A Preliminary Investigation On The Functions Of1-Acylglycerol-3-Phosphate Acyltransferase (AGPAT) And Lysophosphatidylglycerol Acyltransferase (LPGAT) In The Lipid Synthesis Of Eriocheir Sinensis

1-Acylglycerol-3-phosphate acylase(AGPAT),which acylates lysophosphatidic acid(LPA)to phosphatidic acid(PA),is the second key enzyme in the triglyceride(TAG)synthesis pathway,while lysophosphatidylglycerol acyltransferase(LPGAT)is the key enzyme that converts lysophosphatidylglycerol(LPG)to phosphatidylglycerol(PG).The Chinese mitten crab(Eriocheir sinensis)is an important farmed crab in China and also an important model animal for studying triglyceride metabolism in crustaceans.In this study,we firstly cloned the open reading frame sequences of AGPAT and LPGAT in Eriocheir sinensis,and further investigated the expression patterns and localization of AGPAT and LPGAT genes during the molting and ovarian development of Eriocheir sinensis using real-time fluorescence quantitative PCR(q-PCR)and in situ hybridization(ISH).RNAi interference was used to study their effects on triglyceride synthesis gene expression.The main results were as follows.1.Bioinformatics analysis and tissue distribution of AGPAT and LPGAT in the Eriocheir sinensisOne Es-lpgat1 gene and three AGPAT genes(Es-agpat2,Es-agpat3 and Es-agpat4)were identified from the genome of Eriocheir sinensis.The open reading frame(ORF)of Es-lpgat1 is 1125 bp in length,encoding 374 amino acids,with a molecular weight of 44 k Da and a theoretical isoelectric point of 8.31;Es-agpat2 has an ORF of 1113 bp,encoding 370 amino acids,with a molecular weight of 93.15 k Da and a theoretical isoelectric point of 5.05;Es-agpat3 ORF is 1152 bp long,encoding 383 amino acids,with a molecular weight of 95.45 k Da and a theoretical isoelectric point of 5.05;Esagpat4 is 843 bp long,encoding 280 amino acids,with a molecular weight of 70.14 k Da and a theoretical isoelectric point of 5.08.BLAST comparison revealed that these sequences all belong to the LPLAT superfamily,including multiple transmembrane helix regions,the Acyltransferase＿C structural domain,the LPLAT＿LCLAT1-like structural domain,the Pls C structural domain,and the conserved active site.The phylogenetic tree showed that Es-LPGAT1 clustered with the LPGAT1 of Portunus trituberculatus and Chionoecetes opilio,and clustered with the arthropod as one large branch;while Es-AGPAT2 was closely related to AGPAT4 of Portunus trituberculatus and Chionoecetes opilio,Es-AGPAT3 was closely related to AGPAT5 of Portunus trituberculatus and Chionoecetes opilio,and Es-AGPAT4 was closely related to AGPAT1 in Armadillidium vulgare and Homarus americanus,and overall the AGPAT of Eriocheir sinensis were clustered together with crustaceans.The tissue distribution results showed that Es-lpgat1 was highly expressed in the hepatopancreas,heart,muscle and thoracic ganglion,moderately expressed in the eye stalk and stomach,and lowly expressed in the hindgut,midgut and gill;while Es-agpat2 and Es-agpat4 had similar expression profiles in all tissues and were most highly expressed in the hepatopancreas,followed by the heart,muscle,thoracic ganglion,eye stalk,stomach,midgut,hindgut and gill.In contrast,Es-agpat3 was highly expressed in the thoracic ganglion and lowly expressed in other tissues,especially the hepatopancreas.2.Expression patterns and localization of AGPAT and LPGAT during molting and ovarian development in the Eriocheir sinensisThe expression patterns and tissue localization of AGPAT and LPGAT during the molting and ovarian development of Eriocheir sinensis were investigated by real-time fluorescence quantitative PCR and in situ hybridization.During the molting cycle of Eriocheir sinensis,the results showed that the total lipid and TAG contents were lowest in stage AB and highest in stage D.Es-lpgat1,Es-agpat2 and Es-agpat4 had similar expression trends and were all expressed at the highest level in stage C.The expression of Es-agpat3 in the hepatopancreas was significantly lower than that of the remaining three genes.During ovarian development,Es-lpgat1,Es-agpat2 and Es-agpat4 had the same expression trend in the hepatopancreas,with their expressions all rising rapidly from stage I to reach the highest point in stage II,then falling rapidly to reach the lowest point in stage III,and finally showing a slow increase again in stage III-V;while the expression of Es-agpat3 was significantly lower than the other three genes.In the ovary,Es-lpgat1,Es-agpat2 and Es-agpat4 had the highest expression in stage II and the lowest expression in stage I of ovarian development,while Es-agpat3 was highly expressed in the ovary unlike its low expression in the hepatopancreas.In situ hybridization results showed that Es-lpgat1-m RNA and Es-agpat4-m RNA were mainly localized in fibroblasts(F cell)and resorbing cells(R cell)in stage II and IV hepatopancreas,while Es-agpat3 had no signal.In stage II of ovarian development,Eslpgat1-m RNA,Es-agpat3-m RNA and Es-agpat4-m RNA were localized in previtellogenic oocytes(PRO)and endogenous vitellogenic oocytes(EN),but in stage IV of ovarian development,Es-lpgat1-m RNA was localized in a small number of PRO cells,while the rest of the genes showed no signal.3.A preliminary investigation on the functions of AGPAT and LPGAT genes in triglyceride synthesis in the Eriocheir sinensisEs-lpgat1,Es-agpat2 and Es-agpat4 were interfered with using RNAi to examine the effect of in vivo injection of the interfered fragments on the expression of the remaining key genes in the triglyceride synthesis pathway of Eriocheir sinensis after48 hours.The results showed that Es-lpgat1 interference blocked phospholipid synthesis,and Es-gpat2 and Es-dgat2 expression levels were up-regulated by about 40%and 60%,respectively,and increased phospholipid synthesis by increasing the CDPDAG pathway,while Es-agpat4 and Es-dgat1 expression levels were down-regulated by 20% and 60%,respectively.Interfering with Es-agpat2 and Es-agpat4 had broadly similar results.The expression levels of Es-agpat4 and related downstream genes Espap1,Es-dgat1 and Es-dgat2 were significantly decreased when Es-agpat2 was interfered with,and the expression levels of Es-agpat2 and downstream genes Es-pap1 and Es-dgat1 were also significantly decreased.When Es-agpat4 was disrupted,the expression levels of Es-agpat2 and the downstream genes Es-pap1 and Es-dgat1 were also significantly decreased,because the synthesis of CDP-DAG pathway was blocked,so phospholipid synthesis was increased by elevating the expression level of Es-lpgat1.The above results imply that LPGAT and AGPATs may play different roles in triglyceride synthesis in Eriocheir sinensis,and there is a compensatory effect between Es-lpgat1 and Es-agpat4.