CN106520825A - 一种水稻核苷酸片段人工加接头的方法 - Google Patents

一种水稻核苷酸片段人工加接头的方法 Download PDF

Info

Publication number
CN106520825A
CN106520825A CN201611008369.2A CN201611008369A CN106520825A CN 106520825 A CN106520825 A CN 106520825A CN 201611008369 A CN201611008369 A CN 201611008369A CN 106520825 A CN106520825 A CN 106520825A
Authority
CN
China
Prior art keywords
primer
joints
paddy
represented
template
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201611008369.2A
Other languages
English (en)
Inventor
朱子超
李贤勇
王楚桃
何永歆
蒋刚
欧阳杰
黄乾龙
管玉圣
郭爽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chongqing Academy of Agricultural Sciences
Original Assignee
Chongqing Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chongqing Academy of Agricultural Sciences filed Critical Chongqing Academy of Agricultural Sciences
Priority to CN201611008369.2A priority Critical patent/CN106520825A/zh
Publication of CN106520825A publication Critical patent/CN106520825A/zh
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Saccharide Compounds (AREA)

Abstract

本发明公开了一种水稻核苷酸片段人工加接头的方法，步骤为：1)以含目的片段的水稻基因组或者cDNA为模板，用扩增目的片段的前后引物(S1F和S1R表示)、高保真酶进行PCR扩增；经1％琼脂糖胶电泳检测胶回收与预期大小一致的条带；2)将S1F和S1R分别与包含有重组位点同源序列、线性化质粒酶切位点序列的接头连接，进行人工合成；3)以P2为模板，S2F和S2R为引物，用高保真酶进行PCR扩增；经1％琼脂糖胶电泳检测、胶回收与预期大小一致的条带，胶回收产物用P3表示；4)P3就是在目标片段两侧加上接头的目的产物，用它与线性化的质粒连接，进行后面的基因工程操作。本发明可以显著提高给水稻核苷酸片段人工加接头的成功率，加快载体构建进程。

Description

一种水稻核苷酸片段人工加接头的方法
技术领域
本发明属于水稻基因工程领域，可直接应用于水稻转基因载体构建。
背景技术
基因工程中，将目的片段连接到工程载体上是最基础的步骤。目前，在水稻基因工程中，将目的片段连接到载体上的比较常用的方法是同源重组法。该方法需要在目的片段的两端加上重组位点的同源序列和线性化质粒酶切位点序列，长度为16bp左右。这段16bp的序列就是基因工程操作中的接头。通常人工加接头的方法是将扩增目的片段的前后引物的5’端连上这16bp序列一起合成，然后进行PCR扩增。但是用这种传统加接头的方法对于大片段(5000bp以上)成功率较低。
发明内容
针对上述现有技术中存在的不足之处，本发明提供了一种可以显著提高人工加接头成功率的水稻核苷酸片段人工加接头的方法。该方法可以有效解决传统方法加接头成功率低的问题，有力推进载体构建进程。
为了解决上述技术问题，本发明采用了如下技术方案：
一种水稻核苷酸片段人工加接头的方法，该方法包括如下步骤：
1)以含目的片段的水稻基因组或者cDNA为模板，该片段用P1表示，模板用D1表示，用扩增目的片段的前后引物、高保真酶进行PCR扩增，该前后引物分别用S1F和S1R表示；经1％琼脂糖胶电泳检测胶回收与预期大小一致的条带，胶回收产物用P2表示；
2)将S1F和S1R分别与包含有重组位点同源序列、线性化质粒酶切位点序列的接头连接，进行人工合成，前后引物的接头分别用J1和J2表示；合成的带接头的引物用S2F和S2R表示；
3)以P2为模板，S2F和S2R为引物，用高保真酶进行PCR扩增；经1％琼脂糖胶电泳检测、胶回收与预期大小一致的条带，胶回收产物用P3表示；
4)P3就是在目标片段两侧加上接头的目的产物，用它与线性化的质粒连接，进行后面的基因工程操作。
本发明的有益效果是：可以显著提高给水稻核苷酸片段人工加接头的成功率，加快载体构建进程。
附图说明
图1为一种水稻核苷酸片段人工加接头的方法的流程图；
图2为实例扩增的P1产物(B泳道，7421bp)和带有接头的P3产物(A泳道，7453bp)的示意图。
具体实施方式
一种水稻核苷酸片段人工加接头的方法，其流程如图1所示，该方法包括如下步骤：
1)以含目的片段的水稻基因组或者cDNA为模板，该片段用P1表示，模板用D1表示，用扩增目的片段的前后引物、高保真酶进行PCR扩增，该前后引物分别用S1F和S1R表示；经1％琼脂糖胶电泳检测胶回收与预期大小一致的条带，胶回收产物用P2表示；
2)将S1F和S1R分别与包含有重组位点同源序列、线性化质粒酶切位点序列的接头连接，进行人工合成，前后引物的接头分别用J1和J2表示；合成的带接头的引物用S2F和S2R表示；
3)以P2为模板，S2F和S2R为引物，用高保真酶进行PCR扩增；经1％琼脂糖胶电泳检测、胶回收与预期大小一致的条带，胶回收产物用P3表示；
4)P3就是在目标片段两侧加上接头的目的产物，用它与线性化的质粒连接，进行后面的基因工程操作。
实施例
水稻日本晴LOC_OS10g31910基因含启动子及3’端全长7500bp左右，传统方法很难将如此大的片段一次性加上接头。采用了本发明所述方法：
1、以引物S1F：TCTACACACGTAAACTTTCAACT和S1R：TGGCACATTGTAGCTGTAGAC扩增日本晴，目标克隆水稻LOC_OS10g31910基因上游启动子区(1545bp，[001]段序列)、LOC_OS10g31910序列(5004bp，[002]段序列)和下游872bp([003]段序列)，目标片段全长7421bp。经第1轮PCR(Tm＝58℃)，顺利扩增并胶回收P2(图2中的B泳道)。
2、以引物对S2F：TCTACACACGTAAACTTTCAACT(下划线部分为EcoRI酶切位点，斜粗体序列为接头J1)和S2R：TGGCACATTGTAGCTGTAGAC(下划线部分为BamHI酶切位点，斜粗体序列为接头J2)，P2为模板，经第2轮PCR(Tm＝58℃)顺利扩增出含接头的P3(图2中的A泳道，7453bp)。
3、将P3与线性化的空载质粒2300连接，进行互补载体构建。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
TCTACACACGTAAACTTTCAACTTTTCCATCACATCGTTCCAATTTTAACCAAACTTCCAATTTTAGCGTAAACTAAACACACCCACACACAAACTTCTAACTTTTCCATCACGTCATTCCAATTTCAATCAAATTTTCAATTTTACACCCTTGCATATTGCATGCACACGAGCAGTCGACCACACAACAAGGACCAACCAAAGCAATGCATGTCTGAACTCTGAACAAAGCAACACCGGCAGTAGCAGGCTAGCAGCAGAAGTCGTCTTCGGGGTTCGTGGTAACTGAAAAAACAAACACTCCTGTACTCCACTTCGCACGGTTGCCTAGAAGGGAAGGGTGGGAAAAGGGTTCCTGGTCGTGCGCGCCTGTCTTCGCGGTTCGCGATGGAATTCGTTTTTTAGTTTGGGACAAGAAAAGTTTTATAGTTTAGTAATGGGGGAATTTCTTTTTCTTCTGTTTCCCGGTGGTGCCAAATGCTACGTATGCTTTTCTTCGCGCTAGATATCTGAAACGGGGACGGTCGGAAATTCAACCACGCAAGAAATGGACTTGCGGTGCACCTTTGGAAATACAGAGGGAAGAAGATGTTGTTTTGCGTTTATTACTTGGAAAAACAAGGAAGAAAAGTCAACATTACTTTTTATGTAAATTGAGTCGTGGATCTACCGGTATTTTAAGCACTTCCATACAATAGGCTAAATTCTCCCTTAAAGGCTCTTTGATTCAAAGGATTAAAGCTTTACAAATCCTATGAAATTTCTATGGAATGACACATTGTATGTGGATTTTAGAAGAAATTTAGCAAGAGCTCAAACCTCTAGGAAAATTTCCTTGGAGTCCACCTCTCTCATCCGATTCCTGCGCTTTTCCTGCGCTCCAATCAAACGACCATTCCTATGTTTTTCTTGTGTTTTACAATCCTCTGTTTTACACTTCAATTCCTGTCAGAATCATGTGTTAGAGGTCTTCCGGCTGGCTTCACAAGGTGATGGACTAGATGATTTGGATTCGAAGCCTCACTCATTCTAATTATTTGATATTATGTCCTTCCCTGATATTCGCCATCTCAATTCACCTTCCAGGCACCAATAAGAGAAGATTTGCGAGCGCTTCTATGCCGACTTGAGATCAGCATTAACACACGAACACAACAAATCATCATCATGTGCTCAATTTGACAAACTCCATTGATGCCAATCTTCCCCTCCATGAAACATTCTCCATTCTCCCTCCTCCAAAGGCTCAAACAAACCCCCTCTTTTAACAAGCAAGGAAGCCATCTCCCCCAGGTGATTCCATTTCACCTCCTCTCTCTCTCTCTCTCCTCCCCCAGCGAGTTCTCCCCTTCCTCCCCACCATTGCTGTCCAATCCCAAGCTCCAAATTAAATCTCAAGAACTTCACCACCCAGCTGCTGATTCTCTCTCCAAATCCACCATTTTCCACTCCCTCACTGTGCTCCACACTCTCCTGCATTGCTATAAAAATCCACTGCATCTCCAGTTCTCCACTCCCAAATCCACCCAAGAACCAGCCGCCGCC
ATGGCCGGGCCCGTCGAATCCACCCGCATTACCCTCCTCCTCGTCGCCGCCACCCTCCTCCTCCTCCCGCCGCCGCTCGCCGCCTCCCTCAACTCCTCCCTCCCCGACCCCGCCGCCGTCGTCGCCGACTTCCACAGGTAAGCGTGCACCCCCCGCAATGCCGGTGCGATTTTTTCGGAATCCTTTTTCTTGAGGTTTCTTGGCTAATGGCAGCAAGGTGGCGACGTCGCGGCGGCGGATGCAGGAGGCCGGCGGCGGCGGCGGCGGTGGCGGCGGGGGGTGTTTGACGGGGAACCCCATCGACGACTGCTGGCGGTGCGCCGGGACGGACTGGAGGCAGGACCGGCAGCGGCTGGCGGACTGCGGCATCGGGTTCGGGCGCAACGCCATGGGCGGCAAGGGCGGGCCGGTGTACGTCGTCACCGACCCCTCCGACGGCGACCCGGTGAACCCCGCGCCGGGGACGCTCCGGTACGGCGCCATCCAGGAGGGCCCCCTCTGGATCGTGTTCGCCGGCGACATGACCATCCGCCTCAACGAGGAGCTCCTCGTGAACAGCTACAAGACCATCGACGGCCGCGGCGCCAACGTCCACGTCGGCGCCGGCGGCGCGTGCATCACGCTCCAGTACGTCTCCAACGTCATCATCCACAACATCCACGTCCACGACTGCGTCCCCGCCGGCAACGCCAACGTGCGCGCCTCGCCGACGCACTACGGGTGGCGCACCCGCTCCGACGGCGACGGCATCTCGCTCTACTCGGCGCGCGACGTGTGGGTGGACCACTGCGCGCTGTCGCGCTGCGCCGACGGCCTCATCGACGCCATCATGGGGTCCACGGCGATCACCGTGTCGAACAGCTACTTCTCGCACCACAACGAGGTGATGCTGCTGGGGCACAGCGACGGGTACCTGCCGGACTCGGCGATGCAGGTGACCATCGCGTTCAACCACTTCGGGATCCAGCTCGTGCAGCGGATGCCTCGGTGCAGGAGGGGGTACTTCCACATCGTCAACAACGACTACACGGCGTGGGAGATGTACGCCATTGGAGGGAGCGCGAGCCCCACCATCAACAGCCAGGGCAACCGCTACATCGCCCCCGCCGATCCCAACGCCAAGGAGGTAAGCAAGCGACGCCATTGTTGTTTGCCGTGTTCTTGGCTTCTTGCTCTCTTCGTTGTTGCTTTGCTTGTGAGTCGTGAGATGTGATAGCAGAAGGAAAAAAGAAAAATCTTTTTGTGTGTGTGTGTGTGTTGGATTGGTTTTGGTCTGTTTGTTTCTCATTTCGGCTCGAATTGCTGATGCTGTCGGATGTTTGATGCCCATTTTTGGATGATTTTATTTTATTGGGTGGTTATTTCTTGTTCTGTTCATTGGTTTGCTTATGAAAGGAAAAAAAATGTCTTTTATTTAGTTGGTTTTCCTCCGTTTGCTTCTCATTTTGGCTCAAATTGCTGAGGTTGTCACTTGTCAGCTGTTTGATGCCCCAATTTGGGATGAATTCCTGGTTTAAGTGGGGTGTGAAATTTAGAGCAATTTGACCATATTTAGAAAAATTCCCGGTTAAAGTGGGGTGTGAAATTTAGAGCAATCTGACCATATTTAGAAAAATTTCTCAAGTCGTTTGACCTGTTTTTAAGGTGTGAGTAGCGCTCTATGGTCATGTGAATTGCCACGCTTTTAGGGCGAATGTTTGGAGCATCATCATAATAGCGCCTCTTCTTTAAAGAGCAAAAATTTGGGTGAATGTTTTTCCCCCTTGCCCTCGTGATAGCAGCATCCGAGGCAGGCGCGTGCACCGCACGATGCGATGCGATGAGATGAGATGCACCCTCCCCCAAGATTCGGTGAGGCTGACATGTGGGCCCCACACTGGGACAAAAAACGTGCAGGTGACGAAGCGGGTGGACACGGAGGAGGGGCAGTGGGCGGGGTGGAACTGGAGGACGGAGGGGGACATGATGGTCAACGGCGCCTTCTTCGTCCCCTCCGGCGAGGGCCTCGAGGCCATCTACGACAAGGCCTCCAGCACCGACCCCAAGTCGTCGGCGCTCGTCGACCAGCTCACCGCCGGCGCCGGCGTCCTCGGCGGCCCCAGGTACGTACGGCACCCACCCATCCCTCCTACATTGCATCGTCATCGCCTCGCCGGCTCGCCGCTCTCCTTTTGCACGGTCGAATGAATCCGACATGTAGCAGCAGCGTGGGTGTGTGGGCCCCACTGCGTGAAATATCCGCCGGATCCCTCTCGCTGACTGGTGGGGCCCACGTGGGCATTTTTTTTTCTTTTGGGTTGGGCCGTCTTTTTTTTAATGCGGCAGCGGCGCCTTTAAACTGCCACCTCCGGTCGCCGGAGGGGCGCCGCTTGTCGAGTTGTTGTCGGCGCCATTGCCGCGACCTGGATGGGTAGAGAGGGATTTGCGGTAGAACAGCTCGTCGATTTGGTAACATTGAGGTCCCGTTGGAAGCATGAAGATTAGGAAAGGGCACGTAAGACGAGAGTTATTAGCACACATTAATTGATTTTTAATCCCTCCATCCAAAAATATAATGGATTTTGGGTGGATGAGTCGTAGGGTCTGTTTGGTACAGCTCCAACTCCTAAATTTAGAGGAGTTGGGTCTGGAGTGGATCTAATTCATTTTGTGAGAGCTCCATCCAACTCCACTCCCAGTTCTAGTGGAGCTGAAACTGTTTGGCCAAGCTCCAGCTCTAGAAGGGGTGGAGCTGGAGCTGTGCCAAACAGGCCCGTACTATAGTACTATGAATCTTGACAACATGGTACTAGAATATATCGCATCCATCCAAAATCCCTTATATTTTAGGACAGATGGAGTAGATATTATAAACTTAAAAATAAACTTTATTTGATACTAGTTTAGATCAACTTCTATATAAAAATTTTTCGCACGTAATATATTATTTAACAGTTTAAAAAAAGTGCTAATGAAAATTAGGATAGAATCTAGTAGTATGATCTTAGTCAGAAACAAACGGGCCTGAGAGCATGAGAGCAGAGCAGTTTGGCTAGCTAGCAACAAGCGTGGCAGTACAGTAAGAAATGCAGGCCGGCGATGCAGGTGGACTTGTACAAGCACTCTGACTGGAACTGAGCTAACCATGTCGGCGTCCTACCGCGATAATCCTACAATTATTTTCCTGGCCAAATCCTCAATTTATAGACCCTTAGCTGGTAAAAGGCAGGACAGGTTAGGAGGGCCAAAGTAAAGTATTCTTAACAGATGATTACTATTACAAGGCTGAGCACTCAATAGAGAGCCGTACCACTAACACTAGCGGCTGACTGACAAAGTCGGGTTGATTAAACACCGGGATAAAAGTTTATAAAGCAACTTTGTCTTGTACTACCAGTAGGCCCAGTACTTGTATTCTGGTACACTGCTAGTGCTAGTATTCTTTTTAGACAGCGAACTAGACTCTGCTCGTGTGGGATGGGCGAAACGCGAGACTTGGGGAGTGGGCAGCACGGGCAGGCAAATGCACTGCCGCCGTTGCATTTTCGTTCTCCCGTTCTTTCCTCTGAAACCATGGCAAGACAAGAGCAGACTACTCATGTCAAGCTTACAAAAACCTTCCAACTATCATAAAATAAACAAAACGATGTGACCTAGTATGAATCTGGATAGATCGTATTCGTAATAAGTATATGTTTATTTTTGACGGATGGAGTACATTTTTACATGAATGACTTACTCCCTTCGTCCTAAAAAAAATCAACTTTTAAAGAAAAATCTAGACAAATGCATGACCGGATTTATCTTTAGAATTTGATTTTTTTTTTTAAAGAACGAAGAGTGTACATCATTGCGTTAGACTTTAATACTCCATTCGTTTCTAAAAAATAAACTAATACGGAAATTAGGGTTGTTTTTTATACGACGAGGAGGGAATACATTGCAATGCTAGTACGTTGGCACATAAACTCTCTTCAGTCAATCTGCCTCCGGATGTCCGGATTATGAATAAAAAATAATGGACCAGTGTTATCTTTTGGTAGCCGGTCATGTCGATGAGCCATCAACTGAATCCACAGATGGTGGCCGGTCTTTTTTCCTCTTCGCTCGATGCCAAAAGTAGCCGGGATCGAAAAGATCGTCGCATGCTCAGCAACATGCGTGCTATATCCATCGATTGCATAACCCAGGGATCACGGTAGGCCGGCAATCACTCACCATGCATTGCCTTCTTCTTCTCCACTTGCTCGCCACTGACCCCTTCGTTTTGTCGCCGAATCCTGCAAAAGATAGCCAGAAAAGAAAAAAAAGGCTCAAAAGAAGCTAGTACACACAAACACAAACTGGCCCAATGCATTATAGTTGTTGCTGAATTATTCTAGTACTGCGAGAAGCTGTGTTCTTTGGGCGTTTTGGATCTCAAACAGTGTCCTGTACTGGAGACCCTACCATATTGCAATGCGATGACCTTTTCGGATGGAGCCAAGCAAGAAGAGAACGTTGAGGGTCCATTTGCAACGATCCATTAGAAAATGGCGTGCACTTTTTGTCGTTCTGTCCATCACTCTGCAGGAGAAGACCCATGTTCCTCGGCATCCAATTCCTTATTCTGCAGCGTGCATATGCACTTGTGACAGACAGCAGTAGTAGATCTCACCTTAAGTACAGTTTCACTTGAAAGTTTTTGCTACAACTTGTGCCAGTTTTGCTAATGACATACTGGCAGCTACTAGCTCTCTCATGGTAGTACATGAGAAGAGAATATAAGATAGTTTGCTAATGTTTTTGGAGATTTGATCTGGCAGGGACAACGGCGAGGCGGCGGCGTACGCCGGCGTCAACTACGCCGGAGTTGGAACCGGCGGCGGAGGCGGTGGCGGCGCCGGTGCCGGCGGGATGGGGTATGGTTATCTCGGGATGGTCTATGGCAGCGGCGGCAATTGGAGCTGCCGAGCTGACCTGACACTGCAATTGACTTCATTGTTTCTTGCACTCTTTGCTCTCATATGCTTGCACCCATTGTGA
TTGTAAGATGCTTCATTATGATTCAATTGGAAAGGTTAGGAAAAAGGGGAGAAGAAAAAGGAAGCAAAAGGTGTTGACAGTTGACACTTAGGGAGTAGTAGGCAACAGTGTTACAGTGAGATGATATCCACCATGTAATGAGCACATGGATGGGAATATTGCAGGTTTAGCCAATGTAGTTGTTGCAAAGGGGAATGGGATTATTCAGACTAACTTAAGCTATTAACTCTAACCTCTCTGTAATCTCTGTCGAAGGGTAACGAATCTATGGTTTAATGGATAAACAAAGTTGAGCAAATGCTAGAAAGAAACCGTAATTTGGCCGTGCTTGTGACAAGAAAATTTGATCCGGTTGAGAGGTGGCCCAAAATCGTTTAAATTCTAAGGGTGGGCAAATTTGCTCCTGTTCAGAGGTGTATACCAATTTCGATTATGGGCATAATTTGGGACGCCTAATTTTCATGGTCATCGGTTGGCCAATTTTTTGTATGGGCTTTTATATGCACTGATTTAGATCTGGGAATGTGGTCCGCTGCTGGGCTTGAGTATTGAGCCTAAACCAACCCAGACAGAGCCGACTTGGGCTCTAGCAAACTGCTCCAGCTTTCGAGAATGGCGATGATTGAAGAGCCGCAGGTAGTAGAACTGCAAACAAAACTACCATATCACTAAGCTTAATAAACAGCAGCTGAAATCACAAAAATACAGTTAACCACAAAATTCATGTATTTCCAACAAAATACAGTTAACCTCGAGAATTTGTATCAGTGTCAGCTAAACTCACTGAATTCTGATCCTCTCCTAGTGTTTGCAAGTTACATGGAAATATAAGAAAGGTACCAACATTCTCAGTCTACAGCTACAATGTGCCA

Claims (1)

1.一种水稻核苷酸片段人工加接头的方法，其特征在于，该方法包括如下步骤：
1)以含目的片段的水稻基因组或者cDNA为模板，该片段用P1表示，模板用D1表示，用扩增目的片段的前后引物、高保真酶进行PCR扩增，该前后引物分别用S1F和S1R表示；经1％琼脂糖胶电泳检测胶回收与预期大小一致的条带，胶回收产物用P2表示；
2)将S1F和S1R分别与包含有重组位点同源序列、线性化质粒酶切位点序列的接头连接，进行人工合成，前后引物的接头分别用J1和J2表示；合成的带接头的引物用S2F和S2R表示；
3)以P2为模板，S2F和S2R为引物，用高保真酶进行PCR扩增；经1％琼脂糖胶电泳检测、胶回收与预期大小一致的条带，胶回收产物用P3表示；
4)P3就是在目标片段两侧加上接头的目的产物，用它与线性化的质粒连接，进行后面的基因工程操作。
CN201611008369.2A 2016-11-16 2016-11-16 一种水稻核苷酸片段人工加接头的方法 Pending CN106520825A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611008369.2A CN106520825A (zh) 2016-11-16 2016-11-16 一种水稻核苷酸片段人工加接头的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611008369.2A CN106520825A (zh) 2016-11-16 2016-11-16 一种水稻核苷酸片段人工加接头的方法

Publications (1)

Publication Number Publication Date
CN106520825A true CN106520825A (zh) 2017-03-22

Family

ID=58352093

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611008369.2A Pending CN106520825A (zh) 2016-11-16 2016-11-16 一种水稻核苷酸片段人工加接头的方法

Country Status (1)

Country Link
CN (1) CN106520825A (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107502621A (zh) * 2017-09-22 2017-12-22 福建农林大学 一种快速检测体内dna末端连接的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105274109A (zh) * 2015-10-30 2016-01-27 上海交通大学 一种调控基因在非分泌型腺毛中表达的启动子及其应用
CN105505935A (zh) * 2016-01-19 2016-04-20 上海交通大学 AaALDH1基因启动子及其用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105274109A (zh) * 2015-10-30 2016-01-27 上海交通大学 一种调控基因在非分泌型腺毛中表达的启动子及其应用
CN105505935A (zh) * 2016-01-19 2016-04-20 上海交通大学 AaALDH1基因启动子及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
康海岐等: "水稻IIB型拓扑异构酶OsSp11基因的cDNA克隆、表达谱分析及其可变剪接的而检测", 《中山大学学报（自然科学版）》 *
徐勤枝等: "BALB/c小鼠sIL-4受体基因克隆及县病毒载体的构建", 《军事医学科学院院刊》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107502621A (zh) * 2017-09-22 2017-12-22 福建农林大学 一种快速检测体内dna末端连接的方法
CN107502621B (zh) * 2017-09-22 2021-04-13 福建农林大学 一种快速检测体内dna末端连接的方法

Similar Documents

Publication Publication Date Title
Ji et al. Conferring DNA virus resistance with high specificity in plants using virus-inducible genome-editing system
CN107012164B (zh) CRISPR/Cpf1植物基因组定向修饰功能单元、包含该功能单元的载体及其应用
CN105821075B (zh) 一种茶树咖啡因合成酶CRISPR/Cas9基因组编辑载体的构建方法
WO2018099256A1 (zh) 一种CRISPR/nCas9介导的定点碱基替换在植物中的应用
US11767536B2 (en) Method for obtaining glyphosate-resistant rice by site-directed nucleotide substitution
CN108841845A (zh) 一种带有筛选标记的CRISPR/Cas9载体及其构建方法
Ren et al. Bidirectional promoter-based CRISPR-Cas9 systems for plant genome editing
CN105132451A (zh) 一种CRISPR/Cas9单一转录单元定向修饰骨架载体及其应用
CN103981215B (zh) 一种用于基因工程的骨干质粒载体及应用
Wu et al. Increasing cytosine base editing scope and efficiency with engineered Cas9-PmCDA1 fusions and the modified sgRNA in rice
AU2014227831A1 (en) Engineering plant genomes using CRISPR/Cas systems
CN106520825A (zh) 一种水稻核苷酸片段人工加接头的方法
CN106868016A (zh) 一个控制水稻籽粒高镉积累的基因突变位点及其应用
Xu et al. The evaluation of active transcriptional repressor domain for CRISPRi in plants
Hao et al. Introduction of mutations in plants with prime editing
Wu et al. A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification
McCarthy et al. Determinants of translational initiation efficiency in the atp operon of Escherichia coli
CN103276006B (zh) 一种抗黑条矮缩病载体及抗黑条矮缩病转基因水稻培育
CN116622648A (zh) 一种紫藤花叶病毒WiMV及其侵染性克隆载体和应用
KIYOKAWA et al. Detection of rol genes of Ri plasmids by PCR method and its application to confirmation of transformation
CN116286949A (zh) miRNA166o在植物光转录过程中的构建方法
CN116064647A (zh) 一种植物病毒表达载体及其应用
CN102392020A (zh) 玉米逆境胁迫诱导型启动子的克隆及功能分析
CN112375765A (zh) 一种水稻细菌性条斑病的感病基因OsHXK5及其应用
CN105624173B (zh) 一个水稻未知膜蛋白基因OsMP1及其在抗病基因工程中的应用

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20170322