谷子干细胞决定基因SiWUS的克隆及功能分析

Cloning and functional characterization of the stem cell organizer SiWUS gene in setaria italica

[目的]谷子离体再生效率低,遗传转化困难。为了提高谷子离体再生和遗传转化效率,本文对谷子SiWUS基因在拟南芥干细胞维持分化和体细胞胚胎发生过程中的功能进行了深入研究。[方法]利用同源克隆的方法克隆了谷子SiWUS基因;利用MEGA7.0分析了SiWUS蛋白与其它14个物种WUS蛋白之间的遗传进化关系;最后,用农杆菌介导的花絮浸法将SiWUS基因转到拟南芥中。[结果]从谷子中共鉴定了19个WUS基因,其中与拟南芥相似度最高的为SiWUS基因,全长2 161bp,编码325个氨基酸,包含一个典型的同源异型盒结构域。遗传进化分析表明,谷子SiWUS与狗尾草SvWUS基因亲缘关系最近,蛋白序列一致性为99.69%。进一步,将SiWUS基因克隆到雌激素诱导表达载体pER8,并转化拟南芥,获得了14个株系的阳性转基因植株。pER8-SiWUS转基因拟南芥在10μmol·L-1 17-β-雌二醇诱导培养基上生长发育受阻,根部偶尔会出现体细胞胚;类似地,在10μmol·L-1 17-β-雌二醇诱导培养基上离体培养的根和叶片也产生大量体细胞胚胎。[结论]过量表达SiWUS基因可以促进植物体细胞向胚性细胞的转变,从而大幅提高离体再生效率,因此该基因有望解决谷子遗传转化效率低的难题。

[Objective]The regeneration efficiency of millet in vitrois low,and the genetic transformation is difficult.To improve the efficiency of in vitro regeneration and genetic transformation efficiency in foxtail millet,the function of SiWUS gene in Arabidopsis thaliana stem cell maintenance and somatic embryogenesis is studied in detail in this paper.[Methods]The WUSgene in foxtail millet was identified homologs cloning.The evolutionary relationship between SiWUS and WUSs in 14 other species was analyzed using MEGA7.0.The SiWUSgene was transformed into Arabidopsis using flora dip method mediated by agrobacterium.[Results]We identified 19 WUSgenes in millet.Sequence analysis showed that the full length of the SiWUSgene was 2 161 bp,which encoded 325 amino acids.Similar to WUS proteins in other plants,the SiWUS protein contains a typical homeobox domain.While on the evolutionary relationship,the SiWUS is closest to SvWUS,with 99.69% amino acid identity.The SiWUSgene was further cloning into the estrogen inducible vector pER8,and transformed into Arabidopsis.Finally,14 transgenic lines were obtained and used for further analysis.The growth and development of pER8-SiWUStransgenic Arabidopsis was retarded on the inductive medium containing 10μmol·L-1 17-β-estradiol,and somatic embryos were occasionally observed on root tips;Somatic embryos were generated from roots and leaves of pER8-SiWUStransgenic Arabidopsis cultured on inductivemedium.[Conclusion]Overexpression of the SiWUSgene could promote vegetative to somatic transition,and thus improve the regeneration efficiency.Therefore,the application of the SiWUSgene is expected to solve the problem of low efficiency of genetic transformation in foxtail millet.