广藿香PcGA2ox1基因克隆、VIGS载体构建和表达分析

Cloning,VIGS Vector construction and Expression Analysis of PcGA2ox1 Gene in Pogostemon cablin(Blanco)Benth.

赤霉素2-氧化酶(gibberellin 2-oxidases,GA2ox)是赤霉素(GAs)代谢途径关键酶,调节活性GAs降解,在植物株型调控中发挥重要作用。为探寻GA2ox基因在广藿香中的功能,本研究对广藿香PcGA2ox1基因进行克隆,得到561 bp的CDS序列。生物信息学分析结果表明,PcGA2ox1蛋白编码186个氨基酸,为亲水性蛋白,分子量为20.98 kDa,等电点为5.81,不含信号肽,无跨膜结构域,具有GA2ox超基因家族的保守结构域,主要定位在细胞核。系统发育分析结果显示,PcGA2ox1基因与丹参GA2ox7的亲缘关系最为密切。qRT-PCR结果显示,PcGA2ox1基因在广藿香中的表达具有组织特异性,叶中表达量最高,根中表达量最低。用病毒诱导基因沉默(VIGS)技术侵染广藿香的结果显示,侵染两周后,PcGA2ox1基因表达量比阴性对照下降83%,比空白对照下降71%,沉默效果明显。本研究结果可为深入了解PcGA2ox1结构、功能和作用机理奠定基础,并为广藿香优良品种选育提供参考。

Gibberellin 2-oxidases(GA2ox)is a key enzyme in metabolic pathway of gibberellins(GAs),which regulates the degradation of active GAs and plays important roles in plant type regulation.In order to explore the function of GA2ox gene in Pogostemon cablin,we cloned PcGA2ox1 gene of P.cablin and obtained 561 bp of CDS sequence.The results of bioinformatics analysis showed that PcGA2ox1 protein enco-ded 186 amino acids,was a hydrophilic protein with the molecular weight as 20.98 kDa and the isoelectric point as 5.81,contained neither signal peptide nor transmembrane domain,had the conserved domain of GA2ox supergene family,and was mainly located in nucleus.Phylogenetic analysis showed that PcGA2ox1 was most closely related to Salvia miltiorrhiza GA2ox7.The qRT-PCR results showed that PcGA2ox1 gene had tis-sue-specific expression in P.cablin with the highest level in leaves and the lowest level in roots.Infestation of P.cablin by virus-induced gene silencing(VIGS)technology showed that PcGA2ox1 gene expression decreased by 83%compared with negative control and 71%compared with blank control two weeks later,and the silen-cing effect was obvious.The results of this study could lay a foundation for an in-depth understanding of the structure,function and mechanism of PcGA2ox1,and provide a reference for the selection and breeding of ex-cellent P.cablin varieties.