Genes, recombinant vectors and uses for regulating flowering in plants

Abstract

The application belongs to the field of plant growth and relates to a gene for regulating flowering and disease resistance of plants, in particular to a gene for regulating flowering, a recombinant vector and application. In the research process of disease resistance breeding, a NBS-LRR type gene of Nicotiana benthamiana is cloned by the research group NbLOV1 . Bioinformatics analysis results show that the gene is closely related to the response of plants to fungal pathogens, and in the experiment of verifying the gene function by gene editing knockout, the plants created by using the knockout vector for NbLOV1 and the Agrobacterium-mediated transformation method have obvious differences compared with the control: after knocking out NbLOV1 , the budding and flowering time of Nicotiana benthamiana is obviously earlier than that of the control. The mutant plant with the knockout NbLOV1 gene presents strong resistance to fusarium, only with slightly yellow leaf color and slight wilting; while the tobacco of the control presents damaged vascular bundle, seriously wilting leaf surface and necrotic spots.

Description

Technical Field

[0001] This invention belongs to the field of plant growth and relates to genes that regulate plant flowering time, particularly genes, recombinant vectors, and applications used to regulate plant flowering time. Background Technology

[0002] Plant flowering is influenced by a variety of internal factors, including photoperiod and endogenous hormones, as well as external environmental factors. Flowering period is a key factor affecting the length of tobacco's growth cycle and determining the number of effective leaves; it is an important stage in the life cycle of flowering plants, marking the transition from vegetative to reproductive growth. Flowers, fruits, and seeds are the main products of many crops; therefore, flowering determines the market availability and economic value of agricultural products. Through long-term selective evolution, plants have developed a complex and sophisticated regulatory network to respond to various internal and external signals (such as photoperiod, temperature, age, and gibberellins), thereby regulating flowering to ensure that plants bloom at the optimal time.

[0003] Flowering time regulation involves artificially altering the external environment of a plant or modifying its genes or internal components through biotechnology, based on the plant's flowering habits and growth patterns, to advance or delay flowering. Flowering time regulation enhances the economic value of plants. For example, early-flowering fruit trees can be protected from or lessened by early spring frosts; vegetables, fruits, flowers, and crops can be supplied out of season, such as winter cucumbers, tomatoes, and strawberries. Furthermore, for hybridization breeding, flowering time control technology can cause hybrid parents with previously mismatched flowering periods to flower simultaneously, resolving the timing conflict in cross-pollination and facilitating breeding efforts. Therefore, plant flowering time regulation has significant agricultural and economic value. Patent 202110374691.1 discloses a transcription factor LbNAP that delays the flowering period of lilies. By reducing the expression level of the LbNAP transcription factor, the flowering period of plants is prolonged. A virus-induced gene silencing vector containing the LbNAP transcription factor gene fragment is constructed based on tobacco brittle virus TRV. Patent 201911317235.2 discloses the gene NtDUF599, which is related to early flowering of tobacco under low temperature. This gene advances the flowering cycle under low temperature stress. Genes that shorten the flowering cycle of plants have always been a hot research topic in breeding work. Shortening the growth period of germplasm materials by inducing earlier flowering and other methods, thereby achieving multiple generations per year and improving breeding efficiency, is the research direction of this research group. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a gene, a recombinant vector, and its application for regulating plant flowering time.

[0005] The technical solution of this invention is implemented as follows:

[0006] In the course of research on disease-resistant breeding, this application cloned an NBS-LRR gene from Nicotiana benthamiana. NbLOV1, The sequence is shown in SEQ ID No. 1. Bioinformatics analysis results indicate that this gene is closely related to the plant's response to fungal pathogens. Experiments verifying gene function through gene editing and knockout revealed that knockout... NbLOV1 Afterwards, the budding and flowering time of *Nicotiana benthamiana* was significantly earlier than that of the control.

[0007] On the one hand, this application seeks protection for a gene used to regulate the flowering period of plants, said gene being a protein containing nucleotide binding sites and leucine-rich repeat sequences.

[0008] Furthermore, the amino acid sequence of the gene is shown in SEQ ID No. 2.

[0009] The nucleotide sequence encoding the above gene is shown in SEQ ID No. 1.

[0010] Recombinant vectors using the above-mentioned genes as target genes.

[0011] Preferably, the recombinant vector is a CRISPR-based gene editing recombinant vector.

[0012] Furthermore, the recombinant vector is a pORE-Cas9 editing vector or a pDC45 editing vector.

[0013] The above-mentioned recombinant vectors are used to shorten the flowering period of plants.

[0014] The above-mentioned recombinant vectors are used in the cultivation of new varieties that have been developed over a year.

[0015] The above-mentioned recombinant vectors are used to enhance plant resistance to Fusarium diseases.

[0016] The application steps are as follows: The recombinant vector is transferred into the sample plants using Agrobacterium-mediated genetic transformation technology to prepare varieties with shortened flowering period and / or resistance to Fusarium diseases.

[0017] Preferably, the plant is tobacco.

[0018] Furthermore, the plant in question is Nicotiana benthamiana.

[0019] The present invention has the following beneficial effects:

[0020] 1. The newly discovered gene in this application NbLOV1 Not only can it respond to disease-related genes, but knocking out these genes can also shorten the flowering cycle. The gene-edited positive lines created in this application showed significant differences in disease resistance compared to the control. The constructed knockout... NbLOV1Tobacco plants transfected with recombinant gene vectors showed strong resistance to Fusarium: only the leaves turned slightly yellow and wilted slightly, while the control tobacco plants showed vascular bundle damage, more severe wilting on the leaf surface, and necrotic spots.

[0021] 2. Knockout using the method described in this application NbLOV1 The new plants of the gene-derived variety flowered significantly earlier than the control. Under the same culture conditions, the control Nicotiana benthamiana needed to grow to nearly 20 leaves and a height of over 20 centimeters before budding and flowering, while... NbLOV1 Gene-edited mutants will bud and bloom when they have fewer than 10 leaves and are only about 10 centimeters long, shortening the flowering cycle by about half. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 for NbLOV1 Electrophoretic gel image of the gene's CDS region.

[0024] Figure 2 for NbLOV1 A schematic diagram of the gene structure.

[0025] Figure 3 The results show the PCR amplification and sequencing verification of the gene editing vector. A. PCR electrophoresis image, M is the DL2000 marker; 1, 2, and 3 are the Cas9, U26, and gRNA fragments in strain P1, respectively; B. The target site sequence of the sgRNA in the constructed vector is compared with the original vector sequence, and the highlighted part is the target site sequence.

[0026] Figure 4 This is an electrophoresis image of positive plants amplified by PCR, where M is the DL2000 marker; 1-11 are, respectively, [images of positive plants]. NbLOV 5. NbLOV -6、 NbLOV -8、 NbLOV 9. NbLOV -10、 NbLOV 11. NbLOV 15. NbLOV 20. NbLOV 28. NbLOV 31. NbLOV The band at 45; 12 is the positive control, and 13 is the negative control.

[0027] Figure 5 The images show the budding stage of gene-edited positive lines and control lines under the same culture conditions.

[0028] Figure 6 The image shows the gene editing results for NbLOV28 and NbLOV31, where the underlined areas represent the target sites and the red areas represent the editing results.

[0029] Figure 7 This is a comparison chart of the budding stage under potted conditions.

[0030] Figure 8 This is a bioinformatics diagram of the protein structure.

[0031] Figure 9 Images show the condition of leaves from different plant varieties after their petioles were soaked in crude toxin. The left side represents the wild type, and the right side represents the resistant plant. NbLOV1 .

[0032] Figure 10 This is a photograph of tobacco plants 30 days after inoculation with Fusarium oxysporum. The left side shows... NbLOV1 The mutant plant shows disease resistance; the wild-type control on the right shows susceptibility to the disease. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Experimental materials and reagents

[0035] The seeds of Nicotiana benthamiana were kindly donated by Professor Li Dawei of China Agricultural University. Aseptic seedlings were cultured in a light incubator in our key laboratory.

[0036] The culture medium consisted of 2.2 g / L MS basal salt, 20 g / L sucrose, Gamborg's vitamins, and 8 g / L agar. The culture conditions were 16 hours of light followed by 8 hours of darkness, with a temperature set at 25-28°C.

[0037] The pORE-Cas9 editing vector was provided by Southwest University, and the pDC45 editing vector was kindly donated by Professor Dai Changbo of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences (CN 113667689A has been published).

[0038] The restriction endonuclease Bsa I (NEB) was purchased from Geneplus Ltd.

[0039] Plant genome extraction was performed using CTAB reagents, and RNA extraction was performed using a kit. The main reagents were purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd.

[0040] Primers were prepared at Shanghai Bioengineering Co., Ltd., and sequencing was performed at Qingke Biotechnology. PCR instruments, electrophoresis apparatus, and gel imaging systems were purchased from Bio-Rad Laboratories, USA.

[0041] Example 1: Gene Cloning and Structural Analysis

[0042] NbLOV1 Gene cloning: The putative sequence information of the *Nicotiana benthamiana* gene was obtained using the Solanaceae Genome Database (https: / / solgenomics.net) and the *Nicotiana benthamiana* Genome and Transcriptome Database of Queensland University of Technology (QUT) (https: / / benthgenome.qut.edu.au / ). Primers were designed to amplify the sequence using *Nicotiana benthamiana* DNA and cDNA templates, and the sequence correctness was verified by sequencing. Primer sequence information is shown in Table 1.

[0043] Gene structures were drawn using an online tool. Online tool: http: / / gsds.gao-lab.org / index.php

[0044] Table 1. Primers and their sequence information used in this paper

[0045]

[0046] After cloning the gene, NbLOV1 The position of the fragment on the electrophoresis image, such as Figure 1 As shown. NbLOV1 The gene sequence is attached. The CDS region of this gene is 2574 bp, encoding a protein composed of 857 amino acids. By comparing genomic information, this gene contains 2 introns, 3 exons, and 2 UTR regions, as shown below. Figure 2 As shown. Analysis of the protein structure encoded by this gene yielded the following results. Figure 8 As shown, by Figure 8 It can be known that this protein is a protein containing nucleotide binding sites and leucine-rich repeat sequences (NB-LRR).

[0047] Example 2: sgRNA design, vector construction and validation

[0048] Based on gene CDS sequences, editing sites were designed using online tools such as CRISPRdirect (http: / / crispr.dbcls.jp / ) and CRISPR MultiTargeter (http: / / www.multicrispr.net / ). Target sites with the highest scores and lowest off-target probabilities were selected through cross-matching. DNA at the target sites was obtained via an annealing reaction using 4 μl Annealing Buffer for DNA Oligos reagent, 4 μl each of forward and reverse primers, and 8 μl of ultrapure water, under a programmed cooling system (95℃ for 5 min, decreasing by 0.1℃ every 8 s until the temperature reached 25℃).

[0049] CRISPR / Cas9 vector digestion, ligation, and validation: Enzyme digestion was performed using NEB CutSmart buffer, BsaI enzyme, and a 50 μl volume. The digestion and ligation process was performed according to the kit instructions. The digestion and annealing products were ligated to a linearized pORE-Cas editing vector or pDC45 editing vector using T4 ligase. After transformation of DH5α competent cells, the U26-jiance-F sequence on the vector was used as the upstream primer, and the reverse complementary sequence of the target gene site was used as the downstream primer. Colony PCR was used to screen positive clones, followed by sequencing validation to ensure successful insertion of the target fragment into the vector. Electrophoresis results are shown below. Figure 3 As shown, the correctness of the vector was verified by PCR amplification and sequencing.

[0050] Example 3: Genetic transformation of Agrobacterium and Nicotiana benthamiana using editing vectors

[0051] After the vector was sequenced and verified, single clones with completely correct sequence information were selected, expanded for culture, and plasmid DNA was extracted. These clones were then transformed into Agrobacterium GV3101 competent cells using the freeze-thaw method. After shaking, the plasmid was verified for correctness by colony PCR before being used in genetic transformation experiments. Specific steps were performed according to the competent cell instruction manual.

[0052] Agrobacterium-mediated genetic transformation was used to obtain T0 generation *Nicotinus benthamiana*-positive seedlings resistant to kanamycin. After verification by PCR and electrophoresis, 11 transgenic *Nicotinus benthamiana* lines containing the Cas9 sequence were screened out. NbLOV 5. NbLOV -6、 NbLOV -8、 NbLOV 9. NbLOV -10、 NbLOV 11. NbLOV 15. NbLOV 20. NbLOV 28. NbLOV 31. NbLOV 45) of which NbLOV 5. NbLOV- 6. NbLOV -8、 NbLOV 9. NbLOV -10 uses pORE-Cas as the CRISPR / Cas9 editing vector, while the other lines use pDC45 as the CRISPR / Cas9 editing vector. Figure 4 As shown;

[0053] Under the same culture conditions, such as Figure 5 As shown, the gene-edited positive lines that survived and grew well in the later stages of the two experiments... NbLOV-5, NbLOV-6, NbLOV-8, NbLOV9, NbLOV-10, NbLOV20, NbLOV28, NbLOV31 Compared to wild-type Nb-WT plants, they bud and flower earlier; in culture bottles, NbLOV1 The gene-edited Nicotiana benthamiana strains budded when they had only 5-7 effective leaves and were only about 5 cm tall, and flowered about a month later. Under the same culture conditions, the control wild-type Nicotiana benthamiana did not bud when it had 10 effective leaves, and only budded after about 2 months of culture. NbLOV1 The gene-edited plants had a flowering cycle that was about half that of the control wild-type plants. NbLOV28 and NbLOV31 positive plants were selected for hi-tom sequencing analysis. NbLOV1 The results of gene editing, such as Figure 6 As shown, the red text represents the editing results, and it can be seen that both plants are new plants successfully edited by CRISPR.

[0054] When tobacco plants are transplanted and grown in pots, they exhibit the same trend, such as... Figure 7 As shown: the control group of Nicotiana benthamiana needed to grow to nearly 20 leaves and a plant height of over 20 centimeters before it budded and flowered, while... NbLOV1 The gene-edited mutant NbLOV45 will bud and bloom when it has fewer than 10 leaves and is only about 10 centimeters long. NbLOV1 The gene-edited NbLOV45 plants had a flowering cycle that was half that of the control wild type.

[0055] Example 4: Disease resistance experiment of positive plants

[0056] ① Activation of pathogens: Take the strain stored at -80℃ (for long-term storage), inoculate it into PDA (potato dextrose agar) medium, and place it in an incubator at 25-28℃ for 5-7 days in the dark.

[0057] ② Liquid culture of pathogens: Take 1-3 pieces (approximately 5-10 mm in diameter) from the edge of the mycelium in the activated culture dish. 2The mycelium cake is transferred to a 250mL Erlenmeyer flask containing approximately 150mL of sterile PDB (potato glucose liquid) medium. The flask is then placed on a constant-temperature shaker at 25-28℃ and 180 rpm for 3 to 5 days, with 3 days being optimal.

[0058] ③ Preparation of crude toxin: Take the liquid culture from step 2, pour it into a funnel lined with 4 layers of sterile gauze, filter to remove mycelia, transfer the filtrate into a centrifuge tube, and centrifuge at 8000 rpm and 4℃ for 15 min. The resulting supernatant is the crude toxin, which is then filtered through a 0.22-micron filter membrane for later use.

[0059] ④ Apply crude toxin to identify resistance: Take a 300ml glass culture bottle, pour in about 30ml of the above crude toxin solution, and quickly cut off the base of the petiole with a sharp blade to take fresh leaves from the tobacco plant. Place the petiole in the toxin solution and closely observe the changes in the condition of the leaves.

[0060] ⑤ Assessment of Fusarium resistance: 3-7 days after the petioles are immersed in crude toxin, different leaf conditions will be observed in different varieties (strains), such as... Figure 9 As shown, different plant varieties respond differently to toxins, with gene-edited resistant plants exhibiting varying degrees of resistance. NbLOV The leaves of the wild control plant were slightly yellow and slightly wilted, while the leaves of the wild control plant showed vascular bundle damage, more severe wilting, and necrotic spots.

[0061] ⑥ Tobacco plants 30 days after inoculation with Fusarium, such as Figure 10 As shown, the left side is NbLOV1 Gene-edited positive plants NbLOV The right side shows the wild-type control without genetic manipulation. Figure 10 It is clear that the gene-edited positive plants showed strong resistance to Fusarium, while the control group plants showed severe wilting, death and other symptoms.

[0062] Implementation Results Example

[0063] By editing Ben's tobacco NbLOV1 After obtaining the edited strain, observation and analysis of inoculated pathogens revealed that... NbLOV1 Gene editing can lead to: 1. Earlier flowering of Nicotiana benthamiana; 2. Increased resistance to Fusarium.

[0064] show NbLOV1 This gene is involved in regulating flower development (growth period regulation) in Nicotiana benthamiana and its response to Fusarium. These results indicate that this gene has certain application value in breeding work such as regulating crop growth period and improving resistance.

[0065] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A protein used to regulate the flowering period of plants, characterized by: The protein is a protein containing nucleotide binding sites and leucine-rich repeat sequences; The amino acid sequence of the protein is shown in SEQ ID No.

2.

2. A gene encoding the protein of claim 1, the nucleotide coding sequence of which is shown in SEQ ID No.

1.

3. A recombinant vector containing the gene described in claim 2.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector is a CRISPR-based gene editing recombinant vector.

5. The application of the recombinant vector according to claim 4 in shortening the flowering period of plants, characterized in that: The plant in question is *Nicotiana benthamiana*.

6. The application of the recombinant vector according to claim 4 in enhancing plant resistance to Fusarium diseases, characterized in that: The plant in question is *Nicotiana benthamiana*.

7. The application according to claim 5 or 6, characterized in that, The application steps are as follows: the recombinant vector is transferred into the sample plants through Agrobacterium-mediated genetic transformation technology to create varieties with shortened flowering period and / or resistance to Fusarium diseases.

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