Rice lateral floret regulation gene LF3 as well as mutant gene and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly discloses a rice lateral floret regulation gene LF3 as well as a mutant gene and application thereof. Wherein the nucleotide sequence of the rice lateral floret regulation gene LF3 is as shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is as shown in SEQ ID No. 2; the nucleotide sequence of the mutant gene of the rice lateral floret regulation gene LF3 is as shown in SEQ ID No.3, and the amino acid sequence of the encoded protein is as shown in SEQ ID No.4. The rice lateral floret regulation gene LF3 and the mutant gene thereof disclosed by the invention provide more evidence and basis for a'three-flower spikelet 'hypothesis and'three-flower spikelet' breeding, provide a new gene for breeding for increasing the number of grains per ear, and have very important significance for further improving the rice yield.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a rice lateral floret regulating gene LF3 and a mutant gene and application thereof. Background Art

[0002] In grasses such as rice, the basic unit of inflorescence is called "spikelet". In rice and other Oryza species, the pair of secondary guard glumes and terminal florets in the spikelet correspond to the glumes and florets in other grass species, respectively, while the pair of glumes between the secondary guard glumes and terminal florets, the guard glumes, have no corresponding organs in the spikelets of other grasses. The "three-flowered spikelet" hypothesis is a view that is more widely accepted in the controversy over the origin of secondary guard glumes and guard glumes (Hong et al., 2010; Kellogg, 2009; Schmidt and Ambrose, 1998; Terrell et al., 2001; Yoshida et al., 2009). Mutations in a large number of genes, such as G1 / ELE, OsMADS34 / PAP2, ASP1, ASD1, OsVIL2, and NSG1 / LRG1, cause the guard glumes to elongate and acquire lemma characteristics (Gao et al., 2010; Hong et al., 2010; Kobayashi et al., 2010; Li et al., 2014; Wang et al., 2013; Yoon et al., 2018; Yoshida et al., 2012; Yoshida et al., 2009; Zhuang et al., 2020). In plants with ectopic expression of the OsMADS1 / LHS1 gene, the guard glumes are also homologously transformed into lemma-like organs (Prasad et al., 2001; Wang et al., 2017), indicating that the guard glumes and lemmas are homologous organs. These studies provide strong evidence to support the "three-flowered spikelet" hypothesis, which holds that the spikelets in rice ancestors had three florets, and the two lateral florets degenerated during evolution and eventually formed the guard lemma in the current rice spikelets. The spikelets of the gain-of-function mutant lf1 developed lateral florets with floral organs in the axils of the guard lemma (Zhang et al., 2017b), which provided direct evidence for the "three-flowered spikelet" hypothesis and theoretically revealed the possibility of breeding methods to increase the total number of grains per spikelet by increasing the number of florets in the spikelet. However, the mutant lf1 produces a high proportion of lateral florets, but the structure is not complete and cannot bear fruit, so it cannot be directly used to breed "three-flowered spikelets". Therefore, exploring more lateral floret mutants and their regulatory genes, deeply analyzing the molecular mechanism of the production of "three-flowered spikelets", and "restoring" lateral florets to normal fertile florets through molecular design will greatly increase rice yield and will be of great significance in rice breeding research. Summary of the invention

[0003] The purpose of the present invention is to provide a rice lateral floret regulatory gene LF3 and a mutant gene and application thereof.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a rice lateral floret regulatory gene LF3, the nucleotide sequence of which is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No.2.

[0006] The present invention also provides a protein encoded by the rice lateral floret regulatory gene LF3, and its amino acid sequence is shown as SEQ ID No.2.

[0007] The present invention also provides the use of the rice lateral floret regulatory gene LF3 and the protein encoded by the rice lateral floret regulatory gene LF3 in improving rice yield.

[0008] The present invention also provides a mutant gene of the rice lateral floret regulatory gene LF3, whose nucleotide sequence is shown in SEQ ID No.3, and the amino acid sequence of the encoded protein is shown in SEQ ID No.4.

[0009] The present invention also provides a protein encoded by the mutant gene of the rice lateral floret regulatory gene LF3, and the amino acid sequence of the protein is shown in SEQ ID No.4.

[0010] The present invention also provides the use of the mutant gene of the rice lateral floret regulatory gene LF3 and the protein encoded by the mutant gene of the rice lateral floret regulatory gene LF3 in improving rice yield.

[0011] The present invention uses ethyl methanesulfonate EMS to induce the indica rice maintainer variety Xida 1B, and finds a genetically stable lateral floret development mutant, named lf3, and the lateral floret development mutant regulatory gene is named LF3. In the lf3 mutant, the spikelet guard lemma elongation has the characteristic structure of the lemma, and some spikelet guard lemma axils develop lateral florets containing a complete four-round flower organ structure, most lateral florets can bear fruit normally, and in a few cases, lateral florets are developed in the axils of both sides of the guard lemma to form "three-flower spikelets", and can bear fruit. The LF3 gene is located by using the map-based cloning method, and the LF3 gene is located between the markers RM3555 and CY7-3 at the long arm end of the seventh chromosome. The genes in the interval are further sequenced and analyzed, and it is found that the 98th base on the exon of one of the AP2 / ERF family transcription factors (LOC_Os07g47330 / FZP) is replaced by CA, resulting in the 33rd amino acid codon becoming a stop codon, and the translation is terminated prematurely. In order to determine whether LOC_Os07g47330 is the target gene of LF3 gene, the knockout of LOC_Os07g47330 gene by cas9 was verified. The results showed that the transgenic plants could not differentiate into spikelets on the peduncles, and continuously produced the next level of peduncles inflorescence, which was the same as the inflorescence phenotype of lf3 mutant, proving that LOC_Os07g47330 gene was the target gene of LF3 gene. The expression pattern of LF3 gene was analyzed. The qPCR results showed that LF3 gene was almost not expressed in the vegetative growth organs, but was strongly expressed in the inflorescence, and the expression level decreased significantly with the development and elongation of the inflorescence. Among the various floral organs, LF3 gene was expressed most highly in the glumes, and was also highly expressed in the lodicules and pistils. In situ hybridization results showed that LF3 was strongly expressed in various meristems, and obvious expression signals were also detected in the glumes. Further analysis of LF3 encoded protein revealed that LF3 gene encodes an AP2 / ERF family transcription factor, which is homologous to the BD1 gene in maize. The sequences of LF3 and its homologous proteins are highly conserved in Gramineae plants and contain an AP2 / ERF domain. The C-terminus of LF3 protein has an acidic domain, which makes LF3 protein have strong transcriptional activation activity. The truncated protein pGBKT7-LF31-244 of LF3 was further used as bait to screen the rice panicle cDNA library to find the interacting proteins of LF3. Finally, 20 proteins that may interact with LF3 and participate in rice growth and development were screened, including OsMADS1, NAL9, nuclear factor subunits NF-YC6 / OsHAP5D and NF-YC7 / OsHAP5E, and chromatin remodeling complex-related proteins (Os02g0723700, Os11g0545600, Os02g0122000).Further analysis of transcriptome sequencing data of the lf3 mutant showed that the differentially expressed genes were most highly enriched in the plant hormone signaling pathway, which contained more genes in the auxin synthesis pathway and the auxin signal response pathway. In addition, the auxin content in the lf3 mutant was significantly downregulated compared with the wild type, and qPCR results showed that the expression levels of OsYUCCA1 and OsYUCCA6 genes were significantly reduced in lf3, indicating that the LF3 gene may affect the development of rice flowers / panicles by regulating auxin synthesis.

[0012] The beneficial effects of the present invention are as follows: the present invention discloses a gain-of-function mutant lf3, in which the spikelet guard lemma elongates with the characteristic structure of the lemma, and lateral florets containing a complete four-round flower organ structure are developed under the axils of some spikelet guard lemmas, most lateral florets can bear fruit normally, and in a few cases, lateral florets are developed under the axils of both sides of the guard lemma to form "three-flower spikelets", and can bear fruit. The present invention also discloses the control gene LF3 gene of the lf3 mutant trait and its mutant gene of LF3, wherein the nucleotide sequence of LF3 is shown in SEQ ID No.1, the amino acid sequence of the encoded protein is shown in SEQ ID No.2, the nucleotide sequence of the mutant gene of the LF3 gene is shown in SEQ ID No.3, and the amino acid sequence of the encoded protein is shown in SEQ ID No.4. The mutant lf3, the mutant trait control gene LF3 and the mutant gene of LF3 disclosed by the present invention provide more evidence and basis for the "three-flower spikelet" hypothesis and "three-flower spikelet" breeding, provide a new gene for breeding to increase the number of spikelets, and are of great significance to further improve rice yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The inflorescence morphology of the lf3 mutant; A: wild-type inflorescence; B: wild-type primary pedicel; CD: wild-type spikelet; E: surface structure of wild-type spikelet axis; F: wild-type secondary guard glume structure; G: lf3 mutant inflorescence; lf3 mutant primary pedicel; IJ: lf3 spikelet; lf3 mutant secondary guard glume structure; Scale bars: AD and GJ are 1000 μm, EF and K are 100 μm.

[0014] Figure 2 This is the phenotypic analysis of lf3 mutant spikelets; where AD: wild-type spikelet; EH: lf3 spikelet containing lemmaized guard glumes; IP: lf3 spikelet containing lateral florets; QR: lf3 spikelet containing two lateral florets; SV: histological analysis of wild-type and lf3 mutant spikelets; scale bar: AR is 1000μm, SV is 500μm.

[0015] Figure 3This is a scanning electron microscopic observation of the early development of the lf3 mutant spikelet; AC: lf3 mutant branch; D: wild-type spikelet; EG: lf3 mutant spikelet. rg: secondary guard glume; sl: guard glume; le: lemma; pa: palea; lesl: lemma-like guard glume; tf: terminal floret; lf: lateral floret; scale bar: AC is 1000μm, DG is 500μm.

[0016] Figure 4 In situ hybridization analysis of HISTONE H4 in early spikelets; AC: wild-type spikelet; DF: lf3 mutant spikelet; bm: branch meristem; sm: spikelet meristem; fm: floral meristem; lfm: lateral floral meristem; scale bar: 100 μm.

[0017] Figure 5 In situ hybridization analysis of OSH1 and OsMADS15 in early spikelets; AB: expression analysis of OSH1 and OsMADS15; CK: in situ hybridization analysis of OSH1 (CE: wild-type spikelet, FK: lf3 mutant spikelet); LQ: in situ hybridization analysis of OsMADS15 (LN: wild-type spikelet, OQ: lf3 mutant spikelet); bm: branch meristem; fm: floral meristem; lfm: lateral floral meristem; pi: pistil; scale bar, 100 μm.

[0018] Figure 6 This is the map-based cloning of the LF3 gene; A: gene localization of the LF3 gene; B: cas9 knockout verification of the LF3 gene; the scale bars are 2 cm and 1 mm, respectively.

[0019] Figure 7 Expression pattern analysis of LF3 gene; A: quantitative analysis of LF3 gene; B: in situ hybridization analysis of LF3 gene; scale bars: BC is 1000 μm, DE is 100 μm.

[0020] Figure 8 This is the structural analysis of LF3 protein; A: LF3 domain analysis; B: amino acid sequence analysis of LF3 homologous proteins in Poaceae plants.

[0021] Fig. 9 Analysis of LF3 transcriptional activity; A: Auto-activation; B: amino acid sequence analysis of LF3 homologous proteins in Poaceae plants.

[0022] Fig.10 Differential gene enrichment for lf3 transcriptome sequencing.

[0023] Fig.11 GO analysis for lf3 transcriptome sequencing.

[0024] Fig.12 KEGG analysis of lf3 transcriptome sequencing. DETAILED DESCRIPTION

[0025] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above contents of the present invention are covered within the scope that the present invention is intended to protect. It should be noted that the experimental materials in the embodiments of the present invention can all be obtained commercially, and the experimental methods in the embodiments of the present invention that do not specify specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the experimental material manufacturer. The experimental materials involved in the present invention are specifically described as follows:

[0026] 1.1 Plant materials

[0027] The lf3 mutant was derived from the EMS-induced mutant population of Xida 1B (indica rice). The transgenic materials involved were entrusted to Boyuan Biotechnology Company to complete the genetic transformation and obtain plants. All rice materials were planted at the Xiema planting base of the Rice Research Institute of Southwest University. Xida 1B was planted at the same time as the wild-type control.

[0028] 1.2 Bacterial strains and vectors

[0029] The competent strain of Escherichia coli DH5α and the strain Y2HGold for yeast two-hybrid experiment of GAL4 system were provided by Rice Research Institute, College of Agriculture and Biotechnology, Southwest University. The plant expression vector pCAMBIA1300-GFP-NOS, yeast two-hybrid expression vector pGBKT7, pGADT7 were preserved and used by Rice Research Institute, College of Agriculture and Biotechnology, Southwest University; the dual luciferase reporter vector CaMV35S-Ω-GAL4BD-NOS was donated by Qian Qian's laboratory, Chinese Academy of Rice Research.

[0030] 1.3 Kit

[0031] Plasmid mini-extraction kit, endotoxin-free medium-volume plasmid extraction kit, and universal gel DNA recovery kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; plant total RNA extraction kit and dual luciferase detection kit were purchased from Beijing Promega Biotechnology Co., Ltd.; reverse transcription kit and fluorescence quantification kit were purchased from TaKaRa Bioengineering Co., Ltd.; DNA cleaning and recovery kit was purchased from AXYGEN Biotechnology Co., Ltd.; digoxigenin RNA labeling (SP6 / T7) kit and digoxigenin detection kit were purchased from Roche, Germany.

[0032] 1.4 Various enzymes and molecular biology reagents

[0033] High-fidelity enzymes and various restriction enzymes, DNaseI digestion enzymes and T4 rapid ligases used for gene cloning were purchased from TaKaRa Biotechnology Co., Ltd. EasyTaq enzymes and seamless cloning recombinases for general PCR amplification were purchased from Beijing Quanshijin Biotechnology Co., Ltd. Tryptone, peptone, sodium chloride, yeast extract, glucose, agar powder used for the preparation of various culture media; various antibiotics and CTAB, Tris-HCl and other molecular biology reagents were purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.; xylene, formaldehyde, anhydrous ethanol and other reagents were purchased from Chengdu Kelong Chemical Reagent Factory; solid and liquid reagents related to in situ hybridization experiments and protoplast extraction experiments were purchased from Sigma, USA.

[0034] 1.5 Primers used in the present invention

[0035] 1.5.1 Construction of complementary vector and primers for genetic transformation

[0036]

[0037] 1.5.2 Primers for gene expression analysis (qPCR)

[0038]

[0039] 1.5.3 Primers for in situ hybridization

[0040]

[0041] Example 1 Acquisition and phenotypic analysis of lf3 mutants

[0042] Using ethyl methanesulfonate (EMS) to induce the indica rice maintainer variety Xida 1B, a genetically stable lateral floret development mutant was discovered and named lf3. Its lateral floret development regulatory gene was named LF3.

[0043] During the vegetative growth period, there is no obvious difference between the lf3 mutant and the wild-type plants. After entering the reproductive growth period, the lf3 mutant inflorescence development shows multiple defects. The wild-type inflorescence contains a main axis, primary peduncles, secondary peduncles and spikelets on the peduncles. A typical wild-type spikelet contains a terminal floret that can develop into a grain. Below the terminal floret are a pair of guard lemmas and a pair of secondary guard lemmas. In the lf3 mutant, the primary peduncles and secondary peduncles on the main axis can differentiate and develop normally, but the number of spikelets on the peduncles is significantly reduced and the morphology is abnormal. In addition, above the secondary guard lemmas in the wild-type spikelet are the guard lemmas and terminal florets, and there are no other tissues or organs under the axils of the secondary guard lemmas. However, in the lf3 mutant spikelet, secondary peduncles tissues continue to develop under the axils of the secondary guard lemmas, which is manifested as an increase in the number of secondary guard lemmas and peduncles, and usually the axils of the secondary guard lemmas are accompanied by unknown tissues. Spikelets with different morphologies can develop at the top of the peduncles ( Figure 1 , HK).

[0044] The terminal floret of the wild spikelet includes four whorls of floral organs: palea, lodicule, stamen and pistil, which contain one seed when mature. Below the terminal floret is the guard lemma, which has a smooth surface and is only about 1 / 5 the size of the lemma. There is no other tissue or organ under the guard lemma axil ( Figure 2 , AD). Compared with wild-type spikelets, the guard glumes of spikelets in the lf3 mutant are elongated and lemma-like or develop complete lateral florets. The lemma-like guard glumes are similar in shape and size to the wild-type lemmas, and the surface is covered with silicified cells and burrs consistent with the lemmas. Paraffin sections observed that the lemma-like guard glumes developed a four-layer cell structure consistent with the lemma (silicified upper epidermal cells, non-silicified lower epidermal cells, and thick-walled cells and thin-walled cells in the middle). The size and shape of the non-elongated guard glumes are consistent with those of the wild-type guard glumes, and the cell composition is simple, containing a large amount of thin-walled tissue ( Figure 2 , EH, S, T). In addition, the lf3 mutant developed lateral florets with normal floral organ structures. Some lateral florets had complete floral organs inside, but lacked the same lemma structure as the terminal florets. Some of these lateral florets could not be well filled with grain, but a large part of them could still be hardened and form smaller grains ( Figure 2 , IL, U). Some lateral florets had no obvious abnormalities in morphology compared with terminal florets, and contained four whorls of floral organs consistent with terminal florets and were basically able to fill and produce grains normally. Each spikelet contained two grains of similar size but smaller than those of the wild type ( Figure 2 , MP, U). In addition, a very small number of spikelets develop two lateral florets with internal floral organs, namely "trifloret spikelets", which are similar in size to the terminal florets and can produce fruit ( Figure 2 , QR, V).

[0045] At the booting stage, the wild-type and lf3 spikelets were taken and observed under a scanning electron microscope for early spikelet development. The results showed that the spikelet primordium did not form directly on the auxiliary glume of the mutant spikelet in most cases, but developed additional stalks and produced auxiliary glume primordium and stalks in multiple cycles. Finally, the spikelet primordium was formed on the top of the stalk and gradually differentiated into various floral organ primordia ( Figure 3 , AC). The spikelet primordium at the top of the peduncle has a relatively normal developmental process, but compared with the wild-type spikelet ( Figure 3 , D), the development of the guard glume primordium was observed to be accelerated in the lf3 spikelet, and its morphology and size were similar to those of the lemma. The spikelet axis between the secondary guard glume primordium and the guard glume primordium was slightly elongated ( Figure 3 , E), and some lf3 spikelets developed lateral meristems at the lemma primordium on one or both sides, and were able to differentiate into complete floral organ primordia as the spikelets developed. However, the development period of the lateral floral organ primordium was 2-3 periods slower than that of the terminal floret floral organ primordium ( Figure 3, FG). The development of the organ primordia in the early spikelet of lf3 was consistent with the phenotype of the mutant in the mature stage, that is, the mutant trait began to develop at the beginning of the organ primordium.

[0046] The expression of HISTONE H4, a marker gene for active cell division, was further detected in early spikelets by in situ hybridization. The results showed that HISTONE H4 was strongly expressed in tissues with active cell division. During the transformation from BM to SM, the lf3 mutant showed more HISTONE H4 expression signals than the wild type ( Figure 4 , AB; D). In addition, when the organs in the spikelet differentiated and formed, HISTONE H4 was also clearly expressed in the floral organs, and a large amount of HISTONE H4 signals accumulated in the lateral meristems in the axils of the guard glumes in the lf3 mutant, while no lateral meristems were produced in the axils of the guard glumes in the wild type ( Figure 4 , C; EF). These results indicate that lf3 is more active in cell division than the wild type during BM-SM transformation and floral organ differentiation.

[0047] The expression of meristem characteristic genes OSH1 and OsMADS15 in lf3 young panicles was further analyzed. qPCR results showed that OsMADS15 was significantly upregulated in young lf3 inflorescences of different lengths of the mutant, and OSH1 gene was significantly upregulated in inflorescences less than 1 cm in length, but there was no significant difference in expression in inflorescences longer than 1 cm ( Figure 5 , AB). In situ hybridization results showed that the OSH1 gene was strongly expressed in the wild-type spikelet floral meristem, and when the floral meristem formed the pistil primordium, the expression of the OSH1 gene was significantly weakened ( Figure 5 , CE). In the lf3 mutant, lateral meristems were generated in the axils of the guard glumes, and strong OSH1 gene expression signals were detected in the terminal floral meristems and lateral meristems. With the development of spikelets, the lateral meristems differentiated into lateral florets or lateral spikelets / branch peduncles, and the early peduncles were differentiated inside them and strong OSH1 gene expression signals were detected ( Figure 5 , FK). The OsMADS15 gene was strongly expressed in the wild-type floral meristem, and after the floral meristem differentiated into the primordia of various floral organs, it was also strongly expressed in the lodicule and pistil primordium ( Figure 5 , LN). In the lf3 mutant, OsMADS15 was strongly expressed in the terminal and lateral small floral meristems, lodicules, and pistil primordia ( Figure 5 The above results indicate that the ectopic expression of OSH1 and OsMADS15 genes in the lf3 mutant induces the production of lateral florets / spikelets.

[0048] Example 2 Genetic Analysis and Map-Based Cloning of LF3 Gene

[0049] The LF3 gene was located by BSA method and located between two SSR markers RM3555 and INDel marker CY7-3 at the long arm of chromosome 7. Sequencing analysis of the genes in the interval revealed that in the lf3 mutant, a base 98 on the exon of an AP2 / ERF family transcription factor (LOC_Os07g47330 / FZP) in the interval was replaced by CA (the nucleotide sequence is shown in SEQ ID No.3), resulting in the 33rd amino acid codon becoming a stop codon, premature translation termination, and a shortened amino acid sequence ( Figure 6 , A) (the amino acid sequence of the encoded protein is shown in SEQ ID No.4). According to the annotation information provided on the website, LOC_Os07g47330 / FZP contains only one exon, the cDNA sequence is 957-bp in length (the nucleotide sequence is shown in SEQ ID No.1), and encodes 318 amino acids (the amino acid sequence is shown in SEQ ID No.2). Therefore, the LOC_Os07g47330 gene is positioned as a candidate gene of the LF3 gene.

[0050] In order to determine whether LOC_Os07g47330 is the target gene of LF3 gene, complementary verification was performed, and the results showed that LOC_Os07g47330 / FZP gene is the target gene of LF3 gene. CRISPR-cas9 editing sites were designed on the exons of LOC_Os07g47330 gene for knockout verification. Transgenic plants could not differentiate into spikelets on the peduncles, and continuously produced the next level of peduncles inflorescence, which was the same as the inflorescence phenotype of lf3 mutant, proving that LOC_Os07g47330 / FZP gene is the target gene of LF3 gene ( Figure 6 , B).

[0051] Example 3 Analysis of expression pattern of LF3 gene

[0052] In order to determine the expression pattern of the LF3 gene, the expression of LF3 in various rice tissues was first analyzed by qPCR. The results showed that the LF3 gene was expressed at very low or no level in the vegetative reproductive organs, strongly expressed in the inflorescence, and the expression level decreased significantly with the development and elongation of the inflorescence. Among the various floral organs, the LF3 gene was expressed most highly in the glumes, with high expression in the lodicules and pistils, and very low expression in the palea and stamens ( Figure 7 ). The expression of LF3 in spikelets was further analyzed by in situ hybridization. The results showed that LF3 was highly expressed in the peduncle meristem, spikelet meristem and flower meristem, and obvious expression signals were also detected in the guard glumes ( Figure 7 ).

[0053] Example 4 Analysis of LF3 protein structure and transcriptional activity

[0054] The LF3 gene contains only one exon, and the cDNA sequence is 957 bp in length, encoding 318 amino acids. It is a homologous gene of the BD1 gene reported in corn. Analysis of the homologous protein sequences of LF3 in the gramineous plants corn, rice, sorghum and Brachypodium distachyon showed that the sequence of LF3 protein is highly conserved in different gramineous crops, and there is a highly conserved AP2 / ERF domain at the N-terminus. In addition, the PEST domain sequence rich in Ser-Pro is between the 27th and 42nd amino acids of the FZP protein. At the front end of the AP2 / ERF domain, there is a nuclear localization signal NLS. The C-terminal sequence of LF3 and its homologous proteins also has a high degree of consistency ( Figure 8 ). It was previously reported that LF3 protein has strong transcriptional activation activity, and the 246-268 amino acids at its C-terminus are an acidic region, which may be an activation region that leads to its strong transcriptional activation activity (Komatsu et al. (2003)).

[0055] In order to study the transcriptional activity of LF3 protein, the AP2 / ERF domain at the N-terminus and the acidic activation domain at the C-terminus of LF3 protein were further truncated, and the known transcription activator LF1 (Zhang et al., 2017), the full-length LF3 coding frame and each truncated region were fused to the DNA binding domain of yeast GAL4 (BD, vector name pGBKT7), and the pGBKT7-LF1 vector and the pGBKT7 empty vector were used as positive control and negative control to transform yeast Y2HGold competent cells. 1-318 , pGBKT7-LF3 107-318 and pGBKT7-LF3 245-318 Yeast cells carrying pGBKT7 empty vector, pGBKT7-LF3 1-106 and pGBKT7-LF3 1-244 Yeast cells cannot survive on D / -Trp-His-Ade deficient medium ( Fig. 9 , A), indicating that the LF3 protein containing the C-terminal acidic activation domain has transcriptional activation activity.

[0056] Next, the dual luciferase (LUC) reporter gene was used to further study its transcriptional activity, and the activity of LUC was detected in rice protoplasts. LF3 cDNAs of different lengths from the yeast assay were fused to the GAL4 DNA binding domain (BD) and driven by the 35S promoter of cauliflower mosaic virus. The transcriptional activator VP16 was used as a positive control, and GAL4-BD was used as a negative control. VP16, different sizes of GAL4BD-LF3, and GAL4-BD effectors were transiently expressed in rice protoplasts. LF3 1-318 , LF3 107-318 , pGBKT7-LF3 245-318 The luciferase activity of VP16 and GAL4-BD was much higher than that of LF3 1-106 , LF3 1-244 No transcriptional activation activity, consistent with the negative control ( Fig. 9 , B). These findings further demonstrate that LF3 acts as a transcriptional activator and may be involved in transcriptional regulation. The C-terminal region 245-318 is essential for the transcriptional activation activity of LF3 protein.

[0057] Example 5 Screening of LF3 interacting proteins

[0058] In order to understand the molecular function of FZP, the rice panicle cDNA library was screened to find the interacting proteins of LF3. Since the LF3 protein has strong self-activation activity, it cannot be used for the screening and verification of interacting proteins. The truncated protein pGBKT7-LF3 containing amino acids 1-244 in the C-terminal activation domain was selected. 1-244The yeast cDNA library was screened as bait. The yeast clones screened on the SD / -Trp-Leu / -His-Ade selection medium were expanded and sequenced to obtain the gene information of the inserted fragments in different clones. The obtained gene information was summarized and screened to obtain the following 20 proteins that may interact with LF3 and participate in rice growth and development (Table 4-1). Among them, the OsMADS1 gene, which is involved in the development of rice floral organs, causes hull leafization after mutation. The ectopic expression of OsMADS1 in japonica rice Zhonghua 11 causes abnormal spikelet development, with phenotypes such as panicle distortion, reduced number of branches and spikelets, and elongated lemma. The narrow leaf gene NAL9 regulates the development of rice leaves and spikelets. The nal9 mutant exhibits phenotypes such as narrow leaves, dwarfing, smaller panicles, and increased tillering throughout the growth period. Two nuclear factor subunits NF-YC6 / OsHAP5D and NF-YC7 / OsHAP5E, of which NF-YC6 may delay heading date by downregulating the expression of Ehd1, Hd3a, and RFT1 (Zhang et al. al.2019); in addition, proteins related to the chromatin remodeling complex (Os02g0723700, Os11g0545600, Os02g0122000) were also screened, which may affect the growth and development of rice by regulating the expression of related genes by regulating chromatin structure.

[0059] Table 4-1 Screening of LF3 interacting proteins

[0060]

[0061]

[0062] Example 6 Transcriptome Analysis of lf3 Mutants

[0063] At the booting stage, young panicles of wild type and mutant (≤2 cm, 3 replicates each) were collected in the field, and total RNA of young panicles was extracted by Novogene Technology (Beijing) Co., Ltd. After the samples were tested, transcriptome sequencing was performed. According to the obtained sequencing data, the standard of differentially expressed genes was set to q < 0.05 to perform differential expression analysis on wild type and lf3 mutant. Compared with the wild type, there were 2846 differentially expressed genes in lf3 mutant, of which 1270 genes were up-regulated and 1576 genes were down-regulated ( Fig.10), including multiple rice flower organ development regulatory genes, branch development regulatory genes and meristem activity-related genes. Among these genes, some differentially expressed genes such as meristem-related genes OSH1 and OsMADS15, glume-guarding characteristic gene G1, and branch development regulatory genes OsMADS34 and LAX1 were selected for qPCR verification. The results showed that the expression of meristem regulatory genes OSH1 and OsMADS15 was significantly upregulated in the mutant, the expression of glume-guarding characteristic gene G1 was significantly downregulated, the expression of branch development regulatory gene OsMADS34 was downregulated, and the expression of LAX1 gene was significantly upregulated. The expression changes of these genes are consistent with the characteristics of increased branching and development of lateral florets in the mutant.

[0064] The exercise of biological functions in organisms requires the coordinated completion of multiple genes. The GO enrichment and KEGG metabolic pathway analysis were performed on the 2846 differentially expressed genes obtained. The results showed that the differentially expressed genes in the lf3 mutant were concentrated in the biological process (Biological progress), among which the most enriched genes were in the heterocyclic metabolism process (GO:0046483) and the intracellular aromatic compound metabolism process (GO:0006725). At the same time, there were also many differentially expressed genes enriched in processes such as gene expression regulation (GO:0010468), intracellular biosynthesis (GO:0031326), transcriptional regulation (GO:0006355), and biological regulation (GO:0065007). In addition, a small number of genes were enriched in molecular function (Molecular function), mainly in the categories of nucleic acid binding transcription factor activity (GO:0001071), transcription factor activity (GO:0003700), and protein dimer activity (GO:0046983) ( Fig.11 ). KEGG metabolic pathway analysis showed that 950 genes were annotated during the rice panicle development period, involving a total of 111 KEGG metabolic pathways. The enrichment of 20 pathways, including starch and sucrose metabolic pathways, hormone signal transduction pathways, glycolysis, and fatty acid synthesis, was high (P≤0.1). Among them, the most enriched genes were in the plant hormone signal transduction pathway, with 40 genes, involving auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, jasmonic acid, and salicylic acid signal transduction pathways ( Fig.12 The above results indicate that the LF3 gene may affect the development of rice flowers and panicles by regulating auxin synthesis.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A rice lateral floret regulatory gene LF3, the nucleotide sequence of which is shown in SEQ ID No.1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.

2.

2. The protein encoded by the rice lateral floret regulating gene LF3 according to claim 1, whose amino acid sequence is shown in SEQ ID No.

2.

3. Use of the rice lateral floret regulatory gene LF3 according to claim 1 and the protein according to claim 2 in increasing rice yield.

4. The mutant gene of the rice lateral floret regulatory gene LF3 according to claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID No. 3, and the amino acid sequence of the encoded protein is shown in SEQ ID No.

4.

5. The protein encoded by the mutant gene of the rice lateral floret regulating gene LF3 according to claim 4, wherein the amino acid sequence of the protein is shown in SEQ ID No.

4.

6. Use of the mutant gene of the rice lateral floret regulating gene LF3 according to claim 4 and the protein encoded by the mutant gene of the rice lateral floret regulating gene LF3 according to claim 5 in increasing rice yield.

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