Characterization and expression of myo-inositol- 3- phosphate synthase (MIPS) gene in Glycine max
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Date
2013
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IARI,Division of Biochemistry
Abstract
D-myo-inositol-3-phosphate synthase (EC 5.5.1.4; MIPS) is the only isomerase that
catalyzes the conversion of glucose-6-phosphate to D-myo-inositol-3-phosphate, a sole
synthetic source of myo-inositol. Phytic acid (myo-inositol-1,2,3,4,5,6-
hexakisphosphate) which is the principal storage form of phosphorus (60-80%) in plant
seeds, is further generated by a stepwise phosphorylation of myo-inositol. Poorly
digested by monogasterics as it chelates essential mineral cations and proteins thereby
reducing their bioavailability, classifying it as an anti-nutrient. In soybean, transcripts
encoding MIPS1 are expressed early during the cotyledonary stage of seed development
to function in phytic acid biosynthesis. In the present study, we report the cloning and
characterization of the MIPS1 gene from developing seeds of soybean (GmMIPS1). A
full-length GmMIPS1 cDNA (Glycine max L. Merr.) of 1,791 bp, containing an ORF of
1,533 bp, encoding 510 amino acids was cloned and characterized. Nucleotide and
deduced amino acid sequences of GmMIPS1 showed striking homology (80-99%) with
other plant MIPS particularly with the dicots, Vigna radiata and Phaseolus vulgare. The
protein sequence analysis of the predicted GmMIPS cDNA indicated the absence of
signal peptide in the N-terminal region. To validate the expression of the GmMIPS1
coding gene, nucleotide sequence residues from 131 to 1,556 bp were amplified by
high fidelity PCR and fused in frame to a 19 amino acid N-terminal region of 6X Histag
in expression vector pET-28a (+). The E. coli strain BL21 (DE3) transformed with
the recombinant plasmid resulted in the production of a 52 kDa fusion protein under
optimized induction and expression conditions as confirmed by SDS-PAGE and
Western blot analysis. Results of the present study suggested that down-regulation of
GmMIPS1 using a seed specific promoter can be targeted as a great potential for
development of low-phytate soybean without affecting the critical aspects of inositol
metabolism in other tissues of the plant.
Keywords: anti-nutrient, MIPS1, phytic acid, prokaryotic expression vector, soybean.
Abbreviations: DAB-3,3´-diaminobenzidine tetrahydrochloride, IPTG- Isopropyl β-Dthiogalactopyranoside,
MIPS- D-myo-inositol-3-phosphate synthase, ORF- open reading
frame, PVDF- Polyvinylidene difluoride , SDS- PAGE- Sodium Dodecyl Sulphate-
Polyacrylamide gel Electrophoresis, UTR- untranslated region.
Introduction
Soybean (Glycine max (L.) Merr.), one of the world's most important economic crops,
has a steadily increasing agronomical value because of its high protein and vegetable oil
content suitable for human and animal nutrition. While soybean is an important source
of protein, its potential to provide energy and minerals has not fully reached in nonruminants
animals including humans due to their inability to digest certain compounds
such as phytates (Sebastian et al., 2000). Phytate (myo-inositol 1,2,3,4,5,6-
hexakisphosphate), also known as phytic acid (PA) or phytin, is the major form of
phosphorus (P) storage in seeds, comprising over 75–80% of the total P in plant seeds
(Cosgrove 1966; Raboy et al., 2001). In soybean seeds, phytic acid accounts for up to
2% of the seed dry weight (Raboy et al. 1984). It begins to accumulate in seeds after the
cellular phosphate levels have reached maximum levels and continues to increase
linearly throughout seed development and seed filling (Raboy and Dickenson, 1987). It
is usually deposited in protein bodies as a mixed salt (phytin), bound to mineral cations
such as Fe3+, Ca2+, Mg2+, Zn2+ and K+ (Prattley and Stanley 1982, Lott 1984).
Additionally, phytate in seed when broken down by the enzyme phytase, it becomes
inorganic phosphate and myo-inositol, which are then available for seedling growth.
Although an important storage molecule for growing seedlings, PA poses severe
nutritional consequences as it acts as an antinutrient by forming indigestible complexes
with minerals and proteins, decreasing the seeds nutritional quality. It chelates mineral
cations, including calcium, zinc, magnesium and iron from the diet and affects the
bioavailability of these essential minerals (Raboy et al., 2001). It also has the potential
to bind charged amino acid residues of proteins resulting in a concomitant reduction of
protein availability and digestibility. Also the excretion of unused P in the waste makes
its way into the waterways causing environmental hazards. This antinutritional quality
of phytate can be further extended to human health as it contributes to the iron
deficiency suffered by over 2 billion people worldwide (Bouis 2000). The economic,
nutritional, and environmental problems associated with phytate in animal or human
feed can be reduced by developing low phytate soybean (Raboy 2007). The
development of low phytic acid (lpa) crops is an important goal in genetic engineering
programs aimed at improving the nutritional quality as well as at developing
environment friendly and sustainable production. One approach for reduction of plant
seed phytate levels involves the reduction of the expression of enzymes in the
biosynthetic pathway of phytic acid. D-myo-inositol 3-phosphate synthase (MIPS, E.C.
5.5.1.4) catalyzes the NADH-dependent conversion of D-glucose 6-phosphate (G-6-P)
to D-myo-inositol 3-phosphate (MIP) the first and the rate-limiting step of myo-inositol
biosynthesis (Biswas et al., 1984; Loewus and Murthy, 2000). Further a stepwise
phosphorylation (Fig. 4) of myo-inositol generates phytic acid. MIPS has been isolated
and characterized from both prokaryotic and eukaryotic organisms. The structural gene
coding for the MIPS was first identified in yeast (Donahue & Henry 1981; Majumder et
al. 1981). Subsequently, MIPS coding sequences have been cloned and characterized
from widely different organisms, including plants such as Spirodela polyrrhiza (Smart
& Fleming 1993), Citrus paradisi (Abu-abied and Holland 1994), Arabidopsis thaliana
(Johnson 1994; Johnson and Sussex 1995), Mesembryanthemum crystallinum (Ishitani
et al. 1996), wild halophytic rice P. coarctata (Majee et al., 2004), Xerophyta viscosa
(Majee et al. 2005), Passiflora edulis (Abreu & Aragao 2007), Cicer arietinum (Kaur et
al., 2008), etc. Several plants have been found to possess multiple isoforms of MIPS
enzyme, suggesting that each gene copy may be differentially controlled and expressed.
Soybean contains four MIPS isoforms and one of the MIPS cDNAs (GmMIPS1) was
shown to express mainly in developing seeds (Hegeman et al., 2001; Chappell et al.,
2006). Using immunolocalization techniques, a specialized area of GmMIPS-1
expression has been identified in the outer integumentary layer during early soybean
seed development (Chiera and Grabau, 2007). A number of genes homologous to
GmMIPS1 have been reported till date and a “core catalytic structure” conserved across
evolutionary divergent taxa has been identified (Majumdar et al., 2003). Down
regulation of MIPS gene expression in seeds offer a potential approach for developing
low-phytate soybean (Hitz and Sebastian, 1998).
In the present study, we report the isolation, cloning and characterization of full length
GmMIPS cDNAs from developing seeds of soybean and validation of its expression in
Escherichia coli, especially with respect to its involvement in phytic acid biosynthesis.
The fully functional GmMIPS1 gene can further be targeted for genetic manipulation by
advanced gene silencing strategies to develop low phytate soybean seeds with improved
nutritional value.
Materials and methods
Bacterial strains and plant materials
Escherichia coli BL21 (DE3) and DH-5α strains were cultured on LB medium at 37oC.
Cells containing recombinant plasmids, pGEMT-Easy and pET-28a(+) were
supplemented with 100 mg ml-1 ampicillin and 50 mg ml-1 kanamycin. Soybean seeds
(Glycine max) were collected from the Division of Genetics, Indian Agricultural
Research Institute, New Delhi, India. Mercuric chloride (0.02%, 5 min.) sterilized seeds
were sown in pots maintained under controlled environmental conditions at the National
Phytotron Facility, I.A.R.I., New Delhi. The developing seeds (4 to 6 mm) were
harvested and rapidly frozen in liquid nitrogen at -80oC.
RNA isolation and RT-PCR amplification
Total RNA was isolated from developing cotyledons (4 to 6mm seeds) of soybean
samples (100 mg) using the RNeasy Plant Mini Kit (Qiagen) according to
manufacturer’s instructions. Frozen plant tissues were homogenized using pestle and
mortar with liquid nitrogen and one ml of Qiagen lysis buffer added per 100 mg of
tissue in 2 ml micocentrifuge tubes. First strand of cDNA was synthesized from RNA
by using oligo(dT) primer and reverse transcriptase from RevertAidTM H Minus first
strand cDNA synthesis kit (Fermentas, Life Sciences). The full length cDNA for MIPS1
was amplified using oligonucleotide primers designed by BioEdit software based on the
published soybean MIPS sequences (GenBank Accession Number AF293970) available
in NCBI GenBank (forward primer: 5’-ATAGGATTCTCTTC TTTATTCCT-3´;
reverse primer: 5´-TACACAAAATTATACTACATTCAT-3´). The PCR thermal
cycling parameters used were 94○C denaturation for 4 min followed by 35 cycles
Description
t-8793
Keywords
anti-nutrient, MIPS1, phytic acid, prokaryotic expression vector, soybean. Abbreviations: DAB-3,3´-diaminobenzidine tetrahydrochloride, IPTG- Isopropyl β-Dthiogalactopyranoside, MIPS- D-myo-inositol-3-phosphate synthase, ORF- open reading frame, PVDF- Polyvinylidene difluoride , SDS- PAGE- Sodium Dodecyl Sulphate- Polyacrylamide gel Electrophoresis, UTR- untranslated region.