红花二氢吡啶二羧酸合酶基因片段的分离及表达分析

Cloning and expression analysis on gene encoding dihydrodipicolinate synthase from Carthamus tinctorius

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 from Carthamus tinctorius（safflower） seeds and study its expression in different developmental stages of seed. Methods Primers were designed according to Ct DHDPS gene segment which was selected from transcriptome sequencing results of safflower. Taking total RNA of safflower seed as template, Ct DHDPS genes were amplified by RT-PCR and connected to p EASY-T1 carrier, and positive cloning was detected by PCR and then sequenced. Results Sequencing results showed that 396 bp sequence was acquired. The gene had high homology compared with DHDPS from other species. Conclusion The fragment of Ct DHDPS gene is cloned from safflower, and PCR primers of safflower are designed based on Ct DHDPS gene for Real-time PCR in different developmental stages of safflower seed. The results show that the expression of Ct DHDPS genes in DAF 14 in Chuan-hong1 line is the highest.