Isolation of Housekeeping Genes on Durik-durik ( Syzygium sp )

Housekeeping gene is a gene expressed with a fixed level and in abundant amounts under various conditions. After validation, the housekeeping gene can be used as an internal control to normalize gene expression data. This study reports the isolation of several housekeeping genes in Durik-durik plant (Syzygium sp). This plant material in form of fresh leaves from Durik-durik plants are taken from Kajuik Lake, Riau Province. The next stage is total DNA isolation, polymerase chain reaction, electrophoresis, sequencing and data analysis using bioinformatic tools. The isolated housekeeping genes included 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Beta-tubulin and Ubiquitin with sizes of 400 bp, 679 bp, 1134 bp, 836 bp, 1167 bp and 2155 bp, respectively. In addition to 18S rRNA, the five housekeeping genes are the first reported from the genus Syzygium and referable to isolate housekeeping genes in other species in this genus. The six housekeeping genes can be used as internal controls on Durik-durik plants after validation.

2016b; Roslim, 2017;Roslim & Herman, 2017b).One of the peculiarities of the Durik-durik and other plants that grow in Kajuik Lake is their resistance to flooding stress for several months during the rainy season.This resistance is certainly because these plants carry the tolerant genes of flooding stress.To isolate and study these tolerant genes through the study of gene expression requires internal control.However, no single internal control has ever been reported from Durik-durik plants.Therefore, this study reports the isolation of housekeeping genes in Durik-durik plants.

Materials
The fresh leaves of Durik-durik (Syzygium sp) plants were taken from Kajuik Lake, Riau for total DNA isolation.The primer pairs used for isolation of tubulin-encoding genes were designed based on Einax & Voigt (2003), 18S rRNA based on Gantasala et al. (2013), and a 1-alpha elongation factor based on Rosa multiflora mRNA sequence (accession number JN399225).The previous studies have isolated the actin-coding gene from Durik-durik using the degenerate primer designing by McDowell et al. (1996) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) glyceraldehyde gene using primer pairs designing by Gantasala et al. (2013).The sequences of actin and GAPDH obtained were then used to design the Durik-durik specific primers.Primers for the amplification of ubiquitin-coding genes were designed on the basis of conserved regions of the ubiquitin-coding gene in some plants (Table 1).

Extraction of Total DNA
Young and fresh leaves of Durik-durik plants were taken for total DNA isolation using Genomic DNA Mini Kit Plant (Geneaid).A total of 0.1 grams of leaves were cut into small pieces and then inserted into the mortar and crushed using pestel with the help of liquid nitrogen.After the leaves became powder, they were then put into a 1.5 ml tube for continued total DNA isolation by procedure following the manufacturer's instructions from the kit.The total DNA solution obtained was then stored in the refrigerator.

INTRODUCTION
Gene expression studies need some internal controls that commonly come from housekeeping genes group.Housekeeping genes are group of genes expressed constitutively at a constant level under various conditions.This gene group encodes the proteins involved in the various metabolisms that are essential for the survival of a cell (Koonin, 2000).For example, actin and tubulin proteins are involved in cytoskeleton formation because they play a role in maintaining cellular turgidity and structure (Keren et al., 2008;Kueh & Mitchison, 2009).Another example is the ubiquitin protein involved in the recycling of cell components through the ubiquitination process (Callis, 2014;Stone, 2014).Another example is the 1-alpha protein elongation factor which is involved in elongation of the polypeptide chains during translation (Sasikumar et al., 2012).Glyceraldehyde-3-phosphate dehydrogenase and 18S ribosomal RNA are also a member of housekeeping gene group.Some housekeeping genes have been isolated from various plants, such as the actin from the plant of Arabidopsis thaliana (McDowell et al., 1996), Melastoma malabathricum (Hannum et al., 2010), Oryza sativa (McElroy et al., 1990), Zea mays (Staiger et al., 2000), Jatropha curcas (Yuniati et al., 2011) and Elaeocarpus floribundus (Roslim & Herman, 2017a).The tubulin-coding gene has been isolated from the Arabidopsis thaliana plant (Snustad et al., 1992) and Linum usitatissimum (Gavazzi et al., 2017).Another example is the ubiquitin-coding gene which has been isolated from the Mesembryanthemum crystallinum plant (Azad et al., 2013).The gene encoding of glyceraldehyde-3-phosphate dehydrogenase has been isolated from the Haloxylon salicornicum plant (Al Mazrooei & Ghazala, 2017) and the 1-alpha elongation factor from the Rosa hybrid plant (Klie & Debener, 2011).
Isolation of housekeeping genes are important because these gene groups are often used as internal controls for normalizing the expression data of certain genes, such as the abiotic stress tolerant gene.However, to function as an internal control, this group of genes must be validated.The validation of housekeeping genes has been done in some plants (Galeano et al., 2014;Sinha et al., 2015;Hou et al., 2017).
Durik-durik (Syzygium sp) is one of the many plants that grow in Kajuik Lake, Riau Province.This lake is one of the floodplain lakes in Riau.This lake contains the typical flora and fauna that are spesific to Riau (Roslim et al., 2016a, PCR reaction to 50 μl.The PCR process included pre-PCR at 95°C for 5 min, followed by 35 cycles consisting of three stages: denaturation at 95°C for 45 seconds, annealing at the corresponding primer annealing temperatures (Table 1) for 45 seconds and extention at 72°C for 1 minute 30 seconds.The last stage was post-PCR at 72°C for 10 minutes.

Electrophoresis
Total DNA molecules and PCR products were migrated to 1.2% agarose gel containing 5 μg/ml of ethidium bromide to determine their quality and quantity.The electrophoresis buffer used was 1X TBE (Tris Borate EDTA pH 8.0).Electrophoresis was performed at 50 volts for 45 minutes.After that, the gel was placed on top of the UV lamp to visualize the DNA bands.DNA bands were photographed using a digital camera (Olympus SP-500 UZ) for analysis purposes.

Delivery of PCR Products for Cloning and Sequencing
A total of 45 μl of PCR products from housekeeping genes and 30 μl primer per sample were packed and sent to PT Gentika Science Jakarta, Indonesia for gel purification, cloning and sequencing at 1 st Base in Malaysia.Gel purification and sequencing were performed on PCR products of the 18S rRNA (Syz-18S), actin (Syz-act), elongation factor 1-alpha (Syz-ef1a) and glyceraldehyde-3-phosphate dehydrogenase (Syz-gapdh) genes, while cloning and sequencing were performed on PCR products of the betatubulin (Syz-βtub) and ubiquitin (Syz-ubq) genes.
Gel purification using Zymoclean™ Gel DNA Recovery Kit (Zymo Research) and sequencing using forward and reverse PCR primers.Cloning used pTA2 vector with Toyobo Target Clone -Plus-(Toyobo) and tansformed into E. coli Zymo 5a with Mix and Go Competent Cells™ (Zymo Research).The bacteria colony was PCR using T3 and T7 promoter primers by using KOD FX Neo (Toyobo).Plasmid was isolated using ZR Plasmid MiniPrep (Zymo Research) and subsequently plasmid was sequenced in two directions using T3 and T7 promoter primers.

Analysis of Housekeeping Gene Sequences
The lengths of the housekeeping gene sequences from Durik-durik obtained in this study after be sequenced were 421 bp for Syz-18S rRNA, 679 bp for Syz-ACT, 1134 bp for Syz-GAPDH, 836 bp for Syz-EF1α, 1167 bp for Syz-βTUB and 2155 bp for Syz-UBQ.The sequences have already been registered to GenBank database (Figure 2).BLASTn analysis showed that housekeeping genes from Durik-durik had relatively high similarities with fellow housekeeping genes from some plants, especially members of the Myrtaceae family.The identity and query cover values were very high on the BLASTn analysis based on genes encoding the 18S rRNA, actin, EF1α and β-tubulin, ranging from 86%-99% and 80%-100%, respectively.Meanwhile, although the identity values of BLASTn analysis based on the glyceraldehyde-3-phosphate dehydrogenase and ubiquitin genes were very high, ranging from 86% to 95%, but the query cover values were low, i.e. 9%-42% (Table 2).It because the sequences available in GenBank database are mRNA molecules which are consisting of exons only.These results also indicated that the exon regions of both genes were short.The length of Syz-GAPDH and Syz-UBQ genes obtained in this study were quite long, i.e. 1134 bp and 2155 bp, respectively, but the exon lengths were predicted at 456 bp and 192 bp, respectively (Table 3).
The six housekeeping genes obtained in this study can then be used as an internal control on the study of gene expression in Durik-durik plant.The first step in the study of gene expression is determining whether or not genomic DNA contaminants are present in total cDNA (Hannum et al., 2010).The total cDNA molecules contaminated with genomic DNA can not be used for the study of gene expression because the expression data becomes invalid and unreliable (Laurell et al., 2012).
The determination of genomic DNA contaminants can be performed using five housekeeping genes other than 18S rRNA.A simple way to detect the genomic DNA contaminants is to do PCR using a template of total DNA and cDNA separately and the primer pair flanking the introns, then observe the number and size of the PCR product from both (Table 4).If the PCR products of PCR using a template in the form of total The primer pairs of Syz-18S_F/Syz-18S_R, Syz-act-F2/Syz-act-R2, EF1α_F/ EF1α_R and P-bTub3-F/P-bTub4-R (Table 1 and  Table 4) can be directly used in the study of gene expression in Durik-durik because those are annealing in two different exon regions flanking the introns.Exon is the coding region and intron is the non coding region of a gene.Genomic DNA contains both exon and intron, whereas cDNA contains only exon (Rogozin et al., 2005;Zhu et al., 2009;Griffiths et al., 2008).This is the basis for determining the genomic DNA of contaminants in total cDNA (Hannum et al., 2010).
In addition to the Syz-18 gene, the five housekeeping genes obtained in this study were the first reported from the Syzygium genus.Therefore, all five can serve as a basis for isolating housekeeping genes in other species within the genus Syzygium.

CONCLUSION
This research has isolated six housekeeping genes from Durik-durik plant (Syzygium sp), i.e. 421 bp of Syz-18S rRNA, 679 bp of Syz-ACT, 1134 bp of Syz-GAPDH, 836 bp of Syz-EF1α, 1167 bp of Syz-βTUB and 2155 bp of Syz-UBQ.Housekeeping genes from Durik-durik have relatively high similarities with fellow housekeeping genes from several plants, especially members of the Myrtaceae family.In addition to the Syz-18 gene, the five housekeeping genes obtained in this study were the first reported from the Syzygium genus.These six housekeepings may be used in the study of gene expression as internal control after validation and also as a marker of genomic DNA contaminants in total cDNA.

Table 4 .
Primer pairs and their PCR product (bp) and annealing temperature (°C) in gene expression study on Durik-durik (Syzygium sp).