KR100480843B1 - Transgenic organism expressing fungal MRP-like ABC transporters - Google Patents

Abstract

PURPOSE: A transgenic organism capable of expressing fungal MRP-like ABC transporter proteins is provided, thereby improving accumulation of and resistance against toxic material such as heavy metals and herbicides. CONSTITUTION: The gene encoding a fungal MRP-like ABC transporter protein and having resistance against and accumulation of lead is provided, wherein the transporter protein is selected from the group consisting of YCF1, YHL035C, BPT1, YBT1, and YOR1; the YCF1 has the amino acid sequence of SEQ ID NO: 2; the gene encoding the YCF1 has the nucleotide sequence of SEQ ID NO: 1; the YCF1 has resistance against and accumulation of toxic material selected from cadmium, arsenic and herbicides, wherein the herbicide is chloro-dinitrobenzene; the YHL035C has the amino acid sequence of SEQ ID NO: 4; and the gene encoding the YHL035C has the nucleotide sequence of SEQ ID NO: 3. The expression vectors, pESC-YCF1, ENpCambia-YCF1, PBI121-YCF1, pESC-YHL035C and pBI121-YHL035C, are provided. The transgenic organism capable of expressing fungal MRP-like ABC transporter proteins is produced by transformation with the expression vectors.

Description

Transgenic organism expressing fungal MRP-like ABC transporters expressing fungal MRP-based ABC transport proteins

[Technical Field]

The present invention relates to an MRP-based ABC transport gene of a fungus, and to a transgenic organism transformed with the gene. More specifically, the present invention expresses MRP-based ABC transport genes of fungi, including YCF1 or YHL035C, to lead, cadmium, The present invention relates to a transgenic organism having improved resistance to and accumulation of harmful substances such as arsenic and herbicides.

[Prior art]

Heavy metals such as lead, cadmium, and mercury accumulate in people's bodies through natural food chains, causing chronic damage to the brain, nerves, bones, and the like, and the polluted environment and its damage persist for generations. Representative examples of the toxicity of heavy metals include Minamata and Itaitai diseases in Japan. Lead is the most damaging pollutant among heavy metals (Salt, DE, Smith, RD, and Raskin, I. Phytoremediation.Annu . Rev. Plant Physiol. Plant Mol. Biol . 49, 643-668 (1998)) , and it is very important to remove lead from the environment, the US government in order to eliminate lead from the environment in which children grow and spend about 500 million dollars a year (Lanphear, BP the paradox of lead poisoning prevention. Science. 281 1617-1618 (1998). Arsenic is a major problem because it contaminates drinking water, causing skin diseases and cancer.

Genes related to resistance and accumulation of toxic substances such as heavy metals and pesticides known in the art include bacterial P-type ATPase, which functions to release lead from the cell membrane of cells (Rensing) , C., Sun, Y., Mitra, B., and Rosen, BP Pb (ll) -translocating P-type ATPases.J. Biol. Chem . 273: 32614-32617 (1998)), genes involved in cadmium resistance Examples include yeast YCF1 gene, genes related to arsenic resistance, yeast YCF1 gene and ACR genes, and bacteria ArsAB.

On the other hand, organisms have a mechanism to mitigate the toxicity of harmful substances by using biological materials or transport proteins that bind harmful substances introduced into the body. Genes that contribute to the resistance of harmful substances in living organisms can be used to purify the environment contaminated with harmful substances much more economically and environmentally compared to the physical, chemical, and engineering environmental purification methods currently used (Mejare and Bulow). , Trends in Biotechnology; 2001, Raskin I. and Ensley BD Phytoremediaton of Toxic Metals., John Wily & Sons, New York; 2000). In particular, plants express new traits by expressing foreign genes well, and are economically manageable and have good aesthetics.Therefore, researches are actively underway to improve the environment by putting good genes into plants. It is called Phytoremediation.

  In a situation where environmental pollution such as soil, such as lead, cadmium, arsenic, and pesticides is severe, there is an urgent need for transgenic organisms using genes that exhibit resistance and / or accumulation to these toxic substances.

Several transgenic plants that can be used to remove cadmium from the environment have been reported in the literature (Zhu et al., (1999) Plant Physiol. 119: 73-79, Hirschi et al., (2000) Plant Physiol . 124 : 125-33, Dominguez-Solis et al., (2001) J. Biol. Chem . 276: 9297-9302), no plants have been reported that can remove lead or arsenic. In addition, the use of YCF1 to develop not only lead, but also to improve the resistance to cadmium, arsenic, herbicides and has been used for the purification of harmful substances has not been reported yet.

The present invention provides DNA molecules encoding resistance and accumulating resistance to lead and encoding Md-based ATP-binding casette transporter protein (MRP-based ABC transport protein) of fungi. It aims to do it.

Another object of the present invention is to provide a recombinant vector comprising the MRP-based ABC transport gene.

Still another object of the present invention is to provide a transformant that is transformed with the MRP-based ABC transport gene and has improved resistance and / or accumulation to harmful substances.

In order to achieve the above technical problem, the present invention exhibits resistance and accumulation to lead, MRP-associated protein-like ATP-binding casette transporter protein, MRP-based ABC of mold Transport protein).

The present invention also relates to a recombinant vector comprising the MRP-based ABC transport gene of the fungus.

The present invention also relates to a transgenic organism transformed with a DNA molecule encoding the MRP-based ABC transport protein of the fungus.

Hereinafter, the present invention will be described in detail.

The present invention relates to genes exhibiting toxin resistance and / or toxin accumulation, vectors comprising the same, transformed cells, and transformed organisms.

In the present invention, genes that exhibit noxious substance resistance and / or accumulation include a fungal multidrug resistance associated protein (MRP) -based ATP-binding cassette transport protein (hereinafter referred to as MRP-based ABC transport protein). It is a gene that encodes.

The MRP-type ABC transport protein of the fungus is a kind of ABC transport protein, and serves to transport various organic substances in the cytoplasm out of the cytoplasm. ABC transport proteins are present in many organisms, from prokaryotes to human liver cells, and transport a wide variety of substances. Organic materials transported by the ABC transport protein include heavy metals and bile acids bound to glutathionine. It is known that YCF1, which belongs to the MRP-type ATP-binding cassette transport protein of the fungus, imparts resistance to these harmful substances by inserting cadmium, arsenic, and pesticides into vacuoles (Li et al ., (1997) Proc. Natl. Acad Sci. USA 94: 42-47, Ghosh et al ., (1999) Proc. Natl. Acad. Sci. USA 96: 5001-5006). However, there have been no reports of using YCF1 to develop organisms with improved resistance to cadmium, arsenic, and pesticides and to use them to purify harmful substances. Moreover, it is not yet known whether YCF1 contributes to lead resistance, and there have been no reports on the function of YHL035C protein and the transformants expressing it.

Examples of the MRP-based ABC transport gene of the fungus include YCF1 (Yeast cadmium factor1) and YHL035C genes, and MRP-based ABC transport genes having a sequence homology of 28% or more with amino acid sequences of YCF1 protein and YHL035C protein. The YCF1 and YHL035C genes or these proteins have 28% sequence identity. Further, a DNA molecule encoding at least 28% sequence homology, preferably at least 40% homology, more preferably at least 50% homology with the amino acid sequence of the YCF1 or YHL035C protein and encoding the fungal MRP system ABC transport protein It is intended to be included. For example, when comparing the amino acid sequence data of GenBank using the CLUSTRALW program, YCF 1 With YHL035C protein And BPT1 gene, YBT1 gene, and YOR1 gene having at least 28% homology on amino acid sequence. The BPT1 protein has 40% homology with YCF1 in amino acid sequence, the YBT1 protein has 51% homology with YHL035C in amino acid sequence, and the YOR1 protein has 28% homology with YCF1 in amino acid sequence. YBT1 and BPT1 are fungal MRP-based ABC transport proteins, which are known to be able to transport bile acids (Ortiz et al., (1997) Journal of Biological Chemistry 272: 15358-15365, Petrovic et al., (2000) Yeast 16: 561-571), the YOR1 protein is a fungal MRP-based ABC transport protein, which is known to be able to transport various drugs (Decottignies et al., (1998) Journal of Biological Chemistry 273: 12612). -12622 ) .

The MRP-based ABC transport protein according to the present invention is known to have a common domain structure, which is a N-terminal extension domain (N-terminal extension domain), six transmembrane domains that are fairly long at the N-terminus "first transmembrane spanning domain" consisting of domains, cytoplasmic domain "first nucleotide binding fold domain," second transmembrane domain consisting of six transmembrane domains (second transmembrane spanning domain) "and the" second nucleotide binding fold domain ", a cytoplasmic domain located at the c-terminus.

In a preferred embodiment of the invention, the MRP-based ABC transport protein of the fungus is at least 28%, preferably at least 40%, more preferably at least 50% with the amino acid sequence of the YCF1 protein of SEQ ID NO: 2 or the YHL035C protein of SEQ ID NO: 4 It has a sequence homology of more than%, the YCF1 protein or YHL035C protein is N-terminal extension domain (N-terminal extension domain), the first transmembrane spanning domain, the first nucleotide binding domain (first nucleotide binding fold domain) ), A second transmembrane spanning domain, and a domain of a second nucleotide binding fold domain, wherein each domain of the fungal MRP-based ABC transport protein is a YCF1 protein of SEQ ID NO: 2. Or at least 28% of the amino acid sequence of each domain corresponding to YHL035C protein of SEQ ID NO: 4 It may be one having a heat homology.

The present invention is a YCF1 gene comprising a sequence encoding a polypeptide of the YCF1 protein as a gene that simultaneously confers resistance and accumulation to harmful substances, examples thereof include SEQ ID NO. YCF1 polypeptide of SEQ ID NO: 2, in addition to the base sequence encoding the polypeptide, preferably, resistance to lead and accumulation, and resistance to or accumulation of at least one harmful substance selected from the group consisting of cadmium, arsenic, and herbicides. It is a YCF 1 gene containing a nucleotide sequence encoding the, more preferably is a YCF 1 gene having a nucleotide sequence of SEQ ID NO: 1. YCF1 gene is present on chromosome 6 of Saccharomyces cerevisiae . Since the YCF1 gene has a function when expressed as a protein, it has at least 28% homology, preferably at least 40% homology, more preferably at least 50% homology with the amino acid sequence of the YCF1 protein, and It is intended to include both proteins that contribute to resistance and accumulation and DNA molecules encoding them.

YCF1 protein is one of the ABC transport proteins in the vacuole membrane of yeast and is known to mitigate the toxicity of cadmium by transferring cadmium bound to glutathione in the cytoplasm into the vacuole using the energy of MgATP. (Li, ZS et al . A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae : YCF1-catalyzed transport of bis (glutathionato) cadmium. Proc. Natl. Acad. Sci. USA 94, 42-47 (1997).

Examples of genes that are resistant and / or accumulator to another harmful substance include the YHL035C gene, and a preferred example thereof is a nucleotide sequence that shows resistance to lead and encodes a polypeptide of SEQ ID NO: 4, more preferably, a sequence. Provided is a YHL035C gene comprising the nucleotide sequence of No. 3. YHL035C gene is present on chromosome 8 of Saccharomyces cerevisiae , and YHL035C protein is one of the MRP-based ABC transport proteins. Since the YHL035C gene has a function when expressed as a protein, it has at least 28% homology, preferably at least 40% homology, more preferably at least 50% homology with the amino acid sequence of the YHL035C protein, and It is intended to include both proteins that contribute to resistance and accumulation and DNA molecules encoding them.

The present invention also provides a recombinant vector comprising the gene, and preferably provides a recombinant vector comprising the YCF 1 gene or YHL035C gene. Specific examples of such recombinant vectors include pESC- YCF1, ENpCambia-YCF1, or PBI121- YCF1 recombinant vector, or pESC- YHL035C recombinant vector, and pPBI121-YHL035C. Preparation of the recombinant vector may be carried out according to a production method known to those skilled in the art.

In the present invention, the harmful substances may include heavy metals including lead, cadmium, arsenic, or the like, pesticides, and herbicides. The herbicides are generally known as low molecular weight lipophilic compounds that easily pass through the plant cell wall and inhibit plant-specific processes such as photosynthetic electron transport or biosynthetic metabolism of essential amino acids. For example, chlorosulfuron, Axidofluorfen, norflurazone, and chloro-dinitrobenzene (CDNB) can be mentioned.

Therefore, using a DNA molecule containing a nucleotide sequence encoding the polypeptides of the YCF1 protein and YHL035C protein of the present invention or DNA molecules with a homology of 28% or more, a transformation capable of accumulating harmful substances while exhibiting resistance to harmful substances The organisms can be prepared, and the produced transgenic organisms can be used to economically and conveniently purify the pollutant site.

The invention also relates to a transgenic organism transformed with a DNA molecule encoding the fungal MRP-based ATP binding cassette transport protein. The invention also relates to transformed cells, preferably plant cells, transformed with a DNA molecule encoding the fungal MRP-based ATP binding cassette transport protein. The ABC gene includes all of the above-described genes, preferred examples are YCF 1 Genes , and the YHL035C gene.

The transgenic organism is preferably a prokaryote or eukaryote, and examples thereof include plants, animals, yeast, Escherichia coli, and fungi. Transgenic plants contain heterologous DNA sequences by genetic engineering methods and are designed to be expressed in plant cells, plant tissues or plants to express heterologous DNA sequences. Plant transformants can be prepared by known techniques, with Agrobacterium tumefaciens-mediated DNA transfer being representative. More preferably, the recombinant Agrobacterium prepared by a method selected from the group consisting of electroporation, micro-particle injection or gene gun is introduced into plants by dipping. In a specific example of the present invention, a method for producing a transformed plant produces an expression cassette comprising a sequence encoding an MRP-based ABC transport protein linked to be transcribed and translated, and preparing a recombinant vector comprising the expression cassette. In addition, the recombinant vector may be introduced into a plant cell or plant tissue to prepare a transformed plant.

The plants include herbaceous rapeseed, rapeseed, lampshade, tobacco, onion, carrot, cucumber, rapeseed, olive, sweet potato, potato, cabbage, radish, lettuce, tobacco, broccoli, petunia, sunflower, lampshade, grass, rapeseed Plants, and woody plants, including willow, birch, poplar and birch, preferably poplar and cephalopods.

In a preferred embodiment of the invention, the transgenic organism may be YCF1 Arabidopsis (Accession No. KCTC10064BP), YCF1 Poplar, or YHL035C Poplar. The YCF1 Arabidopsis plant of the present invention was deposited on September 5, 2001 by the Biotechnology Research Institute Gene Bank of Yueun -gu, Yuseong-gu, Daejeon, Korea, on September 5, 2001, and was given the accession number KCTC10064BP. The YCF1 Arabidopsis plants can be asexually propagated by tissue culture, and can be grown into plants by conventional plant cell culture methods and differentiation methods. The YCF1 Arabidopsis plants are more resistant to heavy metals and other harmful substances (Fig. 6, 7, 8) and accumulating (Fig. 9) than wild type, and particularly resistant and accumulator to lead, cadmium, arsenic and pesticides Indicates.

YCF1 poplar and YHL035C poplar plants of the present invention can be asexually propagated by tissue culture, and can be grown into plants by conventional plant cell culture methods and differentiation methods. The YCF1 poplar and YHL035C poplar plants have better resistance to lead (FIG. 10, 11) than wild type.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1 Sensitivity to Lead and Cadmium of YCF1 Mutant Yeast

The wild-type yeast (DTY 165) and ycf1 mutant yeast (DTY 167, MATa ura3 leu2 his3 lys2 trp3 ycf suc2 :: hisG) to YPD liquid medium (1% yeast extract, 2% peptone, 2% dextrose) OD ₆₀₀ After incubation at 30 ° C. until reaching 1-2, the same number of yeast cells (1 × 102, 1 × 103, 1 × 104 or 1 × 105) were diluted to 1/2 with 3 mM lead Cultured at 30 ° C. in YPD solid medium for 3 days. In addition, the same culture was carried out in a YPD solid medium diluted to 1/2 with 0.1 mM cadmium. The experimental results are shown in FIG. 1.

As shown in FIG. 1, the ycf1 mutant yeast showed a decrease in growth in a medium containing 3 mM of lead compared to the native yeast, and the ycf1 mutant yeast was hardly grown in a medium containing 0.1 mM of cadmium. Thus, it was confirmed that YCF1 is a gene that makes it resistant to lead or cadmium.

Example 2: Preparation of Cloning Vectors

2-1: Cloning Vector (PESC-YCF1)

For YCF1 gene isolation, native yeast was grown for 3 hours at 3 ml YPD liquid medium at 30 ° C., followed by centrifugation (12,000 rpm, 20-60 seconds). The pellet is suspended in 400 μl of yeast lysis buffer (1 M sobitol, 0.1 M EDTA, 50 mM DTT, pH 7.5) and zymolase (5 mg / 1 ml, 0.9 M sobitol). ) 40 μl was added and left at 37 ° C. for 15-30 minutes. To this was mixed 400 ml of urea buffer (7 M urea, 0.3125 M NaCl, 0.05 M Tris-HCl (pH 8.0), 0.02 M EDTA (pH 8.0), 1% Sarcosine), a mixture of phenol / chloroform and the supernatant Separated. 1 ml of 100% ethanol was mixed in the supernatant and centrifuged (12,000 rpm, 10 minutes) to precipitate the precipitated DNA in TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0). By carrying out PCR with the the isolated DNA as a template, YCFa primer (SEQ ID NO: 3), YCFb primer (SEQ ID NO: 4), and LA taq polymerase kit (LA taq polymerase kit, Takara) was isolated YCF1 gene.

In order to demonstrate that YCF1 gene in yeast is involved in the resistance to the lead, the above-mentioned (1) a gene YCF1 the pESC-URA separated in (yeast shuttle vector, stratagene) it was cloned into the vector. In other words, the YCF1 PCR product was digested with XhoI and ScaI restriction enzymes to prepare YCF1 (Xho I / Sca I), and the pESC-URA vector was cut with Hind III, and then treated with Klenow fragment and dNTPs. Were blunted and digested with Xho I restriction enzymes. The recombinant vector pESC-YCF1 was prepared by linking the YCF1 (Xho I / Sca I) and the cut pESC-URA vector (Xho I / blunt-end) with a T4 DNA ligase.

2-2: Cloning Vector (PESC-YHL035C)

Yeast genomic DNA used in Example 2-1 as a template was used as a template, Y HL035Ca primer (SEQ ID NO: 9), Y HL035Cb primer (SEQ ID NO: 10), and LA taq polymerase kit (LA taq polymerase kit, Takara) ) Was carried out in substantially the same manner as in Example 2-1 except that Y HL035C gene was isolated by PCR.

YHL035Ca: 5'-cgacgcggccgcatgggaacggatccccttattatc-3 '

YHL035Cb: 5'-cgacgcggccgccatcatcttacttgattgcttgg-3 '

In order to express a gene in yeast Y HL035C the YHL035C gene pESC-URA (yeast shuttle vector, stratagene) was cloned into the vector. YHL035C The PCR product was cleaved with Notl and linked to the pESC-URA vector by T4 DNA ligase to prepare recombinant vector pESC- YHL035C .

Example 3: Lead and Cadmium Resistance of YCF1 Recombinant Yeast

ycf1 mutant recombinant yeast introducing the empty vector in yeast (yeast ycf1 -pESC), in which the recombinant yeast overexpressing the native YCF1 the introduction of the empty vector in yeast, recombinant yeast (wt-pESC Yeast), and mutant yeast ycf1 (ycf1 - YCF1 yeast) were prepared respectively and tested for lead or cadmium resistance.

3-1: Preparation of YCF1 Recombinant Yeast

Inoculate yeast into 3 ml liquid YPD medium and incubate at 30 ° C. for 12 hours, then add 0.5 ml of culture solution to 10 ml liquid YPD medium, and incubate for 6-8 hours at 30 ° C. until OD ₆₀₀ 0.5-0.8. It was. The culture was centrifuged (1,500 rpm, 5 minutes) to obtain yeast, and the yeast was suspended in 5 ml of buffer (0.1 M LiOAc, TE, pH 7.5) and then centrifuged. Centrifuged yeast was suspended in buffer (0.1 M LiOAc, TE (pH 7.5)) and incubated for 1 hour in a stirred incubator at 30 ° C., and pESC, pESC-YCF1 or pESC-YCF1 plasmid and salmon DNA (salmon tested DNA) were added. Incubated at 30 ° C. for 30 minutes. 0.7 ml of buffer (40% PEG 3300, 0.1 M LiOAc, TE (pH 7.5)) was mixed with the cultured yeast and shaken at 30 ° C. for 1 hour. The mixture was then placed at 42-45 ° C. for 5 minutes, subjected to thermal shock, and then centrifuged (2500 rpm, 5 minutes) to obtain yeast. Wash yeast with 1 ml of TE buffer (pH 7.5), suspend with 0.2 ml of water or TE buffer (pH 7.5) and incubate for 2-3 days in selection medium (CM Ura ⁻ ) to transform the yeast ( ycf1 -pESC yeast, wt-pESC yeast, ycf1 - YCF1 yeast).

3-2: Resistance of Lead and Cadmium to Recombinant Yeast

The ycf1- pESC yeast, wt-pESC yeast, and ycf1 - YCF1 yeast prepared above were galactose medium containing 1.8 mM of lead (2% galactose, 1% 0.17% YNB, 0.13% dropout powder, 0.5% amonium). sulfate) or 50 uM of cadmium added galactose medium. Control was incubated with galactose medium without heavy metals. Growth of each recombinant ycf1- pESC yeast, wt-pESC yeast, and ycf1 - YCF1 yeast in lead or cadmium-added medium is shown in the photo in FIG.

FIG ycf1 was overexpressed in the YCF1 ycf1 mutant yeast at 2 - YCF1 yeast play an important role in ycf1 -pESC yeast or wt-pESC shown to better growth than yeast, YCF1 gene cadmium resistance in medium containing lead, as well as Cd We also confirmed previous results, and also newly confirmed that this gene is important for lead resistance.

3-3: of recombinant yeast YCF1  Expression

The Northern blot to confirm expression of YCF1 was performed in yeast YCF1-one exemplary ycf1 -pESC yeast, yeast pESC-wt, and ycf1 prepared in Example 3.

Recombinant yeasts were pulverized with liquid nitrogen and total RNA extraction buffer (0.25 M Tris HCl pH 9.0, 0.25 M NaCl, 0.05 M EDTA, 0.345 M p-Aminosalicylic acid, 0.027 M triisopropyl naphthalene sulfonic acid, 0.02% β- mercaptoethanol, 0.024% phenol) and phenol / chloroform were mixed in the same amount. The supernatant was transferred to a new tube by centrifugation at 12,000 rpm for 10 minutes and 400 μl of isopropanol was added. The RNA was precipitated by centrifugation at 1,2000 rpm for 10 minutes and then dissolved in DEPC-treated water and stored in a freezer.

To perform Northern blot, 30 μg of RNA was electrophoresed on agarose gel for RNA and RNA was transferred to nylon membrane. The nylon membrane was reacted by stirring at 42 ° C. for 2 hours on a hybridization reaction solution (6 × SSPE, 0.5% SDS, 10% PEG, 1% nonfat milk, 50% formamide). Then YCF1 labeled with ³² P dCTP was added and reacted at 42 ° C. for 12 hours. After completion of the hybridization, the nylon membrane was washed twice with buffer (2 × SSPE and 0.5% SDS), washed with buffer (0.2 × SSPE, 0.5% SDS), dried, and then exposed to X-ray film. The experimental results are shown in B of FIG. 2.

2B is a northern blot photograph of three recombinant yeasts, and it can be seen that ycf1 - YCF1 yeast overexpresses YCF1 mRNA. That is, the lead and cadmium resistance of the ycf1 - YCF1 yeast shown in Example 3-2 proved to be due to overexpression of YCF1 .

3-4: Lead and Cadmium Resistant Mechanisms

Experiments were conducted to determine whether lead and cadmium resistance by the YCF1 gene was due to intracellular accumulation of heavy metals or by extracellular excretion.

Three yeasts ( ycf1- pESC yeast, wt-pESC yeast, and ycf1 - YCF1 yeast) were incubated for one day in a half galactose solid medium supplemented with 1.5 mM lead or 15 uM cadmium, and the cultured yeast Obtained by scraping. The obtained yeast was dissolved in 1 ml of concentrated nitric acid and dissolved at 200 ° C. for about 6 hours, diluted with 10 ml of 0.5 N nitric acid, and the amount of heavy metals contained in the yeast was measured by an atomic absorption spectrometer (AAS). Measurement results for ycf1 -pESC yeast, wt-pESC yeast, and ycf1 - YCF1 yeast are shown graphically in FIG.

As a result, wt-pESC yeast and ycf1 - YCF1 yeast had higher accumulation of lead and cadmium than ycf1 -pESC yeast, and in particular, wt-pESC yeast and ycf1 - YCF1 yeast were about 2 times higher than ycf1 -pESC yeast. Lead accumulation was shown. Therefore, the lead and cadmium resistance by the YCF1 gene was confirmed to be due to the intracellular accumulation of the heavy metal.

Example 4: Lead Resistance of YLH035C Recombinant Yeast

4-1: Preparation of Recombinant Yeast

By using pESC-YHL035C prepared in Example 2 in substantially the same manner as in Example 3-1, recombinant yeast (yhl035c-v yeast) in which an empty vector was introduced into yhl035c mutant yeast, and a blank vector in natural yeast The recombinant yeast (wt-v yeast) and the recombinant yeast (YHL035C yeast) which overexpressed YHL035C in y hl035c mutant yeast were prepared, respectively, and the transformed yeasts (wt-v yeast, yhl035c-v yeast, and YHL035C yeast). ) Was selected. The resistance to lead was performed using the recombinant yeast.

4-2: Lead Resistance Test of Recombinant Yeast

Lead resistance tests were performed on wt-v yeast, yhl035c-v yeast and YHL035C yeast in substantially the same manner as in Example 3-2. The growth of each of the recombinant wt-v yeast, yhl035c-v yeast and YHL035C yeast in lead-added medium is shown in the photo in FIG.

As shown in Figure 4, yhl035c mutant yeast yhl035c-v was more sensitive than wt-v yeast in a medium containing 1.8 mM lead. However, YHL035C yeast, which expressed YHL035C in yhl035c mutant yeast, regained resistance to lead, showing growth similar to that of wt-v yeast in a medium containing 1.8 mM lead. Thus, the new YHL035C gene plays an important role in lead resistance.

Example 5: Preparation of Transgenic Plants

5-1: Preparation of YCF1 Vector and YHL035C Vector for Plant Transformation

In order to introduce a gene into a plant, YCF1, by inserting a BamH I / SnaB I fragment of 4.6 kb of the YCF1 pESC-YCF1 plasmid (BamH I / Sma I) was prepared in PBI121-YCF1 PBI121 vector. EnPCAMBIA1302-YCF1 vectors were made to enhance the expression of the YCF1 gene in plants. The PCAMBIA1302 vector was cut with Sal I restriction enzyme and treated with Klenow fragment and dNTPs to blunt the end of the vector and insert a 35S enhancer (BamH I / blunt-end) into the vector cut with BamH I to prepare an EnPCAMBIA1302 vector. did. The EnPCAMBIA1302-YCF1 vector was made by inserting the YCF1 gene (BamHI / blunt-end) into the BglII / PmlI site of the EnPCAMBIA1302 vector.

The PBI121-YCF1 vector and EnPCAMBIA1302-YCF1 were introduced into E. coli as an electroporator (BIO-RAD) and incubated in LB solid medium. Single colonies were inoculated in 3 ml LB (Amp) liquid medium, incubated for 12-16 hours and then centrifuged to obtain transformed E. coli. E. coli was suspended by adding 100 ul of solution I (50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA) and 200 ul of solution II (1% SDS, 0.2 N NaOH) was added thereto. The mixture was mixed lightly and put in iced water for 5 minutes. 150 ul of solution III (5 M Potassium acetate) was added to the mixture, mixed slowly 3-5 times, and centrifuged (12,000 rpm, 10 minutes) to obtain a supernatant. 100% ethanol was mixed in the supernatant to precipitate DNA, separated and dried. The obtained PBI121-YCF1 plasmid DNA was dissolved in TE buffer and digested with BamHI and EcoRI restriction enzymes to yield the YCF1 gene. It was confirmed that the orientation was inserted correctly.

In order to introduce the YHL035C gene into the plant, pBI121-YHL035C vector was prepared by substantially the same method as the YCF1 vector for plant transformation, except that the YHL035C gene was cut out from the pESC-YHL035C vector prepared in Example 2, An EcoICR1 restriction enzyme site was made, and the pBI121-YHL035C vector was prepared by inserting it into a pBI121 vector cut with SacI and SmaI restriction enzymes.

5-2: Transformation Arabidopsis Plant Preparation

The PBI121-YCF1 and EnPCAMBIA1302-YCF1 vectors prepared in Example 5-1 were introduced into Agrobacterium LBA4404 as an electroporator (BIO-RAD). Transformed Agrobacterium was screened in Murashige-Skoog (MS) medium containing kanamycin and dipping method; Clough, SJ, and Bent, AF, Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana.Plant J. 16, 735-743 (1988)) infected the flowers of Arabidopsis thaliana and introduced the YCF1 gene into the plant. After 4-5 weeks, the Arabidopsis seeds were harvested, and the plants transformed with the PBI121-YCF1 vector were kanamycin, and the plants transformed with the EnPCAMBIA1302-YCF1 vector were selected as YCF1 Arabidopsis plants with hygromycin.

A total of five YCF1 Arabidopsis plants were obtained, and experiments were performed to determine whether the expression level of YCF1 mRNA and the length of the expressed mRNA were 700 bp of the natural type. First, mRNA was extracted from five YCF1 Arabidopsis plants, and RT-PCR was used as a primer of SEQ ID NO: 5 / SEQ ID NO: 6. The RT-PCR results are shown in FIG. 5A. As shown in FIG. 5A, the expression of 700 bp of YCF1 was confirmed in plants 1, 3, 4, and 5, and in particular, the expression of YCF1 was high in plants 4 and 5.

Sudden blot was performed on the RT-PCR product and the results are shown in B of FIG. 5. As shown in B of FIG. 5, as in the result of A of FIG. 5, YCF1 was expressed in plants 1, 3, 4, and 5, and it was confirmed that native YCF1 mRNA was completely transcribed. The YCF1 Aeggijang plants with high YCF1 expression were deposited on September 5, 2001 with the Biotechnology Research Institute Gene Bank, 52, Eeun -dong, Yuseong-gu, Daejeon, Korea, and received the accession number KCTC10064BP.

5-3: Preparation of Transgenic Poplars

As a transgenic plant, Bonghua 1, a populus alba x P. glandulosa clone, which was not selected in the open air, was propagated. In order to secure sterile materials in the inflight of the poplars, stems were cut from the brine preserved at the cultivation site of the Forest Breeding Laboratory, and then sterilized with 70% ethanol (5 minutes) and 2% NaCl (20 minutes). Stem was used as a transformation sample.

The process of transformation, callus induction, and stem induction using Agrobacterium containing the target gene was carried out by Noh et al. (Poplar's genetic engineering (2002), by Noeun Woon, ISBN # 89-8176-098-5 93520). Agrobacterium tumefaciens containing the PBI121-YCF1 and EnPCAMBIA1302-YCF1 vectors prepared in Example 5-2 were inoculated in LB medium and incubated overnight at 30 ° C., centrifuged at 1,000 g for 10 minutes, and then the medium was poured out and pellets Was suspended in 0.85% NaCl solution. After the suspension was poured into a petri dish, the interstitial tissues of in-flight cultured poplars were dipped for 20 minutes. Then, put it between two sterilized hygroscopic filter papers and press lightly to remove excess agrobacteria, callus induction medium without antibiotics (MS + 2.4-D 1.0mg / L, BA 0.1mg / L, NAA 0.01mg / L) (Murashige and Skoog. 1962) followed by two days of incubation. Transformed cells were selected using selection medium containing 50 mg / L kanamycin and 500 mg / L cefotaxime.

Callus formed was induced by adding 50 mg / L kanamycin to stem induction medium (WPM + zeatin 1.0 mg / L, BA 0.1 mg / L, NAA 0.01 mg / L) (Lloyd and McCown, 1981). Once the stem was induced, the stem was elongated by passage in MS basal medium to which 50 mg / L kanamycin was added, and the root was induced by adding 0.2 mg / L of IBA to the same medium.

Example 6 Growth of Transgenic Arabidopsis Plants in the Presence of Hazardous Substances

Example 5 YCF1 Arabidopsis plants lead (0.9,1, 1.1 mM), cadmium (50, 60,70 μM), herbicide chloro-dinitrobenzene (CDNB) (60 μM), arsenic arsenic (50 μM) was cultured in 1/2 MS medium containing the growth was confirmed. In addition, Arabidopsis plants were compared with the plants transformed with the empty vector (PBI) and wild type (wt) as a control. The experimental results are shown in FIGS. 6 to 8.

Figure 6 is a photograph and graph showing the growth of YCF1 transgenic Arabidopsis plants. As shown in FIG. 6, when the YCF1 Arabidopsis and control plants were grown for three weeks in a medium containing lead, the leaves and roots of the YCF1 Arabidopsis ( 1,3,4,5 ) were PBI empty vector transgenic plants. And less whitening than wild type (wt), and root growth was good (A, B, D). In addition, when YCF1 and wild type (wt) Arabidopsis plants were grown in various concentrations of cadmium medium, wild type (wt) Arabidopsis plants were bleached with yellow leaves and shorter than roots (C, E).

Figure 7 is a photograph showing the resistance of YCF1 transgenic Arabidopsis plants to arsenic. Wild type Arabidopsis (wt) grown for 3 weeks in medium containing 60 μM 5 arsenic showed little growth, but YCF1 transformants (1, 2, 3, 4) were better than wild type.

8 is a photograph showing the resistance of YCF1 transgenic Arabidopsis plants to CDNB . When the YCF1 Arabidopsis and control plants were grown for two months in a medium containing 60 μM of CDNB, the wild type plants did not germinate well and almost died, but the YCF1 Arabidopsis plants grew almost as well as normal plants.

Thus, the YCF1 transgenic Arabidopsis plants of Example 5 were found to be resistant to lead, cadmium, arsenic, and herbicides.

Example 7 Heavy Metal Accumulation of Transgenic Arabidopsis Plants

The YCF1 Arabidopsis plant obtained in Example 5 and PBI, a native Arabidopsis plant, were cultured in 1/2 MS medium containing lead (0.75 mM) and cadmium (70 μM) for 3 weeks to confirm heavy metal accumulation. Accumulated amount of lead or cadmium was confirmed by the same experimental method as in Example 6, the results are shown in Figure 9, A of Figure 9 shows the accumulation of lead and B represents the accumulation of lead and cadmium.

As a result of the experiment, as shown in FIG. 9A, the YCF1 Arabidopsis plants exhibited 2 to 1.4 times higher lead accumulation than the native Arabidopsis and PBI. In addition, as shown in FIG. 9B, YCF1 Arabidopsis plants showed two to three times the accumulation compared to PBI.

Thus, it was confirmed that YCF1 Arabidopsis plants have higher lead and cadmium accumulation than native Arabidopsis.

Example 8: YCF1  Mechanisms to Resist Hazardous Substances in Transgenic Plants

YCF1 transgenic plants were resistant to lead, cadmium, arsenic and herbicides, and accumulated on lead and cadmium. To determine whether this phenomenon was caused by the heavy metals contained in the vacuoles, YCF1 protein was extracted from YCF1 transgenic plants and wild-type plants (wt) to carry out cadmium and herbicide transport experiments. The results of transport experiments for cadmium (Cd + GSH) and herbicides (DNB-GS) in vacuoles of the YCF1 transgenic plants and natural plants are shown in Table 1 below.

 Ingestion of GS-related compounds (unit: pmol / 1 μl vacuole / 20min)  compound Natural plant (wt) YCF1 Transgenic Plant MgATP + MgATP MgATP + MgATP DNB-GS 21 ± 1.1 28 ± 1.3 24 ± 0.9 33 ± 1.2 Cd + GSH 75 ± 3.3 104 ± 4.9 71 ± 3.4 177 ± 9.8 GSH - 70 ± 2.2 - 69 ± 1.1

As shown in Table 1, since YCF1 uses MgATP as energy for transporting materials, the amount of cadmium (Cd + GSH) and herbicide (DNB-GS) in the -MgATP group is YCF1 transformant and natural plant. Did not make a difference. However, when MgATP was added, the YCF1 transformant vacuole was about 1.7 times higher than the wt vacuole in the case of cadmium (Cd + GSH). For herbicides (DNB-GS), the YCF1 transformants were slightly higher in content than the native form.

YCF1 transgenic Arabidopsis plants were found to be more resistant and accumulating to lead, cadmium, arsenic and herbicides than their native counterparts. This phenomenon confirmed that the YCF1 protein expressed in the vacuoles of YCF1 transgenic Arabidopsis plants stabilized lead, cadmium, arsenic and herbicides in the vacuoles, thereby making them resistant and accumulating.

Example 9: Growth of Transgenic Poplar Plants

9-1: YCF1 Transgenic Poplar Plant

YCF1 Poplar of Example 5 The leaves and stem pieces of the plant were placed on a callus induction medium containing 500 ppm of lead, and cultured for 2 weeks to confirm the growth. Wild-type poplar (wt) plants that were not transformed were treated with the same method and compared with the control group, and the experimental results are shown in FIG. 10. 10 is a photograph and a graph showing the growth of YCF1 transgenic poplar plants.

As shown in FIG. 10, when the YCF1 poplar and control plants were grown for two weeks in a medium containing lead, the stem fragments of the YCF1 poplar (1, 2, 3, 4) grew about 2 times wild type (wt). It was about twice as high (A, C), and leaf fragments of YCF1 poplar (1,2,3) were less browned than wild type (wt) and retained the color of blue chlorophyll (B).

9-2: YHL035C Transgenic Poplar Plant

The YHL035C transgenic poplar plants of Example 5 were transferred to pots and grown into pot seedlings. The pot seedlings were transferred to soil soaked in 500 ppm solution of Pb (NO) ₃ for resistance testing. The experimental results are shown in FIG. 11.

FIG. 11 is a photograph showing that wild type poplar (wt) grown for 4 weeks in the soil containing lead was hardly grown, but YHL035C transformants were much better than wild type. Therefore, it was confirmed that the YCF1 transgenic poplar plant and the YHL035C poplar plant of Example 5 were resistant to lead.

As mentioned above, the YCF1 gene of the present invention is to improve the resistance and accumulation of heavy metals and other harmful substances in the organism, the YHL035C gene is to improve the resistance to lead, the transformation that expresses the gene It can be used for the purpose of preparing a sieve to purify the environment contaminated with hazardous substances. The transformant of the present invention can be used for environmentally friendly and economical environmental purification.

1 is a photograph confirming the growth of yeast susceptibility to lead and cadmium of YCF1 mutant yeast,

2 is a ycf1 -pESC yeast, wt-pESC Yeast and photo checking the resistance of the lead or cadmium ycf1-YCF1 yeast growth so (A), and Northern blot results (B),

3 is a graph comparing lead or cadmium accumulation of ycf1 -pESC yeast, wt-pESC yeast and ycf1 - YCF1 yeast,

4 is a photograph confirming the sensitivity of the YHL035C mutant yeast to the growth of the yeast,

5 is a photograph confirming the expression of YCF1 in YCF1 transgenic Arabidopsis by RT-PCR,

6 is a photograph confirming the growth of lead and cadmium resistance of YCF1 transgenic Arabidopsis plants,

Figure 7 is a photograph confirming the growth of the resistance to arsenic of YCF1 transgenic Arabidopsis plants,

8 is a photograph confirming the growth resistance of the herbicide of YCF1 transgenic Arabidopsis plants,

9 is a graph analyzing lead and cadmium accumulation of YCF1 transgenic Arabidopsis plants.

10 is a photograph confirming the growth of lead resistance of YCF1 transgenic poplar plants,

11 is a photograph confirming the growth of the lead resistance of YHL035C transgenic poplar plants.

<110> POSCO Pohang University of Science and Technology <120> Transgenic organism expressing fungal MRP-like ABC transporters <130> DPP2002-2952KR <150> KR10-2001-0063802 <151> 2001-10-16 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 4548 <212> DNA <213> ycf1 gene <400> 1 atggctggta atcttgtttc atgggcctgc aagctctgta gatctcctga agggtttgga 60 cctatatcct tttacggtga ctttactcaa tgcttcatcg acggtgtgat cctaaatcta 120 tcagcaattt tcatgataac cttcggtatc agagatttag ttaacctttg caagaaaaaa 180 cactctggca tcaaatatag gcggaattgg attattgtct ctaggatggc actagttctg 240 ttggagatag cgtttgtttc acttgcgtct ttaaatattt ctaaagaaga agcggaaaac 300 tttaccattg taagtcaata tgcttctaca atgttatctt tatttgttgc tttagcctta 360 cactggatag aatacgatag atcagttgta gccaatacgg tacttttatt ctattggctt 420 tttgaaacat tcggtaattt tgctaaacta ataaatattc taattagaca cacctacgaa 480 ggcatttggt attccggaca aacgggtttc atactaacgt tattccaagt aataacatgt 540 gccagtatcc tgttacttga agctcttcca aagaagccgc taatgccaca tcaacacata 600 catcaaactt taacaagaag aaaaccaaat ccatacgata gcgcaaacat attttccagg 660 attaccttct cttggatgtc aggtttgatg aaaactggct atgaaaaata cttagtggaa 720 gcagatttat ataaattacc gaggaacttt agtagtgaag aactctctca aaaattggag 780 aaaaactggg aaaatgagtt gaagcaaaaa tcaaatcctt cattatcatg ggctatatgc 840 agaacttttg gatctaaaat gcttttagcc gcattcttta aagcaattca tgatgttcta 900 gcatttactc aaccacaact actaaggatt ttaatcaagt tcgtcacaga ctataacagt 960 gagagacagg atgaccattc ttctcttcaa gggtttgaaa ataaccaccc acaaaaatta 1020 cccattgtaa gagggttttt gattgcgttt gctatgtttc tggtgggctt tactcagaca 1080 tctgtcctgc atcaatattt cctgaatgtc ttcaacacag gcatgtatat taagagcgcc 1140 ctaacggctt taatatatca aaaatcctta gtgctatcta atgaggcttc tggactttcc 1200 tctaccggtg acattgtcaa tctcatgagt gtggatgttc aaaaattaca agatttaaca 1260 caatggctaa atttaatatg gtcagggcct tttcaaatca ttatttgctt atattctctg 1320 tataagttgt tgggaaattc catgtgggtt ggcgtgatta tactagttat tatgatgcca 1380 ttgaactcat ttttgatgag gatacaaaag aagttgcaaa aatcccagat gaagtacaaa 1440 gatgaaagga cccgtgttat aagtgaaata ctaaacaata ttaaatcttt gaagttatat 1500 gcatgggaga agccttatag ggaaaagcta gaagaagtaa gaaataacaa agagttaaaa 1560 aatcttacaa aactaggatg ttatatggct gtgacaagtt ttcagttcaa tatagtacca 1620 ttccttgttt catgttgtac ctttgctgta tttgtttata ctgaggatag agcattgact 1680 actgacttag ttttccctgc tttgactctg ttcaacctgc tctcattccc actaatgatt 1740 attcctatgg ttttaaattc ttttatcgaa gcttctgttt ctattggtag attatttaca 1800 ttctttacca atgaagagct acaaccagat tcggttcagc gtttaccaaa agtaaaaaat 1860 attggcgatg tagccattaa cattggagat gatgctacct ttttatggca acggaaaccg 1920 gaatacaaag tagccttaaa gaatattaat ttccaagcta aaaaaggaaa tttgacctgt 1980 attgttggta aagttggcag tggtaaaaca gctctattgt catgcatgtt aggtgatcta 2040 ttcagggtta aaggtttcgc caccgttcat ggttctgttg cttatgtttc acaagttcca 2100 tggataatga atggtactgt aaaggaaaac attttatttg ggcatagata cgacgcggaa 2160 ttttacgaaa aaacgatcaa ggcctgtgcg ttaactattg atcttgcaat tttgatggat 2220 ggagataaga cattagttgg cgagaaaggg atctccttat ctggaggaca aaaagctcgt 2280 ttgtctttag caagagcagt ttatgcgaga gctgacactt atttacttga tgatcctttg 2340 gcagctgttg atgaacacgt tgccaggcac ttgatcgaac atgtgttggg tccaaatggt 2400 ttattacata caaaaacgaa ggtattagcc actaataagg tgagcgcgtt atccatcgca 2460 gattctattg cattattaga taatggagaa atcacacagc agggtacata tgatgagatt 2520 acgaaggacg ctgattcgcc attatggaaa ttgctcaaca actatggtaa aaaaaataac 2580 ggtaagtcga atgaattcgg tgactcctct gaaagctcag ttcgagaaag tagtatacct 2640 gtagaaggag agctggaaca actgcagaaa ttaaatgatt tggattttgg caactctgac 2700 gccataagtt taaggagggc cagtgatgca actttgggaa gcatcgattt tggtgacgat 2760 gaaaatattg ctaaaagaga gcatcgtgaa cagggaaaag taaagtggaa catttaccta 2820 gagtacgcta aagcttgcaa cccgaaaagc gtttgtgtat tcatattgtt tattgttata 2880 tcgatgttcc tctctgttat gggtaacgtt tggttgaaac attggtctga agttaatagc 2940 cgctatggat ctaatccaaa tgccgcgcgt tacttggcca tttattttgc acttggtatt 3000 ggttcagcac tggcaacatt aatccagaca atcgttctct gggttttttg taccattcat 3060 gcctccaaat atttacacaa cttgatgaca aactctgtgt tgagagcccc aatgacgttt 3120 tttgaaacaa caccaatcgg tagaattcta aacagattct caaatgacat atacaaagtg 3180 gatgctttat taggaagaac attttctcag tttttcgtca atgcagtgaa agtcacattc 3240 actattacgg ttatctgtgc gacgacatgg caatttatct tcattatcat tccactaagt 3300 gtgttttaca tctactacca gcagtattac ctgagaacat caagggagtt gcgtcgttta 3360 gactctatta ctaggtctcc aatctactct catttccaag agactttggg tggccttgca 3420 acggttagag gttattctca acagaaaagg ttttcccaca ttaatcaatg ccgcattgat 3480 aataacatga gtgcgttcta tccctctatc aatgctaacc gttggctagc atataggttg 3540 gaacttattg gttcaattat cattctaggt gctgcaactt tatccgtttt tagactaaaa 3600 caaggcacat taacggcagg tatggtgggt ttatcattaa gctatgcttt acaaatcact 3660 caaacgttaa attggattgt tagaatgact gtggaagttg aaacgaatat tgtttcagtg 3720 gaaagaataa aggaatatgc tgatttgaag agcgaggcac ctttaatagt tgaaggccac 3780 agaccaccca aagaatggcc gagccagggt gatataaagt ttaataatta ttccactcgt 3840 tataggccgg agcttgatct tgttctgaag cacattaata tacacattaa accaaatgaa 3900 aaagttggta tcgtgggtag aacgggtgcg ggaaaatcct cattaacgct agcattattc 3960 aggatgattg aggctagcga gggaaacatc gtaatcgaca acattgccat caacgagatt 4020 gggttatatg atttgagaca taaattgtca atcatacctc aggattctca agtttttgag 4080 ggcactgttc gtgagaacat tgatcccatt aaccaataca ctgatgaagc tatttggagg 4140 gcattggaac tttctcattt gaaagaacac gtgctatcaa tgagcaatga cggattagat 4200 gcccaactaa ccgaaggtgg tggcaactta agtgttggac aaagacaatt attatgtctt 4260 gcaagagcaa tgttggttcc atcaaagatt ttggtgcttg atgaagccac ggccgcagtc 4320 gacgtggaga cagataaagt cgtccaagag acgattcgta ctgctttcaa ggacagaact 4380 atcttgacca tcgcgcatag actgaacacg ataatggaca gtgatagaat catagtgttg 4440 gacaatggta aagtagccga gtttgactct ccgggccagt tattaagtga taacaaatca 4500 ttgttctatt cactgtgcat ggaggctggt ttggtcaatg aaaattaa 4548 <210> 2 <211> 1515 <212> PRT <213> Ycf1 protein <400> 2 Met Ala Gly Asn Leu Val Ser Trp Ala Cys Lys Leu Cys Arg Ser Pro 1 5 10 15 Glu Gly Phe Gly Pro Ile Ser Phe Tyr Gly Asp Phe Thr Gln Cys Phe 20 25 30 Ile Asp Gly Val Ile Leu Asn Leu Ser Ala Ile Phe Met Ile Thr Phe 35 40 45 Gly Ile Arg Asp Leu Val Asn Leu Cys Lys Lys Lys His Ser Gly Ile 50 55 60 Lys Tyr Arg Arg Asn Trp Ile Ile Val Ser Arg Met Ala Leu Val Leu 65 70 75 80 Leu Glu Ile Ala Phe Val Ser Leu Ala Ser Leu Asn Ile Ser Lys Glu 85 90 95 Glu Ala Glu Asn Phe Thr Ile Val Ser Gln Tyr Ala Ser Thr Met Leu 100 105 110 Ser Leu Phe Val Ala Leu Ala Leu His Trp Ile Glu Tyr Asp Arg Ser 115 120 125 Val Val Ala Asn Thr Val Leu Leu Phe Tyr Trp Leu Phe Glu Thr Phe 130 135 140 Gly Asn Phe Ala Lys Leu Ile Asn Ile Leu Ile Arg His Thr Tyr Glu 145 150 155 160 Gly Ile Trp Tyr Ser Gly Gln Thr Gly Phe Ile Leu Thr Leu Phe Gln 165 170 175 Val Ile Thr Cys Ala Ser Ile Leu Leu Leu Glu Ala Leu Pro Lys Lys 180 185 190 Pro Leu Met Pro His Gln His Ile His Gln Thr Leu Thr Arg Arg Lys 195 200 205 Pro Asn Pro Tyr Asp Ser Ala Asn Ile Phe Ser Arg Ile Thr Phe Ser 210 215 220 Trp Met Ser Gly Leu Met Lys Thr Gly Tyr Glu Lys Tyr Leu Val Glu 225 230 235 240 Ala Asp Leu Tyr Lys Leu Pro Arg Asn Phe Ser Ser Glu Glu Leu Ser 245 250 255 Gln Lys Leu Glu Lys Asn Trp Glu Asn Glu Leu Lys Gln Lys Ser Asn 260 265 270 Pro Ser Leu Ser Trp Ala Ile Cys Arg Thr Phe Gly Ser Lys Met Leu 275 280 285 Leu Ala Ala Phe Phe Lys Ala Ile His Asp Val Leu Ala Phe Thr Gln 290 295 300 Pro Gln Leu Leu Arg Ile Leu Ile Lys Phe Val Thr Asp Tyr Asn Ser 305 310 315 320 Glu Arg Gln Asp Asp His Ser Ser Leu Gln Gly Phe Glu Asn Asn His 325 330 335 Pro Gln Lys Leu Pro Ile Val Arg Gly Phe Leu Ile Ala Phe Ala Met 340 345 350 Phe Leu Val Gly Phe Thr Gln Thr Ser Val Leu His Gln Tyr Phe Leu 355 360 365 Asn Val Phe Asn Thr Gly Met Tyr Ile Lys Ser Ala Leu Thr Ala Leu 370 375 380 Ile Tyr Gln Lys Ser Leu Val Leu Ser Asn Glu Ala Ser Gly Leu Ser 385 390 395 400 Ser Thr Gly Asp Ile Val Asn Leu Met Ser Val Asp Val Gln Lys Leu 405 410 415 Gln Asp Leu Thr Gln Trp Leu Asn Leu Ile Trp Ser Gly Pro Phe Gln 420 425 430 Ile Ile Ile Cys Leu Tyr Ser Leu Tyr Lys Leu Leu Gly Asn Ser Met 435 440 445 Trp Val Gly Val Ile Ile Leu Val Ile Met Met Pro Leu Asn Ser Phe 450 455 460 Leu Met Arg Ile Gln Lys Lys Leu Gent Lys Ser Gln Met Lys Tyr Lys 465 470 475 480 Asp Glu Arg Thr Arg Val Ile Ser Glu Ile Leu Asn Asn Ile Lys Ser 485 490 495 Leu Lys Leu Tyr Ala Trp Glu Lys Pro Tyr Arg Glu Lys Leu Glu Glu 500 505 510 Val Arg Asn Asn Lys Glu Leu Lys Asn Leu Thr Lys Leu Gly Cys Tyr 515 520 525 Met Ala Val Thr Ser Phe Gln Phe Asn Ile Val Pro Phe Leu Val Ser 530 535 540 Cys Cys Thr Phe Ala Val Phe Val Tyr Thr Glu Asp Arg Ala Leu Thr 545 550 555 560 Thr Asp Leu Val Phe Pro Ala Leu Thr Leu Phe Asn Leu Leu Ser Phe 565 570 575 Pro Leu Met Ile Ile Pro Met Val Leu Asn Ser Phe Ile Glu Ala Ser 580 585 590 Val Ser Ile Gly Arg Leu Phe Thr Phe Phe Thr Asn Glu Glu Leu Gln 595 600 605 Pro Asp Ser Val Gln Arg Leu Pro Lys Val Lys Asn Ile Gly Asp Val 610 615 620 Ala Ile Asn Ile Gly Asp Asp Ala Thr Phe Leu Trp Gln Arg Lys Pro 625 630 635 640 Glu Tyr Lys Val Ala Leu Lys Asn Ile Asn Phe Gln Ala Lys Lys Gly 645 650 655 Asn Leu Thr Cys Ile Val Gly Lys Val Gly Ser Gly Lys Thr Ala Leu 660 665 670 Leu Ser Cys Met Leu Gly Asp Leu Phe Arg Val Lys Gly Phe Ala Thr 675 680 685 Val His Gly Ser Val Ala Tyr Val Ser Gln Val Pro Trp Ile Met Asn 690 695 700 Gly Thr Val Lys Glu Asn Ile Leu Phe Gly His Arg Tyr Asp Ala Glu 705 710 715 720 Phe Tyr Glu Lys Thr Ile Lys Ala Cys Ala Leu Thr Ile Asp Leu Ala 725 730 735 Ile Leu Met Asp Gly Asp Lys Thr Leu Val Gly Glu Lys Gly Ile Ser 740 745 750 Leu Ser Gly Gly Gln Lys Ala Arg Leu Ser Leu Ala Arg Ala Val Tyr 755 760 765 Ala Arg Ala Asp Thr Tyr Leu Leu Asp Asp Pro Leu Ala Ala Val Asp 770 775 780 Glu His Val Ala Arg His Leu Ile Glu His Val Leu Gly Pro Asn Gly 785 790 795 800 Leu Leu His Thr Lys Thr Lys Val Leu Ala Thr Asn Lys Val Ser Ala 805 810 815 Leu Ser Ile Ala Asp Ser Ile Ala Leu Leu Asp Asn Gly Glu Ile Thr 820 825 830 Gln Gln Gly Thr Tyr Asp Glu Ile Thr Lys Asp Ala Asp Ser Pro Leu 835 840 845 Trp Lys Leu Leu Asn Asn Tyr Gly Lys Lys Asn Asn Gly Lys Ser Asn 850 855 860 Glu Phe Gly Asp Ser Ser Glu Ser Ser Val Arg Glu Ser Ser Ile Pro 865 870 875 880 Val Glu Gly Glu Leu Glu Gln Leu Gln Lys Leu Asn Asp Leu Asp Phe 885 890 895 Gly Asn Ser Asp Ala Ile Ser Leu Arg Arg Ala Ser Asp Ala Thr Leu 900 905 910 Gly Ser Ile Asp Phe Gly Asp Asp Glu Asn Ile Ala Lys Arg Glu His 915 920 925 Arg Glu Gln Gly Lys Val Lys Trp Asn Ile Tyr Leu Glu Tyr Ala Lys 930 935 940 Ala Cys Asn Pro Lys Ser Val Cys Val Phe Ile Leu Phe Ile Val Ile 945 950 955 960 Ser Met Phe Leu Ser Val Met Gly Asn Val Trp Leu Lys His Trp Ser 965 970 975 Glu Val Asn Ser Arg Tyr Gly Ser Asn Pro Asn Ala Ala Arg Tyr Leu 980 985 990 Ala Ile Tyr Phe Ala Leu Gly Ile Gly Ser Ala Leu Ala Thr Leu Ile 995 1000 1005 Gln Thr Ile Val Leu Trp Val Phe Cys Thr Ile His Ala Ser Lys Tyr 1010 1015 1020 Leu His Asn Leu Met Thr Asn Ser Val Leu Arg Ala Pro Met Thr Phe 1025 1030 1035 1040 Phe Glu Thr Thr Pro Ile Gly Arg Ile Leu Asn Arg Phe Ser Asn Asp 1045 1050 1055 Ile Tyr Lys Val Asp Ala Leu Leu Gly Arg Thr Phe Ser Gln Phe Phe 1060 1065 1070 Val Asn Ala Val Lys Val Thr Phe Thr Ile Thr Val Ile Cys Ala Thr 1075 1080 1085 Thr Trp Gln Phe Ile Phe Ile Ile Ile Pro Leu Ser Val Phe Tyr Ile 1090 1095 1100 Tyr Tyr Gln Gln Tyr Tyr Leu Arg Thr Ser Arg Glu Leu Arg Arg Leu 1105 1110 1115 1120 Asp Ser Ile Thr Arg Ser Pro Ile Tyr Ser His Phe Gln Glu Thr Leu 1125 1130 1135 Gly Gly Leu Ala Thr Val Arg Gly Tyr Ser Gln Gln Lys Arg Phe Ser 1140 1145 1150 His Ile Asn Gln Cys Arg Ile Asp Asn Asn Met Ser Ala Phe Tyr Pro 1155 1160 1165 Ser Ile Asn Ala Asn Arg Trp Leu Ala Tyr Arg Leu Glu Leu Ile Gly 1170 1175 1180 Ser Ile Ile Ile Leu Gly Ala Ala Thr Leu Ser Val Phe Arg Leu Lys 1185 1190 1195 1200 Gln Gly Thr Leu Thr Ala Gly Met Val Gly Leu Ser Leu Ser Tyr Ala 1205 1210 1215 Leu Gln Ile Thr Gln Thr Leu Asn Trp Ile Val Arg Met Thr Val Glu 1220 1225 1230 Val Glu Thr Asn Ile Val Ser Val Glu Arg Ile Lys Glu Tyr Ala Asp 1235 1240 1245 Leu Lys Ser Glu Ala Pro Leu Ile Val Glu Gly His Arg Pro Pro Lys 1250 1255 1260 Glu Trp Pro Ser Gln Gly Asp Ile Lys Phe Asn Asn Tyr Ser Thr Arg 1265 1270 1275 1280 Tyr Arg Pro Glu Leu Asp Leu Val Leu Lys His Ile Asn Ile His Ile 1285 1290 1295 Lys Pro Asn Glu Lys Val Gly Ile Val Gly Arg Thr Gly Ala Gly Lys 1300 1305 1310 Ser Ser Leu Thr Leu Ala Leu Phe Arg Met Ile Glu Ala Ser Glu Gly 1315 1320 1325 Asn Ile Val Ile Asp Asn Ile Ala Ile Asn Glu Ile Gly Leu Tyr Asp 1330 1335 1340 Leu Arg His Lys Leu Ser Ile Ile Pro Gln Asp Ser Gln Val Phe Glu 1345 1350 1355 1360 Gly Thr Val Arg Glu Asn Ile Asp Pro Ile Asn Gln Tyr Thr Asp Glu 1365 1370 1375 Ala Ile Trp Arg Ala Leu Glu Leu Ser His Leu Lys Glu His Val Leu 1380 1385 1390 Ser Met Ser Asn Asp Gly Leu Asp Ala Gln Leu Thr Glu Gly Gly Gly 1395 1400 1405 Asn Leu Ser Val Gly Gln Arg Gln Leu Leu Cys Leu Ala Arg Ala Met 1410 1415 1420 Leu Val Pro Ser Lys Ile Leu Val Leu Asp Glu Ala Thr Ala Ala Val 1425 1430 1435 1440 Asp Val Glu Thr Asp Lys Val Val Gln Glu Thr Ile Arg Thr Ala Phe 1445 1450 1455 Lys Asp Arg Thr Ile Leu Thr Ile Ala His Arg Leu Asn Thr Ile Met 1460 1465 1470 Asp Ser Asp Arg Ile Ile Val Leu Asp Asn Gly Lys Val Ala Glu Phe 1475 1480 1485 Asp Ser Pro Gly Gln Leu Leu Ser Asp Asn Lys Ser Leu Phe Tyr Ser 1490 1495 1500 Leu Cys Met Glu Ala Gly Leu Val Asn Glu Asn 1505 1510 1515 <210> 3 <211> 4776 <212> DNA <213> Artificial Sequence <220> <223> yhl035c gene <400> 3 atgggaacgg atccccttat tatccgaaat aatggttcat tttgggaagt tgatgatttt 60 actcgtttag gaagaactca gctattgagc tactatttac cattggctat catagcctca 120 attggcattt tcgcactttg tcgcagtgga ttatctcgtt atgtaagatc tgccgagtgc 180 gatttagtga acgaatatct atttggcgca caagaagaga gaaaagaaga taatagtata 240 gaaagacttc tacggaactc aaatacccaa gccaattacg tcaacgtcaa aaagcaagga 300 aggattttga aacttagaca ttttgatata acaactatag atgtcaagca aatcgatgct 360 aaaaatcatg gtggactaac gtttagtaga ccgtctacta gtgaccactt aagaaaatca 420 tctgaaattg tattaatgtc tttacaaata attggccttt cctttttaag agtaacaaaa 480 atcaatattg aattaacgaa cagagatgtt acaactttac tattattttg gttaatacta 540 ctttccctaa gtatcttaag agtttacaag cgttcaacga atctttgggc catctgtttt 600 actgcccata caactatttg gatttcaacc tggattccaa ttcgttcggt ctatattggt 660 aatatcgatg atgtaccctc acagatattt tacatctttg aattcgtaat tacttcaacc 720 ttacagccaa taaagctcac ttcaccgatt aaagacaact catccatcat ctacgttaga 780 gacgaccata cgtctccttc gagggaacac atatcctcaa ttttaagttg cattacttgg 840 agctggatta ccaattttat atgggaggcc caaaagaaca ctattaagtt aaaggatatt 900 tggggcttat caatggaaga ctatagcatt ttcattctaa aagggtttac caggagaaac 960 aagcacatta ataatttgac gctagcactg tttgaatctt tcaaaacata tttactcata 1020 ggaatgttat gggttctggt gaacagtatt gtaaaccttc ttcccacaat tttaatgaaa 1080 agatttttag aaattgtgga taacccaaac cgttcctcat catgcatgaa tttggcgtgg 1140 ctttatatta ttggtatgtt catttgtaga ttgacattag caatttgtaa ttcccaaggt 1200 caatttgttt ctgataagat ttgtttaaga ataagagcca tactcatagg agaaatttat 1260 gcaaaaggct tacgtaggag gctgtttaca tctccaaaaa ccagctctga ttcagatagt 1320 atctccgcaa accttggtac cataattaat ctcatttcta ttgactcatt taaggtatcg 1380 gaactagcaa actaccttta tgtgacagtt caggcagtaa ttatgataat agttgttgta 1440 ggactacttt tcaacttttt aggtgtttca gcttttgcag gaatttcaat tatcttagtg 1500 atgttcccat tgaatttctt gttagcgaat ttgttaggta agtttcaaaa gcaaacactg 1560 aaatgtactg accaaagaat ctcaaaattg aacgagtgct tacagaacat aagaattgtc 1620 aaatattttg cttgggaaag gaatattata aatgaaatca aatcaataag gcaaaaggaa 1680 ttaagatcct tattaaaaaa atctttggtg tggtccgtaa cttcttttct ttggttcgtg 1740 acaccgacct tggtgacagg tgtcactttc gccatctgta catttgttca acatgaagat 1800 ttgaatgccc cgcttgcttt cactactttg tcactcttca ctttgttaaa gacacccctg 1860 gatcaattat caaatatgct aagtttcata aatcaatcaa aagtctctct aaaaagaata 1920 agcgattttt taaggatgga cgatacagaa aaatataatc aactaaccat atctccagac 1980 aaaaataaaa ttgaatttaa aaatgcgact ttaacctgga atgaaaatga cagcgatatg 2040 aatgcattca aattatgtgg tttgaatatt aaatttcaaa ttggtaagtt aaatttgatt 2100 ttgggttcta caggatctgg taaaagtgca ttgctgctgg gtttactggg tgaactaaat 2160 ctaattagtg gctctatcat tgttccgagc ttagaaccaa agcatgattt aattcccgac 2220 tgcgaaggtt taaccaattc cttcgcatat tgttcacaaa gtgcgtggct attaaatgac 2280 acggtaaaaa acaatattat ctttgataac ttctataacg aggataggta caacaaagta 2340 attgatgcat gtgggctgaa aagagacctg gagattttac cagcaggtga cctaacagaa 2400 attggtgaaa agggtataac tttatcagga gggcagaaac agagaatttc cttggcgaga 2460 gctgtttatt cgagtgctaa gcatgtctta ctagatgatt gtttgagcgc tgtcgattca 2520 catactgctg tatggatcta tgaaaattgc atcacaggtc cactaatgaa aaatagaacc 2580 tgcattttag ttacgcacaa tgtttcatta acacttagaa atgcccattt cgcgattgtg 2640 ttggaaaatg gcaaagtgaa gaatcaagga actattacag aattacaaag caaagggctt 2700 tttaaggaaa aatatgttca actttcttct cgagatagca ttaatgaaaa gaacgctaat 2760 agattaaaag ctcccagaaa aaatgactct cagaaaatcg aacctgtcac cgagaacata 2820 aattttgatg caaattttgt caatgatggc cagctaatag aagaggaaga aaaatcaaac 2880 ggtgccataa gccccgatgt ttataaatgg tacctgaaat tttttggagg cttcaaagct 2940 ttaacagccc tgttcgctct ttatatcaca gctcaaattt tgttcatcag tcagtcttgg 3000 tggatacgac attgggtcaa cgataccaat gtacgaataa atgctccagg ttttgcgatg 3060 gacacgctgc cattaaaagg gatgaccgac tcttcgaaaa ataaacataa tgcattttat 3120 tacttaaccg tatattttct tattggtatc attcaggcaa tgctaggtgg ttttaaaaca 3180 atgatgacgt ttttatccgg tatgcgagcc tccagaaaga tctttaataa tctgctagat 3240 ctagttctac atgcccaaat acgatttttc gacgtgacgc cggttggtag aatcatgaat 3300 cgcttttcaa aggacatcga aggtgttgat caagaattga ttccatactt agaagtaact 3360 atattttgcc taattcaatg cgcatcaatt atatttctca ttaccgtaat aactcctcgc 3420 tttttgacag tcgccgttat cgtttttgtt ttatatttct ttgtggggaa atggtactta 3480 acggcaagta gagaattgaa aaggttagat tcaataacca aatcacccat ttttcaacat 3540 ttctcagaga ccttggtagg cgtttgcaca attcgtgcat ttggcgacga gaggagattc 3600 attttagaaa atatgaacaa aattgaccaa aataacagag cattctttta tttatcagtt 3660 actgtcaaat ggttttcttt tagagtcgac atgattggcg cattcattgt tttagcatca 3720 ggttctttta ttctgctcaa tattgcaaat attgactcgg gtcttgccgg catttctttg 3780 acatatgcca ttttgtttac agatggtgct ttatggttag ttagactgta ctcaacattt 3840 gaaatgaaca tgaactctgt tgaaagacta aaagaatatt ctagcattga acaagagaac 3900 tatcttggcc atgatgaagg ccgcattcta cttctaaacg aaccatcgtg gccaaaagat 3960 ggagaaattg aaattgaaaa cttatcttta cgttacgcgc caaatttgcc tcctgtcata 4020 agaaatgtta gtttcaaagt ggatcctcaa agtaagattg ggattgtcgg aagaactggc 4080 gcaggcaaat ctaccataat aacggcatta ttcagattac tagaaccaat aaccggatgt 4140 atcaaaatag atgggcagga tataagtaaa attgatctcg ttacattacg tcgttccatt 4200 actatcatcc ctcaggaccc tattctattt gcaggtacaa tcaaaagtaa tgttgatcca 4260 tatgatgaat atgatgaaaa aaaaatattc aaagcacttt cacaagtaaa tctaatttct 4320 tcacatgaat ttgaagaagt gcttaactcg gaggaacgct ttaacagcac tcataataaa 4380 tttttaaatc ttcacacaga aatagctgag ggcggcttaa atctgtccca aggtgaaagg 4440 caattgcttt ttattgcacg atcattgtta cgcgagccaa agataatact tttggacgag 4500 gctacttcct ctattgatta cgattctgac catttaattc agggtattat aagaagtgag 4560 tttaataaaa gcacaattct tactattgca catcgtttga gatctgttat cgattacgac 4620 aggataattg tgatggatgc cggtgaggta aaagaatatg atcgccctag tgaactgttg 4680 aaagatgaac gcggtatatt ttatagtatg tgtcgtgaca gtgggggcct agagcttttg 4740 aagcaaatag ccaagcaatc aagtaagatg atgaaa 4776 <210> 4 <211> 1592 <212> PRT <213> Artificial Sequence <220> <223> yhl035c protein <400> 4 Met Gly Thr Asp Pro Leu Ile Ile Arg Asn Asn Gly Ser Phe Trp Glu 1 5 10 15 Val Asp Asp Phe Thr Arg Leu Gly Arg Thr Gln Leu Leu Ser Tyr Tyr 20 25 30 Leu Pro Leu Ala Ile Ile Ala Ser Ile Gly Ile Phe Ala Leu Cys Arg 35 40 45 Ser Gly Leu Ser Arg Tyr Val Arg Ser Ala Glu Cys Asp Leu Val Asn 50 55 60 Glu Tyr Leu Phe Gly Ala Gln Glu Glu Arg Lys Glu Asp Asn Ser Ile 65 70 75 80 Glu Arg Leu Leu Arg Asn Ser Asn Thr Gln Ala Asn Tyr Val Asn Val 85 90 95 Lys Lys Gln Gly Arg Ile Leu Lys Leu Arg His Phe Asp Ile Thr Thr 100 105 110 Ile Asp Val Lys Gln Ile Asp Ala Lys Asn His Gly Gly Leu Thr Phe 115 120 125 Ser Arg Pro Ser Thr Ser Asp His Leu Arg Lys Ser Ser Glu Ile Val 130 135 140 Leu Met Ser Leu Gln Ile Gly Leu Ser Phe Leu Arg Val Thr Lys 145 150 155 160 Ile Asn Ile Glu Leu Thr Asn Arg Asp Val Thr Thr Leu Leu Leu Phe 165 170 175 Trp Leu Ile Leu Leu Ser Leu Ser Ile Leu Arg Val Tyr Lys Arg Ser 180 185 190 Thr Asn Leu Trp Ala Ile Cys Phe Thr Ala His Thr Thr Ile Trp Ile 195 200 205 Ser Thr Trp Ile Pro Ile Arg Ser Val Tyr Ile Gly Asn Ile Asp Asp 210 215 220 Val Pro Ser Gln Ile Phe Tyr Ile Phe Glu Phe Val Ile Thr Ser Thr 225 230 235 240 Leu Gln Pro Ile Lys Leu Thr Ser Pro Ile Lys Asp Asn Ser Ser Ile 245 250 255 Ile Tyr Val Arg Asp Asp His Thr Ser Pro Ser Arg Glu His Ile Ser 260 265 270 Ser Ile Leu Ser Cys Ile Thr Trp Ser Trp Ile Thr Asn Phe Ile Trp 275 280 285 Glu Ala Gln Lys Asn Thr Ile Lys Leu Lys Asp Ile Trp Gly Leu Ser 290 295 300 Met Glu Asp Tyr Ser Ile Phe Ile Leu Lys Gly Phe Thr Arg Arg Asn 305 310 315 320 Lys His Ile Asn Asn Leu Thr Leu Ala Leu Phe Glu Ser Phe Lys Thr 325 330 335 Tyr Leu Leu Ile Gly Met Leu Trp Val Leu Val Asn Ser Ile Val Asn 340 345 350 Leu Leu Pro Thr Ile Leu Met Lys Arg Phe Leu Glu Ile Val Asp Asn 355 360 365 Pro Asn Arg Ser Ser Ser Cys Met Asn Leu Ala Trp Leu Tyr Ile Ile 370 375 380 Gly Met Phe Ile Cys Arg Leu Thr Leu Ala Ile Cys Asn Ser Gln Gly 385 390 395 400 Gln Phe Val Ser Asp Lys Ile Cys Leu Arg Ile Arg Ala Ile Leu Ile 405 410 415 Gly Glu Ile Tyr Ala Lys Gly Leu Arg Arg Arg Leu Phe Thr Ser Pro 420 425 430 Lys Thr Ser Ser Asp Ser Asp Ser Ile Ser Ala Asn Leu Gly Thr Ile 435 440 445 Ile Asn Leu Ile Ser Ile Asp Ser Phe Lys Val Ser Glu Leu Ala Asn 450 455 460 Tyr Leu Tyr Val Thr Val Gln Ala Val Ile Met Ile Ile Val Val Val 465 470 475 480 Gly Leu Leu Phe Asn Phe Leu Gly Val Ser Ala Phe Ala Gly Ile Ser 485 490 495 Ile Ile Leu Val Met Phe Pro Leu Asn Phe Leu Leu Ala Asn Leu Leu 500 505 510 Gly Lys Phe Gln Lys Gln Thr Leu Lys Cys Thr Asp Gln Arg Ile Ser 515 520 525 Lys Leu Asn Glu Cys Leu Gln Asn Ile Arg Ile Val Lys Tyr Phe Ala 530 535 540 Trp Glu Arg Asn Ile Ile Asn Glu Ile Lys Ser Ile Arg Gln Lys Glu 545 550 555 560 Leu Arg Ser Leu Leu Lys Lys Ser Leu Val Trp Ser Val Thr Ser Phe 565 570 575 Leu Trp Phe Val Thr Pro Thr Leu Val Thr Gly Val Thr Phe Ala Ile 580 585 590 Cys Thr Phe Val Gln His Glu Asp Leu Asn Ala Pro Leu Ala Phe Thr 595 600 605 Thr Leu Ser Leu Phe Thr Leu Leu Lys Thr Pro Leu Asp Gln Leu Ser 610 615 620 Asn Met Leu Ser Phe Ile Asn Gln Ser Lys Val Ser Leu Lys Arg Ile 625 630 635 640 Ser Asp Phe Leu Arg Met Asp Asp Thr Glu Lys Tyr Asn Gln Leu Thr 645 650 655 Ile Ser Pro Asp Lys Asn Lys Ile Glu Phe Lys Asn Ala Thr Leu Thr 660 665 670 Trp Asn Glu Asn Asp Ser Asp Met Asn Ala Phe Lys Leu Cys Gly Leu 675 680 685 Asn Ile Lys Phe Gln Ile Gly Lys Leu Asn Leu Ile Leu Gly Ser Thr 690 695 700 Gly Ser Gly Lys Ser Ala Leu Leu Leu Gly Leu Leu Gly Glu Leu Asn 705 710 715 720 Leu Ile Ser Gly Ser Ile Ile Val Pro Ser Leu Glu Pro Lys His Asp 725 730 735 Leu Ile Pro Asp Cys Glu Gly Leu Thr Asn Ser Phe Ala Tyr Cys Ser 740 745 750 Gln Ser Ala Trp Leu Leu Asn Asp Thr Val Lys Asn Asn Ile Phe 755 760 765 Asp Asn Phe Tyr Asn Glu Asp Arg Tyr Asn Lys Val Ile Asp Ala Cys 770 775 780 Gly Leu Lys Arg Asp Leu Glu Ile Leu Pro Ala Gly Asp Leu Thr Glu 785 790 795 800 Ile Gly Glu Lys Gly Ile Thr Leu Ser Gly Gly Gln Lys Gln Arg Ile 805 810 815 Ser Leu Ala Arg Ala Val Tyr Ser Ser Ala Lys His Val Leu Leu Asp 820 825 830 Asp Cys Leu Ser Ala Val Asp Ser His Thr Ala Val Trp Ile Tyr Glu 835 840 845 Asn Cys Ile Thr Gly Pro Leu Met Lys Asn Arg Thr Cys Ile Leu Val 850 855 860 Thr His Asn Val Ser Leu Thr Leu Arg Asn Ala His Phe Ala Ile Val 865 870 875 880 Leu Glu Asn Gly Lys Val Lys Asn Gln Gly Thr Ile Thr Glu Leu Gln 885 890 895 Ser Lys Gly Leu Phe Lys Glu Lys Tyr Val Gln Leu Ser Ser Arg Asp 900 905 910 Ser Ile Asn Glu Lys Asn Ala Asn Arg Leu Lys Ala Pro Arg Lys Asn 915 920 925 Asp Ser Gln Lys Ile Glu Pro Val Thr Glu Asn Ile Asn Phe Asp Ala 930 935 940 Asn Phe Val Asn Asp Gly Gln Leu Ile Glu Glu Glu Glu Lys Ser Asn 945 950 955 960 Gly Ala Ile Ser Pro Asp Val Tyr Lys Trp Tyr Leu Lys Phe Phe Gly 965 970 975 Gly Phe Lys Ala Leu Thr Ala Leu Phe Ala Leu Tyr Ile Thr Ala Gln 980 985 990 Ile Leu Phe Ile Ser Gln Ser Trp Trp Ile Arg His Trp Val Asn Asp 995 1000 1005 Thr Asn Val Arg Ile Asn Ala Pro Gly Phe Ala Met Asp Thr Leu Pro 1010 1015 1020 Leu Lys Gly Met Thr Asp Ser Ser Lys Asn Lys His Asn Ala Phe Tyr 1025 1030 1035 1040 Tyr Leu Thr Val Tyr Phe Leu Ile Gly Ile Ile Gln Ala Met Leu Gly 1045 1050 1055 Gly Phe Lys Thr Met Met Thr Phe Leu Ser Gly Met Arg Ala Ser Arg 1060 1065 1070 Lys Ile Phe Asn Asn Leu Leu Asp Leu Val Leu His Ala Gln Ile Arg 1075 1080 1085 Phe Phe Asp Val Thr Pro Val Gly Arg Ile Met Asn Arg Phe Ser Lys 1090 1095 1100 Asp Ile Glu Gly Val Asp Gln Glu Leu Ile Pro Tyr Leu Glu Val Thr 1105 1110 1115 1120 Ile Phe Cys Leu Ile Gln Cys Ala Ser Ile Ile Phe Leu Ile Thr Val 1125 1130 1135 Ile Thr Pro Arg Phe Leu Thr Val Ala Val Ile Val Phe Val Leu Tyr 1140 1145 1150 Phe Phe Val Gly Lys Trp Tyr Leu Thr Ala Ser Arg Glu Leu Lys Arg 1155 1160 1165 Leu Asp Ser Ile Thr Lys Ser Pro Ile Phe Gln His Phe Ser Glu Thr 1170 1175 1180 Leu Val Gly Val Cys Thr Ile Arg Ala Phe Gly Asp Glu Arg Arg Phe 1185 1190 1195 1200 Ile Leu Glu Asn Met Asn Lys Ile Asp Gln Asn Asn Arg Ala Phe Phe 1205 1210 1215 Tyr Leu Ser Val Thr Val Lys Trp Phe Ser Phe Arg Val Asp Met Ile 1220 1225 1230 Gly Ala Phe Ile Val Leu Ala Ser Gly Ser Phe Ile Leu Leu Asn Ile 1235 1240 1245 Ala Asn Ile Asp Ser Gly Leu Ala Gly Ile Ser Leu Thr Tyr Ala Ile 1250 1255 1260 Leu Phe Thr Asp Gly Ala Leu Trp Leu Val Arg Leu Tyr Ser Thr Phe 1265 1270 1275 1280 Glu Met Asn Met Asn Ser Val Glu Arg Leu Lys Glu Tyr Ser Ser Ile 1285 1290 1295 Glu Gln Glu Asn Tyr Leu Gly His Asp Glu Gly Arg Ile Leu Leu Leu 1300 1305 1310 Asn Glu Pro Ser Trp Pro Lys Asp Gly Glu Ile Glu Ile Glu Asn Leu 1315 1320 1325 Ser Leu Arg Tyr Ala Pro Asn Leu Pro Pro Val Ile Arg Asn Val Ser 1330 1335 1340 Phe Lys Val Asp Pro Gln Ser Lys Ile Gly Ile Val Gly Arg Thr Gly 1345 1350 1355 1360 Ala Gly Lys Ser Thr Ile Ile Thr Ala Leu Phe Arg Leu Leu Glu Pro 1365 1370 1375 Ile Thr Gly Cys Ile Lys Ile Asp Gly Gln Asp Ile Ser Lys Ile Asp 1380 1385 1390 Leu Val Thr Leu Arg Arg Ser Ile Thr Ile Ile Pro Gln Asp Pro Ile 1395 1400 1405 Leu Phe Ala Gly Thr Ile Lys Ser Asn Val Asp Pro Tyr Asp Glu Tyr 1410 1415 1420 Asp Glu Lys Lys Ile Phe Lys Ala Leu Ser Gln Val Asn Leu Ile Ser 1425 1430 1435 1440 Ser His Glu Phe Glu Glu Val Leu Asn Ser Glu Glu Arg Phe Asn Ser 1445 1450 1455 Thr His Asn Lys Phe Leu Asn Leu His Thr Glu Ile Ala Glu Gly Gly 1460 1465 1470 Leu Asn Leu Ser Gln Gly Glu Arg Gln Leu Leu Phe Ile Ala Arg Ser 1475 1480 1485 Leu Leu Arg Glu Pro Lys Ile Ile Leu Leu Asp Glu Ala Thr Ser Ser 1490 1495 1500 Ile Asp Tyr Asp Ser Asp His Leu Ile Gln Gly Ile Ile Arg Ser Glu 1505 1510 1515 1520 Phe Asn Lys Ser Thr Ile Leu Thr Ile Ala His Arg Leu Arg Ser Val 1525 1530 1535 Ile Asp Tyr Asp Arg Ile Ile Val Met Asp Ala Gly Glu Val Lys Glu 1540 1545 1550 Tyr Asp Arg Pro Ser Glu Leu Leu Lys Asp Glu Arg Gly Ile Phe Tyr 1555 1560 1565 Ser Met Cys Arg Asp Ser Gly Gly Leu Glu Leu Leu Lys Gln Ile Ala 1570 1575 1580 Lys Gln Ser Ser Lys Met Met Lys 1585 1590 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> YCFa primer <400> 5 actaccgtaa agctcgagaa aatggctggt aat 33 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> YCFb primer <400> 6 cttgcctaag tgacgtgacg tctcctt 27 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> RT-YCF1A primer <400> 7 catgagtgcg ttctatccct ctat 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> RT-YCF1B primer <400> 8 ccaccttcgg ttagttgggc atct 24 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> forward primer: YHL035Ca <400> 9 cgacgcggcc gcatgggaac ggatcccctt attatc 36 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> backward primer: YHL035Cb <400> 10 cgacgcggcc gccatcatct tacttgattg cttgg 35

Claims (21)

A DNA molecule that exhibits resistance to and accumulation of lead and encodes a fungal MRP-associated protein-like ATP-binding casette transporter protein (MRP-based ABC transport protein). The DNA molecule of claim 1, wherein the MRP-based ABC transport protein of the fungus is selected from the group consisting of YCF1 protein, YHL035C protein, BPT1 protein, YBT1 protein, and YOR1 protein. The DNA molecule of claim 2, wherein the YCF1 protein has an amino acid sequence of SEQ ID NO: 2 and has a nucleotide sequence encoding the amino acid sequence. The DNA molecule of claim 3, wherein the DNA molecule encoding the YCF1 protein has a nucleotide sequence of SEQ ID NO: 1. The DNA molecule of claim 2, wherein the YCF1 protein further exhibits resistance and accumulation to one or more harmful substances selected from the group consisting of cadmium, arsenic, and herbicides, in addition to resistance and accumulation to lead. 6. The DNA molecule of claim 5, wherein the herbicide is chloro-dinitrobenzene. The DNA molecule of claim 2, wherein the YHL035C protein has an amino acid sequence of SEQ ID NO: 4 and has a nucleotide sequence encoding the amino acid sequence. The DNA molecule of claim 7, wherein the DNA molecule encoding the YHL035C protein has a nucleotide sequence of SEQ ID NO: 3. 9. According to claim 1, wherein the MRP-based ABC transport protein of the fungus MRP- of the fungus having an amino acid sequence homology of at least 28% of the amino acid sequence of the YCF1 protein of SEQ ID NO: 2, or YHL035C protein of SEQ ID NO: 4 DNA molecule, which is a lineage ABC transport protein. The method according to claim 9, wherein the MRP-based ABC transport protein of the fungus has a sequence homology of at least 28% with the amino acid sequence of the YCF1 protein of SEQ ID NO: 2 or YHL035C protein of SEQ ID NO: 4, The YCF1 protein or YHL035C protein is N-terminal extension domain (N-terminal extension domain), the first transmembrane spanning domain, the first nucleotide binding fold domain, the second transmembrane domain (second transmembrane spanning domain, and a domain of a second nucleotide binding fold domain, wherein each domain of the fungal MRP-based ABC transport protein is linked to the YCF1 protein of SEQ ID NO: 2 or the YHL035C protein of SEQ ID NO: 4 A DNA molecule having at least 28% sequence homology with the amino acid sequence of each corresponding domain. A DNA molecule encoding a fungal MRP-associated ATP-associated protein-like ATP-binding casette transporter gene (MRP-based ABC transport gene) according to any one of claims 1 to 10. Recombinant vector. The method of claim 11, wherein the recombinant vector is pESC- YCF1, ENpCambia-YCF1, or PBI121- YCF1 . Recombinant vector. The recombinant vector of claim 11, wherein the recombinant vector is a pESC- YHL035C recombinant vector, or pPBI121-YHL035C. Transformation with a DNA molecule encoding the MRP-associated protein-like ATP-binding casette transporter gene (MRP-based ABC transport gene) of the fungus according to any one of claims 1 to 10 Transgenic organisms. The transforming organism of claim 14, wherein the transforming organism is a prokaryote or eukaryote. The transforming organism of claim 15, wherein the eukaryotes are animals, plants, or yeasts. The method of claim 16, wherein the plant is Arabidopsis, rapeseed, fresh, tobacco, onion, carrot, cucumber This, rapeseed, olives, sweet potato, potatoes, cabbage, radish, lettuce, tobacco, broccoli, petunia, sea Wishes, lampshade, grass, cephalopod, birch, poplar, willow, and birch Transgenic organisms selected from the group consisting of. The transforming organism of claim 17, wherein the transforming organism is YCF1 Arabidopsis (Accession No. KCTC 10064BP), or YCF1 poplar. 18. The method of claim 17, wherein the transforming organism is YHL035C poplar Transgenic organisms. Transformed with a DNA molecule encoding a multidrug resistance-associated protein-like ATP-binding casette transporter gene (MRP-based ABC transport gene) according to any one of claims 1 to 10 Plant cells. The method of claim 20, wherein the plant is Arabidopsis, rapeseed, fresh, tobacco, onion, carrot, cucumber, rapeseed, olive, sweet potato, potato, Chinese cabbage, radish, lettuce, tobacco, broccoli, petunia, sunflower, fresh, grass, Plant cells selected from the group consisting of cephalopod, birch, poplar, willow, and birch.