木薯海藻糖合成酶基因MeTPS6克隆及其在非生物胁迫下的表达分析

Cloning of trehalose-6-phosphate synthase gene MeTPS6 from cassava and its expression under abiotic stress

【目的】克隆木薯海藻糖合成酶基因MeTPS6,并分析其在干旱、低温、遮荫等非生物胁迫应答中的表达情况,为研究TPS基因功能及抗逆分子机理提供参考。【方法】采用RT-PCR从木薯中克隆MeTPS6基因,并进行生物信息学分析及构建系统发育进化树。采用荧光定量PCR(q PCR)分析MeTPS6基因在干旱、低温和遮荫胁迫下不同木薯品种不同组织中的表达特性及模式。【结果】克隆获得的MeTPS6基因具有一个2565 bp的开放阅读框,编码854个氨基酸,含有TPS家族保守结构域(Glyco_transf_20)。MeTPS6蛋白与杨树(Potri.010G104500)和杞柳(Sapur V1A.0015s0610)同源蛋白氨基酸序列的相似性分别达92.6%和90.0%,表明木薯与杨树和杞柳的亲缘关系较近。MeTPS6基因的启动子序列中存在大量非生物胁迫相关元件(低温相关元件LTR、干旱相关元件MBS等)、激素相关元件(脱落酸相关的元件ABRE和CE3、水杨酸相关元件TCA-element等)及光响应相关元件(ACE、Box I等)。MeTPS6基因在木薯储藏根中的表达量最高,须根次之,二者均显著高于茎、叶柄和叶片中的表达量(P<0.05,下同),且在干旱、低温和遮荫胁迫下,不同组织中的表达模式存在明显差异。对于不同木薯品种,干旱、低温和遮荫胁迫处理均显著诱导其MeTPS6基因表达。在高置信度(Confidence=0.8)的情况下,共找到13个共表达基因,其中有10个为TPS同源基因,表明这些同源基因可能相互协调,共同参与植物生物学过程;另外3个基因(CAT、CAT1和F5M15.5)均编码过氧化氢酶,主要参与保护细胞免受过氧化氢的毒性作用。【结论】MeTPS6基因主要在木薯的根(特别是储藏根)中表达,并从转录水平参与干旱、低温和遮荫胁迫响应,可作为候选基因进一步研究其在木薯抗逆中的功能。

【Objective】Trehalose-6-phosphate synthase gene MeTPS6from cassava was cloned,and its expression under abiotic stresses including drought,cold and shade was analyzed,in order to provide a reference for the study of TPS gene function and its anti-adversity molecular mechanism.【Method】RT-PCR method was applied to clone gene MeTPS6from cassava leaves,bioinformatics analysis was carried out and a phylogenetic tree was constructed.Fluorescence quantitative PCR(qPCR)was used to analyze the expression characteristics and patterns of gene MeTPS6in different cassava varieties and tissues under different abiotic stresses including drought,cold and shade.【Result】Cloned gene MeTPS6had a2565-bp open reading frame,encoded854amino acids,and contained a conserved TPS family domain(Glyco_transf_20).Protein MeTPS6had close genetic relationship with Populus trichocarpa and Salix purpurea,its homologous sequence similarity with P.trichocarpa(Potri.010G104500)and S.purpurea(SapurV1A.0015s0610)was up to92.6%and90.0%respectively.Promoter sequence in gene MeTPS6had a large number of abiotic stress related motifs(cold-related motif LTR and drought-related motif MBS),hormone related motifs(abscisic acid related motif ABRE and CE3,salicylic acid related motif TCA-element),and light response related motifs(ACE and Box I).Gene MeTPS6expressed the highest in storage root,followed by fibrous root,and the expression in these two tissues were significantly higher than those in stem,petiole and leaf(P<0.05,the same below).The expression patterns of gene MeTPS6were obviously different in various tissues under drought,cold and shade stresses,but its expression was significantly induced under these stresses.【Result】At a high confidence level(Confidence=0.8),thirteen co-expressed genes were found,among which ten were TPS homologous genes,suggesting these genes might coordinate with each other and participate in the process of plant biology,while the other three genes(CAT,CAT1,and F5M15.5)encoded catalase and mainly involved in the protection of cells from toxic effects of hydrogen peroxide.【Conclusion】Gene MeTPS6mainly expresses in cassava root(especially storage root)and participate in the transcriptional regulation of response to drought,cold,and shade stress treatments.It can be served as a candidate to further studying its functions in cassava anti-adversity.