2. Title:Construction of FATTY ACID DESATURASE 2 (FAD2) gene knockouts using RNAi
novel technique in the model plant Thlaspi arvense.
Abstract: FATTY ACID DESATURASE 2 and FATTYU ACID ELONGASE 1 (FAE1) are the two genes of interest
present in the plant pennycress. FAD2 is in the pathway that synthesizes the polyunsaturated linoleic
(18:2) and linoleic (18:3) fatty acids from the monounsaturated oleic acid (18:1). Polyunsaturated fatty
acids are undesirable in biodiesel because they confer oxidative instability, but greater energy (J.C.
Sedbrook, et al, 2014). Targeting gene function reduction of FAD2 in Thlaspi arvense using RNA
interference method, lowered linoleic/linoleic acid content and increased oleic acid content in seed oil.
We are hoping to find less unsaturated fatty acids in the seed oil content in FAD2 gene knockout
transgenic Thlaspi arvense. FAD2 function is knocked out only in the seeds and not in vegetative tissues
as it plays an important role in plant’s fitness (J.C. Sedbrook, et al, 2014).
INTRODUCTION:
Pennycressplanthasprovenitselftobe veryuseful intoday’sagricultural industry.Itsagronomicvalueasanoilseedcrop,
animal feed,andcovercrop are highlysatisfyingenvironmentallybutnoteconomically. Thlaspiarvense(Pennycress)isa
close relative of Arabidopsis thaliana. Like Arabidopsis was genetically modified to improve seed dormancy, flowering
time, seed size and yield, oil quality and quantity, and pod shatter. All the knowledge gained from these genetic
modificationresults and implicationscan be applied to Pennycressplant to improve its seed dormancy, oil quality, seed
size and oil content, and pod shatter (Sedbrook, J.C., 2014). Pennycress is an ideal plantbecause of its diploid genome,
which makes it easier to genetically manipulate its gene expressions.
ThisprojectisfocusedmainlyonimprovingPennycressoil contentforitsuseasabiodiesel. FAD2,averyimportantenzyme
in the fatty acid biosynthesis pathways. Pennycress has relativelyhigh Erucic (22:4) acid content, and disadvantagesof
Erucicacid are thatithasa relativelypoorcoldflow properties. Coldflowisthe tendencyof asolidmaterial tomove slowly
or deformpermanentlyunderthe influence of mechanical stress(cold).The goal istoconstructa knockoutof FAD2inthe
seeds,whichwe are hopingwill reduce the amountof unsaturatedfatty acids,makingthe oil contentmore appropriate
for Biodiesel production. Dr. Sedbrook and his team are trying to domesticate Pennycress to make it economically
advantageous.
RNA interference worksbymicroRNA associatingwithRISCforRNA editingtechnique. DroshaandDicerenzymescutthe
pre-miRNA intoits2°Structure whichcanthenassociatewithRISC factortophysicallyblockthe translation of thatprotein.
3. This technique makes it very easy to construct constructs which will adopt miRNA shape and work in a gene knockout
mechanism.
MATERIALS AND METHODS
CREATING AN ENTRY VECTOR
To amplifyFAD2fromthe cDNA isolatedfrompennycressfloweringbud
a Polymerase ChainReactionwassetup.Twosetsof primerswere used
to obtain the DNA of both the open reading frames. A standard set of
protocol was followedtoobtainhighlyamplified openreadingframe of
FAD2, but with an increased annealing temperature of 55 ° C as the
primer’sboilingpointwashighof 75 ° C. The reaction was labelledRNAIFAD2.1andRNAIFAD2.2.The amplifiedPCRDNA
was then run on 1% agarose gel to separate the amplified DNA. The gel was excised after the electrophoresis,and later
subjectedtogel purification torecollectthe RNAIFAD2.1andRNAIFAD2.2DNA usingthe standardprotocol providedwith
the GeneJetGel ExtractionKit. The DNA aftertranscribedinthe cellsof the pennycresswilladopta miRNA shape due to
its complementary sequence flanked by inverted repeats favoring a hairpin formation.
The purifiedgelextracted RNAIFAD2.1andRNAIFAD2.2werethensubjectedtoarestricteddigest usingBAMH1andXHO1
restrictedenzymesalongwithourdonorvector pENTR 2B. The reactionwas incubatedfor1 hour in 37° water bath.The
reaction was then run on 1% agarose gel.
The isolated vector and RNAIFAD2.1, and RNAIFAD2.2 were cleaned and column purified by adding together 100 µl of
pENTR 2B and 100 µl of RNAIFAD2.1andRNAIFAD2.2(2 separate reactionswere performed). Bindingbuffer,isopropanol
and wash buffer was used to purify the DNA. The mix column purified DNA was ethanol precipitation purified and
concentratedpelletwasobtainedforthe ligationreaction.The insertandvectorwereligatedtogetherusingT4DNA Ligase
and incubated at room temperature overnight. Our Entry vector is now ready.
Figure 3
4. TRANSFORMATION INTO COMPETENT CELLS
The entryvectorwastransformedinto NEB5-alphachemically CompetentE.coli cells.The 5µl entryvectorwasaddedto
the 10 µl of competent cells. First the reactionwas incubatedon ice for 20 minutes, heat shock at 42° C for 30 seconds
afterthat. Growth mediawasaddedandincubatedat37° C withmildshakingfor1hour. The reactionwasthenplatedon
LB agar media,which wasthenincubatedat 37 °C overnight. Fourcolonieswere picked the nextday foreach reaction (4
for RNAIFAD2.1 and 4 for RANIFAD2.2) and inoculated on a liquid LB media.
TRANSFORMED DNA
Liquid cultures were isolated and liquid media safely discarded. The cells were then subjected to Plasmid Miniprep Kit
using the standard protocol provided with the kit. Total of 8 reactions were subjected to purification and extraction of
DNA. The DNA was then used to carry out a Sanger sequencing reaction using the ddTNP method.
RESULTS AND DISCUSSION
CREATING AN ENTRY VECTOR
The PCR reactionwassuccessful,we see twobrightbandsdenotingsuccessful application forbothRNAIFAD2.1and
RNAIFAD2.2.Asexpected,forRNAIFAD2.1a 432 base pair bandand for RNAIFAD2.2a 276 base pairband.This isexactly
whatwe see inFigure 1.
Figure 1. PCR reaction for amplification of
RNAIFAD2.1 and RNAIFAD2.2
Lane 1 RNAIFAD2.1
Lane 2 1 KbLadder
Lane 3 RNAIFAD2.2
Figure 2. The gel in which PCR reaction was carried
out
Excision of band can be seen clearly.
5. To recollectthe DNA,forgel purification,the agarose gel wascut and purified.The purifiedDNA wasthenevaluatedfor
ethanol residue andqualityinspection (aswell asquantity).ForRNAIFAD2.1,the concentrationwas101 ng/ µl andfor
RNAIFAD2.2,the concentrationwas90 ng/ µl.These resultsare exceptionalanddenotesthatourDNA iscontamination
free andhighin number. The figure 2clearlyshowsthe excisedgel.
The restrictiondigestwasalsosetupto make the endsof insertandpENTR2B compatible forligationreactiontooccur.
The vector waslinearizedto2.2 kilobyte. All the restof the reactionswere successfully.
The coloniesof competentcellswhichcontainedourRNAIFAD2.1andRNAIFAD2.2constructsgrew ingood amountof
numbers ona selective media.There wasalsoagoodgrowth in the liquidmedia.
The DNA extractionfromE.coli cellswere highlyconcentratedand successful.All the 8reactions’photo-spectrometry
data are as follows:
These data suggestthatthe DNA extractedwashighin
concentrationandpurifiedwithsomeethanol contamination.
The high260/230 numbersuggeststhatthere isa little ethanol
(fromwashsolution) stillinthe reaction.
SEQUENCING
RNAIFAD2.1A sequencingwithoutlyingprimerpENTR2B_3F_sequence andRNAIFAD2.2A sequencingwithoutlying
primerpENTR2B_3F_sequence were performed. Thiswasdone tocompare our sequence withthe consensussequence
and lookforthe successful transformation.Inbothcases,sequencesare perfectandcan be movedforwardinto a RNAi
compatible binaryvector.
CONCLUSION:
The DNA isnowready to be transformedintothe RNAi compatible binaryvectorwhichwill be ourdestinationvectorto
be usedas an expressionvector. Inthe future,thissame experimentwill be conductedusingthe FAE1enzyme tofurther
enhance oil contentbysynthesizingmore 18:1 monounsaturatedfattyacid. FAE1isan enzyme regulating the elongation
mechanismof the fattyacid biosynthesis(figure 3).
DNA Concentration 260/280 260/230
RNAIFAD2.1A 170.8 ng/ µl 1.9 1.9
RNAIFAD2.1B 139.9 ng/ µl 1.87 2.02
RNAIFAD2.1C 195.0 ng/ µl 1.88 2.03
RNAIFAD2.1D 231.9 ng/ µl 1.88 2.12
RNAIFAD2.2A 168.5 ng/ µl 1.88 1.87
RNAIFAD2.2B 197.6 ng/ µl 1.88 1.96
RNAIFAD2.2C 202.4 ng/ µl 1.9 2.06
RNAIFAD2.2D 205.5 ng/ µl 1.9 2.05
6. REFERENCES:
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NguyenHT,SilvaJE, Podicheti R,etal.Camelinaseedtranscriptome:atool for meal andoil improvementand
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SedbrookJC,PhippenWB,MarksMD. Newapproachestofacilitate rapiddomesticationof awildplantto an oilseed
crop: example pennycress(Thlaspiarvense L.).PlantSci.2014;227:122-32.
Sedbrook,J.C.(2014). Presentationonthe FAD2and FAE expressioninfattyacidsynthesis.Unpublished.
