小麦LEAsm基因及其启动子的克隆与功能研究

胚胎发育晚期丰富蛋白(LEA)是一个小的高亲水性的蛋白家族,该蛋白家族在逆境胁迫下大量积累,保护植物免受逆境胁迫。LEA蛋白可分为7组,其中重复的11-氨基酸基序是第3组LEA蛋白的特征。为深入分析第3组LEA蛋白在小麦响应逆境胁迫中的作用机制,利用芯片技术从小麦表达谱中筛选出一个渗透胁迫诱导表达的第3组LEA蛋白基因TaLEAsm,然后根据该基因序列设计引物筛选石麦15的BAC文库,获得1个含有该基因的BAC单克隆,以该BAC单克隆质粒为模板,通过BAC延伸测序克隆了TaLEAsm基因及其启动子序列,并对TaLEAsm序列特征、表达模式和启动子功能进行了初步分析。结果表明,TaLEAsm基因序列仅含有1个105bp的内含子,其开放读码框长675bp,编码224个氨基酸。TaLEAsm含有10个11-氨基酸重复序列,属于第3组LEA蛋白。低温、高盐和渗透胁迫均诱导TaLEAsm基因上调表达,但在根和叶中表达模式不同。在TaLEAsm基因起始密码子上游1 500bp序列中,预测含有14个逆境响应顺式元件。在拟南芥中,TaLEAsm基因启动子能够启动GUS基因表达,渗透胁迫诱导GUS基因明显上调表达。以上结果表明,TaLEAsm为小麦脱水响应基因,其启动子为渗透胁迫诱导启动子。

Isolation and Functional Analysis of Coding and Promoter Regions of LEAsm Gene in Wheat

Late embryogenesis abundant (LEA) proteins are a family of small highly hydrophilic proteins that accumulate markedly in plants under stress conditions and protect plants from the damage caused by stress. LEA proteins can be classified into seven groups,in which group 3 LEA proteins are characterized as a repeating motif of eleven amino acids. In order to analyze the mechanism of group 3 LEA proteins in response to stress conditions deeply,an osmotic-stress induced gene,TaLEAsm,was screened in DNA microarrays for wheat expression profiles. One BAC clone was isolated from Shimai 15 BAC library using primers designed based on TaLEAsm cDNA sequence. TaLEAsm and its promoter sequence were cloned by sequencing walking using BAC clone plasmid as template. Only an 105 bp intron was identified in TaLEAsm. The open reading frame of TaLEAsm is 675 bp long,encoding 224 amino acids.TaLEAsm belonged to group 3 LEA protein containing ten repeating motifs of eleven amino acids. TaLEAsm was strongly up-regulated induced by low temperature,high salt and osmotic stress,showing different expression patterns in root and leaf of wheat. Fourteen stress-response cis-elements were predicted in 1 500 bp sequence upstream of TaLEAsm start codon. TaLEAsm promoter could lead to the expression of GUS gene,which was induced by osmotic stress in Arabidopsis.The results above indicated TaLEAsm may be a dehydration-response gene and its promoter was an inducible promoter.