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从蜡梅(Chimonanthus praecox)花cDNA文库中获得了1个胚胎晚期丰富蛋白(LEA)基因的cDNA全长序列,命名为CpLEA(GenBank登录号:JF412788),该基因cDNA含有一个273 bp编码91个氨基酸的开放阅读框,从蜡梅基因组DNA中扩增该基因发现,该基因含有两个大小分别为35 bp和707 bp的内含子。生物信息学分析显示,CpLEA的编码蛋白含有4个胚胎晚期丰富蛋白第3族LEA蛋白特有的11个氨基酸的基元重复序列,进化树分析表明该编码蛋白与榛子的LEA蛋白亲缘关系最近。通过原核表达获得了与预期大小一致的融合蛋白。实时荧光定量PCR分析表明,该基因在蜡梅成熟种子中表达量最高,在根中几乎不表达;在盛开期花的各个部位中又以雌蕊中的表达量最高;在花发育早期表达量较高,随后下降并保持相对稳定,在衰败期突增并达到最高;检测该基因在脱落酸(ABA 50μmol·L-1)、低温(4℃)、高温(42℃)、干旱(30%PEG6000)和高盐(NaCl 1 mol·L-1)5种外源非生物胁迫因子作用下的表达特性。结果表明,该基因能够被ABA、低温、高温、PEG和NaCl诱导表达,并在随后的时间内呈现出不同的表达模式。表明CpLEA可能在蜡梅花发育和多种非生物胁迫响应中发挥作用。
A complete cDNA sequence of LEA gene was obtained from the cDNA library of Chimonanthus praecox and named CpLEA (GenBank accession number: JF412788). The cDNA contained a 273 bp cDNA encoding 91 Amino acid open reading frame, the Pichia genome amplification of the gene found that the gene contains two sizes were 35 bp and 707 bp intron. Bioinformatics analysis revealed that the encoded protein of CpLEA contained 11 unique amino acid sequence repeats of 4 LEA proteins of group 3 of late embryo abundance, and phylogenetic tree analysis indicated that this protein has the closest genetic relationship with LEA protein of hazelnut. By prokaryotic expression obtained with the expected size of the fusion protein. Real-time PCR analysis showed that the gene was expressed most abundantly in the mature seed of Chimonanthus praecox and almost not expressed in the roots, and the expression was highest in the pistil in all parts of the flower in full blossom stage. High, then decreased and remained relatively stable, and then increased and reached its peak during the decay period. The results showed that ABA 50 μmol·L-1, low temperature (4 ℃), high temperature (42 ℃), drought (30% PEG 6000 ) And high salt (NaCl 1 mol·L-1) under five exogenous abiotic stress factors. The results showed that the gene could be induced by ABA, hypothermia, high temperature, PEG and NaCl, and showed different expression pattern at later time. Suggesting that CpLEA may play a role in waxy plum development and in a variety of abiotic stress responses.