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目的克隆红花种子中的天冬氨酸激酶(aspartokinase,AK)基因并研究其在种子不同发育时期的表达量。方法根据红花转录组文库注释信息筛选与红花天冬氨酸激酶(Ct AK)基因相关的Unigenes,设计引物,以红花种子总RNA为模板,采用RT-PCR的方法扩增Ct AK基因片段并连接到克隆载体上,经PCR及酶切鉴定,筛选阳性克隆进行测序。同时利用荧光定量PCR技术对其进行基因表达量的分析。结果克隆了红花Ct AK基因的核心片段,分离到486 bp的基因序列。根据Ct AK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荧光定量PCR分析,Ct AK基因在红花品种川红1号初花后13 d表达量最高。结论系统发育树分析表明,该基因与其他物种的AK基因具有较高的同源性。
Objective To clone the aspartokinase (AK) gene in safflower seeds and study its expression in different development stages of seeds. Methods The Unigenes related to Ct AK gene were screened based on the annotation information of safflower transcriptome. The primers were designed. The total RNA of safflower seeds was used as a template to amplify Ct AK gene by RT-PCR The fragment was ligated to the cloning vector, identified by PCR and restriction enzyme digestion, and the positive clones were screened for sequencing. Meanwhile, quantitative analysis of gene expression was carried out by using fluorescence quantitative PCR. Results The core fragment of Ct AK gene was cloned and the 486 bp gene sequence was isolated. According to the primers of Ct AK gene fragment, the fluorescent quantitative PCR analysis of safflower seeds at different developmental stages was carried out. The highest expression level of Ct AK gene was detected 13 days after the first flowering of the safflower variety Chuanhong 1. Conclusion Phylogenetic tree analysis shows that this gene has high homology with AK gene of other species.