Application of BjuPR10 gene in regulation and control of plant leaf development and flowering time

Abstract

The invention discloses an application of a BjuPR10 gene in regulation and control of plant leaf development and flowering time, a nucleotide sequence of the BjuPR10 gene is shown as SEQ ID NO.1, and an amino acid sequence coded by the BjuPR10 gene is shown as SEQ ID NO.2. The invention further discloses an application of the BjuPR10 gene in regulation and control of plant leaf development and flowering time. The overexpression technology finds that BjuPR10 gene up-regulation expression promotes brassicaceous plants such as stem nodule mustard to bolt and flower in advance and enables leaves to generate more leaf cracks, and the BjuPR10 gene can be applied to delaying the flowering process and has a good application prospect in genetic engineering breeding. A theoretical basis and gene resources are provided for genetic breeding of quality and yield of tumorous stem mustard and other plants, a new choice is provided for genetic improvement of the plants, and meanwhile, the biological function of the PR10 protein is perfected.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of BjuPR10 gene in regulating plant leaf development and flowering time. Background Art

[0002] Tumored mustard is a variant of Brassica juncea, a subspecies of Brassica juncea in the family Brassicae cruciferae. Its enlarged stems can be eaten fresh or processed into tumored mustard. The tumored mustard industry is a pillar industry in Chongqing. Currently, the city's tumored mustard planting area has reached 100,000 hectares. 2 , with an annual output value of 13 billion yuan, becoming a powerful tool to promote farmers' income growth. Its main edible organ is the enlarged stem of the plant. Bolting and flowering are important growth and development processes of plants. In cruciferous plants, the process from bolting to flowering determines the transition from the vegetative period to the reproductive period. Early bolting means that the reproductive growth period is extended, the vegetative growth time is long, and the tuber swells significantly. After bolting, the tuber stops swelling. Therefore, the early or late time of bolting and flowering has a great influence on the tuber yield.

[0003] In the prior art, there are many studies on the bolting and flowering mechanisms of plants. For example, invention patent CN111996200A discloses the application of Arabidopsis TGA7 gene in regulating the flowering period of plants. Plants with Arabidopsis TGA7 gene mutations have delayed flowering compared with wild-type plants. Invention patent CN106701782A discloses the application of Arabidopsis gene SPOC1 in regulating the flowering period of plants. The invention study found that Arabidopsis plants with SPOC1 gene knocked out have delayed flowering compared with wild-type plants. Although there have been many reports on the regulatory mechanism of plant flowering-related traits, there are still many unknown areas that need to be studied. Identifying and discovering new flowering trait regulatory genes, and studying the regulatory mechanisms and internal connections of these genes not only have important theoretical significance, but also have potential application value for actual plant production.

[0004] Pathogensis related proteins (PRs) are a class of proteins that are induced and accumulated in plants after being subjected to biotic or abiotic stresses. They are an important part of the plant defense system. In recent years, the discovered PR proteins have been divided into 17 subclasses based on their primary sequence structure, serological relationship and biological activity, and are named PR1-PR17. One of the subclasses, PR10, is a protein with RNase-like properties. PR10 has the characteristics of low molecular weight (16-19 kDa), acidic isoelectric point and highly conserved tertiary structure. There is a highly conserved amino acid sequence GXGGXG (X is any amino acid) in its secondary structure, which is called the "P-loop" motif. This is a domain that is widely present in phosphorylated kinases and nucleic acid binding proteins. As an inducible component in the plant defense system, PR10 is induced by a variety of pathogens. For example, the invention patent CN104152470B discloses an application of the Minjiang lily pathogenesis-related protein 10 gene LrPR10-6, and overexpression of the LrPR10-6 gene can improve tobacco resistance to Botrytis cinerea, Fusarium oxysporum, and Sclerotinia sclerotiorum; the invention patent CN102120997A discloses a method for hydrolyzing RNA and antibacterial activity of grape pathogenesis-related protein PR10-1, proving that grape pathogenesis-related gene PR10-1 has a strong inhibitory effect on tobacco brown spot pathogen. At present, there are few reports that pathogenesis-related protein PR10 is involved in the regulation of plant flowering time and leaf development, and there is no report on pathogenesis-related protein PR10 in stem tumor mustard. Summary of the invention

[0005] In view of the defects of the prior art, the purpose of the present invention is to provide an application of the BjuPR10 gene in regulating plant leaf development and flowering time. The present invention reveals that the BjuPR10 gene is a positive regulator of the flowering time of tumorous stem mustard plants, which provides a gene reserve and theoretical basis for the genetic breeding of yield and quality traits of tumorous stem mustard and other plants, and provides a new option for plant genetic improvement.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solution: BjuPR10 protein or a substance regulating the expression of the gene encoding the BjuPR10 protein or a substance regulating the activity or content of the BjuPR10 protein is used in any of the following ways:

[0007] A1) Application in regulating plant leaf development;

[0008] A2) Use in the preparation of products for regulating plant leaf development;

[0009] A3) Application in regulating flowering time of plants;

[0010] A4) Use in the preparation of products for regulating the flowering time of plants;

[0011] A5) Application in plant breeding;

[0012] A6) Use in the preparation of plant breeding products;

[0013] The amino acid sequence of the BjuPR10 protein is as shown in SEQ ID NO.2, or a protein having the same function is obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0014] Another object of the present invention is to provide any one of the following uses of the biomaterial related to the BjuPR10 protein:

[0015] A1) Application in regulating plant leaf development;

[0016] A2) Use in the preparation of products for regulating plant leaf development;

[0017] A3) Application in regulating flowering time of plants;

[0018] A4) Use in the preparation of products for regulating the flowering time of plants;

[0019] A5) Application in plant breeding;

[0020] A6) Use in the preparation of plant breeding products;

[0021] The biological material is a nucleic acid molecule capable of encoding the BjuPR10 protein or an expression box, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

[0022] Furthermore, the sequence of the nucleic acid molecule encoding the BjuPR10 protein is as shown in SEQ ID NO.1 or a nucleotide sequence encoding a protein with the same function obtained by replacing, inserting or deleting one or more bases of the nucleotide sequence shown in SEQ ID NO.1.

[0023] Furthermore, the plant is a dicotyledon or a monocotyledon. Preferably, the plant is Brassica juncea.

[0024] Another object of the present invention is to provide a method for regulating the flowering time of plants, the method comprising regulating the expression level of the gene encoding the BjuPR10 protein in the target plant or regulating the activity or content of the BjuPR10 protein in the target plant.

[0025] Another object of the present invention is to provide a method for regulating plant leaf morphology, the method comprising regulating the expression level of the gene encoding the BjuPR10 protein in the target plant or regulating the activity or content of the BjuPR10 protein in the target plant.

[0026] Another object of the present invention is to provide a method for preparing a transgenic plant with an early flowering time, comprising the following steps: increasing the content and / or activity of the BjuPR10 protein in the target plant to obtain a transgenic plant with an early flowering time.

[0027] Another object of the present invention is to provide a method for preparing a transgenic plant with lobed leaves, comprising the following steps: increasing the content and / or activity of the BjuPR10 protein in the target plant to obtain a transgenic plant with lobed leaves.

[0028] Furthermore, the method for increasing the content and / or activity of the BjuPR10 protein in the target plant is to introduce the BjuPR10 protein encoding gene or homologous gene into the target plant to overexpress it. Specifically, it can be achieved by any technical means that can achieve this purpose, such as by introducing a plasmid containing the gene into the target plant; by increasing the copy number of the gene on the chromosome of the target plant; by operably connecting a strong promoter to the gene; by introducing an enhancer.

[0029] Furthermore, the plant is a dicotyledon or a monocotyledon. Preferably, the plant is Brassica juncea.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention first discovered a key gene BjuPR10 in Brassica juncea that regulates the flowering time and leaf shape of plants. Using overexpression technology, it was found that up-regulating the expression of the BjuPR10 gene promoted early bolting and flowering of Brassica juncea and other cruciferous plants and produced more leaf fissures in the leaves. This indicates that the BjuPR10 gene is a positive regulatory factor for the flowering time of plants, which can be used to regulate the duration of vegetative growth and reproductive growth, ensure the quality and yield of plants, and has good application prospects in genetic engineering breeding.

[0032] 2. The present invention verifies the function of BjuPR10 gene in tumorous stem mustard, which not only helps to clarify the molecular regulatory mechanism of flowering time and leaf development of tumorous stem mustard, but also provides a theoretical basis and gene resources for genetic breeding of yield and quality traits of tumorous stem mustard and other plants, provides a new option for genetic improvement of plants, and has positive significance for the future genetic engineering breeding of some new varieties. At the same time, the biological function of PR10 protein is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A is the amplification of the BjuPR10 gene sequence; A is the gel electrophoresis detection diagram of the BjuPR10 gene amplified by PCR; B is the colony PCR detection result after the BjuPR10-Blunt-T vector was transformed into Escherichia coli; C is the comparison diagram of the BjuPR10 sequencing result in the positive monoclonal clone with the standard sequence.

[0034] Figure 2 To construct an overexpression vector containing the BjuPR10 gene; A is the electrophoresis detection diagram of the BjuPR10-Blunt-T vector digestion product, where a is the Blunt-T vector after digestion, and b is the BjuPR10 gene fragment after digestion; B is the colony PCR detection result after the BjuPR10-pTFGFP vector was transformed into Escherichia coli.

[0035] Figure 3 This is the subcellular localization analysis of BjuPR10 protein in tobacco leaf cells; from left to right, they are dark field image under excitation light, bright field image under white light, and combined image.

[0036] Figure 4 Expression analysis of BjuPR10 gene in wild-type and BjuPR10 transgenic plants.

[0037] Figure 5 The phenotypes of leaves of wild-type and BjuPR10 transgenic plants.

[0038] Figure 6 There are significant differences in bolting and flowering time between wild type and BjuPR10 transgenic plants. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific examples and drawings, but the scope of protection of the present invention is not limited to the above contents. The raw materials described in the examples are all common commercially available products unless otherwise specified. The experimental methods described in the examples are not particularly specified, that is, they are operated according to conventional molecular biology experimental methods.

[0040] Example 1 Cloning and subcellular localization analysis of BjuPR10 gene

[0041] (1) Gene cloning

[0042] According to the kit method, total RNA of Brassica juncea was extracted and reverse transcribed into cDNA. The cDNA was used as a template to amplify the nucleotide sequence of the BjuPR10 gene according to the CDS sequence of the BjuPR10 gene (SEQ ID NO.1). The amplification primers were BjuPR10-F and BjuPR10-R.

[0043] The primer sequences are as follows:

[0044] BjuPR10-F: 5'-CCAAGCTTATGATGATGGAGATGATGGAC-3'

[0045] BjuPR10-R: 5'-CGGGATCCTTCCCTGGAAAAGAGATGTTC-3'

[0046] PCR amplification system: high-fidelity amplification enzyme Prime STAR HS (R010A, TaKaRa) 0.25 μL, 5× PrimeSTAR Buffer (Mg 2+ Plus) 5 μL, forward primer (10 μM) 0.5 μL, reverse primer (10 μM) 0.5 μL, template (DNA) 1 μL, dNTP (2.5 mM) 2 μL, and sterile ddH2O to 25 μL.

[0047] Reaction program: pre-denaturation at 95°C for 5 min; 95°C for 30 s; 60°C for 40 s; 72°C for 30 s, 38 cycles; 72°C for 10 min.

[0048] The obtained PCR products were analyzed by agarose gel electrophoresis ( Figure 1 A), the target gene fragment BjuPR10 was purified according to the gel recovery kit (9672, Takara), the recovered fragment was connected to the entry vector of pEASY-Blunt Simple Cloning Kit (Blunt-T) of Quanshijin Company (Beijing, China), the ligation product was transformed into Escherichia coli DH5α competent cells, and the bacterial solution cultured for 40 minutes after transformation was spread on LB solid medium containing 100 μg / mL ampicillin, and inverted cultured in a 37°C incubator for 16 hours to screen resistant single colonies, and 2Taq DNA polymerase was used to amplify the BjuPR10 gene in the single colony by colony PCR using the single colony grown on the culture medium as a template, and the positive clone containing the BjuPR10 gene fragment was detected by 1% agarose gel electrophoresis ( Figure 1 B). Pick a positive single clone containing the target gene band and expand it in liquid LB medium and sequence it. The E. coli with the correct sequence contains the cloned BjuPR10 gene ( Figure 1 C).

[0049] (2) Construction of a recombinant expression vector of the BjuPR10 gene fused with green fluorescent protein (GFP)

[0050] The BjuPR10-Blunt-T vector plasmid containing the BjuPR10 gene fragment and the target vector pTFGFP plasmid were extracted, and the two plasmids were double-digested with restriction endonucleases Sma I and BamH I from Thermo Company, respectively. The digestion products were detected by 1% agarose gel electrophoresis and the BjuPR10 gene fragment after digestion was recovered ( Figure 2 A), the BjuPR10 gene fragment after restriction digestion was ligated to the pTFGFP vector after restriction digestion using T4 DNA ligase, and the ligation product was transformed into Escherichia coli DH5α competent cells, and the bacterial solution cultured for 40 minutes after transformation was spread on LB solid culture medium containing 100 μg / mL spectinomycin, and inverted cultured in a 37°C incubator for 16 hours to screen resistant single colonies, and 2Taq DNA polymerase was used to perform colony PCR amplification of the BjuPR10 gene in the single colony using the single colony grown on the culture medium as a template, and the positive clone containing the BjuPR10 gene fragment was detected by 1% agarose gel electrophoresis ( Figure 2 B) and sequencing verification, thus obtaining the recombinant expression vector BjuPR10-pTFGFP.

[0051] (3) Genetic transformation of recombinant expression vector and fluorescence detection analysis

[0052] According to conventional methods, the successfully constructed BjuPR10-pTFGFP vector was transformed into Agrobacterium tumefaciens GV3101 cells, and positive clones were screened by PCR. The Agrobacterium injection buffer was prepared according to the method of (Sun Manli et al., Study on Factors Affecting Transient Expression of Tobacco Mediated by Agrobacterium, Journal of Northwest Agriculture, 2015, 24(1): 161-165). Tobacco plants with 8-10 leaves fully expanded and growing normally in a light incubator were selected for injection, and the injection buffer was slowly pushed into the back of the leaves with a syringe without a needle. Then, the transformed plants were put back into the incubator and observed after 36h-48h of culture.

[0053] Carefully cut the transformed tobacco leaves with scissors and place them on a glass slide. Add 1 drop of distilled water to make a slide. Then place it on a fluorescence microscope and observe the fluorescence under the blue light with an excitation wavelength of 488-507nm. The results show ( Figure 3 ), green fluorescence signals could be detected in the plasma membrane and nucleus of tobacco leaf cells, indicating that the BjuPR10 protein had been successfully expressed in tobacco and functioned throughout the cell.

[0054] Example 3 Genetic transformation of tumorous stem mustard and identification of transgenic plants overexpressing BjuPR10 and phenotypic observation

[0055] (1) Agrobacterium-mediated genetic transformation of Brassica juncea

[0056] The constructed recombinant expression vector BjuPR10-pTFGFP was transferred into Agrobacterium strain GV3101 by conventional freeze-thaw method, positive clones were screened by PCR, and then the hypocotyls of seedlings of tumorous mustard cultivar 'Yong'an Xiaoye' were infected with GV3101 Agrobacterium containing BjuPR10-pTFGFP vector by genetic transformation of tumorous mustard hypocotyls.

[0057] (2) Identification of transgenic BjuPR10 tumorous mustard plants

[0058] The herbicide resistance gene (BAR) on the pTFGFP vector was used to screen positive strains of transgenic explants. The positive explants were cultivated into plants and the transgenic plant seeds were harvested to obtain two relatively stable transgenic strains BjuPR10-OE-2 and BjuPR10-OE-3.

[0059] The TRIzol reagent (Invitrogen TM ), total RNA was extracted from wild-type Brassica juncea ‘Yong’anxiaoye’ plants and two transgenic lines BjuPR10-OE-2 and BjuPR10-OE-3 according to the instructions, and DNase I (Invitrogen TM ) to remove residual DNA, and synthesize the first strand of cDNA using cDNA reverse transcription reagent (Takara) according to the instructions. The expression level of the target gene BjuPR10 in the transgenic strains and wild type of B.

[0060] The internal reference Bju18S rRNA gene primers are:

[0061] RT-Bju18S-F:5'-TCTGACGCAAGCACAACTA-3'

[0062] RT-Bju18S-R:5'-TCTTCTCAGCTTTAGCCATT-3'

[0063] The primers for detecting gene BjuPR10 are as follows:

[0064] RT-BjuPR10-F:5'-CAACAAGGTCACGTTCAAGGT-3'

[0065] RT-BjuPR10-R:5'-CACTTCCAACATGTCCTCC-3'

[0066] The results are as follows Figure 4As shown, compared with the wild-type plants (WT), the gene expression level of BjuPR10 in two independent transgenic lines (OE-2 and OE-3) transformed with BjuPR10-pTFGFP was significantly increased, and the expression level of the internal reference 18S rRNA gene remained basically the same, indicating that the BjuPR10 gene had been introduced into the genome of Brassica juncea and successfully transcribed and expressed.

[0067] (3) Phenotypic analysis of transgenic mustard plants

[0068] The transgenic lines BjuPR10-OE-2, BjuPR10-OE-3 and wild-type seeds were planted and observed for plant morphology under the same conditions. Figure 5 As shown, the results showed that compared with the wild-type 'Yong'an Xiaoye' leaves, the leaves of the transgenic plants of Tuber mustard had deeper leaf fissures, indicating that the BjuPR10 gene can change the leaf morphology of the plants.

[0069] Further, the bolting and flowering time of the transgenic strains and wild-type tumorous stem mustard were observed. Figure 6 The results showed that compared with the wild type, the bolting and flowering time of the two BjuPR10 gene overexpression plants BjuPR10-OE-2 and BjuPR10-OE-3 was advanced and the flowering period was not significantly shortened. The flowering time was advanced by about 20 days, indicating that the transgenic plants overexpressing the BjuPR10 gene had an early bolting and early flowering phenotype, and the gene can regulate the flowering time of the plant but has no obvious effect on the flowering period.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Use of BjuPR10 protein or a substance for regulating the expression of a gene encoding the BjuPR10 protein or a substance for regulating the activity or content of the BjuPR10 protein in any of the following: A1) Application in regulating plant leaf development; A2) Use in the preparation of products for regulating plant leaf development; A3) Application in regulating flowering time of plants; A4) Use in the preparation of products for regulating the flowering time of plants; A5) Application in plant breeding; A6) Use in the preparation of plant breeding products; The amino acid sequence of the BjuPR10 protein is as shown in SEQ ID NO.2, or a protein having the same function is obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.

2.

2. Use of the biological material related to the BjuPR10 protein of claim 1 in any of the following: A1) Application in regulating plant leaf development; A2) Use in the preparation of products for regulating plant leaf development; A3) Application in regulating flowering time of plants; A4) Use in the preparation of products for regulating the flowering time of plants; A5) Application in plant breeding; A6) Use in the preparation of plant breeding products; The biological material is a nucleic acid molecule capable of encoding the BjuPR10 protein or an expression box, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

3. The application according to claim 2, characterized in that: The sequence of the nucleic acid molecule encoding the BjuPR10 protein is shown in SEQ ID NO.1 or a nucleotide sequence encoding a protein with the same function obtained by replacing, inserting or deleting one or more bases of the nucleotide sequence shown in SEQ ID NO.

1.

4. The use according to any one of claims 1 to 3, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

5. A method for regulating the flowering time of a plant, characterized in that: The method comprises regulating the expression level of the gene encoding the BjuPR10 protein in the target plant or regulating the activity or content of the BjuPR10 protein in the target plant.

6. A method for regulating plant leaf morphology, characterized in that: The method comprises regulating the expression level of the gene encoding the BjuPR10 protein in the target plant or regulating the activity or content of the BjuPR10 protein in the target plant.

7. A method for preparing a transgenic plant with an early flowering time, characterized in that: The method comprises the following steps: increasing the content and / or activity of the protein in claim 1 in the target plant to obtain a transgenic plant with an early flowering time.

8. A method for preparing a transgenic plant having leaf lobes, characterized in that: The method comprises the following steps: increasing the content and / or activity of the protein in claim 1 in the target plant to obtain a transgenic plant with leaf lobes.

9. The method according to claim 7 or 8, characterized in that: The method for increasing the content and / or activity of the protein of claim 1 in the target plant is to introduce the protein encoding gene or homologous gene of claim 1 into the target plant to overexpress it.

10. The method according to any one of claims 5 to 9, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

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