坛紫菜6-磷酸海藻糖合成酶(TPS)家族基因的克隆及表达特征分析

Cloning and expression analysis of trehalose-6-phosphate synthase(TPS) family genes from Pyropia haitanensis

  • 摘要: 6-磷酸海藻糖合成酶(TPS)是植物海藻糖合成的关键酶,在植物逆境胁迫应答中发挥着重要的作用。实验以坛紫菜转录组测序获得的unigene序列为基础,采用RACE技术克隆获得坛紫菜的3条TPS基因序列:PhTPS1,PhTPS2-1PhTPS2-2。序列分析结果表明,PhTPS1(收录号:KM580358)序列全长3 557 bp,包含一个3 462 bp的开放阅读框,所编码的多肽包含1 153个氨基酸,分子量为124.2 ku,等电点为6.73,属于TPS Ⅰ亚家族;PhTPS2-1(收录号:KM519457)序列全长4 264 bp,包含一个4 044 bp的开放阅读框,所编码的多肽包含1 374个氨基酸,分子量为145.2 ku,等电点为5.96;PhTPS2-2(收录号:KM519458)序列全长3 733 bp,包含一个3 324 bp的开放阅读框,所编码的多肽包含1 107个氨基酸,分子量为120.2 ku,等电点为5.42。PhTPS2-1PhTPS2-2属于TPS Ⅱ亚家族。基因表达水平的定量分析结果表明高温胁迫条件下,3条PhTPS基因的表达模式基本一致,均表现为先上调后下调,再上调的趋势;而PhTPS1PhTPS2-1基因在中低水平的失水条件下,基因表达水平均没有发生显著变化,只在高水平失水胁迫下显著上调,PhTPS2-2基因在中高水平失水条件下,表达水平显著上调。说明PhTPS基因可能只在高度失水胁迫下发挥应激调节作用。

     

    Abstract: Trehalose-6-phosphate synthase(TPS), the key enzyme involved in trehalose biosynthesis, plays particularly important roles in a variety of stress responses.In this study, based on unigene sequences which were obtained from whole transcriptome sequencing of Pyropia haitanensis, three full-length PhTPS genes were obtained by rapid amplification of cDNA ends(RACE), and named PhTPS1, PhTPS2-1 and PhTPS2-2.The full-length cDNA of the PhTPS gene comprised 3 557 nucleotides and contained an open reading frame of 3 462 bp(GenBank accession:KM580358), encoding a protein of 1 153 amino acid residues with the predicted molecular weight of 124.2 ku and theoretical isoelectric point of 6.73;the full-length cDNA of the PhTPS2-1 gene comprised 4 264 nucleotides and contained an open reading frame of 4 044 bp(GenBank accession:KM519457), encoding a protein of 1 374 amino acid residues with the predicted molecular weight of 145.2 ku and theoretical isoelectric point of 5.96;and the full-length cDNA of the PhTPS2-2 gene comprised 3 733 nucleotides and contained an open reading frame of 3 324 bp(GenBank accession:KM519458), encoding a protein of 1 107 amino acid residues with the predicted molecular weight of 120.2 ku and theoretical isoelectric point of 5.42.On the basis of conserved motifs and phylogenetic tree analysis, PhTPS1 belongs to the Ⅰ subfamily of TPS and PhTPS2-1 and PhTPS2-2 belong to the Ⅱ subfamily of TPS.The expressions of the three PhTPS genes, as measured by real-time quantitative PCR, were significantly induced by high-temperature stress and desiccation stress, but had different expression patterns.During high-temperature stress, the expression levels of the three PhTPS genes were all significantly upregulated firstly and then decreased, but as the high-temperature stress continues, the expression levels were upregulated again.However, during desiccation, the expression levels of PhTPS1 and PhTPS2-2 were significantly upregulateded only when the water loss was >60% or 75%, but the expression level of PhTPS2-1 was significantly upregulat when the water loss was >15%.These results suggested that the three PhTPS genes may play important roles in the response to higher desiccation stress.

     

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