海岛棉WOX9基因(GbWOX9)的克隆及表达模式分析

Cloning and expression pattern analysis of WOX9 gene from Gossypium barbadense L. (GbWOX9)

[目的]WOX9基因在植物的胚胎发育过程中发挥一定作用。[方法]根据已公布的雷蒙德氏棉WOX9基因序列的CDS设计一对引物,从海岛棉‘新海16’中克隆一个同源基因GbWOX9。通过实时荧光定量分析其在棉花体细胞胚不同阶段的表达模式。[结果]获得了1 038 bp的GbWOX9基因的开放阅读框(ORF),编码345个氨基酸,预测蛋白分子量约为38 264.54 k Da,等电点为6.7。通过结构域分析,发现GbWOX9中含有一个由65个氨基酸组成的保守同源异型结构域。系统进化树分析结果表明GbWOX9与亚洲棉Ga WOX9亲缘关系较近。亚细胞定位预测该基因可能定位于细胞核。qRT-PCR表明,GbWOX9基因的表达量在‘新海16’胚胎发育阶段的球形胚>鱼雷胚>心形胚>胚性愈伤。[结论]根据相对表达量数值估算,GbWOX9在球形胚的表达量是鱼雷胚的1.3倍,是心形胚的3.1倍,是胚性愈伤的5.2倍。故该基因在‘新海16’体细胞胚阶段的球形胚中表达量最高。

[ Objective ] WOX9 gene plays an important role in the embryo development of plants. [ Methods ] According to the published C, ossypium raimondii WOX9 gene CDS,a pair of primers were designed and a homologous gene GbWOX9 was cloned from Gossypium barbadense‘ Xinhai 16＇ with PC R technology. [ Results ] The 1038 bp GbWOX9 open reading frame （ORF） was contained, which encoded 345 - amino - acid protein, about 38 264.54 kDa. And the isoelectric point of the protein was about 6.7. The amino acid sequence alignment showed that the sequence contains a conserved 65 amino acid home domain ; and the phylogenetic tree analysis demonstrated that GbWOX9 had a close relationship with Gossypium arboretum C, aWOXg. Sub cel- lular localization prediction displayed that GbWOX9 have a high possibility in the nuclear. Real time fluorescence quantitative PCR showed that the expression of GbWOX9 in ‘ Xinhai 16＇ embryonic development stage of globular embryo 〉 torpedo embryo 〉 heartshaped embryo 〉 embryogenic callus. [ Conclusion ] According to relative expression value estimation, the expression of the globular embryo stage was 1.3 times higher than torpedo embryo,3.1 times higher than heart -shaped embryo and 5.2 times higher than embryogenic callus. So the highest expression level of GbWOX9 in the ‘ Xinhai 16＇ somatic embryos stage was globular embryo.