Gene for regulating and controlling metabolism of rice hydroxyl monoacylglycerol and application of gene

Abstract

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

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, in particular to a gene for regulating the metabolism of rice hydroxymonoacylglycerol and an application thereof. Background Art

[0002] As sessile organisms, plants can produce a huge number of metabolites. Plant metabolites can be divided into two categories: primary metabolites and secondary metabolites. Plant primary metabolites, such as sugars, lipids, and amino acids, are the material basis for plant growth and development and are necessary for plant growth and development. 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 limited primary metabolites through different metabolic pathways. These products are often not necessary for plant growth and development, but they play an irreplaceable role in helping plants adapt to different environments. In addition, plant metabolites, as the material basis for various traits such as plant growth and development and stress resistance and disease resistance, are closer to plant phenotypes than genes and proteins. Research on metabolites in the process of growth and development and resistance formation helps to reveal the role of metabolites in plant growth and development and stress resistance, and to reveal the mechanism of plant growth and development and resistance formation.

[0003] Most plant organ epidermises are covered with a cuticle. Cutin is an insoluble cross-linked polyester composed mainly of 16-carbon and 18-carbon long-chain hydroxy fatty acids, and is one of the main components of the cuticle. Hydroxy fatty acids and hydroxymonoacylglycerol (HMG) are fatty acid metabolites, accounting for 30%-60% of cutin monomers. Cutin monomer synthesis involves long-chain acyl-CoA synthetases (LACSs), cytochrome P450 enzymes (CYP450s), and glycerol-3-phosphate acyltransferases (GPATs) to produce mature monoacylglycerol cutin monomers. The order of cutin monomer synthesis reactions is still undetermined and may vary from species to species. It is generally believed that there may be at least the following order: P450-LACS-GAPT, LACS-P450-GPAT, or LACS-GPAT-P450. After cutin monomer synthesis, it is 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. The deposition of the cuticle participates in the growth of organs and the differentiation of epidermal cells to ensure the continuous coverage of the surface and the integrity of the structure. In addition to controlling the loss of water in plants, the cuticle also controls the development of floral organs by affecting organ differentiation, leading to symptoms such as male sterility in the anthers of crops such as rice, and thus the inability to produce or the collection of a small number of seeds, resulting in reduced rice yields. At present, there are some reports on the synthesis of hydroxy fatty acids and hydroxy monoacylglycerol, components of cutin monomers, but their synthesis order, transport and assembly mechanism need to be further studied.

[0004] In bacteria, genes related to the same metabolic pathway are often clustered to form operons, while in eukaryotes, most non-homologous genes involved in the same metabolic pathway are scattered throughout the genome, and only some non-homologous genes in the metabolic pathways of fungi and plants are clustered to form gene clusters. Metabolic gene clusters are clusters of at least three genes encoding different enzymes that participate in the same metabolic pathway that controls the accumulation of metabolites. Most of the reported metabolic gene clusters synthesize secondary metabolites (such as alkaloids and terpenes), and primary metabolic gene clusters in plants have not yet been discovered, and metabolic gene clusters that control the synthesis of cutin monomers have not been reported.

[0005] Therefore, it is necessary to analyze the gene cluster that controls the synthesis of the primary metabolic cutin monomer hydroxymonoacylglycerol in rice and explore its role in regulating rice fertility and yield. This will be of great significance for the in-depth analysis 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 means. Summary of the invention

[0006] The purpose of the present invention is to provide a gene for regulating rice hydroxymonoacylglycerol metabolism and its application to solve the problems existing in the above-mentioned prior art. The present invention has found that the gene has the function of regulating rice hydroxymonoacylglycerol metabolism, thereby regulating rice male development, and then affecting rice yield, providing a new gene resource for genetic improvement breeding.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a gene for regulating rice hydroxymonoacylglycerol metabolism, wherein the gene is an OsABCG22 gene or an 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 a transport function of transporting ω-hydroxy 2-monoacylglycerol (such as 16-hydroxy 2-monopalmitoylglycerol) or ω-hydroxy fatty acid (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-hydroxypalmitic acid) or medium-chain fatty acids (such as palmitic acid) from the endoplasmic reticulum to the apoplast.

[0012] The protein conserved domain analysis of the protein encoded by the OsABCG22 gene showed that it has ABC_ATP_DarD and ABC2_membrane conserved domains, and belongs to the ABC family. The OsABCG22 protein is responsible for transporting ω-hydroxy 2-monoacylglycerol (such as 16-hydroxy 2-monopalmitoylglycerol) and ω-hydroxy fatty acids (such as 16-hydroxypalmitic acid) in rice, controlling the development of rice flower organs and regulating yield.

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

[0014] The present invention also provides application of the above gene in regulating the metabolism of rice hydroxymonoacylglycerol.

[0015] The present invention also provides the use of the above gene in regulating the male development function of rice.

[0016] The present invention also provides a gene knockout vector of OsABCG22 gene or 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 above gene knockout vector.

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

[0020] (1) Rice plants with low synthesis of hydroxymonoacylglycerol;

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

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

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

[0024] The present invention first discovered and identified a gene cluster controlling hydroxymonoacylglycerol metabolism on rice chromosome 3, including OsABCG22 gene and OsLTPL82 gene with hydroxymonoacylglycerol transport function. Experiments have proved that the genes and gene clusters of the present invention can regulate the biosynthesis and transport steps of rice hydroxymonoacylglycerol. The present invention uses transgenic technology to obtain rice mutant plants with these genes knocked out respectively. The experimental results show that the mutant plants obtained by knocking out the OsABCG22 gene and the OsLTPL82 gene have significantly reduced hydroxymonoacylglycerol (HMGs) metabolite content, and all have male reproductive development defects and significantly reduced yield phenotypes. The present invention reveals the role of rice hydroxymonoacylglycerol metabolism genes in regulating rice fertility, provides new gene resources for rice genetic improvement breeding, and can also be used as molecular markers to screen rice plants with normal reproductive development. In summary, the genes disclosed in the present invention and the constructed vectors, host cells and construction methods can improve breeding efficiency and accelerate the rice genome breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 is the candidate gene cluster location map; wherein, ac are schematic diagrams of palmitic acid, 16-hydroxypalmitic acid metabolites and rice agronomic trait single plant yield co-localized to the candidate gene cluster FGC3 in the rice seed population; d and e are the correlation test results of the relative contents of palmitic acid and 16-hydroxypalmitic acid in the rice seed population and single plant yield; f is the schematic diagram of the location of the candidate gene;

[0027] Figure 2 Figure 1 is a functional validation diagram of the FGC3 gene cluster synthesis; a is the result of in vitro enzyme activity assay of the recombinant OsFAR2 protein; b is the result of in vitro enzyme activity of OsKCS11 yeast microsomes; c is the result of in vitro enzyme activity of OsCYP704B2 yeast microsomes; d is the result of in vitro enzyme activity of OsGPAT6 yeast microsomes; e is the synthetic route map of the cutin monomer hydroxymonoacylglycerol;

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

[0029] Figure 4Figure 1 is a validation diagram of the transport function of the FGC3 gene cluster; wherein, a is the result of determining the transport activity of 16-hydroxy 2-monopalmitoylglycerol added to protoplasts of tobacco leaves expressing OsABCG22, OsLTPL82 and vector control; b is the result of determining the transport activity of thiamine added to protoplasts of tobacco leaves expressing OsABCG22, OsLTPL82 and vector control; c is the result of determining the transport activity of 16-hydroxypalmitic acid added to protoplasts of tobacco leaves expressing OsABCG22, OsLTPL82 and vector control; d is the result of determining the transport activity of palmitic acid added to protoplasts of tobacco leaves expressing OsABCG22, OsLTPL82 and vector control;

[0030] Figure 5 It is a synthesis and transport route map of the keratin monomer hydroxymonoacylglycerol;

[0031] Figure 6 Figure 1 is a picture of mutant plants after CRISPR-Cas9 gene editing of FGC3 members; among them, a is a mutant plant after CRISPR-Cas9 gene editing of FGC3 member osfar2; b is a mutant plant after CRISPR-Cas9 gene editing of FGC3 member oskcs11; c is a mutant plant after CRISPR-Cas9 gene editing of FGC3 member oscyp704b2; d is a mutant plant after CRISPR-Cas9 gene editing of FGC3 member osltpl82; e is a mutant plant after CRISPR-Cas9 gene editing of FGC3 member osabcg22; f is a mutant plant after CRISPR-Cas9 gene editing of FGC3 member osgpat6;

[0032] Figure 7 Figures are the toluidine blue staining results of different anthers; wherein, a is the toluidine blue staining results of the wild strain and mutant strain of ZH11; b is the toluidine blue staining results of the wild strain and mutant strain of MH63;

[0033] Figure 8 The anther metabolite analysis diagram of the wild type and FGC3 member mutant plants; wherein, a is the anther metabolite analysis diagram of the wild type and FGC3 member osgpat6 mutant plants; b is the anther metabolite analysis diagram of the wild type and FGC3 member oscyp704b2 mutant plants; c is the anther metabolite analysis diagram of the wild type and FGC3 member oskcs11 mutant plants; d is the anther metabolite analysis diagram of the wild type and FGC3 member osfar2 mutant plants; e is the anther metabolite analysis diagram of the wild type and FGC3 member osabcg22 mutant plants; f is the anther metabolite analysis diagram of the wild type and FGC3 member osltpl82 mutant plants;

[0034] Fig. 9 The figures show the anther organ phenotype observation, viable mature pollen grains and single plant yield statistics of osgpat6, oskcs11, osabcg22 and osltpl82 mutants; a, d, g and j are the anther organ phenotype observation figures of osgpat6, oskcs11, osabcg22 and osltpl82 mutants; b, e, h and k are the viable mature pollen grain observation figures of osgpat6, oskcs11, osabcg22 and osltpl82 mutants; c, f, i and l are the single plant yield statistics of osgpat6, oskcs11, osabcg22 and osltpl82 mutants;

[0035] Fig.10 The figures show the anther organ phenotype observation, viable mature pollen grains and single-plant yield statistics of osfar2 and oscyp704b2 mutants; a and d are the anther organ phenotype observation figures of osfar2 and oscyp704b2 mutants, respectively; b and e are the viable mature pollen grain observation figures of osfar2 and oscyp704b2 mutants, respectively; c and f are the single-plant yield statistics of osfar2 and oscyp704b2 mutants, respectively. DETAILED DESCRIPTION

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

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

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

[0039] In order to analyze the genetic and biochemical basis of natural variation in rice lipidome, the inventors previously conducted metabolite genome-wide association analysis (mGWAS) on 267 lipid metabolites detected in 533 natural rice varieties. The present invention focuses on the content differences of the metabolite ω-hydroxy fatty acids in the population. The results of genome-wide association analysis show that ω-hydroxy fatty acids, fatty acids and rice plant yield are co-localized to the position of 3.6Mb on rice chromosome 3. ω-Hydroxy fatty acids belong to cutin monomers, and the synthesis steps of cutin monomers mainly include fatty acid acyl activation, fatty acid ω hydroxylation, dicarboxylic acid oxidation reaction, acylation, and then transported from the endoplasmic reticulum through the cell membrane to the cell wall cuticle by adenosine triphosphate binding cassette transporters and lipid transporters to play a role. According to the synthesis and transport steps of cutin monomers and combined with the gene annotation information of this region in the rice genome (rice genome database TIGR), the inventors determined the candidate genes involved in the synthesis and transport of ω-hydroxy fatty acids from the massive rice genome, namely the synthase fatty acid reductase gene OsFAR2, the 3-keto-coenzyme A synthetase gene OsKCS11, the glycerol 3-phosphate acyltransferase gene OsGPAT6, the cytochrome oxidase gene OsCYP704B2, the transporter ABC transporter encoding gene OsABCG22 and the lipid transporter encoding gene OsLTPL82. Further, the inventors calculated the relative positions of the genes and found that these genes responsible for synthesis were distributed within a range of 300kb, and all members responsible for synthesis and transport were distributed within a range of 620kb. According to the above content, these candidate genes belong to six different enzyme families, and this feature meets the definition of plant metabolic gene clusters. Therefore, the present invention named this metabolic regulation gene cluster as "hydroxymonoacylglycerol 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 OsGP AT6 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.14; the amino acid sequence of OsABCG22 protein is shown in SEQ ID NO.15; the OsLTPL82 gDNA sequence is shown in SEQ ID NO.16; the OsLTPL82 CDS sequence is shown in SEQ ID NO.17; and the amino acid sequence of OsLTPL82 protein is shown in SEQ ID NO.18.

[0041] The genes contained in the hydroxy monoacylglycerol metabolism gene cluster separated by the present invention include OsFAR2, OsKCS11, OsGPAT6, OsCYP704B2, OsABCG22 and OsLTPL82, wherein the four genes OsFAR2, OsKCS11, OsGPAT6 and OsCYP704B2 are responsible for the synthesis of hydroxy monoacylglycerol, which is mainly carried out in sequence in a head-to-tail manner in two reaction routes of OsFAR2-OsKCS11-OsCYP704B2-OsGPAT6 and OsFAR2-OsCYP704B2-OsKCS11-OsGPAT6. After the hydroxy monoacylglycerol is synthesized, it is transported outside the cell to form cutin under the action of two transport proteins, OsABCG22 and OsLTPL82. Gene editing technology was used to genetically transform the above six genes, and it was found that mutation of any gene in the hydroxymonoacylglycerol metabolism gene cluster would lead to a phenotype of reduced hydroxymonoacylglycerol content, male reproductive development defects in rice, and decreased yield per plant. Therefore, it is proved that the hydroxymonoacylglycerol metabolism gene cluster isolated by the present invention controls the male reproductive development of rice by regulating the content of hydroxymonoacylglycerol.

[0042] Example 1 Cloning and protein expression of the hydroxymonoacylglycerol gene cluster members OsFAR2, OsKCS11, OsGPAT6 and OsCYP704B2

[0043] Hydroxylated monoacylglycerol needs to rely on the four genes OsFAR2, OsKCS11, OsGPAT6 and OsCYP704B2 to be synthesized in sequence in a head-to-tail manner. When cloning the above genes and expressing them, the OsFAR2 recombinant protein was expressed using the Escherichia coli prokaryotic expression system. When analyzing the gene structures of OsKCS11, OsGPAT6 and OsCYP704B2, it was found that all three were membrane-localized proteins. Therefore, the yeast expression system was used to express the proteins of these three genes, as follows:

[0044] According to the location and structure of OsFAR2 gene, OsKCS11 gene, OsGPAT6 gene and OsCYP704B2 gene in the genome of japonica rice "Nipponbare" (Rice Genome Database TIGR), and the predicted full-length open reading frame sequences of these candidate genes, PCR specific primers with homologous recombination linkers (see Table 1, synthesized by Qingke Biotechnology Co., Ltd.) were designed and the full-length CDS fragments of the above four genes were amplified using ultra-fidelity KOD enzyme (Dalian Bao Biotechnology Co., Ltd.).

[0045] Table 1 Primers used in constructing protein expression vectors in the present invention

[0046]

[0047] Note: In the table Shown is the forward E. coli homologous recombination linker; Shown is the E. coli homologous recombination linker in reverse orientation; Shown is the forward yeast homologous recombination linker; Shown is the yeast homologous recombination junction in reverse orientation.

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

[0049] The cDNA of the target OsFAR2 gene (SEQ ID NO. 2) was recombined into the linearized Pjc767 / pGEX6p-1 expression vector (purchased from Invitrogen, USA) with a glutathione S-transferase (GST) tag using the ClonExpress MultiS one-step cloning kit (Nanjing Novigene). The recombinant expression vector was transformed into Escherichia coli BL21 (DE3) competent cells (Shanghai Weidi Biotechnology) for foreign protein expression. Single colonies were selected and cultured in LB medium containing ampicillin antibiotics until A 600nmThe expression of GST in the culture medium was 0.6-0.8, and then IPTG (isopropylthiogalactoside, Shanghai Biotechnology Co., Ltd.) was used at a final concentration of 0.1 mM for induction at 20°C for 16 hours. The cells were collected (6000g, 10 min) and lysed under high pressure. The GST-fused target protein was purified using glutathione sepharose 4B (GE Healthcare, USA) and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The present invention obtains a well-purified OsFAR2 recombinant protein fused with a GST tag protein, which is stored in a -80°C refrigerator for use.

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

[0051] The structure of OsKCS11, OsGPAT6 and OsCYP704B2 genes was analyzed, indicating that all three are membrane-localized proteins. The protein expression of the above 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 sequenced to obtain the recombinant vectors of OsKCS11, OsGPAT6 and OsCYP704B2, respectively. The constructed recombinant vector of OsGPAT6 was transformed into the yeast gat1Δ strain (purchased from EUROSCARF, Germany), and the recombinant vectors of OsKCS11 and OsCYP704B2 were transformed into the yeast WAT11 strain (purchased from EUROSCARF, Germany).

[0052] The construction method of the above-mentioned 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 by the yeast ADH1 promoter to obtain the PYES2 (ADH1) URA expression vector.

[0053] Yeast cultures were grown and microsomes were prepared as follows:

[0054] The recombinant yeast was first cultured in selenite cystine enrichment medium (SC medium) containing 2 wt% glucose at 30°C. After the 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 disrupted with glass beads (0.45 mm in diameter, Sigma, USA) in 50 mM Tris-HCl buffer (containing 1 mM EDTA and 600 mM sorbitol), pH 7.5. The cells were crushed using a mixing mill (model MM400, Retsch, Germany) to obtain a homogenate, which was centrifuged at 12,000 g for 60 min to obtain a supernatant, which was then centrifuged at 120,000 g for 90 min to obtain a precipitate, i.e., microsomal protein (microspheres composed of microsomal membranes), which was finally resuspended in 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 20% (v / v) glycerol, and stored in aliquots at -80°C for use.

[0055] Microsomes are small vesicles that reclose themselves after the membrane structure of the endoplasmic reticulum breaks down when cells are homogenized. In in vitro experiments, they have the basic functions of the endoplasmic reticulum, such as protein synthesis, protein glycosylation, and lipid synthesis. Microsomes are defined in cell biology as small vesicles obtained from fragments of the endoplasmic reticulum.

[0056] Example 2 Regulation of hydroxymonoacylglycerol by gene cluster

[0057] The four genes of the hydroxymonoacylglycerol gene cluster, OsFAR2, OsKCS11, OsGPAT6, and OsCYP704B2, are responsible for the synthesis of hydroxymonoacylglycerol, mainly in the two reaction routes of OsFAR2-OsKCS11-OsCYP704B2-OsGPAT6 and OsFAR2-OsCYP704B2-OsKCS11-OsGPAT6, which are connected head to tail, thereby regulating the synthesis of hydroxymonoacylglycerol. The following in vitro enzyme activity experiment will be used to prove:

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

[0059] In order to verify the function of the candidate gene OsFAR2, the present invention uses the purified OsFAR2 recombinant protein of Example 2 for in vitro enzyme activity determination. The in vitro enzyme activity reaction system of OsFAR2 recombinant protein is 100 μL, with a final concentration of 50 μmol / L palmitoyl carrier protein (C16:0ACP, Sigma, USA) and palmitoyl coenzyme A (C16:0CoA, Sigma, USA) as substrates, 500 ng purified OsFAR2-GST recombinant protein (GST tag protein is set as a negative control at the same time) and 100 mmol / LTris-HCl buffer (pH7.5) are added, and after incubation at 37°C for 30 min, 50 μL of pre-cooled methanol is added to terminate the reaction. The reaction mixture is centrifuged at 4°C 13000 rpm for 10 min, the supernatant is filtered through a 0.22 μM filter (Shanghai Anpu Company), and then LC-MS (LCMS-8060, Shimadzu Corporation, Japan) analysis is performed.

[0060] The detection results showed that the reaction product had an obvious chromatographic peak, and its retention time was the same as that of the standard compound palmitol (C16:0alcohol, Sigma, USA), indicating that OsFAR2 protein can catalyze C16:0ACP or C16:0CoA to produce palmitol ( Figure 2 (a) shows that OsFAR2 protein produces palmitol as the first step of the reaction.

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

[0062] In order to verify the function of the candidate gene OsKCS11, the present invention uses the OsKCS11 microsomal protein prepared in Example 1 for in vitro enzyme activity determination. The in vitro enzyme activity reaction system of OsKCS11 microsomal protein is 100 μL, with a final concentration of 200 μmol / L palmitic acid (C16:0, Sigma, USA) and 16-hydroxypalmitic acid [C16:0 (ω-OH), Sigma, USA] as substrates, 1 mg microsomal protein, 100 mmol / L Bis-Tris-propane buffer (pH 7.6), 10 mM MgCl2, 5 mM adenine nucleoside triphosphate (ATP), 2.5 mM dithiothreitol (DTT) and 1 mM coenzyme A (CoA), incubated in a shaking incubator (120 rpm) at 30°C for 4 hours, and then 50 μL of pre-cooled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 4°C, 13,000 rpm for 10 min to obtain the supernatant, which 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 ultraviolet absorbance at 280 nm on a spectrophotometer.

[0063] The detection results showed that the reaction product had an obvious chromatographic peak and its retention time was the same as that of palmitoyl-CoA standard and 16-hydroxypalmitoyl-CoA (C16:0(ω-OH)CoA, Sigma, USA) standard, indicating that OsKCS11 protein catalyzed C16:0 or C16:0(ω-OH) to generate C16:0CoA or C16:0(ω-OH)CoA( Figure 2 (b)

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

[0065] In order to verify the function of the candidate gene OsCYP704B2, the present invention uses the OsCYP704B2 microsomal protein prepared in Example 1 for in vitro enzyme activity determination. The in vitro enzyme activity reaction system of OsCYP704B2 microsomal protein is 100 μL, with a final concentration of 200 μmol / L palmitic acid and palmitoyl-CoA as substrates, 1 mg microsomal protein, 20 mmol / L Tris-HCl buffer (pH 7.5) and 500 mM reduced coenzyme II (NADPH), incubated in a shaking incubator (120 rpm) at 30°C for 4 hours, and then 50 μL of pre-cooled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 4°C and 13000 rpm for 10 min to obtain a supernatant, and then the supernatant was filtered through a 0.22 μM filter (Shanghai Anpu Company), and then 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 ultraviolet absorbance at 280 nm on a spectrophotometer.

[0066] The detection results showed that the reaction product had an obvious chromatographic peak and its retention time was the same as that of the 16-hydroxypalmitic acid (C16:0(ω-OH), Sigma, USA) standard and the 16-hydroxypalmitic acid coenzyme A standard, indicating that the OsCYP704B2 protein catalyzed C16:0 or C16:0CoA to generate C16:0(ω-OH) or C16:0(ω-OH)CoA( Figure 2 (c).

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

[0068] In order to verify the function of the candidate gene OsGPAT6, the present invention uses the OsGPAT6 microsomal protein prepared in Example 1 to perform in vitro enzyme activity determination. The in vitro enzyme activity reaction system of OsGPAT6 microsomal protein is 100 μL, with palmitoyl-CoA and 16-hydroxypalmitoyl-CoA at a final concentration of 200 μmol / L as substrates, 1 mg of microsomal protein, 20 mmol / L Tris-HCl buffer (pH 7.0), 400 μM 3-phosphoglycerol (G3P), 1 mM dithiothreitol, 5 mM ethylenediaminetetraacetic acid (EDTA) and 2.5 mg / mL bovine serum albumin (BSA), incubated in a shaking incubator (120 rpm) at 30° C. for 4 hours, and then 50 μL of pre-cooled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 4°C, 13,000 rpm for 10 min to obtain the supernatant, which was then filtered through a 0.22 μM filter (Shanghai Anpu Company) and then 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 ultraviolet absorbance at 280 nm on a spectrophotometer.

[0069] The detection results showed that the reaction product had an obvious chromatographic peak, and its retention time was the same as that of the standard 2-monopalmitoylglycerol (MG 16:0, Sigma, USA) and 16-hydroxy-2-monopalmitoylglycerol (HMG16:0, Tianjin Alta Company), indicating that OsGPAT6 protein catalyzed C16:0 or C16:0CoA to generate C16:0(ω-OH) or C16:0(ω-OH)CoA( Figure 2 (d)

[0070] The above results indicate that OsFAR2, OsKCS11, OsCYP704B2 and OsGPAT6 proteins have the function of continuously catalyzing metabolic reactions. At the same time, two synthetic pathways for the biosynthesis of hydroxymonoacylglycerol metabolites (HMGs), namely OsFAR2-OsCYP704B2-OsKCS11-OsGPAT6 and OsFAR2-OsKCS11-OsCYP704B2-OsGPAT6, were demonstrated to form a metabolic network ( Figure 2 (e)

[0071] Example 3 Functional study of the hydroxymonoacylglycerol gene cluster transport member OsABCG22 gene and OsLTPL82 gene

[0072] After the four genes OsFAR2, OsKCS11, OsGPAT6 and OsCYP704B2 are used to synthesize hydroxy monoacylglycerol, it is then 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 play a role. Subcellular localization and tobacco protoplast transport experiments have proven that the gene cluster transport members OsABCG22 and OsLTPL82 separated by the present invention have the function of transporting hydroxy monoacylglycerol.

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

[0074] According to the location and structure of OsABCG22 and OsLTPL82 genes in the genome of japonica rice "Nipponbare" (Rice Genome Database TIGR), and the predicted full-length open reading frame sequences of these candidate genes, PCR specific primers with homologous recombination linkers were designed (Table 2, synthesized by Qingke Biotechnology Co., Ltd.), and the full-length CDS fragments of the above two genes were amplified using ultra-fidelity KOD enzyme (Dalian Bao Biotechnology Co., Ltd.).

[0075] Table 2 Primers used in constructing tobacco expression vectors in the present invention

[0076]

[0077] Note: In the table Shown is the homologous recombination linker of the forward tobacco expression vector; Shown is the homologous recombination junction of the tobacco expression vector in reverse orientation.

[0078] The PCR products of the CDS sequences of the two genes were cloned into the target overexpression vector pH7WGF2 (the vector was purchased from the BioVector plasmid vector strain cell protein antibody gene storage center) to produce 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 mediated by Agrobacterium. After 2 days of transformation, the fluorescence signal was observed using a confocal laser scanning microscope (FV1000, Olympus, Japan).

[0079] Subcellular localization showed that OsLTPL82 and OsABCG22 were mainly localized on the cell membrane ( Figure 3 ), indicating that OsABCG22 and OsLTPL82 genes may have transport functions.

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

[0081] In order to verify the transport activity of the candidate genes OsABCG22 and OsLTPL82, the present invention cloned the PCR products of the CDR sequences of the OsABCG22 and OsLTPL82 genes into the tobacco transient expression vector pEAQ-HT-DEST2 (from the laboratory of Professor George P. Lomonossoff of the John Innes Centre Institute in the United Kingdom), and obtained the 35S:OsABCG22 and 35S:OsLTPL82 vectors after sequencing verification. The constructed 35S:OsABCG22 and 35S:OsLTPL82 vectors were transformed into Agrobacterium, and then the 4-week-old tobacco leaves were infected, and protoplasts were prepared 4 days later.

[0082] The present invention uses OsCYP704B2 protein and commercially available palmitic acid-1-( 13 C)([ 13 C]-C16:0, Sigma Company, USA) was used for biochemical reaction to generate [ 13 C]-C16:0(ω-OH) stable isotope products were prepared by using OsKCS11, OsCYP704B2 and OsG PAT6 proteins to react with commercially available palmitic acid-1-( 13 C)([13C]-C16:0) undergoes biochemical reaction to generate [ 13 C]-HMG16:0 stable isotope product. Add [ 13 C]-HMG16:0, [ 13 C]-C16:0(ω-OH), [ 13 C]-C16:0 stable isotope was used as the experimental group; [ 13 C4]-thiamine (IsoSciences, USA) stable isotope substrate was used as a control group, and its final concentration was 1mmol / L. The mixture was incubated on ice for 15 minutes to allow for the uptake of the substrate. Protoplasts were separated by a gradient of 50%, 25% and 5% Percoll separation solution (Cytiva, Sweden) from bottom to top to remove external metabolites and recover protoplasts from the 25% and 5% Percoll interface. Protoplasts were incubated at 25°C and 100μL were sampled at 0, 5, 10, 15 and 20min respectively. After centrifugation, the supernatant was taken for metabolite detection. The relative output was calculated based on the metabolite content normalized to the first time point (0min). The experiment was repeated three times, and the data are expressed as the standard error of the mean of all technical replicates.

[0083] The results showed that the initial efflux rates of tobacco cells expressing OsABCG22 and OsLTPL82 genes were both over 40% ( Figure 4 This indicates that the export of HMG16:0 has been significantly enhanced. 13 C4]-thiamine, no significant changes in transport activity were observed ( Figure 4 b), which indicates that both OsABCG22 and OsLTPL82 have HMGs transport activity. These results indicate that OsABCG22 and OsLTPL82 are membrane-localized transport proteins for hydroxy monoacylglycerol metabolites of cutin monomers. In order to further study the specificity of OsABCG22 and OsLTPL82 proteins, the present invention used [ 13 The results showed that the expression of OsABCG22 and OsLTPL82 proteins significantly reduced the net import of labeled C16:0(ω-OH) in the control group, and the initial efflux rate decreased from -29% to -19%, indicating that both proteins have the activity of ω-hydroxy fatty acid export. Figure 4 Similarly, compared with the control, the present invention observed that tobacco cells expressing OsLTPL82 had an effect on [ 13 C]-C16:0 efflux activity was enhanced, with the initial efflux rate increasing from 6% to 15% of the control, but OsABCG22 had no significant effect on [ 13 There was no significant change in the export activity of C]-C16:0 ( 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 hydroxymonoacylglycerol and ω-hydroxy fatty acids.

[0084] In summary, the hydroxymonoacylglycerol gene cluster of the present invention includes the synthesis genes OsFAR2, OsKCS11, OsGPAT6, OsCYP704B2 and the transport genes OsABCG22 and OsLTPL82. Based on the experimental results, a working model of the hydroxymonoacylglycerol gene cluster in cutin monomer metabolism was proposed ( Figure 5 ). Fatty acyl carrier protein is first reduced to fatty alcohols by OsFAR2 in the plastid, which in turn produces fatty acids by an unknown protein and then diffuses to the endoplasmic reticulum. Subsequently, hydroxymonoacylglycerol can be produced in the endoplasmic reticulum through either of the two major synthetic pathways, OsCYP704B2-OsKCS11-OsGPAT6 or OsKCS11-OsCYP704B2-OsGPAT6. Hydroxymonoacylglycerol is then transported to the exoplasmic epidermis of the cell wall by OsABCG22 and OsLTPL82, where it is deposited to form cutin.

[0085] Example 4 Effects of Hydroxymonoacylglycerol Gene Cluster Members (OsFAR2, OsKCS11, OsGPAT6, OsCYP704B2, OsABCG22 and OsLTPL82) on Male Reproductive Development and Yield in Rice

[0086] In order to verify the effect of the gene cluster of the present invention on the reproductive development and yield of rice, this example first constructed transgenic mutant materials and then tested the effect of the mutant materials on rice-related phenotypes.

[0087] 1. Construction of transgenic mutant materials

[0088] Construction of osfar2, oskcs11, osgpat6, oscyp704b2, osabcg22 and osltpl82 mutants by CRISPR-Cas9 technology: According to the NCBI database information, the full-length cDNA and genomic sequences of OsFAR2, OsKCS11, OsCYP704B2, OsABCG22 and OsLTPL82 were downloaded and sequenced to find exons. The exon sequences were used to design space amplification primers for CRISPR vectors on the NEB cutter website (http: / / nc2.neb.com / NEBcutter2 / ), and the template was PJE044 (SgRNA) (this vector was modified by the inventor's laboratory, carrying sgRNA and U3 promoter, and can be used for the amplification of CRISPR elements). The amplified fragment was connected to pH-Ubi-cas9-7 (this vector carries the maize ubiquitin gene promoter and cas9 protein with constitutive and overexpression characteristics, can be used for Agrobacterium-mediated gene knockout genetic transformation, and has been disclosed in the document "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. Peer J. 2020 Jan 29; 8: e8491. doi: 10.7717 / peerj.8491. PMID: 32030327; PMCID: PMC6995270") through the Gateway system.

[0089] The generated OsFAR2, OsKCS11, OsCYP704B2, OsABCG22 and OsLTPL82 vectors were introduced into the callus tissue of Zhonghua 11 (ZH11) and the OsGPAT6 vector was introduced into the callus tissue of Minghui 63 (MH63) by Agrobacterium-mediated transformation. After seed propagation, DNA positive identification and gene transcription level expression identification, stable genetically modified materials ( Figure 6 ).

[0090] 2. Phenotypic Analysis of Transgenic Mutant Materials

[0091] In order to further verify the function of candidate genes in regulating cutin monomer metabolites in rice, the present invention selected T2 generation plants of os far2, oskcs11, osgpat6, oscyp704b2, osabcg22 and osltpl82 mutants, respectively, and used 0.05% (w / v) toluidine blue reagent (Sigma, USA) to stain the anthers of the above mutants. The results showed that compared with the wild type ZH11 or MH63, osfar2, oskcs11, osgpat6, oscyp704b2, osabcg22 and osltpl82 mutants were stained darker by toluidine blue dye, showing dark blue ( Figure 7 ). This indicates that mutations in the hydroxymonoacylglycerol gene cluster members all cause different degrees of damage to the anther cuticle barrier, playing an important role in the biosynthesis of the anther cuticle.

[0092] The cutin monomer metabolites in rice anthers, including fatty acids, hydroxy fatty acids and hydroxy monoacylglycerol, were further detected and analyzed by LC-MS. The test results showed that compared with the wild-type material, the contents of hydroxy monoacylglycerol metabolites such as HMG16:0, HMG16:1, HMG18:0, HMG18:1, HMG18:2 in the anthers of the hydroxy monoacylglycerol gene cluster members 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 were 1.2e10 5 , 6.7e10 5 , 1.2e10 6 , 4.7e10 5 , 2.5e10 4 , and the relative contents in the osgpat6 mutant were 8.7e10 4 , 4.9e10 5 , 8.7e105 , 3.4e10 5 , 1.6e10 4 , all of which were significantly reduced. This experimental result proves that the gene cluster of the present invention has the function of regulating the content of hydroxymonoacylglycerol.

[0093] In order to study the physiological function of the hydroxymonoacylglycerol gene cluster, the phenotype of the mutant plants was observed, and the results showed that the floral organ development of all mutant plants was abnormal ( Fig. 9 and Fig.10 Compared with the wild type, the mutant plants were smaller and had pale anthers ( Fig. 9 a, d, g and j, Fig.10 a and d), and showed a significant reduction in viable mature pollen grains ( Fig. 9 b, e, h and k, Fig.10 b and e). The experimental results show that mutation of any member of the gene cluster of the present invention will lead to abnormal development of floral organs of the plant, verifying that the gene cluster of the present invention has the function of regulating male reproductive development of rice.

[0094] Further statistical analysis showed that the yield per plant of mutant plants of hydroxymonoacylglycerol gene cluster members was significantly reduced compared with the wild type, usually with a decrease of 30% to 62% ( Fig. 9 c, f, i and l, Fig.10 In c and f), taking osgpat6 as an example, the yield per plant of wild-type material MH63 is 32 g, while the yields of the three osgpat6 mutant families are 20 g, 14 g, and 19 g, respectively, which are decreased by 39% to 58%. This indicates that the gene cluster of the present invention can regulate rice yield by regulating anther fertility.

[0095] Combined with the above results, it is shown that mutation of any member of the hydroxymonoacylglycerol gene cluster of the present invention will lead to changes in the composition and content of cutin monomers, abnormal development of anthers and pollen grains, and thus significantly reduce yield. This lays a foundation for using this gene cluster to genetically improve rice fertility, and also provides technical reference for the mining of related metabolic gene clusters in other crops and in breeding. It can also be used as a molecular marker to screen rice plants with normal reproductive development.

[0096] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene for regulating the metabolism of hydroxymonoacylglycerol in rice, characterized in that: The gene is OsABCG22 gene or 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 gene according to claim 1, characterized in that The protein encoded by the OsABCG22 gene has a transport function of transporting ω-hydroxy 2-monoacylglycerol or ω-hydroxy fatty acid from the endoplasmic reticulum to the apoplast; The protein encoded by the OsLTPL82 gene has the function of transporting ω-hydroxy 2-monoacylglycerol, ω-hydroxy fatty acids or medium-chain fatty acids from the endoplasmic reticulum to the apoplast.

3. Use of the gene as claimed in claim 1 in regulating the metabolism of hydroxymonoacylglycerol in rice.

4. Use of the gene as claimed in claim 1 in regulating the male development function of rice.

5. Use of the gene as claimed in claim 1 in regulating rice yield.

6. A gene knockout vector of OsABCG22 gene or 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.

7. The gene knockout vector according to claim 6, characterized in that The gene knockout vector is a CRISPR-Cas9 gene knockout vector.

8. A host cell, characterized in that Comprising the gene knockout vector according to claim 6 or 7.

9. Use of the gene knockout vector according to claim 6 or 7 or the host cell according to claim 8 in constructing the rice plant described in (1) or (2) below: (1) Rice plants with low synthesis of hydroxymonoacylglycerol; (2) Rice plants with male reproductive development defects.

10. A method for constructing a rice transgenic plant with male reproductive development defects, characterized in that: The method comprises the steps 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