摘要
【目的】克隆小麦蛋白磷酸酶2A(PP2A)调节亚基(PR55)基因TaBβ-1,分析其在非生物胁迫下的表达特性,为小麦抗逆育种提供候选基因。【方法】以小麦品种旱选10号为材料,通过电子克隆和RT-PCR获得TaBβ-1的全长cDNA序列,采用生物信息学软件分析TaBβ-1及其编码蛋白TaBβ-1的序列特征,预测其功能,利用实时荧光定量PCR(real-time quantitative PCR)技术分析该基因在小麦孕穗期不同组织中、不同生育时期新叶中的表达情况,以及在PEG、NaCl、低温及外源激素ABA等非生物胁迫下的表达模式,检测不同水分条件下成株期转基因拟南芥的叶片细胞膜稳定性。【结果】获得TaBβ-1的全长cDNA序列1931bp,其开放阅读框(ORF)为1539bp。该基因编码512个氨基酸,预测TaBβ-1蛋白分子量为57.1kD,等电点为5.87,含有PP2A调节亚基的1个CDC55保守结构域、1个alpha/beta结构域、2个PR55保守结构域和6个WD重复子。TaBβ-1在小麦孕穗期的根、穗、叶中均有表达,表达量依次为根>穗>叶;在不同生育时期的新叶中,苗期叶片的表达量最高。TaBβ-1的表达明显受PEG、NaCl、低温以及外源ABA的诱导。【结论】小麦蛋白磷酸酶2A调节亚基家族基因TaBβ-1在小麦苗期叶片中的表达量明显高于根、穗以及其它时期的叶片;TaBβ-1参与对高渗、高盐、低温等多种胁迫以及ABA处理的应答反应,但表达模式不同;在水分胁迫条件下,转基因拟南芥比野生型具有较高的细胞膜稳定性。
【Objective】 TaBβ-1,a gene encoding protein phosphatase 2A （PP2A） regulatory subunit （PR55） was cloned and its expression patterns in wheat under abiotic stresses were dissected for the purpose of providing a candidate gene for wheat breeding in response to various abiotic stresses.【 Method】 A wheat （Triticum aestivum L.） cultivar Hanxuan 10 was used as the plant material to clone the full-length cDNA sequence of TaBβ-1 by in silico cloning and RT-PCR.Suitable bioinformatics softwares were adopted to assay the structure of cDNA sequence,and the function of deduced TaBβ-1.The real-time quantitative PCR （qRT-PCR） method was employed to determine the expression patterns of TaBβ-1 in specific tissues and under abiotic stresses in wheat.【Result】 A full-length cDNA sequence encoding regulatory subunit of PP2A was cloned from wheat and designated by the name of TaBβ-1,which is a 1 931 bp sequence with an 1 539 bp open reading frame.The putative amino acid sequence of TaBβ-1 contains 512 amino acids with a molecular mass of 57.1 kD and pI value of 5.87,which contains a CDC55 conserved domain,an alpha/beta domain,two PR55 conserved sites and six WD-repeats of PP2A subunit B.TaBβ-1 was detected in all tissues,including root,leaf and young-spike of wheat.The expression levels of TaBβ-1 at booting stage were root spike leaf.The highest expression level was identified from the seedling leaf among young-leaves at jointing,booting and flag-leaf expansion stages.TaBβ-1 was up-regulated by PEG,NaCl,cold and ABA treatments.Leaf cell membrane stabilities of transgenic Arabidopsis over-expressing TaBβ-1 were detected under well-watered and drought stress conditions.【Conclusion】 TaBβ-1 is a member of PP2A regulatory subunit family.The expression level of TaBβ-1 in seedling leaf is much higher than that in the root and spike at booting stage,and in leaves at different stages.TaBβ-1 responds to multi-abiotic stresses but shows varied expression patterns,implying that TaBβ-1 involves in different signal pathways responding to abiotic stresses.Transgenic TaBβ-1 Arabidopsis lines show significantly higher cell membrane stabilities under drought stress condition than the wild type.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第11期2197-2208,共12页
Scientia Agricultura Sinica
基金
北京市优秀博士学位论文指导教师项目(YB20088210101)
国家转基因重大专项课题(2009ZX08002-012B)
