CN103497954B - A kind of regulate and control the protein of low temperature lower blade color and gene thereof and application - Google Patents

Abstract

A protein regulating the color of leaves at low temperature and its gene and application belong to the technical field of genetic engineering. The invention discloses a nucleotide sequence of a gene regulating the leaf color protein at low temperature and an amino acid sequence of the protein; and provides an application of the gene. The invention utilizes the rice seedling stage temperature-sensitive mutant to clone the YWL1 gene through the map-site cloning method, and the gene encodes a pseudouracil synthase family protein located in the chloroplast. The function of YWL1 gene was identified by transgene complementation experiment. Therefore, the invention can regulate the development of rice chloroplasts to obtain leaf change phenotypes at the seedling stage. Lay the foundation for the further utilization of this gene.

Description

A protein regulating leaf color at low temperature and its gene and application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a protein for regulating the color of leaves at low temperature, its gene and application.

Background technique

Leaf is the main organ for photosynthesis in plants, and more than 2/3 of the dry matter in rice grains is obtained through photosynthesis after flowering (Wang Xujun, Xu Qingguo, Yang Zhijian (2005) Research progress in rice leaf senescence physiology, China Agricultural Science Bulletin 21: 187-190), the efficiency of photosynthesis has a complex relationship with the integrity of chloroplast structure and function, the stability of photosynthetic complex, and the level of chlorophyll content. In recent years, the application value of leaf color has attracted much attention. Leaf color variation can be used as a marker trait, which plays an important role in rice hybrid breeding and improved rice breeding. It can be used to determine the purity of seeds (Zhang Zhixing, Chen Shanfu (2001) Application of leaf marker technology in hybrid rice seed production, Seed Science and Technology 19: 33-34). In addition, the study of leaf color mutants has important theoretical significance and application value for the effective use of genetic engineering to improve the photosynthetic ability of rice, cultivate rice with high light efficiency, and increase rice yield.

At present, using rice leaf color mutants, several genes involved in or regulating chlorophyll metabolism and chloroplast development have been cloned. Through the analysis of gene function, expression pattern, gene interaction and nuclear-cytoplasmic signal transduction, we have a preliminary understanding of rice leaf color. Formation and regulation mechanism. So far, key enzymes in the process of chlorophyll synthesis in Arabidopsis have been identified (Nagata N (2005) Identification of a Vinyl Reductase Gene for Chlorophy11 Synthesis in Arabidopsis thabliana and Implications for the Evolution of Prochlorococcus Species. The Plant Cell Online 17:233-240 ), but only a few genes have been identified in rice, and other genes have yet to be further discovered. In addition, the regulatory mechanism of chlorophyll degradation is still unclear, and the mechanism of nucleoplasmic interaction is still unclear, which needs to be further studied.

Contents of the invention

Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for regulating the protein, gene and application of the color of leaves at low temperature.

The gene encoding and regulating the color of leaves at low temperature is characterized in that it is one of the following nucleotide sequences:

1) It is the nucleotide sequence described in SEQ ID No.1;

2) It encodes a protein composed of the amino acid sequence shown in SEQ ID No.3.

The protein regulating the color of leaves at low temperature is characterized in that the protein has the amino acid sequence shown in SEQ ID No.3.

The said transgenic cell line is characterized by containing the nucleotide sequence shown in SEQ ID No.1.

The said one kind of transgenic recombinant bacterium is characterized in that it contains the nucleotide sequence shown in SEQ ID No.1.

Application of the gene encoding and regulating leaf color at low temperature in cultivating transgenic plants with changes in chloroplast development.

The application of the gene encoding and regulating the color of leaves at low temperature in cultivating transgenic plants with albino leaves at seedling stage.

Concrete implementation steps of the present invention are as follows:

1. Isolation and genetic analysis of rice seedling temperature-sensitive mutant ywl1

The results of chlorophyll content analysis showed that the chlorophyll accumulation rate of the ywl1 mutant was slower than that of the wild type during the growth and development of leaves at 23°C, and the chlorophyll content tended to be consistent with that of the wild type as the leaves matured; The anterior leaves of Yixin were yellowish-white, the chlorophyll content was significantly lower than that of the wild type, and the chlorophyll fluorescence parameters were not significantly different; the phenotype and physiological indicators of the mutant leaves were consistent with those of the wild type at 28°C (Figure 1). Chloroplast ultrastructural analysis showed that thylakoid lamellar structure and grana had not been differentiated in most cells of the white part of ywl1 mutant leaves, and mature chloroplasts could be observed in only a few cells. With the development of leaves, the number of stacked grana lamina increases (Fig. 4).

In order to determine whether the phenotype of the ywl1 mutant is controlled by a single gene, the present invention carried out corresponding genetic analysis on ywl1. In the F2 individual plants obtained by crossing the ywl1 mutant with japonica rice 02428, the normal phenotype individual plants and the ywl1 mutant phenotype individual plants were obtained respectively, and the segregation ratio of the normal phenotype and mutant phenotype individuals was consistent with the segregation ratio of 3 :1 (see Table 1). In addition, the F1 plants obtained by crossing the ywl1 mutant with wild-type 93-11 and crossing the ywl1 mutant with japonica rice 02428 showed normal phenotypes. The results of genetic analysis indicated that the phenotype of the ywl1 mutant was caused by a single recessive nuclear gene mutant.

Table 1 Separation of white-striped leaf plants and normal leaf-color plants in F2 population

combination cross ywl1 /02428 Wild type 1902 Mutant phenotype Mutant type 619 Total 2521 Chi squared χ ² (3:1) 0.24 P valueP-Value 0.62

2. Analysis of chlorophyll content and light characteristics of ywl1 mutant leaves under different temperature environments

In order to clarify the dynamic change process of ywl1 leaf color, we measured the chlorophyll content of leaves from the one-leaf stage to the five-leaf stage (Fig. 2). It can be seen from the measurement results that the chlorophyll content of the mutant increases gradually with the growth of the plants. At 23°C, the total amount of chlorophyll a, chlorophyll b, and chlorophyll in the wild type was always higher than that in the mutant; at 25°C, the total amount of chlorophyll a, chlorophyll b, and chlorophyll in the wild type was higher than that in the mutant, and the amount of chlorophyll in the three-leaf The chlorophyll content of the two tended to be the same after the period; at 28°C, the chlorophyll a, chlorophyll b and the total amount of chlorophyll were basically the same, which was consistent with the phenotype results.

It can be seen from Figure 3 that the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of the ywl1 mutant at the 23°C environment were significantly lower than those of the wild type, showing a more serious decrease in light and capacity characteristics, heading stage recovery was consistent with that of the wild type; in the ywl1 mutant at 25 °C, the net photosynthetic rate (Pn) at the seedling stage was significantly lower than that of the wild type, and the stomatal conductance (Gs) and transpiration rate (Tr) were similar to those of the wild type There was no significant difference, and the three indicators at the heading stage were consistent with those of the wild type; the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr ) are consistent with the wild type.

3. Map-based cloning of the YWL1 gene

1. Molecular localization of YWL1 gene:

Genetic analysis revealed that the white-striped leaf phenotype of the ywl1 mutant is controlled by a pair of recessive nuclear genes. Using 184 extreme individuals of the F2 population derived from crossing the ywl1 mutant with japonica rice 02428, the ywl1 gene was located between RM14327 and RM5474 on the short arm of the third chromosome. Then, by expanding the mapping population, a high-density linkage map of the ywl1 gene locus region was constructed based on the published SSR and self-developed Indel markers, and finally the mutant gene was finely mapped within the 37.1kb interval, as shown in Figure 5.

2. Identification and functional analysis of YWL1 gene

Using gene analysis and prediction software to predict the located 37.1kb region, it was found that there were 5 open reading frames (ORFs), among which ORF5 coded (LOC_Os03g05806) pseudouridine synthase family protein, and the protein sequence encoded by the gene was similar to that of the proposed The pseudouracil synthase family protein sequence encoded by Arabidopsis SVR1 has 59% similarity. The semi-quantitative RT-PCR results showed that the expression of ORF5 (LOC_Os03g05806) was significantly increased in the ywl1 mutant; Arabidopsis svr1 mutation resulted in chloroplast protein Loss of translation and rRNA processing, phenotype suppression of the variegated phenotype of var2 mutants. Sequencing results found LOC_Os03g05806 There is a deletion of 18 bp bases in the ORF region, resulting in a deletion of 6 amino acids at positions Asp214-Ser219 in the protein sequence of the gene. Therefore, the YWL1 gene was identified as a candidate gene for the ywl1 mutant gene. The gene sequence amplified by the wild-type variety is SEQ ID NO.1, named YWL1 gene.

In order to observe the subcellular localization function of YWL1 protein (SEQ ID NO.3), the CDS sequence of YWL1 gene (SEQ ID NO.2) was constructed into PAN580-GFP In the expression vector, the fusion expression vector was obtained, and then YWL1 (SEQ ID NO.2 protein expression). First, the plastids of the fusion expression vector and the gene-free fusion empty vector PAN580-GFP were extracted, respectively wrapped with gold powder, and bombarded with a gene gun into rice preplastid cells. After culturing for 16 hours in a dark environment, observe under a confocal laser microscope. The results showed that the green fluorescence of the control vector without gene fusion was uniformly expressed in the whole rice preplastid cells, while the fusion protein was specifically distributed in the chloroplast (Figure 6).

The invention utilizes the rice seedling stage temperature-sensitive mutant to clone the YWL1 gene through the map-site cloning method, and the gene encodes a pseudouracil synthase family protein located in the chloroplast. The function of YWL1 gene was identified by transgene complementation experiment. Therefore, the invention can regulate the development of rice chloroplasts to obtain leaf change phenotypes at the seedling stage. Lay the foundation for the further utilization of this gene.

Description of drawings

Figure 1 shows the phenotypes of the japonica rice variety Asominori wild type and the seedling temperature-sensitive mutant ywl1 at various temperatures;

Figure 2 shows the chlorophyll content of the ywl1 mutant and wild type at different developmental stages under different temperature conditions;

Figure 3 is the light and characteristics of ywl1 mutant and wild-type leaves under different temperature conditions;

Figure 4 is the transmission electron microscope observation of wild type and mutant, A, D the mesophyll cell and chloroplast structure of the wild type 93-11; B, the mesophyll nucleus chloroplast structure of the white leaf part of the white striped leaf of the Eywl1 mutant; C, F The mesophyll nuclei and chloroplast structure of the green leaf part of the white-striped leaf of the ywl1 mutant;

Figure 5 shows the fine mapping of the YWL1 gene on the rice chromosome 3;

Figure 6 is 35S:: The results of transient expression of YWL1::GFP fusion protein in rice preplastid cells; the upper row is the localization result of 35S::GFP empty vector, the lower row is the localization result of 35S::YWL1::GFP fusion vector; the left is the white light map, The middle is the GFP fluorescence image, and the right is the fusion image of the former two.

detailed description

In order to understand the present invention, the present invention is further illustrated below with examples, but the present invention is not limited.

Example 1: Phenotypic analysis of ywl1 mutants

a) Rice material

Rice (Oryza sativa L) mutant ywl1, the original wild-type material is indica variety 93-11.

b) Determination of chlorophyll content and photosynthetic characteristics

Select plump mutant and wild-type seeds to soak and accelerate germination, plant the germinated mutant and wild-type 93-11 seeds in pots, and set the daily constant temperature in the artificial climate box to 23°C, 25°C and 28°C respectively , the light length was 12h, the dark treatment was 12h, and the light intensity was 5Lax. Observe and record mutant phenotypes. The chlorophyll content of leaves was measured at five stages (one-leaf stage, two-leaf stage, three-leaf stage, four-leaf stage and five-leaf stage) before and after greening.

Determination of chlorophyll content: Take 0.1g-0.2g of the sample to be tested and soak it in 10ml of 95% ethanol, and place it in a refrigerator at 4°C to extract the pigment for about 48 hours. Take a little of the above extract and pour it into a 1cm cuvette, use 95% ethanol as the reference solution, and read the optical densities at 665nm, 649nm, and 470nm under a Beckman Du80 spectrophotometer. The contents of chlorophyll a (Chla), chlorophyll b (Chlb) and carotenoids (Caro) were calculated using the modified formula of Lichtenthaler method.

Chla content (mg/g) = (13.95O.D665-6.88O.D649) V/1000W

Chlb content (mg/g) = (24.96O.D649-7.32O.D665) V/1000W

Caro content (mg/g) = (1000O.D470+811.74O.D665-2851.32O.D649)/245

Note: O.D: Optical density value at the measured wavelength.

V: total volume of chlorophyll extract (mL). If a diluent is used for the determination, it should be multiplied by the dilution factor.

W: fresh weight of material (g).

Determination of photosynthetic characteristics: Li-6400 portable photosynthetic measuring instrument produced by Li-COR Company of the United States is used, which is an open system, using red and blue light sources, with an optical quantum density of 1 200 μmol m ^-2 s ^-1 , and a flow rate of 500 μmol s ^-1 , on a sunny day From 9:00 to 11:00, the net photosynthetic rate of each material under three environmental conditions was measured respectively. Three representative flag leaves were measured for each treatment, and each leaf was repeatedly measured three times (the average value was taken as one repetition). The measurement indexes include leaf net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO2 concentration (Ci).

It can be seen from Figure 2 that the chlorophyll content of the mutant gradually increases with the growth of the plants. At 23°C, the total amount of chlorophyll a, chlorophyll b, and chlorophyll in the wild type was always higher than that in the mutant; at 25°C, the total amount of chlorophyll a, chlorophyll b, and chlorophyll in the wild type was higher than that in the mutant, and the amount of chlorophyll in the three-leaf The chlorophyll content of the two tended to be the same after the period; at 28°C, the chlorophyll a, chlorophyll b and the total amount of chlorophyll were basically the same, which was consistent with the phenotype results.

It can be seen from Figure 3 that the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of the ywl1 mutant at the 23°C environment were significantly lower than those of the wild type, showing a more serious decrease in light and capacity characteristics, heading stage recovery was consistent with that of the wild type; in the ywl1 mutant at 25 °C, the net photosynthetic rate (Pn) at the seedling stage was significantly lower than that of the wild type, and the stomatal conductance (Gs) and transpiration rate (Tr) were similar to those of the wild type There was no significant difference, and the three indicators at the heading stage were consistent with those of the wild type; the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr ) are consistent with the wild type.

c) Electron microscope observation

Using transmission electron microscope (TEM) to observe the chloroplast ultrastructure of the third leaf of the wild type and ywl1 at 23°C, it was found that most of the cells in the white part of the ywl1 mutant leaves had not yet differentiated thylakoid sheet structure and basal cells. Granules, mature chloroplasts can only be observed in a few cells, and the entire chloroplast is underdeveloped, but with the development of leaves, the number of stacked grana lamellae increases, as shown in Figure 4.

Embodiment 2: Cloning of YWL1 gene

a) Genetic analysis and mapping populations

The positive and negative hybridization was used to confirm that ywl1 was a recessive mutant, and the mutant was selected for hybridization with japonica rice 02428, the F1 generation was self-crossed, and a single plant was harvested to plant the F2 population, and 4391 recessive individuals were selected from the segregated population as the positioning population . At the three-leaf stage, about 1 gram of young leaves were taken from each plant to extract total DNA for gene mapping.

b) Preliminary and fine mapping of the YWL1 gene

The rapid extraction method of rice trace DNA was used to extract genomic DNA for gene localization from rice leaves, and the DNA extraction method was SDS method (Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version Ⅱ. Plant Mol Bio Rep 1:19-21). Take about 100mg of rice leaves, cut them into pieces and put them into a 2ml centrifuge tube, add steel balls, freeze them with liquid nitrogen, grind them on a mill, and then extract DNA. sample.

In the preliminary mapping of YWL1 gene, SSR analysis was carried out with 30 F2 individuals with mutant phenotype (strain F2 obtained by crossing ywl1 mutant with japonica rice 02428). Firstly, according to the published molecular genetic maps of japonica and indica rice, select SSR primers approximately evenly distributed on each chromosome for PCR amplification (the reaction system is as follows). Separation by electrophoresis on 8% polyacrylamide gel (gel configuration method is as follows), and by detecting the polymorphism of the band, the gene is initially located on the short arm of chromosome 3, between the two SSR markers RM14327 and RM5474 between.

PCR reaction system:

DNA template 1μL 10╳PCR Buffer 1μL dNTP (10mM) 0.1μL Upstream primer (10 μm) 0.5μL Downstream primer (10μm) 0.5μL Taq enzyme (5U/μL) 0.2 μL ddH ₂ O 6.7μL to 1ml

8% polyacrylamide gel formula:

5╳TBE 6ml 40% Arc-Bis 6 10% AP (μL) 240 TEMED (μL) 30 ddH ₂ O 18 to 30ml

Polyacrylamide gel chromogenic solution formula:

Na ₃ (BO ₄ ) ₄ 0.152 NaOH 12g formaldehyde 3.2ml ddH ₂ O up to 800ml

Note: Formaldehyde is added before use, and the other three are prepared in advance according to the corresponding amount.

Then, by analyzing the BAC sequence between the two markers RM14327 and RM5474, a new Indel marker was developed, and finally the F2 mapping population was expanded to 4291 for fine mapping, and YWL1 was precisely located on the markers RM6829 and RM14400 on the BAC clone OSJNBa0016I15 Within the range of 37.1 kb, as shown in Figure 5, the candidate gene was deduced by analyzing the open reading frame (ORF) of this segment and sequenced to find the mutation site.

Indel marker primer sequence:

RM14327-F GATGCAGTAGGAACACCAAACAGC (SEQ ID NO.4) RM14327-R ATCGAGTACCAAGTGCCTGTGC (SEQ ID NO.5) RM5474-F GTGGGTTTGTGTTTGGAGAGACG (SEQ ID NO.6) RM5474-R GTGTTGGTGAGCATAGCAGTTGG (SEQ ID NO.7) RM6829-F CGATGAAGAGCCAAATCCTTCAGC (SEQ ID NO.8) RM6829-R TGCTCGTCCCTTTCTACAAACAGG (SEQ ID NO.9) RM14400-F GGCAGCGAGTAAGTGTAGATTGG (SEQ ID NO.10) RM14400-R TGTTGGTATAAGACAGGTGCATGG (SEQ ID NO.11)

f) Gene prediction and comparative analysis

According to the results of fine positioning, in the range of 37.1kb according to RiceGAAS (Rice According to the prediction of Automat ed Systrm, http://ricegaas.dna.affrc.go.jp/) and TIGR (http://rice.plantbiology.msu.edu/), there are 5 candidate genes in this interval. It performed sequence comparison analysis and found that 1029-1046 18bp bases of ORF5 were missing, which in turn caused the deletion of 6 amino acids at Asp214-Ser219 in the protein sequence of the gene. The gene sequence amplified by the wild-type variety is SEQ ID NO.1, named YWL1 gene, the nucleotide sequence obtained by sequencing the encoded protein is SEQ ID NO.2.

Example 3: Chloroplast localization experiment of YWL1 (SEQ ID NO.3)

According to the full-length CDS sequence of YWL1 (SEQ ID NO.2), a restriction recognition site containing BamHI was designed, and recombinant primers were designed. The sequence is:

PGXhF: TTTCTCGAGATAAACCCCCTCCCACTCTCCAC (SEQ ID NO.14)

PGSalR: TTTGTCGAGCCAAATTTGATATTGCACAATGGGA (SEQ ID NO.15)

Using the wild-type cDNA as a template, the CDS sequence (SEQ ID NO.2) of the YWL1 gene was amplified with PrimeSTAR high-fidelity enzyme. After the sequence was verified to be correct, the amplified product was ligated with the PAN580-GFP vector to obtain the fusion expression vector 35S ::YWL1::GFP.

Extract and construct the fusion expression vector 35S::YWL1::GFP plasmid and the 35S::GFP control plasmid without gene fusion respectively wrapped with gold powder, and use the PDS-1000/He type gene gun of Bio-rad company under the helium pressure of 1100psi Bombardment of rice preplastid cells. The rice preplastid cells after bombardment were cultured in hypertonic medium in the dark for 16 hours, and then observed under a fluorescent microscope. The YWL1::GFP fusion protein was localized in the chloroplast, while the empty vector GFP was expressed in the whole cell (Figure 6). This result demonstrates that YWL1 (SEQ ID NO.2) function, it is a chloroplast-localized protein.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

sequence listing

<110> China Rice Research Institute

<120> A protein that regulates leaf color at low temperature and its gene and application

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 2402

<212> DNA

<213> (Rice) Oryza sativa

<400> 1

atccccttct tatccgctct gctctctcca cctcgcctcc ccgaaacgaa atggcagtgg 60

cgttccactg aaccaggcca agccagacgg acaagacttc gccgcagccg cgtcctcctc 120

accgcgccgc cgtcgcaatg gctacggcca tcgcagcctc cgcgttcctc tcctccgcct 180

tcgccaggga caggccgctc ccccgccagc ggcgcgcggc gaggcccgcg acccgccgcg 240

ccgccgcggg gggcctgtcg gtgcggtgcg agcagagcga gaagcagaag aggcagccgc 300

tctccgcgct cgtcccccgc gagcagcgat tcatgttcga aggcgacgag ctctgcggcc 360

ccgtactaac cctcacctct ctttctctct cccgtataca ttcttctctt tctcagttca 420

gaaaatgaat agtaatgcta cattccttgg ataaagggaa attttaccgt gatttaggat 480

tgccgcttat ttaggaaagc tcaagattag tcccattcat ttctaaatcc gtaagtcact 540

tcatcttgct ctattgggtc aaatcctttc ttacatgaa taatgttagg gaaaatacac 600

aatgttttta catgttacta taccgtatag accaccatct tattttattt aaagttatct 660

agttgtaatt tattacgaaa gaggattcca taaaatgaac tgaaaataac atggagggaa 720

cgttggtctt tctgaattgg tcgctaacat tgtgttgtat tactcaacag tgttcgcacc 780

tacatgacta ccatatcttt ttatctgtgg atgtttaggt ttgtgaagtt agaattcagt 840

acggatacca ttcttgtgtt ctttctctct tttctgtgca acctgttctg tgtagtgatc 900

ggaccaagga atttaatttg caggacatat ggaacacaac atggtaccct aaggctgcag 960

atcatgtaac tactgagaag acatggtatg ttgttgatgc aacagacaag attctaggta 1020

ggcttgcatc caccattgca gtacacatca gaggaaagaa tgaggccaca tacactccaa 1080

gtgtggacat gggggctttt gttgttgtgg tatgtatgtt tcttgaaatt aatttataaa 1140

gttaaatagt gcacacaata agatcctctg tagtcccaca aaaggaaaag aggtttagca 1200

gcacatacaa ataagcacca aaaggtatct ggaagctgag gtccacctga aggcattttg 1260

aagctgattt catgtatata tattatgtt actgtgtgct tttaatacta atgcatctct 1320

gaagagtaat tgctacgcat atactattgt aagttcctga actaccatgc tgaactctat 1380

gttcacatag gttaatgcag agaaggttgc tgtttctggc aaaaagcggt cacagaagct 1440

cctacaggagg cactctggac ggcctggtgg aatgaaagaa gaaacttttg atcagcttca 1500

gaaaaggatt ccggagagaa ttattgaaca tgcagtgcgt ggcatgcttc ctaagggcag 1560

agtaagacat catctcttag agctcttatc tagtcagttt catcaagtct ttttgacctt 1620

aagtcagcct acctgggtat acatatgctc ttatccttat ttttggtcaa tttctaatac 1680

atatctgaaa atgggtatcc tcgttctgtt ccattatact tcattattgt gtataattcc 1740

ccaactttat ataccctgtt catggaaata ttcgctctaa ttgaatgccg aaacggcgtg 1800

ggctaacgaa gtactagatg tattctccca aaactcctat gttgcctgta agacatctta 1860

tgattgccta aagcctttta cttatgccta agttcattta taaaaaaaaaaaacgtttac 1920

tatgtcaacc tctcaagttc tcatatgtta gtgacactga cacatgtaat ccgaccaact 1980

acagcggttg tacgtgctct atgtagcctt ctcaacattttgaacatttt gcagctggga 2040

agaagactgt ttacccacct caaggtgtac aagggagcag aacatcccca tgaggctcaa 2100

aaacctgttc cactgcctat caaggataaa agaatacaga agtctgagaa gtagataatt 2160

cctgtggaga ctaagaaaga cacaacaatc tcacgtaaat tcctcccctc gagattttgt 2220

ccaatctttg gaatgtgatg catctgtctg tttggggaat ggagtgtaaa attttttgta 2280

atttacttcc tccaaaagtt tgtgtttcta agtcatagtt cttgttgatt acatatcatg 2340

tacctaatac cgtacgattt taagcaatcc gatatttcca tatccaattg agtagtgcaa 2400

tt 2402

<210> 2

<211> 702

<212> DNA

<213> (Rice) Oryza sativa

<400> 2

atggctacgg ccatcgcagc ctccgcgttc ctctcctccg ccttcgccag ggacaggccg 60

ctcccccgcc agcggcgcgc ggcgaggccc gcgacccgcc gcgccgccgc ggggggcctg 120

tcggtgcggt gcgagcagag cgagaagcag aagaggcagc cgctctccgc gctcgtcccc 180

cgcgagcagc gattcatgtt cgaaggcgac gagctctgcg gccccgacat atggaacaca 240

acatggtacc ctaaggctgc agatcatgta actactgaga agacatggta tgttgttgat 300

gcaacagaca agattctagg taggcttgca tccaccattg cagtacacat cagaggaaag 360

aatgaggcca catacactcc aagtgtggac atggggggctt ttgttgttgt ggttaatgca 420

gagaaggttg ctgtttctgg caaaaagcgg tcacagaagc tctacaggag gcactctgga 480

cggcctggtg gaatgaaaga agaaactttt gatcagcttc agaaaaggat tccggagaga 540

attattgaac atgcagtgcg tggcatgctt cctaagggca gactgggaag aagactgttt 600

accccacctca aggtgtacaa gggagcagaa catccccatg aggctcaaaa acctgttcca 660

ctgcctatca aggataaaag aatacagaag tctgagaagt ag 702

<210> 3

<211> 228

<212> PRT

<213> (Rice) Oryza sativa

<400> 3

Met Ala Thr Ala Ile Ala Ala Ser Ala Phe Leu Ser Ser Ala Phe Ala

1 5 10 15

Arg Asp Arg Pro Leu Pro Arg Gln Arg Arg Ala Ala Arg Pro Ala Thr

20 25 30

Arg Arg Ala Ala Ala Gly Gly Leu Ser Val Arg Cys Glu Gln Ser Glu

35 40 45

Lys Gln Lys Arg Gln Pro Leu Ser Ala Leu Val Pro Arg Glu Gln Arg

50 55 60

Phe Met Phe Glu Gly Asp Glu Leu Cys Gly Pro Asp Ile Trp Asn Thr

65 70 75 80

Thr Trp Tyr Pro Lys Ala Ala Asp His Val Thr Thr Glu Lys Thr Trp

85 90 95

Tyr Val Val Asp Ala Thr Asp Lys Ile Leu Gly Arg Leu Ala Ser Thr

100 105 110

Ile Ala Val His Ile Arg Gly Lys Asn Glu Ala Thr Tyr Thr Pro Ser

115 120 125

Val Asp Met Gly Ala Phe Val Val Val Val Ala Val Ser Gly Lys Lys

130 135 140

Arg Ser Gln Lys Leu Tyr Arg Arg His Ser Gly Arg Pro Gly Gly Met

145 150 155 160

Lys Glu Glu Thr Phe Asp Gln Leu Gln Lys Arg Ile Pro Glu Arg Ile

165 170 175

Ile Glu His Ala Val Arg Gly Met Leu Pro Lys Gly Arg Leu Gly Arg

180 185 190

Arg Leu Phe Thr His Leu Lys Val Tyr Lys Gly Ala Glu His Pro His

195 200 205

Glu Ala Gln Lys Pro Val Pro Leu Pro Ile Lys Asp Lys Arg Ile Gln

210 215 220

Lys Ser Glu Lys

225

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<400> 4

gatgcagtag gaacaccaaa cagc

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence

<400> 5

atcgagtacc aagtgcctgt gc

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence

<400> 6

gtgggtttgt gtttggagag acg

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence

<400> 7

gtgttggtga gcatagcagt tgg

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<400> 8

cgatgaagag ccaatccttc agc

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<400> 9

tgctcgtccc ttctacaaac agg

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence

<400> 10

ggcagcgagt aagtgtagat tgg

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence

<400> 11

tgttggtataagacagggtgc atgg

<210> 12

<211> 35

<212> DNA

<213> Artificial sequence

<400> 12

gcaggtcgac ggatcctatc cgataaccga taaac

<210> 13

<211> 35

<212> DNA

<213> Artificial sequence

<400> 13

gaattcccgg ggatcccata agcaggtttg agaag

<210> 14

<211> 35

<212> DNA

<213> Artificial sequence

<400> 14

tttctcgaga taaaccccct cccacactct ccac

<210> 15

<211> 35

<212> DNA

<213> Artificial sequence

<400> 15

tttgtcgagc caaatttgat attgcacaat ggga

Claims (1)

1. A protein regulating the color of leaves at low temperature, characterized in that the protein is the amino acid sequence shown in SEQ ID No.3.