Gene GNR1 for controlling grain number per ear of rice and application of gene GNR1

Abstract

The invention relates to a gene for regulating and controlling the grain number of crops and application thereof. Specifically, the invention provides an application of GNR1 (Grain Number in Rice1) or an allele of the GNR1 in Rice1 in the aspect of improving the yield of rice by regulating and controlling the change of the grain number per spike of the rice. Also disclosed are methods of growing plants with increased grain number per ear, increased stem number and / or increased yield. The GNR1 gene has a wide application prospect in plant breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and specifically to a rice grain number gene GNR1 and its application. Background Art

[0002] Rice yield is composed of factors such as the number of panicles per unit area, the number of grains per panicle, the fruit setting rate and the thousand-grain weight. Among them, the number of tillers determines the number of effective panicles; the number of primary branches, secondary branches and fruit setting rate determine the number of grains per panicle; and the size of the grain (length, width and thickness) determines the thousand-grain weight (Xing and Zhang, 2010).

[0003] In order to improve rice yield, domestic and foreign scholars have cloned a series of rice yield-regulating genes around related agronomic traits in recent years. Some of these genes regulate rice yield mainly by controlling the number of panicles and panicle shape. For example, Gn1a encodes cytokinin oxidase 2 (CKX2), which is mainly responsible for catalyzing the decomposition of cytokinins and reducing the content of cytokinins in plants. Cytokinins regulate plant growth and development by promoting cell division and axillary meristem activity. Therefore, the loss of function or reduced expression of OsCKX2 leads to an increase in the content of cytokinins in inflorescences and reproductive organs, thereby promoting an increase in the number of panicles and yield of rice (Ashikari et al., 2005). DEP1 is a major QTL controlling rice yield traits, encoding the C-class Gγ subunit of rice. The dominant allele dep1 at this site is a functional gain-of-function mutation, and its plants show a phenotype of shorter and upright panicles, increased grain density, and increased number of panicles (Huang et al., 2009; Sun et al., 2014). IPA1 encodes the Squamosa-like promoter binding protein OsSPL14, which is negatively regulated by OsmiR156. Increasing the transcription and protein content of OsSPL14 can reduce the number of rice tillers and increase the number of panicles and 1000-grain weight, while making the stems thicker and enhancing the lodging resistance, thereby increasing rice yield (Jiao et al., 2010; Miura et al., 2010). In addition, IPA1 can also bind to the promoters of OsTB1, a negative regulator of rice tillering, and DEP1, an important gene for rice plant architecture, to inhibit rice tillering and promote the increase of rice plant height and panicle length (Lu et al., 2013). qWS8 / ipa1-2D is an excellent allele of IPA1 derived from Yongyou 12. The slightly higher expression of the IPA1 gene at this site produces an ideal plant architecture and yield increase at the developmental level (Zhang et al., 2017). D53 can directly interact with IPA1 and inhibit its activity, while IPA1 can also bind to the D53 promoter to negatively feedback regulate the level of D53 induced by strigodendron chinense lipids (Song et al., 2017). OsSPL14 protein levels are regulated by ubiquitination at the post-translational level, where IPI1 promotes the degradation of OsSPL14 in the panicle and stabilizes OsSPL14 in the shoot apex, while OsOTUB1 encodes a deubiquitinase that limits the degradation of OsSPL14 by ubiquitination by interacting with OsSPL14 (Wang et al., 2017a; Wang et al., 2017b).GNP1 (Grain Number per Panicle1) encodes a GA20ox1, which increases the number of rice grains per panicle and thus increases rice yield by increasing gibberellin activity (Wu et al., 2016). NOG1 (NUMBER OF GRAINS 1) encodes an enoyl-CoA hydratase / isomerase, which increases rice yield by increasing the number of rice grains per panicle without negatively affecting the number of panicles per plant (Huo et al., 2017). ERECTA1 (OsER1) regulates gibberellin metabolism by regulating the OsMKKK10-OsMKK4-OsMPK6 signaling pathway, thereby reducing the number of grains per panicle (Guo et al., 2020).

[0004] Some genes are involved in regulating tillering and panicle development. Loss-of-function mutants of the genes Lax1 and Moc1 show both few tillers and reduced peduncles (Komatsu et al., 2001; Li et al., 2003). Loss-of-function mutants of the genes APO1 and RFL / Apo2 involved in rice inflorescence development also show changes in tillering and panicle branching (Ikeda-Kawakatsu et al., 2012; Ikeda et al., 2007). OsSPL14 promotes the transformation of peduncles primordia to grains by promoting the expression of MiR172, thereby regulating panicle branching (Wang et al., 2015a). The fzp mutant shows that the primary and secondary peduncles of the rice panicle develop normally, but the peduncles cannot differentiate into spikelets, and the next level of peduncles continue to form at the position where the spikelets should have formed. Recent studies have found that FZP is the main negative regulator of RFL / APO2, which determines the transformation of rice panicle branches to spikelets (Bai et al., 2016; Komatsu et al., 2003).

[0005] Some genes involved in regulating grain size have also been cloned and studied. GS3 encodes the γ subunit of the G protein, which mainly regulates grain length and weight, and has a minor effect on grain width (Mao et al., 2010). GS5 (Grain size 5) encodes a putative serine carboxypeptidase that controls seed size by regulating grain width and weight (Li et al., 2011). GS6 (Grain size6) encodes a GRAS family protein, which can significantly increase grain width and weight after mutation, thereby increasing rice yield (Sun et al., 2013). GL7 / GW7 regulates the longitudinal elongation of cells while reducing transverse cell division, ultimately causing grains to become slender; the SBP domain transcription factor OsSPL16 can directly bind to the GW7 promoter and inhibit its transcription (Wang et al., 2015b; Wang et al., 2015c). GW2 can activate the division of glume cells after losing its function, thereby increasing the glume width. GW5 / GSE5 encodes a calmodulin-binding protein, and base deletions in its promoter region are responsible for the variation in grain width in different rice varieties. GW5 can interact with GSK2 and inhibit its kinase activity, thereby regulating grain width and weight (Duan et al., 2017; Liu et al., 2017). TGW3 encodes OsGSK5 kinase, which can interact with and phosphorylate the auxin response factor OsARF4, negatively regulating rice grain size and weight by negatively regulating auxin signaling (Hu et al., 2018). TGW6 encodes IAA-glucose hydrolase, which not only directly controls the length of the endosperm but also indirectly participates in the transport of carbohydrates from the source to the sink. Loss of function of the tgw6 allele in Kasalath increases rice yield by increasing grain weight through pleiotropic effects on source organs (Ishimaru et al., 2013).

[0006] At present, although many genes that control the number of grains per ear of rice have been cloned, the mechanism by which rice controls the number of grains per ear is still not fully understood. Summary of the invention

[0007] The inventors of the present invention used the mutagen ethyl methanesulfonate (EMS) to induce mutation and genetic screening, and isolated and identified a mutant gnr1 (grain number in rice1) with reduced rice grain number. Then, the key gene GNR1 controlling rice grain number was cloned by using the map-based cloning method. The function of the gene was confirmed by phenotypic analysis of the mutant and genetic complementation experiments.

[0008] Therefore, the purpose of the present invention is to provide a key factor for increasing the number of grains per ear and the yield of rice, and to provide gene resources and theoretical guidance for breeding high-yield rice varieties.

[0009] In a first aspect, the present invention provides a gene GNR1 for controlling the number of grains per rice panicle, wherein the gene encodes a GNR1 protein, and the amino acid sequence thereof is shown in any one of the following:

[0010] 1) the amino acid sequence shown in SEQ ID NO. 3;

[0011] 2) An amino acid sequence having one or more amino acid residue substitutions, deletions and / or insertions compared to the sequence shown in SEQ ID NO. 3 and having the same function as the amino acid sequence shown in SEQ ID NO. 3;

[0012] 3) an amino acid sequence that has at least 90%, preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO. 3, and has the same function as the amino acid sequence shown in SEQ ID NO. 3; or

[0013] 4) An active fragment comprising the amino acid sequence of any one of 1) to 3).

[0014] The protein shown in SEQ ID NO. 3 consists of 290 amino acids.

[0015] The nucleotide sequence of the gene GNR1 is shown in any one of the following:

[0016] 1) The nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2;

[0017] 2) A nucleotide sequence having one or more nucleotide sequence substitutions, deletions and / or insertions compared to the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and having the same function as the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2;

[0018] 3) a nucleotide sequence that has at least 90%, preferably at least 99%, identity with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and has the same function as the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2;

[0019] 4) A nucleotide sequence that is different in sequence from SEQ ID NO. 1 or SEQ ID NO. 2 due to the degeneracy of the genetic code;

[0020] 5) An active fragment comprising the nucleotide sequence described in any one of 1) to 4);

[0021] 6) a nucleotide sequence that hybridizes to the complementary sequence of the nucleotide sequence described in any one of 1) to 5) under moderately stringent hybridization conditions, preferably under highly stringent hybridization conditions; or

[0022] 7) A nucleotide sequence complementary to the nucleotide sequence described in any one of 1) to 5).

[0023] The gene GNR1 controlling the number of rice grains per panicle is an isolated nucleotide sequence. In a preferred embodiment, the gene GNR1 controlling the number of rice grains per panicle is a nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2. Moreover, those skilled in the art should understand that, in a broader sense, the gene GNR1 controlling the number of rice grains per panicle is a nucleotide sequence having a homology of more than 90%, preferably more than 99%, with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and encoding a protein having the same function.

[0024] In a preferred embodiment, the protein encoded by the gene GNR1 for controlling the number of rice grains per panicle in the first aspect is an isolated protein, and its amino acid sequence is shown in SEQ ID NO. 3.

[0025] In a third aspect, the present invention provides a recombinant vector comprising the gene GNR1 for controlling the number of grains per rice panicle as described in the first aspect. The vector comprises a plant expression vector and / or a plant gene editing vector.

[0026] The plant expression vector is preferably pCAMBIA1300, and the plant expression vector / or plant gene editing vector comprises a GNR1 promoter or an enhanced promoter. Preferably, the plant expression vector or the plant gene editing vector further comprises a gene sequence encoding a tag protein; the GNR1 promoter is preferably a sequence 2kb upstream of the GNR1 gene coding region, the enhanced promoter is preferably an Actin promoter, and the gene sequence encoding a tag protein is preferably a GFP tag sequence as shown in SEQ ID NO. 7.

[0027] Preferably, the exogenous nucleotide fragment contained in the recombinant vector encodes the amino acid sequence shown in SEQ ID NO. 3. More preferably, the exogenous nucleotide fragment contained in the recombinant vector is shown in SEQ ID NO. 1.

[0028] In one embodiment, the plasmid used to construct the recombinant vector can be selected from, but not limited to, pCAMBIA1300.

[0029] The present invention also provides a host cell comprising the GNR1 gene or its allele or the recombinant vector.

[0030] In one embodiment, the recombinant vector can be transformed or transfected into cells to obtain a host cell containing the recombinant vector, which can be further used for amplifying the expression vector, expressing the protein, or obtaining transgenic plants.

[0031] The host cell may be selected from, but not limited to, bacterial cells (such as Escherichia coli cells or Agrobacterium cells), fungal cells (such as yeast cells) or plant cells (such as rice cells).

[0032] In a fourth aspect, the present invention provides use of the gene GNR1 in cultivating plants with increased number of grains per ear, increased number of branches and stems, and improved yield, wherein the plant is a grass plant, preferably rice.

[0033] In a fifth aspect, the present invention provides a method for cultivating plants with increased number of grains per ear, increased number of stalks and / or improved yield, the method comprising increasing the expression of the GNR1 gene or its allele or increasing the amount of protein encoded by the GNR1 gene.

[0034] In one embodiment, the method can be achieved by constructing a GNR1 gene overexpression vector containing an enhanced promoter (preferably Actin promoter), and transforming or transfecting the vector into rice to increase the amount of protein encoded by the GNR1 gene.

[0035] In one embodiment, the transformation or transfection of the GNR1 gene expression vector, the GNR1 gene overexpression vector, and the GNR1 gene editing vector can be performed by Agrobacterium-mediated method or gene gun method.

[0036] The present invention provides theoretical guidance and material basis for breeding high-yield crop varieties. When the GNR1 gene of the present invention is used to increase rice yield, the following method can be used: (1) constructing a GNR1 gene overexpression vector containing an enhanced promoter (preferably Actin promoter); (2) transforming the constructed overexpression vector into regenerable rice tissues or organs; (3) culturing the transformed tissues or organs into plants and screening plants with significantly increased GNR1 gene expression.

[0037] The increase in the number of panicles, the number of branches and / or the increase in yield described in the present invention is compared with wild-type plants, preferably compared with wild-type rice, and more preferably compared with japonica rice ZH11. Compared with the prior art, the present invention has the following beneficial effects:

[0038] The invention clones the key gene GNR1 controlling the number of rice grains per panicle, which can be used in rice genetic engineering and molecular breeding to improve rice traits, increase the number of grains per panicle and the number of branches and stalks, and improve rice yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1The phenotypic analysis of ZH11 and gnr1 is shown; A is the phenotype of mature plants of ZH11 and gnr1, with a scale bar of 10 cm; B is the phenotype of rice panicles of ZH11 and gnr1, with a scale bar of 5 cm; CJ are statistical analyses of plant height, number of tillers, panicle length, number of primary stalks, number of secondary stalks, number of grains per panicle, 1000-grain weight, and yield per plant of ZH11 and gnr1; the statistical number n = 15, analyzed using Duncan's test (p < 0.05).

[0040] Figure 2 The gene localization and candidate gene determination of GNR1 are shown; in the positional cloning of GNR1, the horizontal lines represent chromosomes and the vertical lines represent molecular markers; in the gene structure of GNR1, the green squares, gray squares and black lines represent exons, non-coding regions and introns, respectively.

[0041] Figure 3 The GNR1 gene functional complementation vector ( Figure 3 A) and overexpression vector ( Figure 3 B).

[0042] Figure 4 The figure shows the complementary verification of the function of the GNR1 gene. GNR1 can complement the phenotype of gnr1. A is the phenotype of mature plants of ZH11, gnr1 and transgenic complementary line (GNR1 / gnr1), and the scale bar is 10 cm. B is the phenotype of rice panicles of ZH11, gnr1 and GNR1 / gnr1, and the scale bar is 5 cm. CJ are statistical analyses of plant height, tiller number, panicle length, number of primary stalks, number of secondary stalks, number of grains per panicle, 1000-grain weight and yield per plant of ZH11, gnr1 and GNR1 / gnr1. The statistical number n = 15, and the significance analysis was performed using Duncan's test (p < 0.05).

[0043] Figure 5 The phenotypic analysis of gnr1 allelic mutation and GNR1 overexpression plants is shown; A is the phenotype of mature plants of ZH11, gnr1 allelic mutation (gnr1-cr) and GNR1 overexpression line (GNR1-OE), with a scale bar of 10 cm; B is the phenotype of rice panicles of ZH11, gnr1-cr and GNR-OE, with a scale bar of 5 cm; CJ are statistical analyses of plant height, number of tillers, panicle length, number of primary branches, number of secondary branches, number of grains per panicle, 1000-grain weight and yield per plant of ZH11, gnr1-cr and GNR-OE; the statistical numbers of plant height, number of tillers, panicle length, number of primary branches, number of secondary branches and number of grains per panicle were n = 15, and the statistical numbers of 1000-grain weight and yield per plant were n = 10; significance analysis was performed using Duncan's test (p < 0.05). DETAILED DESCRIPTION

[0044] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0045] The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The reagents, kits, and experimental instruments used in the experiments can all be purchased from biological instrument and reagent companies unless otherwise specified.

[0046] Example 1. Isolation and genetic analysis of genes controlling rice grain number per ear

[0047] The inventors first screened a mutant grnr1 (grain number in rice1) with reduced grain number in a panicle from the japonica rice ZH11 (conventional general material) mutant induced by ethyl methanesulfonate (EMS). In order to further determine the phenotype of the mutant, we analyzed the plant height, number of tillers, panicle length, number of primary stalks, number of secondary stalks, number of grains per panicle, 1000-grain weight and yield per plant phenotype of the mutant. The results showed that the panicle length, number of primary stalks, number of secondary stalks, number of grains per panicle and yield per plant of the mutant were significantly lower than those of ZH11, while the plant height, number of tillers and 1000-grain weight were not significantly different from those of ZH11, indicating that the number of grains per panicle and number of stalks of the mutant were abnormally developed ( Figure 1 ).

[0048] In order to study the molecular causes of the gnr1 phenotype, we crossed it with ZH11 and constructed an F2 population, and then counted the number of plants with similar phenotypes to gnr1 and ZH11 in the population. The chi-square fitness test analysis showed that the ratio of the number of gnr1 and ZH11 phenotypes in the F2 population was 3:1, indicating that gnr1 is controlled by a recessive mononuclear gene.

[0049] To further identify the mutant gene controlling the gnr1 phenotype, we crossed gnr1 with indica rice 9311 (a conventional general material) and constructed an F2 population. By positional cloning, we determined that the candidate gene for the gnr1 phenotype was located between molecular markers IM05217 and IM05212 ( Figure 2 ). Combined with the PCR sequencing results of the genes in this interval, it was found that the exon region of the gene GNR1 (Os05g41220) in this interval was missing two bases, causing its protein translation to terminate prematurely ( Figure 2). This gene encodes the β-regulatory subunit OsSnRK1β2 of the plant sucrose non-fermenting 1related protein kinases (SnRK1) in the energy metabolism pathway, so we identified the GNR1 gene as a candidate gene for the gnr1 phenotype. That is, the difference between the mutant gnr1 and the wild-type ZH11 is that the two bases TC are missing at 298bp of the CDS sequence of the gene GNR1 (Os05g41220).

[0050] Example 2. Construction of complementary vector of GNR1 and genetic transformation of rice

[0051] The complete gene of GNR1 from the genome of wild-type ZH11, which is the nucleotide sequence shown in SEQ ID NO. 1 (including its own promoter sequence, genomic sequence and 3' non-coding region sequence), was amplified by PCR using DNA polymerase, and then ligated to the multiple cloning site of pCAMBIA1300 plasmid (purchased from CAMBIA) by restriction digestion and ligation to obtain the complementary vector pGNR1::GNR1 ( Figure 3 A).

[0052] The constructed complementary vector was transformed into E.coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and positive clones were screened using kanamycin. The bacteria were shaken and the plasmids were extracted. The positive clones with completely correct GNR1 sequences were obtained by sequencing, and then the plasmids of the positive clones were electroporated into EHA105 Agrobacterium competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). Next, the successfully transformed clones were genetically transformed using the Agrobacterium infection method with the mutant grnr1 as the receptor.

[0053] The genetic modification operation method is as follows:

[0054] The first step is to sterilize the seeds. Weigh 25-40 g of rice seeds, remove the seed husks, soak the hulled seeds in 70% ethanol for 1 minute, and then soak them in 30% sodium hypochlorite (the original solution is 10% effective chlorine, and 1 drop of Tween 20 is added for every 50 mL) for 30 minutes. Gently shake them on a shaker, and then rinse them with sterile water 5-6 times; then disinfect them once with 30% sodium hypochlorite without Tween20, rinse them with sterile water 5-6 times, and place the sterilized seeds in an ultra-clean workbench to air dry.

[0055] The second step is to induce callus. The seeds are placed on N6D medium (N6D Macro / Micro, MS Organic, Fe-EDTA, 2, 4-D 2 mg / L, CH (acid hydrolyzed casein) 0.3 g / L, L-Proline 2.878 g / L, Sucrose 30 g / L, pH 5.8, Gelrite (sigma) 4 g / L) and cultured in a light incubator with a temperature of 32°C and 24 hours of continuous light for 5-7 days until callus tissue grows on the seeds.

[0056] The third step is Agrobacterium infection. Agrobacterium EHA105 containing the target plasmid is cultured in the dark for 2-3 days on YEB medium (beef extract 5 g / L, yeast extract 1 g / L, peptone 5 g / L, sucrose 5 g / L, MgSO4·7H2O 0.4 g / 100 mL, agar 20 g / L) containing the antibiotics kanamycin (Kan 50 mg / L) and rifampicin (Rif 25-50 mg / L); single clones with good growth are picked and placed in 5-6 mL of liquid YEB medium containing the same resistance as the solid culture medium, and cultured overnight in a shaker at 28°C and 220 rpm. The bacterial suspension was inoculated into 50 mL of AAM medium (AAMMacro, AAM Micro, AAM Organic, AAM Amino acid, Fe-EDTA sucrose 68.5 g / L, CH (acid hydrolyzed casein) 0.5 g / L, glucose 36 g / L, pH 5.2, Acetosyringone (As) 10-20 mg / L) at a ratio of 1:100, and cultured in a shaker at 28°C and 220 rpm until the OD of the bacteria reached 0. 600 When the value is about 0.1, soak the callus in the Agrobacterium solution for 2 minutes, quickly remove the callus from the solution with tweezers and place it on sterile filter paper to air dry. After the callus is air-dried, transfer it to N6D-As medium (N6D Macro, N6D Micro, MS Organic, Fe-EDTA, 2, 4-D 2 mg / L, CH (acid hydrolyzed casein) 0.3 g / L, sucrose 30 g / L, glucose 10 g / L, plant gel 4 g / L, Acetosyringone (As) 10~20 mg / L, pH 5.2) covered with sterile filter paper (soaked in AAM), wrap the culture dish containing the callus with sealing film and tin foil, and culture it in the dark at 25℃ for 2-2.5 days.

[0057] The fourth step is screening and differentiation. The co-cultured callus tissue is rinsed in sterile water for 5-6 times, and then washed with sterile water containing carbenicillin (500 mg / L) for 2-3 times to clean the Agrobacterium on the surface of the callus tissue. The callus tissue is then dehydrated with sterile filter paper and air-dried, and transferred to N6DS medium (N6D Macro, N6D Micro, MS Organic, Fe-EDTA, 2, 4-D 2 mg / L, CH (acid hydrolyzed casein) 0.3 g / L, sucrose 30 g / L, glucose 10 g / L, plant gel 4 g / L, pH 5.2, 50 mg / L hygromycin B, 400 mg / L Carbenicillin) containing antibiotics hygromycin B (50 mg / L) and carbenicillin (400 mg / L), and cultured in a light incubator at 32°C with 24 hours of continuous light for 2-3 weeks. The callus tissue was transferred to regeneration RE medium (MS Salt, RE organic, hygromycin B 50 mg / L, carbobenzyl 250 mg / L, sucrose 30 g / L, Sorbitol 30 g / L, CH: 2 g / L, NAA 0.02 mg / L, Kinetin 2 mg / L, pH 5.8, Gelrite 4 g / L) containing hygromycin B (50 mg / L) and carbobenzyl (250 mg / L), and cultured in a 32°C incubator for 1 month to induce differentiation. The RE medium was replaced every two weeks until green seedlings appeared. The callus tissue that differentiated into green seedlings was transferred to MS (sucrose 30 g / L) medium containing hygromycin B (50 mg / L) and carbobenzyl (200 mg / L) to induce rooting. When the seedlings grew to a certain size, they were transferred to MS medium without antibiotics for culture.

[0058] Table 1 Culture media and their formulations for rice transformation by Agrobacterium infection

[0059]

[0060]

[0061] Example 3. Phenotypic analysis of GNR1 transgenic complemented plants

[0062] In order to test whether GNR1 can complement the phenotype of gnnr1, the phenotypes of the positive plants GNR1 / gnr1 obtained by transgenic experiments, the wild-type plants ZH11 and the mutant plants gnnr1 were analyzed. The method for obtaining the positive plants GNR1 / gnr1 is as follows: using Agrobacterium EHA105 containing the target plasmid (complementation vector pGNR1::GNR1) to transform E. coli DH5α competent cells, using kanamycin to screen positive clones, and using the Agrobacterium infection method to perform genetic transformation operations on the successfully transformed clones using the mutant gnnr1 callus as the recipient. The transgenic operation method is as described in Example 2, and the positive plants GNR1 / gnr1 are obtained.

[0063] The results showed that there were no significant differences between the GNR1 / gnr1 and ZH11 plants in terms of plant height, tiller number, ear length, primary stalk number, secondary stalk number, number of grains per ear, 1000-grain weight, and yield per plant; while there were significant differences between the gnr1 and ZH11 plants in terms of ear length, primary stalk number, secondary stalk number, number of grains per ear, and yield per plant. This indicates that the gene GNR1 can restore the phenotypes of gnr1 such as the number of grains per ear and the number of stalks ( Figure 4 ), indicating that GNR1 is a key gene controlling the number of grains per panicle in rice.

[0064] Example 4. Phenotypic analysis of the gnr1 allele mutation gnr1-cr

[0065] The present inventors constructed the allelic mutant gnr1-cr of gnr1 using CRISPR / Cas9 gene editing technology according to conventional molecular biology and genetic operation methods (refer to "Plant Genetic Engineering", Wang Guanlin, Fang Hongjun, Science Press, 2004, 2nd edition, prepared by conventional methods). Specifically: in the gene gnr1-cr (SEQ ID NO. 4), compared with the GNR1 gene, the 295bp position of the CDS sequence of the gene gnr1-cr is missing 17 bp, resulting in premature termination of protein translation; the gene editing gRNA guide sequence used is GAATTCCAACTTTGATCTCATGG (SEQ ID NO. 5); the gRNA guide sequence is connected to the gene editing vector (gifted by Professor Fu Xiangdong of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and its construction method is shown in Wu et al., 2020) by PCR and enzyme ligation; the constructed vector is transformed into E. coli DH5α competent cells were used to screen positive clones using kanamycin, and the successfully transformed clones were genetically transformed using the Agrobacterium infection method (with ZH11 as the receptor). The transgenic operation method was as described in Example 2.

[0066] To further verify the biological function of GNR1, we analyzed the phenotypes of ZH11 and gnr1-cr. The results showed that the panicle length, number of primary branches, number of secondary branches, number of grains per panicle and yield per plant of gnr1-cr were significantly lower than those of ZH11, while the plant height, number of tillers and 1000-grain weight were not significantly different from those of ZH11. These phenotypes of gnr1-cr were similar to those of gnr1 ( Figure 4 and 5 ) (Significance analysis was performed using Duncan's test (p < 0.05)), further proving that GNR1 is a key gene controlling the number of grains per panicle in rice.

[0067] Example 5. Construction of GNR1 overexpression vector and genetic transformation

[0068] We ligated the Actin promoter sequence (SEQ ID NO. 6), the GNR1 gene CDS coding sequence, and the GFP tag sequence (SEQ ID NO. 7) into the pCAMBIA1300 plasmid by restriction digestion to obtain the overexpression vector pActin1::GNR1-GFP ( Figure 3 B). The constructed overexpression vector was transformed into E.coli DH5α competent cells, and positive clones were screened using kanamycin. The plasmid was extracted and sequenced to obtain positive clones with completely correct Actin promoter sequence, GNR1 gene CDS coding sequence and GFP tag sequence, and then the plasmid of the positive clone was electroporated into EHA105 Agrobacterium competent cells (refer to "Plant Genetic Engineering", Wang Guanlin, Fang Hongjun, Science Press, 2004, 2nd edition, prepared by conventional methods). The successfully transformed clones were genetically transformed using the Agrobacterium infection method with wild-type japonica rice ZH11 callus as the recipient. The transgenic operation method is as described in Example 2. Finally, the GNR1 overexpression strain GNR1-OE was obtained.

[0069] Example 6. Overexpression of GNR1 can increase the number of grains per panicle, the number of branches and the yield per plant in rice

[0070] In order to determine whether increasing the expression of GNR1 can increase the yield per plant of rice, the phenotype of the GNR1 overexpression line GNR1-OE was analyzed. The results showed that the number of primary branches, secondary branches, number of grains per panicle, and yield per plant of the overexpression line GNR1-OE were significantly higher than those of ZH11, and the plant height, number of tillers, panicle length, and 1000-grain weight were similar to those of ZH11 ( Figure 5 ), indicating that increasing the expression of GNR1 can effectively increase the number of grains per panicle, the number of branches and the yield per plant of rice.

[0071] It should be understood that although the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it should be understood by those skilled in the art that various changes in form and details may be made therein and any combination of various embodiments may be made without departing from the spirit and scope of the present invention as defined by the appended claims.

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[0104] Sequence Listing:

[0105] SEQ ID NO. 1: The genomic nucleotide sequence of the rice GNR1 gene, which also includes the promoter and 3'UTR sequence

[0106]

[0107] SEQ ID NO. 2: Nucleotide sequence (CDS) of the protein encoded by the rice GNR1 gene

[0108] ATGGGGAACGCGAGCGGCAAGGAAGGGGAGGAGAACGGCCACGTGGCGGCGGGGGCCGCCGCCGGAGTGGCTGGCTCGGCGGGGGCAGCGGCCCGGGCTCCGCCGCCGCTTATGCCGCCGGACGCCGTGATGCGGGAGCTGCCTCCCCCGGTGCCCTACGTCTTCACGCCGCAGGTTCCAGTAGCCCCACTGCATATACCTACTGAATTTTCTCCTGTTTTCAACAATTCATGGATAAATGAATCGGATGAATCCACCAATAACCATCCCCAGGAGAAGGGAATTCCAACTTTGATCTCATGGAGTCAAGGAGGAAATGAGGTGTTTGTGGAAGGATCATGGGATAACTGGACATCAAGGAGGGTGTTAGAGAAGTCTGGGAAAGACCATACCATATTGCTAGTTCTGCCATCAGGGGTATACCATTACAGGATCATCGTCGATGGGGAACCGAAATATGTCCCTGAACTACCTCATGTGGCTGATGAGGGAGGGCAGGTTGCCAACCTCCTCGATGTCCATGATTATATCCCAGAAAGCCTGGGCAGCGTGGCAGGATTCGACTCTCCTCCGTCGCCTGAACACAGCTATGATCTCCAGCTCCCAGGTGATGAGGAGTTTGCCAAGGAGCCACCTATACTGCCACCTCAGCTTGTAATGTCTGTTCTTGGTGATACTGATAACTCTGAAGAACAAACTCTGAAGCCAAAGCATGTTGTCCTCAACCACCTGTATATCGAGAAAGGATGGGGATCGCAGTCGCTGCTTGCTCTTGGAGTCACTCACCGGTTTCAATCCAAGTATGTAAGCTTCGTGCTGTACAAGCCGCTGCGAAGGTCATCCACGGCGAAGCGAACAAAGAATGGTGGTTAA

[0109] SEQ ID NO. 3: Amino acid sequence of the protein encoded by the rice GNR1 gene

[0110] MGNASGKEGEENGHVAAGAAAGVAGSAGAAARAPPPLMPPDAVMRELPPPVPYVFTPQVPVAPLHIPTEFSPVFNNSWINESDESTNNHPQEKGIPTLISWSQGGNEVFVEGSWDNWTSRRVLEKSGKDHTILLVLPSGVYHYRI IVDGEPKYVPELPHVADEGGQVANLLDVHDYIPESLGSVAGFDSPPSPEHSYDLQLPGDEEFAKEPPILPPQLVMSVLGDTDNSEEQTLKPKHVVLNHLYIEKGWGSQSLLALGVTHRFQSKYVSFVLYKPLRRSSTAKRTKNGG

[0111] SEQ ID NO. 4: Rice gnr1-cr protein encoding nucleotide sequence (CDS)

[0112] ATGGGGAACGCGAGCGGCAAGGAAGGGGAGGAGAACGGCCACGTGGCGGCGGGGGCCGCCGCCGGAGTGGCTGGCTCGGCGGGGGCAGCGGCCCGGGCTCCGCCGCCGCTTATGCCGCCGGACGCCGTGATGCGGGAGCTGCCTCCCCCGGTGCCCTACGTCTTCACGCCGCAGGTTCCAGTAGCCCCACTGCATATACCTACTGAATTTTCTCCTGTTTTCAACAATTCATGGATAAATGAATCGGATGAATCCACCAATAACCATCCCCAGGAGAAGGGAATTCCAACTTTGAGGAAATGAGGTGTTTGTGGAAGGATCATGGGATAACTGGACATCAAGGAGGGTGTTAGAGAAGTCTGGGAAAGACCATACCATATTGCTAGTTCTGCCATCAGGGGTATACCATTACAGGATCATCGTCGATGGGGAACCGAAATATGTCCCTGAACTACCTCATGTGGCTGATGAGGGAGGGCAGGTTGCCAACCTCCTCGATGTCCATGATTATATCCCAGAAAGCCTGGGCAGCGTGGCAGGATTCGACTCTCCTCCGTCGCCTGAACACAGCTATGATCTCCAGCTCCCAGGTGATGAGGAGTTTGCCAAGGAGCCACCTATACTGCCACCTCAGCTTGTAATGTCTGTTCTTGGTGATACTGATAACTCTGAAGAACAAACTCTGAAGCCAAAGCATGTTGTCCTCAACCACCTGTATATCGAGAAAGGATGGGGATCGCAGTCGCTGCTTGCTCTTGGAGTCACTCACCGGTTTCAATCCAAGTATGTAAGCTTCGTGCTGTACAAGCCGCTGCGAAGGTCATCCACGGCGAAGCGAACAAAGAATGGTGGTTAA

[0113] SEQ ID NO. 5: The gRNA guide sequence for obtaining gnr1-cr

[0114] GAATTCCAACTTTGATCTCATGG

[0115] Nucleotide sequence of Actin promoter, SEQ ID NO. 6

[0116] tcgaggtcattcatatgcttgagaagagagtcgggatagtccaaaataaaacaaaggtaagattacctggtcaaaagtgaaaacatcagttaaaaggtggtataaagtaaaatatcggtaataaaaggtggcccaaagtgaaatttactcttttctactattataaaaattgaggatgtttttgtcggtactttgatacgtcatttttgtatgaattggtttttaagtttattcgcttttggaaatgcatatctgtatttgagtcgggttttaagttcgtttgcttttgtaaatacagagggatttgtataagaaatatctttaaaaaaacccatatgctaatttgacataatttttgagaaaaatatatattcaggcgaattctcacaatgaacaataataagattaaaatagctttcccccgttgcagcgcatgggtattttttctagtaaaaataaaagataaacttagactcaaaacatttacaaaaacaacccctaaagttcctaaagcccaaagtgctatccacgatccatagcaagcccagcccaacccaacccaacccaacccaccccagtccagccaactggacaatagtctccacacccccccactatcaccgtgagttgtccgcacgcaccgcacgtctcgcagccaaaaaaaaaaaaagaaagaaaaaaaagaaaaagaaaaaacagcaggtgggtccgggtcgtgggggccggaaacgcgaggaggatcgcgagccagcgacgaggccggccctccctccgcttccaaagaaacgccccccatcgccactatatacatacccccccctctcctcccatccccccaaccct

[0117] SEQ ID NO. 7: Nucleotide sequence of GFP-encoding protein

[0118] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACC TTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGC ATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGC CCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGgatcctctagagtcgacctgcaggcatgccagggctctcaatggagtttga

[0119] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A GNR1 protein, the amino acid sequence of which is shown in any of the following: 1) the amino acid sequence shown in SEQ ID NO. 3; 2) An amino acid sequence having one or more amino acid residue substitutions, deletions and / or insertions compared to the sequence shown in SEQ ID NO. 3 and having the same function as the amino acid sequence shown in SEQ ID NO. 3; 3) an amino acid sequence that has at least 90%, preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO. 3, and has the same function as the amino acid sequence shown in SEQ ID NO. 3; or 4) An active fragment comprising the amino acid sequence of any one of 1) to 3).

2. A GNR1 gene encoding the GNR1 protein according to claim 1.

3. The GNR1 gene according to claim 2, wherein the nucleotide sequence is as shown in any one of the following: 1) The nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; 2) A nucleotide sequence having one or more nucleotide sequence substitutions, deletions and / or insertions compared to the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and having the same function as the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; 3) a nucleotide sequence that has at least 90%, preferably at least 99%, identity with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and has the same function as the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; 4) A nucleotide sequence that is different in sequence from SEQ ID NO. 1 or SEQ ID NO. 2 due to the degeneracy of the genetic code; 5) An active fragment comprising the nucleotide sequence described in any one of 1) to 4); 6) a nucleotide sequence that hybridizes to the complementary sequence of the nucleotide sequence described in any one of 1) to 5) under moderately stringent hybridization conditions, preferably under highly stringent hybridization conditions; or 7) A nucleotide sequence complementary to the nucleotide sequence described in any one of 1) to 5).

4. A recombinant vector comprising the GNR1 gene or its allele according to claim 2 or 3, characterized in that: The recombinant vector includes a plant expression vector and / or a plant gene editing vector, wherein the plant expression vector or the plant gene editing vector comprises a GNR1 promoter or an enhanced promoter, and preferably the plant expression vector or the plant gene editing vector further comprises a gene sequence encoding a tag protein; the plant expression vector is preferably pCAMBIA1300, the GNR1 promoter is preferably a sequence 2kb upstream of the GNR1 gene coding region, the enhanced promoter is preferably an Actin promoter, and the gene sequence encoding the tag protein is preferably a GFP tag sequence as shown in SEQ ID NO.

7.

5. A host cell comprising the GNR1 gene or its allele according to claim 2 or 3 or the recombinant vector according to claim 4, wherein the host cell is a bacterium, a fungus or a plant cell, and the host cell is preferably an Escherichia coli cell, an Agrobacterium cell or a rice cell.

6. A method for obtaining a plant with increased number of grains per ear, increased number of branches and / or increased yield, the method comprising increasing the expression of the GNR1 gene or its allele as defined in claim 2 or 3 or increasing the amount of the GNR1 protein as defined in claim 1 in the plant.

7. The method of claim 6, wherein the method further comprises: The recombinant vector described in claim 4 or the host cell described in claim 5 is transformed or transfected into plant cells to obtain transgenic plants, so that the GNR1 gene defined in claim 2 or 3 is overexpressed in the transgenic plants to obtain plants with increased number of grains per ear, increased number of branches and stalks and / or improved yield.

8. The method of any one of claims 5 to 7, wherein the plant is a grass plant, preferably rice.

9. The method of claim 7, wherein the transformation or transfection is performed by Agrobacterium-mediated method or gene gun method.

10. Use of the GNR1 gene or the protein encoded by the gene according to claim 2 or 3 in obtaining plants with increased number of grains per ear, increased number of branches and stalks and / or improved yield, wherein the plant is a grass plant, preferably rice.

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