Genes for regulating metabolism of hydroxyl monoacylglycerol in rice and application thereof

Abstract

The application discloses a gene for regulating rice hydroxymonoglycerol metabolism and application thereof, and relates to the technical field of plant genetic engineering. The gene is an OsABCG22 gene or an OsLTPL82 gene; the nucleotide sequences of the OsABCG22 gene and the OsLTPL82 gene are shown in SEQ ID NO. 13 and SEQ ID NO. 16 respectively. The application discloses the role of the rice hydroxymonoglycerol metabolism gene in regulating the fertility of rice, provides a new gene resource for genetic improvement and breeding of rice, and can also be used as a molecular marker for screening rice plants with normal reproductive development. The gene, the constructed vector, the host cell and the construction method can improve the breeding efficiency and accelerate the process of rice genome breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to genes that regulate the metabolism of hydroxymonoglycerol in rice and their applications. Background Technology

[0002] Plants, as sessile organisms, produce a vast number of metabolites. Plant metabolites can be broadly classified into primary metabolites and secondary metabolites. Primary metabolites, such as carbohydrates, lipids, and amino acids, are the material basis for plant growth and development and are essential for plant growth. Primary metabolites provide precursors for the synthesis of secondary metabolites and are also an important food source for humans and animals. Plant secondary metabolites generally refer to a series of intermediate or final products generated from a limited number of primary metabolites through different metabolic pathways. These products are often not essential for plant growth and development, but they play an irreplaceable role in plant adaptation to different environments. In addition, plant metabolites, as the material basis for various traits such as plant growth, development, and resistance to stress and disease, are closer to the plant phenotype than genes and proteins. Research on metabolites in the processes of growth, development, and resistance formation helps to reveal the role of metabolites in plant growth, development, and resistance to stress, and to elucidate the mechanisms of plant growth, development, and resistance formation.

[0003] The epidermis of most plant organs is covered by a cuticle. Cuticle is an insoluble cross-linked polyester mainly composed of long-chain hydroxy fatty acids with 16 and 18 carbons, and is one of the main components of the cuticle. Hydroxy fatty acids and hydroxymonoacylglycerols (HMGs), as fatty acid metabolites, account for 30%-60% of cuticle monomers. Cuticle monomer synthesis involves long-chain acyl-CoA synthases (LACSs), cytochrome P450 enzymes (CYP450s), and glycerol-3-phosphoacyltransferases (GPATs), producing mature monoacylglycerol cuticle monomers. The order of cuticle monomer synthesis reactions is not definitively established and may vary by species. It is generally believed that at least the following order may exist: P450-LACS-GAPT, LACS-P450-GPAT, or LACS-GPAT-P450. After synthesis, cuticle monomers are transported from the endoplasmic reticulum across the cell membrane to the cell wall cuticle by adenosine triphosphate-binding cassette transporters (ABCGs), lipid transporters (LTPs), etc. Cuticle deposition participates in organ growth and epidermal cell differentiation, ensuring continuous surface coverage and structural integrity. Besides controlling water loss in plants, the cuticle also influences floral organ development by affecting organ differentiation, leading to symptoms such as male sterility in anthers of crops like rice, resulting in either inability to harvest seeds or a small yield, thus reducing rice production. While the synthesis of the components of cuticle monomers, hydroxy fatty acids and hydroxymonoacylglycerols, has been reported to some extent, their synthetic sequence, transport, and assembly mechanisms require further investigation.

[0004] In bacteria, genes associated with the same metabolic pathway often cluster to form operons. In eukaryotes, most non-homologous genes involved in the same metabolic pathway are scattered throughout the genome. In fungi and plants, only a few non-homologous genes cluster to form gene clusters. A metabolic gene cluster is a group of at least three genes encoding different enzymes involved in the same metabolic pathway that controls metabolite accumulation. Most reported metabolic gene clusters synthesize secondary metabolites (such as alkaloids and terpenes). Primary metabolic gene clusters in plants have not yet been discovered, nor have metabolic gene clusters controlling cutin monomer synthesis been reported.

[0005] Therefore, it is necessary to analyze the gene clusters in rice that control the synthesis of primary metabolic cutin monomer hydroxymonoglycerol and explore their role in regulating rice fertility and yield. This will be of great significance for a deeper understanding of plant primary metabolic gene clusters and their participation in plant reproductive development. It will also lay a theoretical foundation and provide germplasm resources for crop genetic improvement using metabolic engineering, molecular marker-assisted breeding and other methods. Summary of the Invention

[0006] The purpose of this invention is to provide a gene that regulates the metabolism of hydroxymonoglycerides in rice and its application, in order to solve the problems existing in the prior art. This invention has found that the gene has the function of regulating the metabolism of hydroxymonoglycerides in rice, thereby regulating the male development of rice and affecting rice yield, thus providing a new gene resource for genetic improvement breeding.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a gene that regulates the metabolism of hydroxymonoglycerol in rice, wherein the gene is the OsABCG22 gene or the OsLTPL82 gene.

[0009] The nucleotide sequences of the OsABCG22 gene and the OsLTPL82 gene are shown in SEQ ID NO.13 and SEQ ID NO.16, respectively.

[0010] Furthermore, the protein encoded by the OsABCG22 gene has the function of transporting ω-hydroxy-2-monoacylglycerol (such as 16-hydroxy-2-monopalmitoylglycerol) or ω-hydroxy fatty acids (such as 16-hydroxypalmitic acid) from the endoplasmic reticulum to the apoplast.

[0011] The protein encoded by the OsLTPL82 gene has the function of transporting ω-hydroxy-2-monoacylglycerol (such as 16-hydroxy-2-monopalmitoylglycerol), ω-hydroxy fatty acids (such as 16-hydroxypalmitoic acid), or medium- to long-chain fatty acids (such as palmitic acid) from the endoplasmic reticulum to the apoplast.

[0012] Analysis of the conserved domains of the protein encoded by the OsABCG22 gene revealed that it possesses the conserved ABC_ATP_DarD and ABC2_membrane domains, belonging to the ABC family. The OsABCG22 protein is responsible for transporting ω-hydroxy-2-monoacylglycerols (such as 16-hydroxy-2-monopalmitoylglycerol) and ω-hydroxy fatty acids (such as 16-hydroxypalmitic acid) in rice, controlling the development of rice floral organs and regulating yield.

[0013] Analysis of the conserved domains of the protein encoded by the OsLTPL82 gene revealed that it possesses an AAI conserved domain and belongs to the LTP family. The OsLTPL82 protein is responsible for transporting ω-hydroxy-2-monoacylglycerols (such as 16-hydroxy-2-monopalmitoylglycerol) and ω-hydroxy fatty acids (such as 16-hydroxypalmitic acid) in rice, controlling the development of rice floral organs and regulating yield.

[0014] This invention also provides the application of the above-mentioned gene in regulating the metabolism of hydroxymonoglycerol in rice.

[0015] The present invention also provides the use of the above-mentioned genes in regulating male development in rice.

[0016] The present invention also provides a gene knockout vector for the OsABCG22 gene or the OsLTPL82 gene.

[0017] Furthermore, the gene knockout vector is a CRISPR-Cas9 gene knockout vector.

[0018] The present invention also provides a host cell comprising the gene knockout vector described above.

[0019] The present invention also provides the use of the above-mentioned gene knockout vector or host cell in constructing rice plants as described in (1) or (2) below:

[0020] (1) Rice plants with low levels of hydroxymonoacylglycerol synthesis in the body;

[0021] (2) Rice plants with male reproductive development defects.

[0022] The present invention also provides a method for constructing transgenic rice plants with male reproductive development defects, comprising the step of knocking out the rice OsABCG22 gene and / or the OsLTPL82 gene to construct the transgenic rice plants.

[0023] The present invention discloses the following technical effects:

[0024] This invention is the first to discover and identify a gene cluster controlling hydroxymonoglycerol metabolism on rice chromosome 3, including the OsABCG22 and OsLTPL82 genes, which have hydroxymonoglycerol transport functions. Experiments have demonstrated that the genes and gene cluster of this invention can regulate the biosynthesis and transport steps of hydroxymonoglycerol in rice. Using transgenic technology, this invention obtained rice mutant plants with these genes knocked out. Experimental results showed that the mutant plants obtained by knocking out the OsABCG22 and OsLTPL82 genes had significantly reduced levels of hydroxymonoglycerol (HMGs) metabolites and exhibited phenotypes of male reproductive development defects and significantly reduced yield. This invention reveals the role of rice hydroxymonoglycerol metabolism genes in regulating rice fertility, providing new gene resources for rice genetic improvement breeding, and can also be used as molecular markers to screen for rice plants with normal reproductive development. In summary, the genes, constructed vectors, host cells, and construction methods disclosed in this invention can improve breeding efficiency and accelerate the process of rice genome breeding. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Here are the candidate gene cluster localization maps; where ac are schematic diagrams showing that palmitic acid, 16-hydroxypalmitic acid metabolites, and single-plant yield of rice agronomic traits are co-localized to candidate gene cluster FGC3 in rice seed population; d and e are the correlation test results between the relative contents of palmitic acid and 16-hydroxypalmitic acid in rice seed population and single-plant yield; f is a schematic diagram of the candidate gene localization.

[0027] Figure 2 The diagram shows the functional verification of the FGC3 gene cluster synthesis; where a represents the in vitro enzyme activity assay of recombinant OsFAR2 protein; b represents the in vitro enzyme activity assay of OsKCS11 yeast microsomes; c represents the in vitro enzyme activity assay of OsCYP704B2 yeast microsomes; d represents the in vitro enzyme activity assay of OsGPAT6 yeast microsomes; and e represents the synthetic route of the cutin monomer hydroxymonoacylglycerol.

[0028] Figure 3 The image shows the subcellular localization detection results of the OsABCG22 and OsLTPL82 genes.

[0029] Figure 4This diagram validates the transport function of the FGC3 gene cluster. In this diagram, a shows the results of adding 16-hydroxy-2-monopalmitoylglycerol to protoplasts of tobacco leaves expressing OsABCG22, OsLTPL82, and the vector control; b shows the results of adding thiamine to protoplasts of tobacco leaves expressing OsABCG22, OsLTPL82, and the vector control; c shows the results of adding 16-hydroxypalmitic acid to protoplasts of tobacco leaves expressing OsABCG22, OsLTPL82, and the vector control; and d shows the results of adding palmitic acid to protoplasts of tobacco leaves expressing OsABCG22, OsLTPL82, and the vector control.

[0030] Figure 5 This is a schematic diagram of the synthesis and transport route of the keratin monomer hydroxymonoacylglycerol;

[0031] Figure 6 Figure 1 shows mutant plants resulting from CRISPR-Cas9 gene editing of FGC3 members; where a is a mutant plant resulting from CRISPR-Cas9 gene editing of FGC3 member osfar2; b is a mutant plant resulting from CRISPR-Cas9 gene editing of FGC3 member oskcs11; c is a mutant plant resulting from CRISPR-Cas9 gene editing of FGC3 member oscyp704b2; d is a mutant plant resulting from CRISPR-Cas9 gene editing of FGC3 member osltpl82; e is a mutant plant resulting from CRISPR-Cas9 gene editing of FGC3 member saobcg22; and f is a mutant plant resulting from CRISPR-Cas9 gene editing of FGC3 member osgpat6.

[0032] Figure 7 The images show the toluidine blue staining results of different anthers; where a is the toluidine blue staining result of wild-type and mutant ZH11; and b is the toluidine blue staining result of wild-type and mutant MH63.

[0033] Figure 8 Anther metabolite analysis diagrams for wild-type and FGC3 member mutant plants are shown below; where a represents the anther metabolite analysis diagram for wild-type and FGC3 member osgpat6 mutant plants; b represents the anther metabolite analysis diagram for wild-type and FGC3 member oscyp704b2 mutant plants; c represents the anther metabolite analysis diagram for wild-type and FGC3 member oskcs11 mutant plants; d represents the anther metabolite analysis diagram for wild-type and FGC3 member osfar2 mutant plants; e represents the anther metabolite analysis diagram for wild-type and FGC3 member saobcg22 mutant plants; and f represents the anther metabolite analysis diagram for wild-type and FGC3 member osltpl82 mutant plants.

[0034] Figure 9 Figures show the anther organ phenotypes, surviving mature pollen grains, and yield statistics for the osgpat6, oskcs11, osabcg22, and osltpl82 mutants. Specifically, a, d, g, and j represent the anther organ phenotypes of the osgpat6, oskcs11, osabcg22, and osltpl82 mutants, respectively; b, e, h, and k represent the surviving mature pollen grains of the osgpat6, oskcs11, osabcg22, and osltpl82 mutants, respectively; and c, f, i, and l represent the yield statistics for each mutant.

[0035] Figure 10 Figures show the anther organ phenotypes, surviving mature pollen grains, and yield statistics of the osfar2 and oscyp704b2 mutants; where a and d are the anther organ phenotypes of the osfar2 and oscyp704b2 mutants, respectively; b and e are the surviving mature pollen grains of the osfar2 and oscyp704b2 mutants, respectively; and c and f are the yield statistics of the osfar2 and oscyp704b2 mutants, respectively. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] To elucidate the genetic and biochemical basis of natural variations in the rice lipidome, the inventors previously conducted genome-wide association studies (mGWAS) on 267 lipid metabolites detected in 533 natural rice varieties. This invention focuses on the differences in the content of the metabolite ω-hydroxy fatty acids within the population. The mGWAS results show that ω-hydroxy fatty acids, fatty acids, and rice yield per plant are all located at a position of 3.6 Mb on rice chromosome 3. ω-hydroxy fatty acids belong to the cuticle monomers, and the synthesis steps of cuticle monomers mainly include fatty acid acyl activation, fatty acid ω-hydroxylation, dicarboxylation oxidation, acylation, and then transport from the endoplasmic reticulum across the cell membrane to the cell wall cuticle by adenosine triphosphate-binding cassette transporters and lipid transporters to exert their functions. Based on the steps of cutin monomer synthesis and transport, and combined with gene annotation information for this region in the rice genome (rice genome database TIGR), the inventors identified candidate genes involved in the synthesis and transport of ω-hydroxy fatty acids from a vast amount of rice genome data. These candidate genes are: fatty acid reductase gene OsFAR2, 3-keto-coenzyme A synthase gene OsKCS11, glycerol 3-phosphoacyltransferase gene OsGPAT6, cytochrome oxidase gene OsCYP704B2, ABC class transport protein encoding gene OsABCG22, and lipid transport protein encoding gene OsLTPL82. Furthermore, the inventors calculated the relative positions of these genes and found that the genes responsible for synthesis are distributed within a 300kb range, while all members responsible for synthesis and transport are distributed within a 620kb range. Based on the above, these candidate genes belong to six different enzyme families, a characteristic consistent with the definition of a plant metabolic gene cluster. Therefore, based on the fact that its final product is hydroxymonoglycerol, this invention names this metabolic regulatory gene cluster the "hydroxymonoglycerol gene cluster." Figure 1 ).

[0040] The OsFAR2 gDNA sequence is shown in SEQ ID NO.1; the OsFAR2 CDS sequence is shown in SEQ ID NO.2; the amino acid sequence of the OsFAR2 protein is shown in SEQ ID NO.3; the OsKCS11 gDNA sequence is shown in SEQ ID NO.4; the OsKCS11 CDS sequence is shown in SEQ ID NO.5; the amino acid sequence of the OsKCS11 protein is shown in SEQ ID NO.6; the OsGPAT6 gDNA sequence is shown in SEQ ID NO.7; the OsGPAT6 CDS sequence is shown in SEQ ID NO.8; the amino acid sequence of the OsGPAT6 protein is shown in SEQ ID NO.9; the OsCYP704B2 gDNA sequence is shown in SEQ ID NO.10; the OsCYP704B2 CDS sequence is shown in SEQ ID NO.11; the amino acid sequence of the OsCYP704B2 protein is shown in SEQ ID NO.12; the OsABCG22 gDNA sequence is shown in SEQ ID NO.13; the OsABCG22 CDS sequence is shown in SEQ ID NO. The amino acid sequence of OsABCG22 protein is shown in SEQ ID NO.14; the amino acid sequence of OsLTPL82 gDNA is shown in SEQ ID NO.15; the amino acid sequence of OsLTPL82 CDS is shown in SEQ ID NO.16; the amino acid sequence of OsLTPL82 protein is shown in SEQ ID NO.17; and the amino acid sequence of OsLTPL82 protein is shown in SEQ ID NO.18.

[0041] The hydroxymonoglycerol metabolic gene cluster isolated in this invention includes genes OsFAR2, OsKCS11, OsGPAT6, OsCYP704B2, OsABCG22, and OsLTPL82. Among them, OsFAR2, OsKCS11, OsGPAT6, and OsCYP704B2 are responsible for hydroxymonoglycerol synthesis, mainly proceeding sequentially in a head-to-tail manner via two reaction pathways: OsFAR2-OsKCS11-OsCYP704B2-OsGPAT6 and OsFAR2-OsCYP704B2-OsKCS11-OsGPAT6. After hydroxymonoglycerol is synthesized, it is then transported extracellularly to form keratin under the action of two transport proteins, OsABCG22 and OsLTPL82. Genetic transformation of the above six genes using gene editing technology revealed that mutations in any gene within the hydroxymonoglycerol metabolism gene cluster resulted in decreased hydroxymonoglycerol content, leading to phenotypes of male reproductive development defects and reduced yield per plant in rice. Therefore, this demonstrates that the hydroxymonoglycerol metabolism gene cluster isolated in this invention controls male reproductive development in rice by regulating hydroxymonoglycerol content.

[0042] Example 1: Cloning and protein expression of OsFAR2, OsKCS11, OsGPAT6, and OsCYP704B2 genes, members of the hydroxymonoglycerol gene cluster.

[0043] Hydroxymonoglycerols require four genes—OsFAR2, OsKCS11, OsGPAT6, and OsCYP704B2—to be synthesized sequentially in a head-to-tail sequence. When cloning these genes and expressing their proteins, the recombinant OsFAR2 protein was expressed using an *E. coli* prokaryotic expression system. However, analysis of the structures of the OsKCS11, OsGPAT6, and OsCYP704B2 genes revealed that all three are membrane-localized proteins. Therefore, a yeast expression system was used for the protein expression of these three genes, as detailed below:

[0044] Based on the location and structure of the OsFAR2, OsKCS11, OsGPAT6, and OsCYP704B2 genes in the genome of the japonica rice 'Nipponbare' (rice genome database TIGR), and based on the predicted full-length open reading frame sequences of these candidate genes, this invention designs PCR-specific primers with homologous recombination adapters (see Table 1, synthesized by Qingke Biotechnology Co., Ltd.) and uses ultra-fidelity KOD enzyme (Dalian Baosheng Biotechnology Co., Ltd.) to amplify the full-length CDS fragments of the above four genes.

[0045] Table 1 Primers used in constructing the protein expression vector in this invention

[0046]

[0047] Note: In the table The image shows a forward-directed E. coli homologous recombination adapter; The image shows a reverse E. coli homologous recombination adapter; The image shows a forward yeast homologous recombination linker; The image shows a reverse yeast homologous recombination adapter.

[0048] 1. Cloning and protein expression of the OsFAR2 gene

[0049] The cDNA (SEQ ID NO.2) of the target OsFAR2 gene was recombined into a linearized Pjc767 / pGEX6p-1 expression vector (purchased from Invitrogen, USA) tagged with glutathione S-transferase (GST) using the ClonExpress MultiS one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd.). The recombinant expression vector was transformed into *E. coli* BL21(DE3) competent cells (Shanghai Weidi Biotechnology Co., Ltd.) for exogenous protein expression. Single colonies were selected and cultured in LB medium containing ampicillin antibiotics until A... 600nmThe concentration was initially set at 0.6-0.8, then IPTG (isopropyl galactothioglycoside, Shanghai Sangon Biotech Co., Ltd.) was used to induce cell induction at 20°C for 16 h. Cells were collected (6000 g, 10 min) and lysed under high pressure. The target protein of the GST fusion was purified using glutathione agarose gel 4B (GE Healthcare, USA) and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This invention yielded a well-purified OsFAR2 recombinant protein fused with the GST tag protein, which was aliquoted and stored at -80°C for later use.

[0050] 2. Cloning and protein expression of OsKCS11, OsGPAT6, and OsCYP704B2 genes.

[0051] Structural analysis of the OsKCS11, OsGPAT6, and OsCYP704B2 genes revealed that all three are membrane-localized proteins. Protein expression of these genes was performed using a yeast expression system. The PCR products of the purified CDS sequences of OsKCS11, OsGPAT6, and OsCYP704B2 were cloned into the PYES2(ADH1)URA expression vector, and sequencing was performed to obtain the recombinant vectors for OsKCS11, OsGPAT6, and OsCYP704B2, respectively. The constructed OsGPAT6 recombinant vector was transformed into yeast strain gat1Δ (purchased from EUROSCARF, Germany), and the OsKCS11 and OsCYP704B2 recombinant vectors were transformed into yeast strain WAT11 (purchased from EUROSCARF, Germany).

[0052] The above-mentioned method for constructing the PYES2(ADH1)URA expression vector is as follows: Based on the yeast expression vector PYES2 (purchased from Invitrogen, USA), the transformation vector PYES2(ADH1)URA is constructed, and the GAL1 promoter of PYES2 is replaced with the yeast ADH1 promoter to obtain the PYES2(ADH1)URA expression vector.

[0053] The cultivation of yeast culture and the preparation of microsomes are as follows:

[0054] The recombinant yeast was first cultured in selenite-cystine enrichment medium (SC medium) containing 2 wt% glucose at 30°C. After culture, the cells were collected and resuspended in SC medium containing 2 wt% galactose instead of glucose, and cultured at 30°C for 2 days. The cells were harvested by centrifugation and lysed using glass beads (0.45 mm in diameter, Sigma-Aldrich, USA) in 50 mM Tris-HCl buffer (containing 1 mM EDTA and 600 mM sorbitol) at pH 7.5. Cells were homogenized using a hybrid mill (MM400, Retsch, Germany) to obtain a homogenate, which was then centrifuged at 12,000g for 60 min to obtain a supernatant. The supernatant was then centrifuged at 120,000g for 90 min to obtain a precipitate, namely microsomal proteins (microspheres composed of microsomal membranes). Finally, the precipitate was resuspended in 100mM Tris-HCl, pH 7.5, 1mM EDTA and 20% (v / v) glycerol, aliquoted and stored at -80°C for later use.

[0055] Microsomes are small vesicles that re-close themselves after the membrane structures of the endoplasmic reticulum rupture during cell homogenization. In in vitro experiments, they perform essential functions of the endoplasmic reticulum, such as protein synthesis, protein glycosylation, and lipid synthesis. In cell biology, microsomes are defined as small vesicles derived from fragments of the endoplasmic reticulum.

[0056] Example 2: Regulation of hydroxymonoglycerol by gene clusters

[0057] The four genes OsFAR2, OsKCS11, OsGPAT6, and OsCYP704B2, members of the hydroxymonoglycerol gene cluster, are responsible for hydroxymonoglycerol synthesis. The synthesis primarily follows two sequential reaction pathways: OsFAR2-OsKCS11-OsCYP704B2-OsGPAT6 and OsFAR2-OsCYP704B2-OsKCS11-OsGPAT6, thus regulating hydroxymonoglycerol synthesis. In vitro enzyme activity experiments will demonstrate this below.

[0058] 1. In vitro enzyme activity assay of OsFAR2 protein

[0059] To verify the function of the candidate gene OsFAR2, the OsFAR2 recombinant protein purified in Example 2 was used for in vitro enzyme activity assay. The in vitro enzyme activity reaction system for OsFAR2 recombinant protein was 100 μL. Using palmitoyl carrier protein (C16:0ACP, Sigma-Aldrich) and palmitoyl coenzyme A (C16:0CoA, Sigma-Aldrich) at a final concentration of 50 μmol / L as substrates, 500 ng of purified OsFAR2-GST recombinant protein (with GST-tagged protein as a negative control) and 100 mmol / L Tris-HCl buffer (pH 7.5) were added. After incubation at 37°C for 30 min, 50 μL of pre-cooled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 13000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μM filter (Shanghai Anpu Pharmaceutical Co., Ltd.) and then analyzed by LC-MS (LCMS-8060, Shimadzu Corporation, Japan).

[0060] The detection results showed that the reaction product exhibited a distinct chromatographic peak, and its retention time was the same as that of the standard compound palmitol (C16:0ACOol, Sigma-Aldrich, USA), indicating that OsFAR2 protein catalyzes both C16:0ACP and C16:0CoA to generate palmitol. Figure 2 (a) This indicates that the OsFAR2 protein acts as the first step in the reaction, producing palmitol.

[0061] 2. In vitro enzyme activity assay of OsKCS11 protein

[0062] To verify the function of the candidate gene OsKCS11, the OsKCS11 microsomal protein prepared in Example 1 was used for in vitro enzyme activity assay. The in vitro enzyme activity reaction system of OsKCS11 microsomal protein was 100 μL. Using palmitic acid (C16:0, Sigma-Aldrich) and 16-hydroxypalmitic acid [C16:0(ω-OH), Sigma-Aldrich] at a final concentration of 200 μmol / L as substrates, 1 mg of microsomal protein, 100 mmol / L Bis-Tris-propane buffer (pH 7.6), 10 mM MgCl2, 5 mM adenine triphosphate (ATP), 2.5 mM dithiothreitol (DTT), and 1 mM coenzyme A (CoA) were added. After incubation at 30°C in a shaking incubator (120 rpm) for 4 h, 50 μL of pre-cooled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 13,000 rpm for 10 min at 4 °C to obtain the supernatant. The supernatant was then filtered through a 0.22 μM filter (Shanghai Anpu Company) and analyzed by LC-MS (LCMS-8060, Shimadzu Corporation, Japan). A negative control reaction was performed using recombinant yeast microsomes transformed with empty pESC-URA. The total protein content was estimated by measuring the absorbance at 280 nm using a spectrophotometer.

[0063] The detection results showed that the reaction product exhibited obvious chromatographic peaks, and its retention time was the same as that of palmitoyl-CoA standard and 16-hydroxypalmitoyl-CoA (C16:O(ω-OH)CoA, Sigma-Aldrich, USA) standard, indicating that OsKCS11 protein catalyzes the formation of C16:OCoA or C16:O(ω-OH)CoA from C16:O or C16:O(ω-OH). Figure 2 (b)

[0064] 3. In vitro enzyme activity assay of OsCYP704B2 protein

[0065] To verify the function of the candidate gene OsCYP704B2, the OsCYP704B2 microsomal protein prepared in Example 1 was used for in vitro enzyme activity assay. The in vitro enzyme activity reaction system for OsCYP704B2 microsomal protein was 100 μL. Using palmitic acid and palmitoyl-CoA as substrates at a final concentration of 200 μmol / L, 1 mg of microsomal protein, 20 mmol / L Tris-HCl buffer (pH 7.5), and 500 mM reduced coenzyme II (NADPH) were added. The mixture was incubated at 30°C (120 rpm) for 4 h, and then 50 μL of pre-cooled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 13000 rpm for 10 min at 4°C to obtain the supernatant. The supernatant was then filtered through a 0.22 μM filter (Shanghai Anpu Company) and analyzed by LC-MS (LCMS-8060, Shimadzu Corporation, Japan). Recombinant yeast microsomes transformed with empty pESC-URA were used as a negative control. The total protein content was estimated by measuring the 280 nm ultraviolet absorbance using a spectrophotometer.

[0066] The detection results showed that the reaction product exhibited obvious chromatographic peaks, and its retention times were the same as those of 16-hydroxypalmitic acid (C16:O(ω-OH), Sigma-Aldrich) standard and 16-hydroxypalmitic acid-coenzyme A standard, respectively. This indicates that OsCYP704B2 protein catalyzes the formation of C16:O or C16:OCoA from C16:O(ω-OH) or C16:O(ω-OH)CoA. Figure 2 (c)

[0067] 4. In vitro enzyme activity assay of OsGPAT6 protein

[0068] To verify the function of the candidate gene OsGPAT6, the OsGPAT6 microsomal protein prepared in Example 1 was used for in vitro enzyme activity assay. The in vitro enzyme activity reaction system of OsGPAT6 microsomal protein was 100 μL. Using palmitoyl-CoA and 16-hydroxypalmitoyl-CoA as substrates at a final concentration of 200 μmol / L, 1 mg of microsomal protein, 20 mmol / L Tris-HCl buffer (pH 7.0), 400 μM glycerol 3-phosphate (G3P), 1 mM dithiothreitol, 5 mM EDTA, and 2.5 mg / mL bovine serum albumin (BSA) were added. After incubation at 30°C in a shaking incubator (120 rpm) for 4 h, 50 μL of pre-cooled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 13,000 rpm for 10 min at 4 °C to obtain the supernatant. The supernatant was then filtered through a 0.22 μM filter (Shanghai Anpu Company) and analyzed by LC-MS (LCMS-8060, Shimadzu Corporation, Japan). A negative control reaction was performed using recombinant yeast microsomes transformed with empty pESC-URA. The total protein content was estimated by measuring the absorbance at 280 nm using a spectrophotometer.

[0069] The detection results showed that the reaction product exhibited obvious chromatographic peaks, and its retention times were the same as those of 2-monopalmitoylglycerol (MG 16:0, Sigma-Aldrich, USA) and 16-hydroxy-2-monopalmitoylglycerol (HMG16:0, Tianjin Alta Biotechnology Co., Ltd.), indicating that OsGPAT6 protein catalyzed the formation of C16:0 or C16:0CoA from C16:0(ω-OH) or C16:0(ω-OH)CoA. Figure 2 (d).

[0070] The above results indicate that the proteins OsFAR2, OsKCS11, OsCYP704B2, and OsGPAT6 possess the function of continuously catalyzing metabolic reactions. Simultaneously, it demonstrates two synthetic pathways for the biosynthesis of hydroxymonoglycerol metabolites (HMGs): OsFAR2-OsCYP704B2-OsKCS11-OsGPAT6 and OsFAR2-OsKCS11-OsCYP704B2-OsGPAT6, forming a metabolic network. Figure 2 (e).

[0071] Example 3: Functional study of OsABCG22 and OsLTPL82 genes, members of the hydroxymonoglycerol gene cluster transporter.

[0072] After hydroxymonoglycerol is synthesized using the four genes OsFAR2, OsKCS11, OsGPAT6, and OsCYP704B2, it needs to be transported from the endoplasmic reticulum across the cell membrane to the cell wall cuticle by adenosine triphosphate-binding cassette transporter (ABCG) and lipid transporter (LTP) to exert its function. Subcellular localization and tobacco protoplast transport experiments demonstrated that the transport members of the gene cluster isolated in this invention, OsABCG22 and OsLTPL82, have the function of transporting hydroxymonoglycerol.

[0073] 1. Cloning and subcellular localization prediction of OsABCG22 and OsLTPL82 genes

[0074] Based on the location and structure of the OsABCG22 and OsLTPL82 genes in the genome of the japonica rice 'Nipponbare' (rice genome database TIGR), and based on the predicted full-length open reading frame sequences of these candidate genes, this invention designs PCR-specific primers with homologous recombination adapters (Table 2, synthesized by Qingke Biotechnology Co., Ltd.), and uses ultra-fidelity KOD enzyme (Dalian Takara Bio Engineering Co., Ltd.) to amplify the full-length CDS fragments of the above two genes.

[0075] Table 2 Primers used in constructing the tobacco expression vector in this invention

[0076]

[0077] Note: In the table The image shows the homologous recombination linker for the positive tobacco expression vector; The image shows the homologous recombination linker for the reverse tobacco expression vector.

[0078] The PCR products of the CDS sequences of the two genes were cloned into the target overexpression vector pH7WGF2 (purchased from the BioVector plasmid vector strain cell protein antibody gene depository center) to generate OsMADS6-YFP and OsMADS17-YFP fusion constructs (35S:OsMADS6YFP and 35S:OsMADS17-YFP) driven by the constitutive cauliflower mosaic virus promoter. 35S:OsMADS6YFP, 35S:OsMADS17YFP, and 35S:osGhd7CFP were transiently expressed in tobacco leaves via Agrobacterium-mediated transformation. Fluorescence signals were observed using a confocal laser scanning microscope (FV1000, Olympus, Japan) 2 days after transformation.

[0079] Subcellular localization indicated that OsLTPL82 and OsABCG22 were primarily located on the cell membrane. Figure 3 This suggests that the OsABCG22 and OsLTPL82 genes may have a transport function.

[0080] 2. Verification of the transport activity of the OsABCG22 and OsLTPL82 genes

[0081] To verify the transport activity of candidate genes OsABCG22 and OsLTPL82, the PCR products of the CDR sequences of the OsABCG22 and OsLTPL82 genes were cloned into the tobacco transient expression vector pEAQ-HT-DEST2 (from Professor George P. Lomonossoff's laboratory at the John Innes Centre, UK). Sequencing verification yielded the 35S:OsABCG22 and 35S:OsLTPL82 vectors. The constructed 35S:OsABCG22 and 35S:OsLTPL82 vectors were transformed into Agrobacterium, and then 4-week-old tobacco leaves were infected. Protoplasts were prepared 4 days later.

[0082] This invention uses OsCYP704B2 protein and commercially available palmitic acid-1-( 13 C)([ 13 C]-C16:0, Sigma-Aldrich (USA) conducted a biochemical reaction to produce [ 13 [C]-C16:0(ω-OH) stabilizes isotopic products. OsKCS11, OsCYP704B2, and OsG PAT6 proteins were used with commercially available palmitic acid-1-( 13 C)([13C]-C16:0) underwent a biochemical reaction to produce [ 13 [C]-HMG16:0 stable isotope product. Add [ to protoplasts] 13 C]-HMG16:0、[ 13 C]-C16:0(ω-OH), [ 13 [C]-C16:0 stable isotopes were used as the experimental group; [C]-C16:0 stable isotopes were added to the protoplasts. 13 A stable isotope substrate of C4-thiamine (IsoSciences, USA) was used as a control, with a final concentration of 1 mmol / L. The mixture was incubated on ice for 15 min to allow for substrate uptake. Protoplasts were separated using a gradient of 50%, 25%, and 5% Percoll separation buffer (Cytiva, Sweden) from bottom to top to remove external metabolites, and protoplasts were recovered from the 25% and 5% Percoll interfaces. Protoplasts were incubated at 25 °C, and 100 μL samples were taken at 0, 5, 10, 15, and 20 min. After centrifugation, the supernatant was used for metabolite detection. Relative output was calculated based on metabolite concentrations normalized to the first time point (0 min). The experiment was repeated three times, and data are expressed as the standard error of the mean of all technique replicates.

[0083] The results showed that, compared with the initial efflux rate of approximately 20% in the empty vector control, the initial efflux rates of tobacco cells expressing both the OsABCG22 and OsLTPL82 genes exceeded 40%. Figure 4 (a) This indicates that the output of HMG16:0 was significantly enhanced. Meanwhile, the transporter, as a control, [ 13 No significant changes in transport activity were observed when C4]-thiamine was used. Figure 4 (b) This indicates that both OsABCG22 and OsLTPL82 proteins possess HMG transport activity. These results suggest that OsABCG22 and OsLTPL82 are membrane-localized transporters of keratin monomer hydroxymonoglycerides. To further investigate the specificity of OsABCG22 and OsLTPL82 proteins, this invention uses [ 13 The translocation experiment of C]-C16:0(ω-OH) showed that the expression of OsABCG22 and OsLTPL82 proteins significantly reduced the net import of labeled C16:0(ω-OH) in the control group, with the initial efflux rate decreasing from -29% to -19%, indicating that both proteins have translocation activity of ω-hydroxy fatty acids. Figure 4 (c). Similarly, compared to the control, this invention observed that tobacco cells expressing OsLTPL82 showed better response to […]. 13 The exoefflux activity of [C]-C16:0 was enhanced, with the initial efflux rate increasing from 6% to 15% of the control, but OsABCG22 showed increased exoefflux activity against [C]-C16:0. 13 The transeffervescence activity of C]-C16:0 did not change significantly. Figure 4 (d). Therefore, these results indicate that the gene cluster transport members OsABC G22 and OsLTPL82 of the present invention have the function of transporting hydroxy monoacylglycerols and ω-hydroxy fatty acids.

[0084] In summary, the hydroxymonoglycerol gene cluster of this invention includes the synthetic genes OsFAR2, OsKCS11, OsGPAT6, and OsCYP704B2, as well as the transport genes OsABCG22 and OsLTPL82. Based on experimental results, a working model of their role in keratin monomer metabolism is proposed. Figure 5 The fatty acyl carrier protein is first reduced to fatty alcohol by OsFAR2 in the plastid, and then fatty acids are produced by an unknown protein, which then diffuse into the endoplasmic reticulum. Subsequently, hydroxymonoacylglycerols can be produced in the endoplasmic reticulum via either the OsCYP704B2-OsKCS11-OsGPAT6 or OsKCS11-OsCYP704B2-OsGPAT6 sequence. Hydroxymonoacylglycerols are then transported to the ectoplastic epidermis of the cell wall via OsABCG22 and OsLTPL82, where they are deposited to form keratin.

[0085] Example 4: Effects of hydroxymonoglycerol gene cluster members (OsFAR2, OsKCS11, OsGPAT6, OsCYP704B2, OsABCG22, and OsLTPL82) on male reproductive development and yield in rice.

[0086] To verify the effects of the gene clusters of this invention on the reproductive development and yield of rice, this embodiment first constructed transgenic mutant materials, and then tested the effects of the mutant materials on related phenotypes of rice.

[0087] 1. Construction of transgenic mutant materials

[0088] The mutants osfar2, oskcs11, osgpat6, oscyp704b2, oabcg22, and osltpl82 were constructed using CRISPR-Cas9 technology. Referring to the NCBI database, the full-length cDNA and genomic sequences of OsFAR2, OsKCS11, OsCYP704B2, OsABCG22, and OsLTPL82 were downloaded and sequence aligned to identify exons. Space amplification primers for CRISPR vectors were designed using the exon sequences on the NEB cutter website (http: / / nc2.neb.com / NEBcutter2 / ). The template was PJE044 (SgRNA) (this vector was modified by the inventor's laboratory, carrying sgRNA and a U3 promoter, and can be used for the amplification of CRISPR elements). The amplified fragment was ligated into pH-Ubi-cas9-7 (a vector carrying a constitutive and overexpression-characteristic maize ubiquitin gene promoter and cas9 protein, which can be used for Agrobacterium-mediated gene knockout genetic transformation and has been disclosed in the literature "Liu X, Zhou X, Li K, Wang D, Ding Y, Liu X, Luo J, Fang C. A simple and efficient cloning system for CRISPR / Cas9-mediated genome editing in rice. PeerJ. 2020 Jan 29; 8:e8491.doi:10.7717 / peerj.8491.PMID:32030327; PMCID:PMC6995270") using the Gateway system.

[0089] Agrobacterium-mediated transformation was used to introduce the generated vectors OsFAR2, OsKCS11, OsCYP704B2, OsABCG22, and OsLTPL82 into Zhonghua 11 (ZH11) callus, and the OsGPAT6 vector into Minghui 63 (MH63) callus. After propagation, DNA positivity identification, and gene transcription level expression determination, stable transgenic materials were obtained. Figure 6 ).

[0090] 2. Phenotypic analysis of transgenic mutant materials

[0091] To further verify the function of candidate genes in regulating cutin monomer metabolites in rice, this invention selected T2 generation plants of the osfar2, oskcs11, osgpat6, oscyp704b2, osabcg22, and osltpl82 mutants, respectively. The anthers of these mutants were stained with 0.05% (w / v) toluidine blue reagent (Sigma-Aldrich, USA). The results showed that compared with wild-type ZH11 or MH63, the osfar2, oskcs11, osgpat6, oscyp704b2, osabcg22, and osltpl82 mutants were stained more deeply by the toluidine blue solution, exhibiting a deep blue color. Figure 7 This indicates that mutations in members of the hydroxymonoglycerol gene cluster all cause varying degrees of damage to the cuticle barrier of the anther, playing an important role in the biosynthesis of the anther cuticle.

[0092] Further LC-MS detection and analysis were performed on cutin monomer metabolites, including fatty acids, hydroxy fatty acids, and hydroxymonoacylglycerols, in rice anthers. The results showed that, compared to wild-type materials, the contents of hydroxymonoacylglycerol metabolites, such as HMG16:0, HMG16:1, HMG18:0, HMG18:1, and HMG18:2, in anthers of mutant members of the hydroxymonoacylglycerol gene cluster OsFAR2, OsKCS11, OsGPAT6, OsCYP704B2, OsABCG22, and OsLTPL82 were significantly reduced. Figure 8 Taking osgpat6 as an example, the relative contents of HMG16:0, HMG16:1, HMG18:0, HMG18:1, and HMG18:2 in the wild-type material MH63 are 1.2e10. 5 6.7e10 5 1.2e10 6 4.7e10 5 2.5e10 4 The relative abundance in the osgpat6 mutant was 8.7e10. 4 4.9e10 5 8.7e105 3.4e10 5 1.6e10 4 All of these values ​​decreased significantly. The results of this experiment demonstrate that the gene cluster of this invention has the function of regulating the content of hydroxymonoglycerides.

[0093] To investigate the physiological function of the hydroxymonoglycerol gene cluster, phenotypic observations were conducted on mutant plants. The results showed that all mutant plants exhibited abnormal floral organ development. Figure 9 and Figure 10 Compared to the wild type, the mutant plants are smaller and have paler anthers. Figure 9 a, d, g, and j Figure 10 (a and d), and showed a significant reduction in viable mature pollen grains ( Figure 9 b, e, h and k Figure 10 (b and e). The experimental results show that mutations in any member of the gene cluster of this invention will lead to abnormal development of the plant's floral organs, verifying that the gene cluster of this invention has the function of regulating male reproductive development in rice.

[0094] Further statistical analysis showed that, compared with the wild type, the yield per plant of mutant plants belonging to the hydroxymonoglycerol gene cluster was significantly reduced, typically by 30% to 62%. Figure 9 c, f, i and l, Figure 10 In the c and f examples, taking osgpat6 as an example, the yield per plant of the wild-type material MH63 was 32g, while the yields of the three families of the osgpat6 mutant were 20g, 14g, and 19g, respectively, a decrease of 39% to 58%. This indicates that the gene cluster of this invention can regulate rice yield by adjusting anther fertility.

[0095] The above results demonstrate that mutations in any member of the hydroxymonoglycerol gene cluster of this invention lead to changes in the composition and content of cutin monomers, abnormal development of anthers and pollen grains, and thus significantly reduced yield. This lays the foundation for genetic improvement of rice fertility using this gene cluster, provides technical guidance for the discovery of related metabolic gene clusters in other crops and their application in breeding, and can also be used as a molecular marker to screen for rice plants with normal reproductive development.

[0096] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a gene regulating rice hydroxymonoglycerol metabolism in regulating rice hydroxymonoglycerol metabolism, characterized in that, The amount of hydroxymonoglycerol synthesized in rice plants was reduced by knocking out the aforementioned gene. The gene in question is either the OsABCG22 gene or the OsLTPL82 gene. The nucleotide sequences of the OsABCG22 gene and the OsLTPL82 gene are shown in SEQ ID NO.13 and SEQ ID NO.16, respectively.

2. The application of a gene regulating rice hydroxymonoglycerate metabolism in regulating male development in rice, characterized in that, By knocking out the aforementioned gene, rice plants can be made to have male reproductive development defects. The gene in question is either the OsABCG22 gene or the OsLTPL82 gene. The nucleotide sequences of the OsABCG22 gene and the OsLTPL82 gene are shown in SEQ ID NO.13 and SEQ ID NO.16, respectively.

3. The application of a gene knockout vector for the OsABCG22 gene or the OsLTPL82 gene, characterized in that the nucleotide sequences of the OsABCG22 gene and the OsLTPL82 gene are shown in SEQ ID NO.13 and SEQ ID NO.16, respectively; The application is to construct rice plants as described in (1) or (2) below: (1) Rice plants with low levels of hydroxymonoglycerol synthesis in the body; (2) Rice plants with male reproductive development defects.

4. The application according to claim 3, characterized in that, The gene knockout vector is a CRISPR-Cas9 gene knockout vector.

5. An application of a host cell, characterized in that, The host cell includes a gene knockout vector of the OsABCG22 gene or the OsLTPL82 gene. The nucleotide sequences of the OsABCG22 gene and the OsLTPL82 gene are shown in SEQ ID NO.13 and SEQ ID NO.16, respectively; The application is to construct rice plants as described in (1) or (2) below: (1) Rice plants with low levels of hydroxymonoglycerol synthesis in the body; (2) Rice plants with male reproductive development defects.

6. A method for constructing transgenic rice plants with male reproductive development defects, characterized in that, The process includes the step of knocking out the rice OsABCG22 gene and / or the OsLTPL82 gene to construct the transgenic rice plant. The nucleotide sequences of the OsABCG22 gene and the OsLTPL82 gene are shown in SEQ ID NO.13 and SEQ ID NO.16, respectively.

Images