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

Abstract

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.AbbreviationsantiPATE - 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.

Insights from computational analysis of full-length β-ketoacyl-[ACP] synthase-II cDNA isolated from American and African oil palms

“…For the PATE gene silencing, various novel expression cassettes were constructed[3] and genetic transformation work was initiated in our laboratory. [89] However, Eg TKAS-II gene expression cassette for it's over expression and PATE gene-silencing expression cassettes can be incorporated in a single vector to utilize them in genetic transformation of commercially cultivated E. guineensis .…”

“…‘Tenera’ a hybrid of ‘Dura’ (♀) and ‘Pisifera’ (♂) is highly preferred for the commercial cultivation because of its high oil-yielding fruits. [ 3 ]…”

“…The level of C 16:0 content in E. guineensis oil can be reduced by PATE gene silencing and over expression of the KAS-II gene in a mesocarp tissue-specific manner. [ 3 ] Researchers are working hard to transform oil palm for PATE gene silencing and successful oil palm transformation is reported elsewhere. [ 4 8 9 ]…”

Computational analysis of common bean (Phaseolus vulgaris L., genotype BAT93) lycopene ß-cyclase and ß-carotene hydroxylase gene’s cDNA

“…For the suppression of a gene expression, partial sequence of that gene can be utilized to induce posttranscriptional gene silencing (PTGS) [26–28]. However, the full length gene or its cDNA is required for its over-expression in order to increase either the production of desired vital proteins or natural products [29].…”

Nutritional, Medicinal and Toxicological Attributes of Star-Fruits (Averrhoa carambola L.): A Review

“…If we identify and characterize the key genes involved in oxalic acid and CBX biosynthesis, then the genetic alteration of the Star-fruit genome can be considered as one of the strategies to knock-out the oxalic acid and CBX synthesis in fruit specific manner. Various post-transcriptional gene silencing (PTGS) techniques such as antisense, inverted-repeats and or intron-spliced inverted repeats [ 43 ] can be utilized in genetic engineering of the Star-fruit plant to knock-out the biosynthesis of oxalic acid and CBX. If we develop genetically engineered Star-fruit plants which will produce fruits devoid of oxalic acid and CBX then it will certainly help to increase not only its economic value but nutritional value also.…”