Zebrafish (Danio rerio) Lipovitellin Promotes the Expression of EGFP Reporter Gene Induced by L1-ORF2 in Early Embryos
WANG Wenxia, WANG Xiaodie, WU Chongguang, Luqman ALI, GENG Qi, ZHANG Guozhong, FENG Xu, YANG Yuecheng, WANG Xiufang*, LÜ Zhanjun*
To explore the molecular mechanisms of high expression of L1 (long interspersed nuclear element-1) in early embryo, in this study, C1-ORF2 expression vector (in which human L1-ORF2 was inserted into pEGFP-C1 vector) was generated and the inserted ORF2 could affect the expression of EGFP reporter gene. The C1-ORF2 and other vectors were injected into zebrafish (Danio rerio) early embryos. The results showed that only C1-ORF2 induced high expression of EGFP reporter gene. The EGFP fluorescence intensity induced by C1-ORF2 vector significantly decreased with embryonic development. The expression of EGFP reporter in C1-ORF2 was inhibited by histone, but embryo lysate attenuated the histone-induced suppression of EGFP reporter, and Lv (lipovitellin) purified from embryo lysate had a stronger effect than did unmodified embryo lysate. Gel retardation assays showed that ORF2 bound to embryo lysate protein, histone and Lv. Changing the order of histone, Lv and ORF2 fragment addition proved that Lv could bind with ORF2 fragment and histone to interfere the binding of histone to ORF2. This study further found that Lv increased the accessibility of C1-ORF2 DNA to DNase I. It has been concluded that there are a variety of DNA binding proteins in 0 h zebrafish embryos, among which Lv is the most abundant one. Lv can bind to all DNA fragments detected, but binds to ORF2 with a greater affinity than with other DNA fragments. Lv activates EGFP gene expression induced by ORF2 in zebrafish early embryo by enhancing the accessibility of ORF2 DNA.
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