Application of FT gene in negative regulation of plant adaptability to fluctuating light

Abstract

The present invention provides FT ( FLOWERING LOCUS T ) gene in negatively regulating plant light adaptation, the FT The nucleotide sequence of the gene is shown in SEQ ID NO.1. The negative regulation is manifested in knocking out FT Plants with the gene have increased adaptability to fluctuating light, while overexpression FT Therefore, the present invention provides a new method for improving the adaptability of plants to fluctuating light, by reducing the activity of FT protein in the target plant or reducing FT By expressing the gene, plants with stronger tolerance to fluctuating light than the target plants are obtained, providing a new gene resource for molecular breeding and genetic modification of plants.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to FT Application of genes in negative regulation of plant adaptation to fluctuating light. Background Art

[0002] Photosynthesis sustains almost all life on Earth, provides the oxygen and food we breathe, forms the basis of the global food chain, and meets most of humanity's current energy needs by forming fuel. The photosynthesis process in plants is based on light and dark reactions carried out in different parts of chloroplasts. Light reactions occur on the chloroplast thylakoid membrane and include water splitting, the transfer of protons and electrons, and the production of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). ATP and NADPH are then used by the Calvin-Benson cycle (dark reaction) to convert carbon dioxide into carbohydrates in the chloroplast matrix. Currently, most studies on photosynthesis focus on constant light intensity, while in nature, the light intensity received by plants is in a constant process of change due to seasonal changes, circadian rhythms, solar radiation angles, wind, and cloud movement. In addition, the increasing density of crops due to the increasing population and the decreasing area of ​​arable land have led to more intense changes in canopy light intensity, which will pose a huge challenge to crop photosynthesis. In the face of fluctuating light, plants have evolved a series of mechanisms to cope with it, such as cyclic electron transfer, non-photochemical quenching, state transformation, etc., but there is still a lack of systematic research on the response mechanism under different types of fluctuating light. Therefore, the adaptive mechanism basis of photosynthesis to non-steady-state light intensity needs further study.

[0003] Florigin is a systemic signal for plants to start flowering. It is synthesized in leaves and transported to the shoot apex meristem, where it promotes the transition to flowers. Earlier, people discovered that florigen is a signal for the flowering site T (FLOWERING LOCUS T, FT ) gene encodes a protein product that is localized in the cell nucleus. FT The function of florigen can promote new crop breeding techniques to produce varieties that can start the reproductive stage in the best season. In addition, recent studies have also found that florigen has the function of regulating leaf development and tuber formation in addition to flowering period regulation. The pleiotropic effects of florigen mean that FT Genes may also play an important role in plant growth and development or stress. FT Genes are associated with plant tolerance to fluctuating light stress. Summary of the invention

[0004] The object of the present invention is to provide FT Application of genes in negative regulation of plant adaptation to fluctuating light.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is summarized as follows:

[0006] Based on the Arabidopsis thaliana genome sequence published on the National Arabidopsis Tair official website (https: / / www.arabidopsis.org / ), we obtained FT The nucleotide sequence information of the gene (i.e., AT1G65480). FT The nucleotide sequence of the gene coding frame is 660 bp long, encoding 219 amino acids. Its sequence can be obtained through the Arabidopsis Tair official website.

[0007] The present invention also constructs a series of plant expression and knockout vectors. The functions of expression vectors, gene editing vectors or transgenic plant lines containing the above-mentioned genes and host cells containing the vectors in improving the adaptability of plants to fluctuating light also fall within the protection scope of the present invention.

[0008] The functions of the genes protected by the present invention include not only the above FT Genes, including FT Homologous genes with higher homology (eg, homology higher than 80%; more preferably higher than 90%; more preferably higher than 95%; more preferably higher than 98%) have functions in fluctuating light adaptation.

[0009] The present invention is based on FT The CDS gene sequence of the 35S promoter was used to construct the pCAMBIA1300 gene expression vector, which was then infected with Agrobacterium tumefaciens to obtain FT Overexpression strains were obtained by inserting target genes using T-DNA FT Knockout mutant lines were analyzed FT Overexpression lines and ft-10 The biological functions of mutant strains under fluctuating light treatment at the seedling stage can provide new genetic resources for improving the fluctuating light adaptability of crops.

[0010] The present invention discloses FT The biological function of genes in plant tolerance to fluctuating light is specifically manifested in: under fluctuating light conditions, FT The deletion mutant strain is significantly more adaptable to fluctuating light than the wild type. The above application is concluded through fluctuating light treatment experiments.

[0011] According to its function, plants with strong adaptability to fluctuating light can be obtained through genetic modification. Specifically, FTGenes are introduced to obtain transgenic plants, which have stronger adaptability to fluctuating light than the target plants.

[0012] Specifically, the gene editing vector can be introduced into the target plant. In the method, the gene editing vector can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissue can be cultivated into a plant.

[0013] In order to improve the excellent traits of plants, the present invention also protects a new plant breeding method, comprising the following steps (1) or (2):

[0014] (1) By reducing the FT The activity of the protein was studied to obtain plants with stronger adaptability to fluctuating light than the target plants;

[0015] (2) By reducing the FT The expression of genes can produce plants with stronger adaptability to fluctuating light than the target plants;

[0016] “Reducing the FT The implementation of "gene expression" can be as follows (1) or (2) or (3):

[0017] (1) Knockout in target plants FT Gene;

[0018] (2) Using TDNA to insert into target plants FT Gene;

[0019] (3) Other common methods in this field.

[0020] Wherein, the target plant of the present invention is Arabidopsis thaliana.

[0021] Target gene, also known as target gene, is a gene used for gene recombination, changing the characteristics of receptor cells and obtaining the expected expression product in genetic engineering design and operation. It can be from the organism itself or from a different organism.

[0022] In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, flower plants, or forestry plants, etc. The plants can be, for example (but not limited to): dicotyledons, monocotyledons or gymnosperms.

[0023] As a preferred embodiment, the "plant" includes but is not limited to Arabidopsis thaliana, and any plant having the gene or a gene homologous thereto is applicable.

[0024] The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs, and our target gene or nucleic acid is present in these different parts. The "plant" mentioned here also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, and similarly, each of the aforementioned objects contains the target gene / nucleic acid.

[0025] The present invention includes any plant cell, or any plant obtained or obtainable by the method therein, and all plant parts and propagules thereof. This patent also includes transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and the progeny obtained using the method of this patent have the same characteristics.

[0026] The invention also extends to the harvestable parts of the plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It also further relates to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starch or proteins. The invention also relates to foods or food additives obtained from the relevant plants.

[0027] Advantages of the present invention:

[0028] The present invention obtains Arabidopsis thaliana by cloning plant genome FT Gene, constructed overexpression vector pCAMBIA1300, and obtained FT Gene overexpression lines using TDNA insertion FT Gene knockout mutant strains ft-10 , analyzed FT The biological functions of gene knockout mutant strains under fluctuating light treatment at the seedling stage were investigated. The results showed that under fluctuating light conditions, FT The knockout mutant strain is significantly more tolerant to fluctuating light than the wild type; revealing FT Genes negatively regulate the plant's adaptability to fluctuating light, providing new genetic resources for improving the adaptability of crops to fluctuating light. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The wild type and ft-10 Phenotypes of the mutant under normal light and fluctuating light.

[0030] Figure 2 The wild type and ft-10 Fresh weight analysis of mutants under normal and fluctuating light.

[0031] Figure 3 The wild type and ft-10Analysis of the maximum photochemical efficiency of mutants under normal light and fluctuating light.

[0032] Figure 4 The wild type and ft-10 Analysis of chlorophyll fluorescence parameters of mutants under fluctuating light.

[0033] Figure 5 The wild type and ft-10 Analysis of thylakoid membrane protein accumulation of mutants under normal light and fluctuating light.

[0034] Figure 6 For wild type and ft-10 Analysis of the expression of key genes of Fluorescence in mutants; Figure 6 A is the expression analysis of the nuclear-encoded subunits NDHL~NDHU of the NDH complex; Figure 6 B is SIG4 The genes were expressed in WT, ft-10, FT-OE Expression analysis in three materials

[0035] Figure 7 For wild type and FT-OE Analysis of chlorophyll fluorescence induction kinetics of overexpressed materials.

[0036] Figure 8 for FT Analysis of the fluctuating light phenotype of the affected NDH-related mutants. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below by specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples, unless otherwise specified, are conventional methods. Unless otherwise specified, the reagents and materials used can all be obtained through commercial channels.

[0038] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0039] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

[0040] The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited to these. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include a coding sequence in a cDNA, and / or include a cDNA and its regulatory sequences. In specific embodiments, such as with respect to an isolated nucleic acid sequence, it is preferably assumed to be cDNA.

[0041] Before introducing the specific embodiments, in order to facilitate those skilled in the art to understand the relevant research and development of the present application in detail, the principles, some experimental materials, detection methods, etc. of the present invention are described below in conjunction with the accompanying drawings:

[0042] 1. Extraction and reverse transcription of total RNA from plant materials

[0043] Take 0.2 g of plant material and grind it into off-white powder in liquid nitrogen for experiment. Add 600 µL Buffer EL to the powder, shake for 1 min, centrifuge for 5 min (12000 rpm) immediately after the powder is completely mixed, transfer the supernatant to adsorption column III and centrifuge for 1 min (12000 rpm), transfer the supernatant to a new tube and add 300 µL of anhydrous ethanol to adjust the column environment, mix thoroughly and add the mixture to adsorption column V in batches, centrifuge for 30 s (12000 rpm), and discard the filtrate. Then add RWA and RWB to adsorption column V step by step and centrifuge to remove impurities (RWB needs to be washed twice). After washing, replace the new collection tube and centrifuge the empty column for 2 min (12000 rpm), dry at room temperature and add water to elute (high-speed centrifugation at 4℃ for 2 min). After elution, the total RNA is temporarily stored on ice or frozen in liquid nitrogen and stored at -80℃.

[0044] After measuring the concentration of the extracted total RNA with Nanodrop, the RNA template was denatured according to the amount of 5 µg total RNA, and the PCR program was set (heating at 65℃ for 5 min, cooling at 12℃ for 5 min). After the program was completed and cooled, the denatured product was taken out and ice bathed for 2 min. After ice bathing, gDNA wiper Mix was added and mixed, and incubated at 42℃ for 2 min. RT Mix, HiScriptIII Enzyme Mix, Oligo (dT)20VN and RNase-free ddH2O were added to the mixed solution in sequence. After mixing the above liquids with a pipette, the PCR program was set (heating at 25℃ for 5 min, heating at 37℃ for 45 min, and treating at 85℃ for 5s). The cDNA product after removal can be used for subsequent experiments or stored at -80℃.

[0045] 2. Vector Construction

[0046] Overexpression vector construction process:

[0047] RNA was extracted from wild-type Arabidopsis materials, and the total RNA obtained was measured with Nanodrop and then reverse transcribed into cDNA. At the same time, the CDNA sequence of AT1G65480 was downloaded from the Tair official website (https: / / www.arabidopsis.org / ), and specific primers were designed by Primer Premier5.0. The cDNA of Arabidopsis wild-type materials was used as a template and cloned by PCR reaction (PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, 37 cycles; 72℃ extension for 5 min, 12℃ cooling for 5 s) FT The coding sequence of the gene was determined by electrophoresis of the PCR reaction product on 1% agarose gel. The target band was recovered using the DNA recovery and purification kit of Zhuangmeng and the recovered target gene was connected to pCAMBIA1300 using the ClonExpress Ultra One Step Cloning kit of Novizan (connection at 55°C for 30 min).

[0048] 3. Genetic material creation

[0049] The wild type of Arabidopsis thaliana was used as the starting plant. The constructed vector was introduced into Agrobacterium, and the T0 generation seeds were obtained by infecting the Arabidopsis thaliana inflorescence with Agrobacterium, and the resistance screening was performed to obtain FT Overexpression strains ( FT-OE ). ft-10The mutant was kindly donated by Researcher Yu Diqiu from the Xishuangbanna Institute of Tropical Botany, Chinese Academy of Sciences. The mutant was obtained by inserting T-NDA into the target gene.

[0050] 4. FT Wavelight treatment of related materials

[0051] After 7 days of growth on the culture medium, the culture dishes were placed under different fluctuating light treatments. In this experiment, the fluctuating light was set to 500 μmol m -2 s -1 5s / 50 μmol m -2 s -1 25 s and a constant 125 μmol m -2 s -1 Light intensity was used as a control. The materials were placed under different light intensity conditions for one week and then taken out for phenotypic observation and subsequent experiments. For the seedlings for parameter measurement, seedlings grown on the substrate for 3 weeks were used. Plants of the same size were selected and placed under the above different light intensity conditions for one week and then taken out for subsequent parameter and phenotypic observation. FT Deletion mutant ft-10 The wild type was treated with high-frequency light fluctuations, and the results showed that compared with the control, ft-10 It grows better under high-frequency fluctuating light ( Figure 1 ), further testing of the fresh weight and Fv / Fm of several plant materials revealed that although the high-frequency fluctuating light treatment of WT and ft-10 The difference in Fv / Fm is small, but ft-10 The fresh weight of Figure 2 , Figure 3 ), which indicates that FT is involved in the adaptation of plants to high-frequency fluctuating light.

[0052] 5. FT Determination of chlorophyll fluorescence parameters of related materials

[0053] The in vivo PSII and PSI parameters were measured simultaneously using a Dual-PAM-100 modulated fluorescence instrument (Walz, Germany) based on the changes in chlorophyll fluorescence and P700 absorbance, respectively. Before the measurement, the Arabidopsis material was placed in the dark for 20 minutes of dark adaptation, and then its light response curve was measured. First, the actinic light was set to 0, 89, 167, 267, 416, 606, 918, 1173, and 1450 μmol m -2 s -1, each actinic light lasted for 30 seconds. Calculation method of PSII and PSI parameters: maximum quantum yield of PSII Fv / Fm = (Fm - F0) / Fm; effective yield of PSII under actinic light Y (II) = (Fm' - F) / Fm'; non-photochemical quenching NPQ = (Fm - Fm') / Fm'; effective yield of PSI under actinic light Y (I) = 1 - Y (ND) - Y (NA); acceptor side limitation of PSI Y (NA) = (Pm - Pm') / Pm; donor side limitation of PSI Y (ND) = P / Pm. Where P is the P700+ signal under actinic light. Pm is the maximum oxidizable P700 value, which is calculated by applying a saturated white light pulse (4000 μmol m -2 s -1 , duration 500 ms). The maximum fluorescence (Fm') and the maximum oxidizable P700 (Pm') under actinic light were measured periodically with saturation pulses. To calculate Fv / Fm, the cells were placed in the dark for 20 min and the minimum (F0) and maximum fluorescence (Fm) in the dark-adapted state were measured. Regarding the fluctuating light simulation experiment. When simulating fluctuating light with DUAL-PAM-100, the actinic light intensity was adjusted to 500 μmol m -2 s -1 5s / 50 μmol m -2 s -1 25 s and 500 μmol m -2 s -1 100s / 50 μmol m -2 s -1 500s, other conditions remain unchanged, record the changes in fluorescence parameters of plant materials under real-time fluctuating light. Four-week-old Arabidopsis materials were placed under high-frequency fluctuating light for one week, and the treated plant materials were measured. The measurement results are as follows Figure 4 As shown, under high-frequency fluctuating light ft-10 The quantum yield of the mutant's photosystem was significantly higher than that of the wild type, and its donor-side limitation and acceptor-side limitation were further tested. The results showed that ft-10 The Y(ND) of the mutant is not much different from that of the wild type, but the Y(NA) is significantly lower than that of the wild type. ft-10 The electron transfer on the acceptor side of photosystem I was relatively smooth, indicating that the absence of FT under high-frequency fluctuating light may have a positive effect on the photoprotection of photosystem I.

[0054] 6. Arabidopsis Total Protein Extraction and Immunoblotting

[0055] Weigh fresh plant materials and place them in a mortar. Add an appropriate amount of extract according to the ratio of 0.05 g fresh weight / 200 μL of total protein extract (E buffer: 125 mM Tris-HCl, pH 8.8; 50 mM Na2S2O5; 1% (w / v) SDS; 10% (w / v) Glycerol). Grind thoroughly and transfer to a 1.5 mL centrifuge tube. Shake for 15 min. Centrifuge at room temperature at 12,000 g for 10 min. Pipette the supernatant into a new 1.5 mL centrifuge tube, which is the plant total protein solution. If the solution is still turbid, centrifuge several times and take the supernatant. Then perform protein quantification. Use bovine serum albumin (BSA) as the standard. Use an ultraviolet spectrophotometer to measure the absorbance of the protein at 750 nm and make a standard curve. Dilute the plant protein sample to the range of the standard curve, measure the absorbance, and calculate the total protein concentration in the sample according to the standard curve. Prepare SDS-PAGE premix: 0.2% (w / v) SDS, 6 M Urea, 4.5% acrylamide (upper stacking gel, pH 6.8) or 15% acrylamide (lower separation gel, pH 8.8). Prepare 10% or 20% acrylamide lower separation gel according to the size of the protein to be separated. Place the prepared gel in the electrophoresis instrument, add an appropriate amount of electrophoresis buffer (SDS-PAGE electrophoresis buffer: 0.303% (w / v) Tris, 1.44% (w / v) Glycine, 0.1% (w / v) SDS), and wash the sample wells. Add loading buffer (5 mM Tris-HCl, pH 8.8; 1.2% (w / v) SDS; 1% (w / v) Glycerol; 1% (w / v) β-Mercaptoethanol; 0.0001% (w / v) Bromophenol blue), add the sample to the sample well; electrophoresis, 10-20 mA / block, bromophenol blue indicator indicates the front position of the electrophoresis sample, and the electrophoresis time is adjusted according to the size of the target protein; carefully remove the gel and place it in the transfer buffer (1% (v / v) SDS-PAGE electrophoresis buffer, 2% (v / v) Methanol); place the filter paper and nitrocellulose membrane in the transfer buffer and soak them in advance for about 10 min; from the negative electrode to the positive electrode, lay the sponge, three layers of filter paper, gel, nitrocellulose membrane, three layers of filter paper, and sponge flat in the transfer folder in turn, use a glass rod to drive out the bubbles between the layers, and keep the layers moist during the operation; place the transfer folder vertically in the electrophoresis tank and completely immerse it in the transfer buffer, 180mA, 90 min; take out the membrane, immerse it in the blocking solution with the side in contact with the gel facing down, 30-50 rpm, room temperature for 1 h or 4℃ for more than 3 h; transfer the membrane to a 1% (w / v) skim milk powder in TTBS buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% (v / v) Tween-20), add a certain dilution ratio of the target protein specific antibody (primary antibody) according to the antibody titer and the amount of target protein loaded, 30-50 rpm, room temperature for 6-8 h or 4℃ overnight; rinse with TTBS buffer after incubation of primary antibody 3-5 times, 5 min / time; transfer the membrane to TTBS buffer containing 1% (w / v) skim milk powder, add horseradish peroxidase-conjugated specific secondary antibody at a dilution of 1:10,000, 30-50rpm, room temperature for 1 h or 4℃ for 2-4 h; rinse with TTBS buffer after incubation of secondary antibody 3-5 times, 5 min / time; add horseradish peroxidase substrate to the membrane, and perform immunoblotting detection by chemiluminescence. .

[0056] The immunoblotting method was used to analyze the WT and ft-10 The steady-state levels of photosynthetic electron transport chain protein complexes in vivo were examined. Figure 5 As shown, after 1 week of high-frequency light treatment, WT and ft-10 There was no significant change in the content of ATPase in the photosystem II; the content of core protein in the photosystem II did not change significantly. ft-10 The content of photosystem II antenna is lower than that under normal light conditions. It is worth noting that ft-10 The content of photosystem II antenna is higher than that of wild type, indicating ft-10 Compared with normal light treatment, WT and ft-10 The photosystem I antenna proteins decreased, butft-10 Finally, we detected the content of PGRL1 and NDHH, and found that WT and ft-10 The content of PGRL1 in ft-10 The NDHH content in the plants was significantly higher than that in WT, so FT may participate in the adaptation of Arabidopsis to high-frequency fluctuating light by affecting the content of NDH complex.

[0057] 7. Fluorescence quantitative detection of the expression of key genes affecting light fluctuations in FT-related materials

[0058] We wanted to know whether FT affects the protein content of NDH complex subunits by affecting their expression, so we performed fluorescence quantitative experiments on NDH subunits. There are many NDH complex subunits, some of which are encoded by the cell nucleus, such as NDHL~NDHU; others are encoded by the chloroplast nucleoid, such as NDHA~NDHI. These genes are currently known to be regulated by SIG4, and SIG4 Genes are encoded in the nucleus. Due to the nuclear localization of FT, we believe that it directly affects the nuclear-encoded subunits of NDH and SIG4 The possibility of gene is greater, so we carried out the following experiments: extract wild type, ft-10 The RNA was taken and reverse transcribed into cDNA. The cDNA template prepared by the reverse transcription experiment was diluted according to the abundance of the gene to be detected in the body. Generally, it was diluted 50-100 times (except for genes with extremely low abundance). Specific gene primers were designed on Primer Premier5, and the designed primer sequences were handed over to the Zhengzhou branch of Sangon for synthesis. After obtaining the primers, the reaction system was prepared using the SYBR qPCR Master Mix kit provided by Nanjing Novozymes Biotech Co., Ltd.: (20 μL total system: 10 μL SYBR dye, 1 μL primer Mix (forward and reverse primers 1:1 mixed into primer Mix), 2 μL cDNA template and 7 μL RNase-free ddH2O). The mixed reaction system was dispensed into a 96-well plate with a pipette and sealed with a film. The 96-well plate was then centrifuged and placed in a real-time quantitative PCR instrument (Light Cycler480) for amplification. After the experiment was completed, the data was exported. After analysis, we found that in ft-10 The expression of some nuclear-encoded subunits of NDH complex was significantly upregulated. NDHO , NDHT In the FT overexpression materials, the expression levels of the nuclear-encoded subunits of the NDH complex were mostly downregulated; in addition, SIG4 This study also detected the NDH complex in these materials. SIG4The results showed that the overexpression material of FT FT-OE middle SIG4 The transcription of Figure 6 A. Figure 6 B).

[0059] 8. Chlorophyll fluorescence induction dynamics and transient experiments after illumination

[0060] Fully dark-adapted Arabidopsis leaves were placed under actinic light (50 μmol m -2 s -1 ) were used to record the typical induction kinetics of chlorophyll fluorescence. The measurements were made using a measuring light of approximately 0.2 μmol m -2 s -1 , actinic light is about 120 μmol m -2 s -1 , turn off the actinic light after the baseline stabilized under normal air conditions, and record the changes in chlorophyll fluorescence signals at the moment of light off. The results showed that compared with WT, after the light was turned off, the overexpression material FT-OE The transient increase of chlorophyll fluorescence in the medium was smaller, which indicates that the activity of NDH complex in this material is indeed reduced ( Figure 7 ).

[0061] 9. Fluorescence treatment of NDH complex-related materials

[0062] To verify that the phenotype of FT-related materials is caused by the NDH complex, we selected SIG4 Gene deletion mutant sig4 and NDH complex loss-of-function mutants ndho , and perform wave light processing on it 。 After the plant material was grown on the culture medium for 7 days, the culture dish was placed under different fluctuating light treatments. The fluctuating light in this experiment was set to 500 μmol / m -2 s -1 5s / 50 μmol m -2 s -1 25 s and a constant 125 μmol m -2 s -1 Light intensity was used as a control. The materials were placed under different light intensity conditions for one week and then taken out for phenotypic observation and subsequent experiments. For the seedlings for parameter measurement, seedlings grown on the substrate for 3 weeks were used. Plants of the same size were selected and placed under the above different light intensity conditions for one week and then taken out for subsequent parameter and phenotypic observation. The results are shown in Figure 8 As shown in the figure, under constant light, the three genotype materials grew in the same way, with little difference in fresh weight and Fv / Fm; under fluctuating light, the three materials were all affected, but to different degrees, as shown in the following:ndho and sig4 The impact is more serious, so FT should affect the adaptability of Arabidopsis to fluctuating light by affecting the activity of NDH complex. Therefore, the present invention discloses a method for obtaining plants with strong adaptability to fluctuating light by genetic modification. Specifically, by FT The gene is knocked out from the target plant to obtain a transgenic plant, which has a higher adaptability to fluctuating light than the target plant, providing a new way to breed plant varieties with strong adaptability to fluctuating light.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. FT The use of a gene in negatively regulating plant adaptation to fluctuating light is characterized in that: Said FT The nucleotide sequence of the gene is shown in SEQ ID NO.

1. The negative regulation is manifested in reducing FT Plants expressing the gene have improved adaptability to fluctuating light, while overexpressing FT The plant with the gene has reduced adaptability to fluctuating light, the plant is Arabidopsis thaliana, and the fluctuating light condition is 500 μmol m -2 s -1 5s / 50μmol m -2 s -1 25s.

2. The use according to claim 1, characterized in that: reduce FT The gene expression method is to use TDNA to insert the target gene to obtain FT Gene knockout strains, FT The knockout strains were more adaptable to fluctuating light than the wild type.

3. The use according to claim 2, characterized in that: Using wild-type Arabidopsis as the starting plant, the constructed transgenic vector was introduced into Agrobacterium, and the Arabidopsis inflorescence was infected by Agrobacterium, and resistance screening was performed to obtain FT Knockout strains.

4. The use according to claim 1, characterized in that: Said FT The gene is from Arabidopsis thaliana FT Gene.

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