Sugarcane tillering related gene ScFBP5 and application thereof

Abstract

The invention discloses application of a sugarcane ScFBP5 gene in regulation and control of plant tillering or branching, and belongs to the technical field of gene engineering. 1, and the ScFBP5 gene codes an amino acid sequence as shown in SEQ ID NO. 2. According to the invention, the branching or tillering capability of plants can be improved by improving the expression of the ScFBP5 gene. The ScFBP5 gene is expected to regulate and control the plant type of the plant and improve the germplasm resources of the plant, and has high economic and application values.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a ScFBP5 gene and an application thereof in regulating plant type. Background Art

[0002] Regulating plant type is of great significance in agricultural production. On the one hand, a reasonable plant type can optimize space utilization, allow plants to fully receive light and ventilation, and facilitate photosynthesis and growth. On the other hand, it can increase planting density, increase yield, facilitate field management operations, reduce the incidence of pests and diseases, and contribute a lot to the efficient and high-quality development of agricultural production. Tillering or branching is one of the key elements of an ideal plant type. First, tillering or branching can increase biomass. For example, more tillers in rice can increase the number of panicles, thereby increasing grain yield. Secondly, it expands the range of space and resources that plants can obtain, and better utilizes light, water and nutrients. Taking Arabidopsis as an example, branching allows it to occupy more space in the surrounding environment to grow. Furthermore, it enhances adaptability to the environment. When some branches and leaves are damaged, other tillers or branches can maintain the overall growth function and ensure plant reproduction, which is of great significance to the stable continuation of plant populations.

[0003] Genetic engineering technology is of great significance in regulating plant tillering or branching: by regulating key genes, such as OsMYB27 and MOC1, the number of tillers can be effectively increased or decreased, thereby optimizing the plant structure and providing more efficient breeding tools for agricultural production. However, the genetic regulatory network of plant tillering or branching is intricate, and the molecular regulatory mechanisms of plants with different genetic backgrounds vary greatly. Although some genes that regulate plant tillering or branching have been reported, discovering and cloning more new genes that have the function of regulating plant tillering or branching in different genetic backgrounds, and developing new methods for cultivating new varieties with excellent plant types, are still technical problems that need to be solved urgently. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a gene and its application in regulating plant type. The gene is the sugarcane (Saccharum spp. hybrid) ScFBP5 gene. Studies have found that overexpression of the gene in plants such as Arabidopsis and rice improves the tillering / branching ability of plants such as Arabidopsis and rice, suggesting that the gene has a regulatory effect on tillering / branching. The gene is expected to be applied to genetic engineering breeding of plants, providing a theoretical basis for plants to increase the number of branches or tillers, thereby improving production efficiency. Taking into account the degeneracy of codons, modifying the bases of the above nucleotide sequence without changing the amino acid sequence also falls within the scope of protection of the present invention.

[0005] In one aspect, the present invention provides a protein for regulating plant morphology, characterized in that the amino acid sequence of the protein is as shown in SEQ ID NO.2.

[0006] Furthermore, the present invention provides a gene for regulating plant type, characterized in that the gene encodes an amino acid sequence as shown in SEQ ID NO.2.

[0007] Furthermore, the present invention provides a vector for regulating plant type, characterized in that the vector can overexpress a gene encoding an amino acid sequence as shown in SEQ ID NO.2.

[0008] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene or the aforementioned vector in regulating plant type.

[0009] Furthermore, the regulating plant type is to increase the number of plant branches or tillers by overexpressing the aforementioned protein or the aforementioned gene, or to reduce the number of plant branches or tillers by reducing the expression of the aforementioned protein or the aforementioned gene.

[0010] In a preferred embodiment, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0011] Furthermore, the plant is a grass plant or a cruciferous plant.

[0012] Furthermore, the plant is a rice plant, a sugarcane plant or an Arabidopsis plant.

[0013] Furthermore, the plant is rice, sugarcane or Arabidopsis thaliana.

[0014] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene or the aforementioned vector in plant type breeding.

[0015] Furthermore, the plant type breeding is to regulate the number of branches or tillers of the plant.

[0016] Furthermore, in the plant type breeding, the number of plant branches or tillers is increased by overexpressing the aforementioned protein or the aforementioned gene, or the number of plant branches or tillers is reduced by reducing the expression of the aforementioned protein or the aforementioned gene.

[0017] In a preferred embodiment, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0018] Furthermore, the plant is a grass plant or a cruciferous plant.

[0019] Furthermore, the plant is a rice plant, a sugarcane plant or an Arabidopsis plant.

[0020] Furthermore, the plant is rice, sugarcane or Arabidopsis thaliana.

[0021] Compared with the prior art, the present invention has the following advantages: 1) The present invention provides a new protein and its encoding gene, which has the function of regulating the development of plant branches or tillers.

[0022] 2) The present invention provides a new method for regulating the number of plant branches or tillers, namely, increasing the number of plant branches or tillers by increasing the expression of the ScFBP5 gene or the protein encoded by it.

[0023] 3) The present invention provides a new method for cultivating multi-branched or multi-tillering plant varieties, namely, cultivating multi-branched or multi-tillering plant varieties by increasing the expression of the ScFBP5 gene or the protein encoded by it, and through plant variety improvement, achieving the effect of increasing the yield of a single plant, optimizing the plant structure, and improving production efficiency.

[0024] 4) The above method can also effectively improve the resource utilization efficiency of crops and enhance the stress resistance of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0026] Figure 1 Shown is the overexpression vector of the ScFBP5 target gene.

[0027] Figure 2 Shown is the expression level of ScFBP5 gene in transgenic Arabidopsis thaliana.

[0028] Figure 3 Shown is a comparison of branching between ScFBP5-overexpressing Arabidopsis and wild-type Arabidopsis; A is a phenotypic comparison, and B is a comparison of branch numbers.

[0029] Figure 4 The figure shows the comparison of branching between ScFBP5-overexpressing rice and wild-type rice; wherein A is the expression level of the rice overexpressing strain, B is the phenotypic comparison between wild-type rice and overexpressing rice, and C is the comparison of tillering number between wild-type rice and overexpressing rice. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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.

[0031] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0032] The nucleotide sequence of the sugarcane ScFBP5 gene in the embodiment is shown in SEQ ID NO.1: The amino acid sequence of sugarcane ScFBP5 protein is shown in SEQ ID NO.2: MDHAADAHRTDLMTITRHVLNEQSRNPESRGDFTILLSHIVLGCKFVASAVNKAGLAQLIGLAGETNVQGEEQKKLDVLSNEVFVKALVSSGRTCVLVSEEDEEATFVDPKLRGKYCVCFDPLDGSSNIDCGVSIGTIFGIYMIKDKDNVTLSDVLQPGKDMLAAGYCMYG SSCTLVLSTGTGVNGFTLDPSLGEFILTHPDIKIPKKGKIYSVNEGNAKNWDVPVAKFVEKCKYPKDGSPPKSLRYIGSMVADVHRTLLYGGVFLYPADQKSPNGKLRVLYEVFPMSFLMEQAGGQSFTGKERALDLVPTKIHERSPIFLGSYDDVEEIKALYAEQAKSSSA Example Example 1 Construction of Sugarcane ScFBP5 Gene Overexpression Vector The first fully expanded leaf from top to bottom of the sugarcane seedling was collected, and the total RNA was extracted using a plant RNA extraction kit (Magen), and then the target gene was amplified. The specific experiment is as follows: Take 1 μg high quality (OD 260 / OD 280 : 1.8-2.0; OD 260 / OD 230 ≈ 2.0) RNA was reverse transcribed (biorun reverse transcription kit) to obtain the first-strand cDNA. Amplification was performed using cDNA as a template. The reaction system was: Nuclease-free Water 20 uL; Biorun Pfu PCR Mix 25 uL; Primer (+): AACACGGGGGACTTTGCAACatggaccacgcggcggac, 2 uL; Primer (-): TCGAGACTAGTGGTACCCCCAAATACGAATACTCCGGCAGAT , 2 uL; cDNA template 1 μL. The reaction conditions were: 94℃ 5 min; 94℃ 30 sec, 50℃ 45 sec, 72℃ 62sec, 30 cycles; 72℃ 10 min. After the reaction, the PCR product was recovered using a gel recovery kit (biorun).

[0033] The pBWA(V)HS-ccdb-GLosgfp vector was linearized with restriction endonucleases BsaI / Eco31I. The digestion system was: 4 μL of vector, 1 μL of endonuclease, 2 μL of 10×Smartcut buffer, and 20 μL of Nuclease-free Water. The digestion conditions were: 37°C, 60 min. After the vector digestion products and PCR products were purified and recovered, the DNA concentration was determined using NanoDrop 2000, and the recombination kit (Biorun) was used for recombination. The recombination system was: target fragment: 5 μL, vector fragment: 5 μL, Biorun 2*EasyClone Mix 10 μL, a total of 20 μL. The recombination conditions were: 37°C, 60 min.

[0034] Take 10 uL of the product and add it to 100 μL of E. coli DH5α competent cells. Spread the transformation product on LB solid medium (containing kanamycin resistance, the concentration of kanamycin is 50 mg / L). Incubate at 37℃ for 12 h, and select 4 single clones for colony PCR identification. Select 2 positive clones for sequencing, and obtain positive clones containing the ScFBP5 gene sequence, and finally obtain the overexpression vector containing the ScFBP5 target gene ( Figure 1 ).

[0035] Example 2 Acquisition and identification of ScFBP5 transgenic Arabidopsis The overexpression vector constructed in Example 1 was transferred into Arabidopsis Columbia 0 (Col-0) by floral dipping method using Agrobacterium GV3101-mediated genetic transformation method. Experimental method reference: "Exploration of Arabidopsis thaliana transgenic technology mediated by Agrobacterium", Zhang Zhaoyichun, Agricultural Science and Technology, 2017, No. 9.

[0036] When the seedlings are about 20 days old, the genomic DNA of Arabidopsis thaliana is extracted by CTAB method and PCR detection is performed. Eleven positive T1 transgenic seedlings are propagated to obtain T2 generation. The T2 generation seeds are germinated using a culture medium containing hygromycin. If all the seeds can grow normally, it proves that the strain is a homozygous strain.

[0037] The 11 positive transgenic lines (OE1-OE11) were tested by qRT-RCR, and subsequent experiments and analyses were performed. The process of qRT-RCR identification of the overexpression effect of the ScFBP5 gene in transgenic Arabidopsis is as follows: 1. Use the Plant RNA Extraction Kit (Biorun) to extract total RNA from 7-day-old Arabidopsis seedlings, and take 1 μg of high-quality (OD 260 / OD 280 : 1.8-2.0; OD260 / OD 230 ≈ 2.0) RNA was reverse transcribed (Biorun reverse transcription kit) to obtain the first-strand cDNA.

[0038] 2. Using the cDNA in step 1 as a template, the expression of the ScFBP5 gene was detected by the primer pair ScFBP5-qF (GGTCCCAACCAAGATCCACGAG) and ScFBP5-qR (GAGCTTGACTTTGCCTGCT), and the expression of the Arabidopsis Actin gene was detected by the primer pair AtActin-F (GCTAACCGTGAGAAGATGAC) and AtActin-R (CTAGCATAAAGCGACAGGAC) of the Arabidopsis housekeeping gene Actin as an internal reference. The quantitative PCR reagent was SYBR® Premix Ex Taq™ (TAKARA), and the quantitative PCR instrument was CFX 96 (Bio RAD). The reaction system was: 2×PCR buffer 5 μL, qF primer 0.4 μL, qR primer 0.4 μL, cDNA template 1 μL, sterile water 3.2 μL, and the total volume of the reaction system was 10 μL. Reaction program: 95°C 30 sec; 95°C 5 sec, 68°C 30 sec, 45 cycles.

[0039] The results are as follows Figure 2 As shown, the expression levels of the ScFBP5 gene in the 11 strains were significantly increased, among which OE-5 and OE-6 had the highest expression levels, so these two overexpression strains were selected for subsequent experiments.

[0040] Example 3 Analysis of branching phenotype of ScFBP5 overexpressed Arabidopsis Homozygous overexpression and wild-type Col-0 plant seeds were surface sterilized with 2% sodium hypochlorite, sown on 1 / 2 MS fixed medium, and placed in a plant growth room for germination and growth after being cold-treated in a 4°C refrigerator for 3 days. 7-day-old Arabidopsis seedlings were transferred to Arabidopsis planting pots, and the branching phenotype differences were continuously observed and recorded.

[0041] Phenotypic results are shown in Figure 3 A, The results show that ScFBP5-overexpressing Arabidopsis plants have significantly more branches than the wild type. The statistical results of the number of branches at 45 days are shown in Figure 3 B.

[0042] Example 4 Acquisition and identification of ScFBP5 transgenic rice The overexpression vector constructed in Example 1 was transferred into the japonica rice variety Nipponbare by using the genetic transformation method mediated by Agrobacterium EHA105. T0 transgenic seedlings were obtained after selection, culture, differentiation, rooting and seedling hardening. Experimental method reference: "Study on efficient rice genetic transformation system mediated by Agrobacterium", Zheng Jie, Hunan Agricultural Science, 2008, No. 2.

[0043] All transgenic seedlings were identified by PCR, and the GFP vector fragment was amplified. Eleven positive transgenic seedlings were propagated to obtain T1 and T2 generations. The T2 generation seeds were germinated using a medium containing hygromycin. If all the seeds grew normally, it was proved that the strain was a homozygous strain. The four transgenic strains with the highest expression levels (OE-4, OE-5, OE-8, and OE-10) were selected for qRT-RCR detection, and subsequent experiments and analysis were carried out.

[0044] The process of qRT-RCR identification of the overexpression effect of ScFBP5 gene in transgenic rice is as follows: 1. Plant RNA extraction kit (Magen) was used to extract total RNA from rice leaves at the four-leaf stage. 1 μg high-quality (OD 260 / OD 280 : 1.8-2.0; OD 260 / OD 230 ≈ 2.0) was reverse transcribed (Novozyme reverse transcription kit) to obtain the first-strand cDNA.

[0045] 2. Using the cDNA in step 1 as a template, the expression of the ScFBP5 gene was detected by the primer pair ScFBP5-F (TGCTCCCACTAAACTTCACGAC) and ScFBP5-R (GACTCTGAAGCGTACAGT), and the rice internal reference gene Actin gene Actin-F (CTTCGTAGCACAGCCTTCA) and Actin-R (TTCTCCTTGATGTCCCTCTG) primer pair was used to detect the expression of the rice EF1α gene as an internal reference. The quantitative PCR reagent was SYBR® Premix Ex Taq™ (TAKARA), and the quantitative PCR instrument was CFX 96 (Bio RAD). The reaction system was: 2×PCR buffer 5 μL, qF primer 0.4 μL, qR primer 0.4 μL, cDNA template 1 μL, sterile water 3.2 μL, and the total volume of the reaction system was 10 μL. Reaction program: 95°C 30 sec; 95°C 5 sec, 68°C 30 sec, 45 cycles.

[0046] The results are as follows Figure 4As shown in A, the expression levels of the ScFBP5 gene in the four selected strains were significantly increased, and these two overexpression strains were subsequently selected for experiments.

[0047] Example 5 Analysis of tillering phenotype of ScFBP5 overexpressing rice The homozygous overexpressing rice seeds were broken from dormancy, germinated, sown, and then the 3-week-old seedlings were transferred to buckets, and the phenotypic differences were recorded at the rice maturity stage. The rice seedlings were planted at the Sugarcane Research Institute of Guangxi Academy of Agricultural Sciences from March to July in Guangxi, when the weather in Nanning is naturally long-day.

[0048] The statistical results of tillering time are shown in Figure 4 B and C, the tiller number of ScFBP5-overexpressing Nipponbare was significantly higher than that of WT at 90 days after planting.

[0049] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs may also make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the scope of protection of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the present invention.

Claims

1. A protein for regulating plant type, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. A gene for regulating plant type, characterized in that: The gene encodes the amino acid sequence shown in SEQ ID NO.

2.

3. A vector for regulating plant type, characterized in that: The vector can overexpress the gene encoding the amino acid sequence shown in SEQ ID NO.

2.

4. Use of the protein according to claim 1, the gene according to claim 2, or the vector according to claim 3 in regulating plant type.

5. The use according to claim 4, characterized in that: The regulating plant type is to increase the number of plant branches or tillers by overexpressing the protein described in claim 1 or the gene described in claim 2, or to reduce the number of plant branches or tillers by reducing the expression of the protein described in claim 1 or the gene described in claim 2.

6. The use according to claim 4, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

7. Use of the protein according to claim 1, the gene according to claim 2, or the vector according to claim 3 in plant type breeding.

8. The use according to claim 7, characterized in that The plant type breeding is to regulate the number of branches or tillers of the plant.

9. The use according to claim 7, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

10. The use according to claim 6 or claim 9, characterized in that: The plant is a grass plant or a cruciferous plant.

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