CN112708603A - Application of rice ARE2 gene in plant nitrogen metabolism regulation - Google Patents

Abstract

The invention discloses application of a rice ARE2 gene in regulation and control of plant nitrogen metabolism. The invention provides application of ARE2 protein in promoting plant precocity and/or shortening plant growth period. The invention also provides application of the ARE2 gene in breeding transgenic plants with early maturity and/or shortened growth period. The invention also provides an application of the ARE2 protein: regulating and controlling the tolerance of the plant to low nitrogen stress; regulating and controlling the nitrogen metabolism process of plants; affecting the balance between plant nitrogen metabolism and stress response. The inventor researches and discovers that: the rice ARE2 gene is a key factor for regulating and controlling the nitrogen metabolism process of plants; the are2 mutation inhibits the bad shape caused by nitrogen assimilation deficiency, and increases the tolerance of the rice to nitrogen deficiency stress to a certain extent; the over-expression ARE2 gene has the effect of shortening the growth period and promoting the early maturity of rice, and is one of effective strategies for rice breeding in high latitude areas; the ARE2 gene encodes a ppGpp hydrolase. Therefore, the full exploitation and scientific application of the ARE2 gene have potential application value in future breeding work.

Description

Application of rice ARE2 gene in plant nitrogen metabolism regulation

Technical Field

The invention belongs to the technical field of biology, and relates to application of a rice ARE2 gene in plant nitrogen metabolism regulation.

Background

Nitrogen (nitrogen) is one of major elements necessary for plant growth and development, is an important component of substances such as nucleic acid, protein, chlorophyll, hormone and the like, participates in most physiological and biochemical processes in plants, and is very important for the plant growth and development processes. People often improve the crop yield by applying a large amount of nitrogen fertilizer, and because the nitrogen utilization capability of crops is limited, the yield cannot be continuously improved by excessive fertilization, but environmental pollution and resource waste are caused, the human health is harmed, the ecological balance is damaged, and the effect of diminishing returns is generated. In the face of population, resource, and environmental issues, the International Agricultural Research consultant organization (CGIAR) proposed a Second Green Revolution (Second Green recycling) aimed at increasing crop yields by improving nutrient utilization efficiency. Improving the Nitrogen Utilization Efficiency (NUE) of crops is one of important links for realizing agricultural sustainable development, and the cultivation and popularization of nitrogen-efficient utilization varieties can effectively reduce the application of nitrogen fertilizers and reduce the environmental pressure, and simultaneously meet the requirements of people on grains.

Plant nitrogen utilization efficiency is influenced by environmental and genetic factors and their interactions. Generally, the primary nitrogen source absorbed and utilized by plants is soil inorganic nitrogen. Factors such as temperature, humidity and soil pH affect the absorption of inorganic nitrogen by plants, and plants show preference for different forms of nitrogen sources in different environments. For example, plants grown in dry land (high pH oxygen-rich soils) absorb primarily nitrate Nitrogen (NO)₃ ^-) While plants growing in paddy fields (reduced soils with low pH values) absorb mainly ammonium Nitrogen (NH)₄ ⁺). With the progress of research, key components involved in plant nitrogen metabolism are gradually separated and identified, and constitute a basic nitrogen metabolism network. The plant nitrogen metabolism process mainly comprises three rings of absorption and transport (uptake and transport), assimilation (assimilation) and reuse (remobilization) of nitrogenAnd (4) saving. Plants absorb soil inorganic nitrogen and transport in vivo through nitrate transporters (NRT) and ammonium transporters (AMT), and nitrate nitrogen is assimilated into ammonium nitrogen through the action of Nitrate Reductase (NR) and nitrite reductase (NiR) in turn. The ammonium nitrogen is assimilated into organic nitrogen by glutamine synthetase/glutamate synthase (GS/GOGAT) circulation, and then various organic substances are synthesized. After the plant enters the reproductive growth and maturation stage, nitrogen is gradually transported from the aging tissues to young organs and seeds in the form of amino acids to be reused and participate in yield formation, and the nitrogen in the process is mainly derived from leaf protein. In addition, Glutamate Dehydrogenase (GDH) catalyzes both the assimilation and reverse reaction of ammonium nitrogen, a pathway that may be an important way for plants to adapt to different nitrogen environments.

Rice is an important grain crop, provides staple food for the world population and ensures the basic needs of life. A series of important genetic loci related to the utilization efficiency of the nitrogen element of the rice are identified by the research of genetics, molecular biology and biochemistry, and the scientific utilization of corresponding excellent allelic variation can become an important way for improving the yield of the rice.

Nitrogen assimilation plays an irreplaceable role in the growth and development process of plants, and the functional deletion of key genes in the process often causes serious growth and development defects and even death.

Disclosure of Invention

The invention aims to provide application of rice ARE2 gene in plant nitrogen metabolism regulation.

The invention provides application of ARE2 protein in promoting plant precocity and/or shortening plant growth period.

The invention also provides application of the ARE2 gene in breeding transgenic plants with early maturity and/or shortened growth period.

The invention also provides a method for cultivating a transgenic plant with early maturity and/or shortened growth period, which comprises the following steps: the ARE2 gene is introduced into a receptor plant to obtain a transgenic plant with early maturity and/or shortened growth period. The ARE2 gene is specifically introduced into a recipient plant by a recombinant vector.

The invention also provides an application of the ARE2 protein, which is (c1) or (c2) or (c 3):

(c1) regulating and controlling the tolerance of the plant to low nitrogen stress;

(c2) regulating and controlling the nitrogen metabolism process of plants;

(c3) affecting the balance between plant nitrogen metabolism and stress response.

The regulation and control of the low nitrogen stress tolerance of the plant ARE that the ARE2 protein expression is inhibited, so that the low nitrogen stress tolerance of the plant is increased.

The present invention also provides a method for breeding a transgenic plant having increased tolerance to low nitrogen stress, comprising the steps of: inhibiting the expression of ARE2 gene in the target plant to obtain the transgenic plant with high low nitrogen stress tolerance.

The present invention also provides a method for breeding a transgenic plant having increased tolerance to low nitrogen stress, comprising the steps of: the ARE2 gene in the target plant is used as a target gene to be mutated, so that the transgenic plant with high tolerance to low nitrogen stress is obtained.

Any of the above plants is a monocot or a dicot.

Any of the above plants is a gramineae plant. Any of the above plants is a plant of the genus oryza. Any of the above plants is rice. Any one of the plants is japonica rice. Any one of the plants is Nipponbare.

Any of the above plants is a crucifer. Any of the above plants is an arabidopsis plant. Any one of the above plants is Arabidopsis thaliana.

The invention also protects the application of the ARE2 protein as ppGpp hydrolase.

The ARE2 protein is also protected by the invention. The ARE2 protein was obtained from rice.

The present invention also protects the ARE2 gene. The ARE2 gene was obtained from rice.

Any of the ARE2 proteins is (a1), (a2) or (a3) or (a4) or (a5) or (a6) or (a 7):

(a1) protein shown in a sequence 3 in a sequence table;

(a2) a protein derived from rice, having 98% or more identity to (a1) and having the same function;

(a3) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and having the same function;

(a4) a protein encoded by the DNA molecule shown in the 1581-12019 th site in the sequence 1 of the sequence table;

(a5) a protein coded by a DNA molecule shown in the position 1301-12512 in the sequence 1 of the sequence table;

(a6) protein coded by DNA molecule shown in sequence 1 of the sequence table;

(a7) protein coded by DNA molecule shown in sequence 2 of the sequence table.

Any one of the ARE2 genes is a DNA molecule for coding ARE2 protein.

Any one of the ARE2 genes is (b1), (b2), (b3), (b4), (b5) or (b 6):

(b1) the cDNA coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b2) the genome DNA is shown as the DNA molecule at the 1581-12019 site in the sequence 1 of the sequence table;

(b3) the genome DNA is shown as DNA molecule at position 1301-12512 in sequence 1 of the sequence table;

(b4) the genome DNA is a DNA molecule shown as a sequence 1 in a sequence table;

(b5) a DNA molecule derived from rice and having 98% or more identity to (b1) or (b2) or (b3) or (b4) and encoding the ARE2 protein;

(b6) a DNA molecule that hybridizes under stringent conditions to (b1) or (b2) or (b3) or (b4) and encodes the ARE2 protein.

The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

The recombinant vector containing the ARE2 gene can be constructed by using the existing plant expression vector.

When constructing a recombinant vector, any one of an enhanced, constitutive, tissue-specific or inducible promoter may be added before the translation initiation nucleotide, and may be used alone or in combination with other plant promoters. In addition, enhancers, including translational or transcriptional enhancers, may be used in the construction of recombinant vectors, and these enhancer regions may be ATG initiation codons or initiation codons in adjacent regions, but are necessarily in frame with the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plants, the recombinant vector used may be processed, for example, by adding a gene expressing an enzyme or a luminescent compound which produces a color change in a plant, an antibiotic marker having resistance, or a chemical-resistant marker gene, etc. From the viewpoint of transgene safety, the transformed plants can be directly screened for phenotypes without adding any selectable marker gene.

The plant expression vector can be a pCAMBIA1300 binary vector.

The recombinant vector can be specifically a plasmid of a DNA molecule shown in a sequence 4 of a sequence table.

The recombinant vector can be specifically a recombinant plasmid obtained by inserting a DNA molecule shown in a sequence 4 of a sequence table into a pCAMBIA1300 binary vector (specifically, the DNA molecule can be inserted between HindIII and EcoRI enzyme cutting sites).

The recombinant vector can be specifically a plasmid of a DNA molecule shown in a sequence 6 of a sequence table.

The recombinant vector can be specifically a recombinant plasmid obtained by inserting a DNA molecule shown in a sequence 6 of a sequence table into a pCAMBIA1300 binary vector (specifically, the DNA molecule can be inserted between HindIII and EcoRI enzyme cutting sites).

In order to discover key regulatory components of plant nitrogen metabolic pathways and further analyze genetic regulatory networks thereof, the inventors of the present invention performed chemical mutagenesis on abc1-1 mutants and screened a series of abc1-1 inhibiting mutants, abc1 repressors (are). The are1 mutant partially restores the growth and development defects of abc1-1, and indicates that the nitrogen assimilation process of the plant is complexly regulated. Further analysis shows that the ARE1 gene function loss has the effects of improving nitrogen utilization efficiency and increasing yield, and the rice yield is in negative correlation with the expression level of ARE1, which indicates that ARE1 is a negative regulatory factor of nitrogen metabolism. The invention carries out systematic in-depth research on the representative mutant ARE2 on the basis of screening ARE series mutants, tries to determine the action of the ARE2 gene in the nitrogen metabolism process, analyzes the molecular regulation and control mechanism of the nitrogen nutrition of crops such as rice and the like, and provides theoretical basis and molecular strategy for rice breeding and green high-efficiency agriculture.

The research results of the inventor of the invention show that: the rice ARE2 gene is a key factor for regulating and controlling the nitrogen metabolism process of plants; the are2 mutation inhibits the bad shape caused by nitrogen assimilation deficiency, and increases the tolerance of the rice to nitrogen deficiency stress to a certain extent; the over-expression ARE2 gene has the effect of shortening the growth period and promoting the early maturity of rice, and is one of effective strategies for rice breeding in high latitude areas; the ARE2 gene encodes a ppGpp hydrolase. Therefore, the full exploitation and scientific application of the ARE2 gene have potential application value in future breeding work.

Experiments prove that the gene ARE2 related to rice nitrogen metabolism is separated and identified, the nitrogen metabolism process of a plant is negatively regulated, the tolerance of the plant to nitrogen deficiency stress is improved by the arc 2 mutant part, and the early maturing of the rice is promoted by over-expressing the ARE2 gene. The ARE2 gene encodes a ppGpp hydrolase. These results indicate that ARE2 is an effective site for nitrogen efficient breeding of rice. The excellent natural variation of ARE2 or the fixed-point editing of ARE2 gene by plant genetic engineering technology is an effective strategy for breeding new high-efficiency rice nitrogen varieties.

Drawings

FIG. 1 is a genetic screen for the abc1are2 double mutants.

A is Wild type (Wild type, WT), abc1 mutant, abc1are 2-1 mutant, abc1are2-2 mutant and abc1are 2-1/abc 1are 2-2F₁The filling phase phenotype of the plants. The scale is 15 cm.

B-D is the plant height (B), tillering number (C) and leaf relative chlorophyll content (D) of wild type, abc1 mutant, abc1are 2-1 mutant and abc1are2-2 mutant. The values represent mean. + -. standard deviation, sample capacity ≥ 15.

Indicates that the difference reached a very significant level in the t-test (P < 0.01).

FIG. 2 is a phenotypic analysis of the single mutant of are 2.

A-B are the seedling phenotype (A) and leaf chlorophyll content (B) of Wild type (Wild type, WT), are2-1 mutant and are2-2 mutant. The scale is 2 cm. Values represent mean ± standard deviation, 3 technical replicates. Indicates that the difference reached a very significant level in the t-test (P < 0.01).

C is the vegetative phenotype of wild type, are the are2-1 mutants and are2-2 mutants. The scale is 15 cm.

D-E is the leaf phenotype of wild type, are2-1 mutant and are2-2 mutant at the reproductive growth stage. The scale is 2 cm.

F-H is the filling stage phenotype (F) and the maturity stage phenotype (G and H) of the wild type, the are2-1 mutant and the are2-2 mutant. The scale is 15 cm.

FIG. 3 is an analysis of tolerance of the are2 mutants to nitrogen starvation stress.

A-C are the plant phenotype (A), biomass (B) and root-cap ratio (C) of Wild Type (WT) and are2-2 mutants cultured for 20 days under normal conditions (control) and nitrogen deficiency conditions. The scale is 2 cm. Values represent mean ± standard deviation, sample volume 24. Indicates that the difference reached a very significant level in the t-test (P < 0.01).

FIG. 4 is a map-based cloning and genetic verification of the ARE2 gene.

A is the genetic location of the ARE2 gene. In the gene structure diagram, black boxes, white boxes and straight lines represent exons, untranslated regions and introns, respectively. The are2-1 and are2-2 mutants underwent base substitutions at the positions shown. The M1-M5 markers were Indel 11931, Indel 12655, Indel 12713, SNP12758 and Indel 12928, respectively.

B is the electrophoresis result of products obtained by designing primers CDS-1F and CDS-1R flanking the are2-1 and are2-2 mutation sites and amplifying Complementary DNAs (Complementary DNAs, cDNAs) of Wild type (Wild type, WT), abc1 mutant, abc1are2-2 mutant, abc1are 2-1 mutant and are2-1 mutant.

C-E is the seedling phenotype (C and D) and leaf chlorophyll content (E) of a transformant (COMP) with a wild type, an ARE2-2 mutant, an ARE2 genome full-length sequence with an ARE2-2 mutant background and a transformant (ARE2-FLAG) with a FLAG-tagged ARE2 coding sequence with an ARE2-2 mutant background. The scale is 2 cm. Values represent mean ± standard deviation, 3 technical replicates. Indicates that the difference reached a very significant level in the t-test (P < 0.01).

F-G is vegetative phase phenotype (F) and filling phase leaf phenotype (G) of transformants with the full length sequence of the ARE2 genome in the background of wild type, the ARE2-2 mutant and the ARE2-2 mutant. (F) And (G) scales of 15cm and 2cm, respectively.

H is the filling stage phenotype of a transformant (ARE2-FLAG/abc1ARE2-2) with FLAG-tagged ARE2 coding sequence in the background of wild type, abc1 mutant, abc1ARE2-2 mutant and abc1ARE2-2 mutant. The scale is 15 cm.

FIG. 5 is a phenotypic analysis of ARE2 gene over-expressed plants. A-D ARE the filling stage phenotype (A), ear phenotype (B), leaf phenotype (C) and leaf chlorophyll content (D) of Wild type (Wild type, WT) and ARE2 gene Overexpression plants (OE). The scales (A-C) were 15cm, 2cm and 2cm, respectively. (D) Values represent mean ± standard deviation, sample volume 24. Indicates that the difference reached a very significant level in the t-test (P < 0.01).

FIG. 6 is a functional analysis of the ARE2 gene.

A is the phenotype of plants grown for 15 days on sucrose-free 1/2MS medium. The control was cultured normally for 15 days, and the dark treatment was cultured normally for 7 days and then dark treatment for 8 days. Similar to the AtRSH1 gene, the ARE2 gene restored the senescence-accelerating phenotype induced by the rsh1-1 mutant in darkness. The scale is 1 cm.

B is overnight cultured E.coli. Both AtRSH1 and ARE2 restored the slow-growing phenotype of the spoT203 strain (CF 4943).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were repeated 2 to 3 times and the results were averaged. The BAC plasmid (OSJNBb0050D18) was supplied by Arizona Genomics Institute. Arabidopsis thaliana rsh1-1 mutant seeds were provided by Ben Field researchers (Aix Marseille University). Coli CF4941 and E.coli spoT203(CF4943) were supplied by Michael Cashel researchers (National Institutes of Health). The pCAMBIA1300 binary vector is a commercially available vector having a hygromycin resistance gene therein. Unless otherwise specified, the rice wild type (denoted by WT) refers to Nipponbare, and the Arabidopsis wild type (denoted by Col-0) refers to Columbia ecotype Arabidopsis. The method for detecting the chlorophyll content in the examples comprises the following steps: weighing 10-30mg of sample ground in liquid nitrogen, adding 2mL of absolute ethyl alcohol, fully mixing, and standing for 2 hours at 4 ℃ in a dark place. The supernatant was centrifuged and the absorbance A was measured by a spectrophotometer_646.8And A_663.2。C_a＝13.95A_663.2-6.88A_646.8；C_b＝24.96A_646.8-7.32A_663.2(ii) a Chlorophyll content (mg/g) ═ C_a+C_b) X volume of extract/fresh weight of sample. The relative chlorophyll content of the plant leaves is measured by a SPAD chlorophyll measuring instrument.

The ARE2 gene in the rice genome DNA is shown as a sequence 1 in a sequence table. In the sequence 1 of the sequence table, from the 5 'end to the 3' end, the 1 st-1300 th deoxynucleotide is an upstream sequence, the 1301-ine 12512 th deoxynucleotide is an exon/intron region, and the 12513-ine 13012 th deoxynucleotide is a downstream sequence. In sequence 1 of the sequence table, from the 5 ' end to the 3 ' end, the 1 st exon is the 1608 rd and the 1795 st deoxyribonucleotide (wherein the 5 ' untranslated region is the 1301 1580 th deoxyribonucleotide, the translation initiation site is the 1581 rd and the 1583 rd deoxyribonucleotide is the translation initiation site), the 1 st intron is the 1609 nd 1795 th deoxyribonucleotide, the 2 nd exon is the 1796 nd deoxyribonucleotide 2016, the 2 nd intron is the 2017 th deoxyribonucleotide, the 3 rd exon is the 4295 th deoxyribonucleotide, the 3 rd intron is the 4481 st 4977 th deoxyribonucleotide, the 4 th exon is the 4978 th 5043 th deoxyribonucleotide, the 4 th intron is the 5044 th deoxyribonucleotide 5165 th deoxyribonucleotide, the 5 th exon is the 5166 th deoxyribonucleotide 5258 th intron, and the 5 th intron is the 525358 th ribonucleotide, the 5359-position 5490 deoxynucleotide is the 6 th exon, the 5491-position 5624 deoxynucleotide is the 6 th intron, the 5625-position 5726 deoxynucleotide is the 7 th exon, the 5727-position 5823 deoxynucleotide is the 7 th intron, the 5824-position 5901 deoxynucleotide is the 8 th exon, the 5902-position 5978 deoxynucleotide is the 8 th intron, the 5979-position 6050 deoxynucleotide is the 9 th exon, the 6051-position 7015 deoxynucleotide is the 9 th intron, the 7016-position 7123 deoxynucleotide is the 10 th exon, the 7124-position 7198 deoxynucleotide is the 10 th intron, the 7199-position deoxynucleotide is the 7211 th exon, the 7297-position 7734 deoxynucleotide is the 11 th intron, the 7735-position 7795 deoxynucleotide is the 12 th intron, 7796-7888 deoxynucleotide is the 12 th intron, 7889-7951 deoxynucleotide is the 13 th exon, 7952-8058 deoxynucleotide is the 13 th intron, 8059-8115 deoxynucleotide is the 14 th exon, 8116-8192 deoxynucleotide is the 14 th intron, 8193-8294 deoxynucleotide is the 15 th exon, 8295-8401 deoxynucleotide is the 15 th intron, 8402-8560 deoxynucleotide is the 16 th exon, 8561-9140 deoxynucleotide is the 16 th intron, 9141-9272 deoxynucleotide is the 17 th exon, 9273-9346 deoxynucleotide is the 17 th intron, 9347-9433 deoxynucleotide is the 18 th exon, 9434-9497 th deoxynucleotide is the 18 th intron, the 9498-9569 deoxynucleotide is the 19 th exon, the 9570-10729-deoxynucleotide is the 19 th intron, the 10730-10816-deoxynucleotide is the 20 th exon, the 10817-10882-deoxynucleotide is the 20 th intron, the 10883-11333-deoxynucleotide is the 21 st exon, the 11334-11557-deoxynucleotide is the 21 st intron, the 11558-11619-deoxynucleotide is the 22 nd exon, the 11620-11776-deoxynucleotide is the 22 nd intron, the 11777-11845-deoxynucleotide is the 23 rd exon, the 11846-11926-deoxynucleotide is the 23 rd intron, and the 11927-12512-deoxynucleotide is the 24 th exon (wherein the 12017-12019-deoxynucleotide is the translation termination site and the 20-12512-deoxynucleotide is the 1203' -untranslated region).

The coding frame of ARE2 gene in rice cDNA is shown as sequence 2 in the sequence table.

The protein coded by the rice ARE2 gene is shown as a sequence 3 in a sequence table.

The nucleotide sequences of the primers used in the examples are shown in Table 1.

TABLE 1

Example 1 genetic screening of abc1are2 double mutants

Early researches show that the rice ABNORMAL CYTOKININ RESPONSE1(ABC1) gene encodes a key enzyme in the nitrogen assimilation process, namely, reduced ferredoxin-dependent glutamate synthase (Fd-GOGAT), the weak allelic mutation ABC1-1 of the gene causes the activity of Fd-GOGAT to be reduced, and the corresponding mutant shows a series of nitrogen metabolism abnormalities such as plant height reduction, tiller reduction, leaf color yellowing and growth period extension. The gene is highly conserved in different species, and the function-deletion mutant has a lethal phenotype in the seedling stage.

The inventor of the invention screens an inhibiting mutant abc1 repressors (are) of an abc1 mutant by performing EMS (ethyl methyl sulfon) chemical mutagenesis on the abc1-1 mutant (hereinafter, referred to as abc1) of rice and obtains 2 double mutants with similar phenotypes. Hybridizing the two to obtain a first filial generation (F)₁) The plant phenotype was identical to the parent, indicating that they were two allelic variants of the same gene, which the inventors therefore named abc1are 2-1 and abc1are2-2 (a in fig. 1), respectively. The are2 mutation can partially restore the phenotype of the abc1 mutant in the whole growth and development stage, including the characters of plant height, tillering and leaf color and luster (B, C and D in figure 1). The results show that the ARE2 mutant partially inhibits the nitrogen assimilation abnormal phenotype of the abc1 mutant, and suggest that the ARE2 mutant can enhance the low nitrogen stress resistance of rice, and the ARE2 is a key site involved in the nitrogen metabolism of the rice.

Example 2 phenotypic analysis of the Single mutant of are2

Backcrossing the abc1are2 double mutants with the Nipponbare paddy rice, and separating to obtain an are2 single mutant. The are2 mutant exhibited predominantly light green leaf color throughout growth compared to the wild type (A, B, C, D, E and F in fig. 2). In addition, when the rice was in the late stage of grain filling to the stage of maturity, the are2 mutant showed higher sensitivity to low temperature due to the decrease in air temperature (G and H in FIG. 2). These results indicate that ARE2 ARE involved in regulating the rice nitrogen metabolism process and may affect the balance between rice nitrogen metabolism and stress response.

Example 3 analysis of tolerance of the are2 mutants to Nitrogen deficiency stress

Preparation of rice nutrient solution

The mother liquors I to VI were prepared according to Yoshida et al (1976) with minor adjustments of the nutrient solution formulation. The concentration of mother liquors I to IV and VI is 800 times and the concentration of mother liquor V is 1000 times. The mother liquor I contains 38.56g/L ammonium sulfate and 52.72g/L magnesium sulfate (or 51.6g/L potassium sulfate and 52.72g/L magnesium sulfate, and is used for preparing nitrogen-deficiency nutrient solution). The mother liquor II contains 14.8g/L potassium nitrate and 47.92g/L calcium nitrate (or 10.92g/L potassium chloride and 32.42g/L calcium chloride, and is used for preparing the nitrogen-deficiency nutrient solution). The mother liquor III contains 19.84g/L of monopotassium phosphate and 12.72g/L of potassium sulfate. Mother liquor IV contains 2.288g/L boric acid, 0.064g/L copper sulfate pentahydrate, 0.176g/L zinc sulfate heptahydrate, 1.488g/L manganese chloride tetrahydrate and 0.072g/L molybdic acid tetrahydrate. The mother liquor V contained 5.57g/L ferrous sulfate heptahydrate and 7.45g/L disodium EDTA. Mother liquor VI also contained 40g/L sodium silicate. When used, dilute to 0.5 x and adjust pH to 5.5.

Two, phenotype analysis

To determine whether the are2 mutation changes the tolerance of rice to nitrogen deficiency stress, the inventors of the present invention cultured wild-type and are2-2 mutant plants under normal conditions and nitrogen deficiency conditions, respectively, and measured the physiological indexes related to the above-ground and below-ground parts at about 20 days. The following two trends were found to exist steadily: the biomass (as indicated by fresh weight) of the are2-2 mutant was significantly lower than the wild-type under normal conditions, while the trait was not significantly different between the wild-type and the are2-2 mutant under nitrogen deficiency conditions; the root cap ratio of the are2-2 mutant was significantly higher than that of the wild type under nitrogen deficiency conditions (see FIG. 3). These results indicate that nitrogen starvation stress inhibits biomass accumulation to a lesser extent and root growth to a greater extent in the are2 mutant than in the wild type, suggesting that the are2 mutant is tolerant to nitrogen deficiency stress.

Example 4 map-based cloning and genetic verification of ARE2 Gene

Construction of recombinant expression vector

ARE2 genome full-length sequence (genomic DNA, gDNA) genetic complementation vector (pARE2:: ARE 2): two fragments of ARE2 genome sequence were obtained by amplification using BAC plasmid (OSJNBb0050D18) as template and using primer pairs ARE2-F/ARE2-1R and ARE2-2F/ARE 2-R. The two fragments are sequentially inserted between the SalI and KpnI enzyme cutting sites of the pCAMBIA1300 binary vector, and a recombinant plasmid is obtained after the target fragment is correctly sequenced.

Genetic complementation vector with FLAG tag (pARE2:: ARE 2-FLAG-NOS): a rice genome DNA is taken as a template, and a primer pair pARE2-F/pARE2-R is used for amplification to obtain an ARE2 promoter (ARE2 native promoter, pARE2, namely, deoxynucleotides at 1-1580 th position in a sequence 1) fragment. Using rice Complementary DNA (cDNA) as template, ARE2CDS-F/ARE2CDS-R is amplified to obtain ARE2 coding sequence (CDS, i.e. deoxynucleotide 1-2676 in sequence 2). An ARE2 promoter, an ARE2 coding sequence, a FLAG tag sequence and an NOS terminator sequence ARE sequentially inserted between HindIII and EcoRI enzyme cutting sites of the pCAMBIA1300 binary vector, and a recombinant plasmid is obtained after a target fragment is correctly sequenced.

Second, genetic mapping and phenotype analysis of transformants

To determine the nature of the are2 mutation, the inventors crossed the are2 mutant with the wild type, the next generation (F)₁) The plants all showed wild type phenotype, the second generation (F)₂) Segregation of traits occurred in the population, and the ratio of plants with the wild-type phenotype and the mutant phenotype was close to 3:1, indicating that are the single nuclear gene recessive mutation are2 (see table 2). The are2-1 mutant is hybridized with indica rice variety Nanjing 6 to construct F₂The population was mapped and individuals with the are2 mutant phenotype in the population were selected for map-based cloning. The ARE2 candidate gene was located in a 103-kb segment of the long arm of chromosome 3, which contains 11 functionally annotated Open Reading Frames (ORFs), using simple repeat (SSR), insertion-deletion polymorphism (InDel), and Single Nucleotide Polymorphism (SNP) molecular markers. PCR amplification and sequencing of this segment revealed that only one gene (LOC _ Os03g22160) was mutated in the are2 mutant. In the are2-1 mutant, the 1 st base G of the 8 th intron of the gene is mutated into A; in the are2-2 mutant, base G at position 5 of the 5 th intron of the gene was mutated to A (see A in FIG. 4). Both of these mutations cause splicing confusion of the ARE2 gene transcript, resulting in partial loss of its function (see FIG. 4B).

TABLE 2 genetic analysis of the are2 mutation

To verify the correctness of the candidate gene, the inventors transferred the full-length genomic sequence driven by the candidate gene promoter and the coding sequence with the FLAG tag into an are2-2 single mutant for genetic rescue. The transformants exhibited wild type phenotype and the leaf color was normal throughout the growth period (C, D, E, F and G in FIG. 4), indicating that the exogenous ARE2 gene restored the phenotype of the ARE2-2 single mutant. In addition, a transformant carrying an ARE2 coding sequence with a FLAG tag (ARE2-FLAG) in an ARE2-2 mutant background is taken as a male parent, an abc1ARE2-2 double mutant is taken as a female parent for hybridization, and a transformant carrying an ARE2-FLAG transgene in an abc1ARE2-2 double mutant background is separated from offspring and shows an abc1 mutant phenotype (H in figure 4), so that the exogenous ARE2 gene restores the phenotype of the abc1ARE2-2 double mutant.

The above results indicate that the LOC _ Os03g22160 gene is the ARE2 gene.

Example 5 phenotypic analysis of ARE2 Gene overexpressing plants

Construction of recombinant expression vector

Over-expression vector (pUbi:: ARE 2-FLAG-NOS): an exogenous DNA molecule sequentially provided with a Ubiquitin promoter (pUbi), a rice ARE2 gene coding sequence (namely, deoxynucleotides at the 1 st to 2676 th positions in the sequence 2), a FLAG tag coding sequence and an NOS terminator sequence is inserted between HindIII and EcoRI enzyme cutting sites of the pCAMBIA1300 binary vector, and sequencing is correct to obtain a recombinant plasmid. The exogenous DNA molecule is shown as a sequence 4 in a sequence table. In the sequence 4 of the sequence table, the 1 st-1981 th site is ubiquitin promoter, the 1988 th-4663 th site is rice ARE2 gene coding sequence, the 4670 th-4696 th site is FLAG tag coding sequence, and the 4745 th-5009 th site is NOS terminator. In the sequence 4 of the sequence table, the 1988-4699 th site forms a fusion gene.

Secondly, preparing transgenic plants and carrying out phenotype analysis

1. The over-expression vector (pUbi:: ARE2-FLAG-NOS) was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

2. And (2) taking the recombinant agrobacterium obtained in the step (1), carrying out genetic transformation on the embryogenic callus of the Nipponbare rice by adopting an agrobacterium dip-dyeing method, and then sequentially carrying out co-culture, screening culture, differentiation culture and rooting culture to obtain a T0 generation regeneration plant.

3. T0 transgenic plants ARE selfed to obtain T1 plants, and transgenic plants ARE screened from T1 plants (screening transgenic plants method: using genome DNA of tested plants as template, adopting a primer pair composed of two primers corresponding to two different exons of ARE2 gene to carry out PCR amplification, wherein ARE2 gene which is exogenously introduced is cDNA, and has different size with the amplification product of endogenous genome DNA, and whether the ARE2 gene is a transgenic plant is judged according to whether the ARE gene has the amplification product with the expected size).

4. T2 generation plants are obtained by selfing T1 generation transgenic plants, the T2 generation plants are identified with hygromycin resistance, if T2 generation plants obtained by selfing a certain T1 generation transgenic plant have hygromycin resistance, the T1 generation transgenic plant is a homozygous transgenic plant, and the T1 generation transgenic plant is a homozygous transgenic plant after selfing.

Two homozygous transgenic lines were obtained, i.e. the line OE1 and the line OE 2.

5. Phenotypic analysis

Test plants: rice plants of Nipponbare, plants of the T3 generation of the OE1 strain, and plants of the T3 generation of the OE2 strain.

Under parallel conditions, test plants were cultured normally and the phenotype was observed continuously. Compared with Nipponbare plants of rice, the OE1 plant line and the OE2 plant line mainly show the precocious characteristics after entering the grain filling period, and mainly show that the leaf senescence is advanced and the seed maturation is accelerated. As shown in FIG. 5, the seeds of wild plants at this stage are not yet mature, and the ears are overall cyan, while the seeds of plants of line OE1 and lines OE2 begin to mature, and the ears are overall yellow; plants of line OE1 and plants of line OE2 have lighter leaf color and reduced chlorophyll content compared to wild-type plants. The result shows that the over-expression ARE2 gene has the effect of promoting the early maturing of rice, namely shortening the growth period of the rice.

After the rice enters the reproductive growth stage, the cold injury seriously affects the rice yield, so that in high-latitude areas, an overlong growth period is one of important factors for limiting the rice yield. The effect of over-expressing ARE2 gene to shorten the growth period can compensate the limitation of overlength of the growth period on the variety breeding in high latitude areas to a certain extent.

Example 6 functional analysis of ARE2 Gene

Construction of recombinant expression vector

1. Construction of Arabidopsis thaliana genetic complementation vector

pAtRSH1 AtRSH1-FLAG-NOS vector: an exogenous DNA molecule sequentially provided with an AtRSH1 promoter, an Arabidopsis thaliana AtRSH1 gene coding sequence, a FLAG tag coding sequence and an NOS terminator sequence is inserted between SalI and EcoRI enzyme cutting sites of the pCAMBIA1300 binary vector, and sequencing is correct to obtain a recombinant plasmid. The exogenous DNA molecule is shown as a sequence 5 in a sequence table. In the sequence 5 of the sequence table, the 1 st-801 th site is AtRSH1 promoter, the 808 nd-3459 th site is Arabidopsis thaliana AtRSH1 gene coding sequence, the 3466 nd-3492 th site is FLAG tag coding sequence, and the 3523 st-3787 th site is NOS terminator. In the sequence 5 of the sequence table, the 808 th-wall 3495 th site forms a fusion gene.

pAtRSH 1ARE 2-FLAG-NOS vector: an exogenous DNA molecule which is sequentially provided with an AtRSH1 promoter, a rice ARE2 gene coding sequence (namely deoxynucleotide at the 1 st to 2676 th positions in the sequence 2), a FLAG tag coding sequence and an NOS terminator sequence is inserted between HindIII and EcoRI enzyme cutting sites of the pCAMBIA1300 binary vector, and sequencing is correct to obtain a recombinant plasmid. The exogenous DNA molecule is shown as a sequence 6 in a sequence table. In the sequence 6 of the sequence table, the 1 st-801 th site is AtRSH1 promoter, the 808 nd-3483 th site is rice ARE2 gene coding sequence, the 3490 th-3516 th site is FLAG tag coding sequence, and the 3565 th-3829 th site is NOS terminator. In the sequence 6 of the sequence table, the 808-3519 site forms a fusion gene.

2. Construction of genetic complementation vector of Escherichia coli

pBAD/His A-AtRSH1 vector: the coding sequence of the Arabidopsis thaliana AtRSH1 gene (808 th-3459 th site in the sequence 5 of the sequence table, and the termination codon TAA added at the 3' end) is inserted between the PstI and KpnI enzyme cutting sites of the pBAD/His A vector to obtain the pBAD/His A-AtRSH1 vector.

pBAD/His A-ARE2 vector: the rice ARE2 gene coding sequence (shown as sequence 2 in the sequence table) is inserted between KpnI and HindIII enzyme cutting sites of the pBAD/His A vector to obtain the pBAD/His A-ARE2 vector.

II, obtaining and identifying transgenic arabidopsis

To analyze the biochemical function of the product encoded by the ARE2 gene, the inventors performed heterologous genetic rescue experiments on the Arabidopsis mutant rsh 1-1. Arabidopsis thaliana mutant rsh1-1, i.e., Arabidopsis thaliana ppGpp hydrolase gene function-deleted mutant rsh 1-1. Arabidopsis mutant rsh1-1 is described in: plant Cell 28,661- & 679.

1. The recombinant Agrobacterium is obtained by introducing pAtRSH1:: AtRSH1-FLAG-NOS vector or pAtRSH1:: ARE2-FLAG-NOS vector into Agrobacterium EHA 105.

2. And (3) taking the recombinant agrobacterium obtained in the step (1), carrying out genetic transformation on an arabidopsis mutant rsh1-1 plant by adopting a floral dip method, and harvesting seeds, namely T0 generation seeds.

3. Culturing T0 generation seeds into plants, namely T0 generation plants; transgenic plants were selected from T0 generation plants by hygromycin resistance selection.

4. Selfing the T0 transgenic plants to obtain T1 seeds, wherein the plants grown from the T1 seeds are T1 plants; transgenic plants were selected from T1 generation plants by hygromycin resistance selection.

5. Selfing the T1 transgenic plants to obtain T2 seeds, wherein the plants grown from the T2 seeds are T2 plants; and (3) carrying out hygromycin resistance identification on the T2 plant, and if the T2-generation plants obtained by selfing a certain T1-generation transgenic plant have hygromycin resistance, the T1-generation transgenic plant is a homozygous transgenic plant and is a homozygous transgenic plant line after selfing.

pAtRSH 1-FLAG-NOS vector homozygous transgenic plants obtained by carrying out the above-described steps are denoted by AtRSH 1-FLAG. pAtRSH1 the homozygous transgenic plant obtained by carrying out the above procedure with ARE2-FLAG-NOS vector is represented by ARE 2-FLAG.

6. Seeds (Columbia ecotype arabidopsis seeds or arabidopsis thaliana mutant rsh1-1 seeds or T2 generation seeds of AtRSH1-FLAG or T2 generation seeds of ARE2-FLAG) ARE sown on a sucrose-free 1/2MS solid culture medium, the culture dish is wrapped by tin foil paper for dark treatment when the seeds ARE cultured for about 1 week, and the phenotype is observed after the seeds ARE continuously cultured for 7-10 days.

The results are shown in FIG. 6A. Under normal conditions, the growth conditions of the plants of the arabidopsis thaliana strains are similar. Arabidopsis thaliana mutant rsh1-1 has a phenotype of accelerated senescence induced by darkness, while the transgenic plants remain green when treated in the dark for about one week, similar to Columbia ecotype Arabidopsis plants. The result shows that the rice ARE2 gene can restore the phenotype of an arabidopsis rsh1-1 mutant, the ARE2 gene has a conserved function, and the ARE2 gene has the function of an arabidopsis AtRSH1 gene. AtRSH1 is a ppGpp hydrolase, and thus ARE2 also has ppGpp hydrolase activity.

Thirdly, obtaining and identifying recombinant escherichia coli

To analyze the biochemical function of the product encoded by the ARE2 gene, the inventors performed heterologous genetic rescue experiments on the E.coli spoT203 strain. The Escherichia coli spoT203 strain, i.e., Escherichia coli (p) ppGpp hydrolysis-deficient strain spoT203, is described in the following documents: mechold, u., caseel, m., Steiner, k., Gentry, d., and Malke, H. (1996) Functional analysis of a relA/spoT gene homolog from Streptococcus et al, bacterial. 178,1401-1411. The E.coli spoT203 strain (CF4943) is a strain with a spoT203 point mutation in the E.coli CF4941 background and exhibits a slow-growing phenotype due to the accumulation of (p) ppGpp in vivo. Escherichia coli CF4941 as relA^-A genotype strain.

1. And (3) introducing the pBAD/His A vector into an escherichia coli spoT203 strain to obtain a recombinant strain.

2. The pBAD/His A-AtRSH1 vector is introduced into the Escherichia coli spoT203 strain to obtain a recombinant strain.

3. The pBAD/His A-ARE2 vector was introduced into E.coli spoT203 strain to obtain a recombinant strain.

4. Inoculating the test bacteria to LB liquid culture medium and culturing to bacterial liquid OD₆₀₀The same (about 1.0-1.5), after diluting in equal proportion, a certain volume (about 8-10 μ L) of the bacterial liquid is sucked, inoculated in M9 glucose minimal solid culture medium containing 0.2% of hydrolyzed casein and 0.5-0.8% of arabinose, cultured overnight at 37 ℃ and the growth state of the bacteria is observed.

The test bacteria are: escherichia coli CF4941, Escherichia coli spoT203, the recombinant bacterium obtained in step 1, the recombinant bacterium obtained in step 2 or the recombinant bacterium obtained in step 3.

M9 glucose minimal solid medium was prepared according to the method of Xiao et al (1991) with minor adjustments. The method specifically comprises the following steps: 11.28g/L M9 salt (Sigma-Aldrich), 0.2% glucose, 0.1mM calcium chloride, 1mM magnesium sulfate, 0.5. mu.g/mL ferrous sulfate, 1. mu.g/mL vitamin B₁And 1.5% agar powder. Preparing 10 XM 9 salt mother liquor (pH7.0), and performing moist heat sterilization at 121 ℃ for 20 minutes; preparing 100 times of mother liquor of other components, filtering and sterilizing (vitamin B)₁Mother liquor was stored at 4 ℃ and other mother liquors were stored at room temperature). When preparing the culture medium, dissolving agar powder by using single distilled water, carrying out damp-heat sterilization at 121 ℃ for 20 minutes, and then adding each mother solution in proportion. The arabinose is used for inducing and expressing exogenous genes.

The results are shown in FIG. 6B. The growth speed of the recombinant strain introduced with the AtRSH1 gene and the recombinant strain introduced with the ARE2 gene is similar to that of the Escherichia coli CF4941, namely, the AtRSH1 gene and the ARE2 gene can restore the slow growth phenotype of the Escherichia coli spoT 203. The spoT203 mutation results in a decrease in the (p) ppGpp hydrolase activity of spoT, thus the results indicate that ARE2 has ppGpp hydrolase activity.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

Application of rice ARE2 gene in plant nitrogen metabolism regulation

<130> GNCYX210431

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13012

<212> DNA

<213> Oryza sativa

<400> 1

aatggttaca cgaggacaaa cattcttccc ccaggaagtc acaaatctca aaaggttttt 60

gacgggtgaa tgcttatttc ttttttctta aacgcaatcc acattttttt tcttcttttg 120

attagcgata ctatttgtgc ttaagatatt taccatgcaa aatggcttgg agagagagaa 180

tgtctggact gaagttaagg attatttgga gagcttaaaa ttctgataag caactgatta 240

gtagccaact tttgacaatc tggaaaagct gggttttcta gtttttgact tcaagttcat 300

tttctagatt ctacaaatac agtttctcag aatctggacc aaaagctaga ctgttttaga 360

tagtttttga ttctgataga agttgcaaca gccaaaagct ctcccaaact cccaaacagg 420

cccttactat caagtgtcat tgcatagtac tttgatagtt gatggatagg ctccgaaaaa 480

acaactgcga gaagagtgtt gcttgcacaa tatgatccat atacaatggc tctgcccatc 540

aggttatttt ccaaccaatg gctcccgtcc ctttgtcacc cctgagatca ctttcttggt 600

tgaggtggtg gctttgatct gacatgttag cataaggtca ctcgccacac caccattgta 660

aatgctctta agcagcctct ctagcgattc cataatggtg agtcagtgac agagcaaacc 720

atgatttgag agaaggagca tatttggaat gctatgatat cttggtttca tacagggttc 780

aaaatttagg tgaagtatct caaatttcca actgttttat atacaaaaca atttattcga 840

tatacgtttt tttctctctt cgaagagaac aaaatacttg catgaatata gtttaggtcc 900

atacatccat acccttttta catgggcaca caatccatgc ttctccccac agcccaactg 960

ttagctagaa gaagagatta catgacatga gcaaaccatc gattctattc ttccgtagtg 1020

ctaccaggat cattgctgtg tgaaaactga aaagccataa cgaaaaattc catagtgctc 1080

acgaccaaga cgtgtgccac taaaaactaa cgagtacacg agcagaacgt gtgtgtgcct 1140

ctcgagtcac gagctgctgt acaccaggta tgcagatggg ggaggaggag agtacgagac 1200

taccggatgt aaaccagaaa aaatccgtaa aaaagccctc caaaatacta ctcctcctac 1260

tgctgctgct gctgcaaaag cgaaagagga ttagctgaaa ccgggcaaaa gggagggcgt 1320

ttgccaccta tttcaccaca cggccgcggc gttttgtgtg gccttcgtct cgtgttatct 1380

cttctcttct tcccaccccc tctctctctc cttctcctcc cttccgcctt catcatcggc 1440

ggcaaacccc ttcttccatc cacttccccc accgaattcg cgcgcgcgcg ccccgtggct 1500

gcggcggagg ggaaggtgag gacggcggcg ggcacggcca cggcctccga gccgccgatg 1560

agattgcgcg ccaccgcgac atgcaacccc cgacgggcgc cgtgtcaggt gatgatcccc 1620

tcccctcgcc tcccctccac cccgtcatct cggcttgctc cccccgcgcg attcggtccg 1680

cggctgctga tgatgatgcg tcgcccgcgg tggatggact agggattggg ggaattgatg 1740

ggggagccct gatgatggct ttgtttgttt gtttggtggt ggtcgcggcg tgcagggtcg 1800

tcgtcgtcgt cgctggagtg cgtgagctcg tgcaggacgt cgtggagggg cggagggagg 1860

ccgtacgaat gcagcgtgct ctcgtgcgcc tggaacgcgc cgcgggcgct cacgggggcg 1920

ctcgccagca ccaccgcgca gtgctcctcc tgcagccacg ccgaggctgg ggcagggtgg 1980

aggcggcggg gccagtcgca gcggagtaac aattcggtgc gttgctgcct gcataattga 2040

ttctgtcatt tacacgtaat gttgattggg attcgccatt ttggcttact ttagagttaa 2100

cggaatggaa aaatggaggt gcttactagt gttttattcc atttcgtagc atcaggtccc 2160

tgagcatcac tgaaactgaa cgatctcatg actaaagcca atatatcata catgttttgt 2220

agtacaagtc aagttggtca atctggctat caggtcatac tgaagttagt tcacttgggg 2280

gaaaaaggct tccatctatc tcatttcatt attgcatata tgctttggct ataaacacta 2340

caagtagcaa gtcctgctga ttctcttcaa gtttggcttg tacatagaca taggtaatgg 2400

ttttctgtag attttgtaag tgttatttga tgccatcaga acatatttcc ttcttagaaa 2460

ataaatggat agtggttttg tacttatata ttttccttgt taagaccttt tatttagaag 2520

ttatggaaat ttgtgatgaa gccttaacca tatgaagttt agttcaccca tggttcctcc 2580

atctattaat ttaattcttc ccaaagtaca ggattggcac catttattcc ctggttaatg 2640

attcagttac tcggctttct ccttgatatt ttttacggct gcaacaggtt gctagtacat 2700

gaaacaaaga agtctagggg caacattggt actgtgctag agatcgtgaa cggtaaagct 2760

gtgtattgac aacggaatgt ggatttggtt tggtccatct tctttaggaa ctaggatgct 2820

agggaagaag cagggtcaat ttggcgtttg gcaacaaaca ttagtcaatt tggcgtttga 2880

ctaagtttat ttgtctataa acttagtcaa acttaaagca gagtcatctt gagctagcat 2940

gttgtgagcc tgtaaattaa cttgactatg caatgccttg tttgttcctt tattctgtgc 3000

tccttgtagg agtctgacca taatctaata gataaaaatt atatatacaa aaaaaaaagg 3060

tgagcgtgca tgtttaggat agaattgttt ggtacactca tttgtggctt ggatgcgatg 3120

caaatgcgtt gaaatatatt atagacattt ctatgtatat ctttattgta gcgagtatag 3180

gctgttttta gcaagatttt gatggcgatc tagtggaaat gtggaatgac atctgctaaa 3240

atttccatga cttaggttta gcttatatcg taactatttt ccgaatgcca tcagaaggat 3300

tgtcttcacc taacaatcac cattcttcca tatttatctc agctttgttt cctgttaata 3360

ctgagcactc agtatgtaca tgaactaccc tcatgccaaa tagaatggtt ttcccaaacc 3420

atttaggttt catttggttg gaagttgtaa tgtatcacac tggttttgtg acaaagaatt 3480

gagtgtaatg tacgtgggtt acatcagact ttgtgttctc ttggagttct cctgccgtcc 3540

tacttttatt cttaattcat gcatcgggat tagagttttc caaccaaata acaggtaacg 3600

tcttaattgt gcactcaatc atgatctaga ggagaagttg caaaccaaat gccctggttt 3660

aagtgctagt aaatactttc cactttttcc tttatggtcg cttattgacc atgagtcatt 3720

gaaaacatgt ttggccttta atgtaagggg gatttaacca gagtcacctg tttctgatct 3780

acttggctca aaccgatggg gtcaaacctt ggaaaatatc ttataacaaa atactactac 3840

ctccgtttca ggttatagga tgttttgact ttggtcaaag tcaaactgtt tcaagtttga 3900

caaagtttat agaaaaaaat aataacattt tgaacccaag acaaatttat tactatgaaa 3960

atatattcaa ttattgattt aataaaacta atttgatatt ataaatatta ctatatttgt 4020

ctataaactt agtcaaactt aaagtagatt gactttaacc aaagtcaaaa cgtcttataa 4080

cctgaaaacg gagggagtac taacaaatta caattctaag tctgttatga atacagtcaa 4140

atgccatatt gtgtcaaatg tgacatcatt agttttacaa atttgtaagg catcatacca 4200

tcttactttt ttaatggttg ctgtcacgtt cgttatctac atatgaaaca ataattcttt 4260

gcaatgtctt aatactatca acttattttt ccagttactg cacataacct gggcagaagg 4320

catcaacagg gggaagtttg gttatggttc atcagcccat tcttttccca ctggaaattt 4380

tttcaaatct tggtcaactt ccgtggatcc aacatggaga gttttctgct attcatcatc 4440

tgaatcgttc aatcatattt ctccagaaac tttgtgggag gtttgtcact gaattgcaca 4500

attaaacttt tttgctcgtt tttggtacat aattcagttt tgttaaaatt aaatataaga 4560

gttaaagcat ttttaaagga tttatgatta atcatttgtg ggtttgatcc gtgaaacttc 4620

cataaagaag tcaataatca ttttgtgtct ggtagtatag tactacaatc accttcttct 4680

taatgttgta aacacacttc ataattaact agaacatacg gcaatgaaac tcaattcaga 4740

tttggaagat gcggaagtct ccaggttaag cacaatttcc aatagtgctg ccatggtgca 4800

ttttcaaaat gaaaccatta gttgtgactt gttagcataa tttttgtgcc tagtatgaaa 4860

ttttaaatgt actattcaag tatactgccc tcaaatgcaa ctcatggttg atgttcgttt 4920

gaagaaagag gcaattttgt ttgtggcgct tttaaaattt atgcgattgc atgacaggat 4980

ctcaagccag ctatctcgta ccttcaacct gaagaattga actttgtaca cgatgctctg 5040

aaggtaattg aagtgcaata acattcttcc acgtggtttg tggacaatat tctatacttc 5100

ttttattatt cctgggtagt gcatgcacca ggaggttgta tcatgtacct gattgactct 5160

tgcagttggc atatgaggca cataatgggc agaaacgccg aagtggggaa cctttcatta 5220

ttcatcctgt tgaagttgct cgtatccttg gggagcatgt gagtaatata tcgttaccac 5280

tacggtgatg actttctgcc catataaaca taatgaagtt aaattttctg tcgttccatc 5340

ttctgtggac atatttagga acttgactgg gaatcaatag ctgctggttt attgcatgac 5400

actgttgaag atacagacat ggttactttt gaaagaatag aaaatgagtt tggtgtaacc 5460

gtacgccgta ttgttgaagg ggagacaaag gtacattgtt tcacatttta tattttgcac 5520

atggctgcag tgcatgttta tatttggtat atttctgcaa aatgtttgtg atccaggtag 5580

agggatttta atgctataac agacctattt catttcaaat gcaggtatct aagctaggaa 5640

aacttcagtg caaaaatgag ggtaattcaa aacaggacgt caaggctgaa gatttaagac 5700

agatgtttct tgccatgaca gaagaggttt tttctttttt tgtttcgtca tgtagactgt 5760

tatttatcga gagctatttg aacaccctac taggttccat gaaaatttaa ataaaaattg 5820

taggttcgtg taatcattgt gaaactggca gacaggctgc ataatatgcg tacactcaca 5880

catatgcctc agcacaagca ggtaaactct tgttccactg aaaataatat ctgtccgtaa 5940

cttagtttct atttttattg taagcactct ctttgcagta tgccattgcc atggagacac 6000

tgcaggtttt tgcaccccta gctaaacttc tcgggatgta tcgaataaag gtatctgatg 6060

ataaatcatc ttgaaatata actgttattt ctgtttattg tatagactaa cttcattact 6120

gcagtgcttt tatgttattg ctgtgtttgc ttacaaacat tttgtactac catgctgttg 6180

tgttttttcc aataaaattt atacttcttt tttaatgata attgttttcg tgtttgcttt 6240

tttgatttta aaaacctgta tttctgctat aatttgacca gtagacttgt ttgtcctgaa 6300

cttattagta atattaagaa acaagtccat tttatcctta aatattgctc aagttcagaa 6360

atctatgcta aaatttggta tttcacaacc gtgcaaaaac aacctccctt gcttaattca 6420

ccaatttttc gttgacttgg ttttctgatt gccaacttta tgctgatgtg gcatacgaat 6480

cagtaacagc ataaagtgat tcagttgtac agattgaaat attcagattt gttgttgttg 6540

atttggatgc aatgtcatca attacacctc cctcggatat tttagaagtc aacttttcac 6600

ctcagaatga gacatccaag ccccaaaaca gttaaaatct aaccatcgtt ttatccactt 6660

tatggtttta atggtggttt tggagagtta aaagaacata atgtacttcc aaagttcagg 6720

ggccaaagaa atcaattccc cttaatctta tgactgtacg agatattttc atagtagtat 6780

tttgtagttc aaggaccaaa ggaaaaaaaa tccagttcaa ggaccaaagg aaaaaaaatc 6840

cctttatctt atgaatagaa ggggtggttt catactggtg ttttctgtca gtcttagaaa 6900

ggtctcctct gtagttcgtt gcattgtgag aatgtcgatg tgtttcttat gaagcttatc 6960

atagcttctg gtactttgcc tgggtccatt tgaccagttg ctcttattct tgtagtctga 7020

gctagaatat ctgtccttca tgtacatgaa tcctggtgat ttcgctgaac taaagaaaag 7080

agttgaggac ctctacaagg cccatgaaca agaattggaa gaggtacttt gtttttgcta 7140

ttgcactttg atttgcattc tactctgaat gagattcatt ataatttttg aattacaggc 7200

aaatcaaatc ttaggggaaa agattgctga ggatcaattt cttgatcttg ttagtgttga 7260

aacacaagtg cgttcagtct gcaaagaact ctacaggtag agcacacatg atgtcttatt 7320

aattaaaaat aagtattatc ttgaaccttc acttccactt taatactacc cctgcctagt 7380

gatatactgt tggtataaac ttggtttagc ttatttgttt acactactta caaatgcaat 7440

tcatattaga gcacaatgaa tttcctcaca aatttcttta gttatatctt tgtaaaaaag 7500

tggatatttg ttttcactac tttgtcaatt tgcttacaca acatcattaa atagtgtggt 7560

acagttacag actgattgac gttttctgct gcaaaatcct ttttttcttc tcattcacat 7620

tttgcaccta aaaaggtgat caacaattat ttttgcatta ttcattacca agtttgaaat 7680

aggtggcaaa agtgttttct taggcagatt atttatcatt tggtcatatt gcagcattta 7740

taaaactgcg ctgaagtcca agagttcaat aaatgagata aatcaggttg cacaggtatg 7800

attttcctat gtcagataag ttgcctagct ttatcatttt aacgaataac ttattctctg 7860

aaaagtattg tgtttctata ttattcagct gcggatcatc ataaagccaa aatcctgcaa 7920

tggtgtgggg ccattgtgca ctgcacaaca ggtataacat tacaagtgta acataccatc 7980

ttttctgtat gaaattaaaa tttactaatg tgtaaatatg ctcaatccac tgatgcgcaa 8040

gcattcaatt catttcagat ctgctaccat gtccttggtc ttgttcatgg tatctggaca 8100

cctatacctc aagctgtgag ttatctgctt tccttattca ttataatttt tacctctttt 8160

gattttgcca tttctaaaag ccagctgtat aggtgaaaga ttacattgca accccaaagc 8220

ctaatggata ccaaagtcta cacacgacag tgataccatt tctgaatgag agtatgttcc 8280

atttggaagt tcaggtaatg ttggcttagc tgcctgccat ttagctaaat tcatactaca 8340

aactctcaag catgtgctca ttctaatgca agtttcactt tatgttccta ctgtattgca 8400

gataagaaca gaggatatgg atctcatagc agaaagaggc attgctgcgc actacagtgg 8460

aagaggggtt gtttcagggc ctgttcgtcc tggaatatca agtggaagga attcaaatgg 8520

gaaagtgata tgtctgaaca atacaggctt tgctctgagg gtatataatg atgtcaaagc 8580

ttattgcact tcatattaag taccccctct gttctaaaaa ataaggcgtc cttgttttca 8640

ggaaaaatca acatttgaaa actttgactg ataattgatc taaaatattt ttttggtgtc 8700

atgtatgtag taccactaga tttgtatctg aaaatatttt catatcatgt taatttcaaa 8760

gtaccccagt taataatata ttactacccc tgtcctaaaa tgtatgacac cgttgacttt 8820

tcgcataatg tttgaccatt cgtcttaaaa ttttagtgta aatatgaaaa agtataagtt 8880

aatattattt tgatgataaa gcaagtgaca acaaaataaa tgatatttat atcttttttg 8940

aataagacga atggttaaac attatgtaaa aggtcaacgg cgtcatatat tctgggatgg 9000

attataaaag aaatcaatgg tgaaagtata atgttgggat gctgtgaaat aagttagtac 9060

gctttatatt ttagaacgga ggagtattta ttatcacatt tccttaagaa agaactccaa 9120

agacttctca tctttgatag attggttggc tcaatgcaat ccgtgaatgg caagaagagt 9180

ttgttggtaa catgagttct agggaatttg ttgatactat cacccgagat cttctgggaa 9240

gccgtgtctt tgtgtttaca ccaaaaggcg aggtgaggtg ctgacttgga ggacatttct 9300

tccatttgag ttgagcgatt ggtttgaata tagttttcat tggcagatta aaaacttgcc 9360

taagggggcc actgtggttg attatgctta cctgattcac acagaaattg ggaacaaaat 9420

ggtagctgca aaggttagtt atggataata gcattatgat catcattgta tttgtgtgct 9480

catgttcttc ctttcaggtg aatggcaatc tagtttcacc aatccatgta cttgcaaatg 9540

ctgaagttgt ggagattata atttatgatg taagtactca tttcatatat tgcatgattt 9600

atttctgttt tatatctttg tgtcaatttg taactttcaa gatatgttta ttgattgagc 9660

catgtctcga cattgatgca catgcccaca acatacaagt tgcaactttg attgcccttt 9720

ttttgtgtgt atatgggtga taacctccgt cccaaaattt tagggatcct ggcattcaaa 9780

atttgtccaa aagtagtagg gattctagag tactactccc tgctaaccac ctcatttaat 9840

gttcacccaa tcttaccccc aaccaccctc gcactaccca cagacccctc aataagaggc 9900

atcatagtct tttcccttta accttaatct ctccaaaaaa atcttaagat cccttatatt 9960

ttggaactga gggagcaact tacacgccag taggtctgaa attttattcc agcatataac 10020

catccgacca tgtggtctgt agcatcatat atcttttttc atttgcaaat gtactccctc 10080

catttcatat tataagtcat ttgatttttt ttcttagtca aagttcttta ggtttgccta 10140

agtttataga aaaagctagc aacatctaca gcactaaatt aatttcatta aatttagcat 10200

gaaatatatt ttaataatat gtttgtttta tattgaaaat gctactatat ttttctacaa 10260

acttaaagaa gtttgactaa gaaaaaagtc aaaaagactt ataataagaa acagatggag 10320

tacgattcag cttgctacag acaaattcat agcagtatga taaccccctt gggggccagg 10380

atcctggccc atacaaagga gccctaaaag ggacagcagc atgacagctc acattgtccc 10440

tgaatcaatt gaaagaccaa aggtgcattt ccagttcata ccgtgggatc agcccttcat 10500

agttcctaat ttcacacttg aaagttacaa cataattctg agggaaatag atggaagtag 10560

atgtgatgtc tctgtgaggc catttgcgat ttttctcagt ttaaggcata ctgttactct 10620

tgctagtgcc ttttatccat caagaggatt ctagagaact attccattgg cattgcttat 10680

attatacctg tgacatccat ttaaactcat ctgccttttc ttgtttcaga aattatctgc 10740

taaatatgca ttccagcgtc accagcagtg gcttcaacat gcgaaaactc gcagtgcaag 10800

acacaaaatt atgaaagtaa gtctaaaagg atttcttaca ttattagatg gttctggcaa 10860

ctaaccgttt gcctttctcc agttcttgcg ggagcaagct gctctttctg ctgctgaaat 10920

aactgccgac gccgttaata actttgttgc cgatcttgaa gatgaaagtg actatgagca 10980

gtcgattcca agctctgaaa ataaggacta tacattcaac tggcagaaga tattgaattc 11040

tgacaaacta tcctttggaa acaagaagag cgactgcttt ttacctgtta aaaatgtctc 11100

tgtgccaaag gttaatggga agcacaacaa aactgtcaaa gaactgggca tcaaaattaa 11160

tggttcaaca tttcgtggtg atagcttcac tgattttatt caccctggtg tttccagcag 11220

taaagaagtt ctccctagtg tggataactg gaaagctggt aaaatttgtg cgtggcacaa 11280

tacagaaggc agctctatcc aatggctctg catagtgtgt gttgatcgaa aaggtttcac 11340

tcctgccatc taattgttgg acaaagctga aaatatttta ttagctttac ctctataatt 11400

tattagtttt aactaataag acgttgcaaa catgtccaca ttttgttcct ttctgcacaa 11460

ctatttacat ctaatttatt tttgggttat gttgttttga cctttacctg caaatattaa 11520

tataatcact tcaaatggct ttcaatattc atcataggta tggttgcgga agtttcatca 11580

gctctaacag cgtgtggaat caccatatgc tcgtgtgtgg taaatggacc aacttgcctc 11640

aataatttag tatcatcctt ttttatgtga taatcaatgc aacattgcat catttaccct 11700

attaagttcc catcttgaat ttgtcagcca cctctttgta ctgtttgtgt ttgatcattt 11760

ctgctataat tcttaggcgg agcgagataa gagaagggga ataggtgtga tgttgttcca 11820

ctttgagggg gcatatgaga atgtggtgtg cttccttaac tgatctgtat tcctgttcta 11880

attgttgcat tgggcactgt acctaaatag aaaattgcaa ttgcaggtca gtgcatgctc 11940

cggtgttgat atgattcttg gggttcttgg ttggtccgtt ggatgcagct gtaatccttt 12000

gggtgttctt gaatgctagc ggacaacagg catcacgctg catcagttca ccatctccag 12060

caaaaaggag agacaatttt gaagcaccaa gaaatcagtc cgctcaatgg ttcaggagca 12120

gtccgatctt ggttgatgat gctgtgcaac ccataacttg tccttcaaag aaggtagagt 12180

gagggtagag caagctggag ccggatattc tggttcgttt ggaagcgaga ggagtgtaca 12240

tagctgagca agctactagt gaactgttag tccagggatt ggttgggtta tagtattata 12300

caaaagcctc catgaatgac caacaatggc agcttctctc ttctattttt tttgtttgcc 12360

attcgtacac tagttatgta tgacatgtgc agctagaatt ttgatctgat gggattccaa 12420

tgccccaaat tttgcccgta ttcgttattt cactcggtga ttatgactat gctgtagctt 12480

cgatttttca atggtataag ttttcttttc ttactttggg gcctaatgaa atgcgttggc 12540

ttaagaacat ctaactggca gataaataat tcagcgaaga gtcctctgtt ttgcagaggt 12600

aaaaaaacag acgcagtcca tactgtacag taactgaaac aagagaatgg cttgtttgct 12660

acagttaact tatatgaaat gcacctggat tgcgactgat ttagaatgtg gacaatgtgg 12720

accaagccaa gccattttct cacgtcttac cgagacgaat cgctcgtgcc aaggactcat 12780

tgacccttga ttcagttgag aaatgggaga tatttctgtt tggcatttga taaaatagtc 12840

tgttgcccgg cgaaaaataa aatatctgtt aatcgacata gtgagaacat tgatgtgttt 12900

attcaactat aacgcactct aataagcgaa aactacgtgt ttcgatcttg tttcatgtgc 12960

tagcgtgtgt gagccactct tcagctttgg tggcgtcgca ggaatacgca gt 13012

<210> 2

<211> 2679

<212> DNA

<213> Oryza sativa

<400> 2

atgcaacccc cgacgggcgc cgtgtcaggg tcgtcgtcgt cgtcgctgga gtgcgtgagc 60

tcgtgcagga cgtcgtggag gggcggaggg aggccgtacg aatgcagcgt gctctcgtgc 120

gcctggaacg cgccgcgggc gctcacgggg gcgctcgcca gcaccaccgc gcagtgctcc 180

tcctgcagcc acgccgaggc tggggcaggg tggaggcggc ggggccagtc gcagcggagt 240

aacaattcgt tactgcacat aacctgggca gaaggcatca acagggggaa gtttggttat 300

ggttcatcag cccattcttt tcccactgga aattttttca aatcttggtc aacttccgtg 360

gatccaacat ggagagtttt ctgctattca tcatctgaat cgttcaatca tatttctcca 420

gaaactttgt gggaggatct caagccagct atctcgtacc ttcaacctga agaattgaac 480

tttgtacacg atgctctgaa gttggcatat gaggcacata atgggcagaa acgccgaagt 540

ggggaacctt tcattattca tcctgttgaa gttgctcgta tccttgggga gcatgaactt 600

gactgggaat caatagctgc tggtttattg catgacactg ttgaagatac agacatggtt 660

acttttgaaa gaatagaaaa tgagtttggt gtaaccgtac gccgtattgt tgaaggggag 720

acaaaggtat ctaagctagg aaaacttcag tgcaaaaatg agggtaattc aaaacaggac 780

gtcaaggctg aagatttaag acagatgttt cttgccatga cagaagaggt tcgtgtaatc 840

attgtgaaac tggcagacag gctgcataat atgcgtacac tcacacatat gcctcagcac 900

aagcagtatg ccattgccat ggagacactg caggtttttg cacccctagc taaacttctc 960

gggatgtatc gaataaagtc tgagctagaa tatctgtcct tcatgtacat gaatcctggt 1020

gatttcgctg aactaaagaa aagagttgag gacctctaca aggcccatga acaagaattg 1080

gaagaggcaa atcaaatctt aggggaaaag attgctgagg atcaatttct tgatcttgtt 1140

agtgttgaaa cacaagtgcg ttcagtctgc aaagaactct acagcattta taaaactgcg 1200

ctgaagtcca agagttcaat aaatgagata aatcaggttg cacagctgcg gatcatcata 1260

aagccaaaat cctgcaatgg tgtggggcca ttgtgcactg cacaacagat ctgctaccat 1320

gtccttggtc ttgttcatgg tatctggaca cctatacctc aagctgtgaa agattacatt 1380

gcaaccccaa agcctaatgg ataccaaagt ctacacacga cagtgatacc atttctgaat 1440

gagagtatgt tccatttgga agttcagata agaacagagg atatggatct catagcagaa 1500

agaggcattg ctgcgcacta cagtggaaga ggggttgttt cagggcctgt tcgtcctgga 1560

atatcaagtg gaaggaattc aaatgggaaa gtgatatgtc tgaacaatac aggctttgct 1620

ctgaggattg gttggctcaa tgcaatccgt gaatggcaag aagagtttgt tggtaacatg 1680

agttctaggg aatttgttga tactatcacc cgagatcttc tgggaagccg tgtctttgtg 1740

tttacaccaa aaggcgagat taaaaacttg cctaaggggg ccactgtggt tgattatgct 1800

tacctgattc acacagaaat tgggaacaaa atggtagctg caaaggtgaa tggcaatcta 1860

gtttcaccaa tccatgtact tgcaaatgct gaagttgtgg agattataat ttatgataaa 1920

ttatctgcta aatatgcatt ccagcgtcac cagcagtggc ttcaacatgc gaaaactcgc 1980

agtgcaagac acaaaattat gaaattcttg cgggagcaag ctgctctttc tgctgctgaa 2040

ataactgccg acgccgttaa taactttgtt gccgatcttg aagatgaaag tgactatgag 2100

cagtcgattc caagctctga aaataaggac tatacattca actggcagaa gatattgaat 2160

tctgacaaac tatcctttgg aaacaagaag agcgactgct ttttacctgt taaaaatgtc 2220

tctgtgccaa aggttaatgg gaagcacaac aaaactgtca aagaactggg catcaaaatt 2280

aatggttcaa catttcgtgg tgatagcttc actgatttta ttcaccctgg tgtttccagc 2340

agtaaagaag ttctccctag tgtggataac tggaaagctg gtaaaatttg tgcgtggcac 2400

aatacagaag gcagctctat ccaatggctc tgcatagtgt gtgttgatcg aaaaggtatg 2460

gttgcggaag tttcatcagc tctaacagcg tgtggaatca ccatatgctc gtgtgtggcg 2520

gagcgagata agagaagggg aataggtgtg atgttgttcc actttgaggg ggcatatgag 2580

aatgtggtca gtgcatgctc cggtgttgat atgattcttg gggttcttgg ttggtccgtt 2640

ggatgcagct gtaatccttt gggtgttctt gaatgctag 2679

<210> 3

<211> 892

<212> PRT

<213> Oryza sativa

<400> 3

Met Gln Pro Pro Thr Gly Ala Val Ser Gly Ser Ser Ser Ser Ser Leu

1 5 10 15

Glu Cys Val Ser Ser Cys Arg Thr Ser Trp Arg Gly Gly Gly Arg Pro

20 25 30

Tyr Glu Cys Ser Val Leu Ser Cys Ala Trp Asn Ala Pro Arg Ala Leu

35 40 45

Thr Gly Ala Leu Ala Ser Thr Thr Ala Gln Cys Ser Ser Cys Ser His

50 55 60

Ala Glu Ala Gly Ala Gly Trp Arg Arg Arg Gly Gln Ser Gln Arg Ser

65 70 75 80

Asn Asn Ser Leu Leu His Ile Thr Trp Ala Glu Gly Ile Asn Arg Gly

85 90 95

Lys Phe Gly Tyr Gly Ser Ser Ala His Ser Phe Pro Thr Gly Asn Phe

100 105 110

Phe Lys Ser Trp Ser Thr Ser Val Asp Pro Thr Trp Arg Val Phe Cys

115 120 125

Tyr Ser Ser Ser Glu Ser Phe Asn His Ile Ser Pro Glu Thr Leu Trp

130 135 140

Glu Asp Leu Lys Pro Ala Ile Ser Tyr Leu Gln Pro Glu Glu Leu Asn

145 150 155 160

Phe Val His Asp Ala Leu Lys Leu Ala Tyr Glu Ala His Asn Gly Gln

165 170 175

Lys Arg Arg Ser Gly Glu Pro Phe Ile Ile His Pro Val Glu Val Ala

180 185 190

Arg Ile Leu Gly Glu His Glu Leu Asp Trp Glu Ser Ile Ala Ala Gly

195 200 205

Leu Leu His Asp Thr Val Glu Asp Thr Asp Met Val Thr Phe Glu Arg

210 215 220

Ile Glu Asn Glu Phe Gly Val Thr Val Arg Arg Ile Val Glu Gly Glu

225 230 235 240

Thr Lys Val Ser Lys Leu Gly Lys Leu Gln Cys Lys Asn Glu Gly Asn

245 250 255

Ser Lys Gln Asp Val Lys Ala Glu Asp Leu Arg Gln Met Phe Leu Ala

260 265 270

Met Thr Glu Glu Val Arg Val Ile Ile Val Lys Leu Ala Asp Arg Leu

275 280 285

His Asn Met Arg Thr Leu Thr His Met Pro Gln His Lys Gln Tyr Ala

290 295 300

Ile Ala Met Glu Thr Leu Gln Val Phe Ala Pro Leu Ala Lys Leu Leu

305 310 315 320

Gly Met Tyr Arg Ile Lys Ser Glu Leu Glu Tyr Leu Ser Phe Met Tyr

325 330 335

Met Asn Pro Gly Asp Phe Ala Glu Leu Lys Lys Arg Val Glu Asp Leu

340 345 350

Tyr Lys Ala His Glu Gln Glu Leu Glu Glu Ala Asn Gln Ile Leu Gly

355 360 365

Glu Lys Ile Ala Glu Asp Gln Phe Leu Asp Leu Val Ser Val Glu Thr

370 375 380

Gln Val Arg Ser Val Cys Lys Glu Leu Tyr Ser Ile Tyr Lys Thr Ala

385 390 395 400

Leu Lys Ser Lys Ser Ser Ile Asn Glu Ile Asn Gln Val Ala Gln Leu

405 410 415

Arg Ile Ile Ile Lys Pro Lys Ser Cys Asn Gly Val Gly Pro Leu Cys

420 425 430

Thr Ala Gln Gln Ile Cys Tyr His Val Leu Gly Leu Val His Gly Ile

435 440 445

Trp Thr Pro Ile Pro Gln Ala Val Lys Asp Tyr Ile Ala Thr Pro Lys

450 455 460

Pro Asn Gly Tyr Gln Ser Leu His Thr Thr Val Ile Pro Phe Leu Asn

465 470 475 480

Glu Ser Met Phe His Leu Glu Val Gln Ile Arg Thr Glu Asp Met Asp

485 490 495

Leu Ile Ala Glu Arg Gly Ile Ala Ala His Tyr Ser Gly Arg Gly Val

500 505 510

Val Ser Gly Pro Val Arg Pro Gly Ile Ser Ser Gly Arg Asn Ser Asn

515 520 525

Gly Lys Val Ile Cys Leu Asn Asn Thr Gly Phe Ala Leu Arg Ile Gly

530 535 540

Trp Leu Asn Ala Ile Arg Glu Trp Gln Glu Glu Phe Val Gly Asn Met

545 550 555 560

Ser Ser Arg Glu Phe Val Asp Thr Ile Thr Arg Asp Leu Leu Gly Ser

565 570 575

Arg Val Phe Val Phe Thr Pro Lys Gly Glu Ile Lys Asn Leu Pro Lys

580 585 590

Gly Ala Thr Val Val Asp Tyr Ala Tyr Leu Ile His Thr Glu Ile Gly

595 600 605

Asn Lys Met Val Ala Ala Lys Val Asn Gly Asn Leu Val Ser Pro Ile

610 615 620

His Val Leu Ala Asn Ala Glu Val Val Glu Ile Ile Ile Tyr Asp Lys

625 630 635 640

Leu Ser Ala Lys Tyr Ala Phe Gln Arg His Gln Gln Trp Leu Gln His

645 650 655

Ala Lys Thr Arg Ser Ala Arg His Lys Ile Met Lys Phe Leu Arg Glu

660 665 670

Gln Ala Ala Leu Ser Ala Ala Glu Ile Thr Ala Asp Ala Val Asn Asn

675 680 685

Phe Val Ala Asp Leu Glu Asp Glu Ser Asp Tyr Glu Gln Ser Ile Pro

690 695 700

Ser Ser Glu Asn Lys Asp Tyr Thr Phe Asn Trp Gln Lys Ile Leu Asn

705 710 715 720

Ser Asp Lys Leu Ser Phe Gly Asn Lys Lys Ser Asp Cys Phe Leu Pro

725 730 735

Val Lys Asn Val Ser Val Pro Lys Val Asn Gly Lys His Asn Lys Thr

740 745 750

Val Lys Glu Leu Gly Ile Lys Ile Asn Gly Ser Thr Phe Arg Gly Asp

755 760 765

Ser Phe Thr Asp Phe Ile His Pro Gly Val Ser Ser Ser Lys Glu Val

770 775 780

Leu Pro Ser Val Asp Asn Trp Lys Ala Gly Lys Ile Cys Ala Trp His

785 790 795 800

Asn Thr Glu Gly Ser Ser Ile Gln Trp Leu Cys Ile Val Cys Val Asp

805 810 815

Arg Lys Gly Met Val Ala Glu Val Ser Ser Ala Leu Thr Ala Cys Gly

820 825 830

Ile Thr Ile Cys Ser Cys Val Ala Glu Arg Asp Lys Arg Arg Gly Ile

835 840 845

Gly Val Met Leu Phe His Phe Glu Gly Ala Tyr Glu Asn Val Val Ser

850 855 860

Ala Cys Ser Gly Val Asp Met Ile Leu Gly Val Leu Gly Trp Ser Val

865 870 875 880

Gly Cys Ser Cys Asn Pro Leu Gly Val Leu Glu Cys

885 890

<210> 4

<211> 5009

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgcagcgtga cccggtcgtg cccctctcta gagataatga gcattgcatg tctaagttat 60

aaaaaattac cacatatttt ttttgtcaca cttgtttgaa gtgcagttta tctatcttta 120

tacatatatt taaactttac tctacgaata atataatcta tagtactaca ataatatcag 180

tgttttagag aatcatataa atgaacagtt agacatggtc taaaggacaa ttgagtattt 240

tgacaacagg actctacagt tttatctttt tagtgtgcat gtgttctcct ttttttttgc 300

aaatagcttc acctatataa tacttcatcc attttattag tacatccatt tagggtttag 360

ggttaatggt ttttatagac taattttttt agtacatcta ttttattcta ttttagcctc 420

taaattaaga aaactaaaac tctattttag tttttttatt taataattta gatataaaat 480

agaataaaat aaagtgacta aaaattaaac aaataccctt taagaaatta aaaaaactaa 540

ggaaacattt ttcttgtttc gagtagataa tgccagcctg ttaaacgccg tcgacgagtc 600

taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag cagacggcac 660

ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg ttggacttgc 720

tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg gcacggcagg 780

cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc caccgctcct 840

tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttcccc 900

aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa tccacccgtc 960

ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc taccttctct 1020

agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc atgtttgtgt 1080

tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg cgacctgtac 1140

gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc ctgggatggc 1200

tctagccgtt ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt gcatagggtt 1260

tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt 1320

tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc gttctagatc 1380

ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc tgtatgtgtg 1440

tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg atctaggata 1500

ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt tgttcgcttg 1560

gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg agtagaatac 1620

tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg tcatacatct 1680

tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat acatgttgat 1740

gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1800

cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1860

acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1920

ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1980

taagcttatg caacccccga cgggcgccgt gtcagggtcg tcgtcgtcgt cgctggagtg 2040

cgtgagctcg tgcaggacgt cgtggagggg cggagggagg ccgtacgaat gcagcgtgct 2100

ctcgtgcgcc tggaacgcgc cgcgggcgct cacgggggcg ctcgccagca ccaccgcgca 2160

gtgctcctcc tgcagccacg ccgaggctgg ggcagggtgg aggcggcggg gccagtcgca 2220

gcggagtaac aattcgttac tgcacataac ctgggcagaa ggcatcaaca gggggaagtt 2280

tggttatggt tcatcagccc attcttttcc cactggaaat tttttcaaat cttggtcaac 2340

ttccgtggat ccaacatgga gagttttctg ctattcatca tctgaatcgt tcaatcatat 2400

ttctccagaa actttgtggg aggatctcaa gccagctatc tcgtaccttc aacctgaaga 2460

attgaacttt gtacacgatg ctctgaagtt ggcatatgag gcacataatg ggcagaaacg 2520

ccgaagtggg gaacctttca ttattcatcc tgttgaagtt gctcgtatcc ttggggagca 2580

tgaacttgac tgggaatcaa tagctgctgg tttattgcat gacactgttg aagatacaga 2640

catggttact tttgaaagaa tagaaaatga gtttggtgta accgtacgcc gtattgttga 2700

aggggagaca aaggtatcta agctaggaaa acttcagtgc aaaaatgagg gtaattcaaa 2760

acaggacgtc aaggctgaag atttaagaca gatgtttctt gccatgacag aagaggttcg 2820

tgtaatcatt gtgaaactgg cagacaggct gcataatatg cgtacactca cacatatgcc 2880

tcagcacaag cagtatgcca ttgccatgga gacactgcag gtttttgcac ccctagctaa 2940

acttctcggg atgtatcgaa taaagtctga gctagaatat ctgtccttca tgtacatgaa 3000

tcctggtgat ttcgctgaac taaagaaaag agttgaggac ctctacaagg cccatgaaca 3060

agaattggaa gaggcaaatc aaatcttagg ggaaaagatt gctgaggatc aatttcttga 3120

tcttgttagt gttgaaacac aagtgcgttc agtctgcaaa gaactctaca gcatttataa 3180

aactgcgctg aagtccaaga gttcaataaa tgagataaat caggttgcac agctgcggat 3240

catcataaag ccaaaatcct gcaatggtgt ggggccattg tgcactgcac aacagatctg 3300

ctaccatgtc cttggtcttg ttcatggtat ctggacacct atacctcaag ctgtgaaaga 3360

ttacattgca accccaaagc ctaatggata ccaaagtcta cacacgacag tgataccatt 3420

tctgaatgag agtatgttcc atttggaagt tcagataaga acagaggata tggatctcat 3480

agcagaaaga ggcattgctg cgcactacag tggaagaggg gttgtttcag ggcctgttcg 3540

tcctggaata tcaagtggaa ggaattcaaa tgggaaagtg atatgtctga acaatacagg 3600

ctttgctctg aggattggtt ggctcaatgc aatccgtgaa tggcaagaag agtttgttgg 3660

taacatgagt tctagggaat ttgttgatac tatcacccga gatcttctgg gaagccgtgt 3720

ctttgtgttt acaccaaaag gcgagattaa aaacttgcct aagggggcca ctgtggttga 3780

ttatgcttac ctgattcaca cagaaattgg gaacaaaatg gtagctgcaa aggtgaatgg 3840

caatctagtt tcaccaatcc atgtacttgc aaatgctgaa gttgtggaga ttataattta 3900

tgataaatta tctgctaaat atgcattcca gcgtcaccag cagtggcttc aacatgcgaa 3960

aactcgcagt gcaagacaca aaattatgaa attcttgcgg gagcaagctg ctctttctgc 4020

tgctgaaata actgccgacg ccgttaataa ctttgttgcc gatcttgaag atgaaagtga 4080

ctatgagcag tcgattccaa gctctgaaaa taaggactat acattcaact ggcagaagat 4140

attgaattct gacaaactat cctttggaaa caagaagagc gactgctttt tacctgttaa 4200

aaatgtctct gtgccaaagg ttaatgggaa gcacaacaaa actgtcaaag aactgggcat 4260

caaaattaat ggttcaacat ttcgtggtga tagcttcact gattttattc accctggtgt 4320

ttccagcagt aaagaagttc tccctagtgt ggataactgg aaagctggta aaatttgtgc 4380

gtggcacaat acagaaggca gctctatcca atggctctgc atagtgtgtg ttgatcgaaa 4440

aggtatggtt gcggaagttt catcagctct aacagcgtgt ggaatcacca tatgctcgtg 4500

tgtggcggag cgagataaga gaaggggaat aggtgtgatg ttgttccact ttgagggggc 4560

atatgagaat gtggtcagtg catgctccgg tgttgatatg attcttgggg ttcttggttg 4620

gtccgttgga tgcagctgta atcctttggg tgttcttgaa tgccccgggg attacaagga 4680

tgacgacgat aagtgctaag ctagcggatc cactagttct agaggatccc cgggtaccga 4740

gctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg 4800

ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa 4860

ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat 4920

tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc 4980

gcgcggtgtc atctatgtta ctagatcgg 5009

<210> 5

<211> 3787

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttttgaatga gcttattaaa ttatatataa agtatataca accaatatgc atattttctt 60

gtttatattc acctaataat aaccaatgaa ataataaaat acaaatgtat attattttcc 120

ttttggtcac aaatgtattt tcttttccct tcttatatta tatacacaat aaaataaaat 180

aaaaatatga aattatcctt tttttgtgtg gacattttat catttaagaa aatgaaatta 240

ataattctaa attactatct tattacaaat gaaaattgta aattaattaa atatttacaa 300

gcatccatat aattttggtc attcaatata attcctaaat actaaaaact attgactttt 360

tttttttttt tttttttttt tttgtaaacc ctaaaaacta ttgactaaaa aaaagtattt 420

taacagaaaa agaagagaat atataatttt aatgtttgac cacaaaggga aaaaaatatc 480

tcaaagtttt ttctttcttt ggtatatcgg acggtatttg tgtggccagg gagggctacc 540

tgagactcca tatctgtctc tcttcttctt cttcttcctc ttcttcctct gcttcttctt 600

cttcacatca ctgtgtgcgt ctctctatct ctctattcta tcatttcgaa atcctctcta 660

tccgaatttg aactcgtctc ttcttcgtaa agagttctta atctccgtct ctgtgatttc 720

ggaatctgga ggttttgaat ttcttgttcg tatcaatcaa atggtggatc gatttagtga 780

gattgcttta cctcattagc agtcgacatg acttctgctt cttccatgtc cgtgtctgtg 840

gaatgtgtga acatatgtaa tctaacgaaa ggagatggga atgcaagaag cgattgcagt 900

gctctctcct gtgcttggaa agctccaaga gcgttaactg ggtttctagc tagcactgct 960

catccacctg tgtgttctgt gtattcatgt ggcagaaatg gaagaaagag cagaatgaaa 1020

gcttgcgcct ggcagaggta tgaatatgaa gtaggctttt ctgaggctcc ttactttgta 1080

aatgtgagaa atatcttgaa gtccagatta tcttgtggtg gtcataaaag atgggaactg 1140

tattgcgtat cagctgaatc ttcttctggt gcatccagtg atgttaccgt cgaaacattg 1200

tgggaggacc ttttcccatc aatatcttat ctaccccgta aagaattaga atttgttcaa 1260

aagggcctta agttagcgtt tgaggcacat catggtcaaa agagacgtag tggggaacca 1320

ttcattatac atcccgttgc agttgctcgt atccttgggg aacttgaatt ggattgggag 1380

tctattgttg ctggattact acatgacaca gtcgaggata caaatttcat tacttttgaa 1440

aagatagaag aagagtttgg tgcaactgtg cgtcacatcg tagaagggga gaccaaggtg 1500

tcaaaactgg gaaagttaaa gtgtaaaacg gaaagtgaaa caatacaaga tgtaaaagca 1560

gatgatttgc ggcagatgtt tctggcgatg acagacgagg tccgcgtcat tattgtcaaa 1620

ctagctgacc ggttgcataa tatgcgaact ctctgccaca tgcctcccca taagcagtcc 1680

agcattgcag gggagacttt gcaggtcttt gctcctttag caaaattatt gggaatgtat 1740

tcaataaagt ctgaactgga aaatctgtct ttcatgtacg taagtgctga ggattatgat 1800

agagtcacta gcaggattgc taacctctac aaagagcatg aaaaagaact cactgaggca 1860

aacagaattt tggtgaaaaa gattgaagat gatcagtttc tggaccttgt gactgtgaat 1920

actgatgttc gatctgtttg caaggaaact tacagcatct acaaagctgc tctcaaatcg 1980

aaaggatcaa ttaatgatta caaccagatt gctcagcagt tacggattgt tgtaaagcca 2040

aaaccatctg taggggtcgg gcctttgtgc agtccacaac agatatgcta tcacgttctg 2100

gggcttgttc atgagatctg gaaacctatt ccacgaacag taaaagatta cattgcaacc 2160

ccgaagccca atggatacca gagcctccat actactgtga ttccattctt gtatgagagt 2220

atgtttcgac tggaggttca gatcagaacc gaggagatgg acttgattgc tgaaaggggc 2280

attgctgttt actacaatgg caagtctcta tctactggat tagttggaaa cgcggttcct 2340

ttaggtagaa attcaagggg gaagacgggt tgcctcaaca atgcagattt tgcactcagg 2400

gttgggtggc taaatgcaat aagggaatgg caagaggagt ttgtgggtaa catgagctct 2460

agagaatttg tggataccat tacgagggat cttttaggta gtcgtgtgtt tgtattcaca 2520

cctaaaggag agataaagaa cctcccgaaa ggggccaccg ttgttgacta cgcttatctg 2580

attcacaccg aaatcggaaa caagatggta gcagcaaagg tcaatggtaa tcttgtttcc 2640

ccaactcacg ttcttgagaa tgctgaggtc gtggagatag tcacctacaa cgccctctca 2700

agtaaatctg ctttccaaag acataaacag tggttgcaac atgccaaaac aaggagtgca 2760

agacacaaga ttatgaggtt cctaagggag caagctgcac aatgtgctgc cgaaattacc 2820

caggatcaag tgaatgactt tgtggcggac tctgatagtg atgtggaaga tctcacagaa 2880

gattcaagaa agagcctaca atggtgggag aaaatcctcg tcaatgttaa gcaattccag 2940

tcacaagaca aaagtagaga tacaacaccc gctcctcaaa acggaagcgt ttgggcccca 3000

aaggtgaatg gaaaacacaa caaagccata aagaactcga gttctgatga gccagagttc 3060

ctcctacctg gagatggaat tgccaggatt ttacctgcta atatccctgc ttataaggaa 3120

gtgttgcccg gcttagacag ttggcgagac agtaaaattg ccacatggca tcatctcgaa 3180

ggtcagtcca tcgaatggtt atgtgtagta tccatggatc gcaaaggcat aatcgcagag 3240

gttacaacag tcctcgcagc tgaaggcatt gcattatgtt cttgcgtggc cgagattgac 3300

agaggaagag gattagcagt aatgttattt caaatagaag caaacattga aagtttggtt 3360

agtgtttgcg ctaaggtgga tttagtcttg ggtgtgttgg gatggtccag tggttgtagc 3420

tggccaagat caacagagaa tgctcaagtt cttgagtgtc ccggggatta caaggatgac 3480

gacgataagt gctaagctag cggatccccg ggtaccgagc tcgaatttcc ccgatcgttc 3540

aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat 3600

catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt 3660

atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga 3720

aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact 3780

agatcgg 3787

<210> 6

<211> 3829

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttttgaatga gcttattaaa ttatatataa agtatataca accaatatgc atattttctt 60

gtttatattc acctaataat aaccaatgaa ataataaaat acaaatgtat attattttcc 120

ttttggtcac aaatgtattt tcttttccct tcttatatta tatacacaat aaaataaaat 180

aaaaatatga aattatcctt tttttgtgtg gacattttat catttaagaa aatgaaatta 240

ataattctaa attactatct tattacaaat gaaaattgta aattaattaa atatttacaa 300

gcatccatat aattttggtc attcaatata attcctaaat actaaaaact attgactttt 360

tttttttttt tttttttttt tttgtaaacc ctaaaaacta ttgactaaaa aaaagtattt 420

taacagaaaa agaagagaat atataatttt aatgtttgac cacaaaggga aaaaaatatc 480

tcaaagtttt ttctttcttt ggtatatcgg acggtatttg tgtggccagg gagggctacc 540

tgagactcca tatctgtctc tcttcttctt cttcttcctc ttcttcctct gcttcttctt 600

cttcacatca ctgtgtgcgt ctctctatct ctctattcta tcatttcgaa atcctctcta 660

tccgaatttg aactcgtctc ttcttcgtaa agagttctta atctccgtct ctgtgatttc 720

ggaatctgga ggttttgaat ttcttgttcg tatcaatcaa atggtggatc gatttagtga 780

gattgcttta cctcattagc aaagcttatg caacccccga cgggcgccgt gtcagggtcg 840

tcgtcgtcgt cgctggagtg cgtgagctcg tgcaggacgt cgtggagggg cggagggagg 900

ccgtacgaat gcagcgtgct ctcgtgcgcc tggaacgcgc cgcgggcgct cacgggggcg 960

ctcgccagca ccaccgcgca gtgctcctcc tgcagccacg ccgaggctgg ggcagggtgg 1020

aggcggcggg gccagtcgca gcggagtaac aattcgttac tgcacataac ctgggcagaa 1080

ggcatcaaca gggggaagtt tggttatggt tcatcagccc attcttttcc cactggaaat 1140

tttttcaaat cttggtcaac ttccgtggat ccaacatgga gagttttctg ctattcatca 1200

tctgaatcgt tcaatcatat ttctccagaa actttgtggg aggatctcaa gccagctatc 1260

tcgtaccttc aacctgaaga attgaacttt gtacacgatg ctctgaagtt ggcatatgag 1320

gcacataatg ggcagaaacg ccgaagtggg gaacctttca ttattcatcc tgttgaagtt 1380

gctcgtatcc ttggggagca tgaacttgac tgggaatcaa tagctgctgg tttattgcat 1440

gacactgttg aagatacaga catggttact tttgaaagaa tagaaaatga gtttggtgta 1500

accgtacgcc gtattgttga aggggagaca aaggtatcta agctaggaaa acttcagtgc 1560

aaaaatgagg gtaattcaaa acaggacgtc aaggctgaag atttaagaca gatgtttctt 1620

gccatgacag aagaggttcg tgtaatcatt gtgaaactgg cagacaggct gcataatatg 1680

cgtacactca cacatatgcc tcagcacaag cagtatgcca ttgccatgga gacactgcag 1740

gtttttgcac ccctagctaa acttctcggg atgtatcgaa taaagtctga gctagaatat 1800

ctgtccttca tgtacatgaa tcctggtgat ttcgctgaac taaagaaaag agttgaggac 1860

ctctacaagg cccatgaaca agaattggaa gaggcaaatc aaatcttagg ggaaaagatt 1920

gctgaggatc aatttcttga tcttgttagt gttgaaacac aagtgcgttc agtctgcaaa 1980

gaactctaca gcatttataa aactgcgctg aagtccaaga gttcaataaa tgagataaat 2040

caggttgcac agctgcggat catcataaag ccaaaatcct gcaatggtgt ggggccattg 2100

tgcactgcac aacagatctg ctaccatgtc cttggtcttg ttcatggtat ctggacacct 2160

atacctcaag ctgtgaaaga ttacattgca accccaaagc ctaatggata ccaaagtcta 2220

cacacgacag tgataccatt tctgaatgag agtatgttcc atttggaagt tcagataaga 2280

acagaggata tggatctcat agcagaaaga ggcattgctg cgcactacag tggaagaggg 2340

gttgtttcag ggcctgttcg tcctggaata tcaagtggaa ggaattcaaa tgggaaagtg 2400

atatgtctga acaatacagg ctttgctctg aggattggtt ggctcaatgc aatccgtgaa 2460

tggcaagaag agtttgttgg taacatgagt tctagggaat ttgttgatac tatcacccga 2520

gatcttctgg gaagccgtgt ctttgtgttt acaccaaaag gcgagattaa aaacttgcct 2580

aagggggcca ctgtggttga ttatgcttac ctgattcaca cagaaattgg gaacaaaatg 2640

gtagctgcaa aggtgaatgg caatctagtt tcaccaatcc atgtacttgc aaatgctgaa 2700

gttgtggaga ttataattta tgataaatta tctgctaaat atgcattcca gcgtcaccag 2760

cagtggcttc aacatgcgaa aactcgcagt gcaagacaca aaattatgaa attcttgcgg 2820

gagcaagctg ctctttctgc tgctgaaata actgccgacg ccgttaataa ctttgttgcc 2880

gatcttgaag atgaaagtga ctatgagcag tcgattccaa gctctgaaaa taaggactat 2940

acattcaact ggcagaagat attgaattct gacaaactat cctttggaaa caagaagagc 3000

gactgctttt tacctgttaa aaatgtctct gtgccaaagg ttaatgggaa gcacaacaaa 3060

actgtcaaag aactgggcat caaaattaat ggttcaacat ttcgtggtga tagcttcact 3120

gattttattc accctggtgt ttccagcagt aaagaagttc tccctagtgt ggataactgg 3180

aaagctggta aaatttgtgc gtggcacaat acagaaggca gctctatcca atggctctgc 3240

atagtgtgtg ttgatcgaaa aggtatggtt gcggaagttt catcagctct aacagcgtgt 3300

ggaatcacca tatgctcgtg tgtggcggag cgagataaga gaaggggaat aggtgtgatg 3360

ttgttccact ttgagggggc atatgagaat gtggtcagtg catgctccgg tgttgatatg 3420

attcttgggg ttcttggttg gtccgttgga tgcagctgta atcctttggg tgttcttgaa 3480

tgccccgggg attacaagga tgacgacgat aagtgctaag ctagcggatc cactagttct 3540

agaggatccc cgggtaccga gctcgaattt ccccgatcgt tcaaacattt ggcaataaag 3600

tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa 3660

ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt 3720

tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc 3780

aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatcgg 3829

Claims (10)

1. Use of an ARE2 protein for promoting precocity and/or shortening the growth cycle of a plant;

   the ARE2 protein is (a1) or (a2) or (a3) or (a4) or (a5) or (a6) or (a 7):

   (a1) protein shown in a sequence 3 in a sequence table;

   (a2) a protein derived from rice, having 98% or more identity to (a1) and having the same function;

   (a3) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and having the same function;

   (a4) a protein encoded by the DNA molecule shown in the 1581-12019 th site in the sequence 1 of the sequence table;

   (a5) a protein coded by a DNA molecule shown in the position 1301-12512 in the sequence 1 of the sequence table;

   (a6) protein coded by DNA molecule shown in sequence 1 of the sequence table;

   (a7) protein coded by DNA molecule shown in sequence 2 of the sequence table.
2. Use of the ARE2 gene for breeding transgenic plants with early maturity and/or reduced growth period;

   the ARE2 gene is a DNA molecule encoding the ARE2 protein of claim 1.
3. 3. Use according to claim 2, characterized in that:

   the ARE2 gene is (b1) or (b2) or (b3) or (b4) or (b5) or (b 6):

   (b1) the cDNA coding region is a DNA molecule shown as a sequence 2 in a sequence table;

   (b2) the genome DNA is shown as the DNA molecule at the 1581-12019 site in the sequence 1 of the sequence table;

   (b3) the genome DNA is shown as DNA molecule at position 1301-12512 in sequence 1 of the sequence table;

   (b4) the genome DNA is a DNA molecule shown as a sequence 1 in a sequence table;

   (b5) a DNA molecule derived from rice and having 98% or more identity to (b1) or (b2) or (b3) or (b4) and encoding the ARE2 protein;

   (b6) a DNA molecule that hybridizes under stringent conditions to (b1) or (b2) or (b3) or (b4) and encodes the ARE2 protein.
4. 4. A method of breeding a transgenic plant with early maturity and/or reduced growth period comprising the steps of: introducing the ARE2 gene of claim 2 or 3 into a recipient plant to obtain a transgenic plant with early maturity and/or reduced fertility.
5. The application of the ARE2 protein is (c1) or (c2) or (c3) as follows:

   (c1) regulating and controlling the tolerance of the plant to low nitrogen stress;

   (c2) regulating and controlling the nitrogen metabolism process of plants;

   (c3) affecting the balance between plant nitrogen metabolism and stress response;

   the ARE2 protein is the ARE2 protein of claim 1.
6. 6. A method of breeding a transgenic plant with increased tolerance to low nitrogen stress comprising the steps of: inhibiting the expression of the ARE2 gene of claim 2 or 3 in a plant of interest to obtain a transgenic plant with increased tolerance to low nitrogen stress.
7. 7. A method of breeding a transgenic plant with increased tolerance to low nitrogen stress comprising the steps of: a transgenic plant having increased tolerance to low nitrogen stress, which is obtained by mutating the ARE2 gene of claim 2 or 3 as a target gene in a target plant.
8. 8. A protein which is the ARE2 protein according to claim 1.
9. 9. A DNA molecule which is the ARE2 gene according to claim 3.
10. 10. Use of the ARE2 protein of claim 1 as a ppGpp hydrolase.

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