Application of osdfr1 protein and its coding gene in regulating effective panicle of rice

Abstract

The application belongs to the technical field of plant genetic engineering and crop genetic breeding, and particularly relates to application of an OsDFR1 protein and a coding gene thereof in regulating effective panicles of rice. OsDFR1 The application realizes increase of the number of effective panicles of rice and increase of yield per plant. Based on molecular breeding of the gene, the rice tillering characteristics can be optimized from a genetic level, obvious yield-increasing effect is achieved, and the gene has application prospect in yield improvement.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and crop genetic breeding technology, specifically involving the application of OsDFR1 protein and its encoding gene in regulating effective panicles in rice. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is an important food crop, and the yield per rice plant is a complex quantitative trait, composed of three factors: the number of effective panicles per plant, the number of grains per panicle, and the grain weight. Among these, the number of effective panicles per plant depends on the tillering ability of the plant and is the primary factor determining yield. Therefore, elucidating the regulatory mechanisms of tillering development and identifying key regulatory genes is of great significance for overcoming the bottleneck in rice yield.

[0003] Currently, research on genes related to rice tiller regulation mainly focuses on classical hormone signaling pathways, such as D53 and D14 in the styracin signaling pathway, OsPIN1b and PILS6b in the auxin signaling pathway, and transcription factors MOC1 and OsSPL14 / IPA1. While these genes play important roles in tiller regulation, they have the following limitations: First, existing gene resources are mostly derived from traditional hormone or developmental regulatory pathways, resulting in extremely limited understanding of key genes in the cross-regulatory network of light signaling and tiller development; second, some known genes (such as...) OsSPL14 / IPA1 (1) It has multiple effects, regulating tillering while affecting traits such as plant height and ear type, making it difficult to achieve precise and independent improvement of effective ear number; (2) The allelic variations of existing gene resources have been widely used in breeding, and there is an urgent need to explore new functional genes to expand the space for genetic improvement.

[0004] At the cultivation level, rice tillering regulation mainly relies on exogenous chemical agents (such as paclobutrazol and uniconazole). However, chemical regulation has the following prominent problems: First, long-term application can easily cause soil pollution and pesticide residues in agricultural products, which does not meet the requirements of green agriculture development; second, tillering regulation involves a complex hormone signaling network, making it difficult for chemical agents to achieve precise targeting; and third, the effects of chemical regulation are greatly affected by environmental conditions and have poor stability. Therefore, developing environmentally friendly and precisely controllable new technologies for tillering regulation has become an urgent need.

[0005] In recent years, physical regulation technologies have attracted attention due to their environmental friendliness and lack of pesticide residues. Among them, low-intensity laser treatment, as a special form of light regulation, has the characteristics of good monochromaticity, controllable energy density, and significant biological effects. Previous research by the laser agriculture team at Xianghu Laboratory found that low-intensity laser treatment (2 µmol / m² / s PPFD) can increase the effective tillering of rice by 16.8%, demonstrating significant yield potential. However, the key response genes for laser-regulated tillering have not yet been elucidated. This results in a lack of molecular target support for laser physical regulation technology, making it difficult to combine with molecular breeding and restricting its precision application.

[0006] A thorough analysis of the molecular basis of laser signal regulation of rice tillering and the discovery of key target genes from laser-responsive genes has the following significant value: First, it can enrich the gene resources of the cross-regulatory network of light signals and tillering development, compensating for the lack of existing hormone pathway gene resources; second, it can provide new functional targets for modern breeding technologies such as molecular marker-assisted selection, dominant haplotype screening, and gene editing, enabling precise genetic improvement of effective panicle number; third, it can provide molecular theoretical support for laser physical regulation technology, promoting the synergistic application of light regulation and genetic improvement. Therefore, cloning and identifying new genes regulating effective panicle number from low-intensity laser-responsive genes has important theoretical and practical significance for breaking through the bottleneck of rice yield and promoting green and high-yield breeding. Summary of the Invention

[0007] The number of effective panicles in rice is the primary factor determining yield. However, current methods for regulating rice tillering mainly rely on exogenous chemical pesticides, which pose problems such as environmental pollution, pesticide residue risks, and difficulty in precise targeted regulation. Furthermore, existing known tillering regulatory gene resources are limited, and the molecular mechanisms and key target genes for light-induced tillering regulation remain unclear. Therefore, this invention provides an application of the OsDFR1 protein and its encoding gene in regulating the number of effective panicles in rice.

[0008] The specific technical solution is as follows: The present invention provides in a first aspect OsDFR1 The use of genes or related biological materials in at least one of the following: A1) Regulating the effective panicle size of rice or preparing products that regulate the effective panicle size of rice; A2) Cultivate rice varieties with increased effective panicles or prepare products that increase the effective panicles of rice; A3) Preparation of transgenic rice; The OsDFR1 The nucleotide sequence encoding the gene is shown in SEQ ID NO.1.

[0009] Furthermore, the biomaterial includes one or more of the following: B1) OsDFR1 The gene-encoded protein and / or containing the above OsDFR1 Gene recombinant vectors, recombinant microorganisms, or transgenic rice cell lines; B2) contains upregulation OsDFR1 Nucleic acid molecules for gene expression, recombinant vectors, recombinant microorganisms, or gene-edited rice cell lines.

[0010] Furthermore, the aforementioned OsDFR1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0011] Furthermore, the aforementioned OsDFR1 Gene recombination vectors include basic vectors and OsDFR1 The underlying carrier includes the pFLAGHAU carrier.

[0012] Furthermore, the containing OsDFR1 Recombinant microorganisms include Agrobacterium tumefaciens EHA105.

[0013] Furthermore, the regulation of effective panicles in rice includes: by increasing... OsDFR1 Gene expression is used to increase the effective panicle size in rice.

[0014] The present invention provides an upward adjustment in a second aspect. OsDFR1 Application of gene-expressing biological materials in cultivating rice with increased effective panicles; OsDFR1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] Further, the biomaterial includes: OsDFR1 The gene encodes a protein and / or contains upregulated proteins. OsDFR1 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic rice cell lines.

[0016] In a third aspect, the present invention provides a method for increasing the effective panicles in rice, comprising: Will be raised OsDFR1 Gene-expressing biological materials are transferred into rice.

[0017] The present invention provides a method for cultivating rice with increased effective panicles in a fourth aspect, comprising: Will be raised OsDFR1 Gene-expressing biological materials are transferred into rice.

[0018] The present invention provides, in a fifth aspect, a rice variety with increased effective panicle size, the rice comprising an upregulating... OsDFR1 Biological materials for gene expression; The biomaterial includes: OsDFR1 The gene encodes a protein and / or contains upregulated proteins. OsDFR1 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic rice cell lines; The yield per rice plant has increased.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) Gene resource innovation: For the first time, a new gene regulating the effective panicle number in rice was cloned and identified from a gene that responds to low-intensity laser light. OsDFR1 This enriches the gene resources in the cross-regulatory network of light signal and tiller development, providing a new and excellent gene target for high-yield molecular design breeding of rice.

[0020] (2) Great potential for increased production: Functional verification shows that it can improve production. OsDFR1 The expression level of this substance can increase the number of effective panicles in rice by 33.7% to 49.0% and the yield per plant by 12.2% to 39.7%, showing a significant yield-increasing effect and promising application prospects for yield improvement.

[0021] (3) Environmentally friendly: Molecular breeding based on this gene can optimize the tillering characteristics of rice at the genetic level without the need for exogenous chemical drugs to regulate tillering, thus fundamentally avoiding soil pollution and the risk of agricultural product residues, which meets the requirements of green agriculture and sustainable development.

[0022] (4) Low production cost: portable OsDFR1 Varieties with superior alleles can achieve a significant increase in effective panicle number and yield under conventional field cultivation conditions, without the need for additional lighting equipment or special environmental control inputs, thus reducing production energy consumption and planting costs. Attached Figure Description

[0023] Figure 1 The transcriptome sequencing results are for rice seedlings treated with low-intensity laser. Figure 1 Part a is a heatmap of differentially expressed genes clustering analysis before and after low-intensity laser induction; Figure 1 Part b presents a Venn diagram analysis of differentially expressed genes at different time points after low-intensity laser treatment.

[0024] Figure 2 These are candidate genes screened based on transcriptome sequencing.

[0025] Figure 3 for OsDFR1 Functional loss significantly reduces effective panicle and per-plant yield; among which, Figure 3 part a is OsDFR1 Analysis of mutation types in loss-of-function mutants; Figure 3 Part b is OsDFR1 Phenotypic diagram of loss-of-function mutant, scale bar: 20 cm; Figure 3 part c is OsDFR1 Analysis of the number of effective spikes and yield per plant in loss-of-function mutants.

[0026] Figure 4 For the construction and identification of overexpression materials; among which, Figure 4 part a is OsDFR1 Schematic diagram of the overexpression vector; Figure 4 Part b is OsDFR1 Analysis of the relative expression levels of transcription in overexpressing plants; Figure 4 part c is OsDFR1 Analysis of protein expression levels in overexpressing plants.

[0027] Figure 5 For overexpression OsDFR1 Increase the number of effective panicles and yield per plant in rice; among which, Figure 5 part a is OsDFR1 Field phenotypic diagram of overexpressing plants; Figure 5 Part b is OsDFR1 Phenotypic diagram of overexpression plants at maturity, scale bar: 20 cm; Figure 5 Part c is a statistical chart of the number of effective ears per plant; Figure 5 Part d is a statistical chart of yield per plant. Detailed Implementation

[0028] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0029] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0030] In this invention OsDFR1 The coding sequence of the gene is shown in SEQ ID NO.1; in this invention, the amino acid sequence of the OsDFR1 protein is shown in SEQ ID NO.2.

[0031] SEQ ID NO.1:ATGCCGCCGCGGCGCGTGTGCGTCACCGGCGCCGGCGGGTTCATCGGCTCGTGGCTCGTCAATCTCCTCCTCTCCTGCGGCTACTTCTTCCACGGCACCGTCCGCAACCCAGATGATCCCAAGAACGCGTTTCTGAAGCAGCTAGAGAATGCCACGGAGAATCTGCAGCTGTTCAAGGCCGACGTGCTCGACGGCGGCTCGCTGACGGCGGCGTTCGCCGGCTGCGAGGGCGTCTTCCATCCGGCCACTCCGGTGCCGGAAGAACAGATGGTTGATCCAGAGAAGGAGATGATGGCTCCTGCTGTGAAAGGCACCAGGAATATGCTGGAGGCTTGCTCTGCCGCAGGTGTTCAGAAACTCGTCGTGGTCTCCTCCATTGCTGCTGTATTCTTTAACCCGAGCTGGCCTCATGACAGGCCAAAAGATGAGACTTCTTGGTCAGACAAGAAGCTCTGCATGGAAACTGAGAACTGGTACTCTCTTGCCAAAACTGAAGGTGAAGAGATGGCCCTAGAATACGGAAACAGGAATGGTCTGCATGTTGTTACGGTTTGCCCTGGAATTGTTTTTGGCCCAATGTTGCAGACTGTGCAACTCAACACTACCACCAAAGCTCTCCTCTATATCATCCAAGTGCAGGAGGCCATGGCCCTGATACAATGA。

[0032] SEQ ID NO.2:MPPRRVCVTGAGGFIGSWLVNLLLSCGYFFHGTVRNPDDPKNAFLKQLENATENLQLFKADVLDGGSLTAAFAGCEGVFHPATPVPEEQMVDPEKEMMAPAVKGTRNMLEACSAAGVQKLVVVSSIAAVFFNPSWPHDRPKDETSWSDKKLCMETENWYSLAKTEGEEMALEYGNRNGLHVVTVCPGIVFGPMLQTVQLNTTTKALLYIIQVQEAMALIQ。

[0033] In this invention, the rice varieties are Guangluai 4 (GLA4) and Zhonghua 11 (ZH11).

[0034] Example 1: Cultivation of Experimental Materials The rice variety Guangluai 4 (GLA4) was selected as the experimental material. Seed disinfection: Rice seeds were soaked in a 2% (v / v) sodium hypochlorite solution and disinfected by shaking on a shaker at 40 rpm for 1 hour; then the seeds were washed 5-6 times with sterile water until all sodium hypochlorite residue was removed. The disinfected seeds were then soaked in clean sterile water and cultured in an artificial climate chamber under the following conditions: day / night temperature 28℃ / 25℃. The soaking water was changed daily, and the culture continued until the rice seeds showed signs of sprouting.

[0035] Rice seeds with uniform germination were sown in 96-cell hydroponic boxes and cultured using Kimura B nutrient solution. The environmental parameters were set as follows: day / night temperature 28℃ / 25℃, relative humidity 70%, light intensity 250 µmol / m² / s, and photoperiod of 14h light / 10h dark. The nutrient solution was changed every 3 days to ensure a stable nutrient supply.

[0036] Example 2: Low-intensity laser treatment and transcriptome sequencing analysis (1) Dark treatment stage: Select hydroponic seedlings with a seedling age of 10 days and transfer them to a completely dark environment for 3 days of dark treatment. During this period, except for the change in light conditions, other environmental parameters (temperature, humidity, nutrient solution, etc.) are kept consistent with normal culture conditions.

[0037] (2) Laser treatment and sampling: After the dark treatment, the seedlings were randomly divided into two groups: the control group (Dark) continued to be cultured in complete darkness; the treatment group (Laser) was treated under low-intensity laser irradiation, with the laser intensity set at 2 µmol / m² / s PPFD, and the other culture conditions were strictly consistent with those of the control group. Aboveground tissue samples of the two groups of seedlings were collected at four time points: 0 h, 9 h, 24 h and 72 h after laser treatment, and were flash-frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80℃ for later use.

[0038] (3) Transcriptome sequencing and differentially expressed gene screening: Total RNA was extracted from samples at each time point, and cDNA libraries were constructed followed by high-throughput transcriptome sequencing (RNA-seq). Differentially expressed genes induced by low-intensity laser were screened using bioinformatics analysis. Figure 1 (Part a). The screening threshold for differentially expressed genes was |log2(Fold Change)| ≥ 1 and padj ≤ 0.05.

[0039] (4) Identification of sustained-response genes: Venn diagrams were used to cross-analyze differentially expressed genes at different time points to screen differentially expressed genes that sustained-response at each time point of laser treatment. A total of 947 low-intensity laser sustained-response genes were identified. Figure 1 (Part b) provides a candidate gene pool for the subsequent discovery of key target genes.

[0040] Example 3: Candidate gene screening Based on the aforementioned 947 genes with sustained low-intensity laser response, and further combined with gene expression level and gene function annotation information, a total of 12 candidate genes were screened out. Figure 2 By purchasing or constructing loss-of-function mutant materials corresponding to the above 12 candidate genes, and through standardized field planting and phenotypic identification, mutants were found. osdfr1 The number of effective spikes and the yield per plant were significantly lower than those of the wild type. Figure 3 This indicates that OsDFR1 is involved in regulating rice tiller formation and yield development.

[0041] Example 4: Construction and Identification of Overexpression Materials (1) Construction of overexpression vector: Using wild-type rice (Zhonghua 11, ZH11) cDNA as a template, high-fidelity DNA polymerase was used to amplify the expression vector. OsDFR1 The coding sequence (CDS).

[0042] Upstream primer F (SEQ ID NO.3): 5'-GACGATAAG GGTACC ATGCCGCCGCGGCGCGTGTG-3' (underlined is the KpnI restriction site); Downstream primer R (SEQ ID NO.4): 5'-AGAGTCGAC GGATCC TTGTATCAGGGCCATGGCCT-3' (underlined is the BamHI restriction site).

[0043] The plant expression vector pFLAGHAU was linearized by double digestion with restriction endonucleases KpnI and BamHI. The CDS fragment of OsDFR1 was then directionally inserted into the pFLAGHAU vector using a one-step seamless cloning technique, constructing the overexpression vector pFLAGHAU-OsDFR1, driven by the maize ubiquitin promoter, with an N-terminal FLAG tag fused to the N-terminus and a C-terminal HA tag fused to the C-terminus. Figure 4 (Part a). After Sanger sequencing confirmed the inserted sequence was correct, the recombinant vector was transformed into Agrobacterium tumefaciens (Agrobacterium tumefaciens). Agrobacterium tumefaciens EHA105 competent cells.

[0044] (2) Agrobacterium-mediated genetic transformation: The overexpression vector was transformed into rice ZH11 callus tissue using the Agrobacterium-mediated transformation method. After callus induction, co-culture, screening culture, differentiation culture and rooting culture, transgenic resistant seedlings were obtained. Positive transgenic plants were obtained by PCR detection.

[0045] (3) Molecular identification of overexpression lines: Two-week-old overexpressing transgenic seedlings and wild-type (ZH11) control plants were harvested from the aboveground parts. Total RNA was extracted and reverse transcribed into cDNA. Using OsDFR1-RTF (SEQ ID NO.5) and OsDFR1-RTR (SEQ ID NO.6) as amplification primers, real-time quantitative PCR (RT-qPCR) was employed to detect the RNA. OsDFR1 The relative expression level of rice genes. Actin (LOC_Os03g50885) was used as an internal control (the amplification primers were Actin-RTF (SEQ ID NO.7) and Actin-RTR (SEQ ID NO.8), and 2... ^ ΔΔCt The relative expression level is calculated using this method.

[0046] OsDFR1-RTF (SEQ ID NO.5): CCAAAAGATGAGACTTCTTGG; OsDFR1-RTR (SEQ ID NO.6): CCATCTCTTCACCTTCAGTTT; Actin-RTF (SEQ ID NO.7): CGGGAAATTGTGAGGGACAT; Actin-RTR (SEQ ID NO. 8): AGGAAGGCTGGAAGAGGACC.

[0047] The results showed that, compared with the wild type, the three independent overexpression lines (OE-1, OE-2, OE-3) showed higher expression levels. OsDFR1 The transcriptional levels were significantly upregulated by 164.1-fold, 174.8-fold, and 107.3-fold, respectively. Figure 4 (Part b) confirmed that the target gene was efficiently and stably overexpressed at the transcriptional level.

[0048] Simultaneously, total protein was extracted from the above-mentioned overexpression lines and wild-type materials, and the expression of the target protein was detected by Western blotting. The results showed that specific FLAG fusion protein signal bands were detected at the expected molecular weight positions in all three overexpression lines, while no corresponding signal was observed in the wild-type control, indicating that all three overexpression lines successfully expressed the OsDFR1 target protein at the protein level. Figure 4 (Part C).

[0049] Example 5: Field Phenotypic Identification of Overexpression Materials wild type and 3 independent OsDFR1 Overexpression lines were planted in the field. After the materials were fully mature, 10 plants from each line were harvested individually for evaluation and analysis, examining yield-related traits such as effective spikes and yield per plant. The results showed that the wild-type material (ZH11) had an average of 10.4 effective spikes / plant, while the three overexpression lines had averages of 15.5, 13.9, and 14.9 effective spikes / plant, respectively. The wild-type material had an average yield per plant of 23.14 g, while the three overexpression lines had averages of 32.33 g, 27.64 g, and 25.97 g, respectively. Compared with the wild-type material, the three overexpression lines showed an increase of 33.7%–49.0% in the number of effective spikes and an increase of 12.2%–39.7% in yield per plant. Figure 5 The result indicates that... OsDFR1 It is a key positive regulator that promotes the increase of effective panicle number and yield in rice. Overexpression of this gene can significantly increase the effective panicle number and yield per plant, and has important application value in high-yield breeding.

Claims

1. OsDFR1 The use of genes or related biological materials in at least one of the following: A1) Regulating the effective panicle size in rice; A2) Prepare products that regulate the effective panicle size of rice; A3) Cultivating rice varieties with increased effective panicles; A4) Prepare products that increase the effective panicle size in rice; The regulation of effective panicles in rice includes: by increasing... OsDFR1 Gene expression is used to increase the effective panicle size in rice; The OsDFR1 The coding nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The biomaterials include one or more of the following: The OsDFR1 The gene-encoded protein and / or containing the above OsDFR1 Gene recombinant vectors, recombinant microorganisms, or transgenic rice cell lines; The OsDFR1 The amino acid sequence of the protein encoded by the gene includes that shown in SEQ ID NO.2; The encoding nucleotide sequence of the OsDFR1 gene is shown in SEQ ID NO.

1.

3. Upward adjustment OsDFR1 Application of gene-expressing biological materials in cultivating rice with increased effective panicles; OsDFR1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The application as described in claim 3, characterized in that, The biomaterial includes: OsDFR1 Gene-encoded proteins and / or those containing upregulated OsDFR1 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic rice cell lines.

5. A method for increasing the effective panicles in rice, characterized in that, include: Will be raised OsDFR1 Gene-expressing biological materials were transferred into rice; The OsDFR1 The coding nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. A method for cultivating rice with increased effective panicles, characterized in that, include: Will be raised OsDFR1 Gene-expressing biological materials were transferred into rice; The OsDFR1 The coding nucleotide sequence of the gene is shown in SEQ ID NO.1.

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