Plant type related protein, and coding gene and application thereof

Abstract

A plant type related protein, and a coding gene and an application thereof are provided. The protein is: (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) a SEQ ID NO: 1-derived protein having substitution, deletion, and/or addition of an amino acid residue on the sequence of SEQ ID NO: 1, and related to the plant type and/or inactivation of a plant brassinolide type, or (c) a protein having more than 80% homology to the sequence of SEQ ID NO: 1 and related to a plant type and inactivation of a plant brassinolide type. The protein and its coding gene have very important value in improving crop production, improving the visual enjoyability of a green plant, implementing simple cultivation of a plant and improving the breeding efficiency, and has a broad prospective in genetic improvement of a plant, new variety cultivation and an application.

Claims

1. A gene comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, wherein said nucleotide sequence is operably linked to a heterologous promoter.

2. The gene according to claim 1, wherein the coding sequence comprises (a) the nucleotide sequence as set forth at positions 133 to 1704 of SEQ ID NO: 2; (b) the nucleotide sequence as set forth at positions 133 to 1707 of SEQ ID NO: 2; (c) the nucleotide sequence of SEQ ID NO: 2; or (d) each of the nucleotide sequences as set forth at positions 1 to 279, positions 1135-1356, positions 1457-1703, positions 1849-2216, and positions 2356-2814 of SEQ ID NO: 3.

3. An expression cassette, recombinant expression vector, a transgenic cell line or a recombinant strain comprising the gene of claim 1.

4. A method for producing a transgenic plant, comprising: introducing the gene of claim 1 into a target plant; and selecting a transgenic plant having a decreased plant height relative to the target plant.

5. The method of claim 4, wherein the target plant is a dicotyledon or a monocotyledon.

6. The method of claim 4, wherein the gene is overexpressed in the target plant.

7. The method of claim 6, wherein the overexpression of the gene in the target plant is achieved by operably placing the gene under control of a promoter and/or an enhancer.

8. The method of claim 6, wherein the target plant is a dicotyledon or a monocotyledon.

9. A method for producing a transgenic plant, comprising: overexpressing the gene of claim 1 in a target plant; and selecting a transgenic plant defective in brassinosteroid synthesis.

10. The method of claim 9, wherein the brassinosteroid defective transgenic plant, as compared with the target plant, exhibits one or more phenotypes selected from the group consisting of 1) a shortened hypocotyledonary axis; 2) a reduced plant height; 3) a shortened petiole and/or sheath; 4) a prolonged life cycle; and 5) a photomorphogenetic response in the dark.

11. The method of claim 9, wherein the overexpression of the gene in the target plant is achieved by operably placing the gene under control of a promoter and/or an enhancer.

12. The method of claim 9, wherein the target plant is a dicotyledon or a monocotyledon.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-C show comparisons of phenotypes between a cotton variety of CRI 24 and a mutant pagoda1.

(2) FIG. 2 shows responses of the mutant pagoda1 and the cotton variety CRI 24 to brassinosteroids.

(3) FIG. 3A-B show photomorphogenetic responses of the mutant pagoda1 and the cotton variety CRI 24.

(4) FIG. 4A-B show shaping of cotton by locally applying brassinosteroids.

(5) FIG. 5 shows a comparison of relative expression levels of the GhPGD1 gene between the cotton variety CRI 24 and the mutant pagoda1.

(6) FIG. 6 shows a comparison of phenotypes between Arabidopsis thaliana of ecotype Columbia and transgenic Arabidopsis.

(7) FIG. 7 shows a comparison of relative expression levels of the GhPGD1 gene between Arabidopsis thaliana of ecotype Columbia and transgenic Arabidopsis.

(8) FIG. 8 shows a comparison of growth states between the mutant pagoda1 and the cotton variety CRI 24 in mid-October at Anyang, Henan, China.

(9) FIG. 9 shows a comparison of phenotypes between the cotton variety CRI 24 and a transgenic cotton.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

(10) Following examples are intended for better understanding of the invention, but not limitative. All the experimental methods in the following examples are conventional methods, unless otherwise specified. All the experimental materials used in the following examples are commercially available from common biochemical supplies, unless otherwise specified. All the quantitative assays in the following examples are set in triplicate, with the results averaged.

(11) The cotton variety CRI 24 (expressed as WT): a variety of Gossypium hirsutum, which is bred by the Cotton Research Institute, Chinese Academy of Agricultural Sciences, and is commercially available from the Cotton Seed Industry Technology Co., Ltd. or other seed companies.

(12) Arabidopsis thaliana of ecotype Columbia (Col-0): commercially available from ABRC (Arabidopsis Biological Resource Center).

(13) Plant expression vector pCAMBIA2300 (abbreviated as vector pCAMBIA2300): commercially available from Cambia (http://www.cambia.org/daisy/cambia/585.html). Agrobacterium strain LBA4404: commercially available from clontech.

EXAMPLE 1

Acquisition of Cotton GhPGD1 Protein and Coding Gene Thereof

(14) I. Acquisition and Genetic Analysis of a Dwarfed Tightened Mutant of Cotton

(15) Genetic transformation of a cotton variety of CRI 24 was performed using an activation label to obtain a plant of a dwarfed and tightened mutant, designated as mutant pagoda1 (expressed as pagoda1). A comparison of phenotypes between the cotton variety CRI 24 (expressed as WT) and mutant pagoda1 may be seen in FIG. 1. As compared with the cotton variety CRI 24, mutant pagoda1 had an extremely decreased height (FIG. 1A), a reduced floral organ size (FIG. 1B), a shortened petiole (FIG. 1C, a petiole of the top 4th leaf).

(16) The cotton variety CRI 24 was hybridized with mutant pagoda1, to obtain a T.sub.0 hybrid, which was sowed to obtain T.sub.1 plants. The phenotype and segregation ratio of the T.sub.1 plants were observed. The results showed that the T.sub.1 plants exhibited a high-to-short segregation ratio of 1:3 from a seedling stage, and kanamycin application showed that dwarfed and tightened properties were co-separated with a transgenic resistant marker gene NptII, demonstrating that the dwarfed and tightened properties were heritable. In a further genetic assay of T.sub.2 plants, the ratio of tall to dwarf of the T.sub.2 plants was still shown as 1:3 (see Table 2), consisting with genetic performance controlled by a pair of single dominant genes. Accordingly, the dwarfing mutation was a dominant mutation.

(17) TABLE-US-00002 TABLE 2 Separation of T.sub.2 plants Number of Number of Chi- dwarfed plants normal height plants Ratio Square DF Pr > ChiSq 85 31 3:1 0.0725 1 0.7878
II. Response of Mutant Pagoda1 to Brassinosteroids

(18) The mutant pagoda1 and the cotton varietyCRI 24 were identified as below, respectively: Test Group: cultivating cotton plants of the cotyledon stage in a liquid medium containing 500 nM brassinosteroids (Sigma) to the five leaf stage, to measure the length of its hypocotyledonary axis;

(19) Control Group: the same as the Test Group, except that brassinosteroids was replaced with an equal volume of an aqueous solution of 0.2% ethanol.

(20) The length measurements of hypocotyledonary axis of the processed plants from respective groups (an averaged measurement of 20 plants for each group) are shown in FIG. 2. For mutant pagoda1, the hypocotyledonary axis was prolonged by 270% after the treatment with brassinosteroids, comparable to the length of the hypocotyledonary axis of the cotton variety CRI 24. For the cotton variety CRI 24, after the treatment with brassinosteroids, the hypocotyledonary axis was prolonged by only 16%. From the results, it is indicated that mutant pagoda1 may be restored by brassinosteroids from a dwarfed phenotype to the phenotype of the cotton variety CRI 24, that is, mutant pagoda1 is a BRs defective mutant.

(21) III. Photomorphogenetic Response of Mutant Pagoda1

(22) The mutant pagoda1 and the cotton variety CRI 24 were identified as below: Illumination Group: cultivating cotton plants in a continuous light condition for 2 weeks, and taking pictures and measuring the length of hypocotyledonary axis; Dark Group: cultivating cotton plants in a continuous dark condition for 2 weeks, and taking pictures and measuring the length of hypocotyledonary axis.

(23) The pictures of the processed plants from respective groups are seen in FIG. 3A, and the length measurements of hypocotyledonary axis of the processed plants from respective groups (an averaged measurement of 20 plants for each group) are shown in FIG. 3B. In dark condition, the cotton variety CRI 24 had a significantly prolonged hypocotyledonary axis, and exhibited no photomorphogenetic response. In dark condition, as compared with the cotton variety CRI 24, the mutant pagoda1 had a restricted prolonging of hypocotyledonary axis, and exhibited photomorphogenetic responses such as cotyledon expansion () and hook opening (). These further demonstrate that mutant pagoda1 is a BRs defective mutant.

(24) IV. Shaping of Cotton by Local Application of Brassinosteroids

(25) Since brassinosteroids cannot be delivered for a long distance in a plant, a local application thereof may promote the growth of the applied part, without an impact on the far part. With an application of 500 nM brassinosteroids to the top of a seeding of mutant pagoda1, it was found that brassinosteroids was capable of significantly promoting the growth of the top of the seeding, without affect the rest below the applied part (see FIG. 4A). With an application of 500 nM brassinosteroids to a lateral branch of a seeding of mutant pagoda1, the lateral branch was significantly extended, even to a length more than that of the main stem, while the growth of the main stem was not affected (see FIG. 4B).

(26) V. Acquisition of Cotton GhPGD1 Protein and Coding Gene Thereof

(27) For that the dwarfed and tightened phenotypes were co-separated with T-DNA, a hiTAIL-PCR (Yao-Guang Liu, et al., High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Yao-Guang Liu and Yuanling Chen. BioTechniques Vol. 43, No. 5: pp 649-656 (November 2007)) method was used for amplifying the flanking sequence at the T-DNA inserting site. TAIL-PCR, also called thermal asymmetric interlaced PCR, can effectively separate an unknown sequence adjacent to a known DNA sequence, which is simple and practicable, effective and sensitive, and capable of obtaining a target fragment in a short time, and thus is a suitable means in molecular-biological studies. In order to improve the success in attempt to obtain a specific, long-fragmental, target product, a substantial modification was made on TAIL-PCR by Professor Yao-Guang Liu, to develop a new method of hiTAIL-PCR, which produced an excellent amplification effect in many species such as rice, Arabidopsis, insects, etc.

(28) Three runs of PCR reaction were performed with the genomic DNA of mutant pagoda1 as a template, and nested primers RB-1, RB-2 and RB-3 designed depending on a known T-DNA boundary sequence.

(29) TABLE-US-00003 RB-1: (SEQIDNO:6) 5-CGTGACTGGGAAAACCCTGGCGTT-3; RB-2: (SEQIDNO:7) 5-ACGATGGACTCCAGTCCGGCCCAACTTAATCGCCTTGCAGCACATC- 3; RB-3: (SEQIDNO:8) 5-GAAGAGGCCCGCACCGATCGCCCTT-3.

(30) The nested primers were paired with random primers and anchor primers to form a 25 l reaction system, particularly with reference to articles such as Yao-Guang Liu, et al. After three runs of nested PCR, the products were separated with 1% agarose gel, specific bands were recovered with a Promega gel recovery kit, and a T-A cloning was performed according to a pMD18-T kit from TAKARA. The reaction system comprises: 4 l of DNA fragment (25 ng/ul), 1 l of T vector, and 5 l of Solution I, totally 10 l. After kept in a water bath at 16 for 1 hour, competent cells of Escherichia coli DH5 were transformed, and screened on a LB plate containing Ampicillin for a positive clone, which was picked for sequencing. The results of the sequencing showed that a flanking sequence of 1.5 kb at T-DNA inserting site was obtained.

(31) Primers designed for the T-DNA and flanking sequence were:

(32) TABLE-US-00004 (SEQIDNO:9) RB1 5-CAGATTGTCGTTTCCCGCCTTCAG-3, (SEQIDNO:10) FL1 5-TCAGACGAGCAATACTCCACAGCAGG-3.

(33) BAC library of pagoda1 existing in our laboratory was screened by pooled PCR. The PCR reaction system was 25 l, comprising 2.5 l of 10Buffer, 2 l of dNTP Mixture (10 M), 0.5 l of Ex Taq (5 /l), 1 l (125 ng) of BAC library plasmid, 1 l of upstream primer (10 M), 1 l of downstream primer (10 M), made up with ddH.sub.2O to 25 l. Reaction conditions comprise: initial denaturation at 94 C. for 5 min; at 94 C. for 30 s, at 58 C. for 30 s, at 72 C. for 1 min, for 30 cycles; extension at 72 C. for 5 min. 5 positive clones were screened out, 3 of which were sequenced. The results of the sequencing showed that T-DNA was inserted upstream of the promoter of a gene having a nucleotide sequence presented by SEQ ID NO: 3 in the sequence listing, which was designated as a GhPAGODA1 gene (abbreviated as a GhPGD1 gene).

(34) The GhPGD1 gene had a coding region with a sequence of: nucleotides at positions 1 to 279, 1135 to 1356, 1457 to 1703, 1849 to 2216, and 2356 to 2814 from 5-terminus of SEQ ID NO: 3 in the sequence listing.

(35) The GhPGD1 gene had a cDNA presented by SEQ ID NO: 2 (1800 bp) in the sequence listing, with an open reading frame of nucleotides at positions 133 to 1707 (1575 bp) from 5-terminus of SEQ ID NO: 2 in the sequence listing.

(36) The GhPGD1 gene coded the GhPGD1 protein (consisting of 524 amino acid residues) presented by SEQ ID NO: 1 in the sequence listing.

(37) Since the T-DNA region contained a 35S enhancer, mutant pagoda1 with dwarfed and tightened phenotypes might be resulted from an overexpression of the GhPGD1 gene. The leaf total RNA of mutant pagoda1 and cotton variety CRI 24 were extracted, respectively, and reversely transcribed into a cDNA, which was subjected to Real-time PCR with a pair of primers qpgd1-S and qpgd1-A, to identify the expression level of the GhPGD1 gene. With cotton house-keeping gene, Histone 3, as a reference gene, Real-time PCR was performed using a primer pair of Histone3-S and Histone3-A.

(38) TABLE-US-00005 qpgd1-S (SEQIDNO:11) 5-CATTGGAAGAAAATCTATGGTGC-3; qpgd1-A (SEQIDNO:12) 5-ATGATGAGCCCATTTTTCGC-3. Histone3-S (SEQIDNO:13) 5-TCAAGACTGATTTGCGTTTCCA-3; Histone3-A (SEQIDNO:14) 5-GCGCAAAGGTTGGTGTCTTC-3.

(39) With the relative expression level of the GhPGD1 gene in the cotton variety CRI 24 being 1, the relative expression level of the GhPGD1 gene in mutant pagoda1 is shown in FIG. 5. The expression level of the GhPGD1 gene in mutant pagoda1 was higher than that of the cotton variety CRI 24 by 30 folds. Accordingly, it may be confirmed that the dominant dwarfed phenotype of mutant pagoda1 is resulted from an overexpression of the GhPGD1 gene downstream of T-DNA insertion region.

EXAMPLE 2

Acquisition of Transgenic Arabidopsis (Overexpression of GhPGD1 Gene)

(40) I. Construction of a Recombinant Expression Vector

(41) 1. Total RNA of a leaf from cotton variety CRI 24 was extracted and reversely transcribed into a cDNA. 2. PCR amplification was performed with the cDNA obtained in step 1 as a template, and a pair of primers pgd1-s and pgd1-a, to obtain products of the PCR amplification.

(42) TABLE-US-00006 pgd1-s (SEQIDNO:15) 5-GCTCTAGAATGGAGGGTGTTTTACAGTGG-3, pgd1-a (SEQIDNO:16) 5-CGAGCTCTCATGACCCTTGATCTCTTGT-3. 3. The products of the PCR amplification from step 2 were subjected to double-enzyme cleavage with restriction endonucleases XbaI and SacI, to recover cleaved products. 4. A recombinant plasmid pCAMBIA2300-35S-nos was double-cleaved with restriction endonucleases XbaI and SacI, to recover a vector backbone of about 10 kb.

(43) The recombinant plasmid pCAMBIA2300-35S-nos was constructed by a method in which: vector pCAMBIA2300, as a backbone, was inserted with a 35S promoter presented by SEQ ID NO: 4 in the sequence listing between the HindIII and XbaI cleavage sites, and with a nos terminator presented by SEQ ID NO: 5 in the sequence listing between the SacI and EcoRI cleavage sites. 5. The cleaved products from step 3 was linked to the vector backbone from step 4, to obtain a recombinant plasmid pCAMBIA-GhPGD1. As a result of sequencing, the structure of the recombinant plasmidpCAMBIA-GhPGD1 is described as below: having the vector pCAMBIA2300 as backbone, with the 35S promoter presented by SEQ ID NO: 4 in the sequence listing inserted between HindIII and XbaI cleavage sites, with a double stranded DNA molecule presented by nucleotides at positions 133 to 1707 from 5-terminus of SEQ ID NO: 2 in the sequence listing inserted between XbaI and SacI cleavage sites, and with the nos terminator presented by SEQ ID NO: 5 in the sequence listing inserted between the SacI and EcoRI cleavage sites.
II. Acquisition of Transgenic Arabidopsis thaliana 1. A recombinant plasmid pCAMBIA-GhPGD1 was introduced into an Agrobacterium strain LBA4404, to obtain a recombinant Agrobacterium. 2. The recombinant Agrobacterium obtained in step 1 was transformed into Arabidopsis thaliana of ecotype Columbia by floral dip, via specific steps as follows: (1) seeds of Arabidopsis thaliana were sterilized with a solution containing 0.01% (volume ratio) Triton X-100 and 10 g/100 mL NaClO in water for 10 min, and then washed with sterile water in a super-clean bench for 6 times; (2) the seeds from step (1) were sowed into a MS medium containing 3.0 g/100 mL sucrose and 0.8 g/100 mL powdered agar, and vernalized for 3-4 days, and subsequently placed in a climate chamber (22, relative humidity of 70%, light intensity of 150 mol m.sup.2 s.sup.1, 12 h illumination/12 h dark), and cultured for 1 week; (3) seedings from step (2) were transplanted to cultivatable soil (turfy soil and roseite mixed in equal mass; a planter filled with the cultivatable soil was placed in a plastic box containing water prior to transplantation, allowing for water spreading up through a bore at the bottom of the planter, and when the cultivatable soil in the planter was wet through, it was ready for the transplantation), and cultivated as covered by a film for 4 d, and further cultivated with the film removed, for totally 4 weeks from the transplantation (22, 70% relative humidity, a light intensity of 150 mol m.sup.2 s.sup.1, 12 h illumination/12 h dark); (4) the recombinant Agrobacterium obtained in step 1 was suspended in a bacterium suspension (containing sucrose at a concentration of 50 g/L, 200 uL/L silwet-77, and other solutes and concentrations thereof the same as the MS medium), to obtain a bacterium suspension of OD.sub.600 nm=0.8; (5) an entire floral bud of the plant from step (3) was dipped in the bacterium suspension obtained in step (4) for 45 s, and removed and stored in dark for 24 h, and then the plant was normally cultivated for 1 week (22, 70% relative humidity, a light intensity of 150 mol m.sup.2 s.sup.1, 12 h illumination/12 h dark), to harvest T.sub.1 seeds; (6) the T.sub.1 seeds were sowed in a MS medium containing 50 mg/L kanamycin, and normally cultivated, to obtain T.sub.1 plants. (7) the T.sub.1 plants were subjected to selfing, to obtain T.sub.2 seeds. (8) the T.sub.2 seeds were sowed in a MS medium and normally cultivated, to obtain T.sub.2 plants. (9) genomic DNA was extracted from leaves of the T.sub.1 and T.sub.2, and subjected to PCR identification with a pair of primers pgd1-s and pgd1-a, and a target sequence of about 1.6 kb (for a certain T.sub.1 plant, if a corresponding T.sub.2 plant thereto was identified as positive by PCR, the T.sub.2 plant was considered as a homozygous transgenic plant, and the plant and offspring thereof belong to a single homozygous transgenic plant); (10) the homozygous transgenic plant (a T.sub.2 plant) was subjected to selfing, to obtain T.sub.3 seeds.

(44) Totally, 60 homozygous transgenic plants were obtained.

(45) III. Acquisition of Arabidopsis thaliana Transfected with an Empty Vector

(46) With a recombinant plasmid pCAMBIA2300-35S-nos instead of recombinant plasmid pCAMBIA-GhPGD1, and the rest the same as above procedure II, Arabidopsis thaliana transfected with an empty vector was obtained.

(47) IV. Phenotype Identification

(48) T.sub.3 seeds were randomly selected from 5 homozygous transgenic plants (plant 1, plant 2, plant 3, plant 4, and plant 5) (20 seeds of each plant), sowed in soil, and normally cultivated (22, 70% relative humidity, a light intensity of 150 mol m.sup.2 s.sup.1, 12 h illumination/12 h dark), for 30 days counted from the sowing day, and then taken pictures, measured for plant height, and assayed for expression level of GhPGD1 gene. 20 of T.sub.3 seeds of Arabidopsis thaliana transfected with an empty vector and 20 seeds of Arabidopsis thaliana of ecotype Columbia were treated in parallel as controls.

(49) The pictures of the plant are shown in FIG. 6. Plant 1 had an averaged height of 25.111.54 centimeters. Plant 2 had an averaged height of 19.782.05 centimeters. Plant 3 had an averaged height of 12.781.39 centimeters. Plant 4 had an averaged height of 6.331.2 centimeters. Plant 5 had an averaged height of 3.830.71 centimeters. Arabidopsis thaliana transfected with an empty vector had an averaged height of 31.892.15 centimeters. Arabidopsis thaliana of ecotype Columbia had an averaged height of 32.171.46 centimeters.

(50) Total RNA was extracted from a leaf of each of the plants, and reversely transcribed into a cDNA, which was used as a template to perform Real-time PCR with a pair of primers qpgd1-S and qpgd1-A, to identify the expression level of GhPGD1 gene. With actin1 gene as a reference gene, Real-time PCR was performed using a pair of primers actin1-S and actin1-A. The relative expression levels of the GhPGD1 gene in respective plants are shown in FIG. 7. Arabidopsis thaliana of ecotype Columbia and the Arabidopsis thaliana transfected with an empty vector had no expression of GhPGD1 gene. And the transgenic plants had different expression levels of GhPGD1 gene.

(51) TABLE-US-00007 qpgd1-S: (SEQIDNO:17) 5-CATTGGAAGAAAATCTATGGTGC-3; qpgd1-A: (SEQIDNO:18) 5-ATGATGAGCCCATTTTTCGC-3. actin1-S: (SEQIDNO:19) 5-ACTCTCCCGCTATGTATGTCGC-3; actin1-A: (SEQIDNO:20) 5-AGAAACCCTCGTAGATTGGCAC-3.

(52) Above results showed that the plant height of Arabidopsis thaliana was inversely associated with the expression level of the GhPGD1 gene, that is, a higher expression level of the GhPGD1 gene resulted in a more dwarf phenotype of the plant.

EXAMPLE 3

Aging Delaying Function of GhPGD1 Gene of Cotton

(53) The mutant pagoda1 and the cotton variety CRI 24 were identified as follows, respectively:

(54) Cotton plants were sowed in field in Anyang at planting time, and subjected to a normal field management. The phenotype thereof was observed and pictured in early October. The pictures are shown in FIG. 8. The cotton variety CRI 24 had most of leaves yellowing, withered and dropped, and mutant pagoda1 had still dark green leaves, and continuously growable top tissue. The results showed that overexpression of the GhPGD1 gene enables delay of plant aging, prolonging the life cycle of the plant.

EXAMPLE 4

Acquisition of Transgenic Cotton

(55) I. Acquisition of Transgenic Cotton

(56) 1. Seeds of a cotton variety of CRI 24 were stripped off coats, and then dipped with 0.1% mercury bichloride for 5 min for sterilization, washed with sterile water for 3-5 times, and sowed in a sterile seeding cultivation medium (a MS medium+25 g/L of sucrose+6.5 g/L of agar, at pH of 7.0), so as to sprout into sterile seedings. 2. At an aseptic bench, hypocotyledonary axes were excised from the sterile seedings that had grown for 7 days with a scalpel sterilized by an alcohol burner, cut into segments of 0.5-0.8 cm, and dipped with the bacterium suspension of the recombinant Agrobacterium from step II. 1 in Example 2 (OD.sub.600 nm value=0.5) for 5 minutes, followed by sucking off of superficial suspension with a filter paper and placement into a callus induction medium (MS medium+30 g/L glucose+0.01 mg/L 2,4-D+0.05 mg/L IAA+0.05 mg/L KT+50 mg/L kanamycin, at pH of 6.5), for cultivation in dark for 48 hours. 3. The resultants were transplanted to a new callus induction medium, and cultivated in an illumination room for 2 months (cultivation conditions: 28, 16 h illumination/8 h dark, a light intensity of 150 mol m.sup.2 s.sup.1; a subcultivation every 20 days). 4. The resultants were transferred to a regenerate seedling induction medium (MS medium+30 g/L sucrose+0.1 mg/L IAA+0.1 mg/L 6-BA+50 mg/Lkanamycin, at pH of 6.5), and cultivated in the illumination room for 3 months (cultivation conditions: 28, 16 h illumination/8 h dark, a light intensity of 150 mol m.sup.2 s.sup.1; a subcultivation every 20 days; embryoids started to generate in succession after 1.5 months), to obtain regenerate seedlings. 5. The seedings of the cotton variety CRI 24 having 4-5 main leaves already were as stock, after the regenerate seedlings were grafted, they were cultivated in a greenhouse (cultivation conditions: 14 h illumination/10 h dark; 28-35 in day time, with a light intensity of 150 mol m.sup.2 s.sup.1; and 25-28 in night time), to obtain T.sub.1 seeds. 6. The T.sub.1 seeds were sowed and cultivated in a greenhouse (cultivation conditions: 14 h illumination/10 h dark; 28-35 in day time, with a light intensity of 150 mol m.sup.2 s.sup.1; and 25-28 in night time), identified with application of kanamycin, and screened for positive plants, and the T.sub.1 plants were subjected to selfing, to obtain T.sub.2 seeds. 7. The T.sub.2 seeds were sowed and cultivated in a greenhouse (cultivation conditions: 14 h illumination/10 h dark; 28-35 in day time, with a light intensity of 150 mol m.sup.2 s.sup.1; and 25-28 in night time), identified with application of kanamycin, and screened for positive plants. 8. Genomic DNA was extracted from leaves of T.sub.1 and T.sub.2 plants, and identified by PCR with a pair of primers pgd1-s and pgd1-a, with a target sequence of about 1.6 kb. For a certain T.sub.1 plant, if a corresponding T.sub.2 plant was identified by PCR as positive, the T.sub.2 plant was considered as a homozygous transgenic plant, and the plant and offspring thereof belong to a single homozygous transgenic plant. Totally 16 homozygous transgenic plants were obtained. 9. The homozygous transgenic plants (T.sub.2 plants) were selfed, to obtain T.sub.3 seeds.
II. Acquisition of Cotton Transfected with an Empty Vector

(57) Cotton transfected with an empty vector was obtained with a recombinant plasmid pCAMBIA2300-35S-nos instead of recombinant plasmid pCAMBIA-GhPGD1, and the rest the same as procedure I.

(58) III. Phenotype Identification

(59) T.sub.3 seeds were randomly selected from 4 homozygous transgenic plants (plant a, plant b, plant c, plant d) (15 seeds of each plant) and sowed in soil in a sunlight greenhouse. These were observed character, measured, and taken pictures at a full-bloom stage. 20 of T.sub.3 seeds of cotton transfected with an empty vector and 20 seeds of the cotton variety CRI 24 were treated in parallel as controls.

(60) The pictures were as in FIG. 9. All of the transgenic plants exhibited shortened branches and internodes, and dwarfed plants. Plant a had an averaged height of 58.772.55 centimeters. Plant b had an averaged height of 35.922.47 centimeters. Plant c had an averaged height of 29.851.99 centimeters. Plant d had an averaged height of 21.773.03 centimeters. The cotton transfected with an empty vector had an averaged height of 89.463.31 centimeters. CRI 24 had an averaged height of 90.773.14 centimeters. These results showed that cotton with an overexpression of GhPGD1 gene had a plant height substantially less than those of the cotton transfected with an empty vector and of the target plant.

INDUSTRIAL APPLICATION

(61) The present invention discloses a protein from cotton related to plant dwarfing and inactivation of brassinosteroids and a coding gene thereof. Overexpression of the gene in a plant allows for a reduction of the amount of endogenous brassinosteroids, which is realized by a shortened hypocotyledonary axis, a dwarfed plant, shortened petioles, shortened internodes, dark green leaves and a prolonged life cycle, as well as photomorphogenesis even in dark. Taking use of this gene, it is possible to improve and shape a plant type, and to delay plant aging. The gene is dominant, which is essential to improve a plant (specifically a crop), shorten breeding time, and increase breeding efficiency. The genetic regulation of cotton plant height with the dwarfing gene and the breeding of a variety having a proper plant height and a desired plant type provided by the present invention are beneficial for making full use of the production potential of light and heat resources and of the space and time advantages of flowering and boll forming of cotton to improve the economic coefficient of cotton, and allow for reducing chemical and artificial regulations, reducing cotton production costs, and improving economic benefits in cotton planting. The protein and coding gene thereof provided by the present invention have very important value in improving the production of a crop (a fruit tree), improving the visual enjoyability of a green plant, implementing a simple cultivation of a plant, and improving a breeding efficiency, and have a broad prospective in genetic improvement of a plant, and new variety cultivation and application.