research article changes in oleic acid content of...

Research ArticleChanges in Oleic Acid Content of Transgenic Soybeans byAntisense RNA Mediated Posttranscriptional Gene Silencing

Ling Zhang Xiang-dong Yang Yuan-yu Zhang Jing Yang Guang-xun Qi Dong-quan GuoGuo-jie Xing Yao Yao Wen-jing Xu Hai-yun Li Qi-yun Li and Ying-shan Dong

Agro-Biotechnology Research Institute Jilin Academy of Agricultural Sciences Changchun Jilin 130033 China

Correspondence should be addressed to Qi-yun Li qyli1225126com and Ying-shan Dong ysdongcjaascom

Received 16 April 2014 Revised 10 July 2014 Accepted 21 July 2014 Published 13 August 2014

Academic Editor Giuliana Napolitano

Copyright copy 2014 Ling Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The Delta-12 oleate desaturase gene (FAD2-1) which converts oleic acid into linoleic acid is the key enzyme determining thefatty acid composition of seed oil In this study we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1bgene by using antisense RNA in soybeanWilliams 82 By employing the soybean cotyledonary-nodemethod a part of the cDNA ofsoybeanGmFad2-1b 801 bpwas cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoterBCSP Leafpainting LibertyLink strip PCR Southern blot qRT-PCR and fatty acid analysis were used to detect the insertion and expressionof GmFad2-1b in the transgenic soybean lines The results indicate that the metabolically engineered plants exhibited a significantincrease in oleic acid (up to 5171) and a reduction in palmitic acid (to lt3) in their seed oil content No structural differenceswere observed between the fatty acids of the transgenic and the nontransgenic oil extracts

1 Introduction

Vegetable oils form an important part of the human diet pro-viding concentrated sources of energy and essential nutrientsA process is used for the deacidification of a vegetable oil inwhich the major acid of the vegetable oil is from the groupcomprised of epoxy fatty acids hydroxy fatty acids linoleicacid and oleic acid [1] The consumption of oils with higholeic acid content is beneficial because this monounsaturatedfatty acid not only improves the shelf life but also reduces theneed for hydrogenation a process adding to the cost of the oilas well as generating unwanted trans fat that has been linkedto many health problems in humans [2ndash4] The commoditysoybean oil is composed of five fatty acids oleic acid (181)palmitic acid (160) stearic acid (180) linoleic acid (182)and linolenic acid (183) The percentages of these five fattyacids in soybean oil average are 18 10 4 55 and 13respectively [5] Most polyunsaturated fatty acids up to 90in nonphotosynthetic tissues of plants are synthesizedthrough a (181) desaturase in the endoplasmic reticulumTheendoplasmic reticulum-associated oleate desaturase FAD2 (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine Δ 12-desaturase)

is the key enzyme responsible for the production of linoleicacid in plants [6ndash8]

In soybeans two copies of microsomal 120596-6 desaturaseFAD2-1 and FAD2-2 have been cloned [9] Studies haveshown that FAD2-1 was expressed primarily in the develop-ment of seeds while FAD2-2 was expressed in both the veg-etative tissues and the development of seeds [10 11] Alsothe FAD2-2 desaturases consisted of FAD2-2A(Glyma19g32930)FAD2-2B (Glyma19g32940) and FAD2-2C(Glyma03g30070) FAD2-1A and FAD2-1B are most closelyrelated to one another with a shared genomic organizationcontaining a single intron and a 99 identity in encodedamino acid sequenceThereby this confirmed the importanceexpression during peak oil synthesis while a possible role wasalso revealed for FAD2-2C under cool temperature con-ditions [12ndash14] Genetic modification (GM) aimed at regu-lating the FAD2 expression has been applied to produce oilswith a higher C181 in soybeans [15ndash19] For example byantisense suppression of FAD2 in soybeans a transgenic linewas obtained that produced oil with a higher C181 (57)compared to the wild variety [20] Similarly downregulatingFAD2-1 and FatB using interference RNA has enabled the

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2014 Article ID 921950 8 pageshttpdxdoiorg1011552014921950

2 International Journal of Genomics

Table 1 Primers for different experiments

Primer name Primer sequence PCR producer (bp)GmFAD2-1b-F 51015840-AGCCACTAGGCATGGGTCTAGCAAA-31015840 801GmFAD2-1b-R 51015840-GCAATGGCACCCCATAAACACATAG-31015840

EB-R 51015840-GGAAAGCAACCATATCAGCATATCAC-31015840 752EB-F 51015840-TTCTCCAAGGTTGCATTCTTACTGG-31015840

GmActin-R 51015840-TTGACTGAGCGTGGTTATTCC-31015840 402GmActin-F 51015840-GATCTTCATGCTGCTGGGTG-31015840

qRTGmFad2-R 51015840-CACCATTCACTGTTGGCCAA-31015840 163qRTGmFad2-F 51015840-ATGAGGGAAAAGGGGTGAGG-31015840

production of soybeans with a significantly higher oleic acidcontent (up to 9458) and reduced levels of palmitic acid(lt3) [21] Recently Plenish high oleic soybeans have beenin commercial production by DuPont since 2012 They weredeveloped using a biotech process known as gene silencing[22] These results demonstrate that the GmFAD2 familymembers play a very important role in metabolicallyengineered oil seed plants [23]

In this study we report the transformation of soybeanwith GmFad2-1b gene by antisense RNA We analyzed andinvestigated the fatty acid composition of the transgenic linesand also discussed the nature of the transcript produced byGmFad2-1b

2 Materials and Methods

21 Plant Materials and Cotyledonary-Node Method Soy-bean cultivars [Glycine max (L) Merrill cv ldquoWilliams 82rdquo]were grown in a greenhouse at the Jilin Academy of Agri-cultural Sciences Gongzhuling during a 16 h photoperiodat a 35∘C daytime temperature and a 25∘C night-time tem-perature The cotyledonary-node method used in this studyfollowed the procedure described by Paz et al [24] In briefsoybean seeds were surface-sterilized by placing seeds into atightly sealed chamber containing chlorine gas for 16 h Thesterilized seeds were soaked with sterile water at 28∘C duringan 18 h photoperiod for 8ndash12 h The imbibed soybean seedswere placed on sterile filter paper and cut longitudinally alongthe hilum The cotyledons and hypocotyls (sim5mm) from asingle seedwere split evenly into two explants Approximately50 coat-removed explants were then inoculated in a liquidcocultivation medium (CCM) which contains 110 B5 saltsand vitamins 39 gL 2-[N-morpholino] ethanesulfonic acid(MES) 3 sucrose (30 gL) (pH 4) filter-sterilized 025mgLgibberellic acid (GA3) 400mgL L-cysteine 167mgL N-6-benzylaminopurine (BAP) 1542mgL dithiothreitol (DTT)and 200120583molL acetosyringone (As) for 30min After thisinoculation seven explants (adaxial side up) were randomlyplaced in Petri dishes (90mm in diameter times 15mm deep)on sterile filter paper placed on solid CCM containing 05purified agar (BD-Difco) and incubated in the dark at 25∘Cfor 3ndash5 days

After the cocultivation the explants were then transferredinto solidified shoot induction (SI) medium containing B5salts and vitamins 3 sucrose 08 agar (8 gL Sigma)

058mgL MES (pH 58) filter-sterilized 167mgL BAP250mgL ticarcillin (Tic) 100mgL cefotaxime (Cef) and5mgL glufosinate and then incubated in a growth room at28∘C under an 18 h photoperiod for two weeks Then thehypocotyl and shoots were trimmed from the explants andthe remaining cotyledons with developing nodules were sub-cultured to a fresh SI medium for another two weeks Fourweeks after SI the cotyledonswere removed from the explantsand the remaining tissues were transferred into a shoot elon-gation (SE) medium The SE medium was composed of MSsalts and vitamins 3 sucrose 08 agar (Sigma) 058mgLMES filter-sterilized 50mgL L-asparagine 50mgL glu-tamine 01mgL indole acetic acid (IAA) 05mgL GA31mgL zeatin riboside (ZR) 250mgL Tic 100mgL Cef and5mgL glufosinate (pH 58) The explants were then trans-ferred to fresh SE medium every two weeks until the regen-erated shoots were suitable for rooting The incubation con-ditions were the same as the conditions for SI At this pointelongated shoots (3-4 cm in length) were excised and placedinto a rooting medium (RM) containing MS salts andvitamins 3 sucrose 08 agar (Sigma) 058mgL MES(pH 58) filter-sterilized 50mgL L-asparagine 50mgL glu-tamine 01mgL indole butyric acid (IBA) 250mgL Tic and100mgL Cef After a period of 1-2 weeks the roots were fullydeveloped to 2-3 cm in length and eventually transplantedinto the pots to be grown in the greenhouse The transgeneevents were named by EB8001 to EB8067 The T0 generationtransgenic plants were grown in the greenhouse and the T

1

and T2lines were grown under field conditions

22 Nucleic Acid Isolation and Vector Construction The freshsoybean tissues were collected to extract the total RNAwith TRIZOL reagent (Invitrogen) RNA samples (2120583g) weretreated with the amplification grade DNase I (Invitrogen) at37∘C for 15min prior to cDNA synthesis using SuperScriptIII Reverse Transcriptase (Invitrogen) according to the man-ufacturerrsquos instructions

For the assembly of the GmFad2-1b antisense RNAvector a part of a fragment about 801 bp of the GmFad2-1b(GenBank accession XM 003555831) was amplified from thecDNA above by using the primers GmFad2-1b-FGmFad2-1b-R (Table 1) The amplified GmFad2-1b gene product wascloned into the pEASY-T1 cloning vector (TransGene) Theproduct was sequenced using T7 and M13 vector sequencingprimers to confirm the sequence of the ligated product An

International Journal of Genomics 3

801 bp XbaISacI T1-vector digested GmFad2-1b fragmentwas anticloned into a pCAMBIA3300 vector and digestedwith the same enzymes to put the GmFad2-1b gene under theseed 1205721015840 subunit of the 120573-conglycinin promoter (BCSP) withthe different direction and the1198611198621198781198751015840 functionwas discussedby Imoto et al [25] The pCAMBIA3300 binary vector has akanamycin resistance gene for bacterial selection and aphosphinothricin acetyl transferase (bar) resistance gene forplant selection The bar gene was driven by the constitutiveCaMV 35S promoter The Agrobacterium tumefaciens strainEHA101 was used in this study

23 Detection of Transgenic Soybean Plants Transgenic soy-bean plants were verified by leaf painting LibertyLink stripanalysis polymerase chain reaction (PCR) analysis andSouthern blot Leaf painting was performed following theprocedure described by Ma et al [26] Stock of LibertyLinkBasta (135 gL) was diluted by 1000-fold and painted on halfthe upper surface of the tested soybean leaves After 3ndash5 daysthe painted leaves of the negative plants died and positiveplants remained healthy LibertyLink strips (Envirologix)were used to determine the genetically modified plants con-taining the phosphinothricin N-acetyltransferase (PAT) pro-tein following the manufacturerrsquos instructions To summa-rize a circular leaf tissue was isolated by closing the cap of theEppendorf tubes The tissue was ground with a pestle for 20ndash30 sec and 025mL of protein extraction buffer was addedbefore regrinding The development of the control line indi-cated that the strip had functioned properly The second line(test line) would show up when the tested sample waspositive

For the PCR analysis the genomic DNA of transgenicsoybeans was extracted using a simple and quick DNAextracting method by Edwards et al [27] We designed thedetection primers named EB-RF according to the GmFad2-1b gene and the upstream BCSP promoter sequence Thelength of the product was 752 bpThe primer sequence can beseen in Table 1 The PCR reaction was conducted using aninitial denaturation at 94∘C for 5min followed by 30 cycles of94∘C for 45 sec 58∘C for 45 sec and 72∘C for 1min and a finalextension of 10min at 72∘CThe PCR products were analyzedon 10 agarose gels As a positive control aGmFad2-1b geneexpression cassette containing pCAMBIA3300 vector DNAwas also used The genomic DNA from the empty pCAM-BIA3300 vector transformed plant and the untransformedWilliams 82 were used as negative controls

A Southern blot analysis of the PCR positive plants wasperformed by DIGHigh Prime DNA Labeling and DetectionStarter Kit II (Roche cat number 11585614910) accordingto the manufacturerrsquos instructions The genomic DNA oftransgenic T

1and T

2plants was isolated by using the high-

salt CTAB DNA method In summation 20120583g of genomicDNAwas digested withXbaI andHindIII electrophoresed on10 agarose gel respectively The DNA was denatured by analkaline solution and transferred to positively charged Amer-sham nylon membranes (Amersham) according to standardprotocol and fixed by UV-crosslinking at 1200U for 1minSince the GmFad2-1b gene and seed-specific promoter BCSP

were endogenous genes we took the marker gene bar inpCAMBIA3300 vector as the probe for the Southern blotThe probe was labeled using digoxigenin- (DIG-) 11-dUTPwith DIG High Prime DNA Labeling Reagents (Roche)Hybridization was carried out at 42∘C Washing blockingand detection were carried out according to the manufac-turerrsquos instructions The Southern blots were then exposedat room temperature for 15ndash20min and subsequently devel-oped

24 Fatty Acid Analysis The fatty acid compositions of theseeds harvested from T

1and T

2transgenic plants were ana-

lyzed by gas chromatography (GC) First the lyophilized soy-bean seeds were ground to a fine powder and then the lipidswere extracted with heptaneThe supernatant was then trans-ferred into a glass vial and the heptane was evaporated witha flow of dry nitrogen gas at 80∘C At this point an 8mgaliquot of the extracted soybean oil was transesterified withsodium methoxide The fatty acid methyl esters were thenseparated on a Hewlett-Packard 6890 GC supplied with ahydrogen flame ionization detector and a capillary columnHP INNOWAX (30m 025mm id) with an N2 carrier at2mLmin The oven temperature was maintained at 100∘Cfor a minimum of 1 min and then increased linearly to 250∘C(15∘Cmin) The fatty acid methyl esters (FAMEs) from TAGwere identified by comparison of their retention times withthe known standards (37-component FAME mix Supelco47885-U) Relative fatty acid compositions were then calcu-lated as the percentage that each fatty acid represented of thetotal measured fatty acids

25 Quantitative RT-PCR Analysis The total RNA wasextracted by using a TRIZOL reagent kit (Invitrogen) fromthe different issues of the mature T

2transgenic plants and

wild types At this point two 120583g total RNA from each samplewas reverse-transcribed by using the PrimeScript RT reagentkit (Takara) with an oligo (dT) 18 primer Quantitative PCRwas performed on the real-time PCR system (Applied Biosys-tems 7900) with SYBR Premix Ex Taq (Takara) with theconditions of 95∘C for 30 sec followed by 40 cycles of 95∘C for5 sec and 60∘C for 30 secTheGmActin gene (NM 001289231)was used as a positive internal control and the gene primersfor the qRT-PCR were designed using conserved sequencesfor them as listed in Table 1

3 Results and Discussion

The high-frequency plant gene transfer system is one of thekey stages for introducing useful or novel gene(s) into soy-beans In this study we used the cotyledonary explant derivedfrom mature soybean seeds for Agrobacterium-mediatedtransformation The entire procedure for the Agrobacterium-mediated cotyledonary-node transformation system used inthis study is illustrated in Figure 1 and Table 2 Addition-ally high-level expression of recombinant molecules hasbeen consistently achieved by utilizing specific regulatorysequences within the seed tissues The cobombardmenttransformation strategy to generate transgenic soybean


Table 2 Transformation efficiency of soybean cotyledonary-node explants inoculated with Agrobacterium tumefaciensrsquos strain EHA101harboring EB gene (pCamibia3300-BCSP-anti-GmFad2)

Number ofexperiments Genotype Explants infected Plants surviving

on SE mediumPositive T

0

transformationTransformationefficiency ()

1 Williams 82 96 24 16 16732 Williams 82 114 36 18 15843 Williams 82 117 45 21 1796

Total 327 105 55 1684

(a) (b) (c)

(d) (e) (f)

Figure 1Agrobacterium-mediated soybean transformation using the cotyledonary node as explants (a) Seed germination in theGMmediumfor the night (b) Inoculation of explants with Agrobacterium (c) A longitudinal cut between the cotyledons and through the hypocotyl togenerate two identical explants (c) Explants cocultured with Agrobacterium for 5 days (d) Two weeks after selection on shoot induction (SI)medium containing 5mgL glufosinate (e) Elongated shoot in shoot elongation (SE) medium (f) Rooting of the resistant shoot

plants allowed us to evaluate the pCAMBIA3300-BCSP-anti-GmFad2-1b plasmid vector (Figure 2) The gene encodingof the anti-GmFad2-1b was placed under the control of 1205721015840subunit of the 120573-conglycinin (BCSP) soybean promoter andwas introduced into the soybean genome with the aim ofhigh-level suppressions which resulted in a high oleic acidcontent in the soybean seeds The promoter BCSP is tissue-specific and temporally regulated and is not expressed in theroots stems or leaves of soybean In this study the geneGmFad2-1b under promoter BCSP was not expressed in theseed of soybean as expected from the result of the qRT-PCR assay (Figure 5) Regulatory information for the tissue-specific expression mediated by the BCSP promoter and theother elements involved in the modulation of the expressionof genes associated with seed storage protein has beenlocalized to the DNA sequences approximately 250 bpupstream from the transcription start site [28 29]

For the detection of the transgenic soybean plants weused three methods (the leaf painting the LibertyLink stripanalysis and the PCR) to confirm the positive transgeneplants We performed 200 Williams 82 on the seeds whichcontains about 327 explants infected by the cotyledonary-node method Finally we obtained 105 T

0transgenic regen-

eration plants and a total of 55 plants tested positive giving a55 transformation efficiency Figure 3 shows a part ofdetection results by leaf painting LibertyLink strip and PCRanalysis Although the T

0transgenic plants were generated in

the lab while the T1 T2were generated in the field the rapid

and exact method for determining positive transgenic statuswas pivotal The PCR detection of a selected marker gene isa traditional and effective way of doing this although falsepositive results are sometimes observed Therefore wechecked a part of the sequence between the BCSP andthe anti-GmFad2-1b at about 752 bp in the vector for the


Bcsp promoter HindIII XbaI SacI EcoRI

HindIII

XbaI

SacI

EcoRI

XhoI

XhoI

RB

LB

bcsp

T-nos

35S35

35S terBar

NptIIpBR ori

pBR bom

pVSl rep

pVSl sta

anti-GmFad2-1b

GmFad2-1b

anti-GmFad2-1bpCAMBIA3300

pCAMBIA3300

Figure 2 Map of vector pCmbia3300-Bcsp-GmFad2 A schematic representation of the expression cassettes of the anti-GmFad2-1b andpCAMBIA3300 plasmids used forAgrobacterium-mediated transformation of soybean cotyledonary nodeThe anti-GmFad2-1b gene is underthe control of the soybean seed promoter and 30 region (terminator)

identification of any transgenic plants (Figure 3(c)) Howeverthe primary PCR testing is cumbersome and costly Instead ofthe PCR procedures a leaf painting method was chosen Asleaf painting directly detected the effect of a selected markergene it showed fewer false positive results than the PCRprocedures Furthermore the leaf painting method was lowin cost and easy to carry out (Figure 3(a)) Since theGmFad2-1b gene fragment did not code for a functional protein nonovel protein was produced from the GmFad2-1b genecassette However the expressing cassette contains the barmarker gene which is characterized by a resistance to theherbicide bialaphosTheLibertyLink strips could therefore beused to quickly determinate genetically modified plantscontaining the bar gene (Figure 3(b)) By the methods statedabove we identified 55 positive T

0transgenic plants from the

total 105 T0transgenic lines We also used these detection

methods to examine the T1and T

2progenies of every

independently transformed T0line 11 of the 55 positive T

0

lines were further regenerated to T1lines and then T

2lines

All 11 plants which exhibited Basta resistance also showedamplification of the bar gene in the PCR analysis (results notshown) thereby confirming the presence and expression ofthe transgene in transgenic the soybean plants

Consequently because the GmFad2-1b gene and seed-specific promoter BCSP were endogenous genes the markergene bar in the pCAMBIA3300 vector became the probe for

the Southern blot among the 11 T2transgene lines In addi-

tion in order to guarantee the accuracy of the Southern blotwe used two different restriction enzymes XbaI andHindIIIfor digesting genomicDNA Based on the Southern blot anal-ysis and sequence data it was determined that the bar genecassette and the GmFad2-1b gene cassette have been insertedinto the soybean genome (Figure 4) In previous studiesthere have been suggestions that if a high transgene copynumber were integrated into the T

0plants the alternative

DNA copies should be integrated at different loci with a lowrecombination frequency [30] This result was corroboratedin our study by the Southern blot analyses of different T

2

plants lines EB8003 EB8005 EB8006 and EB8065 whichpresented at least four bar copies One concern related tothese high transgene copy numbers that were integrated inthe host genome is the possibility of superfluous tandemintegration which could result in possible gene silencing andlow recombinant protein production levels [29] The linesEB8019 EB8011 EB8057 and EB8067 showed one or twocopies and the lines EB8007 and EB8008 cannot be con-firmed by the two restriction enzymes at same time All of thetransgenic plants regenerated grew normally and set flowersand pods

Increasing the oleic acid content in soybean seed oil is oneof the most effective and efficient ways to enhance the nutri-tional value and practical utilization of soybean oil


(a) (b)

M + minus H2O 1 2 3 4 5 6 7 8 9 10 11 12 13 14

BCSPGmFad2

Actin

752bp

402bp

(c)

Figure 3 Procedure for confirmation of transgenic plants (a) Identification of the herbicide resistance of the leaves of transgenic soybeanplants using 135mgL Basta Left negative transgenic plant right positive transgenic plants (b) Events of the PCR positive lines wererandomly selected and tested using LibertyLink strips (c) PCR analysis of genome DNA of putative transgenic soybean plant The lengthof PCR production was 752 bp M trans2K plus DNA marker + plasmid DNA minus nontransformed soybean H

2O 1ndash14 the transgene line

HindIIIXbaI

M minus 1 2 3 4 5 6 7 8 9 10 11 M minus 1 2 3 4 5 6 7 8 9 10 11

15kb10kb

30 kb

15 kb

Figure 4 Southern blot hybridization of transgenic plants The soybean genomic DNA was sampled from 11 independent events in the T2

generation (1ndash11) and digested with two different restriction enzymes XbaI andHindIII and hybridized with the bar probe labeled with DIG

The overall lipid content of 11 T2transgenic lines of

soybean grain remained unchanged compared to the conven-tional soybean grain Using fatty acid analysis we selectedthree high oleic acid modified materials in the T

2transgene

lines They are EB8011 EB8057 and EB8061 The EB8011soybean grain contains increased levels of oleic acid (up to5171) and lower levels of linoleic acid (2732) linolenicacid (847) and to a lesser extent palmitic acid (872) Inaddition EB8057 oleic acid content is 3823 and EB8061

oleic acid content is 4048 which were significantly higherthan the receptor controls (1770) (Table 3)The lines EB8011and EB8057were presented at two copies in soybean genomicDNA while the line EB8061 was presented at three Inprevious studies FAD2 genes were identified as encoding120596-6fatty acid desaturases which catalyze the conversion of 181oleic acid to 182 linoleic acid in soybeans [6 11] Thisconversion is of particular interest in the soybeans due to thegrowing desire for low linoleic soybean oil [4] Moreover a


Table 3 GC-MS results yielding the oil composition of the transgenic soybean seed line

Sample Generation palmitic stearic oleic linoleic linolenicCK Williams 82 1049 447 1770 plusmn 020 5784 8501 EB8003 1063 358 1496 plusmn 001 6244 8382 EB8005 1050 402 1622 plusmn 012 5985 9413 EB8006 1059 420 1685 plusmn 004 5882 9534 EB8007 1033 395 1707 plusmn 012 5923 9425 EB8008 1053 380 1539 plusmn 005 6019 10106 EB8019 1113 410 1655 plusmn 114 5825 9967 EB8011 872 293 5117 plusmn 025

lowastlowast 2732 8478 EB8057 980 351 3823 plusmn 005


lowastlowast 3744 91210 EB8065 1055 396 1425 plusmn 009 6063 106111 EB8067 1020 367 2193 plusmn 005 5403 1017

0000050010001500200025003000350040004500

Root Leaf Seed

Relat

ive e

xpre

ssio

n le

vel

Williams 82EB8011

Stem

Figure 5 qRT-PCR analysis of GmFad2-1b in the different tissueRNA was extracted from root steam leaf and early flowering seedThe abundance of GmFad2-1b transcripts was normalized to that ofActin transcripts

high oleic acid content is desirable because this monounsat-urated fatty acid not only improves shelf life but also reducesthe need for hydrogenation a process which adds to the costof the oil and generates unwanted trans fat that has beenlinked to many health problems in humans [1] Our studydemonstrated high-level suppression of GmFad2-1b by anti-RNA while also resulting in a high oleic acid in the soybeans

4 Conclusions

Wewere successful in the suppression expressionGmFad2-1bin transgenic soybean seeds Based on the published resultson soybeans the soybean GmFAD2 gene family has beenpreviously characterized at the genome level for structure andexpression [9ndash11] and also in genetic modification [15ndash19]In our study silencing of the endogenous Fad2-1 gene inseeds of the soybean event EB8011 was achieved through theintroduction of a GmFad2-1b gene fragment driven by aseed-specific promoter The transcription of the GmFad2-1bgene fragment in the seed acts to silence transcription of theendogenous FAD2-1 gene The line EB8011 had a result of5117 oleic acid and 872 palmitic acid in contrast to 177oleic acid and 447 palmitic acid in the seeds In future

studies we also expect that the altered ratio of oleic andlinoleic acid will confer desirable properties on the resultingbiodiesel without a significant change in the yield

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ling Zhang and Xiang-dong Yang contributed to this workequally

Acknowledgments

This work is supported by a Project from the Ministryof Agriculture of China for Transgenic Research (Grants2013ZX08004-003 2011ZX08004-003 2008ZX08004-003and 2013ZX08004-004) The authors alone are responsiblefor the content and composition of this paper

References

[1] K L Fritsche ldquoLinoleic acid vegetable oils amp inflammationrdquoMissouri Medicine vol 111 no 1 pp 41ndash43 2014

[2] S Bhardwaj S J Passi and A Misra ldquoOverview of trans fattyacids biochemistry and health effectsrdquo Diabetes and MetabolicSyndrome vol 5 no 3 pp 161ndash164 2011

[3] H Sales-Campos P R de Souza B C Peghini J S da Silva andC R Cardoso ldquoAn overview of the modulatory effects of oleicacid in health and diseaserdquo Mini-Reviews in Medicinal Chem-istry vol 13 no 2 pp 201ndash210 2013

[4] A R Raneses L KGlaser JM Price and J ADuffield ldquoPoten-tial biodiesel markets and their economic effects on the agricul-tural sector of the United Statesrdquo Industrial Crops and Productsvol 9 no 2 pp 151ndash162 1999

[5] T E Clemente and E B Cahoon ldquoSoybean oil genetic appro-aches for modification of functionality and total contentrdquo PlantPhysiology vol 151 no 3 pp 1030ndash1040 2009


[6] J Okuley J Lightner K Feldmann N Yadav E Lark and JBrowse ldquoArabidopsis FAD2 gene encodes the enzyme that isessential for polyunsaturated lipid synthesisrdquo Plant Cell vol 6no 1 pp 147ndash158 1994

[7] J P Spychalla A J Kinney and J Browse ldquoIdentification of ananimal 120596-8 fatty acid desaturase by heterologous expression inArabidopsisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 94 no 4 pp 1142ndash1147 1997

[8] S Cao X Zhou C C Wood et al ldquoA large and functionallydiverse family of Fad2 genes in safflower (Carthamus tinctoriusL)rdquo BMC Plant Biology vol 13 no 1 article 5 2013

[9] J A Schlueter I F Vasylenko-Sanders S Deshpande et al ldquoTheFAD2 gene family of soybean insights into the structural andfunctional divergence of a paleopolyploid genomerdquo Crop Sci-ence vol 47 no 1 pp 14ndash26 2007

[10] M F Miquel and J A Browse ldquoHigh-oleate oilseeds fail todevelop at low temperaturerdquo Plant Physiology vol 106 no 2 pp421ndash427 1994

[11] E PHeppard A J Kinney K L Stecca andGHMiao ldquoDevel-opmental and growth temperature regulation of two differentmicrosomal 120596-6 desaturase genes in soybeansrdquo Plant Physiol-ogy vol 110 no 1 pp 311ndash319 1996

[12] G Tang W P Novitzky H Carol Griffin S C Huber and RE Dewey ldquoOleate desaturase enzymes of soybean evidence ofregulation through differential stability and phosphorylationrdquoPlant Journal vol 44 no 3 pp 433ndash446 2005

[13] E P Heppard A J Kinney K L Stecca and G Miao ldquoDevel-opmental and growth temperature regulation of two differentmicrosomal 120596-6 desaturase genes in soybeansrdquo Plant Physiol-ogy vol 110 no 1 pp 311ndash319 1996

[14] J D Lee MWoolard D A Sleper et al ldquoEnvironmental effectson oleic acid in soybean seed oil of plant introductions withelevated oleic concentrationrdquo Crop Science vol 49 no 5 pp1762ndash1768 2009

[15] M L Oliva J G Shannon D A Sleper et al ldquoStability offatty acid profile in soybean genotypes with modified seed oilcompositionrdquo Crop Science vol 46 no 5 pp 2069ndash2075 2006

[16] J L Alt W R Fehr G A Welke and D Sandhu ldquoPhenotypicand molecular analysis of oleate content in the mutant soybeanline M23rdquo Crop Science vol 45 no 5 pp 1997ndash2000 2005

[17] J Zhang J Zhu Q Zhu H Liu X Gao and H Zhang ldquoFattyacid desaturase-6 (Fad6) is required for salt tolerance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 390 no 3 pp 469ndash474 2009

[18] D Sandhu J L Alt C W Scherder W R Fehr andM K Bhat-tacharyya ldquoEnhanced oleic acid content in the soybean mutantM23 is associated with the deletion in the Fad2-1a gene encod-ing a fatty acid desaturaserdquo JAOCS Journal of the American OilChemistsrsquo Society vol 84 no 3 pp 229ndash235 2007

[19] J D Lee K D Bilyeu and J G Shannon ldquoGenetics and breed-ing formodified fatty acid profile in soybean seed oilrdquo Journal ofCrop Science and Biotechnology vol 10 no 3 pp 201ndash210 2007

[20] T Buhr S Sato F Ebrahim et al ldquoRibozyme termination ofRNA transcripts down-regulate seed fatty acid genes in trans-genic soybeanrdquo Plant Journal vol 30 no 2 pp 155ndash163 2002

[21] A M Murad G R Vianna A M Machado et al ldquoMassspectrometry characterisation of fatty acids from metabolicallyengineered soybean seedsrdquo Analytical and Bioanalytical Chem-istry vol 406 no 12 pp 2873ndash2883 2014

[22] R F Wilson ldquoThe role of genomics and biotechnology in achi-eving global food security for high-oleic vegetable oilrdquo Journalof Oleo Science vol 61 no 7 pp 357ndash367 2012

[23] A T Pham J D Lee J G Shannon and K D Bilyeu ldquoMutantalleles of FAD2-1A and FAD2-1B combine to produce soybeanswith the high oleic acid seed oil traitrdquo BMC Plant Biology vol19 p 195 2010

[24] M M Paz H Shou Z Guo Z Zhang A K Banerjee and KWang ldquoAssessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary nodeexplantrdquo Euphytica vol 136 no 2 pp 167ndash179 2004

[25] Y Imoto T Yamada K Kitamura and A Kanazawa ldquoSpatialand temporal control of transcription of the soybean 120573-con-glycinin 120572 subunit gene is conferred by its proximal promoterregion and accounts for the unequal distribution of the proteinduring embryogenesisrdquo Genes amp Genetic Systems vol 83 no 6pp 469ndash476 2008

[26] LMa L Zhou Z J Hua G X Tang Z C Shen andH X ShouldquoEstablishment of methods to rapidly and precisely identifytransgenic rice and soybean containing herbicide-resistant genebar and EPSPSrdquo Journal of Zhejiang University vol 38 no 6 pp647ndash654 2012

[27] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991

[28] Z L Chen M A Schuler and R N Beachy ldquoFunctional anal-ysis of regulatory elements in a plant embryo-specific generdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 83 no 22 pp 8560ndash8564 1989

[29] T Fujiwara and R N Beachy ldquoTissue-specific and temporalregulation of a beta-conglycinin gene roles of the RY repeat andother cis-acting elementsrdquo Plant Molecular Biology vol 24 no2 pp 261ndash272 1994

[30] Y ZhaoQQianHWang andDHuang ldquoHereditary behaviorof bar gene cassette is complex in rice mediated by particlebombardmentrdquo Journal of Genetics and Genomics vol 34 no9 pp 824ndash835 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Primers for different experiments

Primer name Primer sequence PCR producer (bp)GmFAD2-1b-F 51015840-AGCCACTAGGCATGGGTCTAGCAAA-31015840 801GmFAD2-1b-R 51015840-GCAATGGCACCCCATAAACACATAG-31015840

EB-R 51015840-GGAAAGCAACCATATCAGCATATCAC-31015840 752EB-F 51015840-TTCTCCAAGGTTGCATTCTTACTGG-31015840

GmActin-R 51015840-TTGACTGAGCGTGGTTATTCC-31015840 402GmActin-F 51015840-GATCTTCATGCTGCTGGGTG-31015840

qRTGmFad2-R 51015840-CACCATTCACTGTTGGCCAA-31015840 163qRTGmFad2-F 51015840-ATGAGGGAAAAGGGGTGAGG-31015840

production of soybeans with a significantly higher oleic acidcontent (up to 9458) and reduced levels of palmitic acid(lt3) [21] Recently Plenish high oleic soybeans have beenin commercial production by DuPont since 2012 They weredeveloped using a biotech process known as gene silencing[22] These results demonstrate that the GmFAD2 familymembers play a very important role in metabolicallyengineered oil seed plants [23]

In this study we report the transformation of soybeanwith GmFad2-1b gene by antisense RNA We analyzed andinvestigated the fatty acid composition of the transgenic linesand also discussed the nature of the transcript produced byGmFad2-1b

2 Materials and Methods

21 Plant Materials and Cotyledonary-Node Method Soy-bean cultivars [Glycine max (L) Merrill cv ldquoWilliams 82rdquo]were grown in a greenhouse at the Jilin Academy of Agri-cultural Sciences Gongzhuling during a 16 h photoperiodat a 35∘C daytime temperature and a 25∘C night-time tem-perature The cotyledonary-node method used in this studyfollowed the procedure described by Paz et al [24] In briefsoybean seeds were surface-sterilized by placing seeds into atightly sealed chamber containing chlorine gas for 16 h Thesterilized seeds were soaked with sterile water at 28∘C duringan 18 h photoperiod for 8ndash12 h The imbibed soybean seedswere placed on sterile filter paper and cut longitudinally alongthe hilum The cotyledons and hypocotyls (sim5mm) from asingle seedwere split evenly into two explants Approximately50 coat-removed explants were then inoculated in a liquidcocultivation medium (CCM) which contains 110 B5 saltsand vitamins 39 gL 2-[N-morpholino] ethanesulfonic acid(MES) 3 sucrose (30 gL) (pH 4) filter-sterilized 025mgLgibberellic acid (GA3) 400mgL L-cysteine 167mgL N-6-benzylaminopurine (BAP) 1542mgL dithiothreitol (DTT)and 200120583molL acetosyringone (As) for 30min After thisinoculation seven explants (adaxial side up) were randomlyplaced in Petri dishes (90mm in diameter times 15mm deep)on sterile filter paper placed on solid CCM containing 05purified agar (BD-Difco) and incubated in the dark at 25∘Cfor 3ndash5 days

After the cocultivation the explants were then transferredinto solidified shoot induction (SI) medium containing B5salts and vitamins 3 sucrose 08 agar (8 gL Sigma)

058mgL MES (pH 58) filter-sterilized 167mgL BAP250mgL ticarcillin (Tic) 100mgL cefotaxime (Cef) and5mgL glufosinate and then incubated in a growth room at28∘C under an 18 h photoperiod for two weeks Then thehypocotyl and shoots were trimmed from the explants andthe remaining cotyledons with developing nodules were sub-cultured to a fresh SI medium for another two weeks Fourweeks after SI the cotyledonswere removed from the explantsand the remaining tissues were transferred into a shoot elon-gation (SE) medium The SE medium was composed of MSsalts and vitamins 3 sucrose 08 agar (Sigma) 058mgLMES filter-sterilized 50mgL L-asparagine 50mgL glu-tamine 01mgL indole acetic acid (IAA) 05mgL GA31mgL zeatin riboside (ZR) 250mgL Tic 100mgL Cef and5mgL glufosinate (pH 58) The explants were then trans-ferred to fresh SE medium every two weeks until the regen-erated shoots were suitable for rooting The incubation con-ditions were the same as the conditions for SI At this pointelongated shoots (3-4 cm in length) were excised and placedinto a rooting medium (RM) containing MS salts andvitamins 3 sucrose 08 agar (Sigma) 058mgL MES(pH 58) filter-sterilized 50mgL L-asparagine 50mgL glu-tamine 01mgL indole butyric acid (IBA) 250mgL Tic and100mgL Cef After a period of 1-2 weeks the roots were fullydeveloped to 2-3 cm in length and eventually transplantedinto the pots to be grown in the greenhouse The transgeneevents were named by EB8001 to EB8067 The T0 generationtransgenic plants were grown in the greenhouse and the T

1

and T2lines were grown under field conditions

22 Nucleic Acid Isolation and Vector Construction The freshsoybean tissues were collected to extract the total RNAwith TRIZOL reagent (Invitrogen) RNA samples (2120583g) weretreated with the amplification grade DNase I (Invitrogen) at37∘C for 15min prior to cDNA synthesis using SuperScriptIII Reverse Transcriptase (Invitrogen) according to the man-ufacturerrsquos instructions

For the assembly of the GmFad2-1b antisense RNAvector a part of a fragment about 801 bp of the GmFad2-1b(GenBank accession XM 003555831) was amplified from thecDNA above by using the primers GmFad2-1b-FGmFad2-1b-R (Table 1) The amplified GmFad2-1b gene product wascloned into the pEASY-T1 cloning vector (TransGene) Theproduct was sequenced using T7 and M13 vector sequencingprimers to confirm the sequence of the ligated product An






1and T





1and T












0



Total 327 105 55 1684

(a) (b) (c)

(d) (e) (f)




0transgenic regen-







HindIII

XbaI

SacI

EcoRI

XhoI

XhoI

RB

LB

bcsp

T-nos

35S35

35S terBar

NptIIpBR ori

pBR bom

pVSl rep

pVSl sta

anti-GmFad2-1b

GmFad2-1b


pCAMBIA3300






2progenies of every


0


2lines







2




(a) (b)

M + minus H2O 1 2 3 4 5 6 7 8 9 10 11 12 13 14

BCSPGmFad2

Actin

752bp

402bp

(c)



HindIIIXbaI

M minus 1 2 3 4 5 6 7 8 9 10 11 M minus 1 2 3 4 5 6 7 8 9 10 11

15kb10kb

30 kb

15 kb





2transgene









0000050010001500200025003000350040004500

Root Leaf Seed

Relat

ive e

xpre

ssio

n le

vel

Williams 82EB8011

Stem



4 Conclusions







Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






1and T





1and T












0



Total 327 105 55 1684

(a) (b) (c)

(d) (e) (f)




0transgenic regen-







HindIII

XbaI

SacI

EcoRI

XhoI

XhoI

RB

LB

bcsp

T-nos

35S35

35S terBar

NptIIpBR ori

pBR bom

pVSl rep

pVSl sta

anti-GmFad2-1b

GmFad2-1b


pCAMBIA3300






2progenies of every


0


2lines







2




(a) (b)

M + minus H2O 1 2 3 4 5 6 7 8 9 10 11 12 13 14

BCSPGmFad2

Actin

752bp

402bp

(c)



HindIIIXbaI

M minus 1 2 3 4 5 6 7 8 9 10 11 M minus 1 2 3 4 5 6 7 8 9 10 11

15kb10kb

30 kb

15 kb





2transgene









0000050010001500200025003000350040004500

Root Leaf Seed

Relat

ive e

xpre

ssio

n le

vel

Williams 82EB8011

Stem



4 Conclusions







Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





0



Total 327 105 55 1684

(a) (b) (c)

(d) (e) (f)




0transgenic regen-







HindIII

XbaI

SacI

EcoRI

XhoI

XhoI

RB

LB

bcsp

T-nos

35S35

35S terBar

NptIIpBR ori

pBR bom

pVSl rep

pVSl sta

anti-GmFad2-1b

GmFad2-1b


pCAMBIA3300






2progenies of every


0


2lines







2




(a) (b)

M + minus H2O 1 2 3 4 5 6 7 8 9 10 11 12 13 14

BCSPGmFad2

Actin

752bp

402bp

(c)



HindIIIXbaI

M minus 1 2 3 4 5 6 7 8 9 10 11 M minus 1 2 3 4 5 6 7 8 9 10 11

15kb10kb

30 kb

15 kb





2transgene









0000050010001500200025003000350040004500

Root Leaf Seed

Relat

ive e

xpre

ssio

n le

vel

Williams 82EB8011

Stem



4 Conclusions







Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



HindIII

XbaI

SacI

EcoRI

XhoI

XhoI

RB

LB

bcsp

T-nos

35S35

35S terBar

NptIIpBR ori

pBR bom

pVSl rep

pVSl sta

anti-GmFad2-1b

GmFad2-1b


pCAMBIA3300






2progenies of every


0


2lines







2




(a) (b)

M + minus H2O 1 2 3 4 5 6 7 8 9 10 11 12 13 14

BCSPGmFad2

Actin

752bp

402bp

(c)



HindIIIXbaI

M minus 1 2 3 4 5 6 7 8 9 10 11 M minus 1 2 3 4 5 6 7 8 9 10 11

15kb10kb

30 kb

15 kb





2transgene









0000050010001500200025003000350040004500

Root Leaf Seed

Relat

ive e

xpre

ssio

n le

vel

Williams 82EB8011

Stem



4 Conclusions







Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

M + minus H2O 1 2 3 4 5 6 7 8 9 10 11 12 13 14

BCSPGmFad2

Actin

752bp

402bp

(c)



HindIIIXbaI

M minus 1 2 3 4 5 6 7 8 9 10 11 M minus 1 2 3 4 5 6 7 8 9 10 11

15kb10kb

30 kb

15 kb





2transgene









0000050010001500200025003000350040004500

Root Leaf Seed

Relat

ive e

xpre

ssio

n le

vel

Williams 82EB8011

Stem



4 Conclusions







Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







0000050010001500200025003000350040004500

Root Leaf Seed

Relat

ive e

xpre

ssio

n le

vel

Williams 82EB8011

Stem



4 Conclusions







Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article changes in oleic acid content of...

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