Application of VDAC1 gene in regulation and control of plant flowering phase

文档序号：183832 发布日期：2021-11-02 浏览：29次中文

阅读说明：本技术 Vdac1基因在调控植物开花期中的应用 (Application of VDAC1 gene in regulation and control of plant flowering phase ) 是由秦诚许静雅张玉珍任虹佳余润意袁陈胡一凯刘庆港于 2021-07-27 设计创作，主要内容包括：本发明公开VDAC1基因在调控植物开花期中的应用。VDAC1基因为AT3G01280。突变VDAC1基因的拟南芥植株莲座叶数目在长日照条件下少于野生型植株。突变VDAC1基因在调控拟南芥植株提前开花上的应用。拟南芥VDAC1蛋白与FT蛋白相互作用,调控拟南芥开花。(The invention discloses an application of a VDAC1 gene in regulation and control of plant flowering phase. The VDAC1 gene is AT3G 01280. The number of rosette leaves of the Arabidopsis thaliana plant with the mutant VDAC1 gene is less than that of the wild type plant under long-day conditions. Application of a mutant VDAC1 gene in regulating and controlling early flowering of an arabidopsis plant. The Arabidopsis VDAC1 protein interacts with the FT protein to regulate the flowering of Arabidopsis.)

Use of the VDAC1 gene for regulating the flowering phase of a plant.

2. The use as claimed in claim 1 wherein the VDAC1 gene is AT3G 01280.

3. Use according to claim 1 or 2, characterized in that the number of rosette leaves of an arabidopsis thaliana plant mutated for the VDAC1 gene is lower than that of the wild type plant under long day conditions.

4. Use according to claim 1 or 2, wherein the mutant VDAC1 gene is used to control early flowering in arabidopsis plants.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to an application of a VDAC1 gene in regulation and control of plant flowering phase.

Background

VDAC is the most important voltage-dependent anion channel protein localized in the outer mitochondrial membrane, playing a role in metabolite transport between mitochondria and cytoplasm. VDAC protein accounts for 34.2% of the mitochondrial surface. These transport functions help regulate plant growth. VDAC1 participates in reproductive development, and the mutant reduces seed setting rate. In addition to seeds, pollen development can also be affected, including pollen grain count, pollen germination rate, length of the germinating pollen tube, and the like. The increased zygote or early embryo lethality of the VDAC1 mutant may be the main cause of the decreased seed yield. VDACs play an important role in plant growth, but the specific function of each subtype is still poorly understood, especially in relation to flowering.

Here, we found that VDAC1 plays an inhibitory role in the transformation process of flowers, and that VDAC1 protein interacts with FT protein. Loss of AtVDAC1 function resulted in an early flowering phenotype. The results of the study showed that VDAC1 acts through the FT protein during flower conversion.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of an arabidopsis gene VDAC1 in regulating and controlling the flowering period of a plant.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides a function of an arabidopsis gene VDAC1 in regulating and controlling the flowering period of arabidopsis.

Further: plants with the Arabidopsis thaliana VDAC1 gene insertion mutation flower earlier than wild-type plants.

Further: the number of rosette leaves of an Arabidopsis thaliana plant with a mutant VDAC1 gene was less than that of the wild type under long-day conditions.

Further: arabidopsis VDAC1-GUS has significant expression in all parts of Arabidopsis.

Further: the Arabidopsis VDAC1 protein interacts with the FT protein to regulate the flowering of Arabidopsis.

The invention has the advantages and beneficial effects that:

1. by utilizing the existing plant biotechnology, the invention screens and obtains a homozygous plant of the T-DNA insertion mutant of the Arabidopsis thaliana mutant VDAC1-5, and compares the homozygous plant with the flowering time and the rosette leaf number of a wild plant growing under the same condition to find that the mutant VDAC1-5(AT3G01280) gene causes the positioning change of a flowering related gene (FT) in the plant and promotes the flowering of Arabidopsis thaliana.

Drawings

FIG. 1(a) is a schematic gene diagram of the atvdac1-5 (SALK-058473C) mutant, wherein the black and grey boxes in the locus of the atvdac1-5 mutant represent exons and untranslated regions, respectively, and the black bars represent introns; T-DNA is genome insertion mutation; (b) - (c) PCR electrophorogram and semiquantitative graph for detecting mutant atvdac1-5 and Wild Type (WT), respectively, indicating that VDAC1 gene was not expressed in atvdac1-5 mutant.

FIGS. 2(a) - (b) are statistics for the flowering phenotype and number of rosette leaves at flowering time of the atvdac1-5 mutant under Long Day (LD) conditions, where 13 rosette leaves were wild type Arabidopsis thaliana under LD conditions and 11 rosette leaves were mutant atvdac1-5, respectively, with significant differences (.: P < 0.05); (c) VDAC1-5 gene was transferred into mutant atvdac1-5 transgenic Arabidopsis plants to obtain the gene-replying strain atvdac1-5 gVDAC1-5 of VDAC 1-5. After phenotypic identification, the number of replying strain rosette leaves in the background of the atvdac1-5 mutant was found to be 13, and the phenotype was not significantly different from that of the wild type rosette leaf 13.2.

FIG. 3 shows the expression sites of VDAC1 detected by GUS staining of seedlings at 3,5,7,9,11 and 13 days after germination of Arabidopsis thaliana.

FIGS. 4(a) - (b) are yeast double hybrid and Agrobacterium-mediated tobacco transient transformation experiments, respectively, verifying VDAC1 and FT interaction;

FIG. 5 is a phenotypic drawing of double-nosed Arabidopsis thaliana showing the interaction between VDAC1 and the FT protein and regulating Arabidopsis flowering.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

Reagents used in the above experiments and the like were purchased from TAKARA, Roche, TIANGEN, CWBiO and the like.

Reagents and drug instructions used in the experiments: see molecular cloning, third edition.

EXAMPLE 1 obtaining of homozygous mutants

1. Obtaining of mutants

The atvdac1-5 mutant used was a T-DNA insertion.

2. DNA extraction from Arabidopsis thaliana plant leaves

Taking a proper amount of seeds in wet filter paper, placing the filter paper in a refrigerator at 4 ℃ for dark treatment for 48 hours, finishing dormancy breaking of the seeds, transferring the seeds into soil, placing the seeds in a greenhouse for culture (16 hours of illumination/8 hours of dark, 23 ℃, 10000Lux), dropping five seeds in one pot, covering the seeds with a preservative film to keep stable germination conditions, continuing culturing the seeds for 2 days until 11 days, and taking a proper amount of leaves to extract genomes.

Extracting DNA genome from wild WT and atvdac1-5 mutant Arabidopsis thaliana by using self-prepared TPS extract, and specifically performing the following operations:

firstly, collecting a leaf sample into a 1.5ml centrifuge tube, adding a proper amount of TPS and small steel balls, grinding the leaf sample by using a grinding instrument at 55Hz for 2min, and shaking uniformly.

② centrifuging for 10min at 13000rpm, taking out the supernatant and transferring to a new 1.5ml centrifuge tube, adding equal amount of isopropanol.

③ 13000rpm centrifugation for 5min, abandoning the supernatant, adding 500. mu.L 75% ethanol, and shaking up and down for several times. Centrifuging at 13000rpm for 5min, discarding the supernatant, and repeating the previous step.

13000rpm for 2min, sucking the liquid in the tube as much as possible by a pipette, drying in an oven at 37 ℃, adding 50 mu L of warm ddH2O, and dissolving completely.

TPS extracting solution formula

TPS(100ml)：1M Tris-HCl(pH8.0) 10ml

0.5M EDTA(pH8.0) 40ml

2M KCl 50ml

Identification of the homozygous for the atvdac1-5 mutant

(1) Design of identifying primers

Primer Name Forward(5’–3’)

atvdac1-5 LP: GGGAAAGATCAGTAGTTGCCC, shown in SEQ ID NO.1

atvdac1-5 RP: TCGTTGCTCATAATCTGGCTC, shown in SEQ ID NO.2

SALK-LBb1.3: ATTTTGCCGATTTCGGAAC, shown in SEQ ID NO.3

PCR amplification of the wild type WT genomic DNA extracted above using the atvdac1-5 LP, atvdac1-5 RP primers gave a 1184bp sized fragment, whereas amplification of the genomic DNA of the atvdac1-5 mutant extracted above gave no significant band; no significant band was observed after PCR amplification of the wild type WT genomic DNA extracted above with primers SALK-LB1.3 and atvdac1-5 RP, while a 907bp fragment was obtained after amplification of the atvdac1-5 mutant genomic DNA extracted above.

(2) And (3) PCR reaction system:

(3) PCR reaction procedure:

and detecting by 1% agar gel electrophoresis.

4. Extraction of Total RNA of Arabidopsis thaliana (RNA kit of Tiangen Co.)

The 75% ethanol is used for cleaning and disinfecting the operation table when the glove is worn so as to prevent the pollution in the environment; in order to prevent degradation of RNA enzyme in saliva, the mask is worn in the whole process; in order to reduce the degradation degree of RNA, extraction should be carried out at low temperature as much as possible; furthermore, it should be ensured that all samples directly contacted during the process are RNase-Free. The experiment adopts an RNA kit of Tiangen company, and the specific operation is as follows:

firstly, cutting 22 with clean scissors: 2 pieces of rosette leaves of Arabidopsis thaliana at 00 time point were placed in a 1.5ml centrifuge tube, a small steel ball was added, the tube was rapidly frozen in liquid nitrogen, and ground into powder by grinding with an automatic grinder at 35Hz for 2 min.

Preparing RL with 1 percent of beta-mercaptoethanol, adding a proper amount of the RL into the powder, shaking and uniformly mixing, transferring to a CS column, and centrifuging at a high speed for 5min at a low temperature to obtain clear liquid.

③ transferring the supernatant to a new 1.5ml centrifuge tube which is added with absolute ethyl alcohol with 0.5 times volume in advance, uniformly mixing and transferring to a CR3 column, centrifuging at low temperature and high speed for 1min, and discarding the waste liquid.

And fourthly, adding 350 mu l of RW1, centrifuging at low temperature for 1min, adding 80 mu l of DNaseI working solution after discarding the waste liquid, standing at 28 ℃ for 30min, and adding 500 mu l of RW.

Low-temperature centrifuging for 1min, and discarding the waste liquid.

And sixthly, carrying out air separation for 2min, and discarding the waste liquid.

Seventhly, placing the CR3 column head in a new 1.5ml centrifuge tube, standing at room temperature until the column head membrane is dried, adding 30 mu l of warm eluent or sterilized ddH2O on the membrane, and centrifuging at high speed for 2min at normal temperature to obtain the total RNA.

5. Reverse transcription (Tiangen reverse transcription kit)

The experiment used a reverse transcription kit from Tiangen corporation: FastQuantRT Kit (with gDNase), the specific procedure was as follows:

(ii) 0.2ml RNase-Free EP tube, the following ingredients were added:

② after mixing evenly, standing for 3min at 42 ℃; then placed on ice for 5 min.

③ adding the following components into the RNase-Free EP tube after the reaction:

fourthly, after being mixed evenly, the mixture is placed at 42 ℃ for standing for 30 min; then transferring to 95 ℃ and standing for 3min to obtain the cDNA, and storing the cDNA at-20 ℃.

VDAC1 Gene expression assay of atvdac1-5 mutant

Design of identifying primers

Primer Name Forward(5’–3’)

VDAC 1F: CGCTACCGTTGATGAGG, shown in SEQ ID NO.4

VDAC 1R: TGAAGAATGACTTGGGTTTCC, shown in SEQ ID NO.5

(2) And (3) PCR reaction system:

(3) PCR reaction procedure:

and detecting by 1% agar gel electrophoresis.

The VDAC 1F and VDAC 1R primers are adopted to carry out PCR amplification on WT, then the VDAC1 gene is expressed, and the mutant has no obvious band.

The VDAC 1F and VDAC 1R primers are adopted to carry out PCR amplification on the cDNA to obtain an amplification product; WT had VDAC1 gene expression, while atvdac1-5 mutant VDAC1 gene was not expressed with no apparent band.

FIG. 1(a) is a schematic gene diagram of the atvdac1-5 mutant, wherein the black and grey boxes in the locus of the atvdac1-5 mutant represent exons and untranslated regions, respectively, and the black bars represent introns; T-DNA is genome insertion mutation; (b) - (c) PCR electrophorogram and semiquantitative graph for detecting mutant atvdac1-5 and WT, respectively, indicating that VDAC1 gene was not expressed in atvdac1-5 mutant.

Example 2 statistical analysis of flowering time and Lotus throne number

Growing under the condition of long illumination (16h/8h, illumination/dark) 10000Lux illumination at 23 ℃. Selecting 20 plants of a normal-growing arabidopsis wild type plant WT and atvdac1-5 respectively, and counting the number of rosette leaves after the plants bloom.

As shown in FIG. 2(a-b), the statistical results show that the number of leaves of the Arabidopsis thaliana rosette leaves of the atvdac1-5 plant is about 11.4, the number of leaves of the wild type rosette leaves is about 13, and the significant difference is that the flowering time of the atvdac1-5 mutant plant is earlier than that of the wild type. The VDAC1 gene in Arabidopsis is shown to promote the plant to bloom after being mutated. The atvdac1-5 mutant appeared to flower prematurely under long light conditions. (c) The number of leaves of the reverting line against the background of the mutant, which is similar to that of the wild type, was about 13, indicating that VDAC1 gene reverted to the early flowering phenotype of atvdac1-5 mutant, i.e., VDAC1 gene inhibited flowering in Arabidopsis thaliana.

Example 3 expression site of VDAC1-GUS in Arabidopsis thaliana

FIG. 3 shows GUS staining of Arabidopsis thaliana, and expression of VDAC1 in Arabidopsis thaliana was examined. As a result of GUS staining, VDAC1 was expressed in root, stem, leaf, fruit, flower and the like of Arabidopsis thaliana as VDAC 1. Furthermore, from the results of staining 3 to 13 days after germination, the degree of staining of VDAC1-GUS in each part of Arabidopsis thaliana was gradually increased with the lapse of time, that is, the expression level of VDAC1-5 was gradually increased. The detection of the expression level of VDAC1 shows that VDAC1 has significant expression in the parts of rosette leaves, cauline leaves, roots, fruit pods and the like of Arabidopsis thaliana, and the result is consistent with the result of the dyed parts.

1. Transgenic obtaining GUS positive strain

Positive strains are obtained by constructing pCAMBIA1300-gVDAC1-gus vector and carrying out transgene screening.

Preparation of GUS dye

(1)200mmol/L phosphate buffer (pH 7.0)

The preparation method comprises the following steps:

solution A: weighing NaH₂PO₄·2H₂03.12g of distilled water after sterilization was dissolved, and the volume was adjusted to 100 mL.

And B, liquid B: weighing Na₂HPO₄·12H₂07.17g was dissolved in sterilized distilled water to a constant volume of 100 mL.

Mixing 100mLB liquid and 40mLA liquid, and adding NaH₂PO₄·2H₂The pH of the 0 solution was adjusted to 7.0.

(2) Dyeing liquid

Description of the drawings:

the precise localization of GUS activity requires the presence of ferrous ions. GUS enzyme hydrolyzes its substrate X-Gluc to produce soluble indolyl derivative, which can diffuse to other part and must be oxidized and condensed into dimer to form insoluble blue precipitate, and the dimer is catalyzed by oxidation catalyst (such as potassium ferrocyanide, peroxidase or catalase) without adding Fe ^2-It diffuses only as a soluble intermediate, but does not diffuse upon addition of potassium ferrocyanide due to dimerization of the indolyl group, the faster the insoluble blue precipitate is formed, the more accurate the localization of the GUS enzyme.

Planting of vdac1 gVDAC1-GUS Material

(1)1/2MS culture dish configuration

MS：3.37g/L

Sucrose: 15g/L

Agar: 8g/L

Adjusting the pH to 5.7

After the volume is fixed to 1L, sterilizing at 121 ℃ for 20min, cooling to about 40 ℃, adding 300 mu L of antibiotic hygromycin into the culture medium, wherein the concentration of the antibiotic is 100 mu g/mu L.

(2) Configuring 10% NaClO

Taking 5mL of NaClO solution under the condition of an ultra-clean bench, using absolute ethyl alcohol to fix the volume to 50mL, and wrapping the solution with tinfoil paper to keep the solution in a refrigerator at 4 ℃ in a dark place.

(3) Seed killing device

50 mul of seeds are weighed and filled in a sterilized EP tube with 1.5mL, 700 mul of 10% NaClO suspension is added, and the mixture is continuously mixed up and down for about 10 min. Pouring out 10% NaClO solution waste liquid, adding anhydrous ethanol, shaking up and down for 5min, pouring out waste liquid, and repeating for 5 times. And (4) placing the wet seeds in a super clean bench for drying.

(4) Dibbling seed

And dropping the dried seeds into a culture dish containing antibiotics, and breaking the dormancy in a refrigerator at 4 ℃ in dark and dark for two days. The plates were removed and incubated in the greenhouse at 23 ℃.

GUS staining

Soaking 3,5,7,9,11 and 13 days after germination of arabidopsis thaliana seedling materials in a dye solution, vacuumizing, and placing at 37 ℃ for heat preservation and dyeing. Transferring the green materials such as leaves and the like into absolute ethyl alcohol for decoloring for 2-3 times until the negative control material is white. And observing blue dots on the surface of the leaf under a microscope to obtain GUS expression sites.

FIG. 3 shows the expression sites of VDAC1 detected by GUS staining of seedlings at 3,5,7,9,11 and 13 days after germination of Arabidopsis thaliana.

Example 4 interaction between VDAC1 and FT protein

FIGS. 4(a) - (b) demonstrate the interaction between FT and VDAC1 proteins by yeast double hybrid and Agrobacterium-mediated transient transformation experiments in tobacco. The homozygous lines were obtained by crossing the vdac1 line with FT-related material, SUC2: FT-9myc, SUC2: FLAG-FT, KNAT: FT. FIG. 5 shows that phenotypic identification shows that vdac1 further promotes early flowering performance of lines SUC2: FT-9myc, SUC2: FLAG-FT, KNAT: FT, etc. The double mutant line of ft-10vdac1 inhibited the late flowering trait of ft-10. Thus, VDAC1 inhibited arabidopsis flowering by interacting with FT.

1. Yeast double hybrid

(1) Selecting a yeast monoclonal, dissolving in 1mL LYPDA culture solution, blowing uniformly, transferring into 20mL YPDA (250mL Erlenmeyer flask), shaking at 28 deg.C and 200rpm overnight;

(2) about 13 hours, 1mL of the bacterial solution was taken and OD was measured_600nmMore than 1.6;

(3) taking appropriate amount of bacterial liquid (about 2.5 mL) to 100mLYPDA to make OD_600nmCulturing at 28 deg.C and 200rpm for 3 hr to OD of 0.2-0.3_600nmBetween 0.4 and 0.6;

(4) transferring the bacterial liquid to a 50mL centrifuge tube, centrifuging for 5min at room temperature of 1000g, and taking the supernatant;

(5) Resuspend with 25mL sterile water, 1000g remove supernatant;

(6) gently resuspending the cells with 1.5mL of 1 × TE/1 × LiAc to make competent cells;

(7) subpackaging competent cells, adding mixed solution containing 10ul of carrier DNA (boiled in boiling water for 10min, and on ice for 5min) and AD-VDAC1 and BD-FT plasmid into each tube of 100ul, and gently mixing;

(8) adding 0.6ml PEG/LiAc into each tube, oscillating at high speed for 10s, 28 ℃, 200rpm and 30 min;

(9) adding 70ul DMSO, and gently mixing;

(10) heat shock in 42 deg.C water bath for 15min, and standing on ice for 1-2 min;

(11) centrifuging at 13000rpm for 1min, adding 200ul 1 XTE, and blowing uniformly;

(12) coating 200ul of bacterial liquid on a plate (SD/-Leu-Trp), and culturing at 30 ℃ for 2 days;

(13) after 2 days, a single clone was dissolved in 20ul of sterile water and spotted on a plate (SD/-Leu-Trp-Ade-His).

2. Agrobacterium-mediated tobacco transient transformation experiment

(1) The vector transformed with cLUC-VDAC and FT-nLUC Agrobacterium is cultured again in the presence of Kan⁺,Rif⁺The single colonies are picked up and put in 5mL liquid LB (containing Kan)⁺,Rif⁺) Medium shake (28 ℃, 200rpm) overnight.

(2) The next day, 1mL of the bacterial liquid is taken to 10mL of liquid LB (containing Kan)⁺,Rif⁺) Medium scale up culture, OD_600nmAfter reaching 0.5, the strain is collected by centrifugation (5000 rpm).

(3) The cells were washed twice with a 10mM MS-MES (pH5.6) solution, and finally resuspended in an AS-MS-MES (pH5.6) solution to adjust the OD to 0.5.

(4) And (3) carrying out 1: 1 proportion, standing for 3 hours at room temperature to fully mix the bacteria liquid. Then, injecting a proper amount of the uniformly mixed bacterial liquid on the surface of the healthy and smooth tobacco leaves until a circle is formed.

(5) The tobacco injected with the bacterial liquid is placed in the dark condition and cultured for 48 hours at the temperature of 28 ℃, and then the illumination is recovered for 16 hours.

(6) LUC activity assay: spraying 1mM luciferase on the surface of the tobacco leaf blade, and lightly tapping to ensure that the leaf blade is uniformly stained. And processing in dark for 5 min.

(7) Capturing LUC images with a CCD imaging device pre-cooled to-120 deg.C, wherein the exposure time for each image is 7-9min, and the exposure time for capturing leaf chloroplast autofluorescence imaging is 1min, and then exposing for 2 min.

The above evidence demonstrates that VDAC1 interacts by going through FT. The mutant VDAC1 gene may alter the flowering phase of plants by affecting the function of the FT protein.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not materially depart from the spirit and scope of the invention as claimed.

Sequence listing

<110> university of teachers in Hangzhou

Application of <120> VDAC1 gene in regulation and control of plant flowering phase

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA