| Tetrodotoxin(TTX),a highly toxic neurotoxin,was first found in the ovary of Fugu,and later in terrestrial,aquatic and other unrelated animals and sediments in marine and fresh water Found TTX.Takifugu bimaculatus and T.flavidus are the main species of Fugu culture in Fujian Province,but the water quality and feeding form are different in different breeding areas.At the same time,the distribution of TTX in the two species is not clear in different development periods and seasons.There is a big difference in TTX content between Fugu individuals,which limits the development of Fugu industry chain in Fujian.It is very important to determine the distribution of TTX and to establish a stable genetic breeding system for Fugu with low or no toxicity.At present,the process and origin of TTX biosynthesis are not clear,so it is very difficult to intervene in TTX synthesis to reduce the amount of TTX synthesis,which has become the bottleneck of stable genetic breeding of Fugu.So,we used T.bimaculatus as the research material.On the one hand,by means of LC-MS/MS,we understood the distribution of TTX in its body.On the other hand,we planned to start with the regulation of TTX accumulation pathway to establish a toxic animal model,to understand the distribution,reduction and tissue and cell microscopic of exogenous TTX accumulation in the model.At the same time,through the analysis of the model transcriptome,the accumulation mechanism of exogenous TTX in T.bimaculatus was preliminarily discussed,and the genes related to TTX accumulation were screened,which laid the foundation for future gene intervention and development of stable genetic breeding of low or non-toxic T.bimaculatus.The main research contents and results are as follows:1.Using LC-MS/MS detection method,(1)The TTX in different growth stages(sperm,unfertilized egg,embryo,adult and adult tissues in larval stage)of cultured T.bimaculatus was analyzed qualitatively and quantitatively to clarify the distribution of TTX in different development stages of cultured T.bimaculatus;(2)In order to understand the distribution characteristics of TTX in cultured T.bimaculatus,qualitative and quantitative analysis of TTX content in different tissues of wild T.bimaculatus was carried out and compared with that in corresponding tissues of cultured Takifugu bimaculatus;(3)In order to determine whether there is exogenous TTX in the culture environment of T.bimaculatus in Fujian Province,TTX was detected in the culture water of T.bimaculatus.The results showed:(1)TTX existed in sperm,unfertilized eggs,embryos and juveniles,but its TTX content was lower than the minimum quantitative limit.The distribution of TTX in 18 month old(immature)adults was: skin >liver>(kidney,intestine,muscle,spleen,blood and eyes were lower than the minimum quantitative limit,so TTX content cannot be accurately compared).The distribution of TTX in 36 month old(mature,female)adults was as follows: ovary > blood > liver >skin > intestine > spleen > kidney > eyes > muscle.The content of TTX in the liver higher than that in the skin of 36 month old adults,indicating that TTX was redistributed during the growth of cultured T.bimaculatus.(2)The distribution of TTX in the adult of T.bimaculatus is as follows: ovary > liver > skin > eyes > intestine > spleen > kidney >muscle > blood.The content of TTX in the liver of cultured T.bimaculatus is 171 times lower than that of wild Fugu,the ovary is 137 times lower than that of wild Fugu,the skin is 155 times lower than that of wild Fugu,but the blood is 4.51 times higher than that of wild Fugu.It is suggested that the source of TTX in cultured and wild T.bimaculatus may be different.(3)TTX can be detected in the water for culturing T.bimaculatus,indicating that there is exogenous TTX in the culture environment of T.bimaculatus.2.The 18 month old T.bimaculatus model of exogenous TTX accumulation was established by gavage.The TTX of various tissues in the animal model was detected and analyzed by LC-MS/MS.the regulation of exogenous TTX reduction in T.bimaculatus was traced by comparing the changes of TTX content in different models of toxin accumulation time.The results showed: 24 hours after gavage,the TTX content in all tissues of the animal model increased significantly;exogenous TTX entered the body,the TTX content in the blood increased significantly,indicating that exogenous TTX entered into all tissues through the blood.72 hours after gavage,the content of TTX in all tissues was significantly lower than that in the corresponding tissues of the 24 hours storage animal model,which showed that the accumulation of exogenous TTX in all tissues decreased over time,but it was still significantly higher than that in the blank control group.3.The location of TTX accumulation in liver,ovary and skin cells of 24-hour storage animal model was observed by immunohistochemistry.The results showed:(1)In the liver tissue of the animal model,the cytoplasm of the hepatocytes,the single layer flat epithelial cells of the vein,some blood cells in the vein tube and the inner side of the interlobular bile duct lumen showed brown positive immune reaction.It suggests that exogenous TTX may be transported to the cytoplasm of liver through vein and connected with other tissues through bile duct.(2)In the ovarian tissue of the animal model,the ovarian wall and the connective tissue in the ovary showed Brown positive immunoreaction;the yolk vesicle and nuclear matter of oocytes had brown positive immunoreaction,but the cortical alveoli and nucleolus were negative.It is suggested that exogenous TTX may be transmitted to yolk vesicle and nucleus of oocyte through ovarian wall and ovarian connective tissue.(3)In the skin tissue of the animal model,the flat epithelial cells,saccular cells and basal cells in the epidermis showed brown positive immunoreaction,in which the basal cell layer showed strong immunoreaction,suggesting that exogenous TTX might be delivered from the basal cells to the flat epithelial cell layer through the saccular cells.4.We sequenced,analyzed and screened the differentially expressed genes in the liver tissue of the animal model of toxin storage,and selected some differentially expressed genes for real-time quantitative PCR in order to verify the reliability of the transcriptome sequencing results.The results showed: there were 32 differential genes satisfying the screening conditions,among which 24 were up-regulated genes and 8 were down regulated genes.The functions of gene biological process are mainly concentrated in Toll like receptor signaling pathway,insulin-like growth factor receptor signaling pathway,arginase activity and N-terminal peptide arginine acetylation,glycoprotein synthesis and transport,binding with ubiquitin like protein,vitellogenin activity and vitellogenesis,glutamine(asparagine)enzyme activity,platelet synthesis and secretion,vascular correlation Smooth muscle cell migration,regulation of hyaluronic acid biosynthesis process,organ growth,cell response to leptin stimulation,regulation of mediator assembly,etc.The results showed that the accumulation of exogenous TTX may be closely related to the presence of a large number of microorganisms in the liver,the structure of arginine in the liver and the transport of TTX in the blood vessels of the liver.Three genes related to toxin storage were selected for RT-PCR validation,and the results were consistent with the trend of transcriptome sequencing,indicating that the results of transcriptome sequencing were reliable. |
PDF Full Text Request