棉花GhZIP4基因的克隆及表达分析

Cloning and Expression of GhZIP4 Gene from Gossypium hirsutum

根据EST拼接的序列设计引物,利用RT-PCR和PCR方法,从陆地棉‘苏棉18’-cDNA和基因组DNA中分别克隆获得了GhZIP4基因片段。结果表明:(1)GhZIP4基因cDNA序列全长1 487bp,包含1 269bp ORF,编码422个氨基酸残基,其氨基酸序列具有典型的ZIP蛋白特征,预测具有8个跨膜结构域,第Ⅲ和第Ⅳ跨膜结构域间存在可变区,在可变区有2个富含His的结构域"HRHSHPHG"和"HSHGHGHD"。(2)氨基酸进化树分析显示,GhZIP4同拟南芥ZIP家族AtZIP4的相似性较高。(3)GhZIP4DNA序列编码区全长1 778bp,包含4个外显子和3个内含子,所有外显子/内含子交接点都遵从gt/ag剪接规则。(4)半定量分析显示,GhZIP4基因在茎中表达量最高,表明该基因有可能在某些金属离子地上部和根部的动态平衡分布过程中具有重要作用。

A GhZIP4 gene fragment was obtained from cDNA and genomic DNA of upland cotton Sumian 18 by RT-PCR and PCR with the primers designed according to the assembled ESTs of Gossypium hirsutum.The result showed that：（1）The length of the GhZIP4 cDNA sequence was 1 487 bp,it contained an ORF comprising of 1 269 bp nucleotides which encodes 422 amino acids.The amino acids sequence had the characteristics of the ZIP protein,containing 8 trans-membrane domains and a variable region between the third and the forth trans-membrane domains that included two histidine-rich domains of ＇HRHSHPHG＇ and ＇HSHGHGHD＇.（2）Phylogenic tree analysis showed that the protein sequence of the GhZIP4 was highly similar to AtZIP4 of Arabidopsis ZIP family.（3）The full length of coding region of the GhZIP4 DNA sequence was 1 778 bp,containing three introns and four exons.All exon/intron junctions of the gene contained the gt/ag consensus splicing site.（4）Semi-quantitative analysis of the GhZIP4 expression showed that the gene highly expressed in stem,indicating that it may play an important role in dynamic homeostasis of some metal ion between root and shoot.