CN111218460A - Application of cotton GhACO gene in promoting plant flowering - Google Patents

Abstract

The invention discloses cottonGhACOA gene and application thereof in promoting plant flowering belong to the technical field of plant genetic engineering. The cottonGhACOThe gene has a nucleotide sequence shown in SEQ ID No.3 and can encode an amino acid sequence shown in SEQ ID No. 4. The invention relates to a transgene obtained by transgenic technologyGhACOTransgenic Arabidopsis plants, resulting in bolting of ArabidopsisThe number of rosette leaves is obviously reduced, and the flowering time is obviously advanced, which shows thatGhACOThe gene plays a key role in promoting cotton blossoming. The invention provides favorable gene resources for the cultivation of short-season cotton.

Description

Application of cotton GhACO gene in promoting plant flowering

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a GhACO gene in promoting plant flowering.

Background

Cotton is an important economic crop, is used as an important raw material in the fields of textile, medicine, chemical industry and the like, and plays a very important role in national economy in China. China has more people and less land, and grains and cotton compete for land, so that the planting area and the yield of cotton are ensured while the grain safety is ensured. An effective way to solve the contradiction is the breeding of the early maturing cotton. The early maturing cotton variety can realize direct seeding of wheat (rape) in cotton areas of yellow river and Yangtze river basin; is beneficial to the improvement of quality and efficiency of the cotton in the northwest inland area and ensures the safety of domestic cotton. The early maturity of cotton is a composite character, the flower development is closely related to the early maturity of cotton, and the flowering time determines the early and late of the maturity.

At present, the research on the genes related to the cotton early maturity is still less, and the effective gene resources are lacked for the genetic engineering breeding of cotton early-maturing varieties.

Disclosure of Invention

The inventors discovered a 1-aminocyclopropane-1-carboxylic acid oxidase-like gene (GhACO gene) using the transcriptome data of cotton, and cloned the GhACO gene from upland cotton. The fluorescent quantitative result shows that the gene is related to cotton flower bud development, arabidopsis thaliana is transformed by an inflorescence infection method by constructing an overexpression vector of the gene, and the result shows that the number of rosette leaves is obviously reduced when the transgenic arabidopsis thaliana is bolting, and the flowering time is obviously advanced. Therefore, the inventor thinks that the cotton GhACO gene may play a key role in promoting cotton flowering and can be used as a favorable gene resource for short-season cotton cultivation. Thus, the present invention has been completed.

The invention provides a separated cotton GhACO gene. The GhACO gene has a nucleotide sequence shown in SEQ ID No. 3. The open reading frame of the gene is 888 bp.

In some embodiments of the invention, the nucleotide sequence shown as SEQ ID No.3 is capable of encoding the amino acid sequence shown as SEQ ID No. 4. The amino acid sequence comprises 295 amino acids, the relative molecular weight of the protein is 33.28kDa, and the isoelectric point is 5.11.

The invention also provides application of the GhACO gene in improving and promoting plant flowering.

In some embodiments of the invention, the expression level of the GhACO gene is increased in plants to promote flowering in the plants.

In some embodiments of the present invention, the increasing of the expression level of the GhACO gene in the plant is achieved by: improving the expression of the endogenous GhACO gene of the plant, or over-expressing the exogenous GhACO gene in the plant.

In a specific embodiment of the present invention, the overexpression of the exogenous GhACO gene refers to the expression of the GhACO gene in a plant by agrobacterium-mediated transformation using a plant expression vector.

Further, the GhACO gene is introduced into a plant cell, tissue or organ by a plant expression vector.

Further, the plant expression vector drives the expression of the GhACO gene through a constitutive or inducible promoter.

Still further, the constitutive promoter is a 35S promoter.

In the present invention, the promotion of flowering refers to promotion of the flowering phase of plants to be advanced.

In the present invention, the plant is cotton, corn, rice, wheat or Arabidopsis.

The invention has the advantages of

The invention obtains the GhACO gene-transferred Arabidopsis plant by a transgenic technology, so that the number of rosette leaves is obviously reduced when the Arabidopsis is bolting, and the flowering time is obviously advanced, which shows that the GhACO gene possibly has a key role in promoting cotton flowering. The invention provides favorable gene resources for the cultivation of short-season cotton.

Drawings

FIG. 1 shows an electrophoretogram of a GhACO gene fragment amplified from Gossypium hirsutum No. 2 by the PCR method. The sizes of four bottom-to-top bands of the Marker respectively represent 200bp, 500bp, 800bp and 1200 bp.

FIG. 2 shows T₁And (3) PCR detection results of transgenic arabidopsis strains. 1-2 transgenic arabidopsis thaliana strains, and 5-6 strains are selected from each strain for detection.

FIG. 3 shows T₃And (3) fluorescent quantitative identification results of transgenic arabidopsis strains. WT is non-transgenic Arabidopsis, L1, L2 are two transgenic Arabidopsis lines.

FIG. 4 shows T₃And (5) phenotype identification results of the generation transgenic arabidopsis line. In the figure, arabidopsis thaliana is grown for four weeks, WT is a non-transgenic arabidopsis thaliana, two transgenic arabidopsis thaliana lines L1 and L2.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The reagent solutions mentioned but not listed here were prepared according to the method of the third edition of the molecular cloning instructions, and the biochemical reagents were analytically pure or of higher grade.

The materials, reagents and consumables, medicines, culture media and the like used by the invention are as follows:

1. plant material

The experimental material selected in this example is upland cotton salt Zao No. 2, which is planted in a test field (Henan' an Yandong field) of a key laboratory in the cotton biology of the institute of Cotton, national institute of agricultural sciences, China, and the management measure is normal field management.

Arabidopsis thaliana material: wild type arabidopsis thaliana (colombia type) was used for transformation and phenotypic observation of late overexpression vectors.

2. Reagent and consumable

2.1 enzymes and kits: (1) restriction enzyme, modifying enzyme and kit: the PCR reaction system, the gel recovery kit, the cloning kit and the plasmid test kit are purchased from Bao bioengineering Dalian Limited company, and the DNA extraction kit is purchased from OMEGA company.

2.2 other drugs agarose is Spanish original product, peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride and the like are domestic analytical pure, 5-bromo-4-chloro-3-indoside galactoside (X-gal), isopropyl- β -D-thiogalactopyranoside (IPTG), ampicillin and the like are purchased from Bao bioengineering Ginko Ltd, and Escherichia coli competent cell DH5 α is purchased from Beijing Tiangen Biotech.

2.3 culture Medium: LB liquid medium: 10g/L Tryptone (Tryptone), 5g/L yeast extract (Yeast), and 10g/L sodium chloride (NaCl); LB solid medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract), 10g/L of sodium chloride (NaCl) and 15g/L of Agar powder (Agar), and the volume is fixed to 1L; LB selective medium: before LB plate, adding antibiotic with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 deg.C, shaking up and plating.

2.4 Main instruments: : PCR amplification apparatus (BIO-RAD), high speed centrifuge (Hettich MIKRO 200R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), and fluorescence quantitative PCR apparatus (ABI 7500).

Example cloning of Gene sequence of GhACO

1 sampling method

In the full-bloom stage of cotton, mature cotton leaves are taken, quickly frozen in liquid nitrogen and stored in a refrigerator at minus 80 ℃ for later use.

2 extraction of RNA

All centrifugation steps below were performed at room temperature.

1) Homogenization treatment 100mg of plant leaves were rapidly ground into powder in liquid nitrogen, 700. mu.L of SL (β -mercaptoethanol was added before use) was added, and the sample was immediately mixed by vigorous shaking.

2) Centrifuge at 12,000rpm for 2 min.

3) The supernatant was transferred to the filtration column CS and centrifuged at 12,000rpm for 2min, and the supernatant from the collection tube was carefully pipetted into a new RNase-Free centrifuge tube, the tip being kept from touching the cell debris in the collection tube.

4) Adding 0.4 times volume of anhydrous ethanol, mixing, transferring the mixture into adsorption column CR3, centrifuging at 12,000rpm for 15sec, discarding the waste liquid in the collection tube, and returning the adsorption column CR3 to the collection tube.

5) 350. mu.L of deproteinizing solution RW1 was added to the adsorption column CR3, and centrifuged at 12,000rpm for 15sec, thereby discarding the waste liquid in the collection tube and returning the adsorption column CR3 to the collection tube.

6) DNaseI working solution: mix 10. mu.L DNaseI stock and 70. mu.L RDD gently.

7) 80. mu.L of DNaseI working solution was added to CR3 and allowed to stand at room temperature for 15 min.

8) After standing, 350. mu.l of deproteinizing solution RW1 was added to CR3, centrifuged at 12,000rpm for 15sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

9) To the adsorption column CR3, 500. mu.L of rinsing solution RW (ethanol was added before use), centrifuged at 12,000rpm for 15sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

10) Step 9 is repeated.

11) Centrifuging at 12,000rpm (13,400 Xg) for 2min, placing adsorption column CR3 into a new RNase-Free centrifuge tube, and adding 30-50 μ L RNase-Free ddH dropwise into the middle part of the adsorption membrane₂O, left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to obtain an RNA solution.

Note that: the volume of elution buffer should not be less than 30. mu.L, and too small a volume affects the recovery efficiency. The RNA samples were stored at-70 ℃. If the expected RNA yield is more than 30. mu.g, the RNA solution obtained by centrifugation in step 11 may be added to an adsorption column CR3, and left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to obtain an RNA solution.

To prevent RNase contamination, precautions:

1) the gloves are often replaced with new ones. Because the skin is often bacteria-bearing, RNase contamination may result;

2) the RNase-free plastic product and the gun head are used to avoid cross contamination;

3) RNA is not degraded by RNase while in lysate SL. However, after extraction, plastics and glassware without RNase should be used in the further processing.

4) The preparation solution should use RNase-Free ddH₂O。

3 reverse transcription

The RNA samples obtained above were subjected to the specific procedures described in the reverse transcription kit (DRR037A) using TaKaRa, in which the reaction solution for reverse transcription was set on ice, as follows:

the reverse transcription reaction conditions were as follows:

15min at 37 ℃ (reverse transcription);

5sec at 85 ℃ (inactivation reaction of reverse transcriptase).

Finally, 500ng of RNA sample was reverse-transcribed into cDNA, and the resulting reverse transcription product cDNA solution was diluted 8-fold as a template for PCR reaction.

Amplification of the 4 GhACO gene: comprises primer design, PCR reaction system, program and product detection

1) Design of primers

Acquiring a gene sequence of GhACO (Gh _ A09G0562) from cotton FGD NAU assembly, and designing a Primer of the GhACO gene by using Primer 5 software;

upstream primer F (SEQ ID No. 1):

5'-ATGGCAGAAATAAGCTTAGAACG-3'

downstream primer R (SEQ ID No. 6):

5'-TTAAGGTGGGCTTGCCTGCATCA-3'

2) PCR reaction system

The following systems were formulated on ice:

according to the TaKaRa LA Taq Hot Start Version 2.0(DRR042) enzyme specification, the PCR reaction system is as follows:

3) PCR reaction procedure

Extension at 10 ℃ -

4) Detection of PCR products

And (3) adding 1 mu L of 6 XLoading Buffer into 1 mu L of PCR product, mixing uniformly, spotting on 1% agarose gel, and carrying out electrophoresis detection.

5 gel recovery of PCR products

1) Cutting the band to be recovered from the electrophoresis gel under an ultraviolet lamp, paying attention to the fact that the blade needs to be disinfected, and enabling the gel block to be small as much as possible and to be easy to melt completely;

2) weighing an Eppendorf tube in advance, then putting the rubber block in the tube, and weighing again to obtain the weight of the rubber block;

3) adding Binding Buffer in an amount of 300 mu L per 100mg of the rubber block, and checking whether the rubber block is soaked in the liquid;

4) water bath at 55 deg.C for 10min to melt the gel block and release DNA, and taking out every 2-3min during the period and shaking;

5) after the rubber blocks are completely melted, adding isopropanol in an amount of 150 mu L per 100mg of rubber blocks, and fully shaking and uniformly mixing;

6) mounting a High Pure Filter Tube on a Collection Tube;

7) transferring all the liquid in the Eppendorf tubes into a High Pure Filter Tube, taking care that the volume does not exceed 700 mu L, and if the volume exceeds the volume, centrifuging twice;

8) centrifuging at 12,000rpm for 1min, and pouring out liquid in the collecting pipe;

9) adding 500 μ L of Wash Buffer, and centrifuging for 1min again;

10) pouring out the liquid in the collecting pipe, adding 200 mu L of Wash Buffer again, and centrifuging at 12,000rpm for 1 min;

11) carefully taking down the Filter Tube and then loading the Tube into a new Effendorf Tube;

12) mu.L of Elution Buffer was added to the center of the filter element, and the mixture was left at room temperature for 1min and centrifuged at 12,000rpm for 1 min.

6 glue recovery product is connected with cloning vector PMD-18T

1) The following DNA solutions were prepared in a microcentrifuge tube in a total volume of 5. mu.L.

2) Add 5. mu.L (equal amount) of Solution I;

3) the reaction was carried out at 16 ℃ for 30 min.

7 ligation product transformation of E.coli

1) Add 10. mu.L ligation into 100. mu.L E.coli DH5 α competence;

2) performing ice bath for 30 min;

3) heat shock is carried out on the mixture for 45s in water bath at 42 ℃;

4) ice-bath for 2 min;

5) adding 500 μ L LB liquid culture medium, incubating at 37 deg.C and 190rpm for 1 h;

6) centrifuging at 4,000rpm for 1min, discarding the supernatant, and coating on LB plate containing benzyl amine resistance (Amp) after leaving about 100 μ L of the supernatant;

7) incubated at 37 ℃ overnight (12-16 h).

8 detection and sequencing of Positive clones

1) Picking a normal single colony from the transformation plate, putting the colony into a liquid LB culture medium containing Amp, and carrying out shake culture at the constant temperature of 37 ℃ for 6-8 h;

2) and (5) detecting positive clones by colony PCR, and sending the monoclonals with correct verified fragment sizes to a biological company for sequencing.

3) Preservation of positive bacteria liquid

And adding a certain amount of glycerol into the bacterial liquid which is subjected to PCR verification and sequencing to ensure that the final concentration of the glycerol is about 20 percent and storing the glycerol at-80 ℃.

The results show that:

(1) the PCR method was used to amplify the GhACO gene fragment from Gossypium hirsutum No. 2, and the gene fragment was found to be 800bp or more, as shown in FIG. 1.

(2) Obtaining sequence information of GhACCO gene

1) Sequencing to obtain an open reading frame 888bp of the GhACO gene, wherein the sequence is as follows (SEQ ID No. 3):

ATGGCAGAAATAAGCTTAGAACGAGCTCAAGAATCCATTGTCAATCGACAACAGGAATTGAAAGCTTTCGATGAAACAAAATCAGGAGTGAAAGCGCTGGTAGATTCGGGCTTGTCAAAGATTCCAACGATTTTCACCGATGAACAGTACAAGCTTGAGAGAAACAACATCCTCAACCAGAAACCTGGAAGCCCCACCAACAATGACGGAATCCCAATCATAAACTTGACCGGTGTCGACGATAATCCAAATTTACGTCGCGAAATAGTGAAGAAAGTTGGAGAAGCTTGCGAGAAATGGGGTTTCTTTCAAGTTATCAACCATGGGATTCCGTTGGCTACTACGGATGAAATGATAAACGGGGTTCGCAGGTTTCATGAACAGGATGACGAAGCTAAGAAAGAGATTTATTCTCGAGATTATTCCAAGAAAGTGTATTATAACAGTAACATCGATCTTTACAAGGCGGAAGCAACCAATTGGAGGGATACTTTGTGTTGTGTTATGGCACCTCGCCACCCTCTTCCTCAAGAACTGCCTGCAATTTGCAGAGATATAATGATAGAATATTCAAGCAAAATGATGAAATTAGGGCAGACTTTGTTAGAATTGATGTCGGAAGCCTTGGGTCTGAATCGGAGTTATTTGGAAGATATTGGGTGCGGTGAGGGAATGTTTGTGAAAGGCCATTACTATCCACCGTGCCCTGAACCGGACTTGACATTGGGCACCAGCAGGCACACCGATACCGGCTTCTGCACCGTAATTTTACAAGATGAAATCGGCGGACTTCAAATCCTCCATCAAAATCAATGGCTTGATATTAATCCTGTCCGGGGAGCTCTTGTTGTAAATTTGGGCGATATGATGCAGGCAAGCCCACCTTAA

2) bioinformatics analysis shows that the GhACO gene encodes 295 amino acids, the relative molecular weight of the protein is 33.28kDa, and the isoelectric point is 5.11.

The amino acid sequence coded by the GhACO gene is (SEQ ID No. 4):

MAEISLERAQESIVNRQQELKAFDETKSGVKALVDSGLSKIPTIFTDEQYKLERNNILNQKPGSPTNNDGIPIINLTGVDDNPNLRREIVKKVGEACEKWGFFQVINHGIPLATTDEMINGVRRFHEQDDEAKKEIYSRDYSKKVYYNSNIDLYKAEATNWRDTLCCVMAPRHPLPQELPAICRDIMIEYSSKMMKLGQTLLELMSEALGLNRSYLEDIGCGEGMFVKGHYYPPCPEPDLTLGTSRHTDTGFCTVILQDEIGGLQILHQNQWLDINPVRGALVVNLGDMMQASPP

example 2 construction of PBI121-GhACO plant expression vector

1 obtaining target gene fragment with specific enzyme cutting site

The cloned cDNA sequence of the GhACO gene is designed to contain primers suitable for enzyme cutting sites at the initiation codon ATG and the termination codon respectively. The cleavage sites used were Xba I (T/CTAGA) and Sma I (CCC/GGG).

The primer sequence of the GhACO gene enzyme cutting site is as follows:

upstream primer F (SEQ ID No. 5):

5'-CTAGTCTAGAATGGCAGAAATAAGCTTAGAACG-3'

downstream primer R (SEQ ID No. 6):

5'-TCCCCCGGGTTAAGGTGGGCTTGCCTGCATCA-3'

connecting the target fragment with the enzyme cutting site obtained by amplification to a pGEM-T Easy cloning vector, transforming DH5 α competent cells, and screening the intermediary recombinant with no mutation in sequence through PCR, enzyme cutting verification and sequence determination.

Construction of 2 pBI121-GhACO plant expression vector

The specific process is as follows:

1) the cloning vector of the recombinant GhACO and the pBI121 plasmid are subjected to double enzyme digestion by Sma I and Xba I respectively;

2) electrophoresis, and gel recovery of target fragment and large fragment product of pBI121 vector;

3) target gene fragment and T for digestion of large fragment product of pBI121₄Ligase ligation overnight;

4) the ligation product is transformed into Escherichia coli DH5 α, and cultured overnight at 37 ℃;

5) and (4) selecting monoclonal shake bacteria, and sequencing to verify the correctness of the sequence.

Example 3 transformation of recombinant vector pBI121-GhACO into Arabidopsis thaliana by Agrobacterium mediated method

Preparation of 1 LBA4404 Agrobacterium tumefaciens competence

Using CaCl₂The preparation of the method competent cell comprises the following specific processes:

1) selecting a single colony, inoculating the single colony in 4mL LB liquid culture medium containing antibiotics, and culturing overnight at the temperature of 28 ℃ and the speed of 190 rpm;

2) mixing the raw materials in a ratio of 1: 90 percent of the total weight of the cells were inoculated into 80mL of LB liquid medium containing antibiotics, cultured at 28 ℃ and 170rpm to OD₆₀₀＝0.6；

3) After the bacterial liquid is subjected to ice bath for 30min, transferring the bacterial liquid into a 50mL centrifuge tube, centrifuging the centrifugal tube at 4 ℃ and 5,000rpm for 10min, and discarding the supernatant;

4) 5mL of precooled 70mM CaCl was added₂Gently suspending, standing on ice for 20min, centrifuging at 4 deg.C and 5,000rpm for 5min, and removing supernatant;

5) 2mL of pre-cooled 70mM CaCl with 15% glycerol was added₂Resuspending the pellet;

6) the suspension is subpackaged in sterile centrifuge tubes, 200 mu L of each tube, and is stored at minus 80 ℃ for later use after quick freezing by liquid nitrogen.

2 transformation of Agrobacterium

The agrobacterium tumefaciens LBA4404 competent cells are transformed by a freeze-thaw method, and the specific transformation process is as follows:

1) adding 1 mu g of plasmid into 100 mu L of agrobacterium tumefaciens LBA4404 competent cells, uniformly mixing, and carrying out ice bath for 30 min;

2) quick freezing for 75s by using liquid nitrogen, and thermally shocking for 2-6 min at 37 ℃;

3) ice-bath for 5min, and adding 600 μ L LB liquid culture medium;

4) culturing at 190rpm and 28 deg.C for 4 hr, spreading 100 μ L bacterial liquid on LB screening culture medium containing kanamycin, streptomycin and rifampicin, culturing at 28 deg.C for 36-48 hr until resistant colony is visible;

5) selecting positive clones, culturing at 28 deg.C in LB liquid culture medium for 48 hr, adding glycerol to the positive clones to make the final concentration of glycerol about 20%, and storing at-80 deg.C for use.

3 Agrobacterium mediated transformation of Arabidopsis

The method for transforming the arabidopsis thaliana by adopting the inflorescence dip-dyeing method comprises the following specific operations:

1) inoculating 20 mu L of agrobacterium liquid preserved at-80 ℃ to 1mL of LB liquid culture medium, and carrying out shaking culture at 28 ℃ and 180rpm overnight;

2) adding 200 μ L of activated bacterium liquid into 50mL LB liquid culture medium, performing shaking culture at 28 deg.C and 180 rpm;

3) when the OD value of the bacterial liquid is about 1.2, centrifuging the bacterial liquid at 4,000rpm for 5min, and collecting thalli;

4) the preparation of the transformation medium comprises the following components: 1/2MS (macroelement halved, others unchanged), 5% sucrose, 0.01. mu.g/mL Benzylaminopurine (BAP), 0.03% silwet L-77, 20mg/L acetosyringone, KOH adjusted to pH 5.7.

5) Suspending the thallus with the transformation medium, adjusting OD to 0.8, and starting dip dyeing;

6) placing the arabidopsis inflorescence in a transformation medium for 30-50sec, wrapping the arabidopsis by using a preservative film after dip dyeing, culturing in the dark for one day, and then culturing under normal conditions.

Harvesting mature transgenic Arabidopsis seeds as T₀And (5) seed generation.

4 identification of transgenic Arabidopsis plants

(1) Identification of transgenic Arabidopsis lines

1) T to be harvested₀Sterilizing seed generation, planting on 1/2MS containing kanamycin (Kan), treating at 4 deg.C for 3 days, transferring into artificial climate test box, wherein the positive plant grows normally and the negative plant leaves turn yellow in about 10 daysNo longer growing;

2) transplanting the positive arabidopsis thaliana plant into a small flowerpot for planting, extracting DNA after growing for one month, and detecting by using PCR (polymerase chain reaction), wherein primers used in detection are as follows:

upstream primer F (SEQ ID No. 7):

5'-ATGGAGATCTCAGTGGAGAAGA-3'

downstream primer R (SEQ ID No. 8):

5'-GGCCATCAAACTCCATAACCAA-3'

3) the plants of each generation are tested for positive lines until they are propagated to T₃Generating to obtain homozygous transgenic arabidopsis lines;

identification of expression level of T3-generation strain

The procedure for the fluorescent quantitation (qRT-PCR) validation was as follows:

extracting RNA, performing reverse transcription to obtain cDNA, and respectively designing primers for fluorescence quantification of GhACO gene and Arabidopsis thaliana internal reference gene UBQ 10:

GhACO

upstream primer (SEQ ID No. 9): 5'-TGAAAGCGCTGGTAGATTCGGG-3'

Downstream primer (SEQ ID No. 10): 5'-TCATTGTTGGTGGGGCTTCCAG-3'

AtUBQ1

Upstream primer (SEQ ID No. 11): 5'-AGATCCAGGACAAGGAAGGTATTC-3'

Downstream primer (SEQ ID No. 12): 5'-CGCAGGACCAAGTGAAGAGTAG-3'

And (3) preparing a qRT-PCR reaction system on ice, and carrying out fluorescent quantitative PCR reaction.

The PCR reaction system is as follows:

PCR reaction procedure:

analysis of melting curves

The results show that:

(1)T₁PCR detection result of generation transgenic arabidopsis line

By extracting T₁The DNA of different strains of transgenic Arabidopsis thaliana is subjected to PCR amplification by using specific primers to identify a plurality of positive strains, wherein 2 positive strains are selected, and the PCR identification result is shown in figure 2.

(2)T₃Fluorescent quantitative identification result of generation transgenic arabidopsis line

By extracting T₃RNA of a transgenic arabidopsis line is subjected to reverse transcription, and then fluorescence quantification is performed by using a specific fluorescence quantification primer of the GhACO gene, and the result shows that the expression level of the GhACO gene of the transgenic line is remarkably increased relative to a control (WT) (figure 3).

(3) Phenotypic identification and statistics of T3 generation transgenic Arabidopsis lines

Transgenic T3 generation plants and non-transgenic plants are planted and cultivated under the same condition, the change of the phenotype of the whole arabidopsis plant is observed, the number of rosette leaves is reduced to different degrees compared with the wild type when the transgenic arabidopsis is bolting (table 1), and the flowering time is obviously advanced (figure 4).

TABLE 1 Lotus throne number in bolting

Note: WT is non-transgenic Arabidopsis, P <0.01, P <0.001

The results also prove that the GhACO gene can play a key role in promoting cotton flowering and can be used as a favorable gene resource for short-season cotton cultivation.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Application of cotton GhACO gene in promoting plant flowering

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165 170 175

Gln Glu Leu Pro Ala Ile Cys Arg Asp Ile Met Ile Glu Tyr Ser Ser

180 185 190

Lys Met Met Lys Leu Gly Gln Thr Leu Leu Glu Leu Met Ser Glu Ala

195 200 205

Leu Gly Leu Asn Arg Ser Tyr Leu Glu Asp Ile Gly Cys Gly Glu Gly

210 215 220

Met Phe Val Lys Gly His Tyr Tyr Pro Pro Cys Pro Glu Pro Asp Leu

225 230 235 240

Thr Leu Gly Thr Ser Arg His Thr Asp Thr Gly Phe Cys Thr Val Ile

245 250 255

Leu Gln Asp Glu Ile Gly Gly Leu Gln Ile Leu His Gln Asn Gln Trp

260 265 270

Leu Asp Ile Asn Pro Val Arg Gly Ala Leu Val Val Asn Leu Gly Asp

275 280 285

Met Met Gln Ala Ser Pro Pro

290 295

<210>5

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ctagtctaga atggcagaaa taagcttaga acg 33

<210>6

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

tcccccgggt taaggtgggc ttgcctgcat ca 32

<210>7

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

atggagatct cagtggagaa ga 22

<210>8

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ggccatcaaa ctccataacc aa 22

<210>9

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

tgaaagcgct ggtagattcg gg 22

<210>10

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

tcattgttgg tggggcttcc ag 22

<210>11

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

agatccagga caaggaaggt attc 24

<210>12

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

cgcaggacca agtgaagagt ag 22

Claims (10)

1. Separated cottonGhACOA gene characterized in thatGhACOThe gene comprises a nucleotide sequence shown as SEQ ID number 3.

2. Cotton according to claim 1GhACOThe gene is characterized in that the polypeptide coded by the gene comprises an amino acid sequence shown as SEQID number 4.

3. The method of claim 1GhACOThe application of the gene in promoting plant flowering is disclosed.

4. Use according to claim 3, characterized in that: increase in plantsGhACOExpression level of gene to promote plantAnd (4) flowering.

5. Use according to claim 4, wherein said increase is in plantsGhACOThe expression level of the gene is realized by the following method: enhancement of endogenesis in plantsGhACOExpression of genes, or overexpression of foreign sources in plantsGhACOA gene.

6. The use of claim 5, wherein the overexpression exogenous sourceGhACOThe gene refers toGhACOThe gene is transferred into the plant for expression by agrobacterium mediation by using the plant expression vector.

7. Use according to claim 6, characterized in that saidGhACOThe gene is introduced into a plant cell, tissue or organ by a plant expression vector.

8. Use according to claim 7, wherein said plant expression vector drives said plant expression vector through a constitutive or inducible promoterGhACOExpression of the gene.

9. Use according to claim 8, wherein the constitutive promoter is the 35S promoter.

10. Use according to any one of claims 3 to 9, wherein the plant is cotton, maize, rice, wheat or Arabidopsis thaliana.

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