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(CtAK)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CtAK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CtAK基因的核心片段,分离到486 bp的基因序列。根据CtAK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,CtAK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 aspartatokinase (AK) gene from the seeds of Carthamus tinctorius (Ct, safflower) and study its expression in different developmental stages of seeds. Methods Primers were designed according to CtAK gene segment which was selected from transcriptome sequencing results of safflower. Taking total RNA of safflower seed as template, CtAK gene was amplified by RT-PCR and connected to pEASY-T1 carrier, identified by PCR and enzyme digestion, and positive cloning was screened and sequenced. Results The fragment of AK gene is cloned from safflower, and PCR primers of safflower are designed based on CtAK gene fragment. PCR analysis is performed in the safflower seeds of different developmental stages. The sequence of 486 bp was isolated. The expression of CtAK gene in DAF 13 in Chuan-hong 1 line is the highest. Conclusion The gene has higher homology compared with the AK from other species.

国家高技术研究发展计划(“863”)(2011AA100606);国家自然科学基金资助项目(31401445);吉林省科技厅医药产业推进计划项目(20140311034YY);教育部博士点基金(20122223120002)