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二价铁转运基因(iron-regulated transporter 1 gene,IRT1)是植物缺铁时主要的二价铁转运载体,参与植物铁营养吸收过程。为克隆杜梨(Pyrus betulaefolia)IRT1基因的全长序列并研究其序列及表达特征,本实验以杜梨幼苗为材料,采用反转录PCR(reverse transcription PCR,RT-PCR)、半定量PCR和RACE技术克隆到了杜梨二价铁转运蛋白IRT1基因的c DNA全长序列,命名为PbIRT1(Gen Bank登录号:KX355331)。生物信息学分析表明,该基因全长1 369 bp,含有一个1 095 bp的开放阅读框(open reading frame,ORF),编码364个氨基酸,蛋白序列含有9个跨膜结构,并含有一个完整的锌铁转运蛋白(ZRT/IRTlike protein,ZIP)家族结构域。氨基酸序列的同源关系分析表明,杜梨PbIRT1与小金海棠(Malus xiaojinensis)Mx IRT1(Gen Bank登录号:AAO17059.1)、砀山酥梨(Py.bretschneideri)Pbr IRT1(Gen Bank登录号:XP_009357272.1)蛋白序列的亲缘关系最近,聚为同一小枝。RT-PCR和qRT-PCR表达分析表明,PbIRT1基因在叶片和茎中几乎不表达,具有较强的根组织特异性;在根系中表达量随缺铁胁迫呈逐渐上调趋势,恢复供铁又抑制其表达,正常供铁处理的表达量不随时间变化。以上研究结果表明,杜梨PbIRT1基因的表达受到缺铁胁迫的诱导,可能参与了根系对二价铁的吸收和转运过程,该结果为进一步研究IRT1基因的功能及杜梨缺铁胁迫机制提供了理论依据。
The iron-regulated transporter 1 gene (IRT1) is the main bivalent iron transporter in plant iron deficiency and participates in the iron absorption process of plants. In order to clone the IRT1 gene of Pyrus betulaefolia and to study the sequence and expression characteristics of the IRT1 gene, we used reverse transcription PCR (RT-PCR), semi-quantitative PCR RACE technique was used to clone the full-length cDNA sequence of IRT1 gene of fermented pear iron, named PbIRT1 (Gen Bank accession number: KX355331). Bioinformatics analysis showed that the gene was 1 369 bp in length and contained a 1 095 bp open reading frame (ORF) encoding 364 amino acids. The protein contained 9 transmembrane structures and contained a complete Zinc transporter (ZRT / IRTlike protein, ZIP) family domain. The homology analysis of the amino acid sequence showed that there was no significant difference between PbIRT1 and Mx IRT1 (Gen Bank Accession No .: AAO17059.1) and Pyr.bretschneideri Pbr IRT1 (Gen Bank Accession No .: XP_009357272) of Malus xiaojinensis. 1) The protein sequence has the closest genetic relationship and is clustered into the same branchlet. The results of RT-PCR and qRT-PCR showed that PbIRT1 gene was almost not expressed in leaves and stems and had strong root tissue specificity. The expression level of PbIRT1 in roots was gradually increased with iron deficiency stress, Its expression, the normal expression of iron for treatment does not change with time. The above results showed that the expression of PbIRT1 gene in Pyrus pyrifolia was induced by iron deficiency stress, which may be involved in the biosorption and transport of ferrous iron in the root system. The results provide the basis for further study on the function of IRT1 gene and the mechanism of iron deficiency in Pyrus pyrifolia Theoretical basis.