Application of DIV1 Gene in the Regulation of Plant Flowering Time

Abstract

The present invention relates to the technical field of plant genetic engineering and provides DIV1 the application of a gene in regulating the flowering time of plants. DIV1 The application of the gene is in at least one of the following S1 to S2: S1, regulating the flowering time of plants; S2, cultivating transgenic plants. By the above technical solution, the problems in the related art that the flowering time of plants is late, the growth period is long, and it is difficult to adapt to various farming systems are solved.

Description

Technical Field

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

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

[0003] MYB transcription factors are one of the largest transcription factor families in plants and play an important regulatory role in plant growth, development and physiological metabolism. DIV1 encodes an R-R type MYB transcription factor and plays an important role in regulating plant growth and development. Existing studies have shown that in pepper DIV1 acts as an activator to mediate ABA signal transduction and regulate the synthesis of capsanthin in pepper; in Arabidopsis DIV1 positively regulates seed germination to cope with salt stress.

[0004] Although researchers have currently isolated the genes of Arabidopsis and rapeseed DIV1 and conducted preliminary studies on their functions in the process of plant growth and development, the function of the DIV1 gene in regulating the flowering time of plants remains to be further studied. Summary of the Invention

[0005] The present invention proposes DIV1 the application of the

[0006] gene in regulating the flowering time of plants, which solves the problems of late flowering time, long growth cycle, and difficulty in adapting to multiple farming systems in the related art.

[0007] The technical solution of the present invention is as follows: DIV1 The application of the

[0008] gene in at least one of the following S1~S2:

[0009] S1. Regulating the flowering time of plants;

[0010] As a further technical solution, the DIV1 nucleotide sequence of the gene is as shown in SEQ ID NO:1 or SEQ ID NO:3.

[0011] As a further technical solution, the DIV1 protein encoded by the gene DIV1 has an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:4.

[0012] As a further technical solution, when the application is S1, the regulation of the flowering time of the plant is manifested as: the flowering time of the plant is shortened as the DIV1 expression level of the gene increases.

[0013] As a further technical solution, the DIV1 gene promotes plant flowering by promoting the synthesis of gibberellin.

[0014] As a further technical solution, when the application is S2, the cultivation of the transgenic plant is to cultivate a plant with early flowering.

[0015] As a further technical solution, the plant is Arabidopsis thaliana or rapeseed.

[0016] The present invention also provides a method for cultivating a transgenic plant, comprising the following steps:

[0017] Express the DIV1 gene in a recipient plant to obtain an overexpressing transgenic plant.

[0018] As a further technical solution, the way to express the DIV1 gene in the recipient plant is: ligate the DIV1 gene with an overexpression vector to obtain a recombinant vector, and introduce the recombinant vector into the recipient plant.

[0019] As a further technical solution, the overexpressing transgenic plant has a shorter flowering time compared with the recipient plant.

[0020] The working principle and beneficial effects of the present invention are:

[0021] 1. In the wild type of the model plant Arabidopsis thaliana (Col-0), the present invention reveals the DIV1 function of the gene in regulating the flowering time, which can effectively promote the early flowering of plants and shorten the growth cycle of plants. By exploring the DIV1 biological function of the gene in regulating the flowering time of plants, the DIV1 application of the gene in regulating the flowering time of plants is given. At the same time, the DIV1The gene, along with DIV1 the increase in the expression level of the gene, significantly shortens the flowering time of plants. The present invention fills the DIV1 gap in the application of the gene in genetic engineering.

[0022] 2. The present invention is of great significance for analyzing the transcriptional regulation mechanism during the process of plant flowering time, controlling the flowering period, and cultivating new varieties of crops with short growth periods. In practical applications, the flowering time of plants can be controlled by adjusting the photoperiod and water and fertilizer management at the seedling stage, which can shorten the plant growth cycle. This has important value for cultivating crops with short growth periods in agriculture to adapt to different crop rotation methods, adjust the growth period, and adapt to various tillage systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0024] Figure 1 is for Arabidopsis thaliana AtDIV1 identification results of transgenic plants overexpressing the gene; 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 transgenic plants overexpressing the gene, Cas represents the recombinant vector pGreen-35S:AtDIV1-6HA, which is the positive control of the experiment; Col-0 represents the wild-type receptor plant, which is the negative control of the experiment; Figure 1 C is a statistical bar chart of the transcriptional level of the gene in transgenic plants overexpressing the gene detected by fluorescence quantification; AtDIV1

[0025] Figure 2 is for the effects of Arabidopsis thaliana wild type (Col-0), mutants ( div1-1 and div1-2 ) and AtDIV1 overexpressing plants on the flowering time; among them, Figure 2 A is a photo of the growth status of Arabidopsis thaliana wild type (Col-0), mutants, and AtDIV1 overexpressing plants grown for 27 days; Figure 2 B is a statistical bar chart of the number of rosette leaves and cauline leaves of Arabidopsis thaliana wild type (Col-0), mutants ( div1-1 and div1-2 ) and AtDIV1 overexpressing plants; Figure 2 C is a statistical bar chart of the bolting time of Arabidopsis thaliana wild type (Col-0), mutants ( div1-1 and div1-2 ) and AtDIV1 overexpressing plants; ​

[0026] Figure 3 Effects of Arabidopsis wild type (Col-0) and BnaDIV1 overexpressing plants on flowering time; among them, Figure 3 A shows the growth status photos of Arabidopsis wild type (Col-0) and BnaDIV1 overexpressing plants grown for 23 days; Figure 3 B is a statistical bar graph of the transcriptional levels of genes BnaDIV1 in overexpressing transgenic plants detected by fluorescence quantitative method; Figure 3 C is a statistical bar graph of the number of rosette leaves of Arabidopsis wild type (Col-0) and BnaDIV1 overexpressing plants; Figure 3 D is a statistical bar graph of the bolting time of Arabidopsis wild type (Col-0) and overexpressing plants;

[0027] Figure 4 show DIV1 the effects of genes on the endogenous gibberellin content; among them, Figure 4 A shows the determination results of the content of endogenous gibberellin (GA3) in plants of Arabidopsis wild type (Col-0), mutant ( div1-1 ) and overexpressing line (Col-0 35S:AtDIV1-6HA #4) grown for 17 days; Figure 4 B shows the transcriptional levels of GA3 synthesis genes div1-1 in plants of Arabidopsis wild type (Col-0), mutant ( GA3ox1 ) and overexpressing line (#4) grown for 17 days;

[0028] Figure 5 show div1-1 the effects of exogenous gibberellin on the flowering time of Arabidopsis wild type (Col-0) and mutant ( Figure 5 ); among them, div1-1 A shows the growth status photos of plants of the control group (CK) and the group sprayed with exogenous GA3 of wild type (Col-0) and mutant ( Figure 5 ) grown for 27 days; div1-1 B is a statistical bar graph of the bolting time of plants of the control group (CK) and the group sprayed with exogenous GA3 of Arabidopsis wild type (Col-0) and mutant ( Figure 5 ); div1-1 C is a statistical bar graph of the number of rosette leaves of plants of the control group (CK) and the group sprayed with exogenous GA3 of wild type (Col-0) and mutant (

[0029] Figure 6 show AtDIV1Effect on the flowering time of overexpressing plants (Col-0 35S:AtDIV1-6HA #4); among them, Figure 6 A shows the growth status photos of Arabidopsis wild type (Col-0) in the control group (CK) and the group with exogenous spraying of PAC after 27 days of growth and AtDIV1 the overexpressing plants (Col-0 35S:AtDIV1-6HA #4); Figure 6 B shows the bar graph of the bolting time statistics of wild type (Col-0) in the control group (CK) and the group with exogenous spraying of PAC and AtDIV1 the overexpressing plants (Col-0 35S:AtDIV1-6HA #4); Figure 6 C shows the bar graph of the rosette leaf number statistics of Arabidopsis wild type (Col-0) in the control group (CK) and the group with exogenous spraying of PAC and AtDIV1 the overexpressing plants (Col-0 35S:AtDIV1-6HA #4). Specific implementation manners

[0030] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described, obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0031] In the following embodiments, the reagents used, if not otherwise specified, are all from commercial channels, and the test methods, detection methods, etc. involved, if not otherwise specified, are all conventional test methods and detection methods existing in the prior art.

[0032] Example 1 DIV1 Cloning of genes and construction of overexpression vectors

[0033] 1.1 Preparation of plant materials

[0034] Preparation of Arabidopsis thaliana: Disinfect the mature seeds of Arabidopsis thaliana wild type Col-0 with 75% (v / v) alcohol for 3 times, 3 min each time, then wash with distilled water 2 times, 1 min each time, add 500 μL of distilled water, transfer the water and seeds to MS medium (containing 3% sucrose and 1% agar powder by w / v) with a 1 mL pipette, finally suck out the residual water on the medium, place at 4 °C for 2 days for synchronization treatment, and then transfer to an artificial climate chamber. After 7 days, transplant the seedlings into the culture substrate (substrate: vermiculite = 3:1) and culture in the culture chamber. Among them, the set conditions of the culture chamber are 22 °C, the light cycle is 16 h light / 8 h darkness, and the light intensity is 160 μmol m -2 s -1 ;

[0035] Rape preparation: Disinfect the mature seeds of wild-type ZS11 of rape with 75% (v / v) alcohol for 3 min, wash them 3 times with sterile water for 1 min each time, then wash them with 50% (v / v) sodium hypochlorite for 10 min, wash them 4 times with sterile water and then suck out the moisture. Transfer the rape seeds to 1 / 2 MS medium (containing 1.5% sucrose and 1% agar powder by w / v), place them at 4 °C for 2 days for synchronization treatment, and then transfer them to an artificial climate chamber. After 10 days, transplant the seedlings into the culture substrate (substrate: vermiculite = 3:1) and culture them in the culture chamber. The set conditions of the culture chamber are 25 °C, a photoperiod of 16 h light / 8 h dark, and a light intensity of 160 μmol m -2 s -1 。

[0036] 1.2 Total RNA extraction and cDNA synthesis of plants

[0037] 1.2.1 Total RNA extraction and cDNA synthesis of Arabidopsis thaliana

[0038] Take tissue samples of roots, stems, leaves, flowers and siliques of wild-type Col-0 of Arabidopsis thaliana, quickly freeze them in liquid nitrogen, extract total RNA with the FastPure Plant Total RNA Isolation Kit (source: Nanjing Novoprotein Biological Technology Co., Ltd., product number: RC401), and use the HiScript II 1st Strand cDNA Synthesis Kit (source: Nanjing Novoprotein Biological Technology Co., Ltd., product number: R211) to perform reverse transcription to synthesize cDNA using the total RNA of each tissue sample as a template;

[0039] 1.2.2 Total RNA extraction and cDNA synthesis of rape

[0040] Take tissue samples of roots, stems, leaves, flowers and siliques of wild-type rape, quickly freeze them in liquid nitrogen, extract total RNA with the FastPure Plant Total RNA Isolation Kit (source: Nanjing Novoprotein Biological Technology Co., Ltd., product number: RC401), and use the HiScript II 1st Strand cDNA Synthesis Kit (source: Nanjing Novoprotein Biological Technology Co., Ltd., product number: R211) to perform reverse transcription to synthesize cDNA using the total RNA of each tissue sample as a template.

[0041] 1.3 Primer design and cloning of target genes

[0042] Obtain Arabidopsis thaliana through the Tair and BnIR databases respectively AtDIV1and Brassica napus BnaDIV1 The gene sequence of Arabidopsis thaliana AtDIV1 is shown in SEQ ID NO:1, and the gene sequence of Brassica napus BnaDIV1 is shown in SEQ ID NO:3. Specific primers for the DIV1 gene were designed based on the coding region sequence, and the primers are as follows (5'-3'):

[0043] 35S:AtDIV1-6HA-EcoRI_F:

[0044] 5’-ATAAGCTTGATATCgaattcATGGAGGTTATGAGACCGTCGA-3’;

[0045] 35S:AtDIV1-6HA-PstI_R:

[0046] 5’-GTGGATCCCCCGGGctgcagTAGTTGAAACATTGTGTTTTGG-3’;

[0047] 35S:BnaDIV1-6HA-EcoRI_F:

[0048] 5’-ATAAGCTTGATATCgaattcATGGAGATTCTGAGACCGGC-3’;

[0049] 35S:BnaDIV1-6HA-PstI_R:

[0050] 5’-GTGGATCCCCCGGGctgcagGTATAGCTGAAACATTGTGC-3’;

[0051] Using the mixed sample of cDNA from different tissue samples of wild-type Arabidopsis thaliana as the gene cloning template, with the help of KOD-Plus high-fidelity DNA polymerase (source: Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KOD-201), PCR amplification was carried out using primers 35S:AtDIV1-6HA-EcoRI_F and 35S:AtDIV1-6HA-PstI_R to clone the full-length coding region sequence of Arabidopsis thaliana AtDIV1 ;

[0052] Using the mixed sample of cDNA from different tissue samples of wild-type Brassica napus as the gene cloning template, with the help of KOD-Plus high-fidelity DNA polymerase (source: Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KOD-201), PCR amplification was carried out using primers 35S:BnaDIV1-6HA-EcoRI_F and 35S:BnaDIV1-6HA-PstI_R to clone Brassica napusBnaDIV1 Full-length sequence of the coding region;

[0053] Among them, the PCR reaction system is shown in Table 1, and the PCR reaction program is shown in Table 2:

[0054] Table 1 PCR reaction system

[0055]

[0056] Table 2 PCR reaction program

[0057]

[0058] After the PCR reaction of the high-fidelity DNA polymerase, electrophoresis detection was carried out using 1% agarose gel (w / v). The size of the target gene band was judged according to the DNA marker DL-2000, and the target band was cut and recovered using the Fastpure gel DNA Extracting minikit (source: Nanjing Novoprotein Biological Technology Co., Ltd., product number: DC201) kit, and the concentration of the recovered and purified target fragment was detected using a nucleic acid concentration detector.

[0059] 1.4 Construction of recombinant vectors

[0060] 1.4.1 Construction of recombinant products: Under the condition of a 37 °C water bath, the overexpression vector pGreen-35S-6HA was linearized using the restriction endonucleases EcoRI and PstI from Takara. After 12 h, the double-digested product was cut and recovered using a purification kit (source: Nanjing Novoprotein Biological Technology Co., Ltd., product number: DC201), and then the concentration of the recovered and purified target fragment was detected using a nucleic acid concentration detector. The NovoRec® plus One step PCR Cloning Kit (source: Suzhou Novoprotein Science & Technology Co., Ltd., product number: NR005) kit was used to ligate the purified target gene into the linearized vector respectively to obtain the recombinant vectors pGreen-35S-AtDIV1-6HA and pGreen-35S-BnaDIV1-6HA;

[0061] 1.4.2 Identification of the recombinant vector: Add the above-mentioned recombinant vector into Escherichia coli competent cells (DH5α), mix well, 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 medium, place in a shaker at 37°C and incubate for 1 h, centrifuge at 4000 rmp for 2 min, discard 900 μL of the supernatant, resuspend the pellet, and spread the resuspended liquid on LB solid medium with kanamycin resistance (50 μg / mL kanamycin), invert and culture in an incubator at 37°C until monoclonal colonies grow;

[0062] Resuspend each of the above 6 monoclonal colonies in 6 μL of ddH2O, mix well, take 1.5 μL of the mixture as the colony identification template, and use the vector identification primer 35S-F and DIV1 the specific primer 35S:AtDIV1-6HA-PstI_R or 35S:BnaDIV1-6HA-PstI_R of the gene to configure a PCR reaction system for amplification. The identification PCR reaction system and reaction program are shown in Tables 3 and 4 respectively. After the PCR reaction is completed, perform 1% agarose gel (w / v) electrophoresis on the product, observe the product bands, select a monoclonal bacterial solution with the correct band size and transfer it to 3 mL of LB liquid medium with kanamycin resistance (50 μg / mL kanamycin), and shake in a shaker at 37°C for 12 h for scale-up culture. Take 1 mL of the scale-up cultured bacterial solution for sequence sequencing. The sequencing result sequence is consistent with the CDS sequence of the target gene and the restriction enzyme sites, indicating that the recombinant vectors pGreen-35S-AtDIV1-6HA and pGreen-35S-BnaDIV1-6HA have been successfully constructed. Among them, the schematic diagram of the recombinant vector pGreen-35S-AtDIV1-6HA is as Figure 1 shown in A;

[0063] 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:

[0064] Table 3 Identification PCR reaction system

[0065]

[0066] Table 4 Identification PCR reaction program

[0067]

[0068] 1.5 Obtaining of recombinant vector Agrobacterium and transformation of recipient plants

[0069] 1.5.1 Obtaining Agrobacterium tumefaciens with recombinant vectors: The recombinant vectors pGreen-35S-AtDIV1-6HA and pGreen-35S-BnaDIV1-6HA were respectively added to the thawed competent Agrobacterium tumefaciens cells (GV3101). After mixing, the mixture was left standing on ice for 10 min, quickly frozen in liquid nitrogen for 5 min, rapidly heat-shocked in a 37 °C water bath for 5 min, then left standing on ice for 5 min. 1 mL of antibiotic-free LB liquid medium was added, and the mixture was cultured with shaking in a 28 °C shaker for 2 h. After centrifugation at 4000 rmp for 2 min, 900 μL of the supernatant was discarded. After resuspending the precipitate, the resuspended solution was respectively spread on LB solid medium with kanamycin and rifampicin resistance (50 μg / mL kanamycin and 25 μg / mL rifampicin), and then inverted and cultured in a 28 °C incubator until monoclonal colonies grew. The vector identification primers in step 1.4.2 were used to prepare a PCR reaction system for identifying Agrobacterium tumefaciens colonies.

[0070] The positive Agrobacterium tumefaciens monoclonal bacterial solution with the correct electrophoretic band size was transferred to 100 mL of LB solid medium with kanamycin and rifampicin resistance (50 μg / mL kanamycin and 25 μg / mL rifampicin), and cultured with shaking in a 28 °C shaker for 12 h until the OD of the bacterial solution 600 was approximately 1.8, and the cells were collected by centrifugation at 4000 rpm for 8 min at room temperature;

[0071] 1.5.2 Transformation of recipient plants: The wild-type Arabidopsis thaliana Col-0 at the full-bloom stage was genetically transformed by the floral dip method. The cells were resuspended in an Arabidopsis thaliana transformation solution (5% sucrose (w / v), 0.02% surfactant Silwet L-77 (v / v)) to an OD 600 of 1.0. The flower buds of wild-type Arabidopsis thaliana Col-0 at the full-bloom stage were immersed in the transformation solution for 45 s, then the excess transformation solution was drained off and the flower buds were placed flat in a tray and cultured in the dark. After 2 days, they were taken out and cultured under normal light until the seeds were harvested to obtain T0 generation transgenic seeds.

[0072] 1.6 Screening and identification of transgenic positive seeds

[0073] Spread the above-mentioned T0 generation transgenic seeds on the culture substrate (substrate: vermiculite = 3:1), place them at 4°C for 2 days for synchronization treatment, and then transfer them to a phytotron for light cultivation. After the seeds germinate and grow two true leaves, spray 0.02% herbicide (v / v) every two days. One week later, transplant the vigorously growing seedlings to a new culture substrate and continue to grow for 10 days. Then, take the young leaves of the transplanted seedlings respectively, extract their genomic DNA by the SDS method (the extraction solution formula is 200 mM Tris, 250 mM NaCl, 25 mM EDTA (pH 8.0), 0.5% SDS (w / v) (final pH 7.0)), use it as a template for PCR amplification, and judge the transgenic positive seedlings by electrophoresis on 1% agarose gel (w / v). Use the above vector identification primers 35S-F and 35S:AtDIV1-6HA-PstI_R or 35S:BnaDIV1-6HA-PstI_R to carry out PCR amplification verification respectively. The PCR reaction system and reaction program are shown in Table 3 and Table 4 respectively. The detection results are as Figure 1 shown in Figure 1 Figure B. Among them, BnaDIV1 there are bands in #2, #4 and #6 of Figure B, representing that Arabidopsis Col-0 35S:AtDIV1-6HA #2, #4 and #6 are T1 generation positive lines. The identification method of the transgenic line Col-0 35S:BnaDIV1-6HA #1, #2 and #3 of rapeseed DIV1 is the same as the overexpression transgenic method of Arabidopsis DIV1 . Disinfect the Arabidopsis T1 generation seeds according to the disinfection method in step 1.1, transfer them to the MS medium containing 10 μg / mL glufosinate, finally suck the residual water on the medium, place them at 4°C for 2 days for synchronization treatment, and then transfer them to a phytotron. Seven days later, transplant the seedlings of the above-mentioned T1 generation positive plants to the culture substrate respectively, and cultivate them normally in the culture room until the seeds of each individual plant are harvested (i.e., different transgenic plants of the T2 generation), and continue to plant the T2 generation seeds in the resistant medium until harvest, and homozygous T3 generation transgenic plants can be obtained for subsequent experiments.

[0074] 1.7 Gene expression analysis

[0075] Refer to the method in Step 1.1 to disinfect and plant the T3 generation of 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) in MS medium respectively. After 7 days, take 10 seedlings of wild-type Col-0 and 10 transgenic plants respectively, extract their genomic DNA as templates, and perform fluorescence quantitative PCR experiments with specific fluorescence quantitative primers (AtDIV1-qPCR_F / R or BnaDIV1-qPCR_F / R). EF1aA4 The DIV1 gene is used as an internal reference gene to detect the Figure 1 transcription level of the Figure 3 gene in each plant. The results are shown in DIV1 Figures C and

[0076] B. The expression level of the

[0077] gene in the T3 generation of homozygous transgenic seedlings is significantly higher than that of wild-type Col-0 at the transcriptional level, further confirming that the overexpressed transgenic plants can be used for subsequent experiments;

[0078] AtDIV1-qPCR_F: 5’-AACCGGGTCATCAGGTTTGT-3’;

[0079] AtDIV1-qPCR_R: 5’-AAGTACTTTTGGGCGTGGCT-3’;

[0080] BnaDIV1-qPCR_F: 5’-CGTCATCAAACAGTATAACGAT-3’;

[0081] BnaDIV1-qPCR_R: 5’-ATGTTCCTCCAATCTCCTTTA-3’;

[0082] EF1aA4-qPCR_F: 5’-CTGGAGGTTTTGAGGCTGGTA-3’;

[0083] EF1aA4-qPCR_R: 5’-CCAAGGGTGAAAGCAAGAAGA-3’;

[0084] GA3ox1-qPCR_F: 5’-CCATTCACCTCCCACACTCT-3’;

[0085] Example 2 DIVIEffect of overexpressing transgenic plants / non-transgenic plants / mutant plants on flowering time

[0086] 2.1 Bolting time statistics: Arabidopsis wild type (Col-0), mutants ( div1-1 and div1-2 ), and homozygous overexpressing plants (Col-0 35S:AtDIV1-6HA #2, #4, and #6) were planted into the culture medium according to the method in step 1.1 and cultured under light. Among them, the culture dishes of wild type, mutants, and homozygous overexpressing lines were placed at 4 °C for 2 days. After the synchronization treatment was completed, they were transferred to the artificial climate chamber for light culture and recorded as day 0. When the wild type (Col-0), mutants, and homozygous overexpressing plants began to bolt, the bolting times of 10 plants of each type were recorded respectively. 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), mutants, and homozygous overexpressing plants;

[0087] Among them, the mutant div1-1 is AtDIV1 T-DNA knockout mutant; the mutant div1-2 is a gene-edited mutant;

[0088] 2.2 Leaf number statistics: When the first flower of each of the above 10 wild type (Col-0), mutant, and homozygous overexpressing plants opened, the number of rosette leaves was recorded. The specific operation was to take the leaf closest to the cotyledon as the first rosette leaf, and so on until the topmost leaf at the base of the stem was the number of all rosette leaves (only counting the primary rosette leaf number). For the number of cauline leaves, the operation was to remove all rosette leaves and all lateral branches when the first flower of 10 plants of each type opened. The leaf on the stem closest to the base of the stem was taken as the first cauline leaf, and so on until the leaf on the stem closest to the first flower was the number of all cauline leaves;

[0089] 2.3 Result analysis: As can be seen from Figure 2 A and Figure 2 C, compared with Arabidopsis wild type (Col-0), AtDIV1 T-DNA knockout mutant div1-1 and gene-edited mutant div1-2 showed a late-flowering phenotype, and the overexpressing transgenic plants (Col-0 35S:AtDIV1-6HA #2, #4, and #6) showed an early-flowering phenotype, and the rosette leaves were 2.5 - 3.9 fewer than those of the wild type Col-0; as can be seen from Figure 2 B, AtDIV1 T-DNA knockout mutant div1-1 and gene-edited mutant div1-2The bolting time differed from that of the wild-type Col-0 by at most 1.9 days, and the T-DNA knockout mutants div1-1 and the gene-edited mutants div1- 2 had a relatively larger number of rosette leaves, with the leaf number differing from that of the wild-type Col-0 by at most 1.6. The overexpressing transgenic plants (Col-0 35S:AtDIV1-6HA #2, #4, and #6) had a bolting time advanced by 2.8 - 6.5 days compared to the wild-type Col-0. The above indicates that AtDIV1 has the function of positively regulating the flowering time of plants, that is, shortening the flowering time of plants;

[0090] In addition, for rapeseed BnaDIV1 The bolting time and leaf number of overexpressing transgenic plants / non-transgenic plants were statistically analyzed in the same way as for Arabidopsis thaliana, and the results are as Figure 3 shown;

[0091] It can be seen from the figure that 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 bolting time of the overexpressing transgenic lines was advanced by 4.5 - 4.8 days compared to the wild-type Col-0, and the number of rosette leaves was 3.1 - 3.7 less than that of the wild-type Col-0, indicating that BnaDIV1 also has the function of positively regulating the flowering time of plants, that is, shortening the flowering time of plants.

[0092] Example 3 Arabidopsis thaliana AtDIV1 Determination of phytohormone content in transgenic plants and mutant plants

[0093] To explore DIV1 the mechanism by which the gene causes plants to flower earlier, the gibberellin (GA3) content of 17-day-old seedlings of Arabidopsis thaliana wild-type (Col-0), mutants ( div1-1 ) and homozygous overexpressing lines (Col-0 35S:AtDIV1-6HA #4) was determined:

[0094] 3.1 Determination of gibberellin (GA3) content: 0.3 g of 17-day-old wild-type (Col-0), mutants ( div1-1The seedlings of the mutant line (Col-0 35S:AtDIV1-6HA #4) and the homozygous overexpression line (Col-0 35S:AtDIV1-6HA #4) were ground and crushed in liquid nitrogen and transferred to 2 mL of 80% methanol / water (v / v) for mixing to obtain three mixtures. The three mixtures were incubated overnight at 4°C, centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected, 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. After adjusting the pH of the supernatant to 2-3 with 1 M citric acid solution, extraction was performed, and 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 chromatographic grade methanol, and the content of GA3 was determined using a gas chromatograph (HPLC, Waters 2695); among them, the chromatographic conditions were as follows: the chromatographic column was Rigol Compass C18 (2), with a length of 250 mm and an inner diameter of 4.6 mm. The flow rate was 1 mL / min, the column temperature was 35°C, and elution was performed using phosphoric acid (0.1 wt%) and acetonitrile in a volume ratio of 82:18;

[0095] As can be seen from the results of Example 2, at the 17th day of growth, the overexpression transgenic line (Col-0 35S:AtDIV1-6HA #4) had bolted, while the wild type (Col-0) and the mutant ( div1-1 ) had not bolted, and the determination results of the GA3 content were as Figure 4 shown. It can be seen from the figure that the GA3 content in the seedlings of the mutant ( div1-1 ) plants was only 23.6% of that of the wild type (Col-0), while the GA3 content of the overexpression transgenic line (Col-0 35S:AtDIV1-6HA #4) increased to 1.5 times that of the wild type (Col-0) ( Figure 4 A). It shows that overexpression DIV1 changed the accumulation amount of GA3 in the seedlings. Further detection of the expression of the GA3 synthesis gene in the wild type (Col-0), the mutant ( div1-1 ) and the overexpression line (Col-0 35S:AtDIV1-6HA #4) found that the expression level of div1-1 in the seedlings of the mutant ( GA3ox1 ) was significantly down-regulated, while DIV1 overexpression could significantly increase the expression level of GA3ox1 in the seedlings ( Figure 4 B), proving that DIV1 overexpression could promote the synthesis of GA3 by up-regulating the expression of GA3ox1 ;

[0096] 3.2 Application of exogenous GA3: The wild type (Col-0) and the mutant of Arabidopsis thaliana ( div1-1The plants were planted into the culture medium according to the method in step 1.1 and cultured under light. After one day of culture, every other day, 100 μM GA3 was sprayed on the seedlings of Arabidopsis wild type (Col-0) and mutants ( div1-1 ) The seedlings of the plants were sprayed with 100 μM GA3, and the control group (denoted as CK) was replaced with water instead of GA3 until bolting; the method for counting the bolting time and the number of rosette leaves of the plants was the same as in Example 2;

[0097] 3.3 Application of exogenous paclobutrazol: Arabidopsis wild type (Col-0) and overexpressing transgenic lines (Col-0 35S:AtDIV1-6HA #4) plants were planted into the culture medium according to the method in step 1.1 and cultured under light. After one day of culture, 25 μM paclobutrazol (PAC) was sprayed on the seedlings of Col-0 and Col-0 35S:AtDIV1-6HA #4 twice a week, and the control group (denoted as CK) was replaced with water instead of PAC until bolting. The method for counting the bolting time and the number of leaves of the plants was the same as in Example 2;

[0098] During the plant development process, exogenous GA3 was used to treat wild type (Col-0) and mutant ( div1-1 ) plants, and at the same time, the inhibitor of GA3 synthesis, paclobutrazol (PAC), was used to treat wild type (Col-0) and overexpressing transgenic lines (Col-0 35S:AtDIV1-6HA #4) to explore the role of GA3 in DIV1 regulating the flowering process. The results are as Figures 5 - 6 shown:

[0099] From Figure 5 it can be found that exogenous GA3 can restore the late-flowering phenotype of the mutant ( div1-1 ) to the wild type (Col-0) level, and the bolting time and the number of rosette leaves are both restored to the wild type (Col-0) level;

[0100] 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 expressing transgenic line (Col-0 35S:AtDIV1-6HA #4) is also restored to the wild type (Col-0) level;

[0101] The above results show that DIV1 the flowering time of plants is regulated through the GA3 synthesis pathway.

[0102] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. DIV1 Use of a gene, characterized in that, Application in at least one of the following S1 to S2: S1. Regulation of plant flowering time; S2. Cultivation of transgenic plants with early flowering; The said DIV1 The nucleotide sequence of the gene is as shown in SEQ ID NO:1 or SEQ ID NO:3; The plant is Arabidopsis thaliana or rapeseed.

2. DIV1 Application of the gene-encoded protein, characterized in that, The DIV1 protein encoded by the gene DIV1 has the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4; The said application is in at least one of the following S1 to S2: S1. Regulation of plant flowering time; S2. Cultivation of transgenic plants with early flowering; The plant is Arabidopsis thaliana or rapeseed.

3. The application according to claim 1, characterized in that, When the application is S1, the regulation of the plant flowering time is manifested as follows: the plant flowering time shortens as the DIV1 expression level of the gene increases.

4. A method for cultivating a transgenic plant with early flowering, characterized in that, Including the following steps: Express the gene shown in SEQ ID NO:1 or SEQ ID NO:3 in the application according to any one of claims 1 to 3 in a recipient plant to obtain a transgenic plant with overexpressed DIV1 gene; DIV1 ​ The recipient plant is Arabidopsis thaliana or rapeseed.

5. The method according to claim 4, characterized in that, Link the DIV1 gene with an overexpression vector to obtain a recombinant vector, and introduce the recombinant vector into the recipient plant; The recipient plant is Arabidopsis thaliana or rapeseed.

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