摘要
干旱、盐碱和冻害等非生物逆境严重影响了植物的生长发育。在逆境胁迫下,植物可以启动或增强两类基因的表达来抵御不利环境的危害:(1)基因编码的产物包括传递信号和调控基因表达的转录因子,如bZIP转录因子、MYC转录因子、MYB转录因子及DREB转录因子等;感应和转导胁迫信号的蛋白激酶(如MAP激酶、核糖体蛋白激酶和转录调控蛋白激酶等);以及在信号转导中起重要作用的蛋白酶(如磷酸醋酶、磷脂酶C等)。(2)基因编码的产物包括一些功能蛋白和酶类,如LEA蛋白、水通道蛋白、脯氨酸合成酶等,使细胞各种生理生化代谢活动能维持正常进行。由于植物基因的表达调控大多发生在转录水平上,主要受控于顺式作用元件和反式作用因子。尤其是转录因子基因的表达可提高其下游一系列抗逆功能基因的表达水平,从而提高植物的综合抗逆性,在抗逆转基因育种中具有重要功能价值。因此,分离和筛选鉴定优异的转录因子基因是当前抗逆分子生物学研究的重点。
本研究中,以不同基因型高粱为材料,利用简并引物和RACE技术及RT-PCR研究了一个高粱中由高盐诱导的DREB类转录因子基因。首先根据GenBank中多种植物AP2/EREBP转录因子氨基酸序列的保守区段设计了一对简并引物,通过RT-PCR扩增出一条117 bp的cDNA片段。经Blast比较及测序验证其正确性后,根据此片段序列设计3’端特异性引物,利用3’RACE得到949bp的3’端cDNA片段,命名为SbDREB1001,并对其进行了相关的生物信息学分析。该序列包括789 bp的开放阅读框,编码262个氨基酸,该肽段分子量为28.6 kDa,理论等电点为5.52。生物信息学分析显示该基因具有C端疏水区,并且它的蛋白序列在第83~138氨基酸处包含一个保守的AP2结构域。DREB氨基酸序列与其他植物DREB类基因有很高的同源性,其序列经Blast软件进行同源性分析,表明与已知的植物DREB基因的序列具有很高的同源性,该基因与玉米的一个DREB类基因(No.:NM-001158997.1)同源性高达88%。利用实时定量PCR技术,分析了该基因在高盐胁迫条件下转录表达情况。分析结果表明,DREB类基因参与了高粱对高盐非生物胁迫的应答反应,但在不同物种或胁迫处理下的表达模式不同,其中623B在高盐胁迫下出现了明显较快的应答反应,在623B中的表达量高峰比河农16提早2h左右。推测DREB类转录因子在高粱受到盐胁迫条件下发挥重要功能。
Abiotic stresses such as drought,salt and freezing could produce severe damages on growth and development of plants. Genes upregulated upon these stress conditions were basically classified into two groups:those that directly protect against environmental stresses,and those that regulate gene expression and signal transduction upon stress conditions. The first group of gene products includes transcription factors,protein kinases,and enzymes involved in phosphoinositide metabolism,such as bZIP, MYC,MYB,DREB,MAP, Phosphoric acid vinegar enzyme, et al.The second group includes proteins and enzymes that likely function by protecting cells from dehydration,such as late- embryogenesis-abundant (LEA)proteins,enzymes required for biosynthesis of proline,and aquaporins (AQP), et al, making the cell various physiological and biochemical activity can maintain normal metabolism. In the past few years,scientists have showed great interest in the second group of genes.In plants,the expression of many genes were regulated at transcriptional level,and mostly it’s realized through cis- and trans-acting elements. Therefore,transcription factors could play a key role in stress prevention in plants.
In this study, we obtained a DREB gene from Sorghum bicolor(L.) by RT-PCR using degenerate primers and RACE techonolgy. Through the multiply alignment of amino acid sequences and nucleotide acid sequences of several plant AP2/EREBP in GenBank, a pair of degenerate primers were designed against the conserved regions that was used for RT-PCR to amplify a 117 bp cDNA fragment from Sorghum bicolor(L.). Base on the sequence of the cDNA fragment, primers were designed for 3’RACE and a 949 bp cDNA fragment was amplified. It was named SbDREB1001, and was analyzed with relative bioinformatics methods. The DREB cDNA sequence was 949 bp long with an ORF (open reading frame) of 789 bp, encoding a predicted polypeptide of 262 amino acid with a calculated MW of 28.6 kDa and pI of 5.52. It contain a C-terminal hydrophobic domain. In addition, one AP2 domain between 83 and 138 amino acid. The amino acid sequence compared by NCBI_Blast revealed high homology with that of other plant DREB genes, and the similarity to ethylene response factor 2 of Zea mays(GeneBank Accession No.:NM-001158997.1)was 88%. The real-time fluorescence quantitative PCR was performed to reveal transcript level of DREB under different abiotic stresses. Real-time quantitative polymerase chain reaction (PCR) expression analysis results showed that DREB gene was involved in sorghum response to salt stress, but the response in speed was different in different materials under stress condiction.The response was 2 hours faster in 623B than HN16 under salt stress. The DREB gene insorghum play an important function under salt stress condition.
引文
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