cDNA Actin Isolated From Pandanus Sp

Actin is one of the reference genes that is often used as an internal control in gene expression analysis. This study aimed to isolate actin cDNA from Pandanus sp originated from Riau. Fresh leaves Pandanus sp. Lake Kajuik, Langgam District, Pelalawan Regency, Riau Province. Isolation of RNA, synthesis of total cDNA, amplification of actin genes used McDowell's designed degenerate primer (PlAc46S20/PlAc245N-20), electrophoresis, sequencing, and data analysis. Actin cDNA fragments obtained were 353 pb in size, registered at GenBank and encoded 117 amino acids. Actin cDNA fragment consists of two exons and one introne. Specific actin primers from Riau Pandanus sp. can be designed based on sequences obtained for the purpose of analyzing certain gene expressions. .


Introduction
Pandanus sp. grows on Lake Kajuik, Langgam District, Pelalawan Regency, Riau Province, Indonesia. The genus often growth submerged and sank to 1.5 m below the surface of the water and resistant to flooding stresses and certainly contains tolerant genes inundation stress. To study the expression and role of these genes a reference gene is needed as an internal control.
The reference genes are genes that are expressed in all tissues and the stages of development of eukaryotic plants. The expression is abundant and not affected by external conditions (Thellin et al, 1999). Because of its characteristics, the reference gene after being validated is often used as an internal control for the purposes of gene expression. Some examples of reference genes that have been validated and used as internal controls are actin genes (ACT), cassava (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and tubulin (TUB) (Volkov et al, 2003;Jain et al, 2006;Caldana et al, 2007).
The actin gene encodes the actin protein which is the constituent unit of actin filaments in the cytoskeleton. This actin protein is present in all eukaryotic cells (Thellin et al, 1999). Actin genes have been isolated from Arabidopsis thaliana plants (Volkov et al, 2003;McDowell et al, 1996). Melastoma malabathricum (Hannum et al, 2010) and Populus (Zhang et al, 2010). This study aims to isolate actin cDNA from Pandanus sp. from Riau.

Materials and Methods
Fresh leaves of Pandanus sp. were obtained from Lake Kajuik, Langgam District, Pelalawan

Total RNA Isolation
RNA isolation were treated with DEPC water to protect RNA from nuclease. Total RNA isolation of leaves and roots was carried out using Trizol reagents following the manufacturer's instructions (Invitrogen, Molecular Research Center, Inc., USA). The total RNA molecules obtained were then treated with DNase enzymes to remove DNA that might contaminate.

Total cDNA synthesis
Synthesis of cDNA (reverse transcription) was carried out using Superscript First-Strand synthesis system kit (Invitrogen, USA) and primary oligo (dT) 21. The reaction composition as follows: 5000 ng RNA, 1x first strand buffer, 1 M primer Oligo (dT) 21, 0.5 mM dNTPs, 10 mM DTT, 40 units of enzyme RTase, 0.01% water DEPC to total reaction 20 l. The mixture was incubated on a PCR machine with the following program: 30C for 10 minutes, 42C for 50 minutes, 95 C for 5 minutes, and 20 C for 5 minutes.

Total cDNA amplification using PCR (Polymerase Chain Reaction) Technique
Amplification was carried out with PCR components including 1x PCR buffer (plus Mg2 +), 0. PCR products are sent to PT Genetika Science to be sequenced at 1st Base Malaysia.
Sequencing was performed using a primer for PCR.

Bioinformatics Analysis
The DNA sequence data is then analyzed and aligned using the MEGA6, BLASTn Sequences of several accessions were used to make phylogenetic trees obtained from the GenBank database.

Result and Discussion
The cDNA fragment of actin encoding has been obtained with a size of around 370 pb ( Figure   1). Sequencing the cDNA fragment obtained 353 pb ( Figure 2) and was registered with the GenBank database with accession number MG836260.   (Table 1). But none of these accessions are members of the genus Pandanus.
Therefore the sequences obtained in this study are cDNA sequences of actin encoders which were first reported from the genus Pandanus.

Conclussion
The actin cDNA fragments obtained in this study were 353 pb in size and were registered at GenBank and encoded 117 amino acids. This actin cDNA fragment consists of two exons and one introne. Specific actin primers from Riau Pandanus sp. can be designed based on sequences obtained for the purpose of analyzing certain gene expressions.