| As the central part of the complement system, complement component C3 plays an essential role in activating the whole complement system, which is also significant in immune defense against pathogens in invertebrates.This paper studied the influence of temperature on the expression of AjC3 mRNA and MicroR-137 in sea cucumber coelomic fluid, intestinal and respiratory tree. The sea cucumber with the same size reared at seawater of 23℃,16℃ and 8 ℃ was injected with LPS 2 g/ l 100 L in this experiment. Then extracting coelomic fluid in 3h, 6h, 9h, 12 h, 24 h separately with three sea cucumber. Total RNA extracted from the body fluid, intestine and respiratory trees under the condition of low temperature sterilization would be reversed to cDNA after the quality is qualified. Taking coelomic fluid, intestinal and respiratory tree cDNA as template, design of cytochrome b and mRNA MicroR-137 and AjC3 primers can detect the contents of mRNA AjC3 and MicroR-137 in the body fluid.The result shows that AjC3 mRNA is expressed in sea cucumber Apostichopus japonicus, respiratory tree and intestinal these three tissues after LPS stimulation. Among them, the content of the body fluid is the most, the intestine is the second, and the least is in the respiratory tree. LPS stimulation test results show that expression changes of mRNA AjC3 within body fluid, respiratory tree and intestinal in each period presents a trend of rise to decline. And the expression level of mRNA AjC3 in the body fluid, respiratory tree and intestine reaches the peak at 9h. During the process of LPS stimulation in one day for 8℃, 16℃, 23℃Apostichopus japonicas group, MicroR-137 is expressed in all these three tissues and the expression level has overall similarity-a trend of decline first then rise, but the variation range has big difference. 8 ℃ sea cucumber group decreased significantly(p<0.01) in the stimulation of 6h expression, then reaches the lowest value in the 9h. There is no obvious change(p>0.05) before and after the stage of 9h for 16 ℃ sea cucumber group. With regard to 23℃ group, there also exists great difference(p>0.05) before and after 9h,with a trend of increase to decrease. However, compared with the various tissues, it is obvious that the 16 ℃MicroR-137 content in the body fluid presents the tendency from decrease to increase.23 ℃ and 8 ℃ MicroR-137 would go down in 9h, but its content would float slightly after 9h. In the intestinal and respiratory tree, MicroR-137 content decreases in 9h, but after the period of declining, there also appeares the changes of rise and fall for the content of MicroR-137.The expression level of AjC3 mRNA and MicroR-137 is different under different temperature. Low temperature and high temperature has inhibitory effect on the immune response of sea cucumber. MicroR-137 would decline when the AjC3 showed a rise after LPS stimulation. In the coelomic fluid, AjC3 mRNA showes a trend of increase to decrease, while the MicroR-137 appeares an opposite trend. And in the coelomic fluid, the AjC3 mRNA content changes evidently, but it has no obvious change for the content of MicroR-137 in body fluid, intestinal and respiratory tree, which in an decreasing manner. With regard to the correlation between AjC3 mRNA and MicroR-137(MicroR-137 fallen when AjC3 mRNA rise), there has the highest relevance in the coelomic fluid, followed by the intestine, finally the respiratory tree. Temperature has an effect upon expression of mRNA AjC3. High temperature or low temperature will inhibit the expression of mRNA AjC3, however, the influence of temperature on MicroR-137 expression is unobvious. There exits negative correlation between MicroR-137 and mRNA AjC3, which may regulate the expression of mRNA AjC3 and participate in the immune response. The correlation of MicroR-137 and mRNA AjC3 is different in different tissues. The correlation between AjC3 and mRNA MicroR-137 in the body fluid is the highest, then the intestine and that of respiratory tree is the lowest. |
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