Identification,Expression And Functional Analysis Of Zebrafish Vitellogenin Receptor Gene

For oviparous animals including most fish,amphibians and birds,vitellogenesis is one of the key stages in the process of oogenesis.During vitellogenesis,the egg will accumulate a large amount of yolk proteins(YPs),providing energy and nutrient sources for early embryo development in vitro.YPs are the production of accumulation of vitellogenin(Vtg)after it enters the oocyte through the vitellogenin receptor(VtgR).Vtg,the precursor of YPs through which VtgR enters oocytes has always been a research hotspot.However so far,vertebrate VtgR has not been fully determined,which lacks the direct functional evidence.In this study,zebrafish(Danio rerio)is used as the animal model and systematically identified and analyzed VtgR.(1)Using lipovitelin(Lv)antibody,we confirmed by Western blotting and fluorescence immunohistochemistry that vitellogenesis in zebrafish depends on the intake of Vtg.(2)There was evidence that the VtgR gene found in many species had been confirmed to belong to the low-density lipoprotein receptor(LDLR)family.In order to identify which VtgR mediates the intake of Vtg,we screen all the genes of the LDLR family members of the current zebrafish according to the level of expression in the ovary.We found that vldlr,lrp13 a,and lrp13b(lrp13 and si: dkey-88l16.3 are named as lrp13 a and lrp13 b by phylogenetic analysis)these three genes have the highest expression in the zebrafish ovary.It is speculated that these three genes may be the zebrafish VtgR.(3)Semi?quantitative reverse transcription polymerase chain reaction(Semiquantitative RT-PCR)and Real-time Quantitative polymerase chain reaction(qPCR)results show that vldlr,lrp13 a and lrp13 b are mainly expressed in zebrafish ovaries,and a small amount is also expressed in body tissues of zebrafish,vldlr is mainly expressed in the brain,muscle and kidney,lrp13 a and lrp13 b are mainly expressed in the kidney,muscle and intestine.(4)qPCR results show that vldlr,lrp13 a and lrp13 b are mainly expressed in oocyte of zebrafish ovary.In vitellogenesis,the overall expression of vldlr,lrp13 a and lrp13 b in zebrafish vitellogenesis do not change significantly,but remain at a certain level.(5)To investigate whether these three VtgR candidate genes are regulated by luteinizing hormone(LH),we analyzed the expression of these three candidate genes in human chorionic gonadotropin(hCG)-induced oocyte maturation by qPCR and find that the expression levels of these three genes are not changed significantly,and their expressions are not regulated by LH.(6)Using a novel gene knockout method——four locus knockout of a single gene based on CRISPR/Cas9(The phenotype can be quickly observed in the G0 generation),the functions of these three candidate genes were analyzed.Using this method,we successfully knock out three candidate genes in zebrafish.The survival rate of embryos produced by vldlr G0 adult zebrafish after selfing is the most significantly decreased at 24 hpf.The survival rate of embryos produced by lrp13 a G0 and lrp13 b G0 zebrafish also decrease to a certain extent,and lrp13 a G0 is slightly higher than that of lrp13 b G0.Moreover,it is found that embryos produced by the same G0 adult fish were different in size,and the degree of difference was ranked as vldlr G0> lrp13 a G0> lrp13 b G0.It can be seen that the fertility of adult zebrafish vldlr,lrp13 a and lrp13 b G0 has been affected to a certain extent.(7)To further confirm the function of the three genes vldlr,lrp13 a and lrp13 b.We further use traditional CRISPR/Cas9 gene knockout technology to obtain homozygous knockout line zebrafish.At present,the experiment has successfully obtained F2 generation zebrafish.Through the above research results,we have preliminarily determined that vldlr,lrp13 a and lrp13 b may be the VtgR gene of zebrafish,the function of vldlr gene in vitellogenesis may be more important,and these VtgR may also have multiple receptor compensation effects.This is also the first time to explore the function of VtgR in vertebrates through gene knockout.The findings in this study contribute to understanding the molecular mechanism of vitellogenesis and provide important information for the study of in vitro culture and reconstruction of fish eggs.