| 摘要: |
| 【目的】克隆山黧豆β-腈基丙氨酸合成酶基因（CASase）DNA全长序列，构建CRISPR/Cas9敲除载体，为筛选“低毒、高硫”山黧豆品系奠定基础。【方法】以萌发4 d山黧豆幼苗根部为材料，提取其基因组DNA，利用PCR克隆CASase基因全长；经序列同源性、内含子、外显子、sgRNA靶位点和脱靶分析后，在CASase cDNA上选取5个sgRNA靶位点进行寡核苷酸链设计和体外活性检测；从5个sgRNA靶位点中取活性较高的4个sgRNA，利用酶切连接法构建基因敲除载体pPLHACas9-sgRNA20r、pPLHACas9-sgRNA68r、pPLHACas9-sgRNA225f和 pPLHACas9-sgRNA256f。【结果】 获得了CASase基因3 143 bp的DNA全长序列，该序列编码β-腈基丙氨酸合成酶；在CASase cDNA中选取5个sgRNA靶位点在体外均能有效剪切靶序列；菌落PCR检测结果显示，4个CRISPR/Cas9基因敲除载体构建成功。【结论】克隆了山黧豆CASase基因DNA全长，成功构建了该基因上4个sgRNA靶位点的CRISPR/Cas9敲除载体。 |
| 关键词: 山黧豆 CASase基因 基因克隆 载体构建 CRISPR/Cas9系统 |
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| 基金项目:国家自然科学基金项目（31401910）；中国博士后科学基金面上项目（2016M590975）；陕西省博士后科研项目（2016BSHEDZZ119）；中央高校基本科研业务费资助项目（2014YB040） |
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| Amplification of CASase gene from Lathyrus sativus and construction of its knock-out vector via CRISPR/Cas9 system |
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TAO Yingjie,LIU Fengjuan,BI Chunxiao,et al
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| Abstract: |
| 【Objective】This project cloned full-length DNA sequence of CASase gene from Lathyrus sativus and construct its recombinant knock-out vector via the CRISPR/Cas9 system to provide basis for screening novel lines with low-ODAP and high sulfur-containing amino acids.【Method】The full-length DNA sequence of CASase gene was cloned from radicle DNA of L.sativus via PCR.After alignment,exons and off-target analysis,five target sites on CASase cDNA sequence were selected to design double stranded Oligo DNA and were inserted into pPLatU6-sgRNA,which was then recombined into the expression vector to get pPLHACas9-sgRNA20r,pPLHACas9-sgRNA68r,pPLHACas9-sgRNA225f and pPLHACas9-sgRNA256f.【Result】The full length DNA sequence of CASase gene was obtained with 3 143 bp length and encoded β-cyanoalanine synthase (CASase).All the five selected sgRNA worked with target sequence.Colony PCR and sequencing showed that four knockout vectors were conducted successfully.【Conclusion】The full-length DNA sequence of CASase gene was cloned and four knock-out vectors were conducted successfully via the CRISPR/Cas9 system. |
| Key words: Lathyrus sativus CASase gene gene cloning knock-out vector construction CRISPR/Cas9 system |
