CN110117321B - Application of cotton GhDctpp1-D11 gene in promoting plant flowering - Google Patents

Abstract

The invention discloses application of cotton GhDctpp1-D11 gene in promoting plant flowering, and belongs to the technical field of plant genetic engineering. The GhDctpp1-D11 gene has a nucleotide sequence shown in SEQ ID NO. 1 and can encode an amino acid sequence shown in SEQ ID NO. 2. The invention provides favorable gene resources for the cultivation of short-season cotton.

Description

Application of cotton GhDctpp1-D11 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 GhDctpp1-D11 gene in promoting plant flowering.

Background

The growth and development process of flowering plants comprises two stages of vegetative growth and reproductive growth. Vegetative growth is mainly focused on root, leaf and stem development, while reproductive growth includes flowering and fruit formation and ripening. Thus, flower formation is an important process for the transformation of flowering plants from vegetative to reproductive growth, which is influenced by the genetic control of the plants themselves and by external factors.

Cultivated species upland cotton is a cotton species which is insensitive to illumination, and becomes less and less sensitive to illumination after a long-term evolution process. In China, along with the increasing reduction of the cultivated land area, the contradiction of land competition between grains and cotton is gradually highlighted, and the cultivation of short-season cotton varieties becomes an important target of breeding.

Disclosure of Invention

The inventor clones a cotton GhDctpp1-D11 gene from upland cotton, is homologous with AtDctpp1 in Arabidopsis thaliana, is DCTP pyrophosphatase 1, and has few researches on plant flowering. The expression pattern shows that the expression level of the strain is dominantly expressed in terminal buds of cotton seedlings, the expression level is increased in the trefoil stage (flower bud differentiation stage) of cotton, and the expression level of an early-maturing variety is remarkably higher than that of a late-maturing variety, and the result shows that GhDctpp1-D11 possibly plays an important role in flower bud differentiation, growth stage conversion and flower organ formation; the function of GhDctpp1-D11 in cotton was further verified using virus-induced gene silencing (VIGS) technology, and it was observed that silenced plants appeared to flower significantly later than CK grown under the same growth conditions. Therefore, GhDctpp1-D11 is considered to have 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 application of a GhDctpp1-D11 gene in improving and promoting plant flowering, wherein the GhDctpp1-D11 gene has a nucleotide sequence shown in SEQ ID NO. 1.

The open reading frame of the GhDctpp1-D11 gene is 465 bp.

In some embodiments of the invention, the nucleotide sequence set forth in SEQ ID NO. 1 is capable of encoding the amino acid sequence set forth in SEQ ID NO. 2. The protein comprising the amino acid sequence has a relative molecular weight of 14.45kDa and an isoelectric point of 5.40.

In some embodiments of the invention, the expression level of the GhDctpp1-D11 gene is increased in a plant to promote flowering in the plant.

In some embodiments of the present invention, the increasing of the expression level of the GhDctpp1-D11 gene in the plant is achieved by: improving the expression of the plant endogenous GhDctpp1-D11 gene, or over-expressing the exogenous GhDctpp1-D11 gene in the plant.

In a specific required embodiment of the invention, the overexpression of the exogenous GhDctpp1-D11 gene refers to the expression of the GhDctpp1-D11 gene in a plant through agrobacterium-mediated transformation by using a plant expression vector.

Further, the GhDctpp1-D11 gene is introduced into a plant cell, tissue or organ through a plant expression vector.

Further, the plant expression vector drives the expression of the GhDctpp1-D11 gene by 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

According to the invention, by silencing the GhDctpp1-D11 gene in cotton, the result shows that the GhDctpp1-D11 gene may have a key role in promoting cotton flowering. The invention provides favorable gene resources for the cultivation of short-season cotton.

Drawings

FIG. 1 shows the results of gene amplification gel electrophoresis. Lanes 1 and 2 show the target fragment of GhDctpp1-D11 PCR, 485bp in size, and lane M MARKER III.

FIG. 2A shows the expression levels of GhDctpp1-D11 in different fertile phase materials; FIG. 2B shows the expression levels of GhDctpp1-D11 in different tissues.

FIG. 3 shows virus-induced GhDctpp1-D11 gene silencing and expression analysis.

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.

Example 1

1. Cotton material

The cotton materials selected in this example are upland cotton 50, China Cotton institute 74, Kongxin Cotton 11 and Bohai Cotton 1, and are planted in the national key laboratory test field (Anyang white wall) of the Cotton institute of Cotton, academy of agricultural sciences, and the management measure is normal field management.

2. Reagent and consumable

2.1 enzymes and kits:

GXL DNA Polymerase high-fidelity enzyme, gel recovery kit and PCR product purification kit were purchased from Takara corporation; RNA reverse transcription kit, KOD FX Neo enzyme (code. No. KFX-201) purchased from Toyobo company;

the Ultra One Step Cloning Kit was purchased from Vazyme; the plasmid small quantity extraction kit is purchased from magenta company; restriction enzymes (BamH I, Sac I) were purchased from NEB; the DNA Marker III and the plant total RNA extraction kit are purchased from TIANGEN company; fluorogenic quantitative TransStart Top Green qPCR Supermix was purchased from TransGen.

2.2 other drugs: agarose is Spanish original product, peptone, yeast extract, chloroform, isoamylol, ethanol, isopropanol, sodium chloride, sucrose, silwet L-77, phloroglucinol and the like are domestic analytical purities, kanamycin, streptomycin sulfate, ampicillin and the like are purchased from Botryobo bioengineering (Dalian) Co., Ltd., Escherichia coli competent cell Trans5 alpha is purchased from Beijing holotype gold biotechnology Co., Ltd., and Agrobacterium competent cell LBA4404 is purchased from Shanghai Weiji Biotechnology Co., Ltd.

2.3 culture Medium: LB liquid medium: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), 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 is spread flat, adding antibiotic with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 ℃, shaking up and spreading flat. The reagent solutions mentioned but not listed herein were prepared according to the method of molecular cloning, the third edition of the Experimental guidelines, and the biochemical reagents were analytically pure or of the above grade.

2.4 Main instruments: PCR amplification instrument (Eppendorf), high-speed centrifuge (Eppendorf 5427R), electrophoresis equipment (Hexagon, Beijing), gel imaging system (BIO-RAD), fluorescence quantitative PCR instrument (ABI7500), fluorescence microscope (Olympus BX43), constant temperature culture oscillator (Shanghai Zhicheng), artificial climate test box (Saifu) and the like.

Experimental methods and results

Bioinformatics analysis and cloning of 1 cotton GhDctpp1-D11 gene

1.1 obtaining GhDctpp1-D11 gene sequence from NCBI, designing Primer by using Primer Premier 5.0 software, amplifying from upland cotton 50 by using PCR (polymerase Chain reaction) method, wherein the open reading frame is 465bp, 154 amino acids are coded, the relative molecular weight of protein is 14.45kDa, and the isoelectric point is 5.40. The sequence of the gene cds is (SEQ ID NO: 1):

ATGACTGGGGGAGTTGAGGAACGTGATCAAAGCGTGAGTCTCGATCTTCTTAAGCAGAAAATGGCTAACTTTGCCAAAGAACGGGACTGGGATCAGTTTCACAGCCCTAGAAATCTCCTTTTGGCCCTGGTGGGTGAAGTGGGAGAATTGTCGGAGATATTTCAGTGGAAAGGGGAGGTCCCAAAAGGGTTGCCTGATTGGAAAGAAGAAGAAAAAGTTCACCTTGGTGAAGAACTCTCAGATGTTCTACTCTACCTTGTAAGGCTGTCTGATATATGTGGCATTGATCTGGGCAAAGCTGCTCTGCGCAAAGTTGAACTTAATGCCATCAAGTATCCAGCTTCAAAGAAAAACTTCAATACCAGCAATGGCACTGCACACACTGGTACCACTGCCGTGGAGCGCACAAAAACAGTGGTTCATCCCTTTAACGTCCAATCAGTTGCAGCACCTAAGCCTATTTAG

the amino acid sequence is (SEQ ID NO: 2):

MTGGVEERDQSVSLDLLKQKMANFAKERDWDQFHSPRNLLLALVGEVGELSEIFQWKGEVPKGLPDWKEEEKVHLGEELSDVLLYLVRLSDICGIDLGKAALRKVELNAIKYPASKKNFNTSNGTAHTGTTAVERTKTVVHPFNVQSVAAPKPI

1.2 the process of cloning genes specifically is:

(1) 50 of early-maturing varieties of test materials upland cotton, 74 of middle cotton institute, national Xin cotton No. 11 of late-maturing varieties and Bohai cotton No. 1 of the test materials are planted in an Anyang test base of a cotton research institute of Chinese academy of agricultural sciences, and the test materials are managed according to a common field. Sampling is carried out by taking cotton and a seedling tip from the flattening of cotyledon, sampling each flattened true leaf once, repeating three biology procedures every time, quickly freezing the obtained material in liquid nitrogen, and storing in a refrigerator at-80 ℃ for later use. The flat mark of the sub-leaf is 0TLS (0true-leaf stage), the flat mark of a true leaf is 1TLS (1true-leaf stage), and the analogy is carried out until the fifth true leaf is completely unfolded. The total RNA of the plants is extracted by a TIANGEN reagent kit.

(2) The RNA extraction steps are as follows:

1) and (3) homogenizing treatment: taking a proper amount of fiber sample, quickly grinding the fiber sample into powder in liquid nitrogen, adding 700 mu L SL (adding beta-mercaptoethanol before use), and immediately and violently shaking the mixture to uniformly mix the sample;

2) centrifuging at 12,000rpm for 2 min;

3) transferring the supernatant to a filtration column CS, centrifuging at 12,000rpm for 2min, carefully sucking the supernatant in a collecting tube into a new RNase-Free centrifugal tube, and preventing a suction head from contacting cell fragments in the collecting tube;

4) adding 0.4 times of anhydrous ethanol, mixing, transferring the mixture into adsorption column CR3, centrifuging at 12,000rpm for 15sec, removing waste liquid, and returning the adsorption column CR3 to the collection tube;

5) adding 350 μ L deproteinizing solution RW1 into adsorption column CR3, centrifuging at 12,000rpm for 15sec, pouring off waste liquid in the collection tube, and returning adsorption column CR3 to the collection tube;

6) DNase I working solution: mixing 10 μ L DNase I stock solution and 70 μ L RDD solution gently;

7) adding 80 mu L of DNase I working solution into CR3, and standing at room temperature for 15 min;

8) after standing, 350 μ L deproteinized 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) adding 500 μ L of rinsing solution RW (ethanol before use) into adsorption column CR3, centrifuging at 12,000rpm for 15sec, discarding waste liquid in the collection tube, and returning adsorption column CR3 to the collection tube;

10) repeating the step 9;

11) centrifuging at 12,000rpm (13,400 Xg) for 2min, placing the adsorption column CR3 into a new RNase-Free centrifuge tube, suspending and dripping 30-50 μ L of RNase-Free ddH2O into the middle part of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12,000rpm (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.

(3) And (3) synthesizing cDNA. And (2) carrying out reverse transcription on 500ng of RNA into cDNA by adopting a reverse transcription kit FSQ-201 of Toyobo, wherein a reverse transcription system is as follows:

RT reaction liquid is prepared according to the following components (the reaction liquid is prepared on ice):

the reverse transcription reaction conditions were as follows:

15min at 37 ℃ (reverse transcription reaction),

5s at 98 ℃ (inactivation reaction of reverse transcriptase);

the reverse transcription product cDNA solution was diluted 6-fold as a template for PCR reaction.

(4) PCR amplification of target genes

The following system was prepared on ice, and the desired gene GhDctpp1-D11 was amplified using TM-1 cDNA as a template. According to TaKaRa

GXL DNA Polymerase high-fidelity enzyme instruction, and a PCR reaction system is as follows:

the PCR amplification procedure was:

the primer sequence is as follows:

GhDctpp1-D11-F：5′-ATGACTGGGGGAGTTGAGGAA-3′(SEQ ID NO:3)

GhDctpp1-D11-R：5′-CTAAATAGGCTTAGGTGCTGC-3′(SEQ ID NO:4)

after the reaction is finished, the reaction is stored at 4 ℃, and the detection is carried out by using 1% agarose electrophoresis, and the effective result is considered when the size of a band is in accordance with the expected design (as shown in figure 1).

(5) And (3) cutting and recovering the target fragment by using a gel recovery kit.

(6) The product recovered from the above gum is

The Ultra One Step Cloning Kit is used for connecting T vectors and transforming Escherichia coli.

(7) Overnight culture at 37 ℃ Single clones were picked from the resistant LB medium and cultured with shaking at 37 ℃.

(8) And (3) carrying out PCR verification on bacterial liquid, selecting a positive clone sample, sending the sample to Jinzhi biotechnology limited for sequencing, and adding a certain amount of glycerol into the bacterial liquid with correct sequencing to ensure that the final concentration of the glycerol is about 20 percent and storing the glycerol at-70 ℃.

Construction and infection of cotton VIGS vector of 2GhDctpp1-D11 gene

Virus-induced gene silencing (VIGS) is a RNA-mediated post-transcriptional gene silencing that is generated by a plant's defense mechanism against viruses. A fragment with the length of 322bp of a target gene GhDctpp1-D11 is connected into a shuttle plasmid pCLCrV, a vector (pCLCrV-GhDctpp1-D11) is constructed, and Escherichia Coli (Escherichia Coli) is transformed, monoclone is selected, and the sample is sent for sequencing (Jinzhi, Suzhou). And (4) sequencing successfully, cloning, shaking and extracting the plasmid. A positive control vector (pCLCrV-VA), a negative control vector (pCLCrV), an auxiliary plasmid (pCLCrV-VB) and a constructed vector (pCLCrV-GhDctpp1-D11) containing a target fragment of the GhDctpp1-D11 gene are respectively transformed into an Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain LBA 4404. In a period when cotyledons are flattened but true leaves have not grown, the method of injecting and infecting cotton cotyledons with Agrobacterium involves a detailed procedure in Gao et al (2013) reports on cotton virus-induced gene silencing (Gao et al, Functional genetic analysis of cotton genes with an immobilized virus-induced gene cloning. methods Mol Biol (2013), 975157-65; Gu et al, A top system for Functional analysis of genes and microRNAs in cotton, Plant Biotechnology Journal (2014)12, pp.638-649, incorporated herein by reference in its entirety). And (3) carrying out light-shielding treatment on the infected cotton seedlings for 24h, extracting the total RNA of the cotton leaves after 40 days, and detecting the silencing condition of the genes by utilizing a qRT-PCR technology.

The amplification primers are as follows:

analysis of expression patterns of 3 GhDctpp1-D11 in different growth-stage materials

3.1 test materials of 50 in early-maturing varieties of upland cotton, 74 in middle cotton institute, and No. 1 Bohai cotton of late-maturing varieties, are planted in the Anyang test base of the cotton research institute of Chinese academy of agricultural sciences, and are managed according to a common field. Sampling is carried out by taking cotton and a seedling tip from the flattening of cotyledon, sampling each flattened true leaf once, repeating three biology procedures every time, quickly freezing the obtained material in liquid nitrogen, and storing in a refrigerator at-80 ℃ for later use. The flat mark of the sub-leaf is 0TLS (0true-leaf stage), the flat mark of a true leaf is 1TLS (1true-leaf stage), and the analogy is carried out until the fifth true leaf is completely unfolded.

3.2 taking the different samples, extracting RNA, performing reverse transcription to form cDNA, analyzing the expression level of GhDctpp1-D11 genes in different materials, taking GhACTin as an internal reference gene, and performing fluorescence quantitative PCR by using the following primers:

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

The qRT-PCR reaction system is as follows:

qRT-PCR reaction procedure:

the apical meristem of plants is similar to the stem cells of animals, and constantly differentiates various above-ground tissue organs of plants, and when the internal and external conditions are appropriate, the apical meristem differentiates flower buds. As can be seen from the results of qRT-PCR, the GhDctpp1-D11 gene is expressed predominantly in leaves, terminal buds and stamens, and the expression level is highest in stamens and whole flowers (FIG. 2, B). With the development of terminal buds, the expression level of the GhDctpp1-D11 gene in different growth period materials is in an increasing trend, and the expression level of 50 and SAZAZAO No. 2 in the two early-maturing varieties is obviously higher than that in the late-maturing materials from the third true leaf development period (figure 2, A). It shows that the GhDctpp1-D11 gene may be related to the bud differentiation of upland cotton and even the development of floral organs.

4 Virus-induced GhDctpp1-D11 Gene silencing

Cotyledons of cotton seedlings were injected with Agrobacterium solutions of positive control (pCLCrVA), negative control (pCLCrV) and viral vector of GhDctpp1-D11 (pCLCrV-GhDctpp1-D11), respectively. And detecting the GhDctpp1-D11 gene expression condition of the injected plants after four weeks of injection. As shown in FIG. 3, after infection with the virus vector containing pCLCrV-GhDctpp1-D11, the expression level of GhDctpp1-D11 gene was very significantly down-regulated compared to the empty vector control. Compared with an empty vector pCLCrVA plant, the pCLCrV-GhDctpp1-D11 plant with the GhDctpp1-D11 gene expression quantity remarkably reduced shows a remarkable late-flowering phenotype, and the plant type is loose and the plant height is high (figure 3, A). Research shows that GhDctpp1-D11 may have 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

<110> Cotton institute of agricultural sciences of China

Application of <120> cotton GhDctpp1-D11 gene in promoting plant flowering

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ttggccctgg tgggtgaagt gggagaattg tcggagatat ttcagtggaa aggggaggtc 180

ccaaaagggt tgcctgattg gaaagaagaa gaaaaagttc accttggtga agaactctca 240

gatgttctac tctaccttgt aaggctgtct gatatatgtg gcattgatct gggcaaagct 300

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Claims (6)

1. The application of the GhDctpp1-D11 gene in reducing the late flowering of a plant is characterized in that the nucleotide sequence of the GhDctpp1-D11 gene is shown in SEQ ID NO:1, and the plant is cotton.

2. The use according to claim 1, wherein the nucleotide sequence shown as SEQ ID NO. 1 is capable of encoding the amino acid sequence shown as SEQ ID NO. 2.

3. Use according to any one of claims 1 or 2, characterized in that: the expression level of the GhDctpp1-D11 gene is down-regulated in the plant so as to promote late flowering of the plant.

4. The use of claim 3, wherein the down-regulation of the expression level of the GhDctpp1-D11 gene in a plant is achieved by: the GhDctpp1-D11 gene post-transcriptional silencing is mediated by utilizing a virus-induced gene silencing technology.

5. The use of claim 4, wherein the mediating post-transcriptional silencing of the GhDctpp1-D11 gene is achieved by ligating a 322bp fragment of the GhDctpp1-D11 gene into a shuttle plasmid pCLCrV, constructing a vector pCLCrV-GhDctpp1-D11, and expressing the vector by Agrobacterium-mediated transformation into a plant.

6. The use according to claim 5, wherein the vector pCLCrV-GhDctpp1-D11 is transformed into a plant cell, tissue or organ.

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