PRIMER DESIGN AND OPTIMIZATIONOF ANNEALING TEMPERATURE FOR GENE AMPLIFICATION GSTL2 ON RICE

Glutathione S-Transferase is a superfamily enzyme that has many roles for living things, especially in the detoxification of Reactive Oxygen Species (ROS), one of which is rice plants. One gene of this family that has a role for plants is GSTL2 This gene is known to have a contribution to Arabidopsis and Oryza sativa L. against abiotic stress. To find out how this gene expression is requires a primer to specifically amplify this gene, and an optimal annealing temperature to support the success of the primer. This study aims to design primers and determine the optimal annealing temperature for gene amplification GSTL2. Primers were designed using the Primer3, which were then analyzed with Genious Prime based on good primer criteria and optimizing the annealing temperature which was carried out using gradient PCR. The results of this study obtained a primer pair is forward 5’-TTCGAAGGGCCAGCATTACT-3’

In plant, Glutathione s-transferase has an important role for its growth and development, (Hossain & Fujita, 2013), mainly involved in defense mechanisms against biotic and abiotic stress (Estévez & Hernández, 2020;Vaish et al., 2020).One of the most damaging abiotic stress is drought, because the response to drought stress is a very complex process involving various mechanisms at different levels (Basal et al., 2020).Drought can decrease the rate of photosynthesis, by inducing closure and decrease in total stomata size (Zagoto & Violita, 2019) which has an effect on the rate of photosynthesis due to differences in the production and use of electrons and photosynthetic apparatus which causes plants to experience oxidative stress due to the formation of Reactive Oxygen Species (ROS) (Verma et al., 2019).
One of the plants that is very vulnerable to drought stress is rice.According to Maisura et al. (2014), rice is a semi-aquatic plant that grows normally in waterlogged conditions.Drought can affect the growth of rice plants at both the morphological, anatomical, and even physiological levels (Violita & Azhari, 2021).
There have been many previous studies reporting the role of glutathione s-transferase against drought stress; CaGST on Capsium annum (Islam et al., 2019), ThGSTZ1 in transgenic plants Arabidopsis thaliana (Yang et al., 2014), and OsGSTL2 on Arabidopsis (Kumar et al., 2013).OsGSTL2 or GSTL2 has many synonym in the previous studies like GSTZ5 and Protein IN2 homolog (Hu et al., 2011).
The method that can be used to investigate gene expression is Quantitative Polymerase Chain Reaction (q-PCR) (Maddocks & Jenkins, 2017).The important thing that determine success in gene expression analysis is primer design (Biedendieck et al., 2011).The designed primer must fulfill the criteria of a good primer (Chuang et al., 2013;Rodríguez et al., 2019).In addition, an important aspect that determines the success of q-PCR is determining the annealing temperature (Ta).Annealing temperature (Ta) is an important variable for primer performance (Bustin & Huggett, 2017), especially for obtaining specific genes.
In previous studies, there were primers for GSTL2 gene in rice plant (Hu et al., 2011) but after conducting an analysis on Primer-BLAST it showed that the primer pair has 20 nt with GC content primer forward 45% and primer reverse 50%, temperature melting (Tm) forward 55,73 C and reverse 58,89 C, and produce PCR amplicon 300 bp.In this analysis showed this primer has high amount of GC content especially on primer reverse.This high amount of GC content in primer can impact the primer temperature melting (Tm) (Rodríguez-Lázaro et al., 2013).According to Thornton and Basu (2011) the Tm difference between primer pair should not be more than 1-2 C, and this primer pair has Tm difference up to 3 C.In addition, based on specificity analysis it showed this primer pair could amplify genomic DNA (gDNA) such as Oryza sativa Japonica Changxianggeng chromosome 11 (CP101151.1),Oryza sativa Indica chromosome 3 (CP056054.1),Oryza sativa Japonica chromosome 3 (AP014959.1),and Oryza sativa chromosome 3 BAC OSJNBa0044H10 (AC084405.6)which has the same amplicon of 300 bp, so it can affect the result of gene expression.
In the study of gene expression, a primer design specifically amplified on RNA is needed.One of the solutions to avoid contamination from genomic DNA is DNase treatment (Dotti & Bonin, 2011).However, the application of DNase treatment requires greater costs, so that can be done to avoid gDNA is design primer specific.Furthermore, a primer design is needed to obtain a specific primer design.The purpose of this study was to design a specific primer and optimize the annealing temperature for GSTL2 gene in rice plant.

MATERIALS AND METHODS
This research was conducted in October 2022-January 2023 at the Laboratory of Biotechnology and Genetics, Padang State University.This research was carried out based on molecular biology and computational testing (bioinformatics) and optimization of annealing temperatures through laboratory testing.

Primer Design
Primer design was carried out using Primer3 NCBI.Primers are designed on GSTL2 sequence of Oryza sativa L. (DQ323738) with an amplicon target of 150-250 bp.The primer candidate that was obtained analyzed using Geneious Prime software to fulfill the ideal primer criteria including; primer length and difference, GC content, melting temperature (Tm), and the presence of secondary structure check like dimers and hairpins (Rodríguez et al., 2019).

Primer Specificity Analysis
The selected primer pairs were analyzed in silico to test the specificity of the primers and the similarity of PCR products produced with the database at GenBank NCBI, using Primer BLAST tools on the NCBI sites (Pranata & Ahda, 2022).Primer specificity was tested by alignment the primer sequences using PrimerBLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi(Syamsurizal et al., 2021).Primer specificity was tested with primer pair specificity cheking parameters; specificity check was activated, search mode automatic, database nr, no exclusion, and organism was blank.Primer with the best criteria and specificity will be selected and sent to PT. Genetics Science Indonesia for primer ordering.

RNA Extraction
RNA extraction was carried out by follow the official protocol from GENEzol TM .The 3 days old rice roots were taken and weighed as much as 40 mg, then, a liquid nitrogen added and crushed using a micropestle until smooth.The crushed sample then used in RNA isolation followed the official protocol from GENEzolTM with a few modifications.The isolated RNA then checked for the concentration and purity using the NanoDrop spectrophotometer.The results of RNA isolation are stored at -80 C or can be continued to the cDNA synthesis stage.

cDNA Synthesis
The RNA samples will be synthesized using the SensiFAST TM cDNA Synthesis Kit. cDNA synthesis was carried out by preparing a mixture of cDNA synthesis reactions, then homogenized with a vortex.Samples were run for 30 minutes on a thermal cycler, with annealing settings of 25 C for 10 minutes, the reverse transcription step at 42 C for 15 minutes, and inactivation at 85 C for 5 minutes, then the cDNA samples could be stored temporarily at 4 C.The volume of extracted RNA added to a mixture of cDNA synthesis reaction was calculated using the following formula, RNA volume (µl) = .
Optimization Annealing Temperature Optimization of the primer annealing temperature was carried out by gradient PCR.The cDNA then amplified using GoTaq® (PROMEGA, cat.M7128) by PCR machine.It was set with a pre-denaturation temperature at 95 C for 3 minutes, followed by 35 cycles with a denaturation 95 C for 30 seconds, annealing was carried out by gradient PCR with temperature variation start with temperature around 5 C under Tm both primers, elongation at 72 C for 30 seconds.And the last cycle at 72 C for 5 minutes.The amplification results from the PCR gradient were electrophoresis with 1.5% agarose gel.The electrophoresis results were then observed with UV-TEC.

Primer Design
Primer design performed on GSTL2 sequence of Oryza sativa L. (DQ323738) resulted in six primer candidates (Table 1).Based on the results of the primer analysis based on ideal primer criteria, primer pair number three was selected.Each primer has 20 nt, with temperature melting (Tm) 59-59.4C, with %GC 50%.In the primer forward there is a secondary structure in the form of self-dimer with Tm 6.2 C (Table 2).The results of aligning the primer pair sequences using Primer-BLAST showed that primer pair number three successfully amplified the GSTL2/GSTZ5 gene specifically in rice (Oryza sativa L.) in both Japonica and Indica varieties in the NCBI database (Table 2).

Optimization of Annealing Temperature (Ta)
The results of annealing temperature optimization with PCR gradient visualized using agarose obtained bright and thick DNA bands for each given temperature variation.The band formed corresponds to the length of the target gene PCR product which is 224 bp (Figure 1).Based on the results of the optimization, the Ta used is the highest temperature which is 60 C.

Design Primer
Primer designs are performed on gene sequences of GSTL2 Oryza sativa L. with accession no.DQ323738 using the Primer3 on NCBI produced 6 primer candidates (Table 1).Based on the 300 bp 100 bp a b c d e f g analysis using Genious Prime, primer pair number 3 primer forward GST-f (5'-TTCGAAGGGCCAGCATTACT-3' and reverse GST-r 5'-GCAATGTCCACCAAGCTGAA-3').
The information regarding the characteristics of the design primer results selected from Geneious Prime can be seen in (Table 2).
A good primer must fulfill the ideal primer characteristics such as; primer length, %GC, melting temperature (Tm) and Tm difference between primer pair, the presence of dimers and hairpins (Rodríguez et al., 2019).Primer length should be between 18-24 mer, because shorter primers have a higher efficiency of attachment to their targets than longer primers (Hung & Weng, 2016).
Percentage of GC base is a percentage value that indicates the ratio of G and C base nucleotides.The suitable percentage of GC base is between 40-60%.It is important to know The percentage of GC base in the primer because it will affect the primer annealing process indirectly (Chuang et al., 2013), according to Bustin and Huggett (2017) the % GC should be as close to 50% as possible.Due to the high percentage of GC can make primers "sticky" so as to increase the possibility of mispriming on unwanted parts (Hung & Weng, 2016).Besides that, the oligonucleotides with high %GC will complicate the denaturation process during PCR which can lead to decreased amplification efficiency (Lim et al., 2011), especially repeating Guanine (G) bases should be avoided (Bustin & Huggett, 2017).
The amount of base content of GC and TA can affect the value melting temperature (Tm).Primers that are rich in GC content had higher Tm values than those rich in TA (Rodríguez-Lázaro et al., 2013).According to Thornton and Basu (2011) to optimize the primer work, preferably the selected primer has a Tm in the range of 59-68 C with optimal Tm 63-64 C.In addition, the difference in Tm between the forward and reverse primers should not be more than 1-2 C.Based on the results of the primer candidates obtained that fulfilled the Tm requirements, only primer pair number 5 had a Tm difference of 3 C.
Based on these criteria, primer pair number 3 was chosen.Primer pair no. 3 each has a base length of 20 nucleotides with %GC on 50% and Tm ~59 C.On forward primer found secondary structure form self dimers at a temperature of 6.2, while the reverse primer free from secondary structure hairpin nor self dimers.Although there are self dimers in the forward primer this is acceptable because the dimer formed has a relatively low Tm (Putri et al., 2021) and is far from the Tm of the two primer (Thornton & Basu, 2015) which is equal to 6.2 C.This shows that self dimers formed can separate at this temperature.
This primer pair produces an amplicon of 224 bp.The size of the PCR amplicon taken has a range of 150-250.According to Wang et al. (2012) , this range was chosen because the relatively short size of the amplicon is very important for the efficiency of PCR amplification, especially if the quality of the RNA obtained is low.In addition, if the amplicon size is below 100 bp, it will be difficult to distinguish specific target PCR products from primer dimers during gel electrophoresis.Where all the primer candidates that were successfully designed produced amplicon according to the standard, namely in the range of 150-250 bp.
The results of aligning the primer pair sequences using Primer-BLAST can be seen in (Table 2).Primer-BLAST is a tool that can be used to check primer specificity with pre-existing primers (Ye et al., 2012).Based on the results of checking through Primer-BLAST, the selected primer succeeded in amplifying the gene GSTL2/GSTZ5 specifically for rice (Oryza sativa L.) for both Japonica and Indica varieties in the NCBI database.In addition, it can be seen that the primer pairs are designed not to amplify genomic DNA, so as to prevent genomic DNA amplification from occurring which can affect the resulting PCR product.In addition to B-like IN2-homologous Protein is a synonym of gene GSTL2/GSTZ5 on the NCBI database with accession number NM_001417548 (https://www.ncbi.nlm.nih.gov/nuccore/NM_001417548).It can also be known where this gene can be found on chromosome 3 where the gene GSTL2 to be (Hu et al., 2011).

Optimization of Annealing Temperature (Ta)
Optimization of annealing temperature is carried out to obtain optimal annealing temperature for the primer.Optimization of annealing temperature can be done with gradient PCR by applying several different annealing temperatures.According to Rodríguez et al. (2019) optimizing the annealing temperature by testing several annealing temperatures, it is recommended to start with a temperature of around 5 C under Tm both primers to obtain optimal experimental annealing conditions.The gradient PCR result are visualized in the Figure 1.
Based on the results of the PCR gradient visualized using 1.5% agarose, it shows a single DNA band that is thick and bright for each variation of annealing temperature tested on the primer.This single band indicates its absence as an unspecific band which indicates that the selected primer does not contain genomic DNA contamination, the primer specifically produces a PCR product according to the target with a length of 224 bp.
Although it can be seen at any temperature it produces thick, bright DNA bands.However, the annealing temperature chosen is the highest temperature, which is 60 C.This is because using a higher annealing temperature will reduce the risk of forming DNA bands from non-specific amplicons.According to Thornton and Basu (2015) the annealing temperature (Ta) is usually not far from the primer Tm, normally it is between 55-60 C.However, to increase the specificity of the primer it is recommended to use high temperatures (Erjavec, 2019;Thornton & Basu, 2015).In Patel et al. (2015) the use of a higher annealing temperature can increase the bonding of a greater specific primer.

CONCLUSION
The results of this study obtained a pair of primers designed using Primer3 (NCBI) and analysis with Geneious Prime.The designed primer has succeeded in amplifying GSTL2 gene of Oryza sativa L. specifically by producing a PCR product of 224 bp.The primer has an annealing temperature of 60 C resulting a single thick, bright DNA strand.Therefore, this primer pair can be used in future studies such as to determine the expression GSTL2 gene in rice plant.

Table 2 .
Results of Primer-BLAST using NCBI database