摘要
本研究通过同源克隆技术对三角梅(Bougainvillea glabra Choisy)甜菜色素合成途径中4,5-多巴双加氧酶(4,5-DOPA dioxygenase,DOD)基因进行了克隆分析。随后将获得的三角梅DOD基因克隆到表达载体pET30b(+)上,构建原核表达载体pET30b(+)-DOD,并转入大肠杆菌Rosetta(DE3)进行诱导表达。同时结合实时荧光定量PCR技术分析了遮光处理对三角梅DOD表达量和甜菜红素含量的影响。结果表明,三角梅DOD基因的cDNA序列长度为804 bp,编码268个氨基酸,GenBank登录号为KY676801。系统进化树分析显示该DOD与叶子花(Bougainvillea spectabilis Willd.)和紫茉莉(Mirabilis jalapa L.)DOD亲缘关系比较近;原核表达得到约37 k D的目的蛋白,这与预测结果相符合;实时荧光定量PCR结果表明,遮光处理使DOD表达量降低;同时,甜菜红素含量也表现为下降。这些研究结果为解析三角梅甜菜色素合成途径提供了理论依据,为三角梅花色形成机制的研究提供了基础资料。
This study cloned and analyzed the 4,5-DOPA dioxygenase gene（DOD） in the betalain biosynthesis pathway of Bougainvillea glabra by homologous cloning.Afterwards,the obtained DOD gene was cloned into the expression vector pET30 b（＋） to construct the prokaryotic expression vector pET30 b（＋）-DOD.Then it was transformed into Rosetta（DE3） of E.coli strain to be induced.At the same time,the effect of shading treatment on DOD expression and betacyanin accumulation was analyzed by combining with real-time fluorescence quantitative PCR.The results showed that the length of c DNA sequence of DOD gene was 804 bp,encoding 268 amino acids（GenBank accession No.KY676801）.Phylogenetic tree analysis indicated that the DOD had a close genetic relationship with that of Bougainvillea spectabilis Willd.and Mirabilis jalapa L..Prokaryotic expression resulted in a target protein of about 37 k D,which agreed with the prediction results.The results of qRT-PCR showed that DOD expression decreased after shading treatment.Meanwhile,the betacyanin accumulation also decreased.These results provided a theoretical basis for analyzing the biosynthetic pathway of betalain and basic data for studying the formation mechanism of the flower color in Bougainvillea glabra.
出处
《分子植物育种》
CAS
CSCD
北大核心
2018年第1期54-60,共7页
Molecular Plant Breeding
基金
四川省高等学校重点实验室科研项目(GR-2013-C-04)资助
关键词
三角梅
4
5-多巴双加氧酶
克隆
表达分析
Bougainvillea glabra
4,5-DOPA dioxygenase
Cloning
Expression analysis
