Data on PCR primer design for glucose 6-phosphate dehydrogenase gene and the effects of dietary carbohydrate levels on its expression in the liver of Malaysian mahseer (Tor tambroides)

The enzyme glucose-6-phosphate dehydrogenase (G6PD) catalyses the metabolite glucose-6-phosphate in producing NADPH during the first phase of pentose-phosphate pathway thus provides reducing power to all cells for cellular growth, antioxidant defence, and biosynthetic reactions in all living organism. The deliberate inclusion of starch as carbohydrate source in commercial feed however may affect the G6PD hepatic activity in cultured fish. We designed a set of primers to target G6PD gene in the popular Malaysian aquaculture species, Tor tambroides. For this dataset, the molecular characteristics of obtained T. tambroides G6PD (TtG6PD) nucleotide sequence was analysed using multiple alignments and phylogenetic analyses of the deduced amino acids. The set of primers obtained were then used in a study to evaluate the effect of different dietary carbohydrate inclusion levels on the hepatic TtG6PD mRNA expression of the T. tambroides fingerlings. Four groups of fish were given a dietary treatment of 15%, 20%, 25% and 30% starch at the optimal inclusion level of 23.4% for 10 weeks. The TtG6PD mRNA transcripts were measured using real-time-PCR assays and its expression normalized against β-actin, which acts as the internal control gene. This article provides supportive data in relation between hepatic TtG6PD mRNA gene expression in T. tambroides and how it is influenced by its dietary carbohydrate intake. These data will also assist in further nutritional genomic studies of carbohydrate and energy utilization for all species in the mahseer family.


a b s t r a c t
The enzyme glucose-6-phosphate dehydrogenase (G6PD) catalyses the metabolite glucose-6-phosphate in producing NADPH during the first phase of pentose-phosphate pathway thus provides reducing power to all cells for cellular growth, antioxidant defence, and biosynthetic reactions in all living organism. The deliberate inclusion of starch as carbohydrate source in commercial feed however may affect the G6PD hepatic activity in cultured fish. We designed a set of primers to target G6PD gene in the popular Malaysian aquaculture species, Tor tambroides. For this dataset, the molecular characteristics of obtained T. tambroides G6PD ( TtG6PD ) nucleotide sequence was analysed using multiple alignments and phylogenetic analyses of the deduced amino acids. The set of primers obtained were then used in a study to evaluate the effect of different dietary carbohydrate inclusion lev-els on the hepatic TtG6PD mRNA expression of the T. tambroides fingerlings. Four groups of fish were given a dietary treatment of 15%, 20%, 25% and 30% starch at the optimal inclusion level of 23.4% for 10 weeks. The TtG6PD mRNA transcripts were measured using real-time-PCR assays and its expression normalized against β-actin , which acts as the internal control gene. This article provides supportive data in relation between hepatic TtG6PD mRNA gene expression in T. tambroides and how it is influenced by its dietary carbohydrate intake. These data will also assist in further nutritional genomic studies of carbohydrate and energy utilization for all species in the mahseer family.

Value of the data
• These data provide information on the relationship between hepatic TtG6PD mRNA gene expression and the influence of dietary carbohydrate intake on Malaysian mahseer ( Tor tambroides). • The data are valuable for the researchers interested in the carbohydrate metabolism of mahseer species, particularly on cells adaptation to dietary energy from carbohydrate consumption.
• It facilitates the scientists and researchers to further study the nutritional genomics and carbohydrate metabolism of Mahseer and other freshwater species.
Subsequently, the molecular characterization of TtG6PD was examined. The size of TtG6PD partial open reading frame identified was 225 bp and 74 amino acids ( Fig. 1 ). The predicted molecular weight of TtG6PD is 8.31 kDa and its isoelectric point pI is 6.08. Multiple sequence alignment (MSA) analysis of TtG6PD and other species revealed that the highest identity as Formosan land-locked salmon ( Oncorhynchus masou formosanus ) at 96%, and the lowest identity as bees ( Melipona quadrifasciata ) at 78% ( Fig. 2 ). Phylogenetic tree analysis of the G6PD gene di-  vided clusters of fishes, insects, mammals, and birds, and revealed that TtG6PD is closely related to G6PD from O. masou formosanus ( Fig. 3 ). The effects of different levels of dietary carbohydrate on hepatic TtG6PD mRNA expression were examined using real time PCR assays with TtBact acting as the internal control gene to normalize inefficiencies. Different levels of dietary carbohydrate inclusion influenced the hepatic TtG6PD mRNA transcripts ( Fig. 4 ) . The expression in the liver of fish group fed 20% carbohydrate was found to be the highest ( P < 0.05) between treatments. The groups fed 15%, 25% and 30% carbohydrate indicated low expressions but did not differ significantly among the three treatments ( P > 0.05).

Feeding trial and sampling
Liver samples for real-time PCR assays were collected from a 10 weeks feeding trial commenced at the Wet Laboratory, Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia. Wild T. tambroides fingerlings from Pahang were acclimatized for 3 weeks and then 240 fish were equally distributed at random into twelve 60 L glass tanks (38 cm × 75 cm × 35 cm), pre-prepared with de-chlorinated municipal water equipped with continuous aeration. Water quality was maintained as the following: pH (7.0 ±0.5), dissolved oxygen (5.0 ±0.5 mg l −1 ) and water temperature (26.0 ±1.0 °C). Fish were fed formulated diets of different starch level 15%, 20%, 25% and 30% starch based on published formulation [3] . Feeding frequency were twice daily at 4% body weight. At the end of the feeding trial, 10 fish were sacrificed for each treatment and their livers were extracted. Livers were immediately stored upon extraction in RNA later TM Stabilization Solution (Thermo Fisher Scientific, USA) and kept at −80 °C until further analysis.

Isolation and quantification of total RNA
Samples were thawed and RNAlater TM solution removed from samples before extraction. Extraction of total RNA from liver tissue samples was done using easy-Blue® total RNA extraction kit (Intron Biotechnology, Inc., Korea) according to the manufacturer's specifications and quantified using GeneQuant TM pro RNA/DNA Calculator (Amersham Pharmacia Biotech, UK). The integrity of the RNA extracted was tested by electrophoresis in 1% of agarose gel added with non-toxic EcoDye TM DNA staining dye in 1X TAE running buffer.
PCR reactions were performed in 25 μl volumes using Verso 1-Step RT-PCR ReddyMix Kit (Applied Biosystems, USA) on a PTC-200 Peltier thermal cycler (MJ Research Inc, Canada). Each reaction mixture consisted of 100ng μl −1 RNA template, 2 × 1-Step PCR ReddyMix, 0.4 μM of forward and reverse primers, RT enhancer and 1 U of Verso Enzyme mix. The thermal profile was as follows: initial cDNA synthesis step at 50 °C for 1 min to transcribe RNA template into single strand cDNA; a step of Verso enzyme activation at 95 °C for 2 min to stop reverse-transcription activity; followed by 35 cycles of [95 °C for 20 s, 54 °C for 30 s, 72 °C for 1 min] and a final extension at 72 °C for 5 min. PCR products were resolved by gel electrophoresis before sent for Sanger sequencing on Applied Biosystems 3730 DNA Analyzer (NHK Bioscience Solutions Sdn. Bhd., Malaysia). The sequencing results of the PCR products obtained were ran through BLASTn (NCBI) to further verify the specificity of the primers.

Molecular characterization and sequence analysis
The TtG6PD amino acid sequence was predicted using BLASTX (Basic Local Alignment Search Tool) program [4] from NCBI (National Centre for Biotechnology Information) database. The Prot-Param tool of ExPASy Proteomics Server [5] was used to calculate the theoretical isoelectric point (pI) values and the molecular weight of the protein. To compare the similarity of TtG6PD amino acid sequence with the G6PD amino acid sequence of other species, the multiple sequences alignment (MSA) was analysed using Clustal Omega program [6] . Molecular Evolutionary Genetics Analysis (MEGA) version X was performed to construct the phylogenetic tree using the neighbour-joining (NJ) method with 2,0 0 0 replicates of bootstrap sampling [7] .

Real-time PCR assays
Specificity of amplifications for each primer pairs were initially assessed using melt curve analysis where single peak melt curves indicated the amplification of single gene products. Melt curve analysis was performed with continuous fluorescence acquisitions from 60 to 95 °C at a temperature transition rate of 0.05 °C s −1 and standard curves were generated from the calculated threshold cycle (C T ) C T value the transcripts. Real-time PCR assays were performed in triplicates on 7500 Real-Time PCR Systems (Applied Biosystems, USA). Total PCR mixture volume of 25 μl per reaction contained 100 ng of DNAse treated total RNA, 125 × RT Enzyme mix, 0.2 μM of primers, 2 × RT-PCR mix using Power SYBR® Green RNA-to-C T TM 1-Step kit (Applied Biosystems, USA). The thermal profile was as follows: Holding steps at 48 °C for 30 min to synthesize the first strand cDNA, 95 °C for 10 min for enzyme activation; followed by Cycling steps: denature step at 95 °C for 15 s and anneal/extend step at 60 °C for 1 min for 50 cycles. Data generated were analysed with Gene Expression Macro TM v1.1 (Bio-Rad, USA) using the Comparative C T Method ( CT Method). Average C T values and standard deviations are used in the CT calculations to determine the relative mRNa expression of TtG6PD against TtBact , which acts as the internal control gene to normalize inefficiencies during analyses.

Statistical analysis
Data values were reported as mean ±standard error from three real-time PCR assays ( n = 3) and were subjected to one-way analysis of variance (one-way ANOVA). Differences between means were tested using Duncan's new Multiple Range Test at P < 0.05.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.