[目的]利用高效基因编辑系统(pHDE-Cas9)构建巨桉细胞分裂素氧化酶/脱氢酶(CTX)基因家族规律成簇的间隔短回文重复(CRISPR)载体,为开展巨桉CTX基因家族功能验证研究打下基础.[方法]设计巨桉CTX基因靶位点和引物,扩增pCBC质粒上的目的片段,BsaⅠ酶切pHDE载体并与目的片段连接形成重组质粒,转化DH5α感受态细胞.提取单克隆进行菌液PCR初步验证、重组质粒PCR验证,挑选阳性克隆质粒进行测序分析,构建CTX基因家族CRISPR载体.[结果]重组质粒PCR鉴定及测序结果表明,重组载体序列无突变,与预期序列高度一致.成功构建了巨桉CTX基因家族pHDE-CTXA、pHDE-CTXB和pHDE-CTXC同源表达载体.[结论]利用pHDE-Cas9成功构建了巨桉CTX基因家族CRISPR载体,构建过程快速、高效,为桉树基因组定点编辑打下了基础,也可为其他桉树CRISPR/Cas9表达载体的构建提供借鉴.%[Objective]Interspaced short palindromic repeats(CRISPR/Cas)vectors of Eucalyptus grandis whose cy-tokinin oxidase/dehydrogenase(CTX)gene family was regularly clustered were constructed by efficient gene editing sys-tem(pHDE-Cas9)to lay a foundation for verification of the functions of E.grandis CTX gene family.[Method]Target sites and primers of gene CTX in E. grandis family were designed,target fragment on pCBC plasmid was amplified, pHDE vector was digested with Bsa I,after that they were ligated into target fragment to form recombinant plasmids.The recombinant plasmids were transformed into DH5α competent cells.The monoclonal colonies were picked up for prelimi-nary confirmation by the bacterial liquid PCR,recombinant plasmid PCR verification,and then the positive cloned plas-mids were sequenced,and CRISPR vector of CTX gene family was constructed.[Result]The recombinat plasmids PCR identification and sequencing indicated that,there was no mutation in recombinat plasmid sequence,and it was highly consistent with the expected sequence.Homologous expression vectors pHDE-CTXA,pHDE-CTXB and pHDE-CTXC of CTX family in E.grandis were constructed.[Conclusion]By using pHDE-Cas9,point editing vector of CTX gene family in E.grandis is constructed,and the procedure is rapid and efficient.It establishes a foundation for future studies in ge-nome point editing vector of eucalyptus and construction of other eucalyptus CRISPR/Cas9 expression vectors.
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