长白落叶松过氧化氢酶LoCAT1基因克隆及表达分析

为了解长白落叶松过氧化氢酶(CAT)基因的相关信息,探究该基因在长白落叶松不同组织中及不同逆境胁迫下的表达特性,本研究根据长白落叶松转录组数据库中获得的CAT1基因全长序列设计引物,克隆得到长白落叶松CAT1基因,命名为LoCAT1。该基因完整的开放阅读框(ORF)长度为954bp,共编码317个氨基酸。系统进化树分析结果显示,LoCAT1基因与北美云杉、银杏等CAT基因亲缘关系较近。利用实时定量RT-PCR技术分析了LoCAT1基因在长白落叶松中的组织表达特异性和应对非生物胁迫的表达模式。结果表明:LoCAT1基因在长白落叶松的根、茎、叶中均有表达,其中在茎部表达量最低,在叶中相对表达量最高。在非生物胁迫下,LoCAT1基因在长白落叶松根、茎、叶中的表达均发生了变化,但表达模式不同。在NaCl处理后,根和茎中LoCAT1基因均表现为下调表达,在12h时表达量最低,而叶中LoCAT1基因表达在24h明显受抑制,随后被上调表达,胁迫96h时表达量最高。PEG6000处理后,根和茎中LoCAT1基因的表达在胁迫早期被明显抑制,随后被上调表达。而叶中LoCAT1基因的表达在所有时间点均表现为上调表达。本研究推测长白落叶松LoCAT1基因可能参与了植物响应逆境胁迫的应答。     

Molecular cloning and characterization of the gene encoding 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase from hairy roots of Rauvolfia verticillata

2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MCT) catalyzes the third reaction in the plastidial non-mevalonate pathway, which provides the precursors for ajmalicine. A full-length cDNA encoding MCT (RvMCT) was identified from hairy roots of Rauvolfia verticillata. The full-length 1,499-bp cDNA of RvMCT had a 945-bp coding sequence that encoded a 314-amino-acid protein with an N-terminal chloroplast transit peptide of 67 amino acid residues. RvMCT exhibited homology with other plant MCTs at the levels of sequence and structure. The phylogenetic analysis revealed the plant MCTs could be divided into three separated clusters including gymnosperms, monocotyledons and dicotyledons. Gene expression of ajmalicine metabolism (DXR, MCT, MECS, HDS, HDR, STR and SGD) in hairy roots, roots, stems, old leaves, young leaves and barks was analyzed by quantitative PCR. All the seven genes had higher expression levels in hairy roots than in other plant organs. This suggested hairy roots of R. verticillata possessed more active alkaloid metabolism than other organs and it was the reason that hairy roots produced higher levels of ajmalicine. Furthermore, the expression of DXR, MECS, HDS, HDR, STR and SGD genes was not detected in stems (only MCT detected in stems), so it could be presumed that stem acted as a transporter tissue of ajmalicine. Finally, the colour complementation assay indicated that the function of RvMCT was the same as Arabidopsis MCT. Molecular cloning, characterization and functional identification of RvMCT will be helpful to understand more about the role of MCT involved in ajmalicine biosynthesis at the molecular level.