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目的克隆红花Carthamus tinctorius赖氨酸合成途径关键酶二氢吡啶二羧酸合酶(dihydrodipicolinate synthase,DHDPS)基因并研究其在不同发育时期红花籽粒中的表达量。方法根据红花转录组文库注释信息筛选出与红花DHDPS(Ct DHDPS)基因相关的Unigenes,设计引物,以红花总RNA的反转录产物为模板,采用RT-PCR技术克隆Ct DHDPS基因片段,连接p EASY-T1克隆载体,经PCR和酶切鉴定,筛选阳性克隆并测序。同时利用荧光定量PCR技术对其在红花籽粒不同发育时期基因表达量进行分析。结果分离到长度为396 bp的Ct DHDPS基因片段,系统发育树分析表明该基因与其他物种的DHDPS基因具有较高的同源性。结论克隆了Ct DHDPS基因的核心片段,根据Ct DHDPS基因片段设计引物,对不同品种不同发育时期红花种子进行基因表达量分析,Ct DHDPS基因在红花品种川红1号初花后14 d表达量最高。
Objective To clone the dihydrodipicolinate synthase (DHDPS) gene, a key enzyme involved in the lysine biosynthesis pathway of Carthamus tinctorius, and to study its expression in safflower kernels at different developmental stages. Methods Unigenes related to DHDPS (Ct DHDPS) gene were screened based on the annotation information of safflower transcriptome library. Primers were designed and the reverse transcript of total RNA of safflower was used as a template. The Ct DHDPS gene fragment was cloned by RT-PCR , Connected p EASY-T1 cloning vector, identified by PCR and restriction enzyme digestion, positive clones were screened and sequenced. At the same time, the quantitative analysis of gene expression at different developmental stages of safflower seeds was carried out by using fluorescence quantitative PCR. Results A 396 bp Ct DHDPS gene fragment was isolated. Phylogenetic tree analysis showed that this gene shared high homology with other DHDPS genes. Conclusion The core fragment of Ct DHDPS gene was cloned. According to the primers of Ct DHDPS gene fragment, the expression of Ct DHDPS gene was analyzed 14 days after the first flowering of Chuanhong 1 The highest volume.