花斑裸鲤胚胎/仔鱼型血红蛋白基因家族成员鉴定与时序表达研究

THE SPATIO TEMPORAL EXPRESSION OF EMBRYO/LARVAL HEMOGLOBIN GENE IN GYMNOCYPRIS ECKLONI

为了探讨花斑裸鲤(Gymnocypris eckloni)血红蛋白时序转换利用花斑裸鲤全基因组数据鉴定胚胎/仔鱼型血红蛋白基因家族成员,并通过整胚原位杂交方法,检测花斑裸鲤胚胎/仔鱼型血红蛋白基因在胚胎发育不同阶段的表达及定位。结果表明,花斑裸鲤基因组中共鉴定到5个胚胎/仔鱼型血红蛋白基因,分别为hbae1、hbae4、hbae5、hbbe1和hbbe3,与斑马鱼(Danio rerio)相比,花斑裸鲤基因组缺少hbae3和hbbe2基因,暗示第四轮全基因组复制事件后所经历的小规模基因删除事件在花斑裸鲤特异性血红蛋白基因形成中发挥了重要作用。整胚原位杂交结果显示,hbae1基因在胚胎发育的120h至432h内持续表达,hbbe1基因在96h开始表达持续至432h,hbbe3基因杂交信号出现在胚胎发育120h至384h内,在胚胎发育全过程中未能观察到hbae4和hbae5基因的杂交信号。杂交信号主要位于胚胎正中轴、后部侧向中胚层、背主动脉腹侧区、尾部造血区及卵黄。正义探针作为阴性对照,在胚胎发育阶段均无任何杂交信号。花斑裸鲤具有与其他鱼类不同的胚胎/仔鱼型血红蛋白基因家族成员及血红蛋白转换表达特征;hbae1、hbbe1和hbbe3基因在花斑裸鲤早期胚胎发育过程中发挥重要作用,而hbae4和hbae5基因的生物学功能可能有所弱化。

To investigate the hemoglobin switching and hematopoietic development of teleost fish,the embryo/larval hemoglobin gene repertoire was determined in Gymnocypris eckloni,and the expression and location of embryo/larval hemoglobin genes at different stages of embryonic development was tested by using whole embryo in situ hybridization.The members of the embryo/larval hemoglobin gene family were identified based on the genome data of G.eckloni.Then the RNA probes were prepared according to the obtained CDS sequences.Finally,we used the whole embryo in situ hybridization to investigate the expression patterns of the embryo/larval hemoglobin gene of G.eckloni during early embryonic development.The results showed that there were five embryo/larval hemoglobin genes in G.eckloni genome,including hbae1,hbae4,hbae5,hbbe1and hbbe3.Compared to the zebrafish,the hbae3 and hbbe2 genes have lost in G.eckloni in the genome evolution,suggesting that the recent small-scale gene-deleted events after the latest WGD(4R)have played important roles in shaping the embryo/larval hemoglobin gene repertoire in G.eckloni.The results of the whole embryo in situ hybridization experiment showed that the hbae1 gene expressed at 120h and continued to 432h of embryonic development,while the hbbe1 gene expressed at 96h and continued to 432h.It was observed that the hbbe3 genexpressed at 120h and continued to 384h.The hybridization signal of hbae4 and hbae5 could not be observed in the whole process of embryo development.The hybrid signals were mainly located in the median axis of the embryo,the posterior lateral mesoderm,the ventral area of the dorsal aorta,the caudal hematopoietic tissue and the yolk sac.The sense probe was used as a negative control,the hybridization signal was not observed during embryonic development.Embryo/larval hemoglobin gene expression and localization studies showed that G.eckloni has unique characteristic of hemoglobin switching.Among them,hbae1,hbbe1 and hbbe3 played important roles in the embryo development of the G.eckloni,while the biological functions of hbae4 and hbae5 may be weakened.