JLST

Journal of Life Sciences and Technology, 2012, 1(1):9-13

SITH - ITB

Genetics and Molecular Biology - Research Paper

Isolation of Two Elongation Factor 1 Alpha Promoters Gene from Cassava (Manihot esculenta Crantz)
Sony Suhandono , Ardha Apriyanto , and Tati Kristianti
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1*

School of Life Sciences and Technology, Bandung Institute of Techonology, Jalan Ganesa No. 10 Bandung 40132 West Java Indonesia. Tel./Fax. 62 22 2534107 / 62 22 2511575 Abstract Eukaryotic elongation factor 1 alpha, eEF1A, plays an important role in protein synthesis, catalyzing the binding of aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism. The eEF1A gene promoter is expected to have high expression level and various expression characters. This research was conducted in order to isolate and analyze the eEF1A gene promoter from cassava (Manihot esculenta Crantz). Two promoters from cassava eEF1A gene family was successfully isolated, named as MeEF1A3 (1247 bp) and MeEF1A4 (1168 bp) based on cassava genome sequence available in phytozome. Promoter region of MeEF1A3 and MeEF1A4 was confirmed by alignment of the promoters with the EST sequence available from Genbank. Furthermore, sequence analyses showed that those promoters contain three conserved putative control elements that are located upstream of the transcription start site. These control elements include a TEF1, a TELO and TATA boxes. In addition, both of the promoters also have the 5'UTR intron which usually increases the expression level. Hoping those promoters can be used in plant biotechnology to improve crops quality. Keywords:cassava, elongation factor 1 alpha, promoter

Introduction
Cassava or Singkong (Manihot esculenta Crantz) is one of the most important crops in the world. It is currently the most important food source for carbohydrate, after rice, sugarcane and maize. Over 500 million people consumed cassava in the developing countries of the tropics and sub-tropics (El-Sharkawy 2004). Cassava has storage roots with dry matter containing more than 80% of starch. However, the production of this crop in Indonesia is still low but can be improved using plant biotechnology technique to create genetically modified (GM) crops. The successful engineering of GM crop with desired trait is a complex procedure and depends on many factors. The defined spatial and temporal expression pattern of transgenes has been reported as an important factor, considering that either constitutive as well as overexpression strategies may lead to undesirable abnormal effects in transgenic plants. In some cases, the use of endogenous
*Correspondence author: E-mail: sony@sith.itb.ac.id Received: 30 November 2011/Accepted: 25 December 2011/Published online: 20 April 2012 The authors. Journal of Life Sciences and Technology, SITH ITB

regulatory regions with particular developmental expression or specific patterns has proven to mitigate the problem (Zavallo et al. 2010). Hence, the identification of gene promoters leading to particular expression patterns is crucial for the development of new GM plant generations. Eukaryotic elongation factor 1A (eEF1A), one of the four subunits composing eukaryotic translation elongation factor-1 (eEF-1), is the highly abundant G protein, constituting up to 3-10% of the total soluble protein in cells cytoplasm (Merrick 1992). eEF1A, plays an important role in protein synthesis, catalyzing the binding of aminoacyl-tRNA to the Asite of the ribosome by a GTP-dependent mechanism (Merrick 1992). The studies also revealed that eEF1As are typically encoded by multigene family in plants (Vijaykumar et al. 2002, Xu et al. 2007), including cassava (Suhandono et al. 2001). Since the eEF1As major function is a housekeeping gene, therefore the gene maybe expressed under controls of promoters with different characteristic. External and internal factors were known to influence the eEF1A gene expression such as low temperature

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(Berberich et al. 1995), light (Aguilar et al. 1991), developmental stage (Vijaykumar et al. 2002) and wounding (Morelli et al. 1994). Our previous study, reported that one of the eEF1A gene in cassava (MeEF1A1) was expressed in early stages of plant development and also induced by wounding (Suhandono et al. 2001). Although eEF1A genes in some plant species have been well characterized, the cassava eEF1A genes family member, especially their expression and promoter activity was not reported before and need to be explored. We expected that the promoter from cassava eEF1A gene family member have a high expression level and specific characteristic. Identification, isolation and functional analyses of cassava genes including their corresponding promoter have been speeded up significantly after the cassava genome sequence became available in 2010 (http://www.phytozome.net/cassava). The objective of this research was to isolate and analyze other promoter of eEF1A gene family members from cassava, which may be used to control certain gene of interest and improved the quality of many important crops.

amplified products were resolved in 1% agarose gel (Fermentas), stained with ethidium bromide (10 g/ml) and visualized in gel documentation system. PCR products were purified using GeneaidTM PCR purification kit following the manufacturer's protocol. The PCR product was cloned into pJET1.2/blunt vector (Fermentas) and transformed into Escherichia coli strain DH5 with heat shock method (Sambrook et al., 1989). The plasmid vector was isolated by GeneaidTM plasmid isolation kit and sequenced using pJET 1.2 forward and pJET 1.2 reverse primers by Macrogen Inc, South Korea. Sequence analysis Sequence data set forward and reverse from sequencing results were trimmed using Geneious Software (Drummond et al. 2009). Each sequenced trimmed was entered to the Basic Local Alignment Search Tool (BLAST) available online at www.ncbi.nlm.gov/blast/ to verify sequence similarity to previously identified promoter sequences. Promoter region also confirmed using Expressed Sequence Tag (EST) database from GenBank. The PlantCARE (Lescot et al. 2002) and PLACE (Higo et al. 1999) software were used to identify regulatory cis-elements along the putative promoter regions upstream of the theoretical translation site. Multiple alignments using Clustal W2(Larkin et al. 2007)in Geneiouswas also used to identify the conserved motifs.

Materials and Methods

DNA extraction, PCR amplification, Cloning and Sequencing. Cassava (Manihot esculenta Crantz) plant cultivar Adira was used for isolating the eEF1A promoter region. The total genomic DNA from cassava was extracted from the leaves using Doyle & Doyle method (1987). PCR was carried out in BioradTM Thermal Cycler CFX 9600 with the following cycles 95C for 3 min as initial denaturation followed by 35 cycles of 95C for 30 s, 55C for 30 s, 68C for 1 min, 68C for 2 min as final extension and holding at 4C. PCR was performed in a 50-l total reaction volume using Kapa 2GTM Robust Hotstart Kit that containing 2 l DNA template,0.2 l of 20 M of each forward and reverse primer, 10 L of 5X Kapa 2G Buffer B, 10 L of 5X Kapa 2G Enhancer, 1 l of 10 M dNTPs mix, 0,4 L Kapa 2G Polymerase and 26,2 L Water Free-nuclease. We used MeEF1A-Univ reverse primer 5'GTGAACCTTCTCITTACCCATT-3' and MeEF1A3 forward primer to isolate first promoter and MeEF1A-Univ reverse primer 5'GTGAACCTTCTCITTACCCATT-3'and MeEF1A4 forward primer 5'-AAAGATGGACGGC AAATGGT-3' to isolate second promoter. The

Result and Discussion

Identification and Analyses of Promoter Two fragments were successfully isolated from promoter region of eEF1A gene, a 1168 bp named MeEF1A3 and a 1247 bp named MeEF1A4. In order to identify these fragments, the sequences were analyzed using the blastn database on Genebank (www.ncbi.nlm.nih.gov/blast) and aligned with other eEF1A promoters from M. esculenta. The result showed that MeEF1A3 and MeEF1A4 had some homology with eEF1A promoters from M. esculenta (Figure 1). These sequences were also compared with the EST's of M. esculenta in order to identify putative 5' Untranslated (UTR) intron which may contain in these promoters. The result showed that putative 5'UTR intron was also found in both sequence. MeEF1A3 and MeEF1A4 had 99% similarity with an EST from cassava (DB934938) and (DB936919) respectively. A 496 bp intron was found in the 5'UTR
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Figure 1. Promoter architecture of Manihot esculenta eEF1A gene family. MeEF1A1 (Suhandono et al. 2001), MeEF1A2 (Lydya, 2007), MeEF1A3 and MeEF1A4 (This Research). Nucleotides between motif showed by number.

Table 1. Elongation Factor 1 Alpha Promoters Motif

* The sequence and the relative position of control elements from TSS with (A) adenine nucleotide as first transcribed nucleotide. At: Arabidopsis thaliana, Le: Lycopersicon esculentum, Me: Manihot esculenta, n: number of nucleotide between motifs.

of MeEF1A3 and a 842 bp intron was found in the the 5'UTR of MeEF1A4 9 (Fig. 1). The 5' UTR intron also known as important part of the promoter region that can be enhanced the gene expression level. This phenomenon called IME (Intron mediated enhancement) as reported in Rose & Beliakoff (2000). The mechanism of IME is largely unknown. Some efficiently spliced introns boost expression more than 10-fold, while others have little or no effect, but generally introns could enhance gene expression by increasing the steady state amount of mature mRNA in the cell (Rose et al. 2008). However, recent study also reporting that IME mechanism is highly conserved in plants in order to elevate gene expression (Parra et al. 2011). We also found 5' UTR intron presented in the all of plants eEF1A promoters that have been described here. All four sequences of the EF-1 gene family (A1A4) from Arabidopsis also have 5' UTR Intron (Curie et al. 1991). Similarly, the position of the 5'UTR intron is conserved between these genes andEF-1 gene family from M.esculenta (MeEF1A1-MeEF1A4). This data indicated that the present of 5' UTR intron were
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conserved among eEF1A promoters in plant. Sequence analyses shows that eEF1A promoters have putative promoter control elements are predicted based on sequence similarity and the relative position to the transcription start site (TSS) as we can see on (Table 1). The results from table 1 showed that control elements like TEF1 box, TELO box and TATA box are conserved among eEF1A promoter in plants. Except for MeEF1A1 that contain two putative TATA boxes which important for RNA polymerase binding. Two other elements, TEF1 box (Regad et al. 1995) and TELO box (Curie et al. 1991), were identified in MeEF1A promoters in different position at approximately -110 to 30 bp from the TSS. The TEF1 box and TELO box sequences in MeEF13 and MeEF1A4 are similar to the consensus sequences (Table 1). TELO box (consensus 5'-AACCCTA-3') was known to interacted with AtPur in A. thaliana (Tremousaygue et al. 1999). AtPur is Pur homolog protein, a conserved multifunctional protein in eukaryote and play an important role for activating or

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repressing transcription and translation (Gallia et al. 2000). TELO box usually found in the upstream regulatory of ribosomal protein and other translational related gene (Gaspin et al. 2010). It drives gene expression in root primordial (Manevski et al. 2000). The TELO box alone does not confer specificity and must act with other elements such as TEF1 box to drive expression in root meristem (Manevski et al. 2000). TEF1 box (consensus 5'-ARGGRYANNNNNGTA A-3) was initially identified in A. thaliana elongation factor 1 alpha gene (Curie et al. 1991) and several Arabidopsis RP genes (Regad et al. 1995). TEF1 box is the target for two heteromeric protein complexes C1 and C2 (Manevsky et al., 1999). Unlike the TELO box, the TEF1 box alone can confer specific expression, activating transcription in cells entering cell cycle, undergoing the transition from quiescent to mitotically active stages (Regad et al. 1995). However, the TEF1 box is usually associated and works synergically with TELO box or Motif Site II (Tremousaygue et al. 1999) which active in mersitematical tissue or dividing cell. Result from blastn with EST database from genbank showed that MeEF1A3 and MeEF1A4 had a very high similarity with cassava EST (DB934938; DB936919) that related to stress response (Sakurai et al. 2007). In other words these promoter may induced by environmental stress. Moreover, as observed by PLACE and PlantCARE software, MeEF1A3 and MeEF1A4 have the same control element but they have different upstream region (data not shown) that contain different cis-acting elements, this will lead into resulting of different expression pattern. In conclusions, based on analyses of conserved control element in eEF1A promoters and EST database, MeEF1A3 and MeEF1A4 seem to be active during certain developmentally stage and may also controlled by environmental stress. Currently, the construction of MeEF1A3 and MeEF1A4promoters into plant expression vectors are still in progress. A further study of the MeEF1Apromoter constructs in stably transformed Arabidopsis will give more detailed information about potential use of thesepromoters in plant biotechnology. We hope these promoters have specific character and high expression level that can be used to improve crops quality.

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