Essay: Amplification and study of HMGR gene expression in Kermanian black zira

Biosynthesis of secondary metabolites in medicinal plants is involved by many genes. HMGR is a key gene in the pathway of Mevalonic acid. This gene causes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A to the Mevalonic acid that is the essential component of this route. At the beginning, a review of similar sequences was prepared in other plants including Apiaceae family. Simulated sequences and conserved regions of the gene were examined. Appropriate primers were designed based on conserved regions. PCR was performed by designed primers to optimize the proper temperature, the desired gene fragment was amplified. After observing its electrophoretic pattern and ensuring the quality of PCR product, the amplified fragment was sent for sequencing. After obtained the results of sequencing, fragment sequence examined with BLAST and SeqMan softwares. Results show the high similarity and identity of HMGR gene sequence, obtained from Kermanian black zira (B. persicum), with GenBank sequences. This partial gene fragment was submitted by accession number KJ143741 in GenBank. The results of relative expression analysis of this gene in different growth stages of B. persicum showed the significant different between expression in germination stage and multi-leaf stage, also between germination and flowering stage (p<0.05) but there was not significant different between expression in flowering and multi-leaf stages. Expression analysis by Real Time quantitative PCR indicates that HMGR is differentially expressed among tissues, with a strong expression in the Multi-leaf phase and low expression level in the germination phase. This result show that Expression of HMGR increase from germination to adult plant and then the expression become stable until flowering.
Key words: Kermanian black zira, Bunium persicum, secondary metabolites, HMGR
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Kermanian black zira or mountainous black zira (Bunium persicum) is one of the economical and important medicinal plants in Iran and world (Sofi et al., 2009; Jalilzadeh-Amin et al., 2011). It is non-crop and domestic plant. This plant belongs to the family Apiaceae (Umbeliferae) and naturally in temperate and arid regions of Iran and some other countries can be found (Jalilzadeh-Amin et al., 2011). The terpene compounds are the major active ingredients contained in the B. persicum. It has the largest amount of ??-Terpinene (Oroojalian et al., 2010) that is a monoterpene. In another study, the antioxidant effect of B. persicum and thyme examined and these properties were attributed to compounds in the essential oils of these plants that had both of terpene compounds, and especially the plant, had ??-Terpinene (Shahsavari et al., 2008). Isoprenoides are a diverse group of organic compounds that naturally arise when more than 30,000 of them have been identified (Sacchettini and Poulter, 1997). Isoprenoides biosynthesis of prokaryotic and eukaryotic cells is essential for life, as these compounds are involved in a range of processes (Boucher and Doolittle, 2000). Isoprenoides generally are made through one of two routes: the mevalonate pathway is used by the cells of eukaryotes and some prokaryotes, or alternate path 2-C-methyl-D-erythritol 4-phosphate (MEP), which is used by most prokaryotes (Campos et al., 2001; Goldstein and Brown, 1990; Hecht et al., 2001; McAteer et al., 2001). 3-Hydroxy-3-methylgutary coenzyme A reductase (HMGR, EC catalyzes the NADPH-dependent reduction of HMG-CoA to mevalonate, the first committed step in the isoprenoid pathway, which produces the largest group of contemporary natural products (Wu et al., 2012). Terpenoids are derived from the repeated condensation of isoprenoids, among which mevalonic acid function as a precursor. Within plant cells, the concentration of MVA is strictly controlled by the HMGR activity. HMGR is one of the most highly regulated enzymes that have been identified (Goldstein and Brown, 1990).
Enzyme HMGR, converts the 3-hydroxy-3-methylglutaryl coenzyme A to Mevalonic acid and significantly an important key regulator of isoprene metabolism control in mammals and fungi. The limited nature of this enzyme in plants is discussed in the biosynthetic isoprenoides (Chappell et al., 1995).
The mevalonate pathway, which starts with the synthesis of mevalonate by HMG-CoA reductase (HMGR), provides precursors for the diverse spectrum of isoprenoid compounds produced by a cell. Evidence is accumulating that implicates HMGR as an important control point for the mevalonate pathway in plants (Stermer et al., 1994). Because HMGR is a key enzyme in a pathway leading to compounds with diverse and important functions in plants, it is not surprising that HMGR activity in plants is controlled by a variety of developmental and environmental signals. Higher levels of HMGR activity are usually associated with the rapidly growing parts of the plant, such as apical buds and roots, with much reduced activity found in mature tissues (Brooker and Russell, 1975; Bach et al., 1980). For example, the relative activity of microsomal HMGR from the mature leaves of pea seedlings was only 7% of that observed in the apical buds (Brooker and Russell, 1975).
HMGR activity is controlled not only at the mRNA level but also post translationally. Some studies have suggested that HMGR activity in plants is regulated by reversible phosphorylation, as it is in animals. The plant enzyme is inactivated by incubation with supernatant fractions in the presence of MgATP and can be activated by phosphatase (Russel et al., 1985; Sipat, 1982).
Materials and Methods
The seeds were surface-sterilized and synchronously germinated according to the protocol developed by Sharifi and Pouresmael (2006). Seeds were washed with 0.2% (v/v) Sodium Hypochlorite for 20 minutes followed by 70% (v/v) ethanol for 1 minute, then washed for three consecutive times and each time 4 minutes, with sterile water. After the third wash, the seeds were treated with hormons for 24 hours and then washed with sterile water. The Seeds were transfer into sterile Petri dish and incubated in the dark at 4 ??C for an additional 3 weeks. After this time, Seeds start to germination. After 15 day, sample of seedling kept in a -80 ??C for further analysis.
Other samples were taken from Multi-Leaf flowering stage and kept for further tests in -80 ??C. HMGR sequence searched in the GenBank. Sequences from other plants, specially Apiaceae family, were analyzed by MegAlign software and simulated by using Clastal W method. Conserved areas for HMGR gene were identified and appropriate primers were designed by these areas. Length of 220bp was considered to be appropriate to next study. There was no intron in this fragment. Required features of primers were examined by OligoAnalyser software. Primers were designed from conserved point of genes.
For RNA isolation, 50-100 mg of plant tissue was ground and homogenized with lysis buffer of Ribospin plant Cat. NO: 307-150 Kit according to the manufacturer’s instructions (GeneAll, Korea). The RNA was DNase treated using DNase I (Fermentas) to remove remaining genomic DNA. RNA quality was studied using agarose gel 1/5% as well as its quantity was measured by Nanodrop. RNA (1 ??g) was reverse transcribed using RevertAid’H Minus-MuLV reverse transcriptase (Fermentas) primed with 0.5 ??g oligo(dT)18 primer according to the manufacturer’s instructions. The cDNA was performed by RevertAidTM First strand cDNA Synthesis kit from Fermentase. The PCR conditions employed were 94??C for 30 sec, 55-59??C (Gradient) for 45 sec, 72??C for 1 min (35 cycles) for HMGR. The quality of PCR product was studied using agarose gel 1%.
The target gene fragment was amplified by the primer s, and after observing electrophoresis pattern and ensuring the quality of reproduction, was submitted for the purification and sequencing of the PCR product. After the obtained sequence, it was aligned and assembled. The sequencing results after analyzing by SeqMan and BLAST software were discussed.
Real Time PCR was performed using the Bio-Rad iCycler Real-time PCR system (MiniOpticon). Reagents used for Real Time PCR were supplied as part of the EvaGreen Master mix according to the manufacturer’s specifications. The protocol used was a three-step amplification followed by a melt-curve analysis. For each amplification cycle, there was a denaturation step at 95 ??C, an annealing step at 57 ??C, and an extension step at 72 ??C (Table 1). Fourty cycles were used. Relative fold changes in gene expression were calculated based on the 2-‘?CT comparative method (Livak and Schmittgen, 2000; Sehringer et al., 2005; Cikos et al., 2007). In this method, levels of target gene amplification in an experimental sample are compared to levels of target gene amplification in another sample or standard, both of which are first normalized to levels of amplification of a normalizing gene. For this work, the elongation factor 1-alfa (EF1A) gene was used as a normalizing factor.
In order to investigation of HMGR relative expression by the sensitive quantitative Real Time PCR used from EvaGreen?? qPCR Real Time Master Mix, with ROX and other regent as Table 1. The Real Time PCR program used based on Table 2. and instrument was BioRad MiniOpticon Real-Time PCR System. Relative expression levels were analysed using the REST 2009 software V. 2.0.13 (Qiagen, Hilden, Germany) (Pfaffl et al., 2002).
Results and Discussion
Electrophoretic pattern of RNA was observed using 2??l of the solution containing 1.5% agarose gel (Fig. 1). Bands of ribosomal RNA (28s and 18s) were well visible.
The RevertAid First Strand cDNA Synthesis Kit used for synthesis of first strand cDNA from total RNA. Gene-specific primers were synthesized from the core fragments and used to generate the 5′-end and 3′-end DNA fragment. A DNA fragment encoding the HMG-CoA reductase was obtain by PCR from HM-F (5′-GAT GCD ATG GGA ATG AAC ATG GT-3′) and HM-R (5′-GCA CAG TGG TTT TCA AYA CCT TCT TCA C-3’) as primers. PCR program was taken in the temperature range (Gradient) for checking the best temperature of primers. The optimal temperature for HMGR primers was determined (58 ??C). The quality of PCR product was studied using agarose gel 1% (Fig. 2).
By performing PCR using the temperature 58 ??C (Annealing), target gene fragment was amplified. PCR products were separated on 1% agarose gel, stained with CinnaGen DNA safe stain and visualized in Gel documentation system (Uvitec, UK). For purification and sequencing the PCR product (Fig. 3), 20 l?? of PCR product was sent to the Bioneer Company (Korea). The results of sequencing analyzed by using Cromas software and showed that 217 bases were sequenced (Fig. 4). The nucleotide sequence translated to protein sequence by using SeqMan software. Then, both of them (nucleotide and protein sequences) were analyzed by using BLAST software. The sequence show over 76% similarities and 83% identity with Artemisia annua and 76% similarities and 82% identity with Tanacetum parthenium and a few other sequenced fragments of different plants in GenBank. The partial gene fragment was registered first time for Bunium persicum with the number of KJ143741 in GenBank (NCBI).

Gene Expression Analysis
The sensitive quantitative Real Time PCR was performed using total RNA isolated from various tissues of Bunium persicum. Elongtion factor 1-alfa (EF1A) primer used as housekeeping gene. This gene should be highly, stably, and constitutively expressed in all conditions and tissues that are to be analyzed (Deprez et al., 2002; Thellin et al., 1999; Schmittgen et al., 2000; Brunner et al., 2004). The expression of HMGR could be detected in all of the tissues. However, it is highly expressed in the leaves and abundantly expressed in the flowers and roots.
The relative quantification of target gene expression was evaluated using the ‘?CT method. The results of relative HMGR gene expression analysis show the significant different among expression in germination and Multi-leaf stages (Table 3), also between germination and flowering stage (p< 0.05) but there was not significant different between expression in flowering and Multi-leaf stages (Fig. 5). This result show that Expression of HMGR increase from germination to mature plant and then the expression become stable to flowering. Higher levels of HMGR activity are usually associated with the rapidly growing parts of the plant, such as apical buds and roots, with much reduced activity found in mature tissues (Brooker and Russell, 1975; Bach et al., 1980). HMGR exhibited also lower expression in old leaves compared to other tissues in Artemisia annua L. (Olofsson et al, 2011). Over-expression of both hmgr and ads genes in A. annua L. plants results not only increase in artemisinin content, but also enhances synthesis of other isoprenoid including essential oil (Alam et al., 2014). The stability of HMGR expression at stage 2 to 3 could be happen because the activity of MEP pathway in maturity stage of plant increase and produce the common yield with MVA pathway.
HMGR expression is very highly upregulated in plants that shift into a reproductive growth stage (Vail, 2008). However, HMGR is known to be regulated post-transcription (Hey et al., 2006). Phosphorylation of HMGR by kinase causes inactivation. Although levels of HMGR transcripts are remarkably increased, a consequent increase in enzyme activity and carbon flux may not necessarily accompany increased transcript levels (Re et al., 1995).
During the flowering, the plant is in a vital and vulnerable phase; therefore the need for defense compounds is much higher (Vail, 2008). Our results suggest that leaves are crucial to terpenoid biosynthesis in Bunium persicum.
The plastidic pathway was predicted to play an important role in the shift from vegetative to reproductive growth. Isoprenoids produced in the plastid are important for floral pigmentation (carotenoids) and fragrances (monoterpenes) (Vail, 2008). Further, Towler and Weathers (2007) showed that inhibition of the MEP pathway significantly reduced artemisinin production.
However, results showed that the mRNA levels of key isoprenoid biosynthetic genes in the plastid are unchanged in flower budding plants compared to vegetative plants, and they are also downregulated during full flowering. Photosynthesis in this upper portion of the shoot may likely assume a lesser role as the plant shifts to a reproductive phase. It is also possible that cytosolic IPP may provide a crosstalk source of isoprene (IPP) biosynthesis in the plastid, since the cytosolic pool of IPP is likely very large due to the large increases in HMGR transcripts.
MEP pathway genes are known to be regulated post-transcriptionally (Sauret-Gueto et al., 2006); therefore it is possible that, although levels of transcripts are unchanged as the plant shifts to a reproductive, flowering stage, post-translational regulation is occurring.

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Table 1. Real Time PCR program Table 2. Master Mix and Regent
Volume (??l) Regent (sec)Time (C??)Temp Cycle
2 5X- EvaGreen?? master Mix 60??15 95 1
6.4 DEPC-treated water 20 95 40
0.3 Forward Primer (10 pm) 60 57
0.3 Reverse Primer(10 pm) 30 72
1 Target cDNA 60??5 95-60 1
Figure 1 – Agarose gel electrophoresis 1/5% of leaf RNA of B. persicum with. M- Fermentas marker 1kb, 1 – Leaf RNA.

Figures 2 – Agarose gel electrophoresis 1% of Gradient PCR product of HMGR gene for obtain the better temperature, M-marker 1kb Fermentas. 1-5= 55-59??C and 6=Negatice Control.
Figure 3 – Agarose gel electrophoresis 1% of PCR product of HMGR gene for amplification and sequencing M-marker 1kb Fermentas. 1,2 – PCR product in 58??C.
Figure 4 – sequencing a partial fragment of the HMGR gene in B. persicum.

Table 3. Relative expression of HMGR in B. Persicum at different growth stages
Germination/Flowering Flowering/Multi.leaf Germination/Multi.leaf Stages (F)
Stage 3 Stage 2 Stage 1
*38/1 45/0ns *03/3 HMGR
*- significant in 5% , ns- not significant , F- fold change (gene expression)
Figure 5- Relative expression (Folding) of HMGR gene in three Stages of B. persicum growth: Stage1- Multi-Leaf/Germination, Stage2- Flowering/Multi-Leaf and Stage3- Flowering/Germination

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