CN109161554B - Regulatory gene of rice dwarf fragile-stem mutant DBC1 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a regulatory gene of a rice dwarf fragile-stem mutant DBC1 and application thereof. According to the invention, the rice dwarf fragile stalk mutant DBC1 is cloned and sequenced, the regulatory gene of the rice dwarf fragile stalk is excavated, and experiments prove that the gene has the function of regulating the rice dwarf fragile stalk, so that a powerful tool is provided for rice transgenic research, and breeding research of high-yield and high-quality rice can be promoted.

Description

Regulatory gene of rice dwarf fragile-stem mutant DBC1 and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a regulatory gene of a rice dwarf fragile-stem mutant DBC1 and application thereof.

Background

Rice is the most important food crop in China, and more than half of people use the rice as staple food; meanwhile, rice is a cereal grain crop which is sequenced at the earliest because the genome is small and has higher homology, so that the rice becomes a model plant for molecular biology research of monocotyledons. The green revolution and the application of the hybrid rice greatly improve the single amount of the rice and have important significance for solving the food safety in China. The straw returning field is a soil fertility increasing and yield increasing measure which is generally regarded as important in the world at present, and has the functions of increasing fertilizer and increasing yield while avoiding atmospheric pollution caused by straw burning. In addition, the straw returning can increase soil organic matters, improve the soil structure, loosen the soil, increase the porosity, reduce the capacity and promote the activity of microorganisms and the development of crop roots. The effect of increasing the fertilizer and increasing the yield of the straw returning is obvious, and the yield can be increased by 5 to 10 percent generally. Because the straws are tough, the straw is not easy to crush and return to the field, and the straw treatment is always a troublesome problem. The stalk of the brittle stalk mutant becomes moderately brittle, which is beneficial to returning the stalk to the field, and is one of the important technical measures for solving the contradiction. Rice brittle stalk mutants generally show a content change of cellulose and lignin in cell walls, and the content change often changes the mechanical strength of stalks, thereby influencing the lodging resistance and environmental adaptability of rice. Therefore, the identification of the mutant with the stalk being moderately crisp and the agronomic characters being not influenced or less influenced is carried out, the molecular level research on the mutant is not only helpful for clarifying the biosynthesis mechanism of the rice cell wall, but also has important significance for breeding the novel crisp but not fallen environment-friendly rice variety.

Disclosure of Invention

The invention takes the rice dwarf fragile stalk mutant as a research object, digs out a regulatory gene of the rice dwarf fragile stalk by cloning and sequencing the rice dwarf fragile stalk mutant DBC1, and experiments verify that the gene has the function of regulating the rice dwarf fragile stalk.

In particular, the invention comprises several aspects:

one aspect of the invention relates to a regulatory gene of a rice dwarf fragile stalk mutant DBC1, which has a nucleotide sequence shown as SEQ ID No.1, and a coded protein has an amino acid sequence shown as SEQ ID No. 2.

The invention also relates to the application of the regulatory gene of the rice dwarf fragile-stem mutant DBC1 in the breeding of environment-friendly rice varieties.

The invention also relates to an environment-friendly rice variety breeding method, which comprises the step of transforming the regulatory gene of the rice dwarf fragile-stem mutant DBC1 into rice.

The invention also relates to a reagent for regulating the plant height of rice, which mainly comprises the protein coded by the regulatory gene of the rice dwarf fragile-stem mutant DBC1, a vector containing the regulatory gene of the rice dwarf fragile-stem mutant DBC1 or a host cell containing the regulatory gene of the rice dwarf fragile-stem mutant DBC1 as an active ingredient.

The invention has the beneficial effects that: by cloning and sequencing the rice dwarf fragile stalk mutant DBC1 and digging out the regulatory gene of the rice dwarf fragile stalk, a powerful tool is provided for rice transgenic research, and breeding research of high-yield and high-quality rice can be promoted. Experiments prove that the gene has the function of regulating and controlling the division of rice cells and provides a direction for improving the plant type of rice.

Drawings

FIG. 1 is a morphometric identification of wild type and mutant DBC 1;

FIG. 1A shows wild type and mutant dbc1 sown for 7 days;

FIG. 1B shows the wild type and mutant in seedling stage;

FIG. 1C shows the wild type and the mutant at the tillering stage;

FIG. 1D shows internode lengths of mature wild type and mutant;

FIG. 1E is a statistic of internode length of wild type and mutant;

FIG. 1F shows the comparison of the plant heights of wild type and mutant;

FIG. 2 is a histological analysis of Wild Type (WT) and mutant (DBC 1);

FIG. 2A is a longitudinal cut of the second node stem of wild type at heading stage;

FIG. 2B is a longitudinal cut of the second node stem of the heading stage mutant;

FIG. 2C is a scanning electron micrograph of the inner skin of a second leaf sheath of a wild type;

FIG. 2D is a scanning electron micrograph of the inner skin of the second leaf sheath of the mutant;

FIG. 2E is a statistical plot of the sizes of wild-type and mutant second node stem cells;

FIG. 2F is a histogram of the size of epidermal cells in the sheaths of the wild-type and mutant second leaves;

FIG. 2G is a transverse cross-sectional view of wild type inverted two-node stems;

FIG. 2H is a transverse section of the stem of the inverted two nodes of the mutant;

FIG. 2I is an enlarged view of the area of the red frame of FIG. 2G;

FIG. 2J is an enlarged view of the red frame area of FIG. 2H;

FIG. 3 is a map-based clone of gene DBC 1;

FIG. 3A is the molecular location on chromosome 2;

FIG. 3B shows the gene structure of DBC 1;

FIG. 3C shows the amplified fragment sizes of Wild Type (WT) and dbc 1;

FIG. 3D is a sequencing alignment of wild type and mutant;

FIG. 3E is an alignment of wild type and mutant amino acid sequences;

FIG. 4 is an analysis of the expression pattern of DBC1 regulatory gene;

FIG. 5 shows transgenic plant identification and phenotypic analysis;

FIG. 5A shows mature wild type, mutant and transgenic plants;

FIG. 5B is a friability analysis of wild type, mutant and transgenic plants;

FIG. 5C shows the expression analysis of the DBC1 regulatory gene in wild type, mutant and transgenic overexpression lines;

FIG. 6 shows the response of mutant DBC1 to gibberellin GA3 and related gene analysis;

FIG. 6A is a graph showing the response of a wild-type second leaf sheath to gibberellin;

FIG. 6B is a second leaf sheath gibberellin response experiment with mutant DBC 1;

FIG. 6C is a graph showing the response of the wild-type and mutant second leaf sheaths to the growth of gibberellin GA 3;

FIG. 6D is an analysis of the expression of genes involved in gibberellin biological signaling;

FIG. 6E is an analysis of the expression of genes involved in gibberellin biosynthesis;

FIG. 6F shows the effect of gibberellin GA3 on the expression of GA2ox3 in wild-type and mutant DBC 1;

FIG. 7 shows the cell morphology of the meristematic region of the root tip and cyclin expression analysis of wild type and mutant;

FIG. 7A shows the cell morphology of the meristematic region of the wild type root tip;

FIG. 7B shows the cell morphology of the meristematic region of the root tip of the mutant;

FIG. 7C is an enlarged view of the area of the red frame of FIG. 7A;

FIG. 7D is an enlarged view of the area of the red frame of FIG. 7B;

FIG. 7E is a statistical plot of the number of single cells with two nuclei in the root tip;

FIG. 7F is a cyclin expression analysis;

description of the drawings: "x" indicates significant difference at the 0.05 level, and "x" indicates significant difference at the 0.01 level.

Detailed Description

The present invention will now be described more fully hereinafter with reference to the accompanying specific embodiments, in which some, but not all embodiments of the invention are shown. All other embodiments that can be derived from the embodiments of the present invention by a person skilled in the art are within the scope of the present invention.

The experimental procedures, for which specific conditions are not specified in the examples, are generally carried out according to conventional conditions, for example, as described in the molecular cloning protocols (third edition, J. SammBruk et al, Huangpetang et al, science publishers, 2002), or according to the conditions recommended by the manufacturers.

Materials used in the examples: wild type red silk hui No. 10 and rice dwarf fragile-stalk mutant DBC1 were provided by the rice institute of southwest university; M-MLV reverse transcriptase, high-fidelity DNA polymerase PFU, T4DNA ligase, a Trizol kit, a DNA gel recovery kit and a plasmid extraction kit are purchased from TaKaRa company; ampicillin (Ampicillin, Amp) is a product of Sigma company; primer synthesis and DNA sequencing were performed by Shanghai Junjun Biotechnology Co., Ltd; other chemical reagents were purchased from biotechnology limited liability company of beijing dingguo; coli DH5 α was provided by the Rice research institute of southwest university; the expression vector PTCK303 is provided by rice of southwest university.

In earlier research work, the rice institute of southwest university provided: a genetically stable dwarf and brittle rice stem mutant (named as DBC1) is obtained by mutating the excellent restorer line red silk with EMS, wherein the dwarf and brittle rice stem mutant is characterized in that the plant height is obviously reduced compared with the wild type, the brittleness is obviously increased to a different level, and the mutant is shown in figures 1A-F, wherein in 1E, PL represents the ear length, NL represents the ear neck length, FL represents the 1 st internode length, SL represents the 2 nd internode length, Bar is 5cm, "+" represents that the difference is obvious on the level of 0.05, and "+" represents that the difference is obvious on the level of 0.01. In the sterile line with normal phenotype, flower 11 is crossed with the mutant DBC1, F1 represents normal, and the mutant is indicated to be controlled by recessive genes. Obvious segregation appeared in the population of the F2 generation, and the two generations respectively showed the parent characters. According to the chi-square test, the normal strain and the mutant strain meet the separation ratio of 3: 1, and the DBC1 mutant is controlled by a recessive single gene. Cytological observations by paraffin section and electron microscopy (refer to doctor's academic thesis: map-based cloning and functional analysis of AVB gene, a gene related to rice vascular bundle development, 2016, pages 33-34) revealed that the size of cells was not significantly different from that of wild type, i.e., the plant height was dwarfed by affecting the number of cells, as shown in fig. 2A-F, in which Bar was 100 μm in 2A-2D and n was 50 in 2E-2F. In addition, this gene affects the structure of vascular bundles in the stem and increases xylem relative area, see fig. 2G-J, where 2G-J, Bar 200 μm, and 2I-J, Bar 50 μm. The F2 group of the Chinese flower 11/DBC1 cross is used as a positioning group, and 600 mutant strains are obtained in total and used for gene positioning. 400 pairs of SSR markers uniformly distributed on 12 chromosomes are selected to perform individual verification on F2 generation positioning group mutant individuals (refer to the Master academic paper: genetic analysis and gene positioning of rice leaf margin albino mutant mal, 2014, page 22), and as a result, the Ind markers Indel02-2 and Indel02-7 which are linked and positioned on 2 chromosomes are linked with DBC1 mutation sites. An Indel primer was further designed between the two markers, where Indel02-1-Indel02-7, showed polymorphism between the two parents, and the primer sequences were detailed in Table 1, finally positioning DBC1 between Indel markers Indel02-4 and Indel02-5 at a physical distance of 86.7kb, as shown in FIG. 3A.

Primers and sequences for fine localization of genes of Table 1

Implementation 1: cloning, sequencing and functional verification of rice dwarf fragile-stem mutant gene DBC1

1. Cloning and sequencing of rice dwarf fragile-stem gene DBC1

On the basis of fine positioning of a mutant gene DBC1 in the early stage, the method analyzes the functions through online gene prediction (http:// mendel.cs. rhul. ac. uk), BLAST online comparison (http:// BLAST. ncbi. nlm. nih. gov /) and gene overexpression. Through map-based cloning, sequence alignment shows that the gene is caused by mutation of the 5 th base of the 5 th intron of the gene LOC _ Os02g50550 from normal C to T. Because of the need for modification, splicing and splicing after gene transcription, intronic mutations may affect splicing editing of precursor messenger RNAs. Thus, a pair of primers P1, P2, separated by about 550bp, were designed on the cDNA, containing the mutation site, as shown in FIG. 3B, where the grey box represents an exon, the black line represents an intron, the fifth exon is underlined, and the red base represents the mutation site. The PCR amplified fragment was gel electrophoresed to show that the band of mutant DBC1 was relatively lower than that of wild type, and that there may be deletion of large fragments, as shown in FIGS. 3C-D. Transferring the cloned gene DBC1 into an expression vector pMD19-T, carrying out spot picking, detecting, and sending positive bacteria liquid for sequencing. The alignment shows that the mutant has a deletion of 62bp compared to the wild type, and the deletion of this fragment is located at the fifth exon, as shown in FIG. 3E. That is, a single base mutation of the 5 th intron of mutant DBC1 resulted in an overall deletion of the 5 th exon. The amino acid sequence alignment of the wild type and the mutant shows that the gene mutation causes the change of the GTP structural domain.

2. Functional verification of rice short crisp gene DBC1

Expression analysis of wild type and mutant LOC _ Os02g50550 by real-time fluorescent quantitative qPCR shows that (refer to doctor academic paper: map-based cloning and function analysis of rice spikelet development related gene MFS1, 2013, page 29) the expression level of LOC _ Os02g50550 gene in mutant DBC1 is obviously down-regulated relative to wild type, and LOC _ Os02g50550 is expressed in each part of rice, and is expressed in stem and apical meristem of plants to the highest degree, as shown in figure 4. Then, the invention constructs that the cDNA of LOC _ Os02g50550 gene restored by wild type red silk is taken as a template, the amplified fragment is connected to an expression vector pTCK303, a pTCK303-DBC1OE gene overexpression vector is constructed, and the gene overexpression vector is transformed into a mutant DBC1 through agrobacterium-mediated genetic transformation. Observation of the phenotype of the transgenic lines revealed that their phenotype was similar to that of the wild type and was substantially normal. It is preliminarily determined that LOC _ Os02g50550 is a candidate wild-type gene of rice dwarf fragile mutant gene DBC1, and the gene is shown in FIGS. 5A-C.

Example 2: experimental analysis of hormone response of rice short crisp mutant gene DBC1

1) Accelerating germination of mutant and wild seeds, and sowing seeds with consistent exposure to the white color on seeds containing 0 and 10^-10、10^-8、10^-6And 10^-4Mu M Gibberellin (GA) nutrient solution, and placing in a constant temperature incubator for culture. The nutrient solution with the same amount and the nutrient solution containing gibberellin with the corresponding concentration are added every day. The length change of the second leaf sheath was counted every 24 hours to two leaves and one heart period. And the treated leaves were taken at one-leaf one-heart stage (about day 7), RNA was extracted, and reverse-transcribed into cDNA, which was stored for future use. As a result, it was found that when the gibberellin concentration was less than 10^-8At μ M, wild type and mutationThe growth of the second leaf sheath of the body is almost not different from that of the blank control group; when the concentration rises to 10^-6Mu M, the plant begins to grow rapidly, and the difference between the lengths of wild type leaf sheaths and mutant leaf sheaths is increased; as the concentration continued to increase, the difference in the length of the second leaf sheath between the wild type and the mutant increased, as shown in FIGS. 6A-C.

2) Quantitative PCR analysis

The growth of plants is closely related to hormone anabolism. The method comprises the steps of taking the overground parts of wild type and mutant in the one-leaf one-heart stage, extracting RNA, carrying out reverse transcription to obtain a first cDNA chain, and analyzing the expression difference of genes GA2ox-1, GA2ox-3, GA20ox-1, GA20ox-2/SD1, GA3ox-2/D18 and carbamoyl phosphate synthetase (CPS1) and signaling pathway genes MYB61, NAC29, NAC31, SLR1 and GID1 in the wild type and the mutant by utilizing quantitative PCR. Meanwhile, the expression conditions of the gibberellin inactivation gene GA2ox-3 in the wild type and the mutant under different concentration treatment are detected. The quantitative primer sequences are shown in Table 2. The results show that: MYB61, GID1 and NAC29 were expressed in the mutants at a reduced level and SLR1 and NAC31 at an increased level, as shown in FIG. 6D. In addition, the expression level of GA2ox-3 in the mutant DBC1 was significantly higher than that of the wild type, and in addition, the expression levels of GA20ox-1 and GA20ox-2/SD1 in the mutant were significantly lower than that of the wild type, see FIG. 6E, in which ACTIN gene was used as an internal reference gene. GA2ox-3 is a gibberellin-inactivated gene, and the expression of GA2ox-3 will be increased when the content of exogenous gibberellin or endogenous gibberellin is increased. Thus, wild type and mutant seeds with consistent dew were treated with water and different concentrations of gibberellins and cultured under conditions of appropriate light, temperature and humidity. GA2ox-3 was analyzed for expression difference and expression trend between the two at different concentrations. As can be seen from FIG. 6F, the concentration of the gibberellin was 10, which is consistent with the trend of the gibberellin response experiment results^-6At μ M, the expression level began to vary. As the concentration increased, GA2ox-3 expression increased in both wild type and mutant, and was up-regulated faster in the mutant. This probably explains the phenomenon that exogenous gibberellins increase and the plant height changes slowly.

TABLE 2 quantitative PCR primer sequences

Example 3: cell division Rate assay for mutant DBC1

The paraffin sections were analyzed for cells at the growth point of the root tip, and the average number of binuclear single cells in DBC1 was 7.4 cells and 4.1 cells in wild type at the same cell number, which are shown in FIGS. 7A-E. This suggests that the mutant cells divide at a slower rate. RT-PCR analysis wild type, mutant and overexpression OE-1 in cyclin related gene expression change, RT-PCR quantitative primer sequence is shown in Table 3, results show that mutant in cyclin related gene expression is reduced compared with wild type; expression of CycB, CycD, CycP and CycT was restored in the transgenic lines, see fig. 7F. This further illustrates the reduced cell division rate of mutant DBC 1. That is, mutation of the gene causes a decrease in the cell division rate.

TABLE 3 cyclin RT-PCR quantitative primer sequences

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather as the intention of all modifications, equivalents, improvements, and equivalents falling within the spirit and scope of the invention.

Sequence listing

<110> university of southwest

Regulatory gene of <120> rice dwarf fragile-stem mutant DBC1 and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9497

<212> DNA/RNA

<213> Rice (Oryza sativa)

<400> 1

caaccacatc acatcacacc agacgccacc acgccccccg agatctctcc cacccccgac 60

cagcctcgag atcccagcgc ccatgtcgtc gtcggccatg gaggccatgg acgagctcgt 120

ccagctcgcc gagtccatgc gccaggccgc ctccctcctg gccgacgacg acccctccga 180

cgaggccagc ccgcgccgcc cctccacctt cctcaacgcc gtcgcgctcg gcaacgtcgt 240

aagtgttttg cttccaccgg agccgccgcc gccgccgccc gcggcagctc cccctctctc 300

ctccgcccga tctgactcga tccggcttgt tgttgttccg tgcagggcgc cggcaagtcg 360

gccgtgctca acagcctcat cggccacccc gtgctggtga gtgtgcttcg ggtttagctc 420

gcttctctct cttctgcttt tgtggcattc ggtttttgtg aagctttggg tggtggttgc 480

tgtgtggtga tgcagccgac gggtgagaac ggggcgacgc gggcgccgat agtggtcgac 540

ctgcagaggg atccggggct gagcagcaaa tccatcgtcc tgcagatcga cagcaagtcg 600

cagcaggtgt cggcaagtga gtgagatctt cccttggcta gctggatctg gcaatgggcg 660

ccggctggtt ggtgtgctga gtttggtggc cctttctggt ggcgttgtgg cgtgcaggtg 720

ctctccgcca ttcgctgcag gacaggctga gcaagggggc gtccggtgga tcagggaaag 780

gccgaaacga tgagatttac ctcaagctgc ggacaagtac aggtttgttg ctgggcttca 840

cttttactct tacattgcac aatgttggta agccagatgt atagtgagca tgtgccatgt 900

gctgagtcgt gctaatgttt gcgccactgc ccatactgcc cctgtaattg tcggacattg 960

atgccttgtg tattttatgt gttatagaaa gctgaaatgg taagacaata agtggttgta 1020

gaaaaacggt atcaagagtg tcttctgata cagttgttag aatgtgctct caggtttttg 1080

caacactaca acgattgagt accatactta gaataagcac aattttggat attgtactgc 1140

tacatgtaca ataatttctt ctgtttgctt ccctttgttt atattggtaa tttttagatg 1200

atggaaatgg tttacatctc tggccttggc cgttaggctc acaagtttat attggtaatg 1260

aattctcact tcagcatctt ttgttttttt ccagctcctc caataaaatt ggtcgatttg 1320

cctggcatag accaacgagt tatggatgat tcaactatga atatttgcag tttcttgctc 1380

aattacacaa cgatgattga accgcatgta gaataatcca gactcctcat aattaacata 1440

tgaatgtgtt tatgaattgg ataaacttat tatctaatca atgtattggt ggaaaatatt 1500

ccctttagaa aaggttggaa gcatcatagt aatgacattg tgcatgcact ttgactgaga 1560

agcattgaat gccctccatg gatatattag ttgcttgcac ttattacata aaagagcacg 1620

aatatcttaa gtatctgaac catgtttgtt gcatcttctc acaagtgttg gatattgact 1680

tttctccaca tttcagatca gtgaatatgc tggacataat gatgcaatat tgattgttgt 1740

gataccagca atgcaagctg ctgatgttgc atcatctcga gctctcagac tagcgaagga 1800

tattgatcca gatggtctgt tataaaattc ttataatcct gcccaatagg caattcatac 1860

cattctctcc gtagcaagta gaaactgatg tttatccttg ttcatcttgt tgtaggaacc 1920

agaacaatag gtgttttaag taaaattgat caagcatctg cagatgcaaa aactgtatct 1980

tgtgttcagg ctatcttgtc taacaagggt gcaccacgag cagcagctga aattgagtgg 2040

gttgctctaa tagggcaatc tgtctcaatt gcatcggccc aagctggatc agttggttct 2100

gagaactcac tggaaactgc ctggcaggct gaagcggaga cactcaaatc catcctaact 2160

ggagctcctc gaagtaagct agggagaatt gctctcgtgg ataccattgc taagcagata 2220

cgtaaaagga tgaaaattcg gctacctaac cttttgtccg ggtaagtgca ttgccctttt 2280

ttattatttt tattttccta aataattgaa ctttatttta tttttaaagt aaactaagca 2340

caaccccaac ttttaagaaa actgaaactt agatggtctt ggaatttgga tgtcaaaaca 2400

tctttcacat ggaatttgga tgtcaaaaca tctttcacat ttcatttcac taagatttcc 2460

cttctacagt atcgttgtag ttaaaagtaa acaatatgca tgtagcacaa attgtccagg 2520

ttctatgtcg caatcatatt tcatgtatct agatatcccc tccgcagtac cattattgga 2580

gcttgtacct tctcttgtgt ttatgcacct gtggaatcaa gttgtttttt tgtcagcact 2640

tccaagctcc aatttcctat ttgacctagc ttggtacatt tagatgatgt caaacatggt 2700

tatctactca tttcttctga acttctgtgc atttggccaa tctttttgca tctgcaacta 2760

catacttaca acacacatgc gtccatgaca agagaaatgc caagtataaa agatgtgtga 2820

acagtttcta cttggctaca aaatgtcaag cttaaactac cttttaaact ccaaatttaa 2880

atttatgtag ttcatgaact tttccgcggc ataaatgacc cttatttgat ctatcaatgt 2940

aatgttatca tcacacaatg gatcatggac actctggcct tctgtaaaat aagttgattt 3000

taatttcttg ttaaattgct gatatatgta ccttgctaca attgcaggct tcaggggaag 3060

tctcagatag tgcaaaatga acttgcaagg ttgggagaac agatggtatc aagcgctgaa 3120

ggaaccaggg ctgttgcttt ggagctttgc cgtgaattcg aagacaagtt tcttgcccat 3180

gttacttctg gtgaggttag tgataagctc aagtatggtc tttatatata ccttaattag 3240

tatgtgccaa atgaaatacc tgattacaag gctgttattc aatcaaaagg ggtctggttg 3300

gaaaattgtt gcaagctttg aggggaagtt tccagatagg atcaaacagc ttcctttgga 3360

caggcatttt gatattaaca atgtcaaaag ggtaaattca aaagcccttc tagaattaat 3420

tttagagtgg gcacactttg cttatgatga ttactgttat ctttatattc tagattgtcc 3480

tggaagccga tggttatcaa ccatatttga tatctccaga gaaaggttta aaatccttaa 3540

taaaaggagt acttgaaatg gcaaaggagc catcacggct gtgtgttgaa gaggtatgtc 3600

ttttttgatc ccctatatgt ataatttact aactcactta aaaaggtaaa atatgaatag 3660

ttgagatgca atttcatttt taccctttgg atgaatgtca gaagtggatt ttggtggcta 3720

tatttatcct gctaatttta aactttgtaa cttccaatga agtttatgat gcattaatta 3780

atcattttgt tactgtgcat gggtaggcac ttgccagcat agttattaag accagatcga 3840

tgattgaaca tctgatgggc tcggttcagt ggtttactgg tctaaccggt tagatcgccg 3900

ctttgatccg gtttaaatag acatatgctg cactataatc acaacagctg tcatagccca 3960

acggtggagt atttttggtg ttgtagcttg gtcacgggtt cgaatcccag tcttcaccct 4020

tttttccctc aatttttgtg ttactcgcat gcatggctga cgagtccact ccacttgaca 4080

ggcccacgtg ggcagtcacc ggtttatagc cgacccaaaa gatagagaac cgttcagttt 4140

gatcaggtgt ttaccggtcc gattgcgcac tggtctttta atcgaaccag gccgctaaga 4200

gtgttggttc cggtttattc cgggtgaacc gccggtccgg tctgcttcta ataactatgc 4260

ttgccagtgt agtgtcaata tgtaaactaa atacattttg tcaatatact aggtacaccg 4320

tgtattattg gacatagtaa atgcttctgc aaatgctaca ccaggacttg gaagatatcc 4380

tccattcaaa cgcgaggtac cactttgttc tatttcacca acttacaatg cattctactt 4440

ccctttcacc tgcttcctga gcacaacatc ccatcatctc ttttctgaag gtcgttgaaa 4500

ttgcaaccaa tgcattggat gtcttcaaaa gtgatgcaaa aaagatggta gttgcacttg 4560

ttgatatgga gcgtgccttt gtcccaccac aacacttcat ccgtttagta caaagaaggt 4620

atgtcaggct cttaattgaa taactttaca gttggtttgg tttcaatctg ctctaatgta 4680

acttcagtaa ataatttcca ccacaaaagc tgatagggaa gaataacatt gacttgtagt 4740

cttatagaaa tagcatcaac tgcctaaatc caaatggaaa aaaggccggc atattttctg 4800

tacagagtga tgtgcccacg aagtatacca cttcattaca acttctttgt tagagaaata 4860

tagtttcatc ccttagttga agtggataat gtttccatac tagtgtctgt ctatgagctg 4920

aaagaccttt acgatgtttt catgatttat aatagcattg ttcaatcctt atatttggtt 4980

tgatatgaca ttgcagaatg gaaaggcaac gtcgtgagga tgaggtgaaa aacaggtcat 5040

ctaagaaagc ccaagatgca gaacagtcca acaaggtatg atttctgcct cttgcacctt 5100

gcacaaatgt gcttactgaa aggtcatgct aaaagtaatg actgcaagat tcctttttct 5160

tgcgtttgtt tggccctgaa cattggttgc cttctgtatt agggctcagg cacaggatct 5220

gagcaatctg gtggtgcctt gaaatcgttg aaagaaaaat tcagtcagca agacaaagat 5280

aaagatgcaa aagagggacc aaatttgcag gttgctgggc ctggtggtga aataactgca 5340

ggtataatga tgcgctgatg caccataggt tatcattcct gtagtttttt tttatttact 5400

ctggctcttc acagctagtg taatctttca tttgtagtta caagtaactg cagatggttt 5460

tggttaattc tgtatcctgg tttggctttt ttgcatataa gatgatttct tcttttctac 5520

tcaggctacc ttttgaagaa aagtgcaaaa aataatgaat ggagcaaaag atggtttgtc 5580

ctaaatgaga agagtggaaa ggttaatata tctcgtacac tgatgtactg gtgttcccca 5640

tatttgattt ttctgaataa taacttatga taatcttgtc atttgctgcc gattttcaaa 5700

tgtcctttat ttagttaaga atttggtgtc atttactgtt ttttttttca tgtccagctt 5760

gggtacacca agaaacaaga ggagagacat tttcgaggtg ttattgtcct ggaggtgtgc 5820

acttaacttt ttatacttct gtgttcatgc tcaacagtga tctacgatac tttagtttac 5880

accttagact tgtaatttta cagtccttgt ggttccagtt cacattcgat gattcaaact 5940

aaaaaagttg tgtgcacaac tatgagagat gagatatgaa cttaataaca caatttgctg 6000

cgtgagtcag gaatgtaatc tggaagagat agaagaggaa gagctttcca agagtttgaa 6060

ggattcaaag aaggccaatg gggcagagaa aggcccaagt cttgtattta agattactaa 6120

cagagttgcc tacaaaactg tgctgaaatg ttagtgtctc taaacgttat atgctgaggt 6180

tttaaatgct aattatgttg cttaaagtgt gaatttcatg ttgcagctca tagtgctgtt 6240

atactgaagg ccgagactat tgctgacaag gttgaatgga tgaagaagat aagaggtgtc 6300

attcaaagca aagggggttc tgtgaagggt ttaaatactc ctgagggtgg ttccatgagg 6360

caaagccatt ctgatgggtc ccttgtatgt tccttatttg acatttttta tgctattgtc 6420

attgtaaagc ttttaacatc tacaattctg agttattgcc taagctattt ttaccctctc 6480

ttcagtggat gcttgtggca agttttctac tttaccagtg accatgttta tcatttgaat 6540

ttctctaaga aattaaagcc acttgttttg tgcaactatc ttaactgaac actcatatca 6600

acctgttcca ctttcccacg gccatgatta ttttttgaat gtgtcaaaca caaattttga 6660

attgcctgtg acacaattac tctcccttaa ggatacgatg gcccggaagc ctgctgatcc 6720

tgaagaagaa cttcggtgga tgtctcaaga agttcgtggt tatgtagagg ctgttttgaa 6780

cagtttggca gcaaatgttc caaaggttta catttgaacc tcacaattgt ttcaacagtc 6840

atactatttt ttaaactcaa tttaacatgg tcatagttgc aattgttatt attgtctttt 6900

tcatgatcag gcaatcgtac tttgccaagt ggagaaagca aaggaagata tgctaaatca 6960

gctctacagc aaaataaggt ttgatgcgta ttctttcttt gtatttgtgc attgcataag 7020

gtttcgcttc tttctttttc tccactaaat ggacataccc ctagatggaa ctgcattgac 7080

cttaagtata gatattgtct tttttttttt ccttttcaac tcagttttct cttgttaaca 7140

cgccactgtt aacacttgga tcagttgtta acaagtgaaa actgccaaca caaagacttt 7200

ggtgagcacc tgcagatatt tcactaaatg gatataccct tagttttcct ttttcaactt 7260

aaaggactta ctgcaatgtc tgcaggcgct actgaacatt gttcgcataa ctcatatttt 7320

atttattatt tttatttcta ttcatgacat tccttcacat ggcatatcac aagtagatac 7380

atgacatatg gtgcactaaa tggtagaaat tccattaagt aaagcacacc tgaaaaaata 7440

aaactgttac acgctccaca tacatgttaa gtgtttattt catagtcatt ctgttccttt 7500

tgtaacttgc tgctaatctt gttacaactc tcatcattag tgctcaaacc aatgcaaaga 7560

ttgaagaact tctccaagag gaccacaatg ctaaacgtag acgggagaag taccaaaaac 7620

aatcatctct attgtcaaag cttactcgtc agcttagtat ccatgacaat agagcttctg 7680

ttgcatcgta taccaatgat agttctggag ctggtaagtt ggctacattt caaatactta 7740

gttgatagta gcaaatgcaa tagttttgtg tgcttgcaaa tttgtccatt tcttttgtag 7800

gtgtgttttc ttttcttctg aactttaatt ggttgttttc ccatcataat attgagtttt 7860

cctagtctat tttctgcaag ccaatggaat ttactacgat ggccatgttt gaaaccttgt 7920

tgaattacaa ctcatctgat catgaccata attgttctgc cacatgaaca agcacttcag 7980

gacagcacga gtacatatga tggttcaact ttagcagaat ttgtgtgatg acaacttagc 8040

atgaaaatta agataactaa aacttagcaa gtttgctttg ctgcctcttc atctagggaa 8100

gtattaagta gaacaccacc aatttagtgc aatcactgaa accatcacca aacaaattgt 8160

gtacaaattt gctcaaaatc atatgcaagg gaagtgatac tatattatac aacctaaatt 8220

tcgatatctc aatatcataa tcaatttcgt ttggaagatc aaaatttgaa atttagagat 8280

agtatgctaa gggtattagc acgcatcttc atgttttgca tgatcatgac tgtcagcttc 8340

aaaacttcag atctcattag ttcacaaaca agcctgaata ctgcaatatg ataggttctc 8400

atactaggtg agggttttat ctaactattc tgttgccatt aaggttgggt cgatgatgac 8460

attgcatgca tcattttgcc aaaacataag agggtaatgc tggcctaggt tcatgggtaa 8520

caacatcatc ttggtaatct ggtttatatt catggcccat gtttgccagc tattcttgtc 8580

atagtttggg cattgcaatt attggaaatt ttaatggcac aagtctgcaa ccatgatttg 8640

taaattttaa cagaaatgac gataactgaa caagataaat gacctagctg atatctagct 8700

aataacaact gagcaagaca gaacacttgc tgttcacatt taaattttcc tgcatattct 8760

agaagtgtct actatctgtg actctgtata tggattgaga ctgcgtagat ataatggtga 8820

atgattttgt attttcgttc atatttcttt ggctacctgg taggaaatat caagcactta 8880

atcctgacta aacaaaacaa atcatttgca gagagtagcc cacggacctc tggccagtca 8940

ggtgaggact ggaggtctgc gttcgattct gcatcaaatg gctctgttga tcgatccagt 9000

tcacacaatg aaacaaggtc aagaagtgct gacagccgtg gcaaacgcta tgaaaatggg 9060

gacgtgaatg gtggtaactc tggcagccga cgcacaccaa accgtttgcc accagcacct 9120

ccagggcaaa aatattaagc atgttgatca tcatccgcca acatcatatt ttcatgctag 9180

gtatgaagta caacggaatt gatgcatcga gaaccatttg attcaggggg atggcatgca 9240

tgggctcgaa gaatagagct gacgagagat ttgcatgaat ggtgtgtatc cagaggatct 9300

cagttgttca tagaaatata ctcctatagt ttatctacag ttgtttcacg atcattttgg 9360

catattgatc tgtctgaggc tcgctcagcc tgtgggtgtg ggattttgtt accacagatc 9420

ggttgatctt ggtcttgtac tctgctgttg taattcttcc atcaacttgg caatgtatta 9480

tttcttccat tcttttt 9497

<210> 2

<211> 899

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Cys Arg Arg Arg Pro Trp Arg Pro Trp Thr Ser Ser Ser Ser Ser Pro

1 5 10 15

Ser Pro Cys Ala Arg Pro Pro Pro Ser Trp Pro Thr Thr Thr Pro Pro

20 25 30

Thr Arg Pro Ala Arg Ala Ala Pro Pro Pro Ser Ser Thr Pro Ser Arg

35 40 45

Ser Ala Thr Ser Ala Pro Ala Ser Arg Pro Cys Ser Thr Ala Ser Ser

50 55 60

Ala Thr Pro Cys Cys Arg Arg Val Arg Thr Gly Arg Arg Gly Arg Arg

65 70 75 80

Trp Ser Thr Cys Arg Gly Ile Arg Gly Ala Ala Asn Pro Ser Ser Cys

85 90 95

Arg Ser Thr Ala Ser Arg Ser Arg Cys Arg Gln Val Leu Ser Ala Ile

100 105 110

Arg Cys Arg Thr Gly Ala Arg Gly Arg Pro Val Asp Gln Gly Lys Ala

115 120 125

Glu Thr Met Arg Phe Thr Ser Ser Cys Gly Gln Val Gln Ile Ser Glu

130 135 140

Tyr Ala Gly His Asn Asp Ala Ile Leu Ile Val Val Ile Pro Ala Met

145 150 155 160

Gln Ala Ala Asp Val Ala Ser Ser Arg Ala Leu Arg Leu Ala Lys Asp

165 170 175

Ile Asp Pro Asp Gly Thr Arg Thr Ile Gly Val Leu Ser Lys Ile Asp

180 185 190

Gln Ala Ser Ala Asp Ala Lys Thr Val Ser Cys Val Gln Ala Ile Leu

195 200 205

Ser Asn Lys Gly Ala Pro Arg Ala Ala Ala Glu Ile Glu Trp Val Ala

210 215 220

Leu Ile Gly Gln Ser Val Ser Ile Ala Ser Ala Gln Ala Gly Ser Val

225 230 235 240

Gly Ser Glu Asn Ser Leu Glu Thr Ala Trp Gln Ala Glu Ala Glu Thr

245 250 255

Leu Lys Ser Ile Leu Thr Gly Ala Pro Arg Ser Lys Leu Gly Arg Ile

260 265 270

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

275 280 285

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

290 295 300

Gln Asn Glu Leu Ala Arg Leu Gly Glu Gln Met Val Ser Ser Ala Glu

305 310 315 320

Gly Thr Arg Ala Val Ala Leu Glu Leu Cys Arg Glu Phe Glu Asp Lys

325 330 335

Phe Leu Ala His Val Thr Ser Gly Glu Gly Ser Gly Trp Lys Ile Val

340 345 350

Ala Ser Phe Glu Gly Lys Phe Pro Asp Arg Ile Lys Gln Leu Pro Leu

355 360 365

Asp Arg His Phe Asp Ile Asn Asn Val Lys Arg Ile Val Leu Glu Ala

370 375 380

Asp Gly Tyr Gln Pro Tyr Leu Ile Ser Pro Glu Lys Gly Leu Lys Ser

385 390 395 400

Leu Ile Lys Gly Val Leu Glu Met Ala Lys Glu Pro Ser Arg Leu Cys

405 410 415

Val Glu Glu Val His Arg Val Leu Leu Asp Ile Val Asn Ala Ser Ala

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Gln His Phe Ile Arg Leu Val Gln Arg Arg Met Glu Arg Gln Arg Arg

485 490 495

Glu Asp Glu Val Lys Asn Arg Ser Ser Lys Lys Ala Gln Asp Ala Glu

500 505 510

Gln Ser Asn Lys Gly Ser Gly Thr Gly Ser Glu Gln Ser Gly Gly Ala

515 520 525

Leu Lys Ser Leu Lys Glu Lys Phe Ser Gln Gln Asp Lys Asp Lys Asp

530 535 540

Ala Lys Glu Gly Pro Asn Leu Gln Val Ala Gly Pro Gly Gly Glu Ile

545 550 555 560

Thr Ala Gly Tyr Leu Leu Lys Lys Ser Ala Lys Asn Asn Glu Trp Ser

565 570 575

Lys Arg Trp Phe Val Leu Asn Glu Lys Ser Gly Lys Leu Gly Tyr Thr

580 585 590

Lys Lys Gln Glu Glu Arg His Phe Arg Gly Val Ile Val Leu Glu Glu

595 600 605

Cys Asn Leu Glu Glu Ile Glu Glu Glu Glu Leu Ser Lys Ser Leu Lys

610 615 620

Asp Ser Lys Lys Ala Asn Gly Ala Glu Lys Gly Pro Ser Leu Val Phe

625 630 635 640

Lys Ile Thr Asn Arg Val Ala Tyr Lys Thr Val Leu Lys Ser His Ser

645 650 655

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

660 665 670

Lys Lys Ile Arg Gly Val Ile Gln Ser Lys Gly Gly Ser Val Lys Gly

675 680 685

Leu Asn Thr Pro Glu Gly Gly Ser Met Arg Gln Ser His Ser Asp Gly

690 695 700

Ser Leu Asp Thr Met Ala Arg Lys Pro Ala Asp Pro Glu Glu Glu Leu

705 710 715 720

Arg Trp Met Ser Gln Glu Val Arg Gly Tyr Val Glu Ala Val Leu Asn

725 730 735

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

740 745 750

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

755 760 765

Gln Thr Asn Ala Lys Ile Glu Glu Leu Leu Gln Glu Asp His Asn Ala

770 775 780

Lys Arg Arg Arg Glu Lys Tyr Gln Lys Gln Ser Ser Leu Leu Ser Lys

785 790 795 800

Leu Thr Arg Gln Leu Ser Ile His Asp Asn Arg Ala Ser Val Ala Ser

805 810 815

Tyr Thr Asn Asp Ser Ser Gly Ala Glu Ser Ser Pro Arg Thr Ser Gly

820 825 830

Gln Ser Gly Glu Asp Trp Arg Ser Ala Phe Asp Ser Ala Ser Asn Gly

835 840 845

Ser Val Asp Arg Ser Ser Ser His Asn Glu Thr Arg Ser Arg Ser Ala

850 855 860

Asp Ser Arg Gly Lys Arg Tyr Glu Asn Gly Asp Val Asn Gly Gly Asn

865 870 875 880

Ser Gly Ser Arg Arg Thr Pro Asn Arg Leu Pro Pro Ala Pro Pro Gly

885 890 895

Gln Lys Tyr

Claims (4)

1. The amino acid sequence of the encoded protein of the regulatory gene of the rice dwarf fragile-stem mutant DBC1 is shown in SEQ ID No. 2.

2. The use of the regulatory gene of the rice dwarf straw-stalk mutant DBC1 in the breeding of environment-friendly rice varieties according to claim 1.

3. An environment-friendly rice variety breeding method, which comprises transforming the regulatory gene of the rice dwarf fragile-stem mutant DBC1 as described in claim 1 into rice.

4. A reagent for regulating the plant height of rice, the main active ingredient of which is a protein encoded by a regulatory gene of a rice dwarf fragile-stem mutant DBC1 described in claim 1, a vector containing the regulatory gene of the rice dwarf fragile-stem mutant DBC1 described in claim 1, or a host cell containing the regulatory gene of the rice dwarf fragile-stem mutant DBC1 described in claim 1.

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