FDP is considered as key
FDP is considered as key intermediate in the isoprenoid biosynthetic pathway, since it is the common precursor of polyisoprene units and farnesylated proteins (Cao et al., 2012). In order to understand the role of FDP gene in rubber-producing plants it is of critical importance to ursolic acid and characterize the genes and/or enzymes involved in rubber biosynthesis in different rubber clones. Due to the important role of FDP in isoprenoid biosynthesis, it has already been isolated and characterized from many plant species including H. brasiliensis (clone RRIM 600) (Adiwilaga and Kush, 1996), Artemisia annua (Hemmerlin et al., 2003), Arabidopsis (Closa et al., 2010, Keim et al., 2012), Chimonanthus praecox (Xiang et al., 2010), Withania somnifera (Gupta et al., 2011), Euphorbia pekinensis (Cao et al., 2012), Hedychium coronarium (Lan et al., 2013), Medicago sativa (Sun et al., 2013), Rhizosolenia setigera (Ferriols et al., 2015), Tripterygium wilfordii (Zhao et al., 2015), and Matricaria recutita (Su et al., 2015). It has been reported that FDP synthase catalyzes the synthesis of the last common substrate in the isoprenoid biosynthesis. Hence, it is a branch point and a likely regulatory enzyme in the pathway, the availability of the cloned FDP gene from Indian Hevea clone should be useful for further investigation of the regulation of rubber biosynthesis and generation transgenic rubber plants for enhanced latex yield. However, as far as we know, the cDNA encoding for FDP gene has been reported from RRIM 600 a Malaysian rubber clone, there were no reports from Indian high yielding rubber clone RRII105 till now. The major reason is that RRIM 600 rubber clone is a low latex yielder in India and the RRII105 is the most popular high yielding rubber clone which is being cultivated and occupied in rubber growing regions of India. Therefore, isolation and characterization of FDP gene from Indian popular clone RRII 105 will be highly useful for understanding the molecular mechanism of this gene and also for over-expression of this gene under a constitutive promoter for enhanced latex yield production in transgenic rubber plant in the future.
In this communication, we report the cloning and characterization of an HbFDP gene from genomic DNA as well as cDNA from RRII 105 rubber clone. We also demonstrated the expression pattern of FDP gene in different types of tissues by semi-quantitative RT-PCR analysis. In addition, the HbFDP gene was over-expressed by cloning into a binary vector and expression pattern was studied in transgenic Arabidopsis plants.
Materials and methods
Discussion The major goal of this research is to clone and characterize the FDP gene using PCR based approach. FDP is considered to play an important role in the control of plant isoprenoid biosynthesis, since FPP is the starting point of different branched pathway leading to the synthesis of key isoprenoid end products. Considering the important role of FDP gene in isoprenoid biosynthesis, the present study was mainly focused on molecular cloning and characterization of genomic as well as cDNA fragments encoding HbFDP gene in H. brasiliensis (An Indian rubber clone RRII105). The putative nucleotide sequence results demonstrated that Hevea FDP is an intronless gene as no introns were detected within the coding region of genomic sequence and it showed 100% homology with reported HbFDP cDNA sequence as well. The PCR based strategy was applied for isolation and characterization of FDP genes from various plant species recently (Sun et al., 2013, Ferriols et al., 2015, Su et al., 2015, Zhao et al., 2015). A BLAST search of the NCBI protein database suggest that the deduced amino acid sequence of RRII 105 exhibited only 99% similarity with HbFDP protein sequence of RRIM600 published in NCBI. Similarly, Sun et al. (2013) described the isolation and characterization of farnesyl diphosphate synthase gene from M. sativa (MsFPPS) and the size of the cDNA sequence was 1368bp, which encoded a protein of 342 amino acids. Most recently, Zhao et al. (2015) also reported two FPS genes (TwFPS1 and TwFPS2) that encode 342 deduced amino acid sequences in T. wilfordii. On contrary, Su et al. (2015) identified a cDNA encoding farnesyl diphosphate synthase (MrFPS) from M. recutita, a key enzyme in the pathway of biosynthesis of isoprenoids, and the gene comprises 1032bp size and encodes 343 amino acid residues.