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A Vernonia Diacylglycerol Acyltransferase Can Increase Renewable Oil Production Tomoko Hatanaka, William Serson, Runzhi Li, Paul R. Armstrong, Keshun Yu, Todd Pfeiffer, Xi-Le Li, and David Hildebrand J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02498 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 1, 2016

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Journal of Agricultural and Food Chemistry

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Full Title

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A Vernonia Diacylglycerol Acyltransferase Can Increase Renewable Oil

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Production

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Tomoko Hatanaka1, William Serson2, Runzhi Li3, Paul Armstrong4, Keshun Yu2, Todd

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Pfeiffer2, Xi-Le Li2 & David Hildebrand*2

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1Department

of Bioresource Science, Kobe University, Kobe, Japan

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2Department

of Plant and Soil Sciences, University of Kentucky, Lexington, KY USA

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China, 05000473

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Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University, Taigu,

USDA-ARS, EWERU-CGHAR, Manhattan, KS USA

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*Corresponding author

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Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY USA

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Phone: 859/218-0760

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Fax: 859/257-7125

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E-mail: [email protected]

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ACS Paragon Plus Environment

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Key message

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A DGAT1 from Vernonia galamensis produces unusually high oil accumulation in

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different organisms. Some transgenic soybeans show a 4% increase in oil content without

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reduction in protein levels.

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ABSTRACT:

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Increasing the production of plant oils such as soybean oil as a renewable resource for food

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and fuel is valuable. Successful breeding for higher oil levels in soybean, however, usually

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results in reduced protein, a second valuable seed component. We show that by

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manipulating a highly active acyl-CoA: diacylglycerol acyltransferase (DGAT) the

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hydrocarbon flux to oil in oilseeds can be increased without reducing the protein

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component. Compared to other plant DGATs, a DGAT from Vernonia galamensis

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(VgDGAT1A) produces much higher oil synthesis and accumulation activity in yeast,

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insect cells and soybean. Soybean lines expressing VgDGAT1A show a 4% increase in oil

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content without reductions in seed protein contents or yield per unit land area.

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Incorporation of this trait into 50% of soybean worldwide could result in an increase of 850

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million kg oil/year without new land use or inputs and be worth ~ $1 billion/year at 2012

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production and market prices.

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Key Words: triacylglycerol, lipids, oilseeds, Glycine max, diacylglycerol acyltransferase,

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Vernonia galamensis

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INTRODUCTION

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Various renewable oil sources have been used historically with plant oils

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representing one of the world’s most important renewable resources. In addition to

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extensive food uses, plant oils also have value as renewable chemicals and fuels, such as

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biodiesel. World-wide production of plant oil is dominated by palm, soybeans, rapeseed

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(including canola) and sunflowers 1. Oil palm and soybean production has increased

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rapidly and rapeseed has also shown steady increases and this trend is expected to continue

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with total global plant oil production of ~ 500 million MT in 2012 (Inform Sept. 2013).

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Genetic improvement of oilseed yield per unit land area has shown fairly steady

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progress with continuous soybean yield increases being a good example 2, 3. This has

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occurred with little or no increased inputs, making renewable oil production from plants

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less expensive over time and progressively more competitive with petroleum as an

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industrial chemical feedstock. Furthermore, it is becoming increasingly possible to alter

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hydrocarbon flux into oil in various organisms including oilseeds, increasing oil yield per

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unit land area independent of higher yield. This can be particularly true for low oil oilseeds

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such as soybeans. A number of studies report higher oil content increases with enhanced

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expression of TAG biosynthetic genes 4-7. Elevated expression of regulatory genes that up-

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regulate multiple enzymes for fatty acid biosynthesis also can result in higher oil levels 5.

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Co-expression of the transcription factor WRI1 with DGAT1 is shown to have a synergistic

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effect on TAG biosynthesis in plants 8-10.

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Seed oil is biosynthesized during the second main stage of seed maturation 11-13, concurrent with elevated expression of associated biosynthetic enzymes. In studies of


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expression profiles of TAG biosynthetic enzymes and oil accumulation in developing

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soybeans, DGAT1 show an expression profile suggesting a dominant role in soybean oil

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biosynthesis, but DGAT2 and PDAT (phospholipid:diacylglycerol transferase) do not

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(unpublished data; Li et al., 2013). Two full-length DGAT1s have been cloned from

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developing soybean cDNA designated GmDGAT1A (GenBank # AB257589) and

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GmDGAT1B (GenBank # AB257590). Both soybean DGAT1s are highly expressed at

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developing seed stages of maximal TAG accumulation with DGAT1B showing highest

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expression at somewhat later stages than DGAT1A 14.

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Vernonia galamensis, Euphorbia lagascae and Stokesia laevis accumulate 60% or

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more epoxy fatty acids in their seed oil. The DGAT1 genes of V. galamensis and E.

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lagascae were cloned 15 and it was found that two DGAT1s from V. galamensis

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(VgDGAT1A and VgDGAT1B) are expressed in all plant tissues with highest expression in

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developing seeds 16. When an epoxygenase gene from S. laevis (SlEPX) was expressed in

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soybean seeds, the transgenic seeds exhibited some undesired phenotypic alterations, but

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these side effects are overcome in the VgDGAT1 co-expressing soybeans 17.

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Expression of a Tropaeolum majus DGAT1, TmDGAT1, was reported to increase

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oil content, seed size and yield in Arabidopsis 18. Similar increases in oil content and seed

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yield were reported with higher expression of a Brassica napus (canola) DGAT1,

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BnDGAT1, back in B. napus 19. They also reported enhanced drought tolerance of these

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transgenic canola with increased DGAT expression. Lardizabal et al.6 reported a 1.5%

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increase in oil levels of transgenic soybeans expressing a fungal DGAT2. Unlike traditional

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breeding for high oil seed protein levels were not reduced. A more recent study, which used

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a Corylus americana DGAT1 selected from a screening library, resulted in a 3% increase in


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total oil content and an edited GmDGAT1B caused a similar increase in oil 20. Evidence

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from greenhouse experiments and field trails shows VgDGAT1A can increase oil content at

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least this much.

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We find much greater TAG biosynthesis and accumulation in yeast and insect

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cells expressing VgDGAT1A than several other DGAT1s and report generation of > 20

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independent VgDGAT1A transgenic lines which show robustness in the field. Many of these

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lines show a significant increase in oil content with little or no decrease in protein content

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compared to the parental line.

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MATERIALS AND METHODS

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cDNA cloning

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We have previously cloned full length DGAT1s from Vernonia galamensis

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(VgDGAT1A, VgDGAT1B), Euphorbia lagascae (ElDGAT1A) and soybean cultivar “Jack”

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(GmDGAT1A, GmDGAT1B) 14-16. The procedures were based on designing degenerate

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primers and RT-PCR for V. galamensis and E. lagascae, or EST searching for soybean, and

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a RACE strategy to sequence the full length of cDNAs.

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Expression in insect cells The cDNAs were cloned into the pFastBac1 vector (Invitrogen, CA) and the

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expression in Sf9 cells was performed with the Bac-to-Bac expression system (Gibco

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BRL), and the recombinant baculovirus was prepared following their instruction manual.

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Sf9 cells were then infected by a baculovirus possessing Vernonia or Euphorbia DGAT1

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(VgDGAT1A, ElDGAT1A) and cultured for 4 days and the cells were collected. Another set


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of cultured cells was infected by a baculovirus without cloned genes as a control. The

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cultured cells were freeze-dried and their lipids were extracted with chloroform:methanol

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(2:1). The TAG fractions were separated with thin layer chromatography (TLC) in hexane:

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ethyl ether: acetic acid (90:10:1, v/v/v) solvent system and analyzed with gas

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chromatography (GC) as described below.

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Yeast microsome assays

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Arabidopsis, soybean and Vernonia DGAT1s (AtDGAT1, GmDGAT1A, VgDGAT1A

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and VgDGAT1B) were cloned into the yeast vector pYES2 (Invitrogen, CA). The constructs

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along with the void vector as a control were used to transform yeasts (Saccharomyces

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cerevisiae) strain INVSc1 (Invitrogen, CA). Transformed yeast cells were cultured and the

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microsome fractions were prepared according Dahlqvist, et al. 21.

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The reaction mixture (100 µL) contained 20 mM radiolabeled linoleic acid CoA,

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300 mM dioleyl diacylglycerol, 0.02% Tween 20, 100 mM Tris-HCl (pH 7.1), 1 mM

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MgCl2, 0.5 mM CoASH, 0.5 mM ATP and microsomes (corresponding to 50 µg protein).

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The suspension was incubated at 30 °C with shaking (100 rpm) for 1 hour. The reaction

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was stopped by first placing the test tubes with the reaction mixture on ice and followed by

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adding 100 µg of soybean triacylglycerol as carrier. Lipids were extracted with

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chloroform: methanol (2:1, v/v). Samples were loaded on TLC plates and the radioactive

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bands were detected by phosphorimaging and scintillated. For identification of radioactive

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products, methylated fractions were analyzed by TLC with a hexane: MTBE (Methyl tert-

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butyl ether): acetic acid (85:15:1, v/v/v) solvent system.

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Lipid analysis Samples prepared as described above were frozen in liquid N2, stored at – 80˚C and

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then lyophilized. Samples were transferred to glass test tubes and tri-heptadecanoin (tri-

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17:0) was added at 10 µg/mg tissue as a standard. The samples were finely ground and 1-2

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mL of chloroform and methanol (2:1) containing 0.001% butylated hydroxytoluene (BHT)

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was added and samples were ground further. After brief centrifugation, the chloroform

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phase was transferred into a new glass tube. Samples were divided into two aliquots. One

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was used for TLC and the other directly for GC analysis.

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For separation of individual lipid classes by TLC, the chloroform extracts were

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concentrated to ~ 50 – 100 µL. 10 µL of the sample was loaded in a narrow band in lanes of

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Whatman LK6D Silica gel 60A TLC plates 1 cm from the bottom of the plates. The plates

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were put in a chamber with chloroform: methanol: water (65:25:4, v/v/v) containing

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0.0001% BHT for running until the first solvent reached ½ up the plate (~ 10 cm). Then,

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the plate was moved into the second solvent, hexane: diethyl ether: acetic acid (100:100:2,

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v/v/v) containing 0.0001% BHT and developed until the solvent was ~ 1 cm from the top.

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After development, the plate was dried, and subsequently sprayed with 0.005% primulin in

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80% acetone, followed by visualizing under UV light. The bands of interest were scraped

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and transferred to a Pasteur pipette with a glass wool plug washed with chloroform:

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methanol. The lipid samples were eluted with 0.5 mL of chloroform: methanol containing

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0.0001% BHT twice. Finally, eluted lipid samples were analyzed by GC.

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For GC analysis, samples were dried with N2, and 0.5 mL of 0.5 M sodium methoxide (NaOCH3) in methanol was added and incubated for at least 15 minutes with


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shaking at 22 ˚C. 0.5 mL of isooctane containing 0.001% BHT was added to each tube and

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mixed well. Phase separation was obtained with centrifugation and adding aqueous 0.9%

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potassium chloride if needed. The isooctane layer was extracted and transferred into GC

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auto-sampler vials. The fatty acid methyl esters (FAMEs) were analyzed with gas

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chromatography on a Varian CP-3800 GC with a 24 m x 0.25 mm ID CP-Select CB for

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FAME (Agilent Technologies) fused silica column with a 0.25 µm film thickness. The

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temperature program was 90˚C for 1 min., then to 155˚C at 20˚C/min. with no hold, then to

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175˚C at 3.6˚C /min. with no hold and finally to 250˚C at 12˚C/min. holding for one min.

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Analysis of protein and oil content was also performed by non-destructive means on

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a single seed basis using nuclear magnetic resonance (NMR) and near-infrared

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spectroscopy (NIR) 22, 23. Briefly, the instrument collects spectra on a seed as it falls

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though a short length of illuminated glass tubing. Optic fibers are aligned with the tube

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axis and connected to a spectrometer for collection of seed spectra. The instrument is

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designed for large sample sorting and processes seeds at about 3 per second although seeds

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were hand feed for this study to preserve identity. NMR single-seed oil measurement was

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accomplished using a Minispec mq 20, (Bruker BioSpin, The Woodlands, TX) using the

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manufacturers’ protocol for oil measurement. Seed and oil mass were measured and

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converted to a dry basis oil percentage using a moisture content estimated from room

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equilibrium moisture conditions.

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Seed-specific expression vector construction and soybean somatic embryo

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transformation


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An expression vector for soybean transformation was constructed using

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pCAMBIA1301 containing the hygromycin resistance gene and the GUS reporter gene

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(Cambia, ACT, Australia; GenBank # AF234297). The coding sequence of VgDGAT1A

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was amplified by a high fidelity polymerase (Invitrogen) using end-specific primers

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containing restriction sites. The amplification product was then subcloned into the

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respective sites of pPHI4752 vector containing a phaseolin promoter, which confers strong

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seed-specific expression of transgenes 24. The phaseolin promoter cassette containing the

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coding region of each target gene was transferred into the corresponding sites of the binary

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pCAMBIA1301, T-DNA vector. The recombinant expression vector was subsequently

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introduced into somatic embryos of soybean (cv. `Jack’) using the particle bombardment

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method of transformation.

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Soybean somatic embryo induction and culture was carried out using a protocol

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modified from prior procedures 25-28. Immature soybean seeds at 3-5 mm length were

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dissected, and cotyledons were placed on D40 (40 mg/L 2,4-D in MS media) solid medium

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for a one-month somatic embryo induction. The induced embryos were transferred to D20

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plates for proliferation. The globular embryogenic cultures from D20 (20 mg/L 2,4-D in

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MS media) plates were then moved into FN 25 liquid medium for one-month suspension

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culture. Small embryo clumps were selected for particle bombardment gene delivery.

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Plasmid DNA/gold preparation for the particle bombardment was conducted

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according to standard protocols 27. A DuPont Biolistic PDS1000/HE instrument (helium

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retrofit) was used for all transformations. After bombardment, the embryo clumps were

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transferred into FN liquid medium containing 30 mg/L hygromycin for selective culture for

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four to five weeks. The positive transformed embryos obtained by hygromycin selection


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were then moved into fresh FN liquid medium for culture and simultaneously for GUS test

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and identification of the transgene presence by PCR. The PCR-positive transgenic embryo

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lines were transferred into maturation medium (SHaM) 29 for three to five weeks. For the

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transgenic lines, one set of matured somatic embryos were sampled for lipid extraction and

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subsequent GC analysis. The rest of the matured somatic embryos were desiccated for 4-7

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days, and then were placed on half strength MS solid medium for germination. Germinated

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plantlets were transferred to closed sterile soil cups for growth in a culture room under 23:1

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(light: dark) photoperiod cycle and 25˚C. Once seedlings reached 13 cm, they were

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transferred to a greenhouse for flowering and seed set under a 16:8 (light:dark) cycle,

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25/21˚C. Mature seeds were harvested from each regenerated soybean plant separately.

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Seeds were chipped for genotyping by PCR and fatty acid analysis by GC.

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Upon successful transformation of at least one copy of the transgene into

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background Jack, the seedlings were developed in the greenhouse and subjected to seed

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increase the next year. In 2011, plants were grown out at Spindletop Farm in Lexington,

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KY in 20’ headrows and hand harvested. Seeds were sent to Manhattan, KS for oil and

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protein evaluation via non-destructive single seed NIR and NMR described above.

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Segregating Line Analysis Nine independent transgenic lines which contained at least one copy of the

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VgDGAT1A trait as determined by PCR analysis as described above were grown on at

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Spindletop Research Farm in Lexington, KY in the 2012 growing season. Thirty seeds were

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grown each year from these lines in 2013 and 2014, selected using a single seed descent

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method. In 2014, leaf DNA was randomly collected from 63 of 270 planted soybeans using


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phenol-chloroform extraction and analyzed for the presence of the VgDGAT1A trait by

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PCR as described above. Seeds from these plants were hand harvested during October 2014

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and analyzed for oil and protein content via NIRS as described above. The genotype data

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was connected to the phenotype data and the mean oil and protein content of each genotype

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was compared.

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RESULTS

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In vitro assays of VgDGAT1A We have found that Glycine max, E. lagascae and V. galamensis have at least two

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DGAT1s and have made full-length DGAT1 cDNAs from all three species. We isolated five

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cDNA clones: Soybean DGAT1s (GmDGAT1A and GmDGAT1B), Euphorbia DGAT1

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(ElDGAT1A) and Vernonia DGAT1s (VgDGAT1A and VgDGAT1B). These cDNA clones

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are mainly expressed at the stage of seed development which involves high oil

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accumulation.

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VgDGAT1A resulted in high oil accumulation in Sf9 insect cells (Figure S1). In

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experiments expressing VgDGAT1A and ElDGAT1A in Sf9 insect cells, we found much

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higher accumulation of TAG in the VgDGAT1A expressing cells than the ElDGAT1A

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expressing and vector control cells (Figure S1) 15. One way ANOVA analysis confirms

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that VgDGAT1A incorporation results in a statistically significant higher level of oil

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(p=0.002), but E1DGAT1A did not (p=0.072). In fact, VgDGAT1A demonstrated 20-fold

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increase in total lipids compared to the control, consistent with the results with yeast

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microsome assay below.


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An improved system for microsomal analysis was used which utilizes 300-fold

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lower concentrations of microsomes compared to most studies, resulting in lower

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background and more accurate activity determinations than traditional methods 30. In our

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yeast system, cells expressing GmDGAT1A, VgDGAT1A and VgDGAT1B were analyzed

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for TAG biosynthetic activity along with the Arabidopsis thaliana DGAT1 (AtDGAT1) and

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the vector control. Of these, three showed significant activity in the yeast system:

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GmDGAT1A, VgDGAT1A and VgDGAT1B (Figure 1). GmDGAT1A exhibited twice the

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activity of controls, while VgDGAT1A and 1B showed five to six fold activity over

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GmDGAT1A.

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Oil and protein content of transgenic soybeans expressing VgDGAT1A VgDGAT1A was also expressed in soybean somatic embryos. Transformation

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resulted in about 40 viable lines that stably integrated at least one copy of VgDGAT1A

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(unpublished results). Soybean somatic embryos expressing VgDGAT1A were regenerated,

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grown out in a greenhouse and mature T2 seeds were collected. Seeds of these higher oil

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soybean lines were grown out in both greenhouse and field environments and progeny

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again analyzed for protein and oil contents in the next year, and subsequent years until lines

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were homozygous for the transgene, at T6. As it is seen in Table 1, the protein and oil

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content of mature field-grown seeds were measured and many of the VgDGAT1A

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transformed soybean seeds showed 3 to 4 percent increases in total oil content per seed dry

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weight, most without any significant (p

(PDF) A Vernonia Diacylglycerol Acyltransferase

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