CN117230083A

CN117230083A - Application of over-expression GhVOZ1 gene in promotion of cotton flowering

Info

Publication number: CN117230083A
Application number: CN202311150374.7A
Authority: CN
Inventors: 杨永林; 连卜颍; 马亮; 魏恒玲; 王寒涛; 付小康; 芦建华; 喻晓云
Original assignee: Shihezi Institute Of Agricultural Sciences; Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Shihezi Institute Of Agricultural Sciences; Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2023-09-07
Filing date: 2023-09-07
Publication date: 2023-12-15

Abstract

The invention provides application of over-expressed GhVOZ1 gene in promoting cotton flowering, and belongs to the technical field of genetic engineering. The invention analyzes the expression pattern of GhVOZ1 gene in flower buds of early-late maturing variety 1-5 leaf period, and the result shows that the expression quantity of the gene in 5 periods of early maturing variety is obviously higher than that of late maturing variety. In order to further study the influence of the gene on the flowering period, the GhVOZ1 gene is cloned from upland cotton and is constructed into an over-expression vector to be transformed into Arabidopsis thaliana, and compared with the wild type over-expression strain, the gene is early bolting and flowering for more than 4 days, so that the GhVOZ1 gene plays a positive regulation role on the cotton precocity.

Description

Application of over-expression GhVOZ1 gene in promotion of cotton flowering

Technical Field

The invention relates to the field of genetic engineering, in particular to application of an over-expressed GhVOZ1 gene in promoting cotton flowering.

Background

Upland cotton (Gossypium hirsutum l.) is the most important fiber crop in the world. In the yellow river basin and the Yangtze river basin cotton areas of China, the effect that the early maturing cotton is directly sown after wheat or rape can be achieved, and the reseeding index can be improved. In the northwest cotton field, the air temperature is low in spring, the frost is early in autumn, and the pre-frost flower rate of cotton can be improved and the quality of cotton can be improved. Flower bud differentiation is a sign of the transition of plants from vegetative to reproductive growth. When the reproductive growth is entered, the lateral buds become flower buds, and the flower buds develop into fruit branches. Fruit branch differentiation determines flowering capacity, reproductive capacity and cotton yield. Therefore, the early maturing property and the early maturing cotton variety are particularly important in production.

Flower bud differentiation is an important property affecting early maturity of short-season cotton varieties, and is the basis of cotton bud stage, flowering stage and bell stage development. Flower bud differentiation directly affects flowering time. Flower bud differentiation is a sign of the transition of plants from vegetative to reproductive growth. When the reproductive growth is entered, the lateral buds become flower buds, and the flower buds develop into fruit branches. Differentiation of fruit branches determines flowering capacity, sexual capacity and cotton yield.

To explain the genetic basis of cotton precocity-related traits, a number of genetic linkage maps have been constructed. The results show that precocity is a complex quantitative trait consisting of fertility stage (including sowing, budding, flowering and boll-making stages), pre-frost flower rate (YPBF), first fruit branch position (NFFB) and first fruit branch position Height (HNFFB). Because of this complexity, map-based clones were not isolated from cotton to genes associated with flowering. To date, only a few genes have been cloned reverse genetics and have initially demonstrated that they may control cotton flowering, such as GhFPF1 transgenic Arabidopsis longer than wild-type petioles, lower chlorophyll content, may be involved in plant "shade-avoidance" reactions, ghSOC1 overexpression significantly advances transgenic Arabidopsis flowering, and also some such as GhCEN-DT and GhAI 66.Cheng et al selected two early and two late maturing varieties, collected shoot tip samples before and after flower bud differentiation, and analyzed transcriptional dynamics during the seedling stage development stage. Find a key regulatory gene GhCAL for regulating the transformation from vegetative growth to reproductive growth of cotton.

In conclusion, flower bud differentiation is an important trait affecting early maturity of cotton varieties. The research on cotton bud differentiation is almost limited to the morphology, and key genes and control mechanisms thereof for controlling cotton bud differentiation are not clearly elucidated. Because of the difference in flower development characteristics and structure of upland cotton, we speculate that the molecular mechanism of regulating flower development is different from that of arabidopsis and other mode plants. However, there are few studies on molecular biology related to cotton flower organ development, and the regulatory mechanism is still unclear.

Disclosure of Invention

In order to solve the problems, the invention provides application of over-expressed GhVOZ1 genes in promoting cotton flowering, and the invention analyzes the expression pattern of the GhVOZ1 genes in flower buds of early and late maturing varieties in 1-5 leaf stages, and results show that the expression quantity of the genes in 5 stages of the early maturing varieties is obviously higher than that of the late maturing varieties. In order to further study the influence of the gene on the flowering period, the GhVOZ1 gene is cloned from upland cotton and is constructed into an over-expression vector to be transformed into Arabidopsis thaliana, and compared with the wild type over-expression strain, the gene is early bolting and flowering for more than 4 days, so that the GhVOZ1 gene plays a positive regulation role on the cotton precocity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of over-expressed GhVOZ1 gene in promoting cotton flowering.

The invention also provides application of the over-expression GhVOZ1 gene in promoting cotton reproductive growth.

Preferably, the nucleotide sequence of the GhVOZ1 gene is shown as SEQ ID No. 1.

Preferably, the amino acid sequence of the GhVOZ1 gene is shown as SEQ ID No. 2.

Preferably, the nucleotide sequence of an upstream primer for amplifying the GhVOZ1 gene is shown as SEQ ID No.3, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 4.

The invention has the beneficial effects that:

the invention analyzes the expression pattern of GhVOZ1 gene in flower buds of early-late maturing variety 1-5 leaf period, and the result shows that the expression quantity of the gene in 5 periods of early maturing variety is obviously higher than that of late maturing variety. In order to further study the influence of the gene on the flowering period, the GhVOZ1 gene is cloned from upland cotton and is constructed into an over-expression vector to be transformed into Arabidopsis thaliana, and compared with the wild type over-expression strain, the gene is early bolting and flowering for more than 4 days, so that the GhVOZ1 gene plays a positive regulation role on the cotton precocity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 shows the expression pattern of GhVOZ1 in flower buds of different periods of early and late maturing materials;

FIG. 2 is a flowering phenotype of Arabidopsis over-expressing GhVOZ 1;

FIG. 3 shows the bolting time of Arabidopsis thaliana over-expressing GhVOZ 1;

FIG. 4 shows flowering time of Arabidopsis over-expressing GhVOZ 1.

Detailed Description

In the invention, the nucleotide sequence of the GhVOZ1 gene is shown as SEQ ID No.1, and the nucleotide sequence is specifically shown as follows:

ATGGGGAAGGGTTCGAGAAGCAACTGCAAGTCTGCATCCCACAAACTCTTCAAGGACAGGGCAAAGAACCGGGTGGATGACCTGCAGGGGATGTTCTTGGATCTGCAATTTGCCAGGAAGGAGAGCCGCAGTGTCGATGTTGCTGTCCTTGAGGAGCAAGTCCATCAGATGCTCCGTGAATGGAAAGCAGAACTCAATGAACCTTCTCCAGCATCTTCATTGCAACAAGGAGGGAGTCTTGGTTCATTTTCATCAGACATTTGCCGGCTGCTGCAGCTTTGCGAGGAGGAAGATGATGCAACTAGTGTCCTAGCAGCTCCAAAGCCTGAGCCTGATGACCAAAATCTGCAAGTAGGAGATACTGCTGCTTTCCAAGAGGTTTATGGAGTGAATCAGGGGCAACATGAGCGTGGCTTTCCATTAGTTGATCATTGCAAAGACTCACCTTCAGGAGTCCGTACCATGCCAATTAACAACTTGGATGGAGCTACTCAATTGGAATACCACCAGTTTGATTTGCATCAGGATTTTGAGCACTTCTACACAGGTTTTAATGGTACTGGTTTCTCTGGGGAGGATGCCATGCTTCATACTTCTAGCTATCTGCCAAGTATATGCCTGCCACCATCTGCATTCCTAGGTCCAAAGTGTGCACTTTGGGATTGTCCAAGGCCAGCTCAAGCGTTGGACTGGTCTCAAGACTACTGTAGTAGCTTTCATGCTGCTTTAGCAATGAATGAAGGGCCACCTGGAATGGGCCCGATTCTGCGACCTGGTGGCATAGGCCTCAAGGATGGGCTGCTCTTTGCTGCTCTCAGTGCTAAGGCACAAGGAAAAGATGTGGGTATACCAGAATGCGAGGGGGCTGCAACTGCGAAGTCTCCATGGAATGCTCCTGAGCTCTTTGATCTTTCGGTTCTTGACGGTGAATCAATTAGGGAGTGGCTCTTTTTTGATAAGCCTCGAAGAGCATTTGAAAGTGGGAACAGGAAGCAGAGGTCTTTGCCAGATTACAGCGGGCGTGGCTGGCATGAGTCAAGGAAGCAAGTTATGAATGAATTTGGAGGACTGAAGAGATCATACTACATGGATCCTCAGCCACTGAACCATTTTGAGTGGCACCTTTATGAATATGAAATCAACAAGTGTGATGCTTGTGCCTTGTATAGGTTGGAGTTGAAGCTCATTGACGGCAAGAAAAGTGCAAAGGGGAAATCAGCTAACGATACAGTTGCTGATCTGCAGAAGCAGATGGGAAGGCTCACTGCAGAGTTCCCAACTGACAACAAACGCTATGTCAAAGGACGGGCAAAGATTAATGCAAAGGTCACTGTTGGAAATACTTATTCCACCCAAAATGCGGTGGCACCAACCAGTGAGAAGTTCGATTACGGGCATGGTTTGCAGTACGACTATCTCATCGACGATTTAAGTGGCTATTATCTAACATAG。

in the invention, the amino acid sequence of the GhVOZ1 gene is shown as SEQ ID No.2, and the specific steps are as follows:

MGKGSRSNCKSASHKLFKDRAKNRVDDLQGMFLDLQFARKESRSVDVAVLEEQVHQMLREWKAELNEPSPASSLQQGGSLGSFSSDICRLLQLCEEEDDATSVLAAPKPEPDDQNLQVGDTAAFQEVYGVNQGQHERGFPLVDHCKDSPSGVRTMPINNLDGATQLEYHQFDLHQDFEHFYTGFNGTGFSGEDAMLHTSSYLPSICLPPSAFLGPKCALWDCPRPAQALDWSQDYCSSFHAALAMNEGPPGMGPILRPGGIGLKDGLLFAALSAKAQGKDVGIPECEGAATAKSPWNAPELFDLSVLDGESIREWLFFDKPRRAFESGNRKQRSLPDYSGRGWHESRKQVMNEFGGLKRSYYMDPQPLNHFEWHLYEYEINKCDACALYRLELKLIDGKKSAKGKSANDTVADLQKQMGRLTAEFPTDNKRYVKGRAKINAKVTVGNTYSTQNAVAPTSEKFDYGHGLQYDYLIDDLSGYYLT。

in the invention, the nucleotide sequence of an upstream primer for amplifying the GhVOZ1 gene is shown as SEQ ID No.3, and the nucleotide sequence of a downstream primer is shown as SEQ ID No.4, and the specific steps are as follows:

SEQ ID No.3：GGTTTCTCTGGGGAGGATGC；

SEQ ID No.4：AGACCAGTCCAACGCTTGAG。

the present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.

Example 1

1. Test materials

1.1 Cotton Material

The materials used are upland cotton TM-1, cotton institute 50 in early maturing variety and cotton 11 in late maturing variety, wherein the cotton institute 50 and cotton 11 have extremely remarkable differences in flowering time and growth period (table 1), and the materials are planted in cotton institute test fields (white wall town of Anyang city in Henan province) of China academy of agricultural sciences, and the management measures are normal field management. The sampling mode is that flower buds of three cotton varieties from a one-leaf stage to a five-leaf stage are frozen by liquid nitrogen and then stored at-80 ℃ for RNA extraction.

Significance test of cotton institute 50 and Guoxin cotton 11 traits in Table 1

Traits (3)	Middle 50	Guoxin cotton 11	Differences in
				Growth period (Tian)	108	124	15 ^**
Flowering phase (Tian)	62	70	8 ^**

1.2 reagents and consumables

Restriction enzymes, related enzymes of a PCR reaction system, homologous recombination enzymes, gel recovery kits, cloning kits and plasmid small extract kits are purchased from Novamat biotechnology Co., ltd, fluorescent quantitative kits are purchased from century biological technology Co., ltd, and RNA extraction kits are purchased from Beijing Tiangen biochemical technology Co.

Other drugs: agarose is spanish original product, peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride and the like are domestic analytically pure, kanamycin and other Soilebao biological limited company, and escherichia coli competent cells DH5 alpha and agrobacterium competent cells are purchased from the Optimago biological company.

Culture medium: LB liquid medium: tryptone 10g/L, yeast extract 5g/L, chlorine NaCl

10g/L; LB solid medium: adding 15g/L of agar powder on the basis of a liquid culture medium; 1/2MS solid medium: 1/2MS22g/L, agar powder 8g/L and sucrose 30g/L.

The main instrument is as follows: PCR amplification apparatus (BIO-RAD), high-speed centrifuge (HettichMIKRO 200R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), fluorescent quantitative PCR apparatus (ABI 7500), electrothermal constant temperature incubator (Shanghai Senxin), constant temperature culture oscillator (Shanghai Zhi Cheng), artificial climate test chamber (Saifu), artificial climate chamber.

2. Test methods and results

2.1 bioinformatics analysis of cotton GhVOZ1

The CDS sequence and protein sequence of the GhVOZ1 (Gh_D05G1618) gene, which is 1452bp in CDS sequence and encodes 483 amino acids, was downloaded from the CottonFGD (http:// www.cottonfgd.org /) website, named GhVOZ1, and its function was studied.

GhVOZ1 CDS sequence (SEQ ID No. 1):

GhVOZ1 protein sequence is (SEQ ID No. 2):

2.2 analysis of expression pattern of 2GhVOZ1 in flower buds of early and late maturing variety

Because flower bud differentiation and cotton early-maturing characters are closely related, in order to further research whether the expression mode of the gene in the flower bud development period of the early-maturing variety and the late-maturing variety Guoxin cotton 11 are selected, flower bud RNA of one-leaf period to five-leaf period is extracted, and qRT-PCR is used for carrying out expression mode analysis.

2.2.1RNA extraction

(1) Homogenizing: 700 μl SL (β -mercaptoethanol was added before use) was added to 0.1g of the milled flower bud sample, and immediately vigorously shaken to mix the sample. Centrifuge at 12,000rpm for 2min.

(2) The supernatant was transferred to a filter column and centrifuged at 12,000rpm for 2min, and the supernatant was aspirated into a new RNase-Free centrifuge tube.

(3) Adding 0.4 times of absolute ethyl alcohol with the volume of supernatant, uniformly mixing, transferring the mixture into an adsorption column, centrifuging at 12,000rpm for 15sec, pouring out waste liquid in a collecting pipe, and placing the adsorption column into the collecting pipe.

(5) 350. Mu.l of deproteinized liquid RW1 was added to the adsorption column, centrifuged at 12,000rpm for 15sec, and the waste liquid in the collection tube was discarded, and the adsorption column was returned to the collection tube.

(6) Preparing DNase I working solution: mu.l DNase I stock solution and 70. Mu.l RDD solution were gently mixed.

(7) 80. Mu.l DNase I working solution was added to the column and allowed to stand at room temperature for 15min.

(8) 350 μl of deproteinized liquid RW1 was added, and centrifuged at 12,000rpm for 15sec, the waste liquid in the collection tube was discarded, and the adsorption column was returned to the collection tube.

(9) To the adsorption column, 500. Mu.l of a rinse liquid RW (ethanol was added before use) was added, and the mixture was centrifuged at 12,000rpm for 15sec, and the waste liquid in the collection tube was discarded, and the adsorption column was returned to the collection tube. The operation is repeated once.

(11) Centrifuging at 12,000rpm for 2min, placing the adsorption column into a new RNase-Free centrifuge tube, and suspending and dripping 30-50 μl RNase-Free ddH into the middle part of the adsorption membrane ₂ O, 2min at room temperature, centrifugation at 12,000rpm (13,400Xg) for 1min, to give an RNA solution, which was stored at-80 ℃.

2.2.2 Synthesis of reverse transcribed cDNA

Sample cDNA synthesis was performed using PrimeScript ^TM RT reagent Kit with gDNA Eraser kit (TaKaRa, japan). The test process mainly comprises two steps: (1) removal of genomic DNA (gDNA); (2) reverse transcription of RNA into single stranded cDNA.

(1) Removal of gDNA:

TABLE 2 reaction system

Reagent(s)	Dosage of
		5×gDNA Eraser Buffer	2.0μl
gDNA Eraser	1.0μl
		Total RNA	1μg
RNase Free dH ₂ O	up to 10μl

Placing the prepared system at room temperature for 5-10min, and placing on ice for use.

(2) cDNA Single Strand Synthesis

The system prepared in the following table is set at 37 ℃ for 30min;85 ℃, and 5s after reaction. The cDNA can be stored for a long period of time after being placed at-20deg.C.

Table 3 System

2.2.3 fluorescent quantitative PCR

(1) Specific primers of GhVOZ1 gene are designed, and GhACtin is used as an internal reference gene.

Table 4 primers

(2) Fluorescent quantitative PCR

Completed using UltraSYBR Mixture (Low ROX) kit (well known as century, china) and Applied Biosystems 7500 instruments. The specific process is as follows:

1) Diluting the cDNA stock solution by 10 times;

2) The reaction system:

table 5 System

Fluorescent quantitative PCR was performed using Applied Biosystems 7500: the PCR procedure was set up according to the two-step method: 95 ℃ for 10min;95 ℃ for 5s; at 60 ℃,34s (fluorescence signal is collected), these two steps set 40 cycles; dissolution profile: 95 ℃ for 15s;60 ℃ for 20s;95℃for 15s.

2.2.4 analysis of results

GhVOZ1 in the cotton institute 50 (CCRI-50) and Guoxin Mian 11 (Guoxin Mian-1)1) The relative expression quantity of two materials in flower bud differentiation period is utilized to 2 ^-△△Ct And (5) calculating a method. As can be seen from FIG. 1, the expression level of GhVOZ1 gene in 5 periods from one-leaf period to five-leaf period in the early-maturing cotton variety CCRI-50 is obviously higher than that of the late-maturing cotton variety Guoxin cotton 11, which indicates that the gene is possibly related to the early-maturing property of cotton.

Cloning of 3GhVOZ1 Gene and construction of PBI121-GhVOZ1 plant expression vector

2.3.1GhVOZ1 Gene cloning primer design

In order to amplify the whole length of the coding region of the gene and add a specific enzyme cutting site, primers containing the proper enzyme cutting site are respectively designed at an initiation codon and a termination codon according to the CDS sequence of GhVOZ 1. The cleavage sites used were XbaI and SacI.

The primer sequences of the GhVOZ1 cleavage site are as follows:

table 6 primers

Primer name	Primer sequence (5 'to 3')
		GhVOZ1-F	SEQ ID No.7CACGGGGGACTCTAGAATGGGGAAGGGTTCGAGAAGC
GhVOZ1-R	SEQ ID No.8GATCGGGGAAATTCGAGCTCCTATGTTAGATAATAGCCACTTAAATCG

2.3.2PCR cloning of GhVOZ1 Gene

(1) PCR reaction system

According to PrimeSTAR GXL DNA polymerase instructions, the PCR reaction system is as follows:

table 7 System

Reagent name	Dosage of reagent
		5×PrimeSTAR GXL Buffer	10μl
dNTP Mixture	4μl
		Primer F (10. Mu.M)	2μl
Primer R (10. Mu.M)	2μl
		TM-1cDNA	2μl
ddH ₂ O	Up to 50μl

(2) PCR reaction procedure:

(3) Detection of PCR products

Mu.l of the PCR product was taken, 3. Mu.l of 6×Loading Buffer was added, mixed well, spotted on 1% agarose gel, and the band size was checked by electrophoresis for about 1452 bp.

(4) PCR product purification

The product purification kit (Vazyme, DC 301) was used as follows:

1) Rapidly cutting gel containing target DNA fragment under ultraviolet lamp, weighing gel, wherein 100mg gel is equivalent to 100 μl volume, and taking the gel volume as one gel volume;

2) An equal volume of Buffer GDP was added. Water bath at 50-55 deg.c until the gel is dissolved completely;

3) The adsorption column was placed in a collection tube, and 700. Mu.l or less of the sol was transferred to the adsorption column, and centrifuged at 12,000Xg for 30-60sec.

4) The filtrate was discarded and the column was placed in a collection tube. Add 300 μl Buffer GDP to the column. Standing for 1min. Centrifuge at 12,000Xg for 30-60sec.

5) The filtrate was discarded and the column was placed in a collection tube. 700 μl Buffer GW (absolute ethanol added) was added to the column. Centrifuge at 12,000Xg for 30-60sec.

6) And (5) repeating the step 5.

7) The filtrate was discarded and the column was placed in a collection tube. Centrifuge at 12,000Xg for 2min.

8) The column was placed in a 1.5ml centrifuge tube, 20-30. Mu.l of sterilized water was added to the center of the column, and the column was left for 2min. Centrifuge at 12,000Xg for 1min. The column was discarded and the DNA was stored at-20 ℃.

Construction of 2.3.3PBI121-GhVOZ1 plant expression vector

(1) Double enzyme digestion and glue recovery of PBI121 plasmid

The PBI121 plasmid was digested with XbaI and SacI, and the digested product of the PBI121 vector was purified by agarose gel electrophoresis. The enzyme digestion reaction system is as follows:

table 8 System

Reagent name	Dosage of reagent
		XbaI	1μl
SacI	1μl
		Cut Smart	5μl
PBI121 plasmid	1μg
		ddH ₂ O	Up to 50μl

(2) Ligation of PCR gel recovery product and restriction enzyme digestion PBI121 plasmid

Use of Vazyme homologous recombinase reagentsOne Step Cloning Kit carrying out a ligation reaction:

table 9 System

Reagent name	Dosage of reagent
		5XCE Ⅱ Buffer	2μl
Exnase Ⅱ	1μl
		PBI121 double enzyme cutting carrier	25～100ng
PCR fragment	10～100ng
		ddH ₂ O	Up to 10μl

After the system is completed, the components are blown and evenly mixed, and the reaction is carried out for 30min at 37 ℃.

(3) Ligation product transformation of E.coli

1) Adding 100ul of escherichia coli DH5 alpha competent ligation reaction system, adding ligation reaction product into the ligation reaction system, and ice-bathing for 25min;

2) Heat shock in 42 ℃ water bath for 45s;

3) Ice bath for 2min; 700ul of non-resistant LB liquid medium is added, and the mixture is incubated for 1h at 37 ℃ and 200 rpm;

4) Centrifuging at 5000rpm for 1min, leaving about 100ul of supernatant, mixing, and coating on LB plate containing kana resistance;

5) Culturing at 37 deg.C overnight;

(4) Detection and sequencing of Positive clones

1) Selecting a monoclonal from the transformation plate, putting the monoclonal into a liquid LB culture medium containing Kan, and carrying out shaking culture at a constant temperature of 37 ℃ for 8 hours;

2) Colony PCR verifies positive clones and the correct monoclonal is sent to Shang Ya biotechnology limited for sequencing, 3 replicates per sequence.

(5) Preservation of positive bacterial liquid

And (3) performing PCR verification on bacterial liquid, and adding glycerol into bacterial liquid with correct sequencing until the final concentration is more than 20%, and preserving at-80 ℃. The correctly sequenced plasmid was returned for transformation of Agrobacterium.

(6) Transformation of Agrobacterium

Transformation of Agrobacterium tumefaciens GV3101 competent cells by freeze thawing:

1) The agrobacterium is thawed at-80 ℃ and the ice water is inserted into the ice in a mixed state.

2) Mu.l of competent plasmid DNA was added to 1. Mu.g, and the mixture was stirred by hand to the bottom of the tube, and then allowed to stand on ice for 5min, liquid nitrogen for 5min, and ice-bath for 5min at 37 ℃.

3) Adding 700ul of non-resistant LB liquid medium, and shake culturing at 28deg.C for 2-3 hr

4) 100-150ul of bacterial liquid is placed on an LB plate containing kana and rifampicin, and is placed in an incubator at 28 ℃ for 2-3 days in an inverted manner.

5) Positive clones are selected, cultured for 48 hours at 28 ℃ on LB liquid culture medium with resistance, and the bacterial liquid is preserved for standby at-80 ℃ after the bacterial liquid with correct strips is verified by PCR and glycerol is added.

3.3.4 Agrobacterium-mediated transformation of Arabidopsis thaliana

(1) Arabidopsis thaliana culture

And planting Columbia wild type Arabidopsis thaliana in a phytotron, growing to a full bloom stage, and cutting off pod which is already fruiting.

(2) Transformation of Arabidopsis inflorescence infection

1) Activating bacterial liquid: inoculating 20 μl of Agrobacterium solution stored at-80deg.C into 1ml LB liquid culture medium (corresponding antibiotics: kanamicin and rifampin are added), culturing at 28deg.C and 180rpm for 14-18 hr;

2) Expanding and shaking: adding 500 μl of activated bacterial liquid into 50ml LB liquid culture medium containing corresponding antibiotics, culturing at 28deg.C and 180rpm until bacterial liquid OD ₆₀₀ The value is about 0.8-1.2 (about 18-20 h), 5000rpm, centrifuging for 10min, discarding supernatant, and collecting thallus;

3) Preparation of infection transformation medium: 1/2MS halving, 5% sucrose, 0.02% Silwet L-77, pH adjusted to 5.6-5.7 with NaOH, 0.1mM AS (acetosyringone);

4) Resuspension of the cells with transformation medium, OD ₆₀₀ Adjusting to 0.6-0.8;

5) Dip dyeing: placing the arabidopsis inflorescence into a transformation medium for 60s, and culturing for 24h under the condition of weak light or light shading after dip dyeing;

6) Placing the treated arabidopsis thaliana under normal conditions for culture, and carrying out secondary infection by using the same method after one week;

7) After maturation, the Arabidopsis seeds are harvested, namely the transgenic T ₀ Seed generation.

3.3.5 phenotypic characterization of transgenic Arabidopsis plants

(1) The harvested seeds are planted on 1/2MS containing kanamycin after sterilization, vernalization is carried out at 4 ℃ for 2 days, the seeds are transferred into a manual climate test box, positive plants grow normally about 10 days, and negative plant leaves turn yellow and do not grow any more.

(2) Transplanting the positive arabidopsis plants into nutrient soil, extracting DNA after growing for one month, and detecting the positive plants by PCR, wherein the primers used in the detection are as follows:

table 10 primers

Primer name	Primer sequence (5 'to 3')
		35S	SEQ ID No.9GACGCACAATCCCACTATCC
GhVOZ1-R	SEQ ID No.10TCAACTCAGCTCCTGAGTTA

(3) Breeding to T ₃ And (3) generating, namely obtaining the homozygous transgenic arabidopsis strain.

(4) Will T ₃ Planting the generation plant and wild plant (WT) in nutrient soil, transferring the seedling of Arabidopsis into flowerpot for growing after about 10 days, planting under the same condition, and phenotypic observation to find GhVOZ1 transgenic ArabidopsisThe mustard flower was significantly earlier than the wild type (FIGS. 2-4); the data statistics result shows that the bolting days and the flowering days of the GhVOZ1 transgenic arabidopsis are both earlier than that of the wild type arabidopsis for more than 4 days, and the difference is very obvious. The result shows that the overexpression of GhVOZ1 obviously promotes the flowering and reproductive growth and development of the arabidopsis.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. The application of over-expressed GhVOZ1 gene in promoting cotton flowering.

2. The application of the over-expressed GhVOZ1 gene in promoting cotton reproductive growth.

3. The use according to claim 1 or 2, wherein the nucleotide sequence of the GhVOZ1 gene is shown in SEQ ID No. 1.

4. The use according to claim 3, wherein the amino acid sequence of the GhVOZ1 gene is shown in SEQ ID No. 2.

5. The use according to claim 3, wherein the nucleotide sequence of the upstream primer for amplifying the GhVOZ1 gene is shown in SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4.