| fn1 gene encodes fibronectin?FN?,which participates in cell migration,adhesion,tissue structure,remodeling,wound healing and host defense,and plays an important role in embryonic development and organogenesis,but it is not certain that whether it acts on the formation of fish air-breathing.Dojo loach?Misgurnus anguillicaudatus?,a typical freshwater accessory air-breathing fish,has been engaged in the study of the molecular mechanism of fish air-breathing in our laboratory for a long time,and fn1gene has been concentrated in playing an important role in the air-breathing of Dojo loach,while the specific mechanism is not clear.Therefore,in this study,fn1 gene of Dojo loach was cloned and analyzed firstly,and the expression pattern of fn1 was revealed by qRT-PCR,whole embryo and tissue sections in situ hybridization.Thus the high expression level of fn1 in barbels was discovered.Then,the morphology and histology of Dojo loach barbels under normal and hypoxia were observed and compared to find out its structural characteristics and adaptability to air-breathing.Finally,the model of fn1b+/-was constructed based on CRISPR/Cas9 technology.Combined with hypoxia experiment,the histological and physiological parameters related to air-breathing of fn1b+/+and fn1b+/-were measured,and the related genes of fn1b+/-were quantitatively analyzed.Consequently,it was preliminarily discussed that the mechanism of fn1 gene on air-breathing function of Dojo loach in the perspective of barbels,in order to provide new ideas and clues for studying the molecular mechanism of fish air-breathing.The main results were as follows:1. Two fn1 gene ORF sequences of Dojo loach were obtained by one-step cloning.Then referred to the fn1 sequences of zebrafish and other bony fish,they were named fn1a and fn1b respectively.fn1a encoded 2471 amino acids and fn1b 2398 amino acids.According to the multiple comparative analysis of the full-length amino acid sequence,it was found that both fn1a and fn1b contained three types of repetitive sequences,but fn1b lacked a III repeat module?FN3?,which showed structural specificity.The results of molecular phylogenetic analysis showed that the evolutionary positions of fn1a gene and fn1b gene of Dojo loach were differentiated,and their genetic distances were respectively closest to those of fn1a gene and fn1b gene of zebrafish with the same taxonomic status in Cyprinidae.Spatio-temporal expression showed that two fn1 genes of Dojo loach were highly expressed in barbels,especially fn1b gene,and their expressions were different and complementary in embryonic development.qRT-PCR results of adult tissues indicated that the barbels-specific expression of fn1b gene was significant in Dojo loach.Last,the expression of fn1b gene was located in the vascular structure of barbels in Dojo loach through whole embryo in situ hybridization and barbel-sections in situ hybridization.2. Morphological observation showed that the part of the oral flap of Dojo loach as the inner and outer lip,the outer lip had 3 pairs of barbels,and the inner lip had 2pairs of mental lobes.Morphological measurements showed that there was a significant positive correlation between barbels'length and body length of Dojo loach.Histological observation showed that the structure of barbels of Dojo loach was in the following order:epithelium,collagen fiber layer,muscle fiber layer,loose connective tissue layer and central vascular layer,and erythrocytes were distributed in clusters in the central vascular layer,which was obviously different from the axial cartilage in the center of the barbels of other three fish species with barbels?Large-scale loach,Zebrafish and Yellow catfish?.Under the induction of hypoxia,the barbels of Dojo loach were hyperemia,the number of erythrocytes in the central vascular area was significantly increased,and the diffusion distance of air-blood barrier was significantly shortened,showing adaptability to accessory respiration,can be regarded as the leading organ of air-breathing.After the barbels were removed,the number of air-breathing of Dojo loach decreased significantly,which may be due to the function of hypoxia sensation in the barbels.3. fn1b+/-model of Dojo loach was generated based on CRISPR/Cas9 technology,and embryonic development of fn1b+/-was obviously delayed,with possible phenomenon of“fn1b-/-lethal”.Under hypoxia treatment,the expression of fn1b gene in barbels and posterior intestine of Dojo loach showed a trend of"rising first,falling then",indicating an obvious pattern of hypoxia response.Histological measurements under hypoxia performed that air-blood diffusion distances of maxillary barbel and posterior intestine in fn1b+/-were shortened successively,both of which were higher than that of fn1b+/+,indicating that the knock-out of fn1 gene led to the decrease of air-breathing ability of Dojo loach.Physiological measurements under hypoxia showed that the reaction time of fn1b+/-was longer,the asphyxia point was lower,the average oxygen consumption rate was slightly lower and the air-breathing rate was significantly lower than fn1b+/+after their barbels-excision,indicating that the knock-out of fn1 gene further aggravated the weakening of air respiration in Dojo loach due to lack of barbels;meanwhile,the number of erythrocytes of fn1b+/-decreased significantly under hypoxia,and the concentrations of Hb and Met Hb did not change significantly,compared with the results of the constant count of erythrocytes,the increased Hb concentration and the decreased Met Hb concentration.Combined with the decreased expression of epor and hbb genes in fn1b+/-,it was suggested that the knock-out of fn1 gene may affect the oxygenation ability of Hb by inhibiting the production of erythrocytes under hypoxia.Above all,according to the changes of hypoxia response of related genes expression in fn1b+/-barbels,combined with the changes of hypoxia response of Dojo loach in histology and physiology above mentioned,it was preliminarily discussed that fn1 gene may play its role in three aspects:angiogenesis,erythropoiesis and hypoxia sensation for Dojo loach barbels air-breathing. |
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