蓖麻RcFEN1基因克隆及非生物胁迫下表达模式分析

Cloning of RcFEN1 gene in castor(Ricinus comunis)and its expression pattern under abiotic stress

【目的】克隆蓖麻结构特异性核酸酶1基因(RcFEN1),并检测其组织表达特性及不同非生物胁迫下的时空表达模式,为揭示Rc FEN1基因在蓖麻生长发育和响应低温胁迫等非生物胁迫的功能提供理论参考。【方法】根据蓖麻低温胁迫转录组注释信息,通过RT-PCR对RcFEN1基因编码区(CDS)进行克隆,利用生物信息学软件进行序列特征分析,并利用实时荧光定量PCR(q RT-PCR)检测其组织表达特性及不同非生物胁迫下的时空表达模式。【结果】克隆获得的RcFEN1基因(GenBank登录号OP205366)包含1个1154 bp的开放阅读框(ORF),编码383个氨基酸残基,含有RAD2/XPG核酸酶蛋白家族典型的结构域XPG-N和XPG-I,属于RAD2/XPG蛋白家族成员。RcFEN1蛋白含多个生物活性位点及功能区,但无跨膜区域和信号肽结构,是一个非分泌型亲水蛋白,定位在细胞核。RcFEN1蛋白与特洛花TsFEN1蛋白的氨基酸序列一致性最高,与大叶藻ZmFEN1蛋白序列的一致性最低,分别为90.28%和84.32%。RcFEN1蛋白与一串红和硬皮地星的FEN1蛋白亲缘关系较近。RcFEN1基因在子叶中的相对表达量显著高于根、茎和真叶(P<0.05,下同),在真叶中的相对表达量最低。RcFEN1基因在4种非生物胁迫下均有表达,但表达模式存在明显差异,其中,RcFEN1基因对干旱胁迫响应最敏感,处理2和4 h时相对表达量显著高于对照组(0 h)及其他处理时间;RcFEN1基因在盐胁迫和ABA胁迫下呈现相同的响应表达模式,均在处理4 h时开始表达,随后相对表达量降低;在低温胁迫下,RcFEN1基因在低温胁迫下延迟表达,且处理12 h后才被激活表达,且相对表达量持续增加。【结论】从蓖麻中克隆获得RcFEN1基因属于RAD2/XPG家族成员,是蓖麻低温胁迫下的差异表达基因,参与蓖麻低温胁迫的响应调控。

【Objective】The flap endonuclease 1(RcFEN1)gene was cloned from castor,and its tissue expression characteristics and spatial and temporal expression patterns under different stresses were detected,which provided a theoretical reference for revealing the function of the RcFEN1 gene in castor growth and development and response to abiotic stresses such as low-temperature stress.【Method】According to the transcriptome annotation information of castor cold stress,the coding region(CDS)of the RcFEN1 gene was cloned by RT-PCR,the sequence characteristics were analyzed by bioinformatics software,and the tissue expression characteristics and temporal and spatial expression patterns under different stresses were detected by quantitative real-time PCR(q RT-PCR).【Result】The cloned RcFEN1 gene(GenBank accession number∶OP205366)contained an open reading frame(ORF)of 1154 bp,encoding 383 amino acid residues,it contained the typical domains XPG-N and XPG-I of the RAD2/XPG nuclease protein family and belonged to the RAD2/XPG protein family. RcFEN1 protein contained multiple biologically active sites and functional areas but had no transmembrane region and signal peptide structure. It was a non-secretory hydrophilic protein located in the nucleus. The amino acid sequence identity of RcFEN1 protein with TsFEN1 protein was the highest,and the sequence identity with ZmFEN1 protein was the lowest,which were 90.28% and 84.32%,respectively. The RcFEN1 protein was closely related to the FEN1 protein of Salvia splendens and Dorcoceras hygrometricum. The relative expression of the RcFEN1 gene in cotyledons was significantly higher than that in roots,stems and true leaves(P<0.05,the same below),and the relative expression in true leaves was the lowest. The RcFEN1 gene was expressed under four abiotic stresses,but the expression patterns differed greatly. Among them,the RcFEN1 gene was the most sensitive to drought stress,and the relative expression levels at 2 and 4 h were significantly higher than that of the control group(0 h). The RcFEN1 gene showed the same response expression pattern under salt stress and ABA stress,which began to express at 4 h,and then the relative expression decreased. Under low-temperature stress,the presentation of the RcFEN1 gene was delayed under low-temperature stress,and it was activated after 12 h of treatment,and the relative expression level continued to increase.【Conclusion】The RcFEN1 gene cloned from castor belongs to the RAD2/XPG family. It is a differentially expressed gene under low temperature stress and participates in the response regulation of castor low-temperature stress.