The Effect Of Interference With COX2 Gene Expression On The Growth And Energy Metabolism Of Macrobrachium Rosenbergii

In recent years,with the development of the Macrobrachium rosenbergii aquaculture industry,the scale of aquaculture has become larger and the output has increased,but the problem of aquaculture has also followed.The emergence of "iron shell shrimp" has become a problem that plagues farmers.Regarding the reasons for the slow growth of M.rosenbergii,there is still no clear conclusion.Many scholars have explored the effects on the growth of M.rosenbergii from the aspects of disease,growth genes,hormones,and nutrition.In terms of energy metabolism,the relationship between the growth of crustaceans and the enzymes involved in ATP synthesis in mitochondria has not been reported.The enzymes involved in ATP synthesis in mitochondria include NADH dehydrogenase(ND),cytochrome C oxidase(COX),and cell Color reductase(CCR),ATP synthase.Whether the activity of enzymes involved in energy production in mitochondria affects the growth of M.rosenbergii remains to be explored.In this experiment,siRNA was used to interfere with the expression of COX subunit encoded by the mitochondria of M.rosenbergii to down-regulate gene expression to explore the effect on the activity of enzymes involved in energy production and gene expression.In addition,the COX gene of M.rosenbergii was continuously inhibited for different periods of time to analyze the difference in growth of M.rosenbergii.Three siRNAs were designed in the experiment,namely siRNA-COX1,siRNA-COX2-111,and siRNA-COX2-308.Different siRNAs were injected into shrimp(1.50±0.25g).It was found that siRNA-COX2-111 had the best effect,and the interference effects of the four doses of 0.8μg/g,1.5μg/g,2.0μg/g,and 3.0μg/g were different.The interference effect of 2.0μg/g siRNA-COX2-111 has the best effect.COX2 gene expression can be significantly inhibited in hepatopancreas and muscle within seven days.COX2 expression in hepatopancreas and muscle is only 37% and50% of the control group.In this experiment,siRNA was used to interfere with the COX2 gene of M.rosenbergii,and the COX2 gene expression was down-regulated in the hepatopancreas and muscle,which proved that siRNA can also play the same negative regulatory effect on the genes encoded by mitochondria in crustaceans.In addition,it has been found that the effect of high-dose siRNA is not necessarily more significant than that of low-dose.However,within a certain injection dose range,the higher the injection dose of siRNA,the longer the action time,and the more obvious the inhibitory effect.The 2.0μg/g siRNA-COX2-111 was injected into shrimp(1.50±0.25g)as the experimental group.The control group was injected with the same volume of NgsiRNA and DEPC water as the experimental group.The experiment was carried out for seven days.Fluorescence quantitative detection of ND1,ND2 and ATP6 gene expression in hepatopancreas and muscles,COX,ND,ATP synthase,CCR activities and tissue ATP content were detected.Experimental results showed that the expression of ND1,ND2 and ATP6 genes decreased in hepatopancreas and muscle,while the activities of COX,ND,ATP synthase,and CCR decreased,and the content of ATP decreased in hepatopancreas and muscle.It shows that silencing the COX gene will affect the gene expression and activity of other enzymes involved in energy production in the respiratory chain.The decreased expression of COX2 inhibits COX activity,which may cause certain damage to the function of mitochondria and affect the effect of oxidative phosphorylation.Four experimental groups and four negative control groups were designed.The experimental group and the negative control group were injected with different times of siRNA and DEPC water,and the blank control group was not treated.After cultured for28 days,the net weight gain of shrimps decreased with the increase of COX2 expression inhibition time,and the longer the COX2 expression was inhibited,the more activity of ND,CCR,COX and ATP synthase in hepatopancreas and muscles.Low,the lower the gene expression of ND1,ND2,ATP6,and COX1.The content of ATP decreases with the decrease of enzyme activity,indicating that the enzyme activity involved in ATP synthesis will affect the production of ATP,and the decrease of the activity of these four enzymes has an inhibitory effect on the growth of M.rosenbergii.In the group with high gene expression and enzyme activity in hepatopancreas and muscle,shrimp grows fast;in the group with low expression and enzyme activity,shrimp grows slowly,indicating that the gene expression and activity of enzymes related to energy production are inhibited Time,it will affect the growth of shrimp.This study used RNAi technology to interfere with the expression of COX2 gene encoded by M.rosenbergii mitochondria with siRNA for the first time,and explored the gene expression and enzyme activity of enzymes involved in energy production on the respiratory chain after the COX2 gene expression was down-regulated.The change.Using siRNA to continuously inhibit the COX2 gene expression of M.rosenbergii,it was found that the longer the COX2 gene expression was inhibited,the slower the growth of M.rosenbergii,and the gene expression and enzyme activity of enzymes involved in energy production in the respiratory chain.The longer the inhibition time,the less ATP content in the hepatopancreas and muscles.It shows that the gene expression and enzyme activity of enzymes involved in energy metabolism have a certain influence on the growth of M.rosenbergii.Provide reference for the follow-up study on the slow growth of M.rosenbergii.