| Large yellow croaker(Pseudosciaena crocea), one of commercially important marine fish species in China, belongs to Perciformes, Sciaeni-dae. This study may provide a new thought to improve the freezing resistance of P.crocea by researching the relationship between Stearoyl-Co A desaturase 1(SCD1) gene and changing of membrane fluidity. The SCD1 gene, as a speed-rate biosynthesis enzyme of monounsaturated fatty acid, was studied to improve the freezing resistance of P.crocea in the cold environment. The main results were listed as follows:1. We cloned and identified the SCD1 sequence from P.crocea, which has 98%similarity with SCD gene from Dicentrarchus labrax(Genbank accession no.FN868643.1). The full-length c DNA of SCD1 is 1387 bp, which contains a 92 bp 5’terminal untranslated region(UTR), a 287 bp 3’ terminal UTR and an open reading frame of1008 bp encoding 335 amino acids. This c DNA sequence isolated has been deposited to Genbank under accession No. KP731998.2. The sequence analysis of SCD1 from P.crocea showed that the deduced protein was neither an organelle specific protein nor secretory protein, suggesting that this protein had fully function in the cytoplasm.3. The analysis of tissue-specific expression by semi-RTPCR showed that SCD1 was widely expressed in brain, spleen, liver, kidney, heart and muscle, with liver, kidney and heart the most abundant expression. Then we made the anti-SCD1 polyclonal antibody by prokaryotic gene expression to test the expression in protein level by fluorescence immunochemistry. The immunochemistry result also confirmed that SCD1 was widely expressed in all tested tissues. What is more, the result showed SCD1 was expressed in whole cytoplasm without organelle bias, confirming the prediction of subcellular localization by sequence analysis.4. The SCD1 sequence was subcloned into p EGFP-C1 vector to transfected the Epithelioma papulosum cyprini cells(EPC). The EPC cells were estimated the effect caused by the SCD1 gene overexpression.5. We executed the fluorescence recovery after photobleaching(FRAP) experiment on the transfected EPC cells to test the influence of SCD1 overexpression on the change of membrane fluidity at 28℃. The result showed that the SCD1 overexpresion cells had much higher membrane fluidity than the control group and blank group(P<0.05). At the same time, the control group had no significant difference with blank group(P>0.05).6. We set a series of temperature gradients(16 ℃-22 ℃) to test the relationship between the SCD1 gene and membrane fluidity at lower temperature. The result showed the fluidity of all three groups increased with the temperature raised. However in single temperature gradient, the overexpression group had higher fluidity compared with control and blank groups(P<0.05). The only exception was at 16℃ gradient, one of the parameter suggested no significant difference between overexpression group and others(P>0.05),which may caused by the phase change of membrane. when the temperature is lowered, the membrane is gradually ordered and stiffened and may ultimately undergo transition to a gel phase, which is incompatible with membrane functionality, while a full functioning cell membrane is in the liquid crystalline state.In conclusion, this study tried to prove that the SCD1 gene regulated the ratio of saturated to unsaturated fatty acids of the membrane to change the membrane fluidity,hoping to improving the freeze resistance capability of cells and P.crocea in winter. |
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