Isolation of Partial Housekeeping Genes on Tuntun Angin ( Elaeocarpus floribundus )

Indonesia Abstract Some genes like 18S rRNA , 26S rRNA , elongation factor 1-alpha ( EF1 α ), and beta-tubu-lin ( TUB ) are members of housekeeping genes group that are commonly used as internal control in gene expression study. This study aimed to isolate those four housekeeping genes of tuntun angin ( Elaeocarpus floribundus ). The research material included fresh leaves of E. floribundus that were picked up from Kajuik Lake in Riau Province and four primer pairs. The procedures consisted of total DNA isolation using Genomic DNA Mini Kit Plant (Geneaid), polymerase chain reaction (PCR), electrophoresis on 1% agarose gel, sequencing, and bioinformatic analysis. This study has been isolated 18S rRNA , 26S rRNA , EF1 α , and TUB genes with the size of 422 bp, 922 bp, 856 bp, and 877 bp, respectively. The EF1 α and TUB genes has never been reported in Elaeocarpaceae family. Thus, those partial DNA sequences are the first sequences reported from this species and can be used as reference genes in this plant after

Actin and tubulin proteins are components of the eukaryotic cell cytoskeleton. In plants, the genes encoding actin and tubulin proteins have many variants and are called gene family. They participate in movement of chromosomes during mitosis and meiosis, movement of organells, and intracellular transport (McDowell et al., 1996;Dominguez & Holmes, 2011;Kandasamy et al., 2012;Rebouças et al., 2013). Arabidopsis thaliana has ten actin genes (McDowell et al., 1996) while Pinus taeda (Schwarzerova et al., 2010) and Physcomitrella patens usually have seven to eleven actin variants (Zhang et al., 2010). Meanwhile, there are up to eight isotypes of beta-tubulin in plants, for example, six beta-tubulin isotypes in Physcomitrella patens (Jost et al., 2004), twenty beta-tubulin genes in Salix arbutifolia (Rao et al., 2016), twenty in Populus (Oakley et al., 2007), nine in Arabidopsis thaliana (Cheng et al., 2001), and eight in Oryza sativa (Yoshikawa et al., 2003).
Ubiquitin protein is involved in recycle of damage proteins or other cell components and the process is named ubiquitination (Pickart & Eddins, 2004). Ubiquitin gene in plant also consists of many isotypes. Ubiquitin gene familiy in Arabidopsis thaliana comprises 14 members that are grouped into three types such as polyubiquitin, ubiquitin-like, and ubiquitin extension genes. The UBQ3, UBQ4, UBQ10, UBQ11, and UBQ14 genes are included as polyubiquitin genes and UBQ7, UBQ8, UBQ9, and UBQ12 genes are included as ubiquitin like genes (Callis et al., 1995).
Ribosomal RNA and elongation factor-1-alpha are involved in translation (Sasikumar et al., 2012;Gantasala et al., 2013). Ribosome is composed of ribosomal RNA (rRNA) molecules and ribosomal proteins. In eukaryotic cell, the ribosome is 80S in size and consists of large subunit measuring 2017). The reference genes come from houkeeping genes groups because their expressions are abundant and stable at any developmental stages, tissues, and conditions. Previously, a housekeeping gene was isolated from E. floribundus such as partial actin gene (Roslim & Herman, 2017). However, for the purposes of gene expression studies, more than one reference gene are needed as internal control (Rebouças et al., 2013;Wang et al., 2017;Bao et al., 2016). Moreover, the most widely used housekeeping genes as internal control are 18S rRNA, 26S rRNA, EF1α, GAPDH, actin, and beta tubulin (βTUB) in plants like tea (Camellia sinensis), jute (Corchorus capsularis), and olive (Olea europaea) (Kozera & Rapacz, 2013;Ray & Johnson, 2014;Niu et al., 2015;Wang et al., 2017). Therefore, the objective of this research was to isolate the partial housekeeping genes in E. floribundus such as genes encoding 18S rRNA, 26S rRNA, elongation factor 1 alpha (EF1α), and beta tubulin (βTUB). This research aims to provide information about some housekeeping genes of E. floribundus that have not been reported in the previous studies. The genes can be evaluated for possible using as internal control in gene expression study of this plant.

METHODS
Fresh leaves of tuntun angin (Elaeocarpus floribundus BI) for DNA extraction were collected from Kajuik Lake in Riau Province, Indonesia. The primer pairs used in this study are presented in Table 1.
Procedures in this study were performed based on Roslim et al. (2018). The fresh leaves were used to extract the total DNA by using Genomic DNA Mini Kit (Plant, Geneaid GP100). The total DNA obtained was then checked for quantity and quality using agarose gel electrophoresis. After that, polymerase chain reaction (PCR) was performed for amplification of partial genes of 18S rRNA, 26S rRNA, EF1α, and βTUB. The agarose gel electrophoresis was also done for checking of the existence of PCR products. Fourty five microliters of the PCR products were sent to PT Genetika Science for purification and sequencing at 1 st Base in Malaysia.
The DNA sequences were then analyzed using BioEdit version 7. Aligment using BLASTn was also conducted to determine the similarity to other sequences available in GenBank database. Dendrogram was then constructed using MEGA6 software. These sequences analysis were conducted following a procedure suggested by Roslim et al. (2018).

Profile of PCR Products
The PCR products of 18S rRNA, 26S rRNA, EF1α, and βTUB genes were obtained with the size approximately of 400 bp, 1000 bp, 900 bp, and 900 bp, respectively (Figure 1). Those bands were thick and suitable for sequencing requirements.

Analysis of Partial Housekeeping Gene Sequences
The partial DNA sequences obtained in this study were 422 bp for 18S rRNA, 922 bp for 26S rRNA, 856 bp for EF1α, and 877 bp for βTUB.   BLASTn analysis shows that the four sequences of E. floribundus have approximately 75.39%-99.04% similarity to the sequences deposited in GenBank database. The E. floribundus 18S rRNA and 26S rRNA genes have a higher similarity than EF1α and βTUB genes ( Table 2). The result shows that the four genes are relatively conserved between species. The conserved genes are genes that have the same function in all organisms and the genes which involved in any biological processes, cellular localization, and molecular functions (Jayaswal et al., 2017). The high level conserved genes are actin, ubiquitin, tubulin, PP2A, translation elongation factor, and small subunit ribosomal RNA (Hug et al., 2016;Jayaswal et al., 2017).
The EF1α obtained in this study comprises two exons flanking one intron. Exon is a part of eukaryotic gene encoding protein. In other words, the exon will be transcribed and translated into protein.
While intron is a part of eukaryotic gene which does not encode protein in other words, intron will be transcribed but not be translated. The intron region will be removed after transcription through splicing process. Thus, DNA molecule contains exon and intron while mRNA or cDNA molecule consists only These DNA sequences have already been registered to GenBank database (Figure 2). Based on data in GenBank, the complete coding sequence of those genes were approximately 1754 bp for 18S rRNA, 2801 bp for 26S rRNA, 1350 bp for EF1α, and 1745 bp for βTUB (Emrich et al., 2007) exon (Rogozin et al., 2005;Zhu et al., 2009). Such conditions cause the EF1α to be able to be used as a marker to determine DNA contamination in total cDNA in addition to its potential as internal control in gene expression studies. For this purpose, PCR using DNA or cDNA molecule as template was performed separately and after that the sizes of the PCR products from the two templates were compared (Hannum et al., 2010). In this case, the PCR product from DNA template around 850 bp in length and cDNA template will produce a shorter PCR product which is about 750 bp in length.
Previously, one housekeeping gene of E. floribundus was isolated, namely actin gene. The actin gene also contains two exons and one intron intron (Roslim & Herman, 2017) like the EF1α obtained in this study. Hence, in addition to the EF1α, the actin gene may also be used as DNA contaminant marker in total cDNA of E. floribundus.
Housekeeping genes isolated from E. floribundus must be evaluated further to determine which one is appropriate as the reference gene. One of the validation processes can be done using the quantitative real time PCR (qRT-PCR) technique and the statistical analysis using geNorm, Norm-Finder, Best Keeper, and Rank Aggreg (Andersen et al., 2004;Pfaffl et al., 2004;Pihur et al., 2009;Bao et al., 2016). Some housekeeping genes have been validated using qRT-PCR technique in plants such as poplar (Basa et al., 2009), eggplant (Gantasala et al., 2013), rice (Pabuayon et al., 2016) and pigeonpea (Sinha et al., 2015). It is very crucial to select and validate the housekeeping gene for each treatment because it can thus reduce possible errors and contamination during extraction and manipulation of mRNA or cDNA (Rebouças et al., 2013).
The four housekeeping genes such as 18S rRNA, 26S rRNA, elongation factor 1 alpha (EF1α), and beta tubulin (βTUB) obtained in this study are the first reported from E. floribundus. After selection and validation, they can be used as internal controls in gene expression analysis in E. floribundus. For example, the study of gene expression related to flooding stress uses these genes as internal controls so that the expression level can be trusted. Finally, the mechanism underlying this plant's tolerance to flooding stress can be understood.

CONCLUSION
Four housekeeping genes such as 18S rRNA, 26S rRNA, elongation factor 1 alpha (EF1α), and beta tubulin (βTUB) were isolated from tuntun angin (E. floribundus) and these are the first genes reported from E. floribundus. The sizes of these genes are 422 bp for 18S rRNA, 922 bp for 26S rRNA, 856 bp for EF1α, and 877 bp for βTUB. Then, the four genes should further be validated so they can be used as reference genes. One gene, the EF1, can be used as a marker of DNA contamination in total cDNA in gene expression studies of E. floribundus.