Transient Transformation of Artemisinic Aldehyde ∆ 11 (13) Double Bond Reductase (dbr2) Gene into Artemisia annua L

Global demand of antimalarial drug artemisinin has a gap with production capacity from existing sources since the low content of this compound from Artemia annua L. Genetic engineering­based strategy for A. annua plant on key enzymes in artemisinin biosynthetic pathway is needed. Artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) is one of the key enzyme on artemisinin biosynthesis which was studied in this research. Agrobacterium tumefaciens­mediated transformation of A. annua using dbr2 was carried out. Synthetic dbr2 was ligated into pCAMBIA1303 and transformed into Escherichia coli DH5α. pCAMBIA1303­dbr2 plasmid was transformed to A. tumefaciens AGL1. Leaves of A. annua were infected by positive transformant of recombinant A. tumefaciens (OD600 ≈ 1) supplemented with acetosyringone 50 ppm, and Silwet S­408 0.02%. Samples were incubated in desiccators connected with vacuum pump, this method is called infiltration vacuum. Leaves were covered in dark for 45 min, and co­cultivated on MS co­cultivation media for 3 days. All leaves were washed in 300 ppm cefotaxime and divided into 2 parts; 3 leaves for GUS histochemical assay and 300 mg of leaves for HPLC analysis. Transient transformation was done in triplicate. In GUS histochemical assay, pCAMBIA1303 and pCAMBIA­dbr2 showed positive blue spot where coefficient of variance was less than 5%. PCR analysis for genomic DNA of transformed A. annua showed a positive result of inserted dbr2 recombinant indicated by migration profile and direct sequencing analysis. It could be concluded that pCAMBIA­dbr2 construct and transformation into A. annua have been successfully performed.


Introduction
Malaria has already existed since long time ago and become endemic in the tropical and subtropical countries, particularly in South East Asia region including Indonesia.Chloroquine resistant followed by sulfadoxinepyrimethamine resistant had came up at the end of 1900s causing malaria prevalence increases every year.In early 2000s, Indonesia's Ministry of Health recorded 15 million cases of malaria and 38,000 died every year (Depkes RI, 2008), also that 262 million cases and 839,000 people were death globally (WHO, 2015) due to medical failure.In 2001, World Health Organization issued ACTs (Artemisinin basedcombined therapies) policy as firstline in malaria treatment caused by drug resistance, firstly tried in Africa region and spread after to others endemic area in the world (WHO, 2015).As the result, mortality number could decrease into 0.92%, 0.42%, to 0.2% from 2005to 2007(Depkes RI, 2008).
Indonesia used two ACTs drugs, artesunateamodiaquine and dihydro artemisininpiperaquine.These drugs contain artemisinin derived compound which has antimalarial activity against Plasmodium sp.parasite at endoeritrocytic stage.Artemisinin was found in Artemisia annua L. plant which has 0.05-0.2%dried content (Dewick, 2009) thus inhibit commercial production in common.Many efforts have been made such as semisynthetic to enhance artemisinin production as base compound for ACTs.However peroxide bridge structure was unique and hard to be made with common chemical reaction, therefore artemisinin biosynthetic engineering in A. annua plant was focused for five key enzymes (Brown, 2010).Artemisinic aldehyde ∆ 11 (13)double bond reductase (dbr2) is one of key enzymes which has important role as check point in the path separation to artemisinin.The aim of this study was to transform the dbr2 gene in A. annua leaves explant mediated by A. tumefaciens.

PCR analysis
Dream Taq™ Green Master Mix; nucleasefree water; 6x Loading Dye DNA Thermo Scientific; Mass ruler DNA™ Ready Mix Thermo Scientific; Gene ruler1kb™ Thermo Scientific; Reaction Primers (Table 2).PCR system for dbr2 specific (after optimizing) using 25 cycle, with 95°C for 3 min pre and 30s, 46°C for 30s, and 72°C for 1.5 min and 7 min extension, then CaMV35S -GUS PCR system using 25 cycle, with 95°C for 3 min pre and 30s, 60°C for 30s, and 72°C for 1.5 min and 7 min extension.

Gene isolation
Reagent for plasmid isolation containing:

Plasmid construction
A good sample of pCAMBIA133 and pUC57dbr2 vector was chosen by migration profile from the result of polymerase chain reaction confirmation.Those vector were set as templates for restriction reaction with BglI and SpeI enzymes.The restriction reactions were separated by 1% agarose gel electrophoresis and cut at the desired band size, then purified it with Gel/PCR Purification kit Geneaid.All elution liquids collected from same sample were pooled in one tube, then defined the final concentration using Multidrop Thermo Scientific Multiskan Go™ followed by checking the existence of desired fragment size with gel electrophoresis.Gen and vector were ligated with T4 DNA ligation enzyme, incubated at 22°C for an hour then all of reaction were introduced into E. coli DH5α competent cell and transferred to LB medium with antibiotic selection, incubate at 37°C overnight (Table 4).

Confirmation of plasmid
All positive transformants were grown overnight in LB medium with antibiotic selection.Recombinant plasmids were isolated using solution 1 (GTE solution), 2 (NaOH -SDS solution), and 3 (KOAc solution), then ends up DNA pellet reconstituted with TE RNAse.Plasmid solution was diluted to 20x factor.Furthermore plasmids were confirmed by migration analysis with following procedures put 5 µL of plasmid into 0.8% agarose gel and run electrophoresis 30 minute, 100V; PCR analysis in following procedures: set PCR reaction with dbr2 specific primers and run the system as describe above.Screen the result in 1% agarose gel electrophoresis; restriction analysis with following procedures: set restriction reaction with NheI and SacI enzyme incubate 3 hours at 37°C.Separate by 0.8% agarose gel electrophoresis, and analyze the fragment size pattern; and DNA Sequence Analysis with following procedures 20 µL plasmid (undiluted) was send to Macrogen Inc., South Korea, to take sequence analysis with existing primer for sequencing.

Cloning the plasmids in Agrobacterium tumefaciens
Chemically competent cell of A. tumefaciens AGL1 were prepared using liquid nitrogen method (Chen et al., 1994), then pCAMBIAdbr2 plasmid were transformed into the competent cell with YEP medium (+ rRifampicin 50 ppm, ampicillinA 100 ppm, and kKanamycin 50 ppm).The recombinant cells were incubated for three days covered in dark at room temperature.Positive colonies were analyzsed with crude PCR with CaMV35s -GUS PCR system.

Transient transformation of pCAMBIA1302dbr2 into Artemisia annua L.
Hundreds mg leaves explant near shoot apical were weighed and sterilized with ethanol 70% for 1 min, sodium hypochlorite 0.,75% for 15 min, then rinsed 3 times with sterilized aquadest for 5 min.Overnight culture (OD 600 ≈ 1) of positive A. tumefaciens transformants was pelleted, then mixed with sterile explant in MS medium, acetosyringone 50 ppm, and Silwet S408 0.02%.The mixture was vacuum for 45 min in dark room temperature, and it was placed in MSco cultivation in 3 days covered in dark.After that it was washed in cefotaxime 200 ppm, rinsed 3 times and dried.

Analysis of gene in transformed Artemisia annua L.
A few leaves were vacuumed in GUS staining solution then store in room temperature 3 days and measure blue spot area with ImageJ software.Another leaves were prepared for genomic isolation with Plant Genomic Isolation kit Geneshun.Genomic samples were set in PCR reaction with FCaMV35S and RGUS primer and run system as described above.

Results and Discussion
Plasmid samples in stock were checked by gel electrophoresis to ensure quality and intensity of plasmid band.The presence of DNA ladder was being as a reference of samples migration with each other.A bold and clear band with high intensity like plasmid from C, D, E, and F (Figure 1) indicated a good plasmid.In order to improve efficiency and effectiveness of restriction reaction, a double digest method were done using Buffer B™ Promega with 2fold BglII concentration consequently.This could reduce purification step which tend to increase the concentration at last.pC1 and pC2 fragments could be preconcerted with a complete restriction because of two enzymes have managed to cut pUC57dbr2 plasmid in 2 fragments as desired, since the distance between two restriction sites just 7 bp (out of gel).To obtain high final concentration after purification, total elution buffer was reduced then two or more same fragment gels were purified and collected altogether into one tube, which pC1 with pC2 and D1, D2, D3, with D4 (Figure 2).All purified samples then were checked by its existency (boldness and clearness of fragment band) and quality (concentration and purity) to ensure a fragment condition after purification (Table 5).The purification results were a good with bold and clear band also quite high of concentration and purity (Figure 3), thus the ligation reaction with high success rate would be achieved.Composition of ligation 1:8 (pCAMBIA1303:dbr2) was used regard to the amount of insert (dbr2) should be more than vector (pCAMBIA1303).Based on the comparison of concentration value, dbr2 fragment has to be 8 times more than pCAMBIA1303 to make an effective collision in reaction.After one hour incubation at 22°C temperature, all samples were transformed into E. coli DH5α with suitable antibiotic selection.4 showed that all transformation process were successful due to clean negative transformation control where not contaminated with other microorganism were not found and the cell competency were still good enough due to positive transformation result.Ligation control was made to confirm so the colony grows in ligation sample transformant were not false positive.All of the colony in plate A were picked into LB medium to isolate their plasmid.After isolation, dissolved plasmid was diluted with 20 times factor to set as template for confirmation procedure.
First confirmation would be a comparison of migration profile has a desired result that isolated plasmid were slower (have short migration) than pCAMBIA1303 (Figure 5).This should indicate that a isolated plasmid have weighs heavier because of inserted dbr2 gene, but this result could not be as strong evidence.Next confirmation was restriction analysis between inserted vector with empty vector.As the result, a different pattern of fragment that indicates different size between them (Figure 6).In fact, the reason for using NheI and SacI enzyme provided the same number of fragments (theoretically three) but has different size.Although first and third fragment size were the same (around 5600 bp and 3400 bp), the second fragment size showed differences, for empty vector the fragment size was similar to the third fragment (around 3300 bp) so that it looks like one fragment instead of two (Figure 6).It could be imagined from inserted vector simulation    (Figure 7) that dbr2 insert gene make the fragment becomes longer.This analysis also could not strengthening the existing evidence because the addition of fragment size could be from any genetic material not specifically mention it was dbr2 gene.Therefore, further confirmation analysis was needed.
Further confirmation was PCR analysis using dbr2 specific primer with pUC57dbr2 as a positive control.Optimization of annealing temperature was needed to be done using gradient PCR to get the suitable PCR system.The right annealing temperature has bold and clear amplification band than others, therefore lane H was the right one (Figure 8), then dbr2 specific system was set 46°C for annealing temperature.Isolated plasmid were confirmed using optimized PCR system.As the result, two band samples were linear with positive control at around 1110 bp (Figure 9), it could be ascertained the plasmid samples have dbr2 inserted gene.For the purpose of artemisinin biosynthetic engineering, how the nucleotide sequence and reading position was important, and the previous result was not strong enough to convince that plasmid system would be expressed well.To overcome these doubts, DNA sequencing analysis was performed using Macrogen's service as a third party.Firstly, the samples was analyzed using two primers (CaMV35S Forward and primer 3 DBR Reverse) and the result was identical with Genebank database, then to complete ORF sequences, two other primers (DBR Forward and primer 3 GUS Reverse) were used.These four sequences result was combined using BioEdit program, then use nucleotide BLAST™ tools from NCBI to be compared with database.Identity value reached 100% score for dbr2, it means the   whole sequence of dbr2 was completely similar to sequence from library.Because of similarity with database, it should strongly concluded that gene construct was successfully done.
Confirmed pCAMBIAdbr2 and pCAMBIA1303 plasmid were transformed into A. tumefaciens AGL1 competent cell to get recombinant transformed colonies.To ensure that colonies contain desired gene, crude colony PCR was done with all of grown colonies on the plate medium (Figure 10).All of PCR product indicates all colonies were positive due to comparison with control plasmid.Next step was genetic transformation into A. annua which was mediated by A. tumefaciens AGL1 recombinant.This stage was performed using four groups of transformation: pCAMBIAdbr2, pCAMBIA1303, AGL1 (wild type), and without transformation (plant wild type).The aim of this treatment was to observe the gene expression in short period of time, so called transient transformation.Transient transformation was a preliminary test to determine level of transformation and expression result of transferred gene.The transformation was preceded by harvesting A. tumefaciens AGL1 cultures at OD 600 ≈ 1 to get a best result of transfection (Chamidah, 2013).Strain AGL1 has hypervirulent properties than others and made a tough transformation result using GUS histochemical evaluation (Elfahmi, 2014) so it was chosen.This study used a young leaves (near apical) explant to proceed dbr2 gene transformation, because it has either minimum contaminant or minimum barrier that possible for A. tumefaciens infection and also in this part has amount of artemisinin than others.
Acetosyringone as a phenolic compound would trigger gene delivery system through signal transduction mechanism in A. tumefaciens cell surface.Silwet S408 is organosilicon surfactant acts to lower surface tension between bacteria and plant cell, this  type of surfactant gave the best transformation result compared with other series (Chahyadi, 2011), concomitant use with acetosyringone could increase transformation efficiency.Agrobacteriummediated gene transformation efficiency could be enhanced using vacuum infiltration method by creating negative atmospheric pressure conditions then the air in the space between plant cells reduced and facilitate the bacteria to have direct contact with the cells.After three days cocultivation, leaves explant were washed by cefotaxime to kill excessive bacteria attached to the leaves, and were divided for subsequent expression confirmation.
The blue spot on pCAMBIAdbr2 leaves transformant after GUS histochemical assay showed an expression of GUS reporter gene that linked to the dbr2 gene, it means that two genes has been inframe (Figure 11).Percentage of blue spot area in triplicate indicates level of gene expression area, the wider of blue spot area the better transformation efficiency Table 6).Staining time to get blue spot area for its triplicate were in 3 days, 5 days, and 4 days respectively.The difference of staining time was caused by the sensitivity of stored X Gluc staining solution diminished over time.However, repeatability of transformation method could still be accepted due to CV (coefficient of variance) value was below 5% relatively.
To determine the existence of dbr2 gene recombinant on pCAMBIAdbr2 leaves transformant, DNA genomic isolation was performed.It was used as a template for PCR reaction with primer of FCaMV35S and RGUS in spite of dbr2 specific primer because it would give bias result with the existing dbr2 native gene in wild type genomic DNA.There was PCR product band with 664 bp size at A1A3 lane same as pCAMBIA1303 band control which size of CaMV35S to GUS primer region, yet product band at B2-B5 lane have 1894 bp size (Figure 12) means have extra around 1230 bp which is similar to inserted dbr2 gene size.

Conclusions
Gene construction of artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) into pCAMBIA1303 binary vector has been successfully preconcerted after through four steps confirmation test.GUS histochemical assay after pCAMBIAdbr2 transient transformation provide the blue spot area which means dbr2 gene has been transformed as was inframe to GUS reporter gene and its existence was proved by PCR of genomic DNA.Verification of dihydroartemisinic aldehyde and artemisinin content is needed between nonrecombinant compared to recombinant transformant.

Figure
Figure4showed that all transformation process were successful due to clean negative transformation control where not contaminated with other microorganism were not found and the cell competency were still good enough due to positive transformation result.Ligation control was made to confirm so the colony grows in ligation sample transformant were not false positive.All of the colony in plate A were picked into LB medium to isolate their plasmid.After isolation, dissolved plasmid was diluted with 20 times factor to set as template for confirmation procedure.First confirmation would be a comparison of migration profile has a desired result that isolated plasmid were slower (have short migration) than pCAMBIA1303 (Figure5).This should indicate that a isolated plasmid have weighs heavier because of inserted dbr2 gene, but this result could not be as strong evidence.Next confirmation was restriction analysis between inserted vector with empty vector.As the result, a different pattern of

Figure 3 .
Figure 3. Electrophoregram result to check the presence and size conformity of pC = pCAMBIA1303 and DBR = dbr2 fragment.

Figure 6 .
Figure 6.Electrophoregram of restriction analysis between D = isolated plasmid and B = pCAMBIA1303 relative to uncut plasmid C and A respectively.

Figure 7 .
Figure 7. pCAMBIAdbr2 map simulation with NheI and SacI restriction sites at NOS terminator, pVS1 RepA, and MCS are respectively.

Figure 10 .
Figure 10. A. tumefaciens AGL1 transformant contains A = pCAMBIA1303, and B = pCAMBIAdbr2 as evidenced by electrophoregram of crude PCR C and D respectively.

Table 1 .
Primer sequences for sequencing analysis.

Table 5 .
Absorbance measurement of purified fragment at 260 nm and 280 nm..

Table 6 .
Measurement area of blue spot of leaves using ImageJ software.