茶树5-烯醇式丙酮酰莽草酸-3-磷酸合成酶基因的克隆及表达

Cloning and expression analysis of 5-enolpyruvyshikimate-3-phosphate synthase gene in Camellia sinensis

为探究茶树5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)基因的序列特征及在不同组织和逆境胁迫下的表达模式,利用反转录PCR(RT-PCR)从铁观音茶树中克隆获得CsEPSPS基因及其启动子序列,分析其序列特征,并采用实时荧光定量PCR(qRT-PCR)技术研究CsEPSPS基因在不同组织和不同激素、逆境胁迫处理下的表达情况.结果表明:该基因编码区为1563 bp,编码520个氨基酸,在第83-514个氨基酸具有EPSP;ynthase保守结构域;蛋白信息分析显示,CsEPSPS定位于叶绿体且无跨膜域,有1个无序化区域,存在58个磷酸化位点,其二级结构以不规则卷曲和α-螺旋为主;系统进化树及保守基序分析显示,EPSPS在进化上是一种高度保守的酶类,CsEPSPS与猕猴桃和喜树具有较高同源性,亲缘关系更近;基因结构预测显示,CsEPSPS由8个外显子和7个内含子构成,启动子区域含有与激素及植物抗逆相关的顺式作用元件;实时荧光定量PCR分析显示,茶树CsEPSPS基因具有组织表达特异性,表达量由高到低依次为花、茎、老叶、嫩叶和根,同时赤霉素、茉莉酸甲酯和脱落酸等激素及低温、干旱和草甘膦胁迫处理均能诱导茶树CsEPSPS基因表达,其中草甘膦诱导CsEPSPS基因上调表达的作用最为明显.本研究结果表明CsEPSPS基因可能与茶树抗逆响应密切相关.

To explore the sequence characteristics of the CsEPSPS gene and its tempo-spatial expression patterns under different abiotic stress conditions,the CsEPSPS gene and its promoter sequence was cloned from Camellia sinensis cv.Tie-guanyin and quantitative real time polymerase chain reaction(qPCR)was performed to determine the tempo-spatial changes of the gene transcripts.The result showed that the fulllength coding sequence of this gene is 1563 bp,which encodes 520 amino acids and contains EPSP_synthase conserved domains at the 83;-514;amino acids.The results of protein information analysis showed that CsEPSPS was located in chloroplasts without transmembrane domain,and it contains one disordered region and 58 phosphorylation sites.The secondary structure of CsEPSPS was mainly composed of irregular coil and helix.The phylogenetic tree and conserved base sequence analyses showed that evolutionarily,CsEPSPS is a highly conserved enzyme,highly homologous,and more closely related to Actinidia chinensis and Camptotheca acuminata.Gene structure predictions showed that CsEPSPS consists of eight exons and seven introns and that the region of the promoter contains cis-acting elements related to hormones and plant resistance.Realtime fluorescent quantitative PCR analysis results showed that the CsEPSPS gene of tea plant has tissue expression specificity and the expression level ranges as follows:flowers>stems>old leaves>tender leaves>roots.CsEPSPS expression was induced by the treatments of glyphosate,gibberellin,methyl jasmonate,and abscisic acid,low temperature,and drought,of which glyphosate treatment had the most obvious effect.The study indicated that the CsEPSPS gene might be closely related to the stress tolerance response of tea plants.