香鳞毛蕨DfDXR基因克隆和表达模式分析

Cloning and Expression Pattern Analysis of DfDXR Gene in Dryopteris fragrans

为探索1-去氧-D-木酮糖-5-磷酸还原异构酶(DXR)是否参与香鳞毛蕨对环境的抗逆功能,用PCR克隆并鉴定了香鳞毛蕨DfDXR基因,并通过在线预测网站对其蛋白序列进行生物信息学分析,利用实时荧光定量PCR(qRT-PCR)技术研究了DfDXR基因在不同化学物质和逆境胁迫处理下相对表达量的变化。结果表明,成功克隆出DfDXR基因的CDS序列全长1434 bp,编码477个氨基酸,DXR蛋白二级结构为alpha-beta型。蛋白质多序列比对以及进化树分析表明,DfDXR与铁线蕨AcDXR亲缘关系较近,与苹果和桃等植物的DXR蛋白亲缘关系较远,Motif分析表明,该蛋白含有PLN02696结构域,该结构域为DXR蛋白的保守结构域,亚细胞定位预测DXR蛋白定位于叶绿体上,与其他物种一致。对qRT-PCR数据进行分析显示,DfDXR的相对表达量在茉莉酸甲酯(MeJA)和聚乙二醇(PEG)处理后总体都呈现上调的趋势,并分别在1,12 h达到最高;NaCl处理下呈“降—升—降”的趋势,但各处理时间下的表达量均低于对照组;水杨酸(SA)、脱落酸(ABA)、乙烯利(ETH)、高温(HT)以及低温(LT)处理下,DfDXR的相对表达量也发生明显变化。在香鳞毛蕨响应不同非生物胁迫过程中,DfDXR均发挥调控作用,且DfDXR对不同化学物质和逆境胁迫的响应时间不同。

In order to explore whether 1-deoxygen-D-xylose-5-phosphate reductisomerase(DXR)participates in the environmental resilience of Dryopteris fragrans.It used polymerase chain reaction(Polymerase Chain Reaction,PCR)methods to clone and identify Dryopteris fragrans DfDXR gene,the protein sequences were analyzed by bioinformatics through online prediction website,the relative expression levels of DfDXR gene under different chemical substances and stress were studied by Real-time fluorescence quantitative PCR(qRT-PCR).The experiment further explored how DfDXR gene responded to the stress-resistance process of Dryopteris fragrans,and laid a foundation for studying its mechanism.Experimental results:The full-length CDS sequence of DfDXR gene 1434 bp was successfully cloned,encoding 477 amino acids,and the secondary structure of DXR protein was alpha-beta.Multi-sequence alignment and phylogenetic tree analysis of the proteins suggested that DfDXR was closely related to Adiantum capillus-Veneris AcDXR,but far related to DXR protein of apple and peach.Motif analysis showed that the protein contained PLN02696 domain,which was the conserved domain of DXR protein.Subcellular localization predicted that DXR protein was located in chloroplast.The analysis of qRT-PCR data showed that the relative expression of DfDXR showed an upward trend after treatment with methyl 3-oxo-2-(pent-2-enyl)cyclamate(MeJA)and polyethyleneglycol(PEG),and reached the highest level at 1,12 h respectively.Under NaCl treatment,there was a"down—up—down"trend,but the expression levels under each treatment time were lower than those in the control group.The relative expression of DfDXR also changed obviously under the treatment of salicylic acid(SA),abscisic acid(ABA),ethephon(ETH),high teperature(HT)and low temperature(LT).DfDXR plays a regulatory role in the response of Dryopteris fragrans to different abiotic stresses,and its response time to different chemical substances and stress is different.