斑马鱼卵黄脂磷蛋白在早期胚胎中促进L1-ORF2诱导的EGFP报告基因表达  

Zebrafish(Danio rerio)Lipovitellin Promotes the Expression of EGFP Reporter Gene Induced by L1-ORF2 in Early Embryos

作　　者：王文霞 王晓蝶 武崇光 Luqman ALI 耿琦 张国忠[1] 冯旭[1] 杨悦成 王秀芳[1] 吕占军[1] WANG Wenxia;WANG Xiaodie;WU Chongguang;Luqman ALI;GENG Qi;ZHANG Guozhong;FENG Xu;YANG Yuecheng;WANG Xiufang;LÜZhanjun(Department of Genetics,Hebei Key Lab of Laboratory Animal Science,Hebei Medical University,Shijiazhuang 050017,China)

机构地区：[1]河北医科大学,河北省实验动物学重点实验室,遗传研究室,石家庄050017

出　　处：《中国细胞生物学学报》2024年第5期939-953,共15页Chinese Journal of Cell Biology

基　　金：国家自然科学基金面上项目(批准号:81771499);河北省自然科学基金面上项目(批准号:H2018206099、H2021206460)资助的课题。

摘　　要：为了探讨长散布核元件-1(long interspersed nuclear element-1,L1)在胚胎早期高表达的分子机制,将人L1的第二个开放阅读框(open reading frame 2,ORF2)(L1-ORF2,该文中简称ORF2),插入到pEGFP-C1质粒(C1)的EGFP基因下游,生成C1-ORF2表达载体,插入的ORF2可以影响EGFP报告基因的表达。将C1-ORF2等表达载体注射到斑马鱼(Danio rerio)早期胚胎中,结果表明,在所使用的表达载体中,只有插入ORF2的表达载体诱导EGFP报告基因高表达。C1-ORF2表达载体诱导的EGFP荧光强度随着胚胎的发育而显著降低。EGFP荧光报告蛋白在C1-ORF2中的表达受到组蛋白的抑制,0 h胚胎溶卵液(简称胚胎溶卵液)能减弱组蛋白对EGFP表达的抑制作用,从胚胎溶卵液中纯化的卵黄脂磷蛋白(lipovitellin,Lv)的作用强于胚胎溶卵液。凝胶阻滞实验表明,ORF2 DNA能与胚胎溶卵液蛋白、组蛋白和Lv结合。改变组蛋白、Lv和ORF2片段三者的添加顺序,结果证明Lv可以结合ORF2片段和组蛋白,从而干扰组蛋白与ORF2的结合。进一步研究发现,Lv增加了C1-ORF2 DNA对DNase I消化的敏感性。该研究获得以下结论:Lv可以与所有检测的DNA片段结合,但与ORF2的结合比与其他DNA片段的结合亲和力更高。Lv通过提高ORF2 DNA染色质的易接近性,激活ORF2诱导的斑马鱼早期胚胎EGFP基因的表达。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 in-hibited 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.

关 键 词：长散布核元件-1 斑马鱼(Danio rerio)早期胚胎 卵黄脂磷蛋白 凝胶阻滞实验 染色质重组 体外转录 

分 类 号：Q78[生物学—分子生物学]