| Glycerol-3-phosphate acyltransferase(GPAT)is the committed enzyme catalyzing the first step in triglyceride(TAG)synthesis through the acylation of glycerol 3-phosphate with long-chain fatty acyl-Co A,playing a important role in TAG content.In order to explore the mechanism of high fat accumulation in the hepatopancreas and ovary of Eriocheir sinensis,this study conducted a preliminary study on the GPAT gene.In this study,two GPAT genes(EsGPAT1 and EsGPAT2)were identified and characterized in E.sinensis by molecular biology.The ORF of EsGPAT1 gene were 1404 bp encodeing 467 amino acids with the theoretical p I of 6.99.The EsGPAT2 gene were 1377 bp,encoding458 amino acids encodeing 467 amino acids with the theoretical p I of7.03;Both of two EsGPATs which belong to the Pfam01553 family have the characteristics of this family,multiple transmembrane regions,four highly conserved motifs and corresponding functional residues and the most important active site acyltransferase of Pfam01553 family.Phylogenetic tree analysis indicated that EsGPAT1 and EsGPAT2 were clustered together with a 51% identity,and EsGPAT1 and EsGPAT2 were more closely related to the mammalian GPAT3 and GPAT4 genes.Subcellular localization predicted that EsGPAT1 and EsGPAT2 localized to the endoplasmic reticulum.The expressions pattern of EsGPAT1 and EsGPAT2 in different tissues and ovary development stages of E.sinensis were determined by quantitative real-time PCR(QPCR)and in situ hybridization(In situ hybridization)techniques.The expression levels of EsGPATs in different tissues were shown in follows.Both EsGPAT1 and EsGPAT2 were widely expressed in different tissues.EsGPAT1 highly expressed in TG and moderately expressed in hepatopancreas,ovary and intestine.The low expression level was found in brain ganglia,stomach,gill,muscle.hepatopancreas,triangle membrane and hemocytes.However,the transcript of EsGPAT2 was mainly detected in thoracic ganglia,brain ganglia,intestine and followed by hepatopancreas,ovary,stomach,muscle,hemolymph,triangle membrane,gill and hemocytes.The changes in expression levels of EsGPAT1 and EsGPAT2 in the hepatopancreas and ovary were inconformity during the ovary development.In hepatopancreas,the expression level of EsGPAT1 increased from stage I to reach a maximum at stage IV(P < 0.05)and then declined sharply.On the contrary,the EsGPAT2 expression was highest at stage I and then gradually declined to reach a minimum at stage IV(P < 0.05).Consistent with the expression changes of EsGPATs in hepatopancreas,the transcriptional level of EsGPAT1 in ovary increased from stage I to reach a maximum at stage IV and then declined,while the transcripts of EsGPAT2 in ovary reached the peak at stage I and then declined to reach a minimum at stage III.In situ hybridization was performed to localize the transcripts of EsGPAT1 and EsGPAT2 in the ovary and hepatopancreas during ovary development,respectively.The results showed that signals of EsGPAT1 in ovary was detected in all kinds of cells at stage I,the follicle cells at stage III and the oocyte cells at stage IV.In the hepatopancreas,the antisense probe gave a strong signal in F cells and B cells(Fig.5A,g-h).The localization of the EsGPAT2 was same with the EsGPAT1.To further verify the function of EsGPATs,three different GPAT1 i ds RNA and GPAT2 i ds RNA interference fragments were synthesized by reverse transcription in vitro.The hepatopancreas of E.sinensis were treatmented for interference 1h,2h,4h,8 h in 26°C constant temperature incubator.The result showed that GPAT1 has a segment sustained interference and the concentration of interference fragment was 5μg /m L and GPAT1 had two segments sustained interference and the concentration of interference fragments was 2μg /m L.Meanwhile,knockdown of EsGPAT1 could down-regulate the expression levels of down-stream genes in TAG synthesis pathway,but it was not observed in ds GPAT2 treatment group. |
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