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园艺学报 ›› 2014, Vol. 41 ›› Issue (8): 1663-1672.

• 观赏植物 • 上一篇    下一篇

蜡梅胚胎晚期丰富蛋白基因CpLEA的克隆及表达分析

马 婧,孙文婷,王 晶,眭顺照,李名扬*   

  1. (西南大学园艺园林学院,重庆市花卉工程技术研究中心,南方山地园艺学教育部重点实验室,重庆 400715)
  • 收稿日期:2014-01-17 出版日期:2014-08-25 发布日期:2014-08-25

Cloning and Expression Analysis of a Late Embryogenesis Abundant Protein Gene CpLEA from Chimonanthus praecox

MA Jing,SUN Wen-ting,WANG Jing,SUI Shun-zhao,and LI Ming-yang*   

  1. (Chongqing Engineering Research Center for Floriculture,Key Laboratory of Horticulture Science for Southern Mountainous Regions,Ministry of Education,College of Horticulture and Landscape,Southwest University,Chongqing 400715,China)
  • Received:2014-01-17 Online:2014-08-25 Published:2014-08-25

摘要: 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 50 μmol · 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。

关键词: 蜡梅, 胚胎晚期丰富蛋白, 原核表达, 基因表达分析

Abstract: CpLEA,a new late embryogenesis abundant protein(LEA)gene was identified by randomly cloning and sequencing from Chimonanthus praecox flowers cDNA library. CpLEA has an open reading frame(ORF)of 273 bp encoding a putative 91aa-polypeptide,with two introns of 35 bp and 707 bp respectively. The bioinformatics analysis showed that there were no signal peptide and trans-membrane domain existing in CpLEA protein. Phylogenetic analysis indicated that CpLEA protein can be clustered together with the other known plant group 3 LEA proteins according to its sequence characterizations. Then, the prokaryotic expression vector,which contained the ORF of CpLEA gene,was constructed and the targeted fusion protein at the molecular weight 30 kD coding by CpLEA gene was induced by IPTG. qRT-PCR demonstrated that expression of CpLEA was higher in mature seed compared to that in other tissues and was almost cannot be observed in roots. During the flowering stage,the expression of CpLEA was abundant in the early stage of the flower development and then decreased until the blooming stage and increased significantly at the scence senescence stage. The expression of CpLEA was up-regulated by ABA,cold,salt,drought and hot stresses. It can be to some extent inferred that the CpLEA gene obtained in this study plays an important role in early stage of flower development and abiotic stresses responses.

Key words: Chimonanthus praecox, late embryogenesis abundant protein(LEA), prokaryotic expression, gene expression analysis

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