Development of high-lysine rice via endosperm-specific expression of a foreign LYSINE RICH PROTEIN gene.

BACKGROUND: Lysine (Lys) is considered to be the first limiting essential amino acid in rice. Although there have been extensive efforts to improve the Lys content of rice through traditional breeding and genetic engineering, no satisfactory products have been achieved to date. RESULTS: We expressed a LYSINE-RICH PROTEIN gene (LRP) from Psophocarpus tetragonolobus (L.) DC using an endosperm-specific GLUTELIN1 promoter (GT1) in Peiai64S (PA64S), an elite photoperiod-thermo sensitive male sterility (PTSMS) line. The expression of the foreign LRP protein was confirmed by Western blot analysis. The Lys level in the transgenic rice seeds increased more than 30 %, the total amount of other amino acids also increased compared to wild-type. Persistent investigation of amino acids in 3 generations showed that the Lys content was significantly increased in seeds of transgenic rice. Furthermore, Lys content in the hybrid of the transgenic plants also had an approximate 20 % increase compared to hybrid control. At the grain-filling stage, we monitored the transcript abundance of many genes encoding key enzymes involved in amino acid metabolism, and the results suggested that reduced amino acid catabolism led to the accumulation of amino acids in the transgenic plants. The genetically engineered rice showed unfavorable grain phenotypes compared to wild-type, however, its hybrid displayed little negative effects on grain. CONCLUSIONS: Endosperm-specific expression of foreign LRP significantly increased the Lys content in the seeds of transgenic plant, and the the Lys increase was stably heritable with 3 generation investigation. The hybrid of the transgenic plants also showed significant increases of Lys content in the seeds. These results indicated that expression of LRP in rice seeds may have promising applications in improving Lys levels in rice.

Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa).

RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, altering the amino acid specified by the DNA sequence. Here we report the identification of a critical editing factor of mitochondrial nad7 transcript via molecular characterization of a small kernel 1 (smk1) mutant in Zea mays (maize). Mutations in Smk1 arrest both the embryo and endosperm development. Cloning of Smk1 indicates that it encodes an E-subclass pentatricopeptide repeat (PPR) protein that is targeted to mitochondria. Loss of SMK1 function abolishes the C â†’ U editing at the nad7-836 site, leading to the retention of a proline codon that is edited to encode leucine in the wild type. The smk1 mutant showed dramatically reduced complex-I assembly and NADH dehydrogenase activity, and abnormal biogenesis of the mitochondria. Analysis of the ortholog in Oryza sativa (rice) reveals that rice SMK1 has a conserved function in C â†’ U editing of the mitochondrial nad7-836 site. T-DNA knock-out mutants showed abnormal embryo and endosperm development, resulting in embryo or seedling lethality. The leucine at NAD7-279 is highly conserved from bacteria to flowering plants, and analysis of genome sequences from many plants revealed a molecular coevolution between the requirement for C â†’ U editing at this site and the existence of an SMK1 homolog. These results demonstrate that Smk1 encodes a PPR-E protein that is required for nad7-836 editing, and this editing is critical to NAD7 function in complex-I assembly in mitochondria, and hence to embryo and endosperm development in maize and rice.

Metabolic engineering and profiling of rice with increased lysine.

Lysine (Lys) is the first limiting essential amino acid in rice, a stable food for half of the world population. Efforts, including genetic engineering, have not achieved a desirable level of Lys in rice. Here, we genetically engineered rice to increase Lys levels by expressing bacterial lysine feedback-insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) to enhance Lys biosynthesis; through RNA interference of rice lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase (LKR/SDH) to down-regulate its catabolism; and by combined expression of AK and DHPS and interference of LKR/SDH to achieve both metabolic effects. In these transgenic plants, free Lys levels increased up to ~12-fold in leaves and ~60-fold in seeds, substantially greater than the 2.5-fold increase in transgenic rice seeds reported by the only previous related study. To better understand the metabolic regulation of Lys accumulation in rice, metabolomic methods were employed to analyse the changes in metabolites of the Lys biosynthesis and catabolism pathways in leaves and seeds at different stages. Free Lys accumulation was mainly regulated by its biosynthesis in leaves and to a greater extent by catabolism in seeds. The transgenic plants did not show observable changes in plant growth and seed germination nor large changes in levels of asparagine (Asn) and glutamine (Gln) in leaves, which are the major amino acids transported into seeds. Although Lys was highly accumulated in leaves of certain transgenic lines, a corresponding higher Lys accumulation was not observed in seeds, suggesting that free Lys transport from leaves into seeds did not occur.

Characterization of the spatial and temporal expression of the OsSSII-3 gene encoding a key soluble starch synthase in rice.

BACKGROUND: Starch, the major component of rice grain, consists of amylose and amylopectin. SSIIa, a key soluble starch synthase involved in the biosynthesis of rice amylopectin, is a major factor that controls the gelatinization temperature of rice grain. Extensive work has been done and impressive progress has been made in elaborating the function of the gene encoding SSIIa (OsSSII-3). However, the systematic expression analysis of OsSSII-3 is still rare. RESULTS: In the present study, we performed a comprehensive expression analysis of OsSSII-3 in both the developing seeds and other tissues of indica rice 9311 by using quantitative real-time PCR. The results showed that the gene was dominantly expressed in the developing seeds. In addition, the promoter sequence of OsSSII-3 was cloned and fused with the GUS reporter gene and its expression was carefully monitored in the transgenic rice. The data from both histochemical and fluorometric analyses showed that the OsSSII-3 promoter was capable of driving the target gene to have an endosperm-specific expression, which may be due to the existing of several endosperm-specific motifs in the promoter, including the -300 elements, AACA motifs and GCN4 motifs. This result was quite consistent with that of the endogenous transcription analysis of OsSSII-3. CONCLUSION: This study not only advanced our understanding of the spatial and temporal expression characteristics of OsSSII-3, but also provided a valuable promoter for future application in generating elite rice varieties with high nutritional or medicinal value.

Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth.

Vacuolar sorting receptors (VSRs) are type-I integral membrane proteins that mediate biosynthetic protein traffic in the secretory pathway to the vacuole, whereas secretory carrier membrane proteins (SCAMPs) are type-IV membrane proteins localizing to the plasma membrane and early endosome (EE) or trans-Golgi network (TGN) in the plant endocytic pathway. As pollen tube growth is an extremely polarized and highly dynamic process, with intense anterograde and retrograde membrane trafficking, we have studied the dynamics and functional roles of VSR and SCAMP in pollen tube growth using lily (Lilium longiflorum) pollen as a model. Using newly cloned lily VSR and SCAMP cDNA (termed LIVSR and LISCAMP, respectively), as well as specific antibodies against VSR and SCAMP1 as tools, we have demonstrated that in growing lily pollen tubes: (i) transiently expressed GFP-VSR/GFP-LIVSR is located throughout the pollen tubes, excepting the apical clear-zone region, whereas GFP-LISCAMP is mainly concentrated in the tip region; (ii) VSRs are localized to the multivesicular body (MVB) and vacuole, whereas SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LlVSR small interfering RNAs (siRNAs) significantly reduced the growth rate of the lily pollen tubes. Taken together, both VSR and SCAMP are required for pollen tube growth, probably working together in regulating protein trafficking in the secretory and endocytic pathways, which need to be coordinated in order to support pollen tube elongation.

Production of the 42-kDa fragment of Plasmodium falciparum merozoite surface protein 1, a leading malaria vaccine antigen, in Arabidopsis thaliana seeds.

Malaria is widely associated with poverty, and a low-cost vaccine against malaria is highly desirable for implementing comprehensive vaccination programmes in developing countries. Production of malaria antigens in plants is a promising approach, but its development has been hindered by poor expression of the antigens in plant cells. In the present study, we targeted plant seeds as a low-cost vaccine production platform and successfully expressed the Plasmodium falciparum 42-kDa fragment of merozoite surface protein 1 (MSP142), a leading malaria vaccine candidate, at a high level in transgenic Arabidopsis seeds. We overcame hurdles of transcript and protein instabilities of MSP142 in plants by synthesizing a plant-optimized MSP142 cDNA and either targeting the recombinant protein to protein storage vacuoles or fusing it with a stable plant storage protein. An exceptional improvement in MSP142 expression, from an undetectable level to 5% of total extractable protein, was achieved with these combined strategies. Importantly, the plant-derived MSP142 maintains its natural antigenicity and can be recognized by immune sera from malaria-infected patients. Our results provide a strong basis for the development of a plant-based, low-cost malaria vaccine.

Antiviral activity of daphnoretin isolated from Wikstroemia indica.

The ethanol extract of Wikstroemia indica was fractionated with organic solvents of different polarities, and various fractions were screened for their antiviral activity against respiratory syncytial virus (RSV) using a cytopathic effect (CPE) reduction assay. The ethyl acetate fraction was most active against RSV with 50% inhibition concentration (IC(50)) value < 3.9 microg/mL and a selectivity index (SI) > 64.1. Further isolation and purification of the fraction led to a purified compound, daphnoretin. Daphnoretin was tested for its anti-RSV activity using a plaque reduction assay and found active against RSV, with an IC(50 )value of 5.87 microg/mL and SI value of 28.17. The mode of antiviral action study revealed that daphnoretin could slightly inhibit the early events of the viral infection but its effect was mainly on the later phase of the replication cycle.

Expression and subcellular targeting of human insulin-like growth factor binding protein-3 in transgenic tobacco plants.

Human insulin-like growth factor binding protein-3 (hIGFBP-3) is a multifunctional protein which has high affinity for insulin-like growth factor-I (IGF-I). It combines with IGF-I to form a tertiary complex in circulation, thus regulating the activity of IGF-I. Furthermore, recombinant hIGFBP-3 (rhIGFBP-3) has been found to negatively regulate cell proliferation and induce apoptosis. In this study, we have established an efficient plant bioreactor platform for mass production of rhIGFBP-3. Different expression constructs, driven by the seed-specific phaseolin promoter, were designed and transformed into tobacco plant via Agrobacterium. To enhance protein expression level, the signal peptide (SP) and the C-terminal tetrapeptide AFVY of phaseolin were used to direct rhIGFBP-3 to protein storage vacuole (PSV) in tobacco seed for stable accumulation. Western blot analysis showed that rhIGFBP-3 was successfully synthesized in transgenic tobacco seeds, with the highest protein expression of 800 mug/g dry weight. The localization of rhIGFBP-3 in PSV was also evident by confocal immunofluorescence microscopy. Our results indicated that protein sorting sequences could benefit the expression level of rhIGFBP-3 and it is feasible to use plant as "bio-factory" to produce therapeutic recombinant proteins in large quantity.

Isolation, characterization, molecular cloning and modeling of a new lipid transfer protein with antiviral and antiproliferative activities from Narcissus tazetta.

A fetuin-binding peptide with a molecular mass of about 9kDa (designated NTP) was isolated and purified from the bulbs of Chinese daffodil, Narcissus tazetta var. chinensis L., by gel filtration and high-performance liquid chromatography, after removing the mannose-binding proteins by mannose-agarose column. Molecular cloning revealed that NTP contained an open reading frame of 354bp encoding a polypeptide of 118 amino acids which included a 26-amino-acid signal peptide. An analysis of the deduced amino acid sequence of NTP shows considerable sequence homology to the non-specific lipid transfer proteins (nsLTPs) of certain plants. Model of the three-dimensional (3D) structure of NTP exhibits an internal hydrophobic cavity which can bind lipid-like molecules and transfer a wide range of ligands. As a member of the putative non-specific lipid transfer protein of N. tazetta, NTP did not possess hemagglutinating activity toward rabbit erythrocytes. In a cell-free system, it could arrest the protein synthesis of rabbit reticulocytes. Using the in vitro antiviral assays, NTP could significantly inhibit the plaque formation by respiratory syncytial virus (RSV) and the cytopathic effect induced by influenza A (H1N1) virus, as well as the proliferation of human acute promyelocytic leukemia cells (HL-60).

Antiviral and anti-proliferative glycoproteins from the rhizome of Smilax glabra Roxb (Liliaceae).

The glycoproteins possessing antiviral and anti-proliferative activities were isolated from the Chinese medicinal herb Smilax glabra (known as tufuling), by extraction with 0.2 M NaCl, ammonium sulfate precipitation, fetuin-agarose affinity chromatography and gel filtration. The molecular mass of the fetuin-binding glycoprotein (designated SGPF2) was estimated to be about 58 kDa, with a major protein subunit of 26 kDa. The non-fetuin binding glycoproteins (in the unadsorbed fraction) were further separated into 5 different subfractions (SGPF1a-SGPF1e) with anion-exchange chromatography, all of which also contained the major band at 26 kDa. All the isolated proteins of 26 kDa had similar N-terminal amino acid sequences, implying that they were probably the isoforms originated putatively from a multigene family with different binding affinity and ionic strength. The glycoprotein SGPF2 exhibited antiviral activity against respiratory syncytial virus (RSV) with a median inhibitory concentration (IC(50)) of 62.5 microg/ml and Herpes simplex virus type 1 (HSV-1) had an IC(50) of 31.3 microg/ml. The glycoprotein potencies for antiviral activity appeared to depend on the molecules' binding affinity for fetuin, that is, the fetuin-binding protein was more potent than the non-fetuin binding proteins. Further examination revealed that these glycoproteins also had the ability to suppress the proliferation of MCF-7 cells. The possible mechanism of anti-proliferative action as analyzed by DNA flow cytometry indicated that they could induce apoptosis mediated via sub-G(1) phase of the MCF-7 cell cycle. For example, there was an increase by 75.8% of the control level of apoptosis after incubation with SGPF1a.

Purification and characterization of non-specific lipid transfer proteins from the leaves of Pandanus amaryllifolius (Pandanaceae).

Two proteins were isolated from the saline extract of mature leaves of Pandanus amaryllifolius, using affinity chromatography on fetuin-agarose and Affi-gel Blue gel, anion exchange chromatography as well as gel filtration. The proteins were demonstrated as non-glycoproteins, with molecular mass of 18 and 13 kDa, respectively, comprising of peptide subunits from 6.5 to 9 kDa in the forms of heterodimer and homodimer. All of them have similar N-terminal amino acid sequences with only minor variations and are matched to non-specific lipid transfer proteins (nsLTPs) of the other plants such as wheat LTP using NCBI Blast searching for short, nearly exact matches. Furthermore, they explicated each other as isoforms originated putatively from a multigene family with various molecular weight, binding affinity, ionic strength, and subunits. However, the potencies for antiproliferation of HL-60 cell line and inhibition of the growth of the bacteria Pseudomonas aeruginosa are different in that those of the fetuin-binding protein are greater than non-fetuin binding proteins. The non-specific lipid transfer proteins of P. amaryllifolius exhibit weak to moderate hemagglutinating activity toward rabbit erythrocytes, but, this activity could not be reversed by mannose. They thus could be easily differentiated from the previously reported mannose-binding lectin isolated from this plant, which has subunits with similar molecular weight.

Membrane anchors for vacuolar targeting: application in plant bioreactors.

Transgenic plants are attractive expression systems for producing recombinant proteins. Plant cells compartmentalize and store metabolites and proteins in vacuoles, but foreign proteins need to be targeted to the correct compartments if they are to accumulate in a stable fashion. Here we present a general strategy in which unique transmembrane and cytoplasmic tail sequences are used as anchors for delivering recombinant proteins via distinct vesicular transport pathways to specific vacuolar compartments where stable accumulation can occur.

Purification and characterization of a new antiviral protein from the leaves of Pandanus amaryllifolius (Pandanaceae).

A lectin, designated Pandanin, was isolated from the saline extract of the leaves of Pandanus amaryllifolius, using ammonium sulfate precipitation, affinity chromatography on mannose-agarose and molecular size exclusion by gel filtration. Pandanin is an unglycosylated protein with a molecular mass of 8.0 kDa both after gel filtration and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a single polypeptide chain. The first 10 residues of the N-terminal amino acid sequence are DNILFSDSTL. An analysis of the sequence of first 30 amino acids at the N-terminal region shows that Pandanin has about 50-60% homology to those of mannose-specific lectins reported from monocot plants. Pandanin exhibits hemagglutinating activity toward rabbit erythrocytes, and its activity could be reversed exclusively by mannose and mannan. Pandanin also possesses antiviral activities against human viruses, herpes simplex virus type-1 (HSV-1) and influenza virus (H1N1) with 3 day's EC50 of 2.94 and 15.63 microM, respectively.

New mannose-binding lectin isolated from the rhizome of Sarsaparilla Smilax glabra Roxb. (Liliaceae).

A new mannose-binding lectin, designated SGM2, was isolated from the rhizome of a Chinese medicinal herb Smilax glabra (also known as sarsaparilla in general) by saline extraction, ammonium sulfate precipitation and fractionation, and affinity chromatography on fetuin- and mannose-agarose. SGM2 is shown to have a molecular mass of 37 kDa on gel filtration and 12.5 kDa on SDS-PAGE, indicating that it is a trimeric protein composed of three identical subunits. When the first 30 amino acid residues at the N-terminal were compared, SGM2 had approximately 40% homology with those of some other monocots. SGM2 had the property of hemagglutinating activity toward rabbit erythrocytes, which could be reversed by mannose and mannose polymers. SGM2 exhibited antiviral activities against both herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV) with the same EC(50) of 8.1 microM.

Isolation and characterization of a bioactive mannose-binding protein from the Chinese chive Allium tuberosum.

A mannose-binding protein was isolated from two different cultivars of the Chinese chive Allium tuberosum by extraction with 0.2 M NaCl, ammonium sulfate precipitation, and affinity chromatography on mannose agarose and fetuin agarose. It exhibited hemagglutinating activity toward rabbit erythrocytes. The lectin (agglutinin) was adsorbed on the mannose-agarose column, but not on the fetuin-agarose column. This A. tuberosum lectin (ATL) is unglycosylated, and not sialic acid binding. Lectins isolated from the two cultivars exhibited the same molecular mass of 25 kDa on gel filtration (Superose 12) and 12.5 kDa on SDS-polyacrylamide gel electrophoresis, indicating that they might be a dimeric protein composed of two identical subunits. The N-terminal amino acid sequence analysis of the lectin of various cultivars of A. tuberosum revealed that they were identical and showed 50%, or more, homology to the lectins from Galanthus nivalis (family Amaryllidaceae), Narcissus tazetta (family Amaryllidaceae), and Aloe arborescenes (family Liliaceae).

Inhibition of human MCF-7 breast cancer cells and HT-29 colon cancer cells by rice-produced recombinant human insulin-like growth binding protein-3 (rhIGFBP-3).

BACKGROUND: Insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I) to form a complex (IGF-I/IGFBP-3) that can treat growth hormone insensitivity syndrome (GHIS) and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3) in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL) were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 ± 1.72% vs 45.00 ± 0.86%, p < 0.05; 50.84 ± 1.97% vs 45.00 ± 0.86%, p < 0.01 respectively) and HT-29 colon cancer cells (65.14 ± 3.84% vs 18.01 ± 13.81%, p < 0.05 and 54.7 ± 9.44% vs 18.01 ± 13.81%, p < 0.05 respectively) when compared with wild type rice. CONCLUSION/SIGNIFICANCE: These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future.

A model-based method for gene dependency measurement.

Many computational methods have been widely used to identify transcription regulatory interactions based on gene expression profiles. The selection of dependency measure is very important for successful regulatory network inference. In this paper, we develop a new method-DBoMM (Difference in BIC of Mixture Models)-for estimating dependency of gene by fitting the gene expression profiles into mixture Gaussian models. We show that DBoMM out-performs 4 other existing methods, including Kendall's tau correlation (TAU), Pearson Correlation (COR), Euclidean distance (EUC) and Mutual information (MI) using Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Arabidopsis thaliana data and synthetic data. DBoMM can also identify condition-dependent regulatory interactions and is robust to noisy data. Of the 741 Escherichia coli regulatory interactions inferred by DBoMM at a 60% true positive rate, 65 are previously known interactions and 676 are novel predictions. To validate the new prediction, the promoter sequences of target genes regulated by the same transcription factors were analyzed and significant motifs were identified.

An interaction between BZR1 and DELLAs mediates direct signaling crosstalk between brassinosteroids and gibberellins in Arabidopsis.

Plant growth and development are coordinated by several groups of small-molecule hormones, including brassinosteroids (BRs) and gibberellins (GAs). Physiological and molecular studies have suggested the existence of crosstalk between BR and GA signaling. We report that BZR1, a key transcription factor activated by BR signaling, interacts in vitro and in vivo with REPRESSOR OF ga1-3 (RGA), a member of the DELLA family of transcriptional regulators that inhibits the GA signaling pathway in Arabidopsis thaliana. Genetic analyses of plants with mutations in the genes encoding RGA and BZR1 revealed that RGA suppressed root and hypocotyl elongation of the gain-of-function mutant bzr1-1D. Ectopic expression of proteins of the DELLA family reduced the abundance and transcriptional activity of BZR1. Reporter gene analyses further indicated that BZR1 and RGA antagonize each other's transcriptional activity. Our data indicated that BZR1 and RGA served as positive and negative regulators, respectively, of both the BR and the GA signaling pathways and establish DELLAs as mediators of signaling crosstalk between BRs and GAs in controlling cell elongation and regulation of plant growth.

A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform.

BACKGROUND: Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor. METHODOLOGY/PRINCIPAL FINDINGS: To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL) with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP) fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV). The in vivo uncleaved EiP protein was accumulated up to 2-4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein. CONCLUSION/SIGNIFICANCE: This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry.

A sub-space greedy search method for efficient Bayesian Network inference.

Bayesian network (BN) has been successfully used to infer the regulatory relationships of genes from microarray dataset. However, one major limitation of BN approach is the computational cost because the calculation time grows more than exponentially with the dimension of the dataset. In this paper, we propose a sub-space greedy search method for efficient Bayesian Network inference. Particularly, this method limits the greedy search space by only selecting gene pairs with higher partial correlation coefficients. Using both synthetic and real data, we demonstrate that the proposed method achieved comparable results with standard greedy search method yet saved ∼50% of the computational time. We believe that sub-space search method can be widely used for efficient BN inference in systems biology.