CN112899292B - Upland cotton plant height regulation gene GhGA20ox6 and its application - Google Patents

Abstract

The invention discloses an upland cotton plant height regulation gene GhGA20ox6 and use thereof. The gene has the nucleotide sequence shown in SEQ ID NO: 1. By regulating GhGA20ox6, the plant height of upland cotton can be regulated at the molecular level, so as to improve the upland cotton, dwarf the plant, make the upland cotton have a strong resistance to lodging, improve the population utilization rate of light energy of the crop, and thus improve the crop's efficiency. Yield.

Description

Upland cotton plant height regulation gene GhGA20ox6 and its application

technical field

The invention relates to the technical field of genetic engineering, in particular to an upland cotton plant height regulation gene GhGA20ox6 and use thereof.

Background technique

Gibberellin (GA), as one of the six major plant hormones, is a class of tetracyclic diterpenoid plant hormones, which play an important role in all stages of plant growth and development. Growth, flowering and fruiting, stem elongation, leaf growth and unfolding have important roles. In higher plants, a variety of enzymes are involved in the biosynthesis and metabolism of gibberellin in multi-step enzymatic reactions, and the expression of these enzyme genes can effectively regulate the rate of gibberellin synthesis and metabolism. There are many enzymes involved in the synthesis and metabolism of gibberellin. GA2-oxidase (GA2-oxidase) is a key enzyme in the process of GA degradation in plants. GA2ox can decompose active GAs and its precursors and intermediates in plants. inactivated, thereby maintaining the homeostasis between active GAs in plants. GA3-oxidase (GA3-oxidase) and GA20-oxidase (GA20-oxidase), as the last key enzymes in the phyto-gibberellin synthesis pathway, catalyze the conversion of GA ₁₂ and GA ₅₃ into active GA ₁ and GA ₄ , in addition, GA3ox and GA20ox are negative feedback regulation of active GAs in the receptors. The plant gibberellin oxidase gene family belongs to the 2OG-Fe(Ⅱ)oxygenase subfamily, which can be encoded by multiple gene families. So far, the gibberellin synthesis and metabolism pathway of the model plant Arabidopsis has been thoroughly studied, and its gibberellin-related genes have also been cloned and identified. Studies have shown that the current "green revolution" of major crops is closely related to gibberellin. In plants, the advantage of gibberellin involved in regulating plant growth and development is to promote stem elongation and plant height. For example, in rice, through the cloning and gene silencing of GA20ox-related genes, it was found that these genes affect the stem length of rice, This leads to a dwarf phenotype in rice. Mauriat et al. found that GA20ox-related genes affect the stem elongation of aspen, and enhanced the expression of GA20ox-related genes in aspen seedlings, showing that it promotes the synthesis of active gibberellin in the seedlings .

Cotton, the world's most important source of natural fiber, is one of the most widely cultivated cotton species in the world and remains an important cash crop in many countries. Plant height is an important good agronomic trait in crop breeding. Plants that are too high will cause lodging, which will reduce the yield of crops. The dwarfing plants can have strong lodging resistance and improve the utilization rate of light energy in groups of crops. , thereby increasing crop yields. Upland cotton (G.hirsutum L., AADD) has two subgenomes, A and D. At present, castration and topping are basically used in agriculture to control plant height. On the one hand, it will increase the labor force and increase the working time. It is of great significance to improve upland cotton at the molecular level.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a plant height regulating gene GhGA20ox6 and its application of GhGA20ox6. By regulating GhGA20ox6, the plant height of Upland cotton can be regulated at the molecular level, thereby improving the upland cotton and dwarfing the plant, so that the upland cotton has a relatively high quality. Strong anti-lodging ability, improve the utilization rate of light energy of crops, thereby increasing the yield of crops.

The technical scheme adopted by the present invention to solve its technical problems is:

An upland cotton plant height regulation gene GhGA20ox6, the gene has the nucleotide sequence shown in SEQ ID NO: 1.

Through exploratory research, the inventor obtained 13 candidate genes in the first screening of the whole genome of Upland cotton, and then further exploration and analysis, found and determined the key gene GhGA20ox6 that regulates the plant height of Upland cotton, and verified its function. . And use the discovered functional gene to regulate the plant height of upland cotton at the molecular level, so as to improve the upland cotton germplasm resources.

A plasmid comprising the upland cotton plant height regulation gene GhGA20ox6 of claim 1.

A plant expression vector comprising the upland cotton plant height regulation gene GhGA20ox6 according to claim 1.

A host cell, the host cell comprising the GhGA20ox6 highly regulated gene of upland cotton as claimed in claim 1.

A protein encoded by a plant height regulation gene GhGA20ox6 of Upland cotton has the amino acid sequence shown in SEQ ID NO: 14.

The utility model relates to the use of a plant height regulating gene GhGA20ox6 of Upland cotton, which is used for regulating the plant height of Upland cotton.

A dwarfing method of upland cotton, which reduces the plant height of upland cotton and realizes dwarfing by gene silencing of the upland cotton plant height regulation gene GhGA20ox6.

The beneficial effects of the invention are as follows: by regulating GhGA20ox6, the plant height of Upland cotton can be regulated at the molecular level, thereby improving Upland cotton, dwarfing the plant, making the Upland cotton have stronger lodging resistance, and improving the collective light energy of the crop utilization, thereby increasing crop yields.

Description of drawings

Figure 1 is the expression level of GhGA20ox6 gene in different tissues of Upland cotton.

Figure 2 is the subcellular localization of GhGA20ox6 in N. benthamiana; the subcellular localization of 35S::GhGA20ox6::GFP (A-C) and 35S::GFP (D-F) in N. benthamiana leaf cells. A, D: epidermal cells under 488 nm laser; B, E: epidermal cells under bright field; C, F: epidermal cells under combined laser and bright field, 20 μm scale.

Figure 3 is a phenotype of positive control (CLCrVA::PDS), VIGS plants (Linel, Line2 and Line3) and empty vector control (CLCrVA).

Figure 4 is the plant height and internode length of VIGS-positive strains and the control group; *P<0.05, **P<0.01.

Figure 5 is the relative expression of GhGA20ox6 in different internodes between VIGS-positive strains and the control group; *P<0.05, **P<0.01.

Figure 6 is a phenotype of wild-type Arabidopsis and GhGA20ox6 transgenic Arabidopsis. Note: A is the rosette leaf phenotype of Col-0 and positive plants; B and C are the plant height phenotypes of Col-0 and positive plants.

Figure 7 is the expression level of GhGA20ox6 in transgenic Arabidopsis, **P<0.01; the internal reference gene is AtUBQ10.

Detailed ways

The technical solutions of the present invention will be further described in detail below through specific embodiments.

In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or commonly used in the field. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

Example:

1 Materials and methods

1.1 Experimental materials and processing methods

Upland cotton (G.hirsutum L., AADD) TM-1 material was selected for tissue expression analysis, planted in the greenhouse of Zhejiang Agriculture and Forestry University Pingshan Base (Lin'an District, Hangzhou City, Zhejiang Province), and managed by conventional field mode.

N. benthamiana was selected for transient gene transformation and planted in the climate chamber of Xue 16, Zhejiang Agriculture and Forestry University. Environmental conditions: light for 16 hours, darkness for 8 hours, temperature at 23°C, and relative humidity between 70% and 75%.

Upland cotton TM-1 material was selected for VIGS phenotype statistics and gene expression analysis. It was planted in the climate chamber of Xue 16, Zhejiang Agriculture and Forestry University. The temperature conditions were: light 16h, dark 8h; temperature 24℃;

Arabidopsis thaliana col wild-type material was used for transgenic overexpression experiment, which was planted in the climate chamber of Zhejiang Agriculture and Forestry University, Xue 16, temperature conditions: temperature 25°C; light 16h, dark 8h; relative humidity 65%-70%.

The materials used above were all from the Cotton Breeding Laboratory of Zhejiang Agriculture and Forestry University.

1.2 Carrier and reagents used

Premix Ex Taq^TMII购于TaKaRa公司(中国，大连)。从源叶生物公司购入2-吗啉乙磺酸、乙酰丁香酮和1/2MS固体培养基，引物的合成及基因测序由杭州有康生物科技有限公司完成。本实验所用引物序列见表1。The transgenic expression vectors pBI121, pBINGFP4 and cotton wrinkle virus vectors pCLCrVA (empty vector), pCLCrVB (helper vector), pCLCrVA-PDS (positive control vector) were kept in our laboratory. Escherichia coli DH5α competent and Agrobacterium tumefaciens strain GV3101 competent were purchased from Shanghai Weidi Biological Co., Ltd. Restriction endonucleases XbaI, SpeI and SacI were purchased from TaKaRa Company (Dalian, China), and AscI was purchased from NEB Company. RNA extraction kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Gene cloning vector pMD18-T, MightyAmp high-fidelity enzyme, PrimeScript ^TM RT reagent Kit with gDNA Eraser reverse transcription kit, DNA fragment purification kit and fluorescent reagent TB

Premix Ex Taq ^™ II was purchased from TaKaRa Company (Dalian, China). 2-morpholine ethanesulfonic acid, acetosyringone and 1/2MS solid medium were purchased from Yuanye Biological Company, and the synthesis of primers and gene sequencing were completed by Hangzhou Youkang Biotechnology Co., Ltd. The primer sequences used in this experiment are shown in Table 1.

Primer sequences used in Table 1

Note: The underline is the restriction site.

1.3 RNA extraction and cDNA synthesis

According to the instructions of RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441), 9 tissues RNA of 15DPA fiber, ovule, flower, sepal, terminal bud, stem, leaf, pistil and stamen of TM-1 were extracted using reverse These tissue RNAs were reverse transcribed using a transcription kit to synthesize single-stranded cDNA.

1.4 GhGA20ox6 gene cloning

Using the published protein sequence of Arabidopsis gibberellin 20 oxidase as a reference, the upland cotton whole genome database released by Huazhong Agricultural University was downloaded from ( https://cottonfgd.org/ ), and a local Blast database was constructed. After screening, 13 13 candidate genes are: GhGA20ox1 (SEQ ID NO: 2), GhGA20ox3 (SEQ ID NO: 3), GhGA20ox4 (SEQ ID NO: 4), GhGA20ox5 (SEQ ID NO: 5), GhGA20ox6 (SEQ ID NO: 5) ID NO: 1), GhGA20ox7 (SEQ ID NO: 7), GhGA20ox8 (SEQ ID NO: 8), GhGA20ox9 (SEQ ID NO: 9), GhGA20ox10 (SEQ ID NO: 10), GhGA20ox11 (SEQ ID NO: 11) , GhGA20ox12 (SEQ ID NO: 12), GhGA20ox13 (SEQ ID NO: 13).

Through further exploration experiments, the GhGA20ox6 gene with high expression in the stem was finally obtained. Specific primers GhGA20ox6-F and GhGA20ox6-R were designed, and the cDNA of the stem tissue of the TM-1 material was used as the template to amplify GhGA20ox6. The fragment was ligated into the pMD18-T vector, the recombinant plasmid was transformed into E. coli DH5α, and the correctly sequenced monoclonal bacterial solution was saved for subsequent laboratory use.

Bioinformatics analysis of 1.5GhGA20ox6

The obtained GhGA20ox6 protein sequence was used ExPASy ( https://web.expasy.org/protparam/ ) online analysis website to predict its physicochemical properties, including protein length, isoelectric point and molecular mass. The amino acid sequence of the target gene was analyzed by DNAMAN for conservation. The conserved domain of GhGA20ox6 was obtained using the SMART online website.

1.6 Spatial pattern expression of GhGA20ox6

Premix Ex Taq^TMII试剂盒的反应体系和对应程序进行实时荧光定量PCR(Real-time quantitative PCR，RT-PCR)，仪器型号为Roche LightCycler 96，操作方法依据仪器说明书进行。所使用的定量引物为GhGA20ox6-qF和GhGA20ox6-qR，选用Actin7基因作为棉花的内参基因。利用2^-△△Ct方法分析基因的相对表达量，保证3次生物学和技术性重复，得到的数据利用Excel以及Graphpad软件，分析并绘制基因相对表达图。By TB

The reaction system and corresponding program of the Premix Ex Taq ^TM II kit were used for Real-time quantitative PCR (RT-PCR). The instrument model was Roche LightCycler 96, and the operation method was carried out according to the instrument manual. The quantitative primers used were GhGA20ox6-qF and GhGA20ox6-qR, and the Actin7 gene was selected as the internal reference gene of cotton. The 2- ^△△Ct method was used to analyze the relative expression of genes, and three biological and technical replicates were guaranteed. The data obtained were analyzed and plotted using Excel and Graphpad software.

1.7 Subcellular localization of GhGA20ox6

GhGA20ox6-SF and GhGA20ox6-SR primers were used to connect the target gene fragment to the GFP fluorescent protein vector, and the constructed overexpression vector 35S::GhGA20ox6::GFP was transformed into Agrobacterium strain GV3101 by Agrobacterium transformation method. According to the transient transformation method of N. benthamiana, the Agrobacterium solution was injected into tobacco leaves cultivated for 20 days. The infected tobacco leaves were dark-treated for 10 hours, and the GFP cell position signal in tobacco leaves was observed under LSM880 confocal microscope after culturing for 28 hours.

1.8 Construction and infection of virus-induced gene silencing vector (VIGS)

Put the full seeds of TM-1 material on the seedling block and wait until the two cotyledons are fully opened. The partial gene fragment of GhGA20ox6 was recombined with the pCLCrVA viral vector through the pair of enzyme digestion primers GhGA20ox6-VF and GhGA20ox6-VR, and the constructed vectors pCLCrVA-GhGA20ox6, pCLCrVA-pCLCrVB, PDS-pCLCrVB were transferred into Agrobacterium CV3101, and the agricultural The bacillus liquid was injected into the cotyledons with the same growth potential, and dark-treated for 24 hours, and then placed in a climate chamber for normal culture. The changes of internodes and the total length of the six-leaf stage were observed and recorded, and the stem tissue was taken to detect the gene expression to verify the silencing effect.

1.9 Overexpression of transgenic Arabidopsis

The correctly sequenced pBI121-GhGA20ox6 overexpression vector was transferred into the GV3101 Agrobacterium strain for amplification, and the col Arabidopsis thaliana, which had been cut from the pods, was transformed by the Agrobacterium dipping method, and was darkened in a climate room for 20 hours. After treatment, the harvested seeds were T ₀ generation seeds. The seeds of the T ₀ generation were subcultured until the pure generation. The flowering cycle and height difference of homozygous transgenic Arabidopsis were observed and recorded, and the gene expression of GhGA20ox6 in transgenic Arabidopsis was detected.

2 Results and Analysis

2.1 Sequence analysis of GhGA20ox6 gene

GhGA20ox6 is located on chromosome 9 of subgroup D, with an open reading frame of 1155 bp, an amino acid sequence length of 384 (SEQ ID NO: 14), 2 introns and 3 exons. The relative molecular mass of the GhGA20ox6 protein sequence was predicted to be 42.32kDa and the isoelectric point was 6.37 using ExPASy online software. The DIOX_N domain is located in the 63-172nd amino acid sequence, and the 2OG-Fe(II) oxygenase domain is located in the 228-323rd amino acid sequence. The amino acid sequence analysis of the proteins corresponding to the 13 candidate genes showed that the corresponding proteins of the 13 candidate genes, including GhGA20ox6, all had a common conserved protein sequence: a highly conserved motif that binds to substrates, LPWKET, which binds to 2-keto. The conserved sequence NYYPXCQKP related to glutaric acid has an Fe ²⁺ binding site.

2.2 Spatial expression pattern analysis of GhGA20ox6 gene

In order to study the tissue expression characteristics of GhGA20ox6, the expression level of GhGA20ox6 in 9 tissues of TM-1 upland cotton material 15DPA fiber, ovule, flower, sepal, terminal bud, stem, leaf, pistil and stamen was analyzed by qRT-PCR. . The results showed that among these nine tissues, GhGA20ox6 was predominantly expressed in stems, while the expression levels were lower in ovules and flowers (Fig. 1). It is further speculated that GhGA20ox6 plays a role in the growth and development of stems.

2.3 Analysis of subcellular localization of GhGA20ox6 protein

The protein sequence of GhGA20ox6 was predicted by WoLF PSORT ( https://wolfpsort.hgc.jp/ ) online software, and the result showed that it was located in the cell membrane. In order to verify whether the GhGA20ox6 protein is localized on the cell membrane, GhGA20ox6 was transiently expressed in leaf cells of N. benthamiana (Fig. 2). The results showed that there was a bright green distribution in the cell membrane, indicating that there were a lot of green fluorescence signals in the cell membrane, which was consistent with the predicted results, indicating that the GhGA20ox6 protein was a membrane-localized protein.

2.4 Phenotype observation and expression level analysis of GhGA20ox6 gene after silencing

The cotton plants of the positive control pCLCrVA-PDS showed an albino phenotype. Compared with the control group, the cotton plants of pCLCrVA-GhGA20ox6 had shortened stem internodes to varying degrees compared with the control (Fig. 3). The plant height data were plotted as a histogram (Figure 4). The results showed that the plant height of the virus-induced gene silencing of the positive strains was significantly reduced, the length of the first internode and the third internode were slightly shortened, while the length of the second, fourth and sixth internodes showed Significant shortening, indicating that GhGA20ox6 silencing can lead to dwarf plant type. The expression levels of GhGA20ox6 in the six internodes were measured, and the real-time fluorescence quantitative results showed that there was no significant difference in the expression of GhGA20ox6 in the first and third internodes compared with the inoculated empty control group. Compared with the control group, the expression levels of GhGA20ox6 in the internode, the fifth internode and the sixth internode were significantly different (Fig. 5). It is speculated that silencing of the GhGA20ox6 gene results in shortening of different internodes in the stem.

2.5 Functional verification of GhGA20ox6 gene in Arabidopsis

Under long-day conditions, wild-type and transgenic Arabidopsis were grown simultaneously and growth was observed (Figure 6). The results showed that wild-type Arabidopsis started bolting 22 days after seedling transplant, with an average bolting time of 25 days, and transgenic Arabidopsis started bolting 18 days after transplanting, with an average bolting time of 20 days, which was significantly earlier than that of the control wild type. The average plant height of the transgenic Arabidopsis line was about 31 cm, which was significantly higher than that of the control Arabidopsis 24 cm, and the number of rosette leaves of the transgenic Arabidopsis was also significantly increased (Table 2). The stems of transgenic Arabidopsis thaliana were sampled, RNA was extracted, and the expression of GhGA20ox6 gene in Arabidopsis thaliana was detected (Figure 7). GhGA20ox6 was highly expressed in transgenic Arabidopsis thaliana, which was consistent with the 35S promoter-driven exogenous gene transfer. The results indicated that GhGA20ox6 was integrated into the Arabidopsis chromosome, indicating that the GhGA20ox6 gene promoted stem growth.

Table 2 Statistics of bolting time, plant height and number of rosette leaves in wild-type and transgenic Arabidopsis

Material Plant height Number of rosette leaves bolting time Materials Plant height(cm) Number of rosette leaves Bolting time(d) WT 24.00+0.26 9.90+0.26 25.44+0.71 Line1 31.46+0.47** 11.80+0.21** 20.22+0.70** Line2 30.21+0.29** 11.55+0.17** 20.56+0.56** Line3 29.67+0.35** 11.40+0.18** 20.67+0.60**

.

3 Discussion

The gibberellin 20 oxidase family plays an important role in plant growth, development and environmental adaptation, and is an important key enzyme for active gibberellin synthesis in plants.

In the present invention, a gibberellin oxidase gene GhGA20ox6 is obtained by cloning from TM-1 upland cotton material. After multiple amino acid sequence comparison, it is found that the GhGA20ox6 protein sequence has a highly conserved motif LPWKET that binds to the substrate, and binds 2- Ketoglutarate-related conserved sequence NYYPXCQKP, and GhGA20ox6 gene expression in different tissues of TM-1 was significantly different, and the expression level was higher in stem tissue. It is believed that GhGA20ox6 gene is involved in the growth and development of cotton stems and affects plant height traits.

At present, castration and topping are basically used in agriculture to control plant height. On the one hand, it will increase the labor force and increase the working time. Using VIGS experiment and transgene overexpression experiment, the expression level of GhGA20ox6 gene in plants was studied at the molecular level, indicating that GhGA20ox6 gene plays a regulatory role in plant height traits.

4 Conclusion

The present invention clones the GhGA20ox6 gene from the stem of the upland cotton material TM-1 by means of T-A cloning method. The amino acid sequence analysis of the GhGA20ox6 gene has 2OG-Fe(II)oxygenase and DIOX_N domains. The cDNA of GhGA20ox6 gene is 1155bp, encoding 384 amino acids. Subcellular localization prediction and experimental results show that it is localized to the cell membrane. The expression level of TM-1 showed that the GhGA20ox6 gene had obvious tissue specificity, and the expression level was the highest in the stem. The results of VIGS experiment showed that the silencing of GhGA20ox6 gene in cotton caused different degrees of dwarfing phenotypes in different stem nodes of cotton compared with the control group, and the expression of GhGA20ox6 gene in different nodes decreased to different degrees. The results of heterologous overexpression of transgenic Arabidopsis showed that compared with wild type, transgenic Arabidopsis had more rosette leaves, earlier bolting time, and significantly increased plant height, indicating that GhGA20ox6 gene plays an important role in plant height shape.

The above-mentioned embodiment is only a preferred solution of the present invention, and does not limit the present invention in any form, and there are other variations and modifications under the premise of not exceeding the technical solution recorded in the claims.

sequence listing

<110> Zhejiang Agriculture and Forestry University

<120> Upland cotton plant height regulation gene GhGA20ox6 and its application

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gtaccactca ttgacattaa aggtttcctc tctggtgacc ctgttgctgc gatggaagct 240

tcaaggcttg tcggcgaggc atgtcaacag catggtttct ttcttgtagt taatcatgga 300

gtcgatgcca cactcgtggc cgatgctcat aggtacatgg ataacttctt tgaattgcca 360

ctctgcgaaa agcaatgggc tcagagaaaa gttggtgagc actgtggata tgccagcagc 420

ttcaccggca ggttctcctc caagttgcct tggaaagaaa cactttcttt tcgctactca 480

gctcagagaa acgcatccac gatggttgaa gactaccttg ttaataaaat gggagatgaa 540

ttcaggcatt tcggaagggt ataccagagc tactgcgagg ctatgagcaa gctttctcta 600

gggattatgg agcttttagc catcagtctg ggcgtaggca gagcgcattt cagggaattt 660

tttgaggaaa atgattcaat aatgaggctg aattactatc cgctttgcca aaaaccagac 720

ctcactttag gaacggggcc tcattgcgat ccaacgtctt taaccatcct tcaccaagac 780

cgagttggtg gtcttcaagt gtttgtagac aacgaatggc gttcaatcag cccaaatttc 840

gaagcatttg ttgttaacat tggcgacacc ttcatggctt tatcaaatgg aagatacaaa 900

agttgcttgc accgggcggt tgtgaacagt cacaccccaa gaaaatccct tgctttcttt 960

ttgtgcccaa agggtgataa agtggttacc ccaccaaaag agttggtgga cgcaaatagc 1020

cccagagtat atccagattt tacatggcct atgctgcttg aattcacaca aaagcactac 1080

agagctgaca tgaatacgct tgaagtattc tcaaactggg ttcaacagag aagccgctga 1140

<210> 5

<211> 1158

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 5

atggtacatt gtctgccaaa ggtttctgtc attcaagaca agccgatgct tccaccaact 60

gctgctgtcg caaagaataa gcatcgccct ttagctttcg atgcatcaat ccttggatct 120

gagatatcct ctcagttcat atggcctgac gacgacaagc catgcctgga tgccccggaa 180

cttgtaattc caaccattga cttgggagct ttcttgcttg gggactctct tgctgtatca 240

aaagcggcag aggcggtgaa tgaggcatgc aagaagcatg ggttttttct ggttgtaaac 300

catggggtgg attcagggct tatagacaaa gctcatcagt acatggaccg cttcttcagc 360

ctgcaactct ctgagaagca aaaggctaag agaaaagtag gagaaagcta tgggtatgca 420

agcagtttcg ttgggaggtt ttcctccaag ttgccatgga aagaaacact gtcttttcgg 480

tactgtcctc acactcaaaa catcgtgcaa cactatatgg tgaattggat gggtgaagat 540

tttagagatt tcgggaggct ttaccaggaa tattgtgaag ccatgaacaa ggtttcccaa 600

gagataatgg ggctgctagg gataagtcta ggacttgacc aagcctactt caaagacttt 660

ttcgacgaaa atgattcaat cctgagactg aatcactacc ctccatgcca aaagcctgag 720

ttgactctgg ggactggtcc ccacaccgat cccacttcct tgacaatcct tcatcaggat 780

caagttggag gccttcaagt gtttgcagat gaaaagtggc actccgttgc tcccatccca 840

cgagctttcg ttgtcaacat tggcgacaca ttcatggctt tgacaaacgg tatttacaag 900

agctgcttgc acagagcagt ggtgaatact gaaacggtga gaaaatcact tgtattcttt 960

ctatgtccca agctggagag accggtgaca ccagcagctg gcctagttaa tgccgcaaat 1020

tcgaggaagt acccagactt tacttgggca gcactgcttg aatttacaca gaaccattac 1080

cgtgctgaca tgaaaaccct tgttgcattc tccaaatggg ttcaagaaca agaatcgaac 1140

aacaaattaa taccttag 1158

<210> 6

<211> 1128

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 6

atggttcatt gtctgccaaa ggtttctgtc attcaagaca agccgatgct tccatcaact 60

tctgctgtcg caaaggatga gtattggccg cctttagctt tcgatgcatc gatccttgga 120

tctgaatcca acataccctc tcagttcata tggcctgacg acgagaagcc atgcctggat 180

gccccagaac ttgtaattcc aaccattgac ttgggagctt tcttgcttag gtactctctt 240

gctgtatcaa aagcggcaga ggtggtgaat gaggcatgca agaagcatgg gttttttctg 300

gttgtaaacc atggggtgga ttcagggctt atagacaaag ctcatcagta catggaccgc 360

ttcttcagcc tgcaactttc tgagaagcaa aaggctaaga gaaaagtagg agaaagctat 420

gggtatgcaa gcagtttcgt tgggaggttt tcctccaagt tgccatggaa agaaacactg 480

tcttttcggt actgtcctca cactcaaaat atcgtgcaac actatatggt gaatttgatg 540

ggtgaagatt ttagagattt cgggttccaa gagataatgg ggctgctagg gataagtcta 600

ggacttgacc aagcctactt caaagacttc ttcgaagaaa atgattcaat cctgagactg 660

aatcactatc ccccatgcca aaagcctgag ttgactctgg gaaccggtcc ccacaccgat 720

cccacttcct tgacaatcct tcatcaggat caagtgggag gcctttcagg ttttgcagat 780

gaaaagtggc actccgttgc tcccatccca ggagctttcg ttgtcaacat tggcgacaca 840

ttcatggctt tgacaaacgg tatttacaag agctgcttgc acagagcagt ggtgaatact 900

gaaacggtga gaaaatcact tgcattcttt ctatgtccca agctggagag accggtgaca 960

ccagcagctg gcttagtaaa tgccgcaaat ccgaggaaat acccagactt cacttgggca 1020

gcactgctga aatttacaca gaaccattac cgtgctgaca tgaaaaccct tgttgcattc 1080

tccaaatggg ttcaagaaca agaatcgaac aacaaattaa taccttag 1128

<210> 7

<211> 1104

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 7

atggcaattg attgcgtttc caagatatcc atgcctcatc atcctaaaga tgaaaaaaga 60

gaggagcaaa aacaactggt ttttgatgcc tcggtgctta aataccaacc cgacatacca 120

aaacaattta tatggcccga caaggaaaag cctagtgtta atgcaccaga actccaagtg 180

ccattcattg acctaggagg gttcctttcc ggtgaccctg tttctgcaat ggaagcatcg 240

aggctggtgg gcgaggcatg tcggcagcat ggtttcttcc ttgtggttaa tcatggagtg 300

gatgcaacac tggtggctga tgctcacagt tatatggcaa aggctcagag gaaacttggt 360

gagcactgtg gatatgctag tagcttcact ggtagattct cctccaagct gccatggaag 420

gaaacgcttt cctttcggta ttcagctgac aaaaactcat ccaagattgt tgaagactac 480

cttgttggca aattgggaga tgaattcaag catttcggga gggtttacca agattactgc 540

gaggcaatga gcaagctatc tctagggata atggagctat tagccattag tcttggcgta 600

ggaagatcac atttcaggga attttttgag gaaaatgaat caataatgag gctcaattac 660

tacccaccat gccaaaaacc agacctcacc ttaggaacag ggcctcattg cgatccaacg 720

tcattaacca tccttcacca agaccgagtt ggtggtcttc aagtgtttgt agacaatgaa 780

tggcgttcaa ttagcccaaa tctcgaagca tttgtcgtta acattggcga caccttcatg 840

gcactttcaa atgggcgata caagagttgc ttgcaccggg cagtggtgaa ccgccacatc 900

ccaagaaaat ctctggcttt cttcctatgt cccaagggtg ataaagtggt agccccacca 960

acagagttgg tcgacgccta taatcccaga gtatatccag attttacttg gcctatgctg 1020

cttgaattca cacaaaagca ttatagagct gatatgaaca cactcgaagt cttctcaaac 1080

tgggttcaac agagaaacag ctga 1104

<210> 8

<211> 1140

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 8

atggcaattg attgcgtttc caagataccc tccatgcctc atcatcctaa agatgaaaaa 60

agagaggagc aaaaacaact ggtttttgat gcctcggtgc ttaaatacca acccgacata 120

ccaaaacaat ttatatggcc cgaccacgaa aagcctaatg ttaatgcacc agaactccaa 180

gtgccattca ttgacctagg agggttcctt tccggtgacc ctgtttctgc aatggaagca 240

tcgaggctgg tcggcgaggc atgtcggcag catggtttct tccttgtggt taatcatgga 300

gtggatgcaa cactggtggc tgatgctcac agttatatgg gtaacttctt tgaattgcca 360

ctcaatgata agcaaagggc tcagaggaaa cttggtgagc actgtggata tgctagtagc 420

ttcactggta gattctcctc caagctgcca tggaaggaaa cgctttcctt ccggtattca 480

gctgacaaaa agtcatccaa gattgttgaa gactaccttg atggcaaatt gggagatgaa 540

ttcaagcatt tcgggagggt ttaccaagat tactgcgagg caatgagcaa gctatctcta 600

gggataatgg agctattagc cattagtctt ggcgtaggaa gatcacattt cagggaattt 660

ttcgaggaaa atgaatcaat aatgaggctg aattactacc caccatgcca aaaaccagac 720

ctcactttag gaacagggcc tcattgcgat ccaacctcat taaccatcct tcaccaagac 780

cgagttggtg gtcttcaagt gtttgtagac aatgaatggc gttcaattag cccaaatgtc 840

gaagcatttg tcgttaacat tggcgacacc ttcatggcac tttcaaatgg gcgatacaag 900

agttgcttgc accgggcagt ggtgaaccgc cacatcccaa gaaaatctct ggctttcttc 960

ctatgtccca agggtgataa agtggtagcc ccaccaacag agttggtcga cgcctataat 1020

cccagagtat atccagattt tacttggcct atgctgcttg aattcacaca aaagcattat 1080

agagctgata tgaacacact tgaagtcttc tcaaactggg ttcaacagag aaacagctga 1140

<210> 9

<211> 1152

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 9

atggattcaa cgcatctcct ttcctctccg cttgagattc aagatcagac actagtcgac 60

catcataatt cttctatcgg ctcatctttt ctccaaaacc aaaccaacgt ccccaaagaa 120

tttctttggc ccaaagttga tttagttaat gctcatcaag agctgttgga gccacttgta 180

gatcttgaac gcttctttag aggtgatgaa ttggcgattc agcagtctgc caaggtcatt 240

agggctgctt gtctaaccca cggttgcttt caagtcatca atcatggggt tgattcccac 300

ctcatcaatg ctgcatatta tcacctcaat cgtttcttcc atttgccact tagccacaaa 360

ttaagggctc gaagggccac tactgctgga ttaaacacct tgaactattc aggtgcacat 420

tcggatcgtt tctcttcgaa tttgccatgg aaggaaactt taactttccg ggtccatgag 480

aatctcaagg aatccagtgt cgtagacttg ttcaaatcca gtttaggaga tgattttgaa 540

gaaatgggca taacatatca aaagtactgt gaaggaatga agagcttagc tctagcagtg 600

atggaaatac tagcaatcag cttgggagtt gatcgattgc actataaaaa ttattttcag 660

gacggtgggt ctataatgcg atgtaactat tatccgccat gccccgagcc aggacttacc 720

ttcggcactg gtcctcattg tgacaccaca tctttaacaa ttctccacca agacgaagtt 780

gggggcctgg aaatctttgc aaacaacaaa tggcagattg ttcgacctcg tcaggatgcc 840

ctagtaatca acatcggtga gaccttcacg gcattaacaa atgggagata caagagttgc 900

ctgcataggg cagtagtaaa cagcgagagg gcgagaaaat cattggtata ctttgtttgc 960

ccacgagaag acaaggtggt gagaccccca gaggatcttg tacaaggtga tcaactccca 1020

agagcttacc ctgatttcac atggtccgat ttcctccatt tcacccaaaa ctactacaga 1080

gctgacgctc atactctcca tagcttcatc aaatggctct catcttccaa ccccattcat 1140

cacaaccgtt aa 1152

<210> 10

<211> 1164

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 10

atggtacatt gtctgccaaa ggttttttgtc attcaagaca agccgatgct tccatcaact 60

gctgctgtcg caaaggatga gcatcggcct ttagctttcg atgcatcgat ccttggatct 120

gaatccaaca taccctctca gttcatatgg cctgacgacg agaagccatg cctggatgcc 180

ccggcacttg taattccaac cattgacttg ggagctttct tgcttgggga ctctcttgct 240

gtatcaaaag cggcagaggt ggtgaatgag gcatgcaaga agcatgggtt ttttctggtt 300

gtaaaccatg gggtggattc agggcttata gacaaagccc atcagtacat ggaccgcttc 360

ttcagcctac aactctctga gaagcaaaag gctaagagaa aagtaggaga aagctatggg 420

tatgcaagca gtttcgttgg caggttttcc tccaagttgc catggaaaga gacactgtct 480

tttcggtact gtcctcacac tcaaaatatc gtgcaacact atatggtgaa ttggatgggt 540

gaagatttta gagatttcgg gaggctttac caggaatatt gtgaagccat gaacaaggtt 600

tcccaagaga taatggggct gctagggata agtctaggac ttgaccaagc ctacttcaaa 660

gactttttcg aacaaaatga ttcaatcctg agactgaatc actatccccc atgccaaaag 720

cctgagttga ctctgggaac cggtccccac accgatccca cttccttgac aatccttcat 780

caggatcaag ttggaggcct tcaggtgttt gcagatgaaa agtggcactc cgttgctccc 840

atcccaggag cttttgttgt caacgttggc gacacattca tggctttgac aaacggtttt 900

tacaagagct gcttgcacag agcagtggtg aatactgaaa cggtgagaaa atcacttgca 960

ttctttctat gtcccaagct ggagagaccg gtgacaccag cagctggctt agttactgcc 1020

gaaaatccga ggaaataccc agacttcact tgggcagcac tgctgaagtt cacacagaac 1080

cattaccgtg ctgacatgaa aacccttgtt gcattctcca aatgggttca agaacaagaa 1140

tcgaactaca aattaatacc ttag 1164

<210> 11

<211> 1155

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 11

atgtctctct ctgtcttaat ggattcaagt tctccaacca ttcttttaca tccatccata 60

gaaccaagag atgagacggg tagtgtcctt tttgacccat ccaagttgca aaaacaatca 120

agtttaccct cagagttcat atggccatgt ggggacttgg ttcacaccca agaagagctt 180

aacgagccat tgatagactt gggtgggttc atcaaaggag acgaagaagc tactgcacat 240

gcagtcgacc ttgttaagac tgcctgttct aaacatggtt tcttccaagt tacaaaccat 300

ggtgttgatt caaatcttat ccaagctgct taccaagaga ttgatgctgt gtttaagtta 360

ccccttaaca agaagctcgg tttccaaaga aagcctggtg gtttttcagg ctattctgct 420

gcccatgctg accggttttc cgctaagttg ccatggaagg aaacattttc ttttggctac 480

caaggactta actccgatcc ttctgtggtt cattatttca gctctgcttt gggggaagat 540

tttgagcaca ccgggaggat ttaccaaaag tattgtgaaa agatgaggga gctgtcgcta 600

gtgatcttcg aactgttggc aatcagcttg gggatagatc gtttacacta cagaaaattt 660

ttcgaagatg ggaattcaat aatgaggtgt aattactatc ccccatgcaa taattctggc 720

ctcacccttg gcactggccc tcactctgat cctacttcct taacaattct tcaccaagat 780

caagtgggag ggctcgaagt tttcatcaat aacaaatggt atgctgttcg acctcgacaa 840

gatgcctttg tcattaacat tggtgatact ttcatggcat tatgtaacgg aagatacaag 900

agttgcctgc atagagcagt ggtgaacaag gagagagaga ggagatcatt ggtatacttt 960

gtgtgcccaa aagaagacaa aatagtgaga cccccacaag atctaatgtg cagatcagga 1020

gggccaagag tgtatcctga tttcacatgg tctgatttgt tggacttcac tcaaaatcac 1080

tatagagctg acgttgctac actccaaagc ttctttcctt ggctcctttc ttctaaccct 1140

acttccaact tctag 1155

<210> 12

<211> 1083

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 12

atggcaattg attgcgtttc caagataccc tccatgcctc atcatcctaa agatgaaaaa 60

agagaggagc aaaaacaact ggtttttgat gcctcgccta atgttaatgc accagaactc 120

caagtgccat tcattgacct aggagggttc ctttccggtg accctgtttc tgcaatggaa 180

gcatcgaggc tggtcggcga ggcatgtcgg cagcatggtt tcttccttgt ggttaatcat 240

ggagtggatg caacactggt ggctgatgct cacagttata tgggtaactt ctttgaattg 300

ccactcaatg ataagcaaag ggctcagagg aaacttggtg agcactgtgg atatgctagt 360

agcttcactg gtagattctc ctccaagctg ccatggaagg aaacgctttc cttccggtat 420

tcagctgaca aaaagtcatc caagattgtt gaagactacc ttgatggcaa attgggagat 480

gaattcaagc atttcgggag ggtttaccaa gattactgcg aggcaatgag caagctatct 540

ctagggataa tggagctatt agccattagt cttggcgtag gaagatcaca tttcagggaa 600

tttttcgagg aaaatgaatc aataatgagg ctgaattact acccaccatg ccaaaaacca 660

gacctcactt taggaacagg gcctcattgc gatccaacct cattaaccat ccttcaccaa 720

gaccgagttg gtggtcttca agtgtttgta gacaatgaat ggcgttcaat tagcccaaat 780

gtcgaagcat ttgtcgttaa cattggcgac accttcatgg cactttcaaa tgggcgatac 840

aagagttgct tgcaccgggc agtggtgaac cgccacatcc caagaaaatc tctggctttc 900

ttcctatgtc ccaagggtga taaagtggta gccccaccaa cagagttggt cgacgcctat 960

aatcccagag tatatccaga ttttacttgg cctatgctgc ttgaattcac acaaaagcat 1020

tatagagctg atatgaacac acttgaagtc ttctcaaact gggttcaaca gagaaacagc 1080

tga 1083

<210> 13

<211> 1140

<212> DNA

<213> Upland cotton (G.hirsutum L.)

<400> 13

atggccatcg actgcatctc caacatagcc tccatgactc accatccgaa agatgaaaaa 60

aaagatgagc agaaaaagct ggtttttgat gcctcggtgc tcaagttcga atcccagata 120

ccaaaagaat ttatatggcc tgatgacgaa aagccttccg ctaatgcacc agaactccaa 180

gtaccactca ttgacctagg aggcttcctt tctggtgacc ctgttgctac aatggaagct 240

tcaaggttta tcagcgaggc atgtcagcag catggtttct tccttgtagt taatcatgga 300

gtcgatgcaa aactcttggc tgatgcccac aagtacatgg ataacttctt tctattgcca 360

cttagacaaa agcaaagggc tcaaagaaaa cttggtgagc actgtggata tgccagtagc 420

ttcactggca ggttctcgac caagctccca tggaaggaaa cactttcttt tcgctattca 480

gccgagaaca actcatccaa gatggtggaa gactaccttg ttaataaaat gggaaatgaa 540

ttgaggcaac tcgggagggt ttaccaggac tactgcgagg cgatgagcaa gctttctctg 600

gggataatgg agcttttagc cattagtctg ggcgtaggca gagcacattt ccgggagttt 660

tttgataaaa atgattcaat aatgagacta aactactatc ccccttgtca aaaaccagac 720

ctcactttag gaacagggcc tcattgcgat ccaacgtctt taaccatcct tcaccaagac 780

agagttggtg gccttcaagt gtttgtagac aacgaatggc attcaattag cccaaatttc 840

gaagcatttg tcgttaacat tggcgacacc tttatggcac tgtcaaatgg gagatataaa 900

agttgcttgc accaagcagt ggtgaacagc cataagccaa gaaaatctct ggctttcttt 960

ttgtgtccag agggggataa agtggtaacc ccaccagcag agttggtgag ccagaacagt 1020

cccagagtat atccagattt tacatggcct atgctgcttg aattcacaca aaagcattac 1080

agagctgaca tgaacactct tcaagaattc tcaaactggg ttcaacagag aaacagctga 1140

<210> 14

<211> 384

<212> PRT

<213> Upland cotton (G.hirsutum L.)

<400> 14

Met Ser Leu Ser Val Leu Met Asp Ser Gly Ser Pro Thr Ile Leu Leu

1 5 10 15

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

20 25 30

Pro Ser Lys Leu Gln Asn Gln Ser Ser Leu Pro Ser Glu Phe Ile Trp

35 40 45

Pro Cys Gly Asp Leu Val His Thr Gln Glu Glu Leu Asn Glu Pro Leu

50 55 60

Ile Asp Leu Gly Gly Phe Ile Lys Gly Asp Glu Glu Ala Thr Ala His

65 70 75 80

Ala Val Gly Leu Val Lys Thr Ala Cys Ser Lys His Gly Phe Phe Gln

85 90 95

Val Thr Asn His Gly Val Asp Ser Asn Leu Ile Gln Ala Ala Tyr Gln

100 105 110

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

115 120 125

Gln Arg Lys Pro Gly Gly Phe Ser Gly Tyr Ser Ala Ala His Ala Asp

130 135 140

Arg Phe Ser Ala Lys Leu Pro Trp Lys Glu Thr Phe Ser Phe Gly Tyr

145 150 155 160

Gln Gly Leu Asn Ser Asp Pro Ser Val Val His Tyr Phe Ser Ser Ala

165 170 175

Leu Gly Glu Asp Phe Glu Gln Thr Gly Trp Ile Tyr Gln Lys Tyr Cys

180 185 190

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

195 200 205

Ser Leu Gly Ile Asp Arg Leu His Tyr Arg Lys Phe Phe Glu Asp Gly

210 215 220

Asn Ser Ile Met Arg Cys Asn Tyr Tyr Pro Pro Cys Asn Asn Ser Gly

225 230 235 240

Leu Thr Leu Gly Thr Gly Pro His Ser Asp Pro Thr Ser Leu Thr Ile

245 250 255

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

260 265 270

Trp Tyr Ala Val Arg Pro Arg Gln Asp Ala Phe Val Ile Asn Ile Gly

275 280 285

Asp Thr Phe Met Ala Leu Cys Asn Gly Arg Tyr Lys Ser Cys Leu His

290 295 300

Arg Ala Val Val Asn Lys Glu Arg Glu Arg Arg Ser Leu Val Tyr Phe

305 310 315 320

Val Cys Pro Lys Glu Asp Lys Ile Val Arg Pro Pro Gln Asp Leu Met

325 330 335

Cys Arg Ser Gly Gly Pro Arg Val Tyr Pro Asp Phe Thr Trp Ser Asp

340 345 350

Leu Leu Glu Phe Thr Gln Asn His Tyr Arg Ala Asp Val Ala Thr Leu

355 360 365

Gln Ser Phe Phe Pro Trp Leu Leu Ser Ser Asn Pro Thr Ser Asn Phe

370 375 380

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence

<400> 15

atgtctctct ctgtcttaat g 21

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> 16

ctagaagttg gaagtagggt 20

<210> 17

<211> 27

<212> DNA

<213> Artificial sequence

<400> 17

tgctctagag caatgtctct ctctgtc 27

<210> 18

<211> 23

<212> DNA

<213> Artificial sequence

<400> 18

cgagctcgct agaagttgga agt 23

<210> 19

<211> 30

<212> DNA

<213> Artificial sequence

<400> 19

cgagctcgat gtctctctct gtcttaatgg 30

<210> 20

<211> 23

<212> DNA

<213> Artificial sequence

<400> 20

ggactagtga agttggaagt agg 23

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence

<400> 21

ggactagtgt gaacaaggag agagaga 27

<210> 22

<211> 28

<212> DNA

<213> Artificial sequence

<400> 22

ttggcgcgcc aagtagggtt agaagaaa 28

<210> 23

<211> 22

<212> DNA

<213> Artificial sequence

<400> 23

tgcacatgca gtcgaccttg tt 22

<210> 24

<211> 22

<212> DNA

<213> Artificial sequence

<400> 24

ggtcagcatg ggcagcagaa ta 22

<210> 25

<211> 19

<212> DNA

<213> Artificial sequence

<400> 25

atcctccgtc ttgaccttg 19

<210> 26

<211> 19

<212> DNA

<213> Artificial sequence

<400> 26

tgtccgtcag gcaactcat 19

<210> 27

<211> 24

<212> DNA

<213> Artificial sequence

<400> 27

agatccagga caaggaaggt attc 24

<210> 28

<211> 22

<212> DNA

<213> Artificial sequence

<400> 28

cgcaggacca agtgaagagt ag 22

Claims (1)

1. a dwarfing method of upland cotton, is characterized in that, by carrying out gene silencing to upland cotton plant height regulation gene GhGA20ox6 , thereby reduces upland cotton plant height, realizes dwarfing; GhGA20ox6 has the nucleus shown in SEQ ID NO:1 nucleotide sequence.

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