Construction of efficient and effective transformation vectors for palmitoyl-acyl carrier protein thioesterase gene silencing in oil palm

Palm oil obtained from E. guineensis Jacq. Tenera is known to have about 44% of palmitic acid (C16:0). Palmitoyl-Acyl Carrier Protein Thioesterase (PATE) is one of the key enzymes involved in plastidial fatty acid biosynthesis; and it determines the level of the C16:0 assimilation in oilseeds. This enzyme's activity in oil palm is responsible for high (> 44 % in E. guineensis Jacq. Tenera and 25 % in E. oleifera) content of C16:0 in its oil. By post-transcriptional PATE gene silencing, C16:0 content can be minimized for nutritional value improvement of the palm oil. The objective of this study was the construction of novel transformation vectors for PATE gene silencing. Six different transformation vectors targeted against PATE gene were constructed using 619 bp long PATE gene (5' region) fragment (from GenBank AF507115). In one set of three transformation vectors, PATE gene fragment was fused with CaMV 35S promoter in antisense, intron-spliced inverted repeat (ISIR), and inverted repeat (IR) orientations to generate antisense mRNA and hair-pin RNAs (hpRNA). In another set of three transformation vectors with same design, CaMV 35S was replaced with Oil palm mesocarp tissue-specific promoter (MSP). The expression cassette of antisense, ISIR, and IR of PATE gene fragments were constructed in primary cloning vector, pHANNIBAL or its derivative/s. Finally, all 6 expression cassettes were sub-cloned into pCAMBIA 1301 which contains the Hygromycinr and the GUS reporter genes for transformant selection and transformation detection respectively. The results of the RE analyses of the constructs and sequence analyses of PATE and MSP shows and confirms the orientation, size and locations of all the components from constructs. We hypothesize that 4 (pISIRPATE-PC, pIRPATE-PC, pMISIRPATE-PC and pMIRPATE-PC) out of 6 transformation vectors constructed in this study will be efficient and effective in palmitoyl-ACP thioesterase gene silencing in oil palm. Abbreviations antiPATE - Antisense Palmitoyl-acyl carrier protein thioesterase, BCV - Binary cloning vector, cDNA - Complementary deoxyribonucleic acid, hpRNA - hair-pin RNA, ihpRNA - intron containing hair-pin RNA, IR - inverted repeat, ISIR - intron-spliced inverted repeat, MCS - Multiple cloning site, MSP - Oil palm mesocarp tissue-specific promoter, nt - Nucleotide/s, PATE - Palmitoyl-acyl carrier protein thioesterase, PCR - Polymerase chain reaction, PCV - Primary cloning vector, pDNA - Plasmid deoxyribonucleic acid, PTGS - Post-transcriptional gene silencing, RE - Restriction enzyme.

[4]. This yield is almost three times the yield of coconut, and more than ten times that of soybean [5]. Elaeis oleifera a close relative of the E. guineensis is low oil yielding and not preferred for the commercial plantation, even though its oil contains more (69 %) oleic acid in comparison to commercially cultivated oil palm, E. guineensis Jacq. Tenera. In the world market of fats and oils, palm oil is considered as a market leader, constituting about 35 % of the world trade in fats and oils. Malaysia is the largest producer and exporter of the palm oil, and accounts for about 50 % of the world's palm oil output and 62 % of the net export trade in palm oil [5][6]. Palm oil has a great potential to fulfill the increasing demand for vegetable oil from both food and non-food industries. However, like other vegetable oils such as soybean oil, to get a good price in the market it is important to develop different varieties with desired fatty acid composition. To achieve this goal, conventional crop improvement methods are not only tedious but also time-consuming [5]. Oil palm genetic engineering may save 80-90% of the time required for insertion of gene/s for new trait/s in it through conventional crop breeding [7]. Therefore, out of all existing methods available for crop improvement, perhaps genetic engineering is the best method to manipulate fatty acid profile in oil palm in a short period. Palm oil and palm kernel oil are the main commercial products of the oil palm fruits. Palm kernel oil is the main source of lauric acid (C12:0), which is mainly used to fulfill the needs of soap, detergent, and cosmetic industries [8].
Oil obtained from mesocarp of commercially cultivated oil palm (E. guineensis Jacq. Tenera) fruits contains 53.3% saturated fatty acids, while commercially less important E. oleifera contains 28% saturated fatty acids [5]. Among the saturated fatty acids, palmitic acid (C16:0) is predominantly accumulated. Elaeis guineensis Jacq. Tenera oil typically contains 44.0% C16:0, while in E. oleifera oil, it is 25.0% [5]. Lowering down the saturated fatty acid content in palm oil is one of the ways for palm oil nutritional value improvement.
The PATE enzyme is known to have C16:0-ACP substrate specificity [9][10][11]. Therefore, it is necessary to knockout the expression of PATE gene in order to minimize the percentage of C16:0 in palm oil. Palm oil will be healthier for the human consumption and will get more price in the market if the C16:0 percentage in palm oil could be reduced to make it low palmitate [12 -14]. The silencing of PATE gene can be accomplished through any one of the several different methods of gene silencing such as, transcriptional gene silencing (TGS), cosuppression, post-transcriptional gene silencing (PTGS) using antisense, direct inverted repeats (IR), intron-spliced inverted repeat (ISIR) mediated gene silencing, and site-directed mutagenesis (SDM) [15][16][17][18][19]. Cosuppression and PTGS with antisense, IR and/or ISIR trans-genes markedly reduces the steady state mRNA levels of endogenous genes similar in transcribed sequence. These methods are proved to be very effective for gene silencing in oil producing, and other model plants like Arabidopsis [18][19][20][21][22][23][24]. A generic primary cloning vector, pHANNIBAL developed by CSIRO is very useful for the construction of novel transformation vectors with antisense, inverted repeats, and intron-spliced inverted repeats of the gene of interest [18][19][24][25]. This paper reports the construction of novel transformation vectors with constitutive and oil palm fruit mesocarp-tissue-specific promoter for the oil palm PATE gene silencing using pHANNIBAL, pCAMBIA 1301 and 619 bp long 5' region of oil palm PATE gene fragments.

Methodology: Plasmid vectors and bacterial strains:
Two cloning plasmid vectors, pHANNIBAL and pCAMBIA 1301 were used in the construction of 6 different transformation vectors for oil palm PATE gene silencing. The PCV, pHANNIBAL, a derivative of cloning vector pART7 was kindly provided by CSIRO, Australia [25][26]. The BCV, pCAMBIA 1301 was available in our laboratory (UKM, Bangi, Malaysia) [27]. The restriction enzymes map of PCV and BCV is shown in Supplementary Figure 1. The PCR cloning vector, pGEM®-T Easy was used for the cloning of PCR amplified antisense and sense PATE gene fragments. Bacterium, E. coli strain DH5-α was used for preparation of the competent cells to harbor the plasmids. Prepared competent cells were stored at -70 °C until the use.

Construction of transformation vectors for PATE gene silencing:
Plasmid, pPATE-RT is a recombinant PCR cloning vector (pGEM®-T Easy), which carries a 629 bp long (GenBank AF507115) fragment of E. guineensis Jacq. Tenera PATE gene previously isolated in our laboratory. We used this PATE gene fragment as template to synthesize antisense and sense PATE fragments in the vector construction. For the construction of 6 different transformation vectors for PATE gene silencing, standard gene cloning methods were used [28]. Construction of expression cassettes with antisense, ISIR and IR of PATE gene fragment was completed using pHANNIBAL. The expression cassettes from PCVs were then sub-cloned as a SacI-PstI fragment into pCAMBIA 1301.

Plasmid DNA extraction:
During preparation of constructs in PCV, selected and well-isolated colonies from the LB agar plates were inoculated aseptically and separately in universal bottles containing 10 ml LB medium, supplemented with 50 μg/ml Ampicillin (Ampicillin Sodium Salt, MF = C16H18N3NaO4S; Amersham Life Science). But, the BCV carries a Kanamycin resistant gene, and hence cultures were supplemented with 50 μg/ml Kanamycin. Cultures were incubated at 37 °C, 250 RPM for 16 hours. Alkaline lysis method was used for pDNA preparation. For confirmation of the intactness, and quality of the extracted pDNA, it was electrophoresed on 1% agarose gel.

PCR for RE sites addition and PATE amplification:
To incorporate PATE gene fragment in antisense orientation in pHANNIBAL, XbaI and BamHI restriction sites were used. The forward primer [F-XbaI (5'-AGCTCTAGAATCTTTGGTCTTTCATTCCC-3')] and reverse primer [R-BamHI (5'-ATTGGATCCTTCCAATCAAGAAGGGTCC-3')] were designed with flanking sequence of the XbaI, and BamHI restriction site (underlined nucleotides) respectively and used in amplification of the antisense PATE gene fragments. For amplification of 619 bp long sense PATE gene fragment, forward primer [F-XhoI (5'-ATTCTCGAGATCTTTGGTCTTTCATTCCC-3')] and reverse primer [R-EcoRI (5'-AACGAATTCTTCCAA TCAAGAAGGGTCC-3')] were designed with XhoI, and EcoRI restriction site (underlined) flanking sequence, respectively. The PCR for amplification of antisense and sense PATE gene fragments was completed under following conditions. Hot start 94 °C for 5 min, followed by 35

PCR product purification:
The PCR products were purified from the PCR reaction mixture using NucleoSpin ® Extraction Kit (Ready-to-use system for fast purification of nucleic acids), BD Biosciences Clontech, USA. The DNA bands of expression cassettes and plasmid DNA were excised from agarose gel with the help of surgical blade, and DNA was purified using NucleoSpin ® Extraction Kit. Quantitative estimation of purified DNA was calculated using UV-160 A, UVvisible recording spectrophotometer (SHIMADZU).

Ligation reaction:
Ligation reactions for incorporation of 619 bp long antisense and sense PATE gene fragments and MSP into pHANNIBAL and ligation reactions to incorporate expression cassettes into pCAMBIA 1301 were assembled in 10.00 μl volume by adding: ~ 200 ng vector, ~ 600 ng insert, 10X Ligase buffer, 3 units of T-4 DNA Ligase; sterile distilled water was added finally to adjust the final volume of reaction to 10 μl. Recombination reactions for cohesive ends were incubated at 16 (+1) °C, whereas blunt end ligation reactions were incubated at 8 (+1) °C for 18 h.

Transformation of E. coli competent cells:
The preparation of frozen stocks of competent cells (using the protocol-I) and transformation of E. coli strain DH5-α competent cells with ligated product by using heat shock method was performed [28].

Analysis of recombinant plasmids by restriction enzymes:
To confirm integration of antisense and sense PATE gene fragments, extracted recombinant plasmid DNA (20 μg pDNA) samples were double-digested with respective REs. For instance, for the confirmation of the antisense PATE gene fragment insert in pAPATE-H, it was double digested with XbaI-BamHI by incubating digestion reactions at 37°C, for 4 h. For the characterization of the recombinant plasmids, pDNAs were double-digested with different combinations of RE to confirm the size, orientation and location of the inserts and the components of the cassettes.

Nucleotide sequencing:
To confirm the identity of the PATE gene fragment or MSP promoter in constructs, nucleotide sequencing of the inserts was carried out using automated DNA sequencer. The pair of primers used for synthesis of PATE (antisense or sense) and MSP by PCR technique was used in sequencing reactions for the sequencing of respective inserts. Nucleotide sequence was analyzed by using nucleotide-nucleotide blast (blastn) program [29].

Construction of transformation vectors using MSP:
The MSP was isolated previously in our laboratory from Elaeis oleifera [30]. Three transformation vectors (with antisense, ISIR and IR of PATE gene fragment) were constructed for PATE gene silencing using MSP. From the already prepared expression cassette, CaMV 35S promoter was replaced with MSP to control expression of the constructs designed against oil palm PATE gene. MSP was used in the constructs to express constructs in oil palm mesocarp tissue-specific manner.

Transfer of expression cassettes from PCV into BCV:
The SacI and PstI restriction sites were used to separate expression cassettes with antisense, ISIR and IR of PATE gene fragment from the respective PCV. The separated and purified expression cassettes were incorporated as SacI-PstI fragment into BCV (pCAMBIA 1301). The BCV carries a Kanamycin resistant gene, and hence transformed bacteria were selected on LB-agar plates supplemented with 50 μg/ml Kanamycin.

Results:
Six different types of transformation vectors for PATE gene silencing were constructed. A set of three constructs (with antisense, ISIR and IR of PATE) was constructed using constitutive (CaMV 35S) promoter. Another set of three constructs was with the same design of expression cassettes except that the CaMV35S promoter was replaced with MSP. Figure 1 shows the six different constructed expression cassettes and their design for PATE gene silencing. The expression cassette with antisense PATE gene fragment was prepared using 619 bp long PATE gene fragment from plasmid, pPATE-RT and pHANNIBAL. Based on the restriction enzymes map of PCV and PATE gene fragment, antisense PATE gene fragment was incorporated in pHANNIBAL using XbaI and BamHI REs without discarding intron sequence. The intron sequence was kept as it is because it will be spliced out during the mRNA maturation; and hence its removal was not necessary. The strategy used for the construction of transformation vector (pAPATE-PC) with CaMV 35S promoter driven antisense PATE gene fragment is shown in Supplementary Figure 2. Double-digestion of pAPATE-H with SacI-ClaI released ~ 2173 bp long DNA fragment (lane 5, Figure 2A). This fragment contains CaMV 35S promoter and an intron. Double-digestion of pAPATE-H with XhoI-ClaI released DNA fragments of ~ 812 bp in length (lane 6, Figure 2A). This fragment is of intron. The DNA fragment of ~ 1447 bp in size was released from pAPATE-H as a result of its double-digestion with XhoI-XbaI (lane 7, Figure 2A). This fragment comprises an intron, and antisense PATE gene fragment. The doubledigestion of pAPATE-H with SacI-PstI released 3531 bp long entire expression cassette with CaMV 35S driven antisense PATE gene fragment (lane 8, Figure  2A). After completion of the REs analysis of prepared antisense PATE gene fragments expression cassette, it was sub-cloned into pCAMBIA 1301 to have a selection marker, reporter gene, and other elements of the BCV.
The REs analysis of the plasmid, pAPATE-PC was carried out to confirm the location, orientation, and length of the expression cassette and its elements. Digestion of plasmid, pAPATE-PC with EcoRI released 1373 bp long DNA fragment of CaMV 35S promoter (lane 3, Figure 2B). As expected double digestion of pAPATE-PC with EcoRI-PstI released two DNA fragments, smaller one was of 1373 bp long CaMV 35S promoter; and another bigger one was of 2164 bp in size (lane 4, Figure 2B). This fragment contains intron, antisense PATE gene fragment and OCS terminator of the expression cassette.
The double-digestion of pAPATE-PC with SacI-KpnI released one DNA fragment of 1377 bp in size (lane 5, Figure 2B). This DNA fragment is of CaMV 35S promoter. Double-digestion of pAPATE-PC with SacI-PstI released the entire 3531 bp long 'antisense PATE gene fragment expression cassette' (lane 6, Figure 2B). Once the antisense PATE gene fragment is inserted in pHANNIBAL using XbaI and BamHI sites, the resulting plasmid can be used to make expression cassettes with ISIR and IR of PATE for the PATE gene silencing. For the construction of hpRNA generating transformation vector for PATE gene silencing, construct should have PATE gene fragments sequence either in ISIR or in an IR in its expression cassette. The strategy used for the construction of transformation vector (pISIRPATE-PC) with CaMV 35S promoter driven ISIR of PATE gene fragment is shown in Supplementary Figure 3. The REs analysis of pISIRPATE-H and pISIRPATE-PC is depicted in Figure 2C & 2D, respectively. The pISIRPATE-H was used in construction of expression cassette with IR of PATE gene fragment. The intron fragment was taken out from pISIRPATE-H to make an expression cassette with direct IR of PATE gene fragments (Supplementary Figure 4). The REs analysis was carried out for both pIRPATE-H and pIRPATE-PC. The REs analysis results for pIRPATE-H and pIRPATE-PC are shown in Figure 2E & 2F, respectively.   The pMISIRPATE-H carries the ISIR of PATE gene fragments. Hence to make IR of PATE gene fragments in the expression cassette, intron was taken out by using KpnI and ClaI REs (Supplementary Figure 7). The sticky ends were made blunt by using T-4 DNA polymerase; and blunt ends were ligated to make IR of existing PATE gene fragments from the pMISIRPATE-H. The ligated plasmid was designated as pMIRPATE-H. The REs analysis of this plasmid and transformation vector (pMIRPATE-PC) was carried out; and the results are depicted in Figure 3E & 3F.  . In construction of novel transformation vectors two (CaMV 35S and MSP) promoters were used. The use of the tissue-specific gene promoters enables tissue-specific expression of the gene/s. Therefore kernel and mesocarp tissue-specific gene promoters were isolated previously in our laboratory after isolation of tissue-specific genes by differential display method [30][31][32]. Fatty acid biosynthesis pathway in oil palms (E. guineensis Jacq. Tenera and E. oleifera) can be manipulated genetically at different levels using key genes and tissue-specific promoters. The reduction in level of the saturated fatty acid content in palm oil is one of the ways to improve nutritional value of palm oil. To achieve this goal, down-regulation of the PATE gene holds the key, since reducing palmitate content lowers down level of the saturated fatty acids significantly in palm oil.
The research findings reported by Smith et al. (2000) showed that PTGS using antisense and/or cosuppression construct usually leads to modest proportion of silencing and silenced individuals in comparison to gene silencing induced by constructs with ISIR and IR [19,25]. Therefore, a new generation of transformation vectors with ISIR, and IR of PATE gene fragments for PATE gene silencing was constructed. The construction of 2 transformation vectors with antisense construct (pAPATE-PC and pMAPATE-PC) was carried out along with the constructs with ISIR (pISIRPATE-PC and pMISIRPATE-PC) and IR (pIRPATE-PC and pMIRPATE-PC) of PATE for PATE gene silencing to compare the efficiency of the constructs in PATE gene silencing in oil palm. The CaMV 35S promoter was used for three reasons. Firstly, to determine whether there is an effect of PATE gene silencing on fatty acid profile in the leaves instead of waiting for flowering and fruiting of the transgenic oil palm to be obtained. Secondly, to determine the effect of PATE gene silencing on fatty acid profile of the palm oil obtained from mesocarp. And, the third reason was to compare the efficiency of CaMV 35S promoter with MSP [30].   Figure 4. The PCV (pHANNIBAL) is a generic vector that allows simply, single PCR product from a gene of interest to be easily converted into a highly effective and efficient ihpRNA silencing construct [35]. Because of this PCV's proven effectiveness and efficiency of resulting construct, this vector is a choice in constructing effective and efficient constructs for PTGS of targeted gene/s. This PCV is used in peanut to facilitate its genetic engineering for alleviating peanut allergy [36] and in other plants for PTGS of different genes [24,35,37]. By realizing the efficiency and effectiveness of the construct designs which pHANNIBAL enables, pHANNIBAL-like silencing vectors are also developed for gene silencing in fungi [38]. Therefore, we strongly believe that the integration of expression cassettes (containing ISIR and IR of PATE gene fragments) into oil palm genome will lead to the effective PATE gene silencing. As a result of it, C16:0 content will be minimized significantly in the palm oil. It is reported that the silencing effect of such kind of constructs are stably inherited over many generations [35], and oil palm should not be exception for it. . Therefore, ISIR, IR and antisense construct of PATE gene fragments from pMISIRPATE-PC, pMIRPATE-PC, and pMAPATE-PC will express only in the fruit mesocarp tissues of the oil palm after its transformation. Whereas, ISIR, IR and antisense construct of PATE gene fragments from pISIRPATE-PC, pIRPATE-PC, and pAPATE-PC will express constitutively. This will help to determine and compare the efficiency and effectiveness of the 2 promoter used. However, we strongly believe that genetic engineering of oil palm with pISIRPATE-PC, pIRPATE-PC, pMISIRPATE-PC and pMIRPATE-PC vectors will facilitate development of low saturated fatty acids producing oil palms [7]. Based on the literature and our understanding, we hypothesize that 4 (pISIRPATE-PC, pIRPATE-PC, pMISIRPATE-PC and pMIRPATE-PC) out of 6 transformation vectors constructed in this study will be efficient and effective in PATE gene silencing in oil palm. Nevertheless, these constructs with efficient and effective construct design for PATE gene silencing are bound to facilitate the efforts of genetic engineering in oil palm to produce palm oil with low saturated fatty acids.

Conclusion:
Six different transformation vectors namely, pAPATE-PC, pISIRPATE-PC, pIRPATE-PC, pMAPATE-PC, pMISIRPATE-PC, and pMIRPATE-PC are constructed for the PTGS of the PATE gene. The results of the RE sites analyses and nucleotide sequence analyses of the PATE gene fragments and MSP confirms that 619 bp long PATE gene fragments insert are at the right locations and orientations in the constructs. The PATE gene fragment insert is in antisense orientation in pAPATE-PC and pMAPATE-PC, in ISIR orientation in pISIRPATE-PC and pMISIRPATE-PC, and in direct IR orientation in pIRPATE-PC and pMIRPATE-PC. In all six transformation vectors, all the elements of the respective expression cassettes targeted against PATE gene are at the right place and orientation. But, to take this research forward, transformation of E. guineensis Jacq. Tenera and E. oleifera with constructed constructs needs to be done in order to obtain transgenic oil palms for the low C16:0 palm oil in a reasonable time.

Disclosure:
Authors attest that there are no conflicts of interest to declare.