青天葵独脚金内酯信号转导关键基因D14的克隆与亚细胞定位分析

Cloning and Expression Analysis of D14, the Key Gene in the Signal Transduction of Strigolactones from Nervilia fordii

D14基因是独脚金内酯信号转导途径的关键基因,其编码的蛋白能够使SLs释放出活性小分子物质而发挥调节腋芽起始的功能。本研究根据其他物种的D14基因从青天葵转录组数据中筛选获得2条高度同源的片段,命名为NfD14a和NfD14b。应用RT-PCR和RACE技术,克隆获得NfD14a和NfD14b的cDNA全长序列,GenBank登录号分别为MH028026、MH028027。NfD14a基因全长1206 bp,ORF为861 bp,编码286个氨基酸;NfD14b基因全长1082 bp,ORF为813bp,编码270个氨基酸。对NfD14的编码蛋白序列进行了生物信息学分析,结果表明:NfD14a和NfD14b均属于a/b折叠蛋白水解酶超家族成员(Abhydrolase superfamily),但系统进化分析结果表明两者同源性不高。利用一步快速克隆的方法构建了植物表达载体35S::NfD14a-EGFP和35S::NfD14b-EGFP,并分别获得其工程菌。瞬时表达结果表明,NfD14a和NfD14b均定位在烟草原生质体的细胞核和细胞质。本研究通过对NfD14基因全长cDNA序列的克隆与亚细胞定位分析,为青天葵独脚金内酯信号转导途径调控植物分枝生长发育机制的研究奠定基础。

D14 as a key gene encoding strigolactones(SLs) receptor in the signal transduction of SLs, enables SLs to release active small molecules and regulate axillary bud initiation. In this study, two highly homologous fragments in the transcriptome data were selected according to the sequences of D14genes from other species, named NfD14a and NfD14b. Using RT-PCR and RACE technologies, the full-length cDNA sequences of NfD14a and NfD14b were cloned and the GenBank accession numbers of them are MH028026 and MH028027 respectively. The full-length of NfD14a was 1206 bp, which contained an 861 bp open reading frame(ORF) encoding 286 amino acids. The full-length of NfD14b was 1082 bp, and it contained an 813 bp ORF encoding 270 amino acids. Bioinformatics analysis of the proteins encoded by NfD14a and NfD14b revealed that both NfD14a and NfD14b belonged to the Abhydrolase superfamily. However, the phylogenetic analysis showed a low homology of these two proteins. Plant expression vectors containing 35S::NfD14a-EGFP and 35 S:: NfD14b-EGFP were successfully constructed using the one-step rapid cloning method, and transformed into bacteria. Transient expression of these two plasmids showed that both NfD14a and NfD14b located in the nucleus and cytoplasm of tobacco protoplasts. The cloning and subcellular localization analysis of full-length cDNA sequences of NfD14 genes would lay a foundation for study in signal transduction of SLs and mechanism of plant branching in Nervilia fordii.