龙眼体细胞胚胎发生过程中DlWUS的克隆与表达分析

Cloning of DlWUS from Embryogenic Callus and Its Expression Analysis during Somatic Embryogenesis in Dimocarpus longan Lour.

以龙眼‘红核子’LC2悬浮细胞系诱导的胚性愈伤组织为基本材料,按照龙眼体细胞胚胎同步化方法诱导获得龙眼体胚不同阶段材料,并以龙眼体细胞胚胎发生不同阶段混合材料作为试验材料,采用RT-PCR结合RACE技术分离并克隆龙眼中编码同源异型结构域蛋白的转录因子WUSCHEL(简称DlWUS)的cDNA全长及DNA序列,并进行序列分析与表达分析。结果表明:DlWUS的cDNA全长1 110bp,开放阅读框(ORF)858bp,共编码285个氨基酸(GenBank登录号为KM017506),DlWUS的DNA包含2个内含子。序列分析表明,DlWUS是一个不稳定的亲水蛋白,不含信号肽,亚细胞定位于细胞核,具跨膜结构和Homeodomain超级家族的保守结构域以及WUS转录因子家族特有的WUS box和EAR-like结构域,推测该目的基因确实为WUS转录因子。系统进化分析显示,龙眼DlWUS与脐橙WUS归为一个分支,亲缘关系较近。实时荧光定量PCR分析结果表明,在龙眼体细胞胚胎发生整个过程中,DlWUS均有表达,但仅在球形胚时期表达量较高,说明DlWUS可能主要在球形胚阶段发挥作用,并且在一定浓度范围内,外源施加IAA和GA3能够促进DlWUS基因的表达,而外源施加SA则抑制DlWUS基因的表达。

Different stages of somatic embryos were obtained in embryogenic callus from embryogenic cell suspension culture of longan,and used these somatic embryos as the test material. The RT-PCR combined with RACE method was used to clone the complete cDNA sequences and DNA sequences of homeodomain transcription factor WUSCHEL （DlWUS） from embryogenic callus of Dimocarpus longan Lout.. The complete cDNA sequence of DlWUS was 1 110 bp,encoding 285 amino acids DNA sequence analysis indicated that it contains 2 introns. DlWUS located in nucleus;it had no signal peptide,had transmembrane structure,homeobox d omain,WUS box and EAR-like domain. Anglicizing phylogenetic tree of WUS in plants indicated that DlWUS and CsWUS belonged to the same branch,therefore,DlWUS belonged to WUS family. Real-time quantitative PCR results indicated that DlWUS had expression at all stages,but it expressed at the highest levels in the globular embryos, suggesting that DlWUS play a major role at longan somatic embryogenesis. In a certain concentration range, DlWUS showed up-regulated expression patterns under exogenous IAA and GA3 and down-regulated expression patterns under exogenous SA.