| Retinoic acid(RA)is an endogenous signal molecule that plays its regulatory role mainly through binding to nuclear receptors RAR and RXR.Abnormalities of retinoic acid signaling pathway can cause congenital diseases such as heart disease and nerves.System defects and body malformations.Aldhlal,Aldhla2,Aldhla3,and Aldhla4 in Aldehyde dehydrogenases1a(Aldhla)family can encode retinal dehydrogenase and participate in biosynthesis of retinoic acid in vivo.However,whether Aldehyde dehydrogenases4al(Aldh4a1)can encode retinal dehydrogenase and participate in biosynthesis in vivo has not been reported yet.The results obtained in this dissertation are as follows:(1)The zebrafish Aldh4al was knocked out by CRISPR/Cas9 technology,and the homozygous mutant F2 generation with frameshift mutation(-5bp)was screened to generate a truncated protein encoding 143 amino acids.(2)RT-PCR and qPCR were used to study the expression of Aldh4al in various stages of the wild zebrafish embryos and their tissues and organs.The results showed that it was maternally expressed,and the amount of mRNA gradually decreased with time,and zygotes developed at 24h.The gene expression reaches a peak,and is higher in the testis and muscle,and lower in the ovary and other tissues and organs.(3)Aldh4al heterozygous and F1 mutants crossed each other,and some F2 zebrafish died when they developed to the fourth or so day.The genome of the dead zebrafish was extracted individually and its genotypes were detected and found to be homozygous for Aldh4al-/-.Mutants,the statistical results show that it accounts for 25%of all F2 embryos,The embryos that were randomly tested for normal phenotype had wild-type or heterozygous genotypes and no mutant homozygote,which proves that Aldh4al is a recessive lethal gene.(4)Observed Aldh4al homozygous mutant phenotype,found that pericardial cysts,the head of the eye becomes smaller,the body becomes shorter and curved,less pigment,abnormal morphology.(5)In situ hybridization showed that the expression of ventricular myosin heavy chain gene vmhc(ventricular myosin heavy chain)in the 36hpf Aldh4al homozygous mutant ventricle expands to the rear,and the expression of the cardiac myosin heavy chain gene increases in the precordial region.The amhc(atrial myosin heavy chain)expression level in the atrium of the 36 hpf Aldh4al homozygous mutant is reduced and the expression range is reduced,showing abnormal heart development.(6)Statistical analysis of Aldh4al-/-heart rate under the microscope revealed that the heart rate was significantly lower than that of the wild type and the blood circulation was weakened.(7)In situ hybridization showed that the expression of HuC in the zebrafish brain of 48hpf was significantly reduced,and the expression of the expression in the hindbrain was most significantly reduced,indicating abnormal brain development.(8)72 hpf Aldh4al homozygous mutant and 72 hpf wild-type zebrafish were subjected to the same external stimulus under the microscope.It was found that Aldh4al homozygous mutants could not respond to external stimuli and should be almost completely extinguished.(9)The myod(myogenic differentiation)marker segment in in situ hybridization co-localization,the krox20 marker rhombohedral primitive segment 3,and the rhombohedochordial primary segment 5,show that in the Aldh4al homozygous mutant,between the rhombohedral primitive segment 5 and the first somite The distance is relatively short.(10)Alzheimer's staining analysis showed that the Aldh4a1 homozygous mutant had abnormal phacoarchal cartilage development.The mandibular arch and cartilage cartilage completely disappeared,the hyoid bone volume decreased,and there was no pectoral fin.(11)The retinoic acid compensation experiment showed that the exogenously added dose of retinoic acid,Aldh4al homozygous mutant aberrant phenotypic disappeared,and proved that Aldh4al is also a retinoic acid dehydrogenase,involved in retinoic acid Biosynthesis process.In this thesis,Cldn7 gene was knocked out using CRISPR/Cas9 technology to study its effect on male zebrafish reproduction.A-4 bp Cldn7b hybrid mutant was obtained... |
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