| Portunus trituberculatus is an important marine aquaculture animal in China and is widely distributed in China.Salinity is an important environmental factor for the culture of Portunus trituberculatus.At present,the culture is mainly cultured outdoors.The salinity is an important environmental factor in the culture of the crab.It is often caused by drastic changes in the salinity of the culture water due to the rainy weather and large water changes.The three-spotted crab has a stress response,physiological metabolic disorder,and thus decreased immunity,which can easily cause disease outbreaks and even a large number of deaths.Due to the large-scale construction of the factory,the farming area has been decreasing year by year.In order to change this situation and make full use of the newly developed large-scale low-salt ponds,the Yellow Sea has carried out the breeding of new varieties with low salt tolerance.However,the study on the apoptosis of Portunus trituberculatus has not been reported under different low salt conditions.Therefore,the low salt tolerance of different growth stages of Portunus trituberculatus was compared,and the molecular mechanism of apoptosis under different low salts was discussed and analyzed.It is of great significance to the breeding and breeding of the crab.In addition,due to the aggravation of water environment pollution,the disease is becoming more and more serious.Studies have shown that Vibrio parahemolyticus and white spot syndrome virus(WSSV)can cause tissue damage and even death of the crab.When the pathogen is infected,the number of crustacean cell apoptosis increases in the tissue,which causes the body to produce an immune response,indicating that apoptosis can play a role in coping with viral infection.Currently,no apoptosis pathway has been found in the crab.It is reported that it is of guiding significance to study the apoptosis of the pathogen-infected P.trituberculatus.The research contents include the following three parts: 1.Cloning and sequence analysis of P53,Apaf-1 and Bcl-2 genes of Portunus trituberculatus.P53 gene is an important tumor suppressor gene.It was first reported in 1979.However,with the development of research,P53 gene has gradually been found to function as a tumor suppressor gene.As a multifunctional transcription factor,053 gene plays a key role in cell response to DNA damage,hypoxia and carcinogenic signals.Once activated,53 promotes transcription of more than 150 genes involved in apoptosis induction,pelvic cell cycle regulation,tumor suppression,genomic stability,autophagy and DNA repair.Bcl-2 gene is a very important gene in the Bcl-2 family.Its function is to inhibit apoptosis.Bcl-2 family proteins participate in the mitochondrial apoptosis pathway mainly by affecting the permeability of mitochondrial extracorporeal membrane.Bcl-2 proteins regulate apoptosis by changing the changes of mitochondrial membrane potential,thus inhibiting apoptosis.Apoptotic protease activator-1(Apaf-1)plays a central role in mitochondrialmediated apoptosis.Many signaling pathways must first act on Apaf-1 and then induce downstream apoptotic events.Apaf-1 is an important component of developmental programmed cell death.Full-length DNA sequences of P53,Apaf-1 and Bcl-2 genes of Portunus trituberculatus were obtained by RACE cloning technique and analyzed.The results showed that the full length of P53 gene of Portunus trituberculatus was 1544 bp,of which ORF was 1314 bp,encoding 437 amino acids(GenBank NO.MH155954).The sequence contained 168 BP 5’-terminal non-coding region(UTR)and 230 BP 3’-terminal UTR,and polyadenylate polyA tail at 3’-terminal.ExPASy ProtParam tool online software analysis showed that p53 gene encodes a protein composed of 437 amino acids.The isoelectric point of the protein prediction theory is 5.47 and the molecular weight is 49.6 kDa.The unstable coefficient is 51.61,which belongs to the unstable protein.The full length of the Bcl-2 gene of Portunus trituberculatus is 215 bp,of which the ORF is 849 bp,encoding 282 amino acids(GenBank NO.MH155953).The sequence contains 565 BP 5’-terminal non-coding region(UTR)and 602 BP 3’-terminal UTR.Polyadenylate A tail exists at the 3’-terminal.It contains Bcl-2 domain and a transmembrane region.The cDNA sequence of Apaf-1 gene of Portunus trituberculatus is 2032 bp.the ORF is 1050 bp.it encodes 349 amino acids,and the predicted molecular weight is 39.13 kDa.The theoretical isoelectric point is 7.67.Homology and phylogenetic analysis showed that the highest homology between Apaf-1 and Litopenaeus vannamei was 51.27%,which indicated that Apaf-1 was conservative in evolution.The relative expression of P53 gene in gill and eyestalk was the highest,followed by hepatopancreas and heart,Bcl-2 gene was the highest in muscle and lowest in epidermis,Apaf-1 gene was the highest in hepatopancreas and lowest in epidermis.2.Functional Study of P53,Bcl-2 and Apaf-1 Genes in Portunus trituberculatus under Salinity Stress The expression of P53 gene in stage II juvenile crab was up-regulated at 3 hours and reached its peak at 12 hours under low salt stress.The expression of Bcl-2 gene showed a trend of first increasing,then decreasing,and then increasing.The expression of Apaf-1 gene increased at 3 hours,and then increased with the passage of time.Tunel detection of gill tissue under low salt stress by paraffin section showed that low salt stress could change the apoptotic rate.At 80 days of age,the expression of P53 gene in gill tissue showed an upward and downward trend,which was higher than that of control group.The expression of P53 gene in hepatopancreas showed a downward and then upward trend;at 80 days of age,the expression of Bcl-2 gene showed a downward and upward trend under low salt stress.Bcl-2 in hepatopancreas increased first and then decreased,and reached the control level in 72 hours.After salinity stress,the expression of Apaf-1 gene in gill tissue increased first and then decreased,and the expression of salinity 11 was lower than that in control group.The expression of Apaf-1 gene in hepatopancreas increased first and then decreased at 20 salinity,reaching its peak at 12 hours and returning to the level of control group at 72 hours.After 11 salinity stress,the expression of Apaf-1 gene in hepatopancreas changed significantly,reaching its peak at 12 hours.The functional relationship between P53,Bcl-2 and Apaf-1 genes in Portunus trituberculatus was studied by siRNA interference.The results showed that siRNA designed by direct injection of P53 gene had better interference effect.After P53 is disturbed,Apaf-1 can be down-regulated and Bcl-2 can be up-regulated significantly.It is speculated that Apaf-1 plays a dominant role in the regulation of P53.At the same time,Bcl-2 has a negative feedback regulation on P53.Western blot experiments confirmed the specificity of P53 polyclonal antibody,and the results of gray value analysis of P53 protein were basically consistent with the expression of P53 protein after low salt stress,indicating that it participated in the reaction process.These results suggest that P53,Apaf-1 and Bcl-2 genes may play an important role in the response of Portunus trituberculatus to environmental stress.3.Functional Analysis of P53,Apaf-1 and Bcl-2 Genes in Portunus trituberculatus after Infection with Viruses and Bacteria.White spot syndrome virus(WSSV)and Vibrio parahaemolyticus were used to infect Portunus trituberculatus.The gene expression in blood cells and hepatopancreas was detected by fluorescent quantitative RT-PCR.The results showed that the expression of P53 gene in Portunus trituberculatus hemocytes infected with Vibrio parahaemolyticus increased first and then decreased.The overall expression of P53 gene was up-regulated.After WSSV infection,it reached its peak at 72 hours.After infection with Vibrio parahaemolyticus,P53 increased first and then decreased,reaching its peak at 12 hours,4.27 times that of the control group,and reaching its peak at 24 hours after infection with WSSV virus,1.5 times that of the control group.After infection with Bcl-2 gene of Portunus trituberculatus,the expression of Vibrio parahaemolyticus in Portunus trituberculatus was significantly lower than that in control group.The expression of WSSV gene reached its peak at 24 hours,which was 1.58 times higher than that in control group.After infection with Vibrio parahaemolyticus,the expression of Bcl-2 in hepatopancreas was up-regulated as a whole.The peak value reached 6.36 times in 3 hours,6.36 times in control group,and 5.92 times in 48 hours after WSSV infection,which was up-regulated as a whole.After infected with Vibrio parahaemolyticus and WSSV virus,the former reached its peak value at 48 hours,2.76 times in control group,and the latter reached its peak value at 12 hours,1.25 times in control group.After hepatopancreas was infected with Vibrio parahaemolyticus,Apaf-1 reached its peak at 72 hours,which was 5.44 times higher than that of the control group.After WSSV infection,Apaf-1 showed a trend of first rising and then declining,reaching its peak at 12 hours,which was 5.89 times higher than that of the control group.Western blot experiments confirmed that the gray value of P53 protein was basically consistent with the expression of the pathogen after infection.It is concluded that P53,Bcl-2 and Apaf-1 genes are involved in the immune defense process of Portunus trituberculatus.It is speculated that this gene plays an important role in the process of low salt induction and innate immunity of Portunus trituberculatus,which provides theoretical guidance for the study of apoptotic pathway of Portunus trituberculatus and other crustaceans. |
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