胞（DH5a）的制备....................................................13
1.2.8连接产物转化DH5α感受态（热激法）..................................................13
1.2.9 LB平板的转板............................................................................................14
1.2.10对连接产物假阳性的菌落PCR检测......................................................14
2结果..........................................................................................................................16
2.1r Tag酶对水稻4CL5进行全长扩增和分段扩增...............................................16
2.2野生型Os4CL5的PCR扩增（高保真酶）....................................................16
2.3重叠延伸PCR对突变型Os4CL5的PCR扩增...............................................17
2.4 PET载体的双酶切及电泳检测..........................................................................19
2.5野生型4CL5与载体的连接、转化及菌落PCR鉴定.............................20
2.5.1野生型4CL5与载体的连接、转化............................................................20
2.5.2野生型单菌落的转板...................................................................................21
2.5.3野生型连接产物的菌落PCR检测.............................................................21
2.6野生型4CL5与载体的连接、转化及菌落PCR鉴定. ...........................22
2.6.1突变型4CL5与载体的连接、转化...........................................................22
2.6.2突变型单菌落的转板...................................................................................22
2.6.3突变型连接产物的菌落PCR检测............................................................23
2.7野生型及突变型构建载体的测序结果..............................................................24
3讨论..........................................................................................................................25
3.1重叠延伸PCR....................................................................................................25
版权保护: 本文由 hbsrm.com编辑，转载请保留链接: www.hbsrm.com/swgc/swgc/978.html
