Prokaryotic Expression And Subcellular Localization Of DGAT2 Gene In Peanut (Arachis Hypogaea L.)

Peanut is an important economic oil crop. Our country is one of the main peanutproducing countries, the production accounted for 40% of global. Peanut oil is a kindof cooking oil that is more easily digest, and accounts for 30% of the cooking oils inour country. In plant, triacylglycerol (TAG) is the most important lipid storage form.Diacylglycerol acyltransferases (DGAT) is the key enzyme of the TAG biosyntheticpathway and catalyzes fatty acyl group to the sn-3 of diacylglycerol (DAG). In thisprogress, it plays a role of rate-limiting. The data collectively show that the oilaccumulation rate and the oil content are positively correlated with DGAT activity. Inour early study, we identified two types of DGAT2 from peanut cultivar‘Luhua 14’(named as AhDGAT2a and AhDGAT2b respectively), and analyzed their molecularcharacteristics. In this article, we put emphasis on the AhDGAT2 prokaryotic proteinexpression and subcellular localization. As we all know that it is difficult to extractand purify a protein from plants and the yield is lower, whereas it is convenient andefficient to obtain the recombinant protein in E. coli. Prokaryotic expression ofAhDGAT2 protein provides a theoretical and experimental basis for the antibodypreparation and AhDGAT2 function analysis.The construction and expression of pET28a-AhDGAT2: According to the AhDGAT2ORF and the plasmid pET28a vector sequence, we selected NcoI and XhoI as theinsertion site, and inserted 1002 bp AhDGAT2 ORF that did’nt include the terminationcodon into pET28a prokaryotic expression vector that glued a 6×His Tag to theC-terminal of AhDGAT2. pET28a-AhDGAT2 recombinant plasmid was transformedinto BL21(DE3), but results showed that the recombinant protein was not expressed.So we tried using other bacterial strains BL21(DE3)pLysS and Transetta(DE3) to express the recombinant proteins, and we failed again. It was maybe that the pET28awas not suitable for the procarytic expression of AhDGAT2.The construction and expression of the pET28a-AhDGAT2(que): The analysis oftransmembrane domain found that AhDGAT2 had two transmembrane domains. Westill selected NcoI and XhoI as the restriction enzyme site and inserted the amplifiedfragments into pET28a. pET28a-AhDGAT2(que) were transformed into BL21(DE3)for induction. Results showed that AhDGAT2(que) fusion protein were successfullyexpressed. The molecular weight of AhDGAT2a(que) fusion protein was about 33.78kDa, and that of AhDGAT2b(que) fusion protein was about 33.82 kDa. To obtainlarge amount of the fusion proteins, the induction temperature and IPTG finalconcentration were optimized. Results of SDS-PAGE revealed that the fusion proteinsexisted mainly in the form of inclusion bodies. We purified AhDGAT2a andAhDGAT2b fusion proteins from inclusion bodies with the Ni-Agarose His-TaggedProtein Purification Kit.The construction and expression of pGEX-4T-1-AhDGAT2: Prokaryotic expressionvector pGEX-4T-1 carries a 26.97 kDa GST tag. The molecular weight ofGST-AhDGAT2a and GST-AhDGAT2b were about 64.47 kDa and 64.51 kDarespectively. In this experiment，we inserted AhDGAT2 ORF into pGEX-4T-1prokaryotic expression vector and transformed the plamids into BL21(DE3),BL21(DE3)pLysS and Transetta(DE3) for the induction of the fusion proteins.SDS-PAGE results showed that fusion proteins expressed in all the three bacterialstrains. As only activated GST can bind to glutathione resin, we optimized thecondition of the fusion protein expression before purifying them. It was found that thefusion proteins were induced both in the supematant and precipitation at 30℃.In order to confirm AhDGAT2 whether it will work in E. coli, we detected thecontent of fatty acid in recombined strains. The results showed that the total contentof fatty acids in recombined strains in BL21(DE3) were increased. But the totalcontent of fatty acids in recombined strains in Transetta(DE3) were decreased. In these two kinds of recombinant strains, saturated fatty acids and unsaturated fattyacids didn’t show respective change pattern.The subcellular localization of AhDGAT2: We constructed three kind of thesubcellular localization vectors pBSK-35S-AhDGAT2-EGFP,pBSK-35S-AhDGAT2(que)-EGFP, pBSK-35S-AhDGAT2-RFP. By biolisticbombardment, AhDGAT2 and AhDGAT2(que) were transiently expressed in theonion epidermal cells. Results showed that when EGFP as a reporter gene,AhDGAT2a and AhDGAT2b are located in cytoplasm, plasma membrane and nucleus.The location of AhDGAT2a(que) and AhDGAT2b(que) were the same as that ofAhDGAT2a and AhDGAT2b. While RFP as a reporter gene, in the onion epidermalcells that AhDGAT2b transformed into, we found that there were some granularmaterial that existed fluorescent signals in cytoplasm. However the fluorescent signalsdidn’t find in the plasma membrane and nucleus.