斑马鱼deltaD基因对血管内皮生长因子家族基因(vegfaa和flt4)的调控作用  

Regulation of deltaD Gene on Vascular Endothelial Growth Factor Family Genes(vegfaa and flt4)in Zebrafish(Danio rerio)

作　　者：杜璇 王子睿 包林珠 程瑶 任建峰 李伟明[3] 张庆华 Du Xuan;Wang Zirui;Bao Linzhu;Cheng Yao;Ren Jianfeng;Li Weiming;Zhang Qinghua(International Research Center of Ministry of Science and Technology for Marine Biosciences,Shanghai Ocean University,Shanghai,201306;Key Laboratory of Ministry of Education for Exploration and Utilization of Aquatic Genetic Resources,Shanghai Ocean University,Shanghai,201306;Deparment of Fisheries and Wildlife,Michigan Sate University,East Lansing,48824)

机构地区：[1]上海海洋大学,科技部海洋生物科学国际联合研究中心,上海201306 [2]上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海201306 [3]密歇根州立大学渔业与野生生物系,东兰辛48824

出　　处：《基因组学与应用生物学》2022年第4期742-751,共10页Genomics and Applied Biology

基　　金：教育部留学回国人员科研启动基金项目(D-8002-15-0042);水产动物疾病与基因编辑育种的平台建设和前沿科学研究项目(A1-3201-19-3013);中美海洋研究中心基金项目(A1-0209-15-0806)共同资助。

摘　　要：为了探究斑马鱼(Danio rerio)deltaD基因对血管内皮生长因子(vascular endothelial growth factor,VEGF)家族基因vegfaa和flt4的调控作用,本研究通过红色荧光转基因斑马鱼系的成像发现deltaD纯合突变体的节间血管(intersegmental vessels,ISVs)出现了生长紊乱的现象,RT-qPCR实验检测到受精后1 dpf(day post fertilization)大小的deltaD纯合突变体中VEGF家族的vegfaa配体和flt4受体基因的表达量均下调,且flt4基因的表达量显著性下调。之后应用双荧光素酶(Luciferase)实验体外验证deltaD基因对vegfaa和flt4的调控作用。利用加州大学圣克鲁兹分校基因组浏览器数据库获得斑马鱼deltaD、vegfaa及flt4基因序列,并成功构建表达载体p3xFLAG-CMV-deltaD和双荧光素酶报告基因表达载体pGL3-vegfaa-Luc、pGL3-flt4-Luc。双荧光素酶报告基因实验结果显示,pGL3-vegfaa-Luc和pGL3-flt4-Luc报告基因载体在HEK293T细胞中的荧光素酶活性分别约为对照组的92倍和3.9倍。进一步将p3xFLAG-CMV-deltaD分别与pGL3-vegfaa-Luc、pGL3-flt4-Luc转染至HEK293T细胞,发现与对照组相比其荧光素酶活性分别约为2.3倍和3.7倍。本研究明确了Notch信号通路的配体基因deltaD可以通过调控血管内皮生长因子家族中的vegfaa配体和flt4受体基因而影响血管发育,为深入研究Notch信号通路在血管发育中的调控机制提供实验材料和科学依据。In order to explore the regulatory effect of deltaD gene on vascular endothelial growth factor(VEGF)family genes vegfaa and flt4 in zebrafish(Danio rerio),in this study,through the imaging of red fluorescence transgenic zebrafish line,we found that the defected angiogenesis of intersegmental vessels(ISVs)in deltaD homozygous mutants,and RT-qPCR experiments detected that the expression of vegfaa and flt4 of VEGF family genes were down-regulated in deltaD homozygous mutant at 1 dpf(day post fertilization),and the expression of flt4 gene was significantly down-regulated.The sequence of deltaD,vegfaa and flt4 genes of zebrafish were obtained from genome browser database of University of California,Santa Cruz,and the expression vector p3 xFLAG-CMV-deltaD and luciferase reporter gene vectors pGL3-vegfaa-Luc and pGL3-flt4-Luc were successfully constructed.The luciferase assay showed that the activity of pGL3-vegfaa-Luc and pGL3-flt4-Luc reporter gene vectors in HEK293 T cells was about 92 times and 3.9 times higher than that of the control group,respectively.Furthermore,p3 xFLAG-CMV-deltaD was co-transfected with pGL3-vegfaa-Luc and pGL3-flt4-Luc into HEK293 T cells,comparing with the control group,the luciferase activity of p3 xFLAG-CMV-deltaD was 2.3 times and 3.7 times,respectively.This study confirmed that the ligand gene deltaD of Notch signaling pathway can play a role in vascular development by regulating vegfaa ligand and flt4 receptor genes in vascular endothelial growth factor family,which provides experimental materials and scientific basis for studying the regulatory mechanism of Notch signaling pathway in vascular development.

关 键 词：斑马鱼 deltaD vgefaa FLT4 双荧光素酶报告基因 

分 类 号：Q953[生物学—动物学]