Genome sequence analysis of predicted polyprenol reductase gene from mangrove plant kandelia obovata

It has been previously reported that dolichols but not polyprenols were predominated in mangrove leaves and roots. Therefore, the occurrence of larger amounts of dolichol in leaves of mangrove plants implies that polyprenol reductase is responsible for the conversion of polyprenol to dolichol may be active in mangrove leaves. Here we report the early assessment of probably polyprenol reductase gene from genome sequence of mangrove plant Kandelia obovata. The functional assignment of the gene was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI using Blastx. The degree of sequence identity between DNA sequence and known polyprenol reductase was confirmed using the Blastx probability E-value, total score, and identity. The genome sequence data resulted in three partial sequences, termed c23157 (700 bp), c23901 (960 bp), and c24171 (531 bp). The c23157 gene showed the highest similarity (61%) to predicted polyprenol reductase 2- like from Gossypium raimondii with E-value 2e-100. The second gene was c23901 to exhibit high similarity (78%) to the steroid 5-alpha-reductase Det2 from J. curcas with E-value 2e-140. Furthermore, the c24171 gene depicted highest similarity (79%) to the polyprenol reductase 2 isoform X1 from Jatropha curcas with E- value 7e-21.The present study suggested that the c23157, c23901, and c24171, genes may encode predicted polyprenol reductase. The c23157, c23901, c24171 are therefore the new type of predicted polyprenol reductase from K. obovata.


Introduction
Mangrove forests have long been a rich source of phytochemical compounds producing polyisoprenoid alcohols [1,2] and other secondary metabolites [3,4]. Polyisoprenoid alcohols are classified to polyprenol and dolichol according to stereochemistry. A number of studies have been reported that the major polyisoprenoid in plant leaves were polyprenols rather than dolichols [5,6]. By contrast, in the animal world (especially in livers), dolichols were predominant, and few polyprenols occurred [7,8]  However, very recently, we are the first group to report that primary polyisoprenoid alcohols of Okinawan and Indonesian mangrove plants were not polyprenols but dolichols [1,2]. Therefore, the occurrence of larger amounts of dolichols even in the leaves of mangrove plants imply that polyprenol reductase is responsible for the polyprenol conversion to dolichol, may be active in mangrove plant leaves. In spite of the ever-present distribution of polyisoprenoid in the plant kingdom, their function plants world remain unclear, especially in mangrove plants. Two polyprenol reductase genes from Arabidopsis thaliana have been characterized [9]. However, information on the polyprenol reductase from mangrove species is limited. It, therefore, became necessary to understand the DNA sequence of polyprenol reductase in mangrove plants to obtain more insight into the physiological significance and biosynthetic pathway of these compounds. Here we report the early assessment of probably polyprenol reductase gene from the genome sequence of mangrove plant Kandelia obovata.

Sample collection
Fresh leaves of K. obovata were collected at Okukubi River, Okinawa, Japan. Total RNA was extracted from K. obovata leaves using cetyl trimethyl ammonium bromide (CTAB) procedure with minor modification to increase yields as previously described [10]. The total RNA was used for genome sequence.

Genome sequence analysis of probably polyprenol reductase
Genome sequence of K. obovata used shotgun sequencing for sequencing as previously reported [11]. The principle of genome sequencing consists of three steps, short fragment DNA from K. obovata were sequenced to a given length of coverage, then continue with genome assembly, and the last step was annotation [11]. Genome sequence of K. obovata using a BLAST search led to 3 nucleotide sequences that showed the highest similarity to polyprenol reductase.

Sequence and data analysis
Three nucleotide fragments named as c23157, c23901, and c24717 then were used for prediction of polyprenol reductase. The functional assignment of three DNA sequences was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using BLASTX [12]. The degree of sequence identity producing significant alignments between DNA sequences and a known protein database was confirmed by the BLASTX probability score more than 50 bit or an E-value < 10 -10 was considered to have significant similarity [13].
Furthermore, C24171 has the shortest DNA sequence; producing significant alignment to known, predicted polyprenol reductase from various plants (Table 3). Among them, the highest similarity of E-value (7e -21 ), 79% identity, and 93.6 bits score was indicated to polyprenol reductase 2 isoform X1 from J. curcas. These results suggested that c23517, c23901, and c24171 encoded probably polyprenol reductase. The c23157, c23901, and c24171 are therefore the new type of predicted polyprenol reductases from K. obovata. It has been reported that a common rule of two sequences are homologous if they are more than 30% matching over their whole lengths, the 30% condition misses many easily identified homologs [13]. In this context, three partial sequences showed more than 61% similarities to known polyprenol reductase in the plant kingdom. These results support previous results that primary polyisoprenoid alcohol in mangroves are dolichol, but not polyprenols where dolichols were found in examined mangrove samples [1][2]. Besides, the composition of polyisoprenoid may be an expression of the distribution of tissues in these mangrove plants [2].  K. obovata green and yellow leaves have been shown to contain polyprenols C75-100, C45-95, respectively, while dolichol was C70-100, C75-100, respectively. In contrast, in the roots, no polyprenols detected, with 100% dolichols were C80-105 [1]. This study indicated dolichol is more dominant than polyprenols in green leaves in the ratio of 76.3:23.7 and in the yellow leaves in proportions 42.9:57.1 [1]. In this context, the predominance of dolichol over polyprenol in K. candel and the majority of mangrove species suggested that existence of polyprenol reductases in mangrove leaves, which catalyze the conversion of polyprenol to dolichol corresponding to the SRD5A3 protein in animals [14], differ from those of other mangrove plants in reduction activity.

Conclusion
The current study suggested that the c23157, c23901, and c24171 genes may encode predicted polyprenol reductase from K. obovata. A detailed molecular cloning of the c23157, c23901, and c24171 genes are required further investigation to provide product confirmation of these genes and biosynthetic pathway of polyisoprenoid in mangrove plants.