Expression level of DREB gene of local corn cultivars from Kisar Island-Maluku , Indonesia , using quantitative real time polymerase chain reaction

The research objective was to determine the expression level of dehydration responsive element binding (DREB) gene of local corn cultivars from Kisar Island Maluku, Indonesia. The study was designed as randomized block design with single factor consist of six local corn cultivars obtained from farmers in Kisar Island and one reference varieties which has been released as a drought-tolerant varieties and obtained from Cereal Crops Research Institute Maros South Sulawesi. Isolation of total RNA from the second leaf after the flag leaf at the 65 days after planting were carried out according to the protocols of the R & ABlueTM Total RNA Extraction Kit, and was used as a template for cDNA synthesis. Amplification of cDNA from total RNA was carried out according to the protocol of One-Step Reverse Transcriptase PCR Premix Kit. Real Time-PCR was performed usingcDNA from reverse transcription following the procedures of Real MODTM Green Real-Time PCR Master Mix Kit. The real time-PCR data were analyzed using relative quantification method based on the critical point/cycle threshold. The highest DREB gene expression was showed by Deep Yellow local corn cultivar, and the lowest one was showed by Rubby Brown Cob cultivar. The DREB gene expression level of deep yellow local corn cultivar was even higher than Srikandi variety as a reference variety.


Introduction
][9] DREB gene is a member of the APETALAe/ethylene-responsive elementbinding protein (AP2/EREB) family.The AP2 family consist of genes that function to encodes a transcription factor protein.DREB gene encodes a transcription factor protein DREB which involved in the mechanism of plant adaptation to drought.Transcription factor proteins encoded by the genes of a sub-family AP2 has a sustainable domain (conserved domain) AP2/ERF consisting of 50-60 amino acids.][12][13][14][15][16] Kisar Island is one area in the district of South West Maluku Regency which has the potential for developping corn.In the development strategy of corn in Maluku, Kisar island determined to be in region I of the development strategy.This determination is based on the high utilization of corn as a staple food by the public, but planted in dry climates. 17An exploration and documentation of corn germplasm in the Kisar island was conducted and found that there are seven local cultivars of corn that are specific to that Island namely: i) Rubby Brown Cob cultivar, ii) White Brown Cob cultivar, iii) Red Blood, iv) White, v) Waxy, vi) Early Maturing Yellow, and vii) Deep Yellow cultivar.These local corn cultivars have potential to be developed, because it has an ability to adapt to the local dry environment. 18n order to increase the production and to develop a plant as a superior crop, the properties of plants related to its ability to adapt to the environment, especially the environment with limited water availability, absolutely must be known.Some reference was stated that the first step to obtain cultivars tolerant to abiotic stresses such as drought is evaluation and selection of germplasm collection available. 19While, another researcher was reported that germplasm can be either local cultivars (landraces), 20 or can be accession including individual pure lines that has not been tested to determine its properties. 21he properties of plants associated with adaptability to limited water availability conditions can be observed in molecular level, such as observation of genes involved in adaptation to drought.For the local corn cultivars in Kisar Island, study on the response of the corn cultivars in its natural condition including molecular responses has not been done.The purpose of this study was to determine the level of DREB gene expression on local corn cultivars in Kisar Island Maluku.

Materials and Methods
In this research, there are two leaf samples.One from the field and one is the control.Control is corn cultivars planted in the normal condition during 30 days (not under drought stress condition) and used to compare gene expression with samples from the field (under drought stress condition).Isolation of total RNA (samples from field) was conducted using the second leaf after flag leaf at the 65 days after planting, while for control using the second leaf from tip at the 30 days after planting.
Isolation of total RNA for both samples (from field and control) were carried out according to the protocols of the R&A-Blue TM Total RNA Extraction Kit (Intron Biotechnology, Seongnam, Korea).The RNA then used as a template for cDNA synthesis according to the protocol of the One- Step ReverseTranscriptase PCR Premix Kit (Intron Biotechnology).A total of 4 mL reagent, 2 mL samples of RNA isolated, and 4 mL of ddH2O was inserted into the thin wall and prepared for cDNA synthesis by reverse-transcriptase PCR reaction.
Reverse transcriptase polymerase chain reaction (PCR) was programmed; it includes reverse transcription reaction at 45 °C for 30 minutes, and denaturation at 94°C for 10 minutes for one cycle.To ensure that the reverse transcription performed successfully, a standard PCR reaction was carried out.Real Time-PCR was performed using the cDNA obtained from the reverse transcription process following the procedure of Real MOD TM Green Real-Time PCR Master Mix Kit (Intron Biotechnology).A total 1μL cDNA plus 1μL ZmDBP2 forward primer, 1μL primary gene ZmDBP 2 reverse primer, 10 mL Real MOD Real-Time PCR Master Mix Kit, and 5 mL of Bovine Serum Albumine (400 ng/mL) were inserted into cappilary tube 20 mL volume, and prepared for the real time-PCR reactions.Real time-PCR was performed using Light Cycler Real Time PCR Instrument (Roche, Basel, Switzerland) with the program as shown in Table 1.
The real time-PCR data were analyzed using relative quantitative methods based on the critical point/cycle threshold (C P /C T ) between the target gene (gene DREB) normalized to the housekeeping genes β-actin as a reference gene. 22DREB gene expression in samples in the field, expressed of how many times compared with control. 23

Results and Discussion
The results of experiments for sample from the field (Figure 1A) and the control (Figure 1B) both show that the reverse transcriptase-PCR were successfully performed the cDNA, and the resulted 150 bp cDNA was in accordance in length with the product of ZmDBP2 primer.The success of cDNA synthesis process is influenced by the purity of RNA obtained.Factors that influence the success of cDNA synthesis is the purity of RNA template (free of contaminant such as proteins, polysaccharides, and DNA) and RNA integrity. 23cDNA obtained was used for real time PCR process to measure the mRNA expression level of DREB gene.Real Time-PCR was performed on cDNA samples from reverse transcriptase PCR experiment results in the field and a control sample that has been done before.The results of agarose gel electrophoresis of cDNA in real time-PCR showed that the DREB gene also successfully amplified with 150 bp of PCR product (Figure 2).mRNA expression levels of the DREB gene were quantitatively analyzed based on theresults of real time PCR.Quantitative analysis is based on the calculation of the value of cycle threshold (C T ). mRNA expression of the DREB gene levels were analyzed by comparing the C T value of the target gene normalized with housekeeping gene (β-actyn gene).C T value data of the target gene (DREB gene), and the reference gene (β-actin gene) as house keeping genes in the sample results of experiments in the fieldand control samples (Figure 3).
Based on Figure 3, it can be seen that the C T value of targeted gene and reference gene were vary on both control and sample from field.There are cultivars with high value of C T on targeted gene, and low on reference gene.Indeed, there are low value of C T level on targeted gene, but show high level of C T on reference gene.C T value is the value which show the amplification cycle when fluorescent signal overcome the threshold and denote by the increasing of amplicon number.By using relative quantification based on the Livak Method the expression level was done and can be seen in Table 2.

Conclusions
When compared with control, the DREB gene of local cultivars and reference variety show a high level of gene expression till hundred times.This is in line with the statement of Silvera et al. 25 that plant which adapt to drought environment, their gene that control plant traits related to drought will higher expressed.The high level of gene expression of corn cultivars in drought condition shows that plant has molecular mechanism to copy to, by induction and expression of some gene related to plant defense against drought condition.The induction and expression of some gene allow the plant to live and also develop or growth during drought.Even though all cultivars show high level of gene expression, but the highest level of gene expression was showed by Deep Yellow Local Cultivar.This is means that Deep Yellow local cultivars is the most tolerant or the most adaptable of local cultivars to the drought environment condition in Kisar Island.According to Hu et al. 26 rice growth on drought condition, it was showed the high level of gene expression, especially for reg-ulatory gene, and this can increase the tolerance of that plant to drought.As same Silvera et al. 25 that tolerant rice varieties has high expression of gene that control plant mechanism related to drought.Based on the result, it can be conclude that the expression level of DREB gene was highest in the Deep Yellow corn cultivar and the lowest one obtained by Rubby Brown Cob cultivar.

Figure 3 .
Figure 3. Cycle threshold value of target and reference gene from corn cultivars (control and sample from the field) resulting from real time polymerase chain reaction.

Table 2 . Expression level of mRNA in DREB gene. Cultivars Expression level Information Rubby
Brown Cob 4.14 mRNA of the DREB gene of sample from the field was expressed 4.1 times than the control Red Blood 183.54 mRNA of the DREB gene of sample from the field was expressed 183.54 times than the control Waxy 64.44 mRNA of the DREB gene of sample from the field was expressed 64.44 times than the control Early Maturing Yellow 29.446 mRNA of the DREB gene of sample from the field was expressed 29.446 times than the control Deep Yellow 962.071 mRNA of the DREB gene of sample from the field was expressed 962.071 times than the control White 843.3572 mRNA of the DREB gene of sample from the field was expressed 843.3572 times than the control Srikandi 57.28 mRNA of the DREB gene of sample from the field was expressed 57.28 times than the control