柔细束丝藻土臭素合成酶基因的克隆及表达分析

Cloning and expression analysis of geosmin synthase gene from Aphanizomenon gracile

土臭素(Geosmin)是一种具有土霉异味的次级代谢产物,对水体具有严重的影响.为探讨柔细束丝藻(Aphanizomenon gracile)土臭素的合成机制以及环境因子对其土臭素产量的影响,利用高效热不对称交错PCR(High-effi ciency thermal asymmetric interlaced PCR,hi TAIL-PCR)技术对柔细束丝藻藻株WH-1的土臭素合成酶基因进行克隆,并对该基因在不同温度和光强下的表达情况进行实时荧光定量PCR(RT-PCR)分析.结果显示本研究成功克隆柔细束丝藻藻株WH-1的土臭素合成酶基因(geo),geo全长2 262 bp,编码753个氨基酸.低温(15℃)、强光(35μmol m-2 s-1)以及不间断光照处理均有利于geo的表达.这表明hi TAIL-PCR技术作为一种高效、低廉的染色体步移技术具有较好的应用价值,同时为异味物质的监测及防治提供部分理论基础.

Geosmin is a secondary metabolite responsible for earthy odors.The occurrence of geosmin has great impact on the quality of water environment.The gene essential for geosmin biosynthesis have been identified in several species.But little is known about the mechanism of geosmin synthesis in Aphanizomenon gracile.This study attempted to clone the gene involved in geosmin biosynthesis of Aphanizomenon gracile and analyze the geosmin production under different environments.The high-efficiency thermal asymmetric interlaced PCR（hiTAIL-PCR） was used to amplify the full-length of geosmin synthase gene from Aphanizomenon gracile（WH-1）.Real time PCR（RT-PCR） was applied to quantify the geosmin production in different light and temperature.As a result,geo,a geosmin synthase gene from Aphanizomenon gracile（WH-1） was cloned by hiTAIL-PCR.The full-length open reading frame（ORF） of geo was 2 262 bp,coding for a protein of 753 amino acids.Meanwhile,WH-1 was treated with different environment conditions and mRNA expression levels of geo were quantified by RT-PCR.It was found that low temperature（15 ℃）,high light intensity（35 μmol m^-2 s^-1） and continuous light illumination were beneficial to the expression of geo.The successful amplification of geosmin synthase gene verified that hiTAIL-PCR is an effective and simple procedure of low cost.The result provides fundamental knowledge on the monitoring and prevention for odorants.