Calmodulin Expression Mechanism Of Gill And Intestinal Tissues And Primary Cultured Its Cells Of Leuciscus Waleckii In The Environment Of Alkali Stress Tolerance

The study aimed to establish the primary and subculture system of gill and intestinal cells of Leuciscus waleckii,a dominant natural species with high alkali tolerance,to explore the effects of alkali stress on the proliferation and function of primary cells and to analyze the expression of Calmodulin（CAM）in gill and intestine.Thus,the mechanism of calmodulin is preliminarily analyzed with the perspective of the alkaline tolerance mechanism.Based on the above,this study mainly evaluated the acute and chronic alkali tolerance of gill and intestinal cells,the growth and development status of gill and intestine tissue,and observed the serum content of Ca²⁺.The main results are as follows:1.Isolation and primary culture of gill and intestine cells from Leuciscus waleckiiBy choosing the culture conditions,gill and intestine cells’optimal primary culture conditions were determined,and a stable and reliable primary cell model of gill and intestine was established.In this study,trypsin digestion and density gradient centrifugation isolate and purify gill and intestinal cells.The cell suspension was evenly placed in DMEM（Dulbecco’s modified Eagles medium）,MEM（minimum essential medium）,and L-15（Leibovitz medium）.In this process,the cell proliferation was measured by blood cell counting plate,and cell proliferation rate was measured by cell counting method.Simultaneously,the cell source and activity were identified,and the growth status of primary gill and intestinal cells was analyzed.After long-term culture and morphological observation of the two cell lines,which was found that DMEM medium could obtain stable and good culture effect in primary culture of gill and intestine cells,and the growth state of cells was significantly better than that of L-15and MEM.After primary culture for 36-72 hours,gill and intestinal cells grew and metabolized vigorously,and the proliferation law was following the classic"S"growth curve.According to the characteristics of the camk2g2 gene（Locus Tag Prefixes:J5810）,which can be expressed in the whole body,it was confirmed that the two cells were isolated from Leuciscus waleckii.2.Effects of alkali stress on the activity and function of gill and intestine cells of Leuciscus waleckiiThe model of cultured gill and intestinal cells in vitro was established to investigate the effects of alkali stress on the activity and function of primary gill and intestinal cells of Leuciscus waleckii.NaHCO₃ was used as the main tolerance factor（hydrolysis equation:HCO₃^-+H₂O（?）H₂CO₃+OH^-）.The experimental process is as follows:DMEM with10 mmol/L NaHCO₃ was incubated in the CO₂ incubator for 1 hour and 2-5 hours.The absorbance value was detected at 660 nm（Dual-wavelength way）,and the standard curve was made to determine the fluctuation range to facilitate the subsequent determination of alkali gradient experimental indicators.Then,according to the above experimental results,the concentration gradient of NaHCO₃ was set as different levels.The gill and intestinal cell lines were tolerant to 25-75 mmol/L and then incubated in the incubator for 3 hours.The viability and survival rate of the two cells were detected by the CCK-8 method.DNA ladder method was used to detect apoptosis and necrosis of cells in the gill and intestine,to analyze cell growth and metabolism.The results showed that the optimal tolerance time of gill and intestinal cells was 3 hours.The best tolerance concentration of NaHCO₃ was 25-50 mmol/L.Under the above condition,the cells of the gill and intestine showed apoptosis.The results showed that cultured gill and intestinal cells could tolerate 25-50 mmol/L NaHCO₃ high concentration alkali habitat in vitro,and the survival conditions could cause different degrees of apoptosis on the function and activity of the two cell lines.3.Calmodulin on gill,intestinal tissue and cell function of Leuciscus waleckiiThe experiment was divided into two levels:histology and cytology.（1）Histological experiment:the camk2g2 gene was cloned from the early transcriptome data of the laboratory,and RT-QPCR detected the camk2g2 gene in gill and intestine of the experimental fish,and the results showed that under the condition of 50 mmol/L alkali stress,the camk2g2 gene was cloned and sequenced.There was no significant difference in serum calcium content of FW（P>0.05）.The results showed that some base pairs were not complementary and missing.The ORF of the camk2g2gene was 684 bp,encoding 228 amino acids（Locus Tag Prefixes:J5810）.RT-QPCR analysis showed that the expression of the camk2g2 gene in gills and intestines decreased briefly at 7 days and reached a relatively stable level at 30 days.There was no significant difference between the two groups（P>0.05）.Compared with the gene expression levels of xbp1u and gsr,which was found that all three genes were expressed in the gill and intestine,and the changing trend of gene expression levels with tolerance time was similar to the two genes as the counterparts,and there was no significant difference（P>0.05）.（2）Cytological experiment:the primary gill and intestinal cells of Leuciscus waleckii were selected as the experimental objects.Three concentrations of NaHCO₃（0,25,and50 mmol/L）were selected for alkaline tolerance.After incubation for 3 hours,the intracellular Ca²⁺content,the camk2g2 gene,endoplasmic reticulum stress response gene xbp1u,and oxidative stress response key gene gsr expression were determined.To study the effect of alkali stress on the camk2g2 gene of Leuciscus waleckii in vitro,and to explore the potential relationship between this mechanism and the stress response mechanism of abiotic stress factors at the cellular level.The results showed that under the condition of alkali tolerance for 3 hours,the expression of the camk2g2gene was highly significant in gill cells with 25 mmol/L NaHCO₃ tolerance（P<0.01）and significantly increased in gill cells with 50 mmol/L NaHCO₃ tolerance（P<0.05）.However,the expression of the camk2g2 in intestinal cells increased with alkalinity,but there was no significant difference（P>0.05）.The above results indicate that the expression of the camk2g2 gene is generally interfered with by alkaline factors in gill and intestinal cells,but gill cells are more sensitive to it.In addition,analyzing the gene expression trends of the critical genes of endoplasmic reticulum stress xbp1u and oxidative stress gsr,which was found that the expression trends of the camk2g2 gene were similar to those of xbp1u and gsr at the cellular level,and there was no significant difference among the three genes（P>0.05）.There may be a correlation between the role of the camk2g2 gene and the stress response mechanism.