Patent Application
20070162998
1. Field of the Invention
The present invention relates to a novel polypeptide, designated “CeCPI,” and more particularly to CeCPI fusion protein, nucleic acid molecules encoding such polypeptides and proteins, methods of using these amino acid and nucleotide sequences, and composition including these amino acid sequences.
2. Description of the Prior Art
Cystatins are proteinaceous inhibitors of cystein proteases identified in animals, as well as in monocotyledoneous and dicotyledoneous plants. Moreover, plants cystein protease inhibitors are thought to play an important role in defense mechanisms against insect and pathogen attack. In fact, several cystatins can inhibit in vitro digestive proteases from coleopteran insects (Zhao et al., Plant Physiol 111: 1299-1306 (1996)), and transgenic plants overexpressing cystatins and showing enhanced resistance against insects and nematodes have been reported, for example, by Urwin et al., Plant J. 12:455-461 (1997) which is incorporated herein by reference. These proteins are ubiquitous in the plant kingdom and have attracted the attention of researchers due to their capacity to inhibit proteases that occur not only in many herbivorous insect species but also in pathogenic fungi (Ryan, Annu Rev Phytopathol 28:425-449 (1990)). In general, these proteins are specially present in storage organs, and their synthesis might be induced systemically or locally by cell damage that contributes to the complex defense mechanisms of plants.
Cystatins inhibit sulfhydryl proteinase activities and have mainly been studied in animal cells. Similarities in their primary structures and functions show that cystatins form a single evolutionary superfamily (see, for example, Barrett, Trends Biochem Sci 12:193-196 (1987), incorporated herein by reference) that comprises three families: family-I cystatins (stefins) are about 100 aa long with no disulfide bonds; family-II cystatins (cystatin II) are about 150 aa long with two disulfide bonds in the carboxy-terminal region of the protein; and family-III cystatins (the kininogens) three regions with two disulfide loops, similar to the carboxy terminal domain found in members of the cystatin family.
In the plant kingdom, a large number of cysteine proteinase inhibitors have been discovered and these proteinase inhibitors of plant origin have been grouped into a fourth cystatin family, the “phytocystatin,” based on sequence similarities and the absence of disulfide bonds (see, Abe et al., J Biol Chem 262:16793-16797 (1987); Abe et al., Eur J Biochem 209:933-937 (1992)). Phytocystatins are single polypeptide chains with molecular masses from 12 kDa to 16 kDa and share three conserved sequence motifs. Three important regions of the mature cystatin are: a conserved Gly in the vicinity of the N terminal region, a highly conserved Gln-Xaa-Val-Xaa-Gly motif in a central loop segment, and a Pro-Trp residue in what could be the second hairpin loop. In addition, phytocystatins possess a conserved LARFAVDEHN sequence in the N-terminal region that is absent in animal cystatins.
Several phytocystatin members have been isolated from many species such as rice seeds, soybean, maize, tomato, potato, Chinese cabbage, and chestnut. Phytocystatins show variable expression patterns during plant development and defense responses to biotic and abiotic stresses (see, Felton G W and Korth K L, Curr Opin Plant Biol 3:309-314 (2000)). The physiological function of these proteins is not well understood. However, at least two functions have been proposed: regulation of protein turnover and protecting plants against insects and pathogens (see, Turk V, and Bode W, FEBS Lett 285:213-219 (1991)).
The ingestion of protease inhibitors interferes with the protein degradation process in the insect's midgut. Cystatins have been shown to inhibit the activity of digestive proteases from coleopteran pests in vitro, as well as larval development in vivo. Thus, cystatins function as “toxins” by targeting the major proteolytic digestive enzymes of herbivore insects (see Hines et al., J Agric Food Chem 39:1515-1520 (1991); Leplé et al., Mol Breed 1:319-328 (1995); Zhao et al., Plant Physiol 111:1299-1306 (1996)). Moreover, cysteine proteases play an important role in virus replication, and this has been proved in induced virus resistance in tobacco by the expression of rice cystatin (see, Gutierrez-Campos et al., Nat Biotechnol 17:1223-1226 (1999)).
The taro, Colocasia esculenta (Kaoshiung no. 1), is an important staple food of Taiwan aborigines, and is widely cultivated in local mountainous farms. This crop is popular for its high productivity and less pathogen attacks. The reason behind our investigation was the resistant mechanism in taro. In a preliminary survey on proteinase inhibitors from taro, a cysteine proteinase inhibitor with a copious amount in tuber organ was discovered.
Proteins capable of inhibiting the growth of fungus are thought to be useful in agriculture and human life.
Accordingly, the present invention provides a novel polypeptide which has antifungal activity, designated “CeCPI.” The present invention also provides CeCPI polypeptides and CeCPI fusion proteins, nucleic acid molecules encoding such polypeptides and proteins. Moreover, the present invention provides methods of obtaining these amino acid and nucleotide sequences.
The invention now being generally described, the same will be better understood by reference to the following detailed description of specific embodiments in combination with the figures that form part of this specification, wherein:
Methods used in the present invention are described by inventors Yeh and co-workers in Planta 221:493-501 (2005), the entire contents of which are hereby incorporated by reference.
In the present invention, degenerated primers (SEQ ID NO: 3, SEQ ID NO:4) were used to amplify cDNA fragments which were initially reverse-transcribed from the poly(A)+ RNA of taro (C. esculenta cv. Kaoshiung no. 1). The specific cDNA fragments were further extended to full-length cDNA genes by using 5′- and 3′-RACE. Based on the sequence analysis data, a cDNA clone, denoted as CeCPI (SEQ ID NO: 1), was confirmed as a phytocystatin gene. The primary structure of CeCPI exhibits all the consensus sequences conserved in all phytocystatins, such as glycine residue and a conserved LARFAVDEHN (SEQ ID NO:5) in the N-terminal region, a general active motif of QXVXG (Gln49-Val50-Val51-Ser52-Gly53) (SEQ ID NO: 6), and several specific amino acid residues of phenylalanine, tyrosine, and tryptophan in β-sheet regions. The ORF of CeCPI was subcloned into expression vector, pGEX-2TK, to produce recombinant protein (SEQ ID NO: 2). The overexpressed protein was assayed on 0.1% gelatin/SDS-PAGE, and a conspicuous concentration-dependent inhibitory activity towards papain was observed. This demonstrated that CeCPI is certainly a novel phytocystatin from taro. Most plant cystatins are reported as 12-16 kDa in size, and occur commonly in Monocots, such as wheat, rice, maize, and sugarcane. However, it is interesting to note that CeCPI (SEQ ID NO: 2), a Monocot cystatin from taro, is predicted to exceed 22 kDa. Phylogenetic analysis of sequence alignments showed that the primary structure has a closer relationship with Eudicots than with Monocots, and with a longer extension of the C-terminal amino acid cluster than Eudicots. A hypothetical interpretation for this case is natural horizontal gene transfer and this mechanism, enabling a gene transfer across groups of seed plants, has been reported in higher plants.
Sclerotium rofsii Sacc. is a severe phytopathogenic fungi in tropical regions that causes great southern blight damage to tomato, peanut and banana. Therefore, it is a prime target in the antifungal survey. The antifungal activity of tarocystatin protein showed a striking retardation on mycelium growth of S. rofsii Sacc. greater than 80 μml, preferred greater than 150 μg/ml, and most preferred greater than 200 μg/ml. In this antifungal test, recombinant fusion protein, composed of GST and CeCPI protein, was used for assay. Initially, GST was suspected of toxicity action synergistic with CeCPI protein in inhibiting hyphae growth. Eventually, it was proved ineffective, because assays employing GST protein alone did not exhibit this effect. In fact, the effective dosage of antifungal activity of tarocystatin on S. rofsii is half of the above-mentioned concentration. In the antimicrobial assays, it was shown that tarocystatin is unable to inhibit the growth of E. carotovora. This is coincident with the condition that E. carotovora is the major bacterial pathogen of taro in the farm, which causes leaf blight. On the other hand, S. Rofsii causes southern blot in the tuber; it is only at the post-harvest stage that the stored condition is in a warm and humid state. In this case, tarocystatin is gradually degraded; tuber thus loses the resistance to fungal attack. It could be the case that S. Rofsii becomes a pathogen to taro. Therefore, our conclusion is that the effectiveness of cystatin is closely correlated with pathogenic resistance.
In this invention, cysteine protease is discovered to exist in S. rofsii mycelium. Additionally, an inhibitory effect of tarocystatin on fungal cysteine protease was clearly confirmed from 0.1% gelatin/SDS-PAGE assay. It is to say that CeCPI exhibits strong antifungal activity on several ubiquitous phytopathogenic fungi, such as S. rofsii Sacc. etc. Moreover, CeCPI is able to block the endogenous cysteine protease of the fungal mycelium. These results imply that the CeCPI gene has the potential to be developed into a fungicidal compound.
The taro, cultivar C. esculenta cv. Kaoshiung no. 1, was used in this study. The plants were maintained at the experimental farm of the Kaoshiung District Agricultural Improvement Station, Taiwan. Corms ˜0.5 kg were harvested, frozen in liquid nitrogen, and stored at −75° C. until used for protein and RNA extraction.
Total RNA was extracted from mature taro corms following the method described by Yeh et al. Focus 13:102-103 (1991) which is incorporated herein by reference. Poly (A)+ RNA was isolated using the mRNA purification kit (Amersham Pharmacia Biotech, USA). For the molecular cloning of tarocystatin gene, a strategy was performed as follows: a 0.7 kb cDNA fragment was pre-amplified from mRNA using RT-PCR with one adaptor-primer, supplied with the commercial kit (Marathoon, Clontech, Calif., USA), and one of the two cystatin degenerated primers GSP-1 and GSP-2.
Degenerated primers were designed based on the conserved sequence, LARFAVDEHNKK, commonly present in most of these phytocystatins. The 0.7 kb cDNA fragment was cloned into pGEM-T easy vector, and the sequence was determined and confirmed to be a tarocystatin gene. Then, 5′-RACE/3′-RACE methods were employed to extend the fragment into a full-length cDNA gene. The obtained and characterized cDNA clone, denoted as CeCPI, was deposited in GenBank under the accession number AF525880.
Please refer to
The coding region of tarocystatin gene, CeCPI, was amplified by PCR with the following primers:
The forward primer, CeCPI-F: 5′-TTGATCCATGCTTGATGGGGGG CAT-3′ (SEQ ID NO: 7); and
the reverse primer, CeCPI-R: 5′-TTGAATCCTTTCCAGAGTCTGAAT GATC-3′ (SEQ ID NO: 8).
In the amplification reaction, 20 ng template cDNA, 0.75 U Taq DNA polymerase (New England Biolabs), 1×PCR buffer, 1 mM MgCl2, and 0.2 mM dNTPs were used. The reaction was performed in a TouchDown research thermocycler programmed for 30 cycles at 94° C. for 1 min, 46° C. for 40 s, 72° C. for 2 min, and a final extension at 72° C. for 4 min. A DNA fragment of 618 bp was purified and cleaved with the restriction enzymes BamHI and EcoRI and inserted into the expression vector pGEX-2TK (Pharmacia, USA). The recombinant clones obtained in E. coli were identified by sequence determination using an ABI Prism 377 DNA sequencer.
Furthermore, transformed E. coli cells harboring expression vector pGEX-CeCPI were cultured in LB broth containing ampicillin (100 μg/ml) and incubated at 37° C. overnight under continuous agitation. When the culture reached of OD600=0.5-1.0, isopropyl-b-D-thiogalactocide (IPTG) was added to a final concentration at 0.1 mM to induce expression of recombinant tarocystatin protein. Four hours after IPTG induction, the cell culture was harvested and the cell pellet was suspended in 1×PBS buffer. Total soluble protein was obtained by rupturing the cell, and the soluble recombinant glutathione-S-transferase-taro cysteine protease inhibitors (GST-CeCPI) fusion protein was purified by glutathione affinity chromatography following the instructions of the B-PER GST spin purification kit (Pierce Biotechnology, USA). Furthermore, digestion by thrombin to separate CeCPI from GST was performed for 16 h. SDS-PAGE analysis of recombinant CeCPI and Western blot with anti-CeCPI antiserum were carried out as the standard molecular methods.
Expression plasmid pGEX-CeCPI, which harbors the open reading frame of CeCPI cDNA gene, was introduced into E. coli strain XL1-blue. The overexpression of CeCPI protein was induced by adding IPTG (0.1 mM, final conc.) to the culture medium. Total soluble proteins were harvested at 3-4 h after induction. After purification by glutathione affinity chromatography and cleavage by thrombin, the recombinant CeCPI proteins were analyzed on 15% SDS-PAGE. Electrophoresis of recombinant proteins clearly showed a highly productive expressed protein approximately 29 kDa in size (
To determine whether CeCPI recombinant protein, produced from E. coli, retains an inhibitory activity against papain (cysteine protease), 0.1% gelatin/SDS polyacrylamide gel electrophoreses was employed. Different amounts of recombinant CeCPI proteins from 10 to 200 μg were pre-mixed with papain (20 nmol, 0.5 μg), incubated for 15 min at 37° C., and then resolved on 0.1% gelatin/SDS-PAGE to observe the residual protease activity of papain. The mixtures were first subjected to electrophoresis using a Hoefer SE250 system. After migration, the gels were transferred to a 2.5% v/v aqueous solution of Triton X-100 for 30 min at room temperature to allow renaturation, and incubating at active buffer (100 mM sodium phosphate pH 6.8; 8 mM EDTA; 10 mM L-cysteine and 0.2% Triton X-100) for 75 min at 37° C. Subsequently, they were rinsed with water and stained with Coomassie brilliant blue. As shown in
Various concentrations of CeCPI protein samples in 0.2 ml were mixed with 0.1 ml sodium phosphate buffer (0.5 M sodium phosphate/10 mM EDTA, pH 6.0), 0.1 ml of 2-mercaptoethanol (50 mM), and 0.1 ml papain solution (25 μg/ml), and the mixture was incubated at 37° C. for 10 min. After that, 0.2 ml of 1 mM N-benzoyl-DL-arginine-2-naphthylamide (BANA) was added to start the reaction, and the mixture was incubated for another 20 min at 37° C. The reaction was terminated by adding 1 ml of 2% HCl/ethanol and 1 ml of 0.06% p-dimethylaminocinnamaldehyde/ethanol, and the mixture was allowed to stand at room temperature for 30 min for color development and finally measured at OD540 nm. The inhibitory activity of CeCPI was recorded as an inhibition percentage (%), and the inhibition percentage (I%) of papain by CeCPI was calculated using the following equation:
where T denotes the OD540 in the absence of CeCPI and T* that in the presence of CeCPI. One inhibition unit was defined as the amount of inhibitor required to completely inhibit 2.5 μg of papain.
The heat stability of recombinant CeCPI at different temperatures was also investigated. This activity was observed from its residual inhibitory activity against papain after treating the protein samples (from 10 μg to 500 μg GST-CeCPI fusion protein) at 25, 60 and 100° C. for 5 min, respectively. This demonstrated that GST-CeCPI recombinant protein lost significant inhibitory activity only when treated at 100° C. for 5 min (
Two taro pathogens were preferentially chosen for the growth inhibition assay. One is Sclerotium rolfsii, a fungal pathogen causing stored tubers southern blot in humid and warm conditions. The other is Erwinwa carotovora subsp. carotovora, a bacterial pathogen causing soft rot of leaf and stem during farm growth. For a general survey of the antimicrobial toxicity of tarocystatin, several widespread phytopathogenic fungi were further chosen for study. They were Alternaria brassicae, Glomerella cingulata, Fusarium oxysporum, Pythium aphanidermatum and Rhizoctonia solani.AG4.
Fungal strains from the laboratory collection were grown in potato dextrose agar (PDA) medium for 7˜10 days. With the exception of S. rolfsii, which was inoculated to 2 ml of ⅓×potato dextrose broth (PDB) with five pieces of sclerotinia, a spore suspension (asexual spore) of the other fungal strains was collected for the inoculum by washing the mycelium colony with sterile ddH2O. Approximately 103 spores of each fungal strain were inoculated in 2 ml of ⅓×PDB, which contained various amounts of purified GST-cystatin fused proteins (with concentrations of 0, 20, 60, 80, 150, or 200 μg/ml respectively). They were incubated at 28° C. under continuous shaking (200 rpm/min) for 24˜72 h. Pathogenic bacteria (Erwinia carotovora) of taro soft rot was cultured in 1 ml of ⅓×PDB, and allowed to grow until it reached OD600=0.2˜0.4. Then, various concentrations of fusion protein were added and incubated at 28˜30° C. for 20˜24 h. An inhibition test of tarocystatin on fungal cysteine protease activity was carried out as follows. Sclerotinia of 5-day-growing fungal cultures were harvested (0.2 g), ground in liquid nitrogen, and extracted in 500 μl of 100 mM citrate phosphate buffer at pH 6.0. After incubation on ice for 30 min, the homogenate was centrifuged at 12,000 g for 30 min at 4° C., and the supernatant was measured for protein quantification following the method of Bradford (1976). A protein sample (30 μg) of the mycelium extract was used to react with different concentrations of recombinant GST-CeCPI and E64 (Michaud et al. 1996) for 15 min at 37° C. Then, it was analyzed on 0.1% gelatin/SDS-PAGE for protease activity.
Please refer to TABLE 1. The results revealed that there was no inhibitory effect on the bacterial pathogen, E. carotovora subsp. carotovora. However, a varied inhibitory level was present among the fungal pathogens (as shown in TABLE 1). This means that there is a diversity of effective toxic dosages among fungal pathogens.
As the example of S. rolfsii, a quantitative growth inhibition of fungal mycelium was performed by incubating it with increasing amounts of purified recombinant GST-CeCPI fusion protein (20, 40, 60, 80, 100, 150 and 200 μg/ml). Abundant mycelia growth in PDB medium was found in both control cultures—without adding recombinant CeCPI or adding GST protein only (
The result clearly showed that crude protein sample extracted from fungal mycelium might contain cysteine proteinase. Therefore, it was able to digest gelatin contained in the running gel (
Obviously, the CeCPI of the present invention exhibits strong antifungal activity on several ubiquitous phytopathogenic fungi, such as S. rofsii Sacc. etc. These results imply that the CeCPI gene has the potential to be developed into a fungicidal agent. Furthermore, it is easy for the person skilled in the art to transform the CeCPI gene to a plant cell, so as to obtain a transgenic plant cell with antifungal activity.
It should be noted that all publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.
