Application of DIV1 gene in regulation and control of flowering time of plant

Abstract

The invention relates to the technical field of plant genetic engineering, and provides application of a DIV1 gene in regulation and control of plant flowering time. The application of the DIV1 gene is applied to at least one of the following S1-S2: S1, regulating and controlling the flowering time of a plant; and S2, cultivating a transgenic plant. By means of the technical scheme, the problems that in the prior art, plants are late in flowering time, long in growth cycle and difficult to adapt to various cultivation systems are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and specifically to DIV1 Application of genes in regulating flowering time in plants. Background Art

[0002] In higher plants, flowering is a key stage in the transition from the vegetative stage to the reproductive stage, and plays an important role in the reproduction of offspring and ecological adaptation. In agricultural production, timely flowering of crops is of great significance for breeding of improved varieties, introduction and breeding, stable yield and increased yield. The flowering time of plants is regulated by external environmental factors and their endogenous signals. At present, many pathways involved in flowering regulation have been discovered, including photoperiod pathway, vernalization pathway, autonomous pathway and gibberellin pathway. However, in plant breeding work, there are still problems such as difficulty in adapting to various farming systems and long growth cycles. It is necessary to explore how to shorten the flowering time of plants and make plants bloom earlier, so as to obtain offspring with excellent traits faster and accelerate the cultivation and promotion of new varieties.

[0003] MYB transcription factors are one of the largest transcription factor family members in plants and play an important regulatory role in plant growth, development and physiological metabolism. DIV1 Encodes an RR-type MYB transcription factor, which plays an important role in regulating plant growth and development. DIV1 Acts as an activator to mediate ABA signaling and regulate the biosynthesis of capsanthin; Arabidopsis thaliana DIV1 Positive regulation of seed germination in response to salt stress.

[0004] Although researchers have isolated Arabidopsis and rapeseed DIV1 Genes have been preliminarily studied for their functions in plant growth and development. DIV1 The function of genes in regulating plant flowering time needs further study. Summary of the invention

[0005] The present invention proposes DIV1 The application of genes in regulating the flowering time of plants solves the problems in related technologies such as late flowering time, long growth cycle and difficulty in adapting to various farming systems.

[0006] The technical solution of the present invention is as follows: The present invention proposes DIV1 Application of genes, application of at least one of the following S1-S2: S1. Regulation of flowering time in plants; S2. Cultivate transgenic plants.

[0007] As a further technical solution, the DIV1The nucleotide sequence of the gene is shown in SEQ ID NO:1 or SEQ ID NO:3.

[0008] As a further technical solution, the DIV1 Gene-encoded protein DIV1 The amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0009] As a further technical solution, when the application is S1, the plant flowering time regulation is as follows: the plant flowering time changes with the DIV1 The expression of the gene increases and shortens.

[0010] As a further technical solution, the DIV1 The gene promotes flowering in plants by promoting the synthesis of gibberellins.

[0011] As a further technical solution, when the application is S2, the cultivating of transgenic plants is cultivating plants that bloom early.

[0012] As a further technical solution, the plant is Arabidopsis thaliana or rapeseed.

[0013] The present invention also provides a method for cultivating transgenic plants, comprising the following steps: The DIV1 The gene is expressed in the recipient plant, resulting in overexpression transgenic plants.

[0014] As a further technical solution, the DIV1 The gene expression in the recipient plant is achieved by: DIV1 The gene is connected to the overexpression vector to obtain a recombinant vector, and the recombinant vector is introduced into the recipient plant.

[0015] As a further technical solution, the overexpressing transgenic plant has a shortened flowering time compared with the recipient plant.

[0016] The working principle and beneficial effects of the present invention are: 1. The present invention reveals in the wild type (Col-0) of the model plant Arabidopsis thaliana DIV1 The function of genes in regulating flowering time can effectively promote early flowering of plants and shorten the plant growth cycle. DIV1 The biological function of genes in regulating flowering time in plants is given DIV1 Application of genes in regulating flowering time of plants, and at the same time, introduction into recipient plants DIV1 Genes, along with DIV1 The expression level of the gene increases, and the flowering time of the plant is significantly shortened. This invention fills the gap DIV1Gaps in the application of genes in genetic engineering.

[0017] 2. The present invention is of great significance for analyzing the transcriptional regulation mechanism during the flowering time of plants, controlling the flowering period, and cultivating new varieties of short-growing crops. In practical applications, the flowering time of plants can be controlled by adjusting the photoperiod and water and fertilizer management during the seedling stage, which can shorten the plant growth cycle. This is of great value for cultivating short-growing crops in agriculture to adapt to different crop rotation methods, adjust the growth period, and adapt to crops in various farming systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0019] Figure 1 Arabidopsis thaliana AtDIV1 Identification results of gene overexpression transgenic plants; among them, Figure 1 A is AtDIV1 Schematic diagram of the recombinant overexpression vector of the gene; Figure 1 B is AtDIV1 Agarose gel electrophoresis results of DNA level identification of gene overexpression transgenic plants. Cas represents the recombinant vector pGreen-35S:AtDIV1-6HA, which is the positive control of the experiment; Col-0 represents the wild-type recipient plant, which is the negative control of the experiment; Figure 1 C is the fluorescence quantitative detection of overexpression transgenic plants AtDIV1 Gene transcription level statistical histogram; Figure 2 Arabidopsis thaliana wild type (Col-0), mutant ( div1-1 and div1-2 )and AtDIV1 Effects of overexpression on flowering time of plants; Figure 2 A shows the wild type (Col-0), mutant and AtDIV1 Photos of the growth status of overexpressing plants; Figure 2 B is Arabidopsis wild type (Col-0), mutant ( div1-1 and div1-2 )and AtDIV1 Statistical bar graph of the number of rosette leaves and cauline leaves in overexpressing plants; Figure 2 C represents Arabidopsis wild type (Col-0), mutant ( div1-1 and div1-2 )and AtDIV1 Statistical bar graph of bolting time of overexpressing plants; Figure 3 Arabidopsis wild type (Col-0) and BnaDIV1 The results of the effects of overexpression on flowering time in plants; Figure 3A shows the wild type Arabidopsis thaliana (Col-0) and BnaDIV1 Photos of the growth status of overexpressing plants; Figure 3 B is the fluorescence quantitative detection of overexpression transgenic plants BnaDIV1 Gene transcription level statistical histogram; Figure 3 C represents Arabidopsis wild type (Col-0) and BnaDIV1 Statistical bar graph of the number of rosette leaves in overexpressing plants; Figure 3 D is a statistical bar graph of bolting time of Arabidopsis wild type (Col-0) and overexpression plants; Figure 4 for DIV1 The influence of genes on the content of endogenous gibberellins; among them, Figure 4 A shows the wild type (Col-0) and mutant ( div1-1 The results of determination of endogenous gibberellin (GA3) content in plants of the overexpression line (Col-0 35S:AtDIV1-6HA #4); Figure 4 B is the Arabidopsis wild type (Col-0), mutant ( div1-1 GA3 biosynthesis genes in plants of the overexpression line (#4) GA3ox1 The transcription level of Figure 5 The effects of exogenous gibberellins on Arabidopsis wild type (Col-0) and mutant ( div1-1 ) The impact of flowering time; among them, Figure 5 A shows the wild type (Col-0) and mutant ( div1-1 ) Photos of the plant’s growth status; Figure 5 B shows the control group (CK) and the group sprayed with GA3 for Arabidopsis wild type (Col-0) and mutant ( div1-1 ) Statistical bar chart of plant bolting time; Figure 5 C is the wild type (Col-0) and mutant ( div1-1 ) Statistical bar graph of the number of rosette leaves of the plants; Figure 6 The effect of exogenous paclobutrazol (PAC) on the wild type (Col-0) and AtDIV1 Effects on flowering time of overexpression plants (Col-035S:AtDIV1-6HA #4); Figure 6 A shows the wild type Arabidopsis thaliana (Col-0) and the control group (CK) and the group sprayed with exogenous PAC for 27 days. AtDIV1 Photos of the growth status of the overexpressing plant (Col-0 35S:AtDIV1-6HA #4); Figure 6B shows the wild type (Col-0) and AtDIV1 Statistical bar graph of bolting time of overexpressing plants (Col-0 35S:AtDIV1-6HA #4); Figure 6 C is the control group (CK) and the group sprayed with exogenous PAC. AtDIV1 Statistical bar graph of the rosette leaf numbers of the overexpressing plant (Col-0 35S:AtDIV1-6HA #4). DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] In the following examples, the reagents used are all from commercial channels unless otherwise specified, and the test methods, detection methods, etc. involved are all conventional test methods and detection methods available in the prior art unless otherwise specified.

[0022] Example 1 DIV1 Gene cloning and overexpression vector construction 1.1 Plant material preparation Arabidopsis preparation: Mature seeds of Arabidopsis wild type Col-0 were sterilized with 75% (v / v) alcohol for 3 times, each time for 3 min, then washed with distilled water for 2 times, each time for 1 min, and 500 μL of distilled water was added. The water and seeds were transferred to MS medium (containing 3% sucrose and 1% agar powder in w / v) with a 1 mL pipette. Finally, the residual water on the medium was sucked out and placed at 4°C for 2 days for synchronization. Then, the seeds were transferred to an artificial climate culture room. After 7 days, the seedlings were transplanted into the culture matrix (matrix: vermiculite = 3:1) and cultured in the culture room. The culture room was set at 22°C, with a photoperiod of 16 h light / 8 h dark and a light intensity of 160 μmol m -2 s -1 ; Preparation of rapeseed: Mature seeds of wild-type ZS11 of rapeseed were sterilized with 75% (v / v) alcohol for 3 min, washed with sterile water 3 times for 1 min each time, then washed with 50% (v / v) sodium hypochlorite for 10 min, washed with sterile water 4 times, and then the water was removed. The rapeseed seeds were transferred to 1 / 2MS medium (containing 1.5% sucrose and 1% agar powder in w / v), placed at 4°C for 2 days for synchronization, and then transferred to an artificial climate culture room. After 10 days, the seedlings were transplanted into the culture matrix (matrix: vermiculite = 3:1) and cultured in the culture room. The culture room was set at 25°C, with a photoperiod of 16 h light / 8 h dark, and a light intensity of 160 μmol m -2 s -1 .

[0023] 1.2 Plant total RNA extraction and cDNA synthesis 1.2.1 Arabidopsis total RNA extraction and cDNA synthesis Tissue samples of roots, stems, leaves, flowers and siliques of Arabidopsis wild type Col-0 were taken and snap-frozen in liquid nitrogen. Total RNA was extracted using FastPure Plant Total RNA Isolation Kit (Source: Nanjing Novozyme Biotech Co., Ltd., Catalog No.: RC401). Total RNA of each tissue sample was used as a template and reverse transcribed using HiScript II 1st StrandcDNA Synthesis Kit (Source: Nanjing Novozyme Biotech Co., Ltd., Catalog No.: R211) to synthesize cDNA. 1.2.2 Total RNA extraction and cDNA synthesis from rapeseed Tissue samples of roots, stems, leaves, flowers and siliques of wild-type rapeseed were collected and quick-frozen in liquid nitrogen. Total RNA was extracted using the FastPurePlant Total RNA Isolation Kit (Source: Nanjing Novozymes Biotech Co., Ltd., Catalog No.: RC401). The total RNA of each tissue sample was used as a template and reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit (Source: Nanjing Novozymes Biotech Co., Ltd., Catalog No.: R211) to synthesize cDNA.

[0024] 1.3 Primer design and target gene cloning Arabidopsis thaliana was obtained through Tair and BnIR databases. AtDIV1 and rapeseed BnaDIV1 The gene sequence of Arabidopsis thaliana AtDIV1 The gene sequence of rapeseed is shown in SEQ ID NO: 1. BnaDIV1The gene sequence is shown in SEQ ID NO: 3, and the coding region sequence is designed DIV1 Gene-specific primers, primers are as follows (5'-3'): 35S:AtDIV1-6HA-EcoRI_F: 5'-ATAAGCTTGATATCgaattcATGGAGGTTATGAGACCGTCGA-3'; 35S:AtDIV1-6HA-PstI_R: 5'-GTGGATCCCCCGGGctgcagTAGTTGAAACATTGTGTTTTGG-3'; 35S:BnaDIV1-6HA-EcoRI_F: 5'-ATAAGCTTGATATCgaattcATGGAGATTCTGAGACCGGC-3'; 35S:BnaDIV1-6HA-PstI_R: 5'-GTGGATCCCCCGGGctgcagGTATAGCTGAAACATTGTGC-3'; The mixed sample of cDNA of different tissue samples of Arabidopsis wild type was used as a gene cloning template, and PCR amplification was performed with primers 35S:AtDIV1-6HA-EcoRI_F and 35S:AtDIV1-6HA-PstI_R using KOD-Plus high-fidelity DNA polymerase (source: Toyobo (Shanghai) Biotechnology Co., Ltd., catalog number: KOD-201) to clone Arabidopsis AtDIV1 The full-length sequence of the coding region; The mixed sample of cDNA of different tissue samples of the wild type of rapeseed was used as a gene cloning template, and PCR amplification was performed with primers 35S:BnaDIV1-6HA-EcoRI_F and 35S:BnaDIV1-6HA-PstI_R using KOD-Plus high-fidelity DNA polymerase (source: Toyobo (Shanghai) Biotechnology Co., Ltd., catalog number: KOD-201) to clone the rapeseed gene. BnaDIV1 The full-length sequence of the coding region; Among them, the PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2: Table 1 PCR reaction system

[0025] Table 2 PCR reaction procedure

[0026] After the PCR reaction of high-fidelity DNA polymerase is completed, 1% agarose gel (w / v) is used for electrophoresis detection, the size of the target gene band is determined according to the DNA marker DL-2000, and the target band is cut and recovered using the Fastpure gel DNA Extracting minikit (Source: Nanjing Novogene Biotech Co., Ltd., Catalog No.: DC201) kit, and the concentration of the recovered and purified target fragment is detected using a nucleic acid concentration meter.

[0027] 1.4 Construction of recombinant vector 1.4.1 Construction of recombinant products: In a 37°C water bath, the overexpression vector pGreen-35S-6HA was linearized using restriction endonucleases EcoRI and PstI from Takara. After 12 hours, the double-enzyme digestion product was recovered by gel excision using a purification kit (source: Nanjing Novogene Biotech Co., Ltd., catalog number: DC201). The concentration of the recovered and purified target fragment was then detected using a nucleic acid concentration meter. The purified target gene was ligated into the linearized vector using the NovoRec® plus One step PCR Cloning Kit (source: Suzhou Nearshore Protein Technology Co., Ltd., catalog number: NR005) to obtain the recombinant vectors pGreen-35S-AtDIV1-6HA and pGreen-35S-BnaDIV1-6HA. 1.4.2 Identification of recombinant vectors: Add the above recombinant vectors to competent E. coli cells (DH5α), mix well and place on ice for 10 min, heat shock in a 42°C water bath for 60 s, quickly place on ice for 5 min, add 1 mL of antibiotic-free LB liquid culture medium, place in a 37°C shaker for 1 h, centrifuge at 4000 rpm for 2 min, discard 900 μL of supernatant, resuspend the precipitate, apply the resuspended solution to kanamycin-resistant LB solid culture medium (50 μg / mL kanamycin), and invert in a 37°C incubator for culture until monoclonal colonies grow; The above six monoclonal colonies were added to 6 μL ddH2O, mixed, and 1.5 μL of the mixture was taken as a template for colony identification. DIV1The specific primers 35S: AtDIV1-6HA-PstI_R or 35S: BnaDIV1-6HA-PstI_R of the gene were combined to configure the PCR reaction system for amplification. The identification PCR reaction system and reaction procedure are shown in Table 3 and Table 4, respectively. After the PCR reaction, the product was subjected to 1% agarose gel (w / v) electrophoresis to observe the product bands. A monoclonal bacterial solution with the correct band size was selected and transferred to 3 mL of kanamycin-resistant LB liquid medium (50 μg / mL kanamycin) and shaken in a 37°C shaker for 12 hours for expansion culture. 1 mL of the expanded culture bacterial solution was taken for sequence sequencing. The sequencing result sequence was consistent with the CDS sequence and restriction site of the target gene, indicating that the recombinant vectors pGreen-35S-AtDIV1-6HA and pGreen-35S-BnaDIV1-6HA have been successfully constructed. The schematic diagram of the recombinant vector pGreen-35S-AtDIV1-6HA is shown in Figure 1 As shown in A; Among them, the sequence of the vector identification primer 35S-F is 5'-GACCCTTCCTCTATATAAGGAAGTTC-3', the identification PCR reaction system is shown in Table 3, and the identification PCR reaction program is shown in Table 4: Table 3 Identification PCR reaction system

[0028] Table 4 Identification PCR reaction procedure

[0029] 1.5 Obtaining recombinant vector Agrobacterium and transforming recipient plants 1.5.1 Obtaining recombinant vector Agrobacterium: Add recombinant vectors pGreen-35S-AtDIV1-6HA and pGreen-35S-BnaDIV1-6HA to thawed Agrobacterium competent cells (GV3101), mix well, place on ice for 10 minutes, freeze in liquid nitrogen for 5 minutes, heat shock in a 37℃ water bath for 5 minutes, place on ice for 5 minutes, add 1mL of antibiotic-free LB liquid medium, place in a 28℃ shaker for 2 hours, centrifuge at 4000rmp for 2 minutes, discard 900μL of supernatant, resuspend the precipitate, and apply the resuspended liquid to kanamycin and rifampicin resistant LB solid medium (50μg / mL kanamycin and 25μg / mL rifampicin), respectively, and invert in a 28℃ incubator for culture until monoclonal colonies grow. Use the vector identification primers in step 1.4.2 to configure the PCR reaction system for Agrobacterium colony identification.

[0030] Transfer the positive Agrobacterium monoclonal bacterial suspension with the correct electrophoresis band size to 100 mL of kanamycin and rifampicin resistant LB solid medium (50 μg / mL kanamycin and 25 μg / mL rifampicin) and culture in a shaking incubator at 28°C for 12 h until the bacterial suspension OD reaches 600 The value was about 1.8, and the cells were collected by centrifugation at 4000 rpm for 8 min at room temperature; 1.5.2 Transformation of recipient plants: The wild-type Arabidopsis Col-0 at the flowering stage was genetically transformed using the inflorescence dip method. The cells were resuspended in Arabidopsis transformation solution (5% sucrose (w / v), 0.02% surfactant Silwet L-77 (v / v)) to an OD of 600 The flower buds of wild-type Arabidopsis Col-0 at the flowering stage were immersed in the transformation solution for 45 seconds, and then the excess transformation solution was drained and placed flat on a tray for dark culture. After 2 days, they were taken out and cultured under normal light until the seeds were harvested to obtain T0 transgenic seeds.

[0031] 1.6 Screening and identification of transgenic positive seeds The T0 transgenic seeds were spread in the culture matrix (matrix: vermiculite = 3:1), placed at 4°C for 2 days for synchronization, and then transferred to the artificial climate culture room for light culture. After the seeds germinated and grew two true leaves, 0.02% herbicide (v / v) was sprayed every two days. One week later, the healthy seedlings were transplanted to the new culture matrix and continued to grow for 10 days. Then, the young leaves of the transplanted seedlings were taken and their genomic DNA was extracted by SDS method (the extraction solution formula was 200mM Tris, 250mM NaCl, 25mM EDTA (pH 8.0), 0.5% SDS (w / v) (final pH 7.0)). It was used as a template for PCR amplification, and the transgenic positive seedlings were judged by 1% agarose gel (w / v) electrophoresis. The above-mentioned vector identification primers 35S-F and 35S: AtDIV1-6HA-PstI_R or 35S: BnaDIV1-6HA-PstI_R were used for PCR amplification verification. The PCR reaction system and reaction procedure are shown in Table 3 and Table 4, respectively. The test results are shown in Figure 1 B. Among them, Figure 1 B #2, #4 and #6 have bands, indicating that Arabidopsis Col-0 35S:AtDIV1-6HA #2, #4 and #6 are T1 generation positive lines. BnaDIV1 Identification of transgenic lines Col-0 35S:BnaDIV1-6HA #1, #2 and #3 and Arabidopsis thaliana DIV1The overexpression transgenic method is the same. The Arabidopsis T1 seeds were disinfected according to the disinfection method in step 1.1, and transferred to the MS medium containing 10 μg / mL glufosinate. Finally, the residual water on the medium was aspirated and placed at 4°C for 2 days for synchronization. Then, they were transferred to the artificial climate culture room. After 7 days, the seedlings of the above T1 positive plants were transplanted into the culture matrix and cultured normally in the culture room until the seeds of each single plant were harvested (that is, different transgenic plants of the T2 generation). The T2 seeds were continued to be planted in the resistance culture medium until harvest, and homozygous T3 transgenic plants were obtained for subsequent experiments.

[0032] 1.7 Gene expression analysis Referring to the method in step 1.1, the T3 generation Arabidopsis wild-type Col-0 and transgenic plants (Col-0 35S:AtDIV1-6HA #2, #4 and #6 or Col-0 35S:BnaDIV1-6HA #1, #2 and #3) were sterilized and planted in MS medium. After 7 days, 10 seedlings of wild-type Col-0 and 10 transgenic plants were taken, and their genomic DNA was extracted as templates. Fluorescence quantitative PCR experiments were performed with specific fluorescence quantitative primers (AtDIV1-qPCR_F / R or BnaDIV1-qPCR_F / R). EF1aA4 The gene was used as an internal reference gene to detect DIV1 The transcription level of the gene Figure 1 C and Figure 3 As shown in B, in the T3 generation homozygous transgenic seedlings DIV1 The expression level of the gene at the transcriptional level was significantly higher than that of the wild-type Col-0, further confirming that the overexpressing transgenic plants can be used for subsequent experiments; The fluorescent quantitative primers involved in the present invention are as follows: AtDIV1-qPCR_F:5'-AACCGGGTCATCAGGTTTTGT-3'; AtDIV1-qPCR_R:5'-AAGTACTTTTTGGGCGTGGCT-3'; BnaDIV1-qPCR_F:5'-CGTCATCAAACAGTATAACGAT-3'; BnaDIV1-qPCR_R:5'-ATGTTCCTCCAATCTCCTTTA-3'; EF1aA4-qPCR_F:5'-CTGGAGGTTTTGAGGCTGGTA-3'; EF1aA4-qPCR_R:5'-CCAAGGGTGAAAGCAAGAAGA-3'; GA3ox1-qPCR_F:5'-CCATTCACCTCCCACACTCT-3'; GA3ox1-qPCR_R:5'-GCCAGTGATGGTGAAACCTT-3'.

[0033] Example 2 DIVI Effects of overexpressing transgenic plants / non-transgenic plants / mutant plants on flowering time 2.1 Bolting time statistics: Arabidopsis wild type (Col-0), mutant ( div1-1 and div1-2 ) and homozygous overexpression plants (Col-0 35S:AtDIV1-6HA #2, #4 and #6) were planted in the culture matrix according to the method in step 1.1 and cultured under light. The culture dishes of wild type, mutant and homozygous overexpression lines were placed at 4°C for 2 days. After the synchronization treatment, they were transferred to the artificial climate culture room for light culture and recorded as day 0. When each plant of wild type (Col-0), mutant and homozygous overexpression plants began to bolt, the bolting time of 10 plants of each type was recorded. When 3 / 4 of the wild type (Col-0) plants began to bolt, photos were taken to record the growth status of wild type (Col-0), mutant and homozygous overexpression plants; Among them, the mutant div1-1 for AtDIV1 T-DNA knockout mutant; mutant div1-2 for gene-edited mutants; 2.2 Leaf count: When the first flower of the above 10 wild-type (Col-0), mutant and homozygous overexpression plants opens, the number of rosette leaves is recorded. The specific operation is to remove the leaf closest to the cotyledon as the first rosette leaf, and then the leaf on the top of the stem base is the number of all rosette leaves (only the primary rosette leaves are counted). The number of cauline leaves is to remove all rosette leaves and all side branches when the first flower of 10 plants of each type opens, and then the leaf on the stem closest to the stem base is the first cauline leaf, and then the leaf on the stem closest to the first flower is the number of all cauline leaves; 2.3 Results Analysis: Figure 2 A and Figure 2 C shows that compared with the wild type Arabidopsis thaliana (Col-0), AtDIV1 T-DNA knockout mutant div1-1 and gene-edited mutants div1-2The overexpressing transgenic plants (Col-0 35S:AtDIV1-6HA #2, #4, and #6) showed an early flowering phenotype, and the number of rosette leaves was 2.5 to 3.9 less than that of the wild-type Col-0. Figure 2 B can be seen, AtDIV1 T-DNA knockout mutant div1-1 and gene-edited mutants div1-2 The bolting time of the wild type Col-0 was 1.9 days different at most, and the T-DNA knockout mutant div1-1 and gene-edited mutants div1- 2 The number of rosette leaves was relatively large, with the maximum difference of 1.6 leaves between the wild type Col-0 and the overexpressing transgenic plants (Col-0 35S:AtDIV1-6HA #2, #4 and #6) being 2.8 to 6.5 days earlier than that of the wild type Col-0. AtDIV1 It has the function of positively regulating the flowering time of plants, that is, shortening the flowering time of plants; In addition, for rapeseed BnaDIV1 The bolting time and leaf number statistics of overexpressing transgenic plants / non-transgenic plants were the same as those of Arabidopsis thaliana. Figure 3 As shown; As can be seen from the figure, compared with the wild type (Col-0), the overexpressing transgenic plants (Col-0 35S:BnaDIV1-6HA #1, #2 and #3) showed an early flowering phenotype. Specifically, BnaDIV1 The overexpression transgenic lines had an earlier bolting time of 4.5-4.8 days than the wild type Col-0, and the rosette leaves were 3.1-3.7 less than the wild type Col-0, indicating that BnaDIV1 It also has the function of positively regulating the flowering time of plants, that is, shortening the flowering time of plants.

[0034] Example 3 Arabidopsis thaliana AtDIV1 Determination of plant hormone content in transgenic plants and mutant plants To explore DIV1 The mechanism of gene-induced early flowering in plants was investigated in Arabidopsis wild type (Col-0), mutant ( div1-1 The gibberellin (GA3) content of seedlings of the homozygous overexpression line (Col-0 35S:AtDIV1-6HA #4) on the 17th day of growth was measured: 3.1 Determination of gibberellin (GA3) content: 0.3 g of wild type (Col-0) and mutant ( div1-1) and the homozygous overexpression line (Col-0 35S:AtDIV1-6HA #4) were ground in liquid nitrogen and transferred to 2 mL of 80% methanol / water (v / v) to obtain three mixtures. The three mixtures were incubated at 4°C overnight, centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected and concentrated to obtain a residue. The residue was extracted and centrifuged at 8000 rpm for 10 min in a centrifuge, and the supernatant was collected. The pH of the supernatant was adjusted to 2-3 using 1 M citric acid solution, and then extracted, the organic phase was collected, concentrated, and dried to obtain a precipitate containing GA3. Then, the precipitate containing GA3 was resuspended in 0.2 mL of chromatography-grade methanol and the GA3 content was determined using a gas chromatograph (HPLC, Waters2695); wherein, the chromatographic conditions were: chromatographic column Rigol Compass C18 (2), length 250 mm, inner diameter 4.6 mm. The flow rate was 1 mL / min, the column temperature was 35 °C, and phosphoric acid (0.1 wt %) and acetonitrile were used in a volume ratio of 82:18 for elution; From the results of Example 2, it can be seen that the overexpression transgenic line (Col-0 35S:AtDIV1-6HA #4) has bolted on the 17th day of growth, while the wild type (Col-0) and the mutant ( div1-1 ) did not bolt, and the GA3 content determination results were as follows Figure 4 As shown in the figure, it can be seen that the mutant ( div1-1 ) plant seedlings had only 23.6% of that in the wild type (Col-0), while the GA3 content in the overexpressing transgenic line (Col-0 35S:AtDIV1-6HA #4) increased to 1.5 times that in the wild type (Col-0). Figure 4 A). Explanation of overexpression DIV1 The accumulation of GA3 in seedlings was changed, and the wild type (Col-0), mutant ( div1-1 ) and overexpression strain (Col-0 35S:AtDIV1-6HA #4) showed that the mutant ( div1-1 ) Seedlings GA3ox1 The expression level of DIV1 Overexpression can significantly increase GA3ox1 The expression level ( Figure 4 B), prove DIV1 Overexpression can be achieved by upregulating GA3ox1 expression to promote the synthesis of GA3; 3.2 Application of exogenous GA3: Arabidopsis wild type (Col-0) and mutant ( div1-1) plants were planted into the culture matrix according to the method in step 1.1 and cultured under light. After one day of culture, Arabidopsis wild type (Col-0) and mutant ( div1-1 ) The seedlings of the plants were sprayed with 100 μM GA3, and the control group (denoted as CK) was treated with water instead of GA3 until bolting; the bolting time and the number of rosette leaves of the plants were counted in the same manner as in Example 2; 3.3 Application of exogenous paclobutrazol: Arabidopsis wild type (Col-0) and overexpression transgenic strain (Col-0 35S:AtDIV1-6HA #4) plants were planted in the culture matrix according to the method in step 1.1 and cultured under light. After one day of culture, 25 μM paclobutrazol (PAC) was sprayed twice a week on the seedlings of Col-0 and Col-0 35S:AtDIV1-6HA #4. The control group (denoted as CK) was replaced with water instead of PAC until bolting. The bolting time and leaf number statistical method of the plants were the same as in Example 2; During plant development, exogenous GA3 was used to treat the wild type (Col-0) and mutant ( div1-1 ) plants were treated with paclobutrazol (PAC), an inhibitor of GA3 synthesis, to explore the role of GA3 in DIV1 The role of regulating the flowering process, the results are as follows Figures 5 and 6 As shown: from Figure 5 It can be found that exogenous GA3 can transform the mutant ( div1-1 ) The late-flowering phenotype was restored to the wild type (Col-0) level, and the bolting time and rosette leaf number were restored to the wild type (Col-0) level; from Figure 6 It can be found that exogenous PAC can significantly delay the bolting time of the overexpressing transgenic line (Col-0 35S:AtDIV1-6HA #4), and the number of rosette leaves of the overexpressing transgenic line (Col-0 35S:AtDIV1-6HA #4) is also restored to the wild type (Col-0) level; The above results show that DIV1 Regulating the flowering time of plants through the GA3 synthesis pathway.

[0035] The above are only preferred embodiments of the present invention and are 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 in the protection scope of the present invention.

Claims

1. DIV1 The application of genes is characterized in that In at least one of the following applications: S1. Regulation of flowering time in plants; S2. Cultivate transgenic plants.

2. The use according to claim 1, characterized in that: Said DIV1 The nucleotide sequence of the gene is shown in SEQ ID NO:1 or SEQ ID NO:

3.

3. The use according to claim 1, characterized in that: Said DIV1 Gene-encoded protein DIV1 The amino acid sequence is shown in SEQ ID NO:2 or SEQ ID NO:

4.

4. The use according to claim 1, characterized in that: When the application is S1, the plant flowering time regulation is as follows: the plant flowering time changes with the DIV1 The expression of the gene increases and shortens.

5. The use according to claim 4, characterized in that: Said DIV1 The gene promotes flowering in plants by promoting the synthesis of gibberellins.

6. The use according to claim 1, characterized in that: When the application is S2, the cultivating of transgenic plants is cultivating early flowering plants.

7. The use according to claim 1, characterized in that: The plant is Arabidopsis thaliana or rapeseed.

8. A method for cultivating transgenic plants, characterized in that: The steps include: The method according to any one of claims 1 to 7 DIV1 The gene is expressed in the recipient plant, resulting in overexpression transgenic plants.

9. A method for cultivating transgenic plants according to claim 8, characterized in that: Said DIV1 The gene expression in the recipient plant is achieved by: DIV1 The gene is connected to the overexpression vector to obtain a recombinant vector, and the recombinant vector is introduced into the recipient plant.

10. A method for cultivating transgenic plants according to claim 9, characterized in that: The overexpressing transgenic plants have shortened flowering time compared to the recipient plants.

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