Figure S4. Pictures of wild-type and sdp1/D1/W1 plants grown on soil. Supplemental Table S1. Root TAG content and DGAT1 and WRI1 expression in transgenic lines. Supplemental Table S2. Fatty acid composition of TAG from sdp1/D1/ W1 roots.Lipid AnalysisTotal lipids had been extracted from homogenized freeze-dried tissue making use of the strategy of D mann et al. (1995), except that tripentadecanoic acid (15:0 TAG) was added to the homogenized tissue to act as an internal regular. A proportion of the total lipid extract was subjected straight to transmethylation as outlined by the approach of Browse et al. (1986), as well as the fatty acid methyl esters had been quantified by gas chromatography-flame ionization detection with reference for the common (Kelly et al., 2011). The remaining lipid extract was applied to silica TLC plates, and neutral lipids were separated applying a hexane: diethyether:acetic acid (70:30:1, v/v) solvent system. The lipids had been visualized below UV light by staining with 0.05 (w/v) primuline in 80 (v/v) acetone, the TAG band was scraped in the plate and transmethylated, and the fatty acid methyl esters had been quantified as above. ESI-MS/MS analyses had been performed on a 4000 QTRAP (ABSiex) liquid chromatography-MS/MS apparatus coupled with a TriVersa NanoMate mounted with an ESI chip (Advion). For all analyses, the NanoMate was operated in good ion mode with a spray voltage of 2 kV, vented headspace, and pressure of 0.4 p.s.i. A 50-mL aliquot of total lipid plus 15:0 TAG common was mixed in 1 mL of 1:1 (v/v) chloroform:methanol with 10 mM ammonium acetate (James et al., 2010). A 10-mL aliquot of this mix was then loaded around the ESI chip to carry out the analyses. Complete scans had been performed employing the Q1/MS mode to obtain the spectra. Molecular species of TAG had been profiled applying the method described by Krank et al. (2007), except that 12 periods of two min had been employed for neutral loss scans. The data had been then analyzed in Lipidview (version 1.1). The volume of fatty acid was normalized by gas chromatography-flame ionization detection.ACKNOWLEDGMENTSWe are extremely grateful to Dr. Richard Haslam for his invaluable assistance with mass spectrometry procedures and to Prof.Fmoc-D-Cys(Trt)-OH supplier Ljerka Kunst for delivering the dgat1-1 mutant.BuyEthyl 2-bromooxazole-5-carboxylate Received April 19, 2013; accepted May perhaps 16, 2013; published May 17, 2013.PMID:33715956 Creation of DNA Constructs, and Arabidopsis TransformationDGAT1, WRI1, and 1.5 kb of the SDP1 promoter have been amplified by PCR from either Arabidopsis complementary DNA or genomic DNA working with the following primer pairs: DGAT1 (59-CACCATGGCGATTTTGGATTCTGCTGGCG-39 and 59-TCATGACATCGATCCTTTTCGGTTCATCAGG-39), WRI1 (59-CACCATGAAGAAGCGCTTAACCACTTCC-39 and 59-TTATTCAGAACCAACGAACAAGCCC-39), and SDP1p (59-CACCTTCGAGTTTTATTTTCGTTACTTCCA-39 and 59-TATTGATTCGAAGATGAATTTGGGTGTGT-39). The goods have been cloned in to the pENTR/D-TOPO vector then (applying the Gateway LR Clonase enzyme mix) transferred towards the proper destination vector, according to the manufacturer’s guidelines (Invitrogen). SDP1p was transferred to pBGWFS7, DGAT1 to pB2GW7, and WRI1 to pK2GW7 (Karimi et al., 2002). The constructs were transformed into Agrobacterium tumefaciens strain GV3101 by heat shock and into Arabidopsis by the floral dip technique (Clough and Bent, 1998). Transformants containing transfer DNA insertions were selected by means of antibiotic or herbicide resistance.LITERATURE CITEDAndrianov V, Borisjuk N, Pogrebnyak N, Brinker A, Dixon J, Spitsin S, Flynn J, Matyszczuk P, Andryszak K, Laurelli M, et al (2010).
