CN113846105A

CN113846105A - Application of GhAIF3 gene in regulation of plant phenotype and method for regulating plant phenotype

Info

Publication number: CN113846105A
Application number: CN202110895187.6A
Authority: CN
Inventors: 王寒涛; 李梦宇; 喻树迅; 魏恒玲; 马亮; 付小康; 芦建华; 康萌
Original assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2021-12-28
Anticipated expiration: 2041-08-05
Also published as: CN113846105B

Abstract

The invention provides application of a GhAIF3 gene in regulation of plant phenotype and a method for regulating plant phenotype, and relates to the technical field of genetic engineering. The GhAIF3 gene provided by the invention is a valuable gene resource provided for cultivating new plant varieties with appropriate epidermal hair length, plant height, root length and fruit branch initial node position.

Description

Application of GhAIF3 gene in regulation of plant phenotype and method for regulating plant phenotype

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of a GhAIF3 gene in regulation of plant phenotype and a method for regulating the plant phenotype.

Background

Cotton is one of important economic crops and strategic substances all over the world, and plays a very important role in the national economic system of China. The elongation of the cells plays a considerable role in the whole growth and development stage of cotton, and particularly, the plant type of the cotton can be effectively regulated and controlled by regulating the elongation of the cells, the internode length of the main stem and the fruit branches of the cotton is controlled, the plant height is reduced, and the fruit branches are shortened.

The Basic helix-Loop-helix (Basic helix) transcription factor family is widely present in eukaryotes and is one of the largest regulatory protein families. In addition, researches show that the bHLH transcription factor plays an important role in biosynthesis and signal transduction, plant growth and development, adversity growth (stress such as low temperature, high salt, drought, iron deficiency, phosphorus deficiency and the like), and the like. Some studies have aimed at dividing the vast number of members of the bHLH family into smaller sub-groups based on sequence homology. Members of these subfamilies often have similar or overlapping functions. According to the bHLH subgroup described in the Carretero-Paule et al and the Buti et al reviews, the major subfamilies included are: the subfamilies PIF, BEE, BIM, PAR, AIF and PRE.

Studies have shown that members of the AIFs family are actively involved in plant growth and development. In BR-mediated organ elongation, AIF1 may be a negative regulator of ATBS1 of the BR signaling pathway. Overexpression of AIF1 in the double mutant of atbs1-D bri1-301 and in wild type plants using the 35S promoter both led to the dwarf phenotype of the plants. In both contexts, the severity of the dwarf phenotype correlates well with the expression level of p35S: AIF 1. In addition, in RNAi mediated gene silencing, although no obvious morphological change is observed in p35S AIF1i transgenic plants, which is probably caused by functional redundancy of 4 AIFs, in bri1-301, qRT-PCR analysis shows that the knockout part of AIF1 gene inhibits the bri1 weak mutant. In addition, there have been several studies that have shown that AIFs are also actively involved in stress response. In two independent studies by Covington et al, circadian rhythm regulator genes were selected for further screening of their promoter sequences for the presence of the CCA1 binding site ee (cbs). This analysis identified a series of defense-related genes that may be direct targets of CCA1/LHY and may be regulated by circadian rhythms, including AIF 1. AIF2 interacts with ICE1 through its C-terminus to form an AIF2-ICE1 complex to upregulate expression of CBFs negatively regulating dark-triggered BR-induced leaf senescence. Previous studies have shown that RITF1(AIF2) binds directly to elements in the RAS1 regulated gene promoter, forming a new calcium sensing and signaling pathway: the RSA1-RITF1 complex positively regulates the expression of a Na +/H + antiporter SOS1 located on the plasma membrane, which is important for gene regulation and salt stress tolerance.

A total of 16 GhAIFs were previously identified by whole genome identification and analysis of members of the AIFs subfamily of Gossypium hirsutum. The functional studies of the GhAIFS gene in cotton are not clear at present.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The main object of the present invention is to provide the use of the GhAIF3 gene in the regulation of plant phenotype to alleviate at least one of the technical problems of the prior art.

It is a second object of the present invention to provide a method for modulating a phenotype of a plant.

The invention provides application of GhAIF3 gene in regulation and control of plant phenotype, wherein the plant phenotype comprises one or more of leaf epidermal hair length, plant height, root length and fruit branch primary node position;

the GhAIF3 gene has a nucleotide sequence shown in SEQ ID NO. 3.

Further, the GhAIF3 gene expresses a protein having an amino acid sequence shown in SEQ ID NO. 4.

Further, the expression of the GhAIF3 gene in the plant is up-regulated, so that the epidermal hair length of the plant leaves is shortened, the plant is dwarfed and the root length is shortened.

Further, the expression of the GhAIF3 gene in the plant is reduced, so that the fruit branch node position of the plant is reduced.

Further, the plant includes arabidopsis and/or cotton.

In addition, the invention also provides a method for regulating the phenotype of the plant, which comprises the steps of regulating the expression level of the GhAIF3 gene in the plant;

the plant phenotype comprises one or more of leaf epidermal hair length, plant height, root length, and fruit branch inception position;

the GhAIF3 gene has a nucleotide sequence shown in SEQ ID NO. 3.

Further, the GhAIF3 gene in the plant is over-expressed, so that the epidermal hair length of the plant leaves is shortened, the plant is dwarfed and the root length is shortened;

preferably, the plant comprises arabidopsis thaliana.

Further, the GhAIF3 gene is constructed on an expression vector, then the obtained recombinant expression vector is used for transforming agrobacterium, the transformed agrobacterium is used for dip-dyeing arabidopsis inflorescence, and a positive transgenic plant is screened, so that the overexpression of the GhAIF3 gene is realized;

preferably, the expression vector comprises pBI 121.

Further, the expression of GhAIF3 gene in the plant is silenced, so that the fruit branch starting position of the plant is reduced;

preferably, the plant comprises cotton.

Further, constructing a GhAIF3 gene lentiviral interference vector, and injecting cotton seedlings with the GhAIF3 gene lentiviral interference vector to realize the silencing of the GhAIF3 gene;

preferably, the lentiviral interference vector for the GhAIF3 gene comprises CLCrVA-GhAIF 3.

Compared with the prior art, the invention has the following beneficial effects:

the inventor clones cotton GhAIIF 3 gene from upland cotton on the basis of AtAIFs function and main effect QTL qNFFB-D2-4 related to fruit branch initial node by the prior people, and finds that the leaf epidermal hair length of a plant over-expressing the gene is shorter, the plant is dwarf and the root length is shorter than that of a wild plant, which indicates that the gene possibly has an effect on cell elongation, and the gene can influence the plant height or root development in the plant. The position of the fruit branch primary node of the plant silencing the gene is reduced compared with that of a wild plant, which indicates that the gene can regulate and control the development of the fruit branch primary node of the plant. The GhAIF3 gene provided by the invention is a valuable gene resource provided for cultivating new plant varieties with appropriate epidermal hair length, plant height, root length and fruit branch initial node position.

Compared with the traditional method of applying exogenous substances such as auxin, cytokinin, abscisic acid and the like to plants, the method for regulating and controlling the plant phenotype provided by the invention has the advantages that the phenotype of the plants can be regulated and controlled more accurately by directly regulating and controlling the plant genes, and the regulation and control efficiency is high.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a structural diagram of an exon intron of the GhAIF3 gene provided in example 1 of the present invention;

FIG. 2 is a graph comparing the transcription levels of the GhAIF3 gene in transgenic plants and non-transgenic Arabidopsis as provided in example 5 of the present invention;

FIG. 3A is a comparison of the length of the coat hairs of the leaves of transgenic plants and non-transgenic plants according to example 5 of the present invention;

FIG. 3B is a comparison of flowering and plant height of transgenic and non-transgenic plants provided in example 5 of the present invention;

FIG. 3C is a graph comparing the compactness of rosette leaves of transgenic plants and non-transgenic plants provided in example 5 of the present invention;

FIG. 4A is a graph showing the results of expression levels of GhAIF3 gene silencing induced by viruses according to example 5 of the present invention;

FIG. 4B is a comparison of fruit branch start positions of GhAIF3 gene silencing induced by the virus provided in example 5 of the present invention.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the invention, the invention provides the use of the GhAIF3 gene in the modulation of plant phenotype including one or more of leaf epidermal hair length, plant height, root length and fruit branch inception position;

the GhAIF3 gene has a nucleotide sequence shown in SEQ ID NO. 3.

The expression "having" means that the nucleotide sequence of the GhAIF3 gene can be the nucleotide sequence shown in SEQ ID NO.3 only, or can be composed of the nucleotide sequence shown in SEQ ID NO.3 and other nucleotide sequences, such as a nucleotide sequence encoding functional units for protein purification, fluorescent protein markers, DNA binding sites and the like, or encoding elements having a regulating effect on gene transcription and expression, including but not limited to promoters, strong promoters, enhancers or transcription factor binding sites and the like; the "having" may also mean that the nucleotide sequence shown in SEQ ID NO.3 is discontinuous in the GhAIF3 gene, but cDNA of the nucleotide sequence shown in SEQ ID NO.3 can be produced.

In some alternative embodiments, the GhAIF3 gene expresses a protein comprising the amino acid sequence set forth in SEQ ID No. 4.

The protein containing the amino acid sequence shown in SEQ ID No.4 means that the amino acid sequence of the protein can be the whole amino acid sequence shown in SEQ ID No.4, and also can be composed of the amino acid sequence shown in SEQ ID No.4 and other amino acids, and the functions of other amino acids in the protein include, but are not limited to, the combination sites of a label for protein purification, a fluorescent protein marker and DNA, and the like, and in some specific examples, the protein can be, but is not limited to, HIS, GST, MyC, FLAG, HSV, V5, HA, GFP, RFP, BFP, CAT, DHFR, MBP, T7, thioredoxin, and the like.

The inventor clones cotton GhAIIF 3 gene from upland cotton on the basis of AtAIFs function and main effect QTL qNFFB-D2-4 related to the fruit branch initial node position of the prior person, and finds that the leaf epidermal hair length of a plant over-expressing the gene is shorter than that of a wild plant, the plant is dwarf and the root length is shorter, and the fruit branch initial node position of the plant silencing the gene is lower than that of the wild plant.

Wherein the plant comprises Arabidopsis thaliana and/or cotton.

For cotton crops, the fact that the length of the epidermal hair of the leaf is shortened, the plant is dwarf and the root length is shortened means that the gene can participate in the elongation of cotton cells, the cotton plant height is influenced, and the fact that the initial node position of the fruit branch is reduced means that the gene can participate in the regulation of the development of the initial node position of the cotton fruit branch, so that the gene provides gene resources for the breeding of an ideal plant type for cotton cultivation.

The invention also provides a method for regulating the phenotype of the plant, which comprises regulating the expression level of the GhAIF3 gene in the plant;

the GhAIF3 gene has a nucleotide sequence shown in SEQ ID NO. 3.

Because the GhAIF3 gene is related to the plant phenotype, the regulation of the expression level of the GhAIF3 gene in the plant can realize the regulation of the plant phenotype, compared with the traditional method of applying exogenous substances such as auxin, cytokinin, abscisic acid and the like to the plant, the direct regulation of the plant gene can more accurately regulate the plant phenotype, and the regulation efficiency is high.

In some alternative embodiments, the GhAIF3 gene is overexpressed in plants to shorten leaf coat length, stunt the plant, and shorten root length.

Specifically, the GhAIF3 gene can be constructed on an expression vector, then agrobacterium is transformed by the obtained recombinant expression vector, an arabidopsis inflorescence is infected by the transformed agrobacterium, and a positive transgenic plant is screened, so that the overexpression of the GhAIF3 gene is realized.

Wherein, the expression vector comprises pBI 121.

In some alternative embodiments, expression of the GhAIF3 gene in the plant is silenced such that the plant has reduced fruit shoot internodes.

Specifically, a GhAIF3 gene interference vector can be constructed, and the GhAIF3 gene interference vector is used for injecting cotton seedlings to realize the silencing of the GhAIF3 gene.

Wherein, the GhAIF3 gene interference vector comprises a lentivirus interference vector, preferably CLCrVA-GhAIF 3.

After the GhAIF3 gene is silenced or overexpressed, the transgenic arabidopsis and/or cotton both show corresponding phenotype change. In order to facilitate the identification and selection of transgenic plants, vectors used may be processed, for example, by adding a plant selectable marker or a resistant antibiotic marker.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The material sources used in the embodiment of the invention are as follows:

1. cotton material

The cotton material selected in the embodiment is the number 50 of the Zhongmiao institute. For field experiments (different tissues and fiber stages), the variety is planted in a key laboratory test field (Anyang white wall) in the national cotton biology of the institute of Cotton, Chinese academy of agricultural sciences, and the management measure is normal field management.

For virus-induced gene silencing experiments, the No. 24 cotton institute is planted in a greenhouse, and when the cotton grows to the cotyledon and is flattened, bacterial liquid injection treatment is carried out. And performing apparent observation on the original node of the fruit branch in the bud stage.

2. Reagent and consumable

Restriction enzyme, ligase, PCR reaction system related enzyme, gel recovery kit, cloning kit and plasmid test kit are purchased from Bao bioengineering Dalian Co Ltd, and DNA extraction kit is purchased from OMEGA

Other drugs: agarose is Spanish original product, peptone, yeast extract, chloroform, isoamylol, ethanol, isopropanol, sodium chloride, etc. are domestic analytical pure, kanamycin, etc. are from Bao bioengineering Dalian, Inc., and Escherichia coli competent cell DH5 alpha is from Beijing Tiangen Biochemical technology company

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, 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 ℃, shaking up and then plate; 1/2MS solid culture medium: 1/2MS 22g/L, agar powder (agar powder)8g/L, sucrose (sucrose)30 g/L.

The main apparatus is as follows: PCR amplification apparatus (BIO-RAD), high speed centrifuge (Hettich MIKRO 200R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR apparatus (ABI7500), electric heating constant temperature incubator (Shanghai Sensin), constant temperature culture oscillator (Shanghai Zhicheng), artificial climate test chamber (Saifu), and artificial climate chamber.

Example 1 cloning and bioinformatic analysis of the Cotton GhAIF3 Gene and promoter

The gene sequence of GhAIF3(ID: Gh _ D02G0604) was obtained from CottonFGD. The primer is designed by adopting Oligo 7 software, and the cDNA No. 50 of the gossypium hirsutum is amplified by adopting a PCR (polymerase Chain reaction) method, wherein the open reading frame is 663bp, and 220 amino acids are coded. The GSDS website (http:// GSDS. gao-lab. org /) predicts that the GhAIF3 structure contains two exons, one intron (FIG. 1).

An upstream primer F5'-ATGGCATCGTTGATAACGAATC-3' (SEQ ID NO. 1);

the downstream primer R5'-TCATTGGCTGGTCGGCGG-3' (SEQ ID NO. 2).

The open reading frame sequence is as follows:

ATGGCATCGTTGATAACGAATCCAGTGACGAACACAAATTCTGATCGTAGCAAAAGGAAAAAGAAGAAGAATTCTATGGTGAAACAGAATCTTCAACGGAGCCAAAAGGAAGATCATGCCAGATGGAAGTCAGAGGCGCAGCAGCAAATCTATTCCTCAAAACTTCTTCAAGCTTTGAGTCAGGTCAGTCTCGGTACTCCCTCTCCTTCGGCTCCACGCGGTGGTCGAGCTGTCCGTGAAGCCGCTGATAGAGTGTTGGCGGTTGCTGCTAAAGGGAGAACCAGGTGGAGCCGGGCCATTTTGACGAGCCGGCTTAAACTGAAATTTAGAAAGCAGAAGAGATCACAGAGAGGATCCGCTGCTGCCGTTGCTGCCGCAACAAGGACTAGCCGGTCGAAGAAACCGAGAGTTAGCGTTTTGAAATTGAAAGCGAAAAGTGTACCGAATGTTCAAAGAAAAGTAAAGGTCCTAGGCAGATTGGTTCCCGGTTGCCGGAAGCAACCGTTACCGGTAATTCTTGAAGAAGCAACTGATTACATAGCGGCACTTGAGATGCAGGTTCGAGCCATGAGCGCTCTCGCAGATCTGTTATCTGGCTCCGGAGCCGCAAGCTCTAGCTCGGCTCCTCCGCCTGAGTGGCCAATGCCGCCGACCAGCCAATGA(SEQ ID NO.3)。

the encoded amino acid sequence is:

MASLITNPVTNTNSDRSKRKKKKNSMVKQNLQRSQKEDHARWKSEAQQQIYSSKLLQALSQVSLGTPSPSAPRGGRAVREAADRVLAVAAKGRTRWSRAILTSRLKLKFRKQKRSQRGSAAAVAAATRTSRSKKPRVSVLKLKAKSVPNVQRKVKVLGRLVPGCRKQPLPVILEEATDYIAALEMQVRAMSALADLLSGSGAASSSSAPPPEWPMPPTSQ(SEQ ID NO.4)。

the specific process of cloning the gene is as follows:

1. sampling method

During full-bloom stage of cotton, taking mature cotton leaves, quickly freezing in liquid nitrogen, and storing in-80 deg.C refrigerator for use

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 (added beta-mercaptoethanol before use) was added, and the sample was immediately mixed by vigorous shaking. Note that 1: for plant samples with an expected RNA yield of less than 10. mu.g, please use a starting sample size of 100 mg; for starch-rich samples or mature leaves, please increase the amount of lysate SL to 700. mu.l. Note that 2: because of the rich diversity of plants and the different RNA contents of different growth stages and tissues of the same plant, please select the appropriate amount of plant material according to the specific experimental conditions.

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, 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.

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

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 was added 500. mu.l of the 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) And repeating the steps.

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 dH 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 reverse transcription of RNA was carried out according to the instructions of the reverse transcription kit DRR037A using TaKaRa, and the preparation of the reaction solution of RT was carried out on ice, as follows:

the reverse transcription reaction conditions were as follows:

15min at 37 ℃ (reverse transcription reaction),

5s at 85 ℃ (inactivation reaction of reverse transcriptase),

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

4. PCR reaction system, program and product detection for gene cloning

1) PCR reaction system

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

2) PCR reaction procedure

Extension at 10 ℃ -

3) Detection of PCR products

And adding 11. mu.l of 10 Xloading Buffer into 1. mu.l of PCR product, mixing uniformly, spotting on 1% agarose gel, and carrying out electrophoresis detection.

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 eppendorf tube, and weighing again to obtain the weight of the rubber block;

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

4) carrying out water bath at 56 ℃ 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 block is completely melted, adding isopropanol in an amount of 150 mu l per 100mg of rubber block, and fully shaking and uniformly mixing;

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

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

8) centrifuging at 12000rpm for 1min, and pouring out liquid in the collecting pipe;

9) adding 500. mu.l of Wash Buffer and then centrifuging for 1min again;

10) pouring out the liquid in the collecting pipe, adding 200 mul of Wash Buffer again, and centrifuging at 12000rpm for 1 min;

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

12) adding 30 μ l of Elution Buffer at the center of the filter element, standing at room temperature for 1min, and centrifuging at 12000rpm for 1min

6. Connecting the recovered product with cloning vector

1) The following solutions were prepared in a microcentrifuge tube in a total amount of 10. mu.l.

Reagent	Amount of the composition used
		5X CE Buffer	2μL
Exnsae	1μL
		Vector	50ng
Gene	35ng
		Sterilized water	up to 10μL

2) The reaction was carried out at 37 ℃ for 30 minutes.

Note) that the ligation reaction was normally carried out at room temperature (25 ℃ C.), but the reaction efficiency was slightly lowered.

② the ligation reaction was normally carried out in 5 minutes, but the reaction efficiency was slightly lowered.

③ when the long fragment PCR product (more than 2 kb) is used for DNA cloning, the ligation reaction time is required to be prolonged to several hours.

7. Transformation of E.coli by ligation products

1) Adding 100 μ l of Escherichia coli DH5a competent solution into the ligation reaction system, and ice-cooling for 30 min;

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

3) ice-bath for 2 min; adding 900 mul LB liquid culture medium, 37 deg.C, 150rpm, incubating for 1 h;

4) centrifuging to collect bacteria, collecting the bacteria at 4000rpm for 3min, discarding the supernatant, and leaving about 100 μ l of the supernatant, mixing uniformly, and coating an LB plate containing benzyl ammonia resistance;

5) culturing at 37 deg.C overnight;

8. detection and sequencing of Positive clones

1) Picking white colonies from the transformation plate, putting the white colonies into a liquid LB culture medium containing Kna, and carrying out shake culture at the constant temperature of 37 ℃ for 8 hours;

2) positive clones were verified by colony PCR, and the correctly verified monoclonals were sent to Shanghai Biotechnology, Inc. for sequencing, 3 replicates per sequence.

9. 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-70 ℃.

Example 2 construction of plant expression vectors for PBI121-GhAIF3 and CLCrVA-GhAIF3

1. Obtaining target gene segment with specific enzyme cutting site

In order to amplify the entire coding region of the gene and add a specific cleavage site, primers containing appropriate cleavage sites were designed at the start codon ATG and the stop codon, respectively, based on the cloned cDNA sequence of GhAIF 3. The cleavage sites used were XbaI (T/CTAGA) and SmaI (CCC/GGG).

The primer sequence of the GhAIF3 enzyme cutting site is as follows:

an upstream primer F: 5'-CACGGGGGACTCTAGAATGGCATCGTTGATAACGAATC-3' (SEQ ID NO. 5);

a downstream primer R: 5'-GGGGAAATTCGAGCTCTCATTGGCTGGTCGGCGG-3' (SEQ ID NO. 6).

To clone the specific 300bp coding sequence of GhAIF3, the primer sequence was provided with Spe I and AscI linkers, and the CLCrVA-GhAIF3 vector was constructed:

the primer sequence of the GhAIF3 enzyme cutting site is as follows:

an upstream primer F5'-ATGCCTGCAGACTAGTGCTGCCGTTGCTGCCGCAAC-3' (SEQ ID NO. 7);

the downstream primer R5'-AGACCTAGGGGCGCGCCTTGGCTGGTCGGCGGCAT-3' (SEQ ID NO. 8).

2. Construction of plant expression vectors

The specific process is as follows: carrying out double enzyme digestion on pBI121 or CLCrVA plasmids by using corresponding enzymes respectively, and carrying out electrophoresis to recover large-fragment products; connecting the enzyme digestion large fragment product of the target gene fragment and the carrier with the amplified target fragment with the enzyme digestion site by using a Norrespect homologous recombinase Kit Clon ExpressII One Step Cloning Kit; the ligation product is transformed into Escherichia coli DH5 alpha, and cultured overnight at 37 ℃; and (4) selecting monoclonal shake bacteria, and sequencing to verify the correctness of the sequence.

EXAMPLE 3 transformation of Agrobacterium with recombinant vector

1. Preparation of Agrobacterium 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 of LB liquid culture medium containing antibiotics, and culturing at 28 ℃ and 190rpm overnight;

2) mixing the raw materials in a ratio of 1: 90 percent of the total amount of the culture medium is transferred into 80ml of LB liquid culture medium containing antibiotics, and the culture is carried out at 28 ℃ and 170rpm until the OD is reached₆₀₀＝0.6；

3) Ice-cooling the bacterial liquid for 30min, transferring the bacterial liquid into a 50ml centrifuge tube, centrifuging the centrifugal tube at 4 ℃ and 5000rpm for 10min, and removing the supernatant;

4) 5ml of precooled 70mM CaCl were added₂Lightly suspending, standing on ice for 20min, centrifuging at 4 deg.C and 5000rpm for 5min, and removing supernatant;

5) 2ml of pre-cooled 70mM CaCl containing 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; quick freezing for 75s by using liquid nitrogen, and thermally shocking for 2-6 min at 37 ℃;

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

3) 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;

4) selecting positive clones, culturing on LB liquid culture medium at 28 deg.C for 48 hr with glycerol concentration of about 15%, and storing at-20 deg.C.

EXAMPLE 4 Agrobacterium-mediated transformation of Arabidopsis thaliana

Transformation of Arabidopsis thaliana by inflorescence dip-dyeing

1) Inoculating 20 μ l of Agrobacterium liquid stored at-20 deg.C into 1ml LB liquid culture medium, performing shake culture at 28 deg.C and 180rpm overnight, adding 200 μ l of activated bacteria liquid into 50ml LB liquid culture medium, performing shake culture at 28 deg.C and 180 rpm;

2) when the OD value of the bacterial liquid is about 1.2, centrifuging the bacterial liquid at 3000 rpm to collect thalli;

3) the formula of the transformation medium is as follows: 1/2MS (macroelement halved, otherwise unchanged), 5% sucrose, 0.01. mu.g/ml Benzylaminopurine (BAP), 0.03% silwet L-77, 20mg/L acetosyringone, KOH adjusted to pH 5.7(Steven et al 1998).

4) The cells were suspended in the above transformation medium and the OD was adjusted to 0.8 to start the exhaust.

5) Placing the arabidopsis inflorescence in a transformation medium for 30-50s, wrapping the arabidopsis by using a preservative film after dip dyeing, and culturing under normal conditions after dark culture for one day.

6) After the seeds are mature, the seeds are harvested and are T0 generation seeds.

Example 5 phenotypic characterization of transgenic Arabidopsis plants

1. The harvested seeds are sterilized and planted on 1/2MS containing kanamycin, then vernalization is carried out for 3 days at 4 ℃, the seeds are transferred to a climatic test box, positive plants grow normally in about 10 days, and negative plants turn yellow in leaves and do not grow any more.

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:

an upstream primer F5'-GACGCACAATCCCACTATCC-3' (SEQ ID NO. 9);

the downstream primer R5'-GGGGAAATTCGAGCTCTCATTGGCTGGTCGGCGG-3' (SEQ ID NO. 6).

3. The plants of each generation were tested for positive lines until propagation to T3 generations to obtain homozygous transgenic Arabidopsis lines. qRT-PCR detection is carried out on T3 strain, and the process of fluorescent quantitative verification is as follows:

extracting RNA, performing reverse transcription to obtain cDNA, and designing primers for fluorescence quantification of GhAIF3 and an arabidopsis thaliana internal reference gene UBQ10 respectively:

GhAIF3：

an upstream primer: 5'-GGTCAGTCTCGGTACTCC-3' (SEQ ID NO. 10);

a downstream primer: 5'-AGTTTAAGCCGGCTCGTCAA-3' (SEQ ID NO. 11).

UBQ10：

An upstream primer: 5'-AGATCCAGGACAAGGAAGGTATTC-3' (SEQ ID NO. 12);

a downstream primer: 5'-CGCAGGACCAAGTGAAGAGTAG-3' (SEQ ID NO. 13).

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

The PCR reaction system is as follows:

analysis of melting curve:

95℃15 s 60℃1 min 95℃15 s 60℃15 s。

the fluorescent quantitative verification result proves that the transcription level of the GhAIF3 gene in the transgenic plant is remarkably higher than that of the non-transgenic Arabidopsis, as shown in figure 2.

4. And (3) broadcasting the transgenic T3 generation plant to a 1/2MS plate under the same condition with the non-transgenic plant, transplanting the seedling to nutrient soil after 7 days, and observing the length of the epidermal hair of the wild type plant and the transgenic plant line under a common optical microscope by taking rosette leaves overexpressing arabidopsis thaliana and the wild type plant as observation objects after 25 days. The coat of transgenic Arabidopsis thaliana over-expressing GhAIF3 was observed to be significantly shorter than that of wild type, and we calculated the average length of coat of wild type leaf 317.7222 μm, that of L1 leaf 191.2978 μm, that of L2 leaf 231.7656 μm and that of L3 leaf 191.9156 μm by statistical measurement of the length of the leaf coat (FIG. 3A).

FIGS. 3B and 3C show that there is a more pronounced difference in phenotype between wild type and transgenic lines L1, L2 and L3. In FIG. 3B, the wild type Arabidopsis thaliana was flowering 30 days after the transplantation, and transgenic Arabidopsis thaliana L1 and L2 overexpressing GhAIF3 did not flower at the same time, and L3 showed a phenotype of about to flower. Meanwhile, the three strains of the transgenic arabidopsis have obvious dwarfing symptoms, and the higher the expression quantity of the strains is, the more obvious the dwarfing characteristic is. Figure 3C top view shows that the transgenic lines all showed a significantly different phenotype with compact rosette leaves, oval leaves and dark green color compared to wild type.

5. In order to further research the effect of GhAIF3, a virus interference vector of CLCrVA-GhAIF3 is constructed, pCLCrVA (no-load), CLCrVA-GhAIF3 (target gene) and pCLCrVA: (PDS) (positive control) are respectively injected in the bacterial liquid injection process, and after the positive control shows albino phenotype (about 20-30 days after injection), cotton seedling DNA injected by pCLCrVA (no-load) and CLCrVA-GhAIF3 (target gene) is respectively extracted for PCR positive identification. Transplanting the positive single plant into a large pot for planting, extracting leaves of different strains, carrying out fluorescence quantitative PCR, and detecting the silencing efficiency of GhAIF3 in the plant, wherein the result shows that compared with pCLCrLVA no-load control, the pCLCrVA shows that the expression quantity of a virus-induced strain of GhAIF3 is obviously reduced, and the silencing efficiency is about 47.6 percent (figure 4A); comparison of pCLCrLVA unloaded and CLCrVA-GhAIF3 virus-induced lines for fruitshoot internodal sites was observed after budding. The fruit branch primary node of CLCrVA-GhAIF3 was found to be 5, while pCLCrLVA was found to be 6 in no-load (positive control was also 6). Therefore, CLCrVA-GhAIF3 has a lower fruit branch internodal level than pCLCrLVA (FIG. 4B). These results indicate that the expression of GhAIF3 is reduced in cotton, and the fruit branch primary node of the corresponding strain is reduced, which indicates that the gene may have negative regulation effect on the fruit branch primary node.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Cotton research institute of Chinese academy of agricultural sciences

Application of <120> GhAIF3 gene in regulation of plant phenotype and method for regulating plant phenotype

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence

<400> 1

atggcatcgt tgataacgaa tc 22

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence

<400> 2

tcattggctg gtcggcgg 18

<210> 3

<211> 663

<212> DNA

<213> cotton institute No. 50 of upland cotton

<400> 3

atggcatcgt tgataacgaa tccagtgacg aacacaaatt ctgatcgtag caaaaggaaa 60

aagaagaaga attctatggt gaaacagaat cttcaacgga gccaaaagga agatcatgcc 120

agatggaagt cagaggcgca gcagcaaatc tattcctcaa aacttcttca agctttgagt 180

caggtcagtc tcggtactcc ctctccttcg gctccacgcg gtggtcgagc tgtccgtgaa 240

gccgctgata gagtgttggc ggttgctgct aaagggagaa ccaggtggag ccgggccatt 300

ttgacgagcc ggcttaaact gaaatttaga aagcagaaga gatcacagag aggatccgct 360

gctgccgttg ctgccgcaac aaggactagc cggtcgaaga aaccgagagt tagcgttttg 420

aaattgaaag cgaaaagtgt accgaatgtt caaagaaaag taaaggtcct aggcagattg 480

gttcccggtt gccggaagca accgttaccg gtaattcttg aagaagcaac tgattacata 540

gcggcacttg agatgcaggt tcgagccatg agcgctctcg cagatctgtt atctggctcc 600

ggagccgcaa gctctagctc ggctcctccg cctgagtggc caatgccgcc gaccagccaa 660

tga 663

<210> 4

<211> 220

<212> PRT

<213> cotton institute No. 50 of upland cotton

<400> 4

Met Ala Ser Leu Ile Thr Asn Pro Val Thr Asn Thr Asn Ser Asp Arg

1 5 10 15

Ser Lys Arg Lys Lys Lys Lys Asn Ser Met Val Lys Gln Asn Leu Gln

20 25 30

Arg Ser Gln Lys Glu Asp His Ala Arg Trp Lys Ser Glu Ala Gln Gln

35 40 45

Gln Ile Tyr Ser Ser Lys Leu Leu Gln Ala Leu Ser Gln Val Ser Leu

50 55 60

Gly Thr Pro Ser Pro Ser Ala Pro Arg Gly Gly Arg Ala Val Arg Glu

65 70 75 80

Ala Ala Asp Arg Val Leu Ala Val Ala Ala Lys Gly Arg Thr Arg Trp

85 90 95

Ser Arg Ala Ile Leu Thr Ser Arg Leu Lys Leu Lys Phe Arg Lys Gln

100 105 110

Lys Arg Ser Gln Arg Gly Ser Ala Ala Ala Val Ala Ala Ala Thr Arg

115 120 125

Thr Ser Arg Ser Lys Lys Pro Arg Val Ser Val Leu Lys Leu Lys Ala

130 135 140

Lys Ser Val Pro Asn Val Gln Arg Lys Val Lys Val Leu Gly Arg Leu

145 150 155 160

Val Pro Gly Cys Arg Lys Gln Pro Leu Pro Val Ile Leu Glu Glu Ala

165 170 175

Thr Asp Tyr Ile Ala Ala Leu Glu Met Gln Val Arg Ala Met Ser Ala

180 185 190

Leu Ala Asp Leu Leu Ser Gly Ser Gly Ala Ala Ser Ser Ser Ser Ala

195 200 205

Pro Pro Pro Glu Trp Pro Met Pro Pro Thr Ser Gln

210 215 220

<210> 5

<211> 38

<212> DNA

<213> Artificial sequence

<400> 5

cacgggggac tctagaatgg catcgttgat aacgaatc 38

<210> 6

<211> 34

<212> DNA

<213> Artificial sequence

<400> 6

ggggaaattc gagctctcat tggctggtcg gcgg 34

<210> 7

<211> 36

<212> DNA

<213> Artificial sequence

<400> 7

atgcctgcag actagtgctg ccgttgctgc cgcaac 36

<210> 8

<211> 35

<212> DNA

<213> Artificial sequence

<400> 8

agacctaggg gcgcgccttg gctggtcggc ggcat 35

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

gacgcacaat cccactatcc 20

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence

<400> 10

ggtcagtctc ggtactcc 18

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<400> 11

agtttaagcc ggctcgtcaa 20

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<400> 12

agatccagga caaggaaggt attc 24

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence

<400> 13

cgcaggacca agtgaagagt ag 22

Claims

Use of the GhAIF3 gene for modulating a plant phenotype including one or more of leaf epidermal hair length, plant height, root length and fruit branch internode location;

the GhAIF3 gene has a nucleotide sequence shown in SEQ ID NO. 3.
2. The use of claim 1, wherein the GhAIF3 gene expresses a protein having an amino acid sequence as set forth in SEQ ID No. 4.
3. The use of claim 1, wherein the expression of the GhAIF3 gene in plants is up-regulated to shorten the epidermal hair length, stunt the plant and shorten the root length of the plant leaves.
4. The use of claim 1, wherein the expression of the GhAIF3 gene in the plant is downregulated such that the plant fruit shoot internodes are reduced in position.
5. Use according to any one of claims 1 to 4, wherein the plant comprises Arabidopsis thaliana and/or cotton.
6. A method for controlling a phenotype of a plant, comprising controlling the expression level of GhAIF3 gene in the plant;

the plant phenotype comprises one or more of leaf epidermal hair length, plant height, root length, and fruit branch inception position;

the GhAIF3 gene has a nucleotide sequence shown in SEQ ID NO. 3.
7. The method of claim 6, wherein the GhAIF3 gene in the plant is overexpressed so that the plant leaf coat hair length is shortened, the plant is dwarfed, and the root length is shortened;

preferably, the plant comprises arabidopsis thaliana.
8. The method of claim 7, wherein the GhAIF3 gene is constructed on an expression vector, then agrobacterium is transformed by the obtained recombinant expression vector, an arabidopsis inflorescence is infected by the transformed agrobacterium, and a positive transgenic plant is screened to realize the overexpression of the GhAIF3 gene;

preferably, the expression vector comprises pBI 121.
9. The method of claim 6, wherein the expression of the GhAIF3 gene in the plant is silenced such that the plant has reduced fruit shoot internodes;

preferably, the plant comprises cotton.
10. The method of claim 9, wherein the GhAIF3 gene interference vector is constructed, and the silencing of the GhAIF3 gene is achieved by injecting cotton seedlings with the GhAIF3 gene interference vector;

preferably, the GhAIF3 gene interference vector comprises a lentiviral interference vector, preferably CLCrVA-GhAIF 3.