Download Biosynthetic Pathway

Characterization and Modification of a Biosynthetic
Pathway in Glycine max
Project Goals
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Identify and characterize genes in the phytate
biosynthetic pathway in soybean.
Clone, purify, and characterize each soybean
kinase enzyme using a bacterial expression
system.
Generate silencing vectors to allow individual
down-regulation of each kinase gene in
embryogenic soybean culture using RNA
interference (RNAi) technology.
Gene Identification
• 7 full length coding regions were identified and amplified from
soybean mRNA.
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1 myo-inositol kinase gene
4 Ins(1,3,4)P3 5/6-kinase genes
1 Ins(1,4,5)P3 6/3/5-kinase gene
1 Ins(1,3,4,5,6)P5 2-kinase gene
Protein ID
Amino Acids
Expected
Size (kD)
A1
339
38.5
A2
315
35.0
A3
354
40.1
A4
341
38.4
Ins(1,4,5)P4 6/3/5kinase
B
279
30.9
Ins(1,3,4,5,6)P5 2kinase
C
456
51.2
Protein
Ins(1,3,4)P3 5/6kinase
Table 1.
(MIK)
(A1 – A4)
(B)
(C)
Gene Expression of Late Pathway
Kinases in Developing Seeds
Improving the nutritional characteristics of soybeans
Stiles AR, Grabau EA, [email protected]
Department of Plant Pathology, Physiology and Weed Science
Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
seed size (mm)
A1
Phytate or myo-inositol hexakisphosphate is the major storage form of phosphorus present in plant seeds. Bound phytate
phosphorus and chelated mineral cations are largely unavailable in the diets of non-ruminants due to a lack of digestive enzymes to
remove phosphate groups. Phytate phosphorus is excreted in manure, accumulates in soil, and eventually enters watersheds. As a
result, phytate is considered both an antinutrient and an environmental pollutant. Altering seed composition to reduce phytate, improve
nutrient availability, and reduce phosphorus pollution will require an understanding of the phytate biosynthetic pathway. We have
isolated several myo-inositol phosphate kinase genes in soybean as possible candidates for steps in the biosynthetic pathway. We
have characterized the genes for four myo-inositol (1,3,4)P3 5/6-kinases, one myo-inositol(1,4,5)P3 6/3/5-kinase, one myoinositol(1,3,4,5,6)P5 2-kinase and one myo-inositol kinase. We have examined expression in developing seeds and other tissues by
Northern blot analysis and quantitative RT-PCR. We have expressed all seven genes as GST fusion proteins in E. coli, and verified
enzyme activity on the proposed substrates. We are conducting biochemical characterization to determine enzyme kinetics and
substrate specificities. We are utilizing soybean embryogenic cultures to test for alterations in inositol phosphate profiles as a result of
down-regulating the kinase genes by RNA interference.
The Phytate Problem
B
A
A2
A3
A4
B
C
Figure 2.
A) Northern Blots of RNA from developing soybean seeds. Total RNA was extracted from
soybean seeds of 0-4 mm, 5-6 mm, 7-8 mm, and 9-10 mm. The blots were probed with full
length cDNA sequences and detected using chemiluminescence. B) Real-time PCR
results quantifying mRNA expression levels of genes A1-A4, B and C in developing seeds.
Reverse primers were generated from the 3’UTR region of each gene for specificity.
Protein Expression and
Characterization
The phytate molecule
chelates minerals.
Table of the predicted protein masses based on the amino acid coding
region for each protein (GST tag region not included).
Biosynthetic Pathway
Soybeans store phosphorus and
minerals in their seeds in the form of
phytate.
Figure 3.
SDS-PAGE gels showing the purification of GST-fusion proteins (A)
Proteins A1-A4 (B) Protein B. Lane 1 in each gel contains the Mark-12
protein size marker (Invitrogen).
Activity Assays
The excess phosphorus is released into
the environment where it contributes to
phosphorus pollution.
Harmful effects of excess phosphorus runoff into a pond.
Soybeans are one of the main components of
livestock feed. However, non-ruminant animals
cannot break down the phytate molecule. They
are unable to use the phosphorus and minerals
stored in phytate, and farmers must supplement
their feed.
Each protein was incubated with its proposed
substrate and the products analyzed using High
Performance Liquid Chromatography (HPLC).
Phytate passes through the
animal….
Figure 4.
Into the soil and groundwater where it is
broken down by bacteria.
Figure 1. Two predicted pathways for phytic acid biosynthesis. Figure
modified from Raboy (2001).
Studies conducted in various organisms have demonstrated two
potential routes for the phosphorylation of myo-inositol; a
phosphatidylinositol (PtdIns)-dependent and a PtdIns-independent
pathway. In yeast, the PtdIns-dependent pathway includes an
Ins(1,4,5)P3 6/3/5-kinase. In Zea mays an Ins(1,3,4)P3 5/6-kinase has
been identified that is characteristic of an independent pathway [1]. A
mutation in this Zea mays 5/6-kinase gene creates a low phytic acid
(lpa) phenotype. In Arabidopsis, genes encoding both kinases have
been identified [2,3].
Our Goal
To understand how the soybean plant synthesizes phytate. We can then work to develop
a plant with decreased levels of phytate and higher levels of available phosphorus. This
will generate a more nutritious soybean and decrease phosphorus pollution from
livestock farms.
HPLC elution profile of the products of the
Ins(1,4,5)P3 6/3/5-kinase assay. (A) Trace
showing the IP3 and IP4 standards. (B) Trace
showing the products of the B activity assay.
Figure 5.
HPLC elution profiles of the products of the
Ins(1,3,4)P3 5/6-kinase activity assays. (A)
Trace showing the control reaction lacking
enzyme. (B – E) Traces showing the
products of the A1-A4 activity assays.
References
1. Shi, J., Wang H., Wu Y., Hazebroek J., Meeley R., Ertl D. (2003) The Maize
Low-Phytic Acid Mutant lpa2 Is Caused by Mutation in an Inositol Phosphate
Kinase Gene. Plant Physiol. 131:1-9.
2. Wilson, M.P., Majerus, P.W. (1997) Characterization of a cDNA Encoding
Arabidopsis thaliana Inositol 1,3,4-trisphosphate 5/6-Kinase. BBRC 232:
678-681.
3. Stevenson-Paulik J., Odom A.R, York J.B. (2002) Molecular and
Biochemical Characterization of Two Plant Inositol Polyphosphate 6-/3-/5Kinases. J. Biol Chem. 277: 42711-42781.