Application of BpSPL2 gene in regulating and controlling early flowering capability of plant

Abstract

The invention provides application of a BpSPL2 gene in regulating and controlling the early flowering capability of plants, and belongs to the technical field of gene engineering. The nucleotide sequence of the BpSPL2 gene is as shown in SEQ ID NO. 1; according to the application, the BpSPL2 gene is inhibited in a plant, so that the early flowering capability of the plant is improved. The invention finds that the BpSPL2 gene negatively regulates the germination of betula platyphylla spores, and the ability of betula platyphylla to bloom in advance can be obviously improved by inhibiting the expression of the BpSPL2 gene. The method is beneficial to shortening the cultivation time of betula platyphylla improved seeds, enables betula platyphylla plants to bloom in advance, and provides a theoretical basis for research of betula platyphylla germplasm resources.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of a BpSPL2 gene in regulating the early flowering ability of a plant. Background Art

[0002] SPL (SQUAMOSA promoter-binding protein-like) is a type of plant-specific transcription factor that exists in all green plants, including unicellular green algae, mosses, gymnosperms and angiosperms. SPL transcription factors were first cloned by Huijser et al. from Antirrhinum majus. At that time, two closely related genes, AmSBP1 and AmSBP2, were found. They can bind to the promoter of the floral meristem characteristic gene SQUAMOSA (SQUA) and were named Squamosa promoter binding protein (SBP). Afterwards, cDNA cloning of the SBP transcription factor family was performed in Arabidopsis and moss, and it was found that there were multiple copies of SBP transcription factor genes in these two plants. In recent years, such genes have also been found in birch, corn, tomato and strawberry.

[0003] Studies have shown that SPL is involved in regulating multiple important biological processes, such as developmental stage transitions, leaf, plant height and tillering, plant stress response, and copper ion homeostasis in the body. The prior art indicates that Arabidopsis AtSPL9 and AtSPL10 can upregulate the expression of miR172, and then the target gene of miR172, TOE1 / 2 gene, is inhibited, which in turn promotes the transition from juvenile to adult stage; Arabidopsis spl8 mutant has small anthers and reduced fertility; Rice OsSPL14 and OsSPL16 play a decisive role in regulating rice grain size, shape and color; Arabidopsis SPL8 plays a local regulatory role in the GA-dependent developmental process, and the GA response of transgenic plants overexpressing AtSPL8 is changed, resulting in anthers that do not crack and reduced fertility; Overexpression of AtSPL8 gene is insensitive to GA treatment, such as after GA treatment, its seed germination rate is reduced and the elongation growth of seedling roots is inhibited; In the case of copper deficiency, AtSPL7 gene plays a positive role in maintaining copper homeostasis in plants.

[0004] White birch (Betula platyphylla) has the characteristics of good renewal, fast growth, strong adaptability, wide distribution area, fine material and white color, and is one of the preferred tree species for cultivating single-board artificial boards and fast-growing and high-yield forests. However, due to long-term predatory management, the germplasm resources of white birch in the natural state have seriously degraded and cannot meet the demand for white birch improved varieties. Therefore, how to cultivate white birch improved varieties in a relatively short period of time is a problem that those skilled in the art urgently need to solve. In view of this, the purpose of the present invention is to provide the application of the BpSPL2 gene in regulating the early flowering ability of plants, so as to provide a theoretical basis for the cultivation of white birch improved varieties. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the BpSPL2 gene in regulating the early flowering ability of plants, so as to provide a theoretical basis for shortening the plant growth cycle and cultivating improved plant varieties.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of a BpSPL2 gene in regulating the early flowering ability of a plant. The nucleotide sequence of the BpSPL2 gene is shown in SEQ ID NO.1.

[0008]

[0009] Preferably, the amino acids of the protein encoded by the BpSPL2 gene are shown in SEQ ID NO.2.

[0010] SEQ ID NO.2:MESWSCSSEGKGLLLSDEMDLQVDAFARSKKTLME WDNKPTYYFESNGLVSDREAVEGMELLDLGFSDLVRKPFHGNRGMEMLSGEVGSGSSQRAVTPTCMVTSNSCFEEVGSEAKLSSSSMEANSQDLSLIDLKLGTLADCKDAENIKVLKEKPV LSSVSPCLQRKRARKTSSHSQTAFCQVHGCNKDLSSSKDYHKRHKVCDVHSKTAKVIVNGIEQRFCQQCSRFHLLVEFDDGKRSCRKRLAGHNERRRKPQLDTLSGTQYLGTSLQKRTPLVF PDIFRAGIICPGKNEEANWFRHTKLEGESIYSPQSAIPITNGHLLPKSFLHLHGIGKQHCSGVPSSSGSEDYTFTASTVQELPGASISSCALSLLSAQSQDLSSHLTGRPMASPQIMQGRSA HHILGQTDKPVRVSSVEQYGSNGLYSYGMNSMEVDKIGSVMLSDASHAADFQVHTDGIFQESDILNANYFSSTEYGPTVDWLQLSSHLQRVERQRNSIQVKQENGDSCYFPTFRAVCNQQGV

[0011] Preferably, the plant includes birch.

[0012] Preferably, the application is to inhibit the BpSPL2 gene in plants to enhance the ability of the plants to bloom early.

[0013] The present invention also provides a method for improving the early flowering ability of plants, comprising the following steps:

[0014] (1) constructing the inhibitory expression vector of the BpSPL2 gene as described in claim 1, and transforming it into Agrobacterium to obtain an engineered bacterium;

[0015] (2) After infecting plant tissue with the engineered bacteria obtained in step (1), plant tissue culture is performed to obtain transgenic plants with early flowering ability.

[0016] Preferably, the method for constructing the inhibition expression vector of the BpSPL2 gene is: amplifying the BpSPL2 gene with primers BpSPL2F and BpSPL2-SRDXR to obtain the BpSPL2-SRDX target fusion fragment containing a restriction site; connecting the BpSPL2-SRDX target fusion fragment with a plasmid vector to obtain the inhibition expression vector of the BpSPL2 gene.

[0017] Preferably, the nucleotide sequence of the BpSPL2F is shown as SEQ ID NO.3, and the nucleotide sequence of the BpSPL2-SRDXR is shown as SEQ ID NO.4.

[0018] SEQ ID NO.3: CGGGATCCATGGAGTCTTGGAGTTGCAG

[0019] SEQ ID NO.4: GGGGTACCTCAAGCGAAACCCAAACGGAGTTCTAGATCCAGATCCAGCACACCTTGTTGGTTGCAC

[0020] Preferably, the restriction enzyme cutting sites are BamHI and KpnI restriction enzyme cutting sites, and the plasmid vector is pROKⅡ.

[0021] Preferably, the infection site includes plant leaves.

[0022] The present invention finds that the BpSPL2 gene negatively regulates the germination of birch spores, and the ability of birch to bloom early can be significantly improved by inhibiting the expression of the BpSPL2 gene. The present invention is conducive to shortening the time of birch breeding, making birch plants bloom early, and providing a theoretical basis for the research of birch germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0024] Figure 1 This is the growth conditions of different plants in Example 2. DETAILED DESCRIPTION

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example 1

[0027] Construction of BpSPL2 gene suppression expression vector pROKII-BpSPL2-SRDX:

[0028] The 1563bp sequence of the BpSPL2 gene shown in SEQ ID NO.1 after removing the stop codon TGA was transferred into the pMD18-T plasmid. The pMD18-T plasmid containing the BpSPL2 gene was used as a template, and BpSPL2F (SEQ ID NO.3) and BpSPL2-SRDXR (SEQ ID NO.4) were used as primers to obtain the BpSPL2-SRDX target fusion fragment containing BamHI and KpnI restriction sites by PCR amplification. The PCR bands were detected by 1% agarose gel electrophoresis to obtain the BpSPL2-SRDX target fragment. The PCR amplification reaction system was: 30.75 μL sterile water, 5 μL TaqBuffer, MgCl 2 3μL, dNTPs 4μL, primers F and R 2μL each, template 2μL, Taq enzyme 1.25μL; the reaction program was: 98℃ pre-denaturation for 3min, 98℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 2min, 72℃ extension for 7min, for a total of 35 cycles.

[0029] The BpSPL2-SRDX target band was purified and recovered using a gel recovery kit, and the purified product was double-digested (BamHI and KpnI enzymes) according to the instructions of the restriction endonucleases. At the same time, the pROKⅡ plasmid was extracted using a plasmid extraction kit, and the plasmid was double-digested (BamHI and KpnI enzymes), and the digestion products were separated, detected and purified by 1% agarose gel electrophoresis.

[0030] The purified BpSPL2-SRDX gene sequence and the digestion product of the pROKⅡ vector were digested according to the T4 DNA ligase digestion system (10×T4 DNAligasebuffer 1.0μL, T4 DNAligase 1.0μL, target fragment: vector (molar mass) = 3:1, ddH 2 The ligation was carried out under the conditions of 4% paraformaldehyde (2% paraformaldehyde, 0.04% paraformaldehyde and 1.04% paraformaldehyde) and the reaction was carried out at 4°C overnight. The ligation product was then transformed into competent E. coli cells, and a single clone was picked and subjected to bacterial liquid PCR. The positive single clone pROKⅡ-BpSPL2-SRDX was obtained and the plasmid was extracted from it.

[0031] Example 2

[0032] Obtaining transgenic birch with pROKⅡ-BpSPL2-SRDX and pGWB5-BpSPL2-GFP

[0033] The pROKⅡ-BpSPL2-SRDX suppression expression vector plasmid prepared in Example 1 was transferred into EHA105 Agrobacterium competent cells by liquid nitrogen method. The specific operation steps are as follows:

[0034] Take out the competent EHA105 strain of Agrobacterium and melt it on ice. Add 2 μL of pROKⅡ-BpSPL2-SRDX plasmid to 20 μL of competent strain, mix gently with a pipette tip, and ice bath for 10 minutes; freeze in liquid nitrogen for 5 minutes, quickly put in a 37℃ water bath for 5 minutes, and continue ice bathing for 4 minutes after taking it out; add 800 μL of LB liquid culture medium without antibiotics to the centrifuge tube, and shake and culture in a 28℃ incubator (180rpm) for 2 hours; after taking out the centrifuge tube from the shaker, centrifuge at 5000rpm for 5 minutes, retain 100mL of supernatant and gently blow and mix, spread on LB solid plate (50mg / LKan, 50mg / L Rif), and grow single colonies after inverting and culturing at 28℃ for 72 hours. Randomly pick 4 single clones for bacterial liquid PCR detection, and Agrobacterium with positive detection can be used for subsequent genetic transformation by leaf disc method.

[0035] The prepared pROKⅡ-BpSPL2-SRDX Agrobacterium single colony and the overexpressing pGWB5-BpSPL2-GFP Agrobacterium single colony preserved in this laboratory were cultured in LB liquid culture medium at 28°C overnight. The next day, when the OD600 of the bacterial solution was 0.8-1.0, the bacterial solution was diluted 20 times and cultured at 28°C until the OD600 of the bacterial solution was between 0.6 and 0.8. The bacteria were resuspended in liquid WPM (6-BA 2.0 mg / L + NAA 0.2 mg / L) as the infection engineering bacterial solution.

[0036] Select the leaves of the rooted seedlings with bright green leaves and large leaf area (the third and fourth leaves from the top) as explants, and cut them at the part with the strongest ability to divide (about 0.3 cm from the base of the petiole, that is, the bifurcation of the total veins), and ensure that the leaves are less mechanically damaged during cutting. Then infect with the prepared infection engineering bacterial solution for 10 minutes, take out the leaves and absorb the excess bacterial solution with sterile filter paper. The infected explants and Agrobacterium are dark-cultured in a culture medium (WPM+6-BA2.0mg / L+NAA 0.2mg / L) for 2 days to obtain co-cultured leaves.

[0037] The co-cultivated leaves were placed in sterile water containing 200 mg / L cephalosporin to wash the bacteria, excess water was absorbed with absorbent paper, and the leaves were cultured on selective medium (WPM+6-BA2.0 mg / L+NAA 0.2 mg / L+cefotaxime sodium 400 mg / L+Kan 50 mg / L). After about 20 days of cultivation, callus tissue could be seen swelling at the wound site. The callus was moved to WPM+6-BA 0.8 mg / L+NAA 0.02 mg / L+GA30.5 mg / L medium. After the callus grew resistant buds and grew into small plantlets of 3 to 4 cm, they were cut off and placed in rooting medium (WPM+IBA 0.4 mg / L) for rooting culture. Two pROKII-BpSPL2-SRDX transgenic birch tissue culture seedlings (referred to as 35S::BpSPL2-SRDX-1 and 35S::BpSPL2-SRDX-4, respectively) and two pGWB5-BpSPL2-GFP transgenic birch tissue culture seedlings (referred to as 35S::BpSPL2-3 and 35S::BpSPL2-5, respectively) were obtained.

[0038] 3 cm long stem segments with terminal buds at the top of 35S::BpSPL2-3, 35S::BpSPL2-5, 35S::BpSPL2-SRDX-1, 35S::BpSPL2-SRDX-4 transgenic birch seedlings and wild-type birch (WT) seedlings were cut and inserted into WPM rooting medium at 24 ± 2 °C, relative humidity of 70%, and light intensity of 46 μmoL / m 2 / s, and cultured for 40 days under the conditions of 16h light / 8h dark photoperiod. The WPM rooting medium uses distilled water as solvent, contains 2.14g / L WPM powder, 20g / L sucrose, 0.56g / L calcium salt, 0.4mg / L IBA, 8g / L plant agar, and the pH is adjusted to 5.8 with 5mol / L NaOH, and is packaged into 100mL / bottle for standby use.

[0039] The transgenic tissue culture seedlings grown under normal culture conditions for 40 days were transplanted into a soil matrix of vermiculite:flower soil = 1:1 (the culture medium at the roots of the tissue culture seedlings was rinsed clean when transplanting into the soil). After the seedlings were cultured indoors until they were stable, they were replaced with larger soil pots and moved outdoors (natural environment) for cultivation.

[0040] The BpSPL2 transgenic and wild-type birch plants moved outdoors were followed up and observed. In spring (May), the overall growth of different plants was observed and counted, and the inflorescences of BpSPL2 transgenic and wild-type birch plants were compared. Figure 1(The red box shows the inflorescence of birch plants), it can be seen that 35S::BpSPL2-SRDX-1 and 35S::BpSPL2-SRDX-4 transgenic birch plants grew inflorescences compared with wild-type birch plants, 35S::BpSPL2-3 and 35S::BpSPL2-5 transgenic birch plants, indicating that the BpSPL2 gene negatively regulates the germination of birch spores. By silencing or knocking out the BpSPL2 gene, the ability of birch to bloom early can be improved.

[0041] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of the BpSPL2 gene in regulating the early flowering ability of plants, characterized in that: The nucleotide sequence of the BpSPL2 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acids of the protein encoded by the BpSPL2 gene are shown in SEQ ID NO.

2.

3. The use according to claim 2, characterized in that The plants include birch.

4. The use according to claim 3, characterized in that The application is to inhibit the BpSPL2 gene in plants to enhance the ability of the plants to flower early.

5. A method for improving the early flowering ability of plants, characterized in that: The steps include: (1) constructing the inhibitory expression vector of the BpSPL2 gene as described in claim 1, and transforming it into Agrobacterium to obtain an engineered bacterium; (2) After infecting plant tissue with the engineered bacteria obtained in step (1), plant tissue culture is performed to obtain transgenic plants with early flowering ability.

6. The method according to claim 5, characterized in that The method for constructing the inhibition expression vector of the BpSPL2 gene is as follows: amplifying the BpSPL2 gene with primers BpSPL2F and BpSPL2-SRDXR to obtain a BpSPL2-SRDX target fusion fragment containing a restriction site; connecting the BpSPL2-SRDX target fusion fragment with a plasmid vector to obtain the inhibition expression vector of the BpSPL2 gene.

7. The method according to claim 6, characterized in that The nucleotide sequence of the BpSPL2F is shown in SEQ ID NO.3, and the nucleotide sequence of the BpSPL2-SRDXR is shown in SEQ ID NO.

4.

8. The method according to claim 7, characterized in that The restriction sites are BamHI and KpnI restriction sites, and the plasmid vector is pROKⅡ.

9. The method according to any one of claims 5 to 8, characterized in that: The infection sites include plant leaves.

Images