玉米蜕皮激素合成酶基因ZmCYP92A1的克隆和表达分析
玉米蜕皮激素作为一类天然的植物次生代谢产物，在植物抵抗植食性昆虫的为害中扮演着重要的角色。CYP92A1基因编码的酶参与植物蜕皮激素的合成，但玉米CYP92A1基因在逆境胁迫下的相关研究还未见报道。本研究采用RT-PCR结合RACE技术，从玉米中克隆ZmCYP92A1， GenBank 登录号为KY270496.1。 该基因全长1713bp，开放阅读框为1551bp，编码517个氨基酸。预测ZmCYP92A1蛋白分子量为58.58kD，理论等电点为6.44，存在2个跨膜结构域，1个信号肽，有1个p450保守结构域。多序列比对表明ZmCYP92A1蛋白与其它物种的CYP92A1蛋白具有极高的相似性，系统发生分析揭示CYP92A1蛋白与属于CYP75亚家族的类黄酮3-单加氧酶的亲缘关系较近；实时荧光定量PCR结果发现：非生物胁迫(干旱、高盐、低温和脱落酸) 均诱导了ZmCYP92A1基因的表达，表明该基因参与植物抵御非生物胁迫；同时首次揭示该基因参与虫害和茉莉酸甲酯的响应，为研究植物蜕皮激素和茉莉酸甲酯诱导虫害应答的分子机制奠定基础。
英文摘要:
Phytoecdysone as the natural secondary metabolite plays an essential role in plant resistance to phytophagous insect. The enzyme encoded by CYP92A1 is involved in the phytoecdysone biosynthesis. However, the evidence for the relationships between the CYP92A1 gene in Zea mays and adversity stresses remains limited. Consequently, in our research, ZmCYP92A1 was cloned from Zea mays via RT integrated with RACE technology. The predicted protein encoding a 517 amino acid peptide, whose molecular weight is 58.58 kD and isoelectric point is 6.44, which has a full-length sequence of 1713 bp with a complete open reading frame of 1551 bp. It possesses two transmembrane domains, one signal peptide and one conserved domain of p450. Genbank number of the ZmCYP92A1 gene is KY270496.1. Multiple sequence alignment analysis indicated the predicted protein shared high sequence similarities with CYP90B1 from other plants. Meanwhile, phylogenetic analysis revealed that ZmCYP92A1 was more close to flavonoids 3-monooxygenase belonging to CYP75 subfamily. The expression of ZmCYP92A1 was induced by treatments of abiotic stress (drought, high salt, low temperature and ABA), which indicated that this gene involved in abiotic stresses. Meanwhile, ZmCYP92A1 was first revealed to response to insect attack (Spodoptera exigua) and Methyl jasmonate (MeJA), which laid the foundation for studying the molecular mechanism of insect response induced by phytoecdysone and MeJA.
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