Application of transcription factor OsEPR1 as a negative regulator in increasing the content of volatile metabolites in rice

Abstract

The present invention relates to the field of plant biotechnology, and discloses the use of transcription factor OsEPR1 as a negative regulatory factor in increasing the content of volatile metabolites in rice, wherein the amino acid sequence of the transcription factor OsEPR1 is shown in SEQ ID NO. 41. The present invention significantly reduces the content of multiple volatile metabolites in rice grains by overexpressing the OsEPR1 gene driven by a constitutive promoter in rice, including but not limited to volatile metabolites in the lipoxygenase pathway; indicating that OsEPR1, as a negative regulatory factor, inhibits the synthesis of multiple volatile metabolites including the rice lipoxygenase pathway, and therefore the synthesis of multiple volatile metabolites in rice can be increased by knocking out the OsEPR1 gene in rice, and can be applied to the research on improving the aroma quality of rice.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to application of transcription factor OsEPR1 as a negative regulatory factor in increasing the content of volatile metabolites in rice. Background Art

[0002] Plants produce a large number of low molecular weight, lipophilic molecules with high vapor pressure, which are called Volatile Organic Compounds (VOCs). These chemically and biologically diverse volatile metabolites contribute to the interaction between plants and the environment by acting as aroma and flavor molecules through interactions with human receptors and insect receptors. They are of commercial importance in the food and agricultural industries as a resource for olfactory pleasure, flavoring agents, and medicines. For example, β-ionone is a key aroma in food and beverages and is often added for its pleasant floral aroma, which is sensed by the human olfactory receptor OR5A1. Cis-3-hexen-1-ol is a key flavor in many fresh fruits and vegetables and is widely used in processed foods as an added flavor, providing a fresh grassy aroma, which is sensed by the olfactory receptor OR2J3. Rice is an important food crop in the world. Rice aroma, as one of the important quality characteristics and value-added indicators of rice, not only has nutritious edible significance, but also can bring rich economic value. However, for a long time, the goal of domestic rice breeding has been mainly to achieve important agronomic traits such as high yield and disease resistance, while less attention has been paid to the excellent quality of rice, especially to aroma substances and aroma genes, resulting in slow progress in rice aroma research. Therefore, breeding aromatic rice varieties with strong aroma and good quality is of great significance to improving the economic income of Chinese farmers and the international competitiveness of rice.

[0003] Fragrant rice types include indica, japonica and glutinous rice, all of which have a special fragrance, are rich in a variety of essential nutrients for the human body, and have nourishing and medicinal effects. Its fragrance can eliminate fatigue and help increase appetite, and it enjoys a special status in the international market, such as India's "Basmati", the United States' "Oella", Cambodia's "Jasmine Fragrant Rice", Yunnan's "Ivory Fragrant Rice", "Babao Fragrant" in my country, Guizhou's "Xianghe", Guangdong's "Wanjiaxiang", and are known as "one family cooks and ten thousand families smell the fragrance". Therefore, fragrant rice has always been regarded as a precious rice variety in my country and is sold as high-end rice in the modern market. In the international market, fragrant rice is also one of the key factors affecting global rice trading. Studies have shown that more than 100 volatile metabolites have been detected in cooked fragrant rice, among which 2-acetyl-1-pyrroline (2-AP) can significantly change the aroma of rice even at low concentrations due to its low odor threshold, and 2-AP is considered to be the main compound responsible for its popcorn aroma. To date, research on the mechanism of rice aroma has mainly focused on betaine aldehyde dehydrogenase 2 (OsBADH2). Many mutations within the OsBADH2 gene can transform non-fragrant rice into fragrant varieties. For example, there is a 2 bp deletion in exon 1, a 7 bp deletion in exon 2, an 803 bp deletion between exons 4 and 5, an 8 bp deletion and 3 single nucleotide polymorphisms (SNPs) in exon 7, a 7 bp insertion in exon 8, 1 SNP each in exon 10 and exon 13, and 1 bp insertion / deletion / SNP in exon 14. All of these mutations lead to a reduction or loss of OsBADH2 activity, preventing the conversion of γ-aminobutyraldehyde to γ-aminobutyric acid (GABA), thereby promoting the accumulation of 2-AP. Recent studies have shown that a 22 bp deletion in the coding region of ornithine decarboxylase (OsODC) leads to an increase in 2-AP content in rice, thereby identifying OsODC as a rice aroma enhancer. In addition to 2-AP, many fatty acid-derived volatile compounds (FAVs) have also been detected in rice. FAVs include straight-chain aldehydes, alcohols, esters, lactones and ketones, which are mainly synthesized through the lipoxygenase (LOX) pathway. In the lipoxygenase (LOX) pathway, linoleic acid and linolenic acid are converted into hydroperoxide isomers, which are then cleaved by hydroperoxide lyases and then reduced to the corresponding alcohols by alcohol dehydrogenases (ADH). Finally, alcohol acyltransferases promote the esterification reaction of these alcohols with acyl groups to form esters. Rice volatile metabolites are closely related to aroma quality and resistance, but the specific molecular mechanism of their regulation is not clear, and the regulatory network formed has always been one of the hot and difficult points of research at home and abroad.Therefore, studying the application of OsEPR1, a transcription factor involved in negatively regulating the synthesis of rice volatile metabolites, is of great significance for improving the aroma quality of rice. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide an application of a transcription factor OsEPR1 as a negative regulatory factor in increasing the content of volatile metabolites in rice; a second object of the present invention is to provide a method for increasing the content of volatile metabolites in rice.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention discloses an application of transcription factor OsEPR1 as a negative regulatory factor in increasing the content of volatile metabolites in rice. The amino acid sequence of the transcription factor OsEPR1 is shown in SEQ ID NO.41.

[0007] Preferably, in the present invention, the nucleotide sequence of the transcription factor OsEPR1 is shown as SEQ ID NO.40.

[0008] Preferably, the volatile metabolite of the present invention is a volatile metabolite of the lipoxygenase pathway.

[0009] Preferably, the volatile metabolites are: vinyl acetate (Acetic acid ethenylester), 2,3-butanediol (2,3-Butanediol), hexanal (Hexanal), butyl acetate (Acetic acid, butylester), 2,2,5-trimethylhexane (Hexane, 2,3,5-trimethyl-), 4-methyloctane (Octane, 4-methyl-), 1-hexanol (1-Hexanol), heptane (Heptanal), alpha-Thujene, trans-2-hexene-4-oxo-aldehyde (4-Oxohex-2-enal), 3-ethyl-3-methylheptane (Heptane, 3-ethyl-), 2,3-dimethyloctane (Octane, 2,3-dimethyl-), isopentyl propionate (1-Butanol, 3-methyl-, propanoate), 1-octen-3-ol, 1-octen-3-one, 2-pentylfuran, 6-Methylhept-5-en-2-ol, Octanal, alpha-Terpinene, 2-Hexen-1-ol acetate, (E)-, 4-methyl-decane, 1-Hexanol, 2-ethylhexanol, 2-ethyl-), gamma-Terpinene, 1-Octanol, 3,5-Octadien-2-one, Undecane, Isophorone, trans-Isopiperitenol, Decanal, trans-Carveol, Perilla Alcohol, Cyclosativene, Vanillion, Dihydro-beta-ionone, trans-Geranylacetone, Caryophyllene, (5R, 10R, E)-2-ethylidene-10-methyl-6-methylene[4.5] One or more of β-Vetispirene, cis-3-Hexenal, Heptadecane, and Hexadecanoic acid, methyl ester. .

[0010] A method for increasing the content of volatile metabolites in rice comprises: reducing or knocking out the expression of transcription factor OsEPR1 in rice, wherein the amino acid sequence of the transcription factor OsEPR1 is shown in SEQ ID NO.41.

[0011] Preferably, in the present invention, the nucleotide sequence of the transcription factor OsEPR1 is shown as SEQ ID NO.40.

[0012] Preferably, the CRISPR / Cas9 gene editing system is used to knock out the transcription factor OsEPR1.

[0013] More preferably, the sgRNA target sequence of the CRISPR / Cas9 gene editing system is shown as SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11.

[0014] More preferably, the amplification primers for amplifying the OsEPR1 editing sequence by the CRISPR / Cas9 gene editing system are shown as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.19.

[0015] More preferably, the method of knocking out the transcription factor OsEPR1 using the CRISPR / Cas9 gene editing system further comprises: connecting the amplified product into pHUE411 to obtain a plant transformation plasmid containing the OsEPR1 gene editing function of CRISPR / Cas9, and obtaining a recombinant plasmid is the knockout vector.

[0016] The beneficial effects of the present invention are:

[0017] The present invention discloses the application of rice transcription factor OsEPR1 in negatively regulating the content of rice volatile metabolites. Studies have found that OsEPR1 inhibits the synthesis of volatile metabolites by inhibiting the expression of OsLOXs genes in the rice LOX pathway. Knocking out the gene can significantly increase the content of volatile metabolites, indicating that the OsEPR1 transcription factor has important guiding significance and broad market prospects for improving the aroma quality of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0019] Figure 1 The expression of OsEPR1 gene in rice leaves of OsEPR1 overexpressing lines was detected by real-time fluorescence quantitative PCR. The heat map of the content of volatile metabolites in leaves of OsEPR1 overexpressing lines was determined by gas chromatography-mass spectrometry (GC-MS).

[0020] Figure 2 Real-time fluorescence quantitative PCR was used to detect the expression of OsEPR1 in rice leaves. OsLOX1 , OsLOX11 , OsLOX12 and OsLOX13 The expression of genes.

[0021] Figure 3 for OsLOX1 , OsLOX11 , OsLOX12 and OsLOX13 Promoter analysis.

[0022] Figure 4 To verify OsEPR1 binding by dual-luciferase reporter assay (Dual-LUC) proOsLOX1 , proOsLOX12 , thereby inhibiting OsLOX1 , OsLOX12 expression (A: Dual-LUC pattern diagram; B / C: Dual-LUC verification results).

[0023] Figure 5 To verify OsEPR1 binding by yeast one-hybrid assay (Y1H) proOsLOX1 , proOsLOX12 domain. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0025] Example 1. Gene cloning of rice OsEPR1

[0026] (1) Extraction of total RNA from rice leaves

[0027] Fresh rice leaves were taken, quickly frozen in liquid nitrogen, and fully ground; 0.1 g of powder was added to 1 ml of Trizol, and total RNA was extracted according to the kit method of Quanshijin Company. The RNA quality was detected by agarose gel electrophoresis, and the RNA concentration was detected by NanoDrop2000 spectrophotometer.

[0028] (2) Cloning of rice genes

[0029] The extracted total RNA was used as a template to synthesize cDNA using the one-step reverse transcription kit of Beijing Quanshijin Company; PCR amplification primers were designed according to the OsEPR1 gene sequence, and the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, see Table 1.

[0030] Table 1. Cloning primers for rice OsEPR1 gene

[0031]

[0032] The obtained cDNA was used as a template for PCR amplification, and the procedure was: 94℃ pre-denaturation for 5min, 94℃ denaturation for 15s, 58℃ annealing for 20s, 72℃ extension for 1min, repeated 32 cycles; 72℃ final extension for 5min; stored at 25℃. PCR amplification was performed with the OsEPR1 gene primers in Table 1, and the products were recovered by gel excision and sequencing to obtain the rice OsEPR1 gene sequence. The sequencing results showed that the obtained OsEPR1 gene was 1356bp in length, and its nucleotide sequence was shown in SEQ ID NO.40.

[0033] (3) Cloning of rice promoter

[0034] OsLOX1 , OsLOX11 , OsLOX12 and OsLOX13 The promoter analysis is shown in Figure 3 . Refer to the CTAB extraction method of plant genome to extract rice leaf DNA. proOsLOX1 , proOsLOX12 PCR amplification primers were designed based on the 2000 bp sequence of the promoter. The primer sequences are shown in Table 2, such as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0035] Table 2. Rice proOsLOX1 , proOsLOX12 Amplification primers

[0036]

[0037] The obtained DNA was used as a template for PCR amplification, and the procedure was 94℃ pre-denaturation for 5min, 94℃ denaturation for 15s, 58℃ annealing for 20s, 72℃ extension for 2min, repeated 32 cycles; 72℃ final extension for 5min; stored at 25℃. The PCR amplification product was excised and recovered and sequenced to obtain rice proOsLOX1 , proOsLOX12 The sequencing results showed that the obtained proOsLOX1 , proOsLOX12 The total length of the promoter sequence is 2000 bp, and its sequence is shown in SEQ ID NO.42 and SEQ ID NO.43.

[0038] Example 2: Construction of plant overexpression vector

[0039] The construction of plant overexpression vectors was carried out using the Gateway system. First, the desired gene fragment was amplified using rice cDNA as a template. The primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, as shown in Table 3. The gel recovery product with the pDONR207 recombination linker was obtained by PCR. The BP (LR) enzyme, pH2GW7+pUbi (Smedley MA, Harwood WA. Gateway-compatible plant transformation vectors. Methods Mol Biol. 2015;1223:3-16.doi: 10.1007 / 978-1-4939-1695-5_1. PMID: 25300827.), and water were used to form a 2 µL system (see Table 4). The system was connected in a 25°C dry bath for 4 hours and transformed into DH5α. The final vector pH2GW7_OE was obtained after sequencing verification.

[0040] Table 3. Amplification primers for rice overexpression vector

[0041]

[0042] Table 4. BP and LR reaction system

[0043]

[0044] Example 3: Construction of vector for plant gene knockout

[0045] To construct a plant gene knockout vector, the gene editing target site was first designed using the online software CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2), and a three-target site strategy was adopted. A 50 μL PCR amplification system was used, and transformation and colony PCR identification were performed after restriction digestion and ligation. The target site sequences of OsEPR1 are shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11, as shown in Table 5. The OsEPR1 editing sequence was amplified using SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19, and then connected to pHUE411 (purchased from Addgene; catalog number: 62203) to obtain a plant transformation plasmid containing the OsEPR1 gene editing function of CRISPR / Cas9, and the recombinant plasmid was obtained as the knockout vector (the vector was constructed by Boyuan Biotechnology Co., Ltd.).

[0046] Table 5. Target site design information of OsEPR1

[0047]

[0048] Table 6. Primers for amplification of OsEPR1 editing sequences

[0049]

[0050] Example 4: Stable genetic transformation of rice mediated by Agrobacterium tumefaciens

[0051] The overexpression vector and knockout vector of OsEPR1 were introduced into Agrobacterium tumefaciens EHA105 for stable genetic transformation of rice. The specific steps are as follows:

[0052] Agrobacterium positive single clone identification: Take 1 μL of plasmid and add it to 50 μL of EHA105 Agrobacterium tumefaciens competent cells, mix well, add 1 mL of YEB liquid medium after electroporation, shake and culture for 30 min at 30°C and 180 rpm on a shaker, inoculate it into YEB solid medium, and culture it in the dark at 28°C for 48 h. After PCR identification, positive Agrobacterium single clones were obtained, and the identification primer sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, see Table 3 and Table 6.

[0053] Rice genetic transformation: Select complete, plump and uniform rice grains, disinfect with 70% alcohol for 2 minutes, wash with sterile water, disinfect with 30% sodium hypochlorite for 10 minutes, wash with sterile water, place in 1 / 2MS medium, and culture under light for 7-8 days. The grown callus is infecting with positive Agrobacterium bacteria solution for 25 minutes, placed on sterile filter paper to dry in the shade, spread on co-culture medium, and co-cultured in the dark at 25℃ for 2.5-3 days. The callus is evenly placed on the screening medium, screened and cultured in the dark at 25℃ for 2-3 weeks, transferred to the pre-differentiation medium, cultured at room temperature with 14h / 10h light and dark alternation for two weeks, and transferred to the differentiation medium. The rice callus that has grown to 2 leaves or 2 leaves and one heart is transferred to the rooting medium. When the rice seedlings grow to about 10cm, they are placed in clean water for 3-4 days of seedling hardening and moved into soil pots (the transgenic material is created by Baige Gene Technology Co., Ltd.).

[0054] The T0 generation positive plants were identified by PCR, and the identification primer sequences were shown in SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25 as shown in Table 7. The T1 generation positive plants were obtained by self-pollination for subsequent experiments and analysis of the content of related metabolites.

[0055] Table 7. Primers for PCR identification of positive plants

[0056]

[0057] Quantitative PCR was used to determine the expression of OsEPR1 and OsLOX1 , OsLOX11 , OsLOX12 and OsLOX13 The gene expression conditions and the detection primer sequences are shown in SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34 and SEQ ID NO.35, see Table 8.

[0058] Table 8. qPCR identification primers

[0059]

[0060] The results are as follows Figure 1 and Figure 2 As shown. The expression levels of OsEPR1-OE1, OsEPR1-OE10, and OsEPR1-2OE5 increased compared to the wild type (about 30 to 60 times), and OsLOX1 , OsLOX11 , OsLOX12 , OsLOX13 The gene expression levels decreased by different folds (about 0.2-0.8 times) in the OsEPR1-OE1, OsEPR1-OE10, and OsEPR1-OE25 strains.

[0061] Example 5: Determination of the content of volatile metabolites in rice leaves by gas chromatography-mass spectrometry (GC-MS)

[0062] Gas chromatography-mass spectrometry (GC-MS) can meet the requirements for accurate qualitative and quantitative determination of volatile metabolites. Its operation process is briefly as follows:

[0063] Sample preparation: Harvested mature kernels were dehulled and 300 μL ddHO was added. 2 O 1 g sample was extracted for GC-MS analysis.

[0064] Volatile metabolites were detected by gas chromatography system (7890AGC, Agilent Technologies) and triple quadrupole mass spectrometry system (Agilent 7000D triple quadrupole mass detector). HP-5 mass spectrometry capillary column (30 mm* 0.25 mm id, 0.25 mm film thickness; Agilent Technologies) was used to separate volatile metabolites. The initial column temperature was 50 °C, maintained for 2 min, the temperature was increased by 5 °C / min to 200 °C, maintained for 2 min, and then the temperature was increased by 20 °C / min to the final temperature of 250 °C, maintained for 2 min. The injection temperature was 230 °C, the splitless mode was used, and the He flow rate was 1.0 ml / min (99.999%). In full scan mode, the scan range was 35-500 m / z.

[0065] The results are as follows Figure 1The results showed that in the OsEPR1 overexpression transgenic line, 40 volatile metabolites including acetic acid ethenyl ester, 2,3-butanediol, hexanal, acetic acid, butyl ester, 2,2,5-trimethylhexane, 4-methyloctane, 1-hexanol, heptane, alpha-thujene, 4-oxohex-2-enal, 3-ethyl-3-methylheptane, 2,3-dimethyloctane, 1-butanol, 3-methyl-, propanoate), 1-octen-3-ol, 1-octen-3-one, 2-pentylfuran, 6-Methylhept-5-en-2-ol, Octanal, alpha-terpinene, 2-Hexen-1-ol acetate (E)-), 4-methyl-decane, 2-ethylhexanol (1-Hexanol, 2-ethyl-), gamma-Terpinene, 1-Octanol, 3,5-Octadien-2-one, Undecane, Isophorone, trans-Isopiperitenol, Decanal, trans-Carveol, Perilla Alcohol, Cyclosativene, Vanillion, Dihydro-beta-ionone, trans-Geranylacetone, Caryophyllene, (5R, 10R, E)-2-ethylidene-10-methyl-6-methylene[4.5] The levels of β-Vetispirene, cis-3-Hexenal, Heptadecane, and Hexadecanoic acid, methyl ester were significantly reduced compared with the wild type, indicating that OsEPR1 negatively regulates the synthesis of multiple volatile metabolites in rice grains.

[0066] Example 6: Interaction between OsEPR1 and proOsLOX1 and proOsLOX12 and verification of their action sites

[0067] The OsEPR1 cloned in Example 1 was connected to the vectors pGADT7 (Cat. No.: HG-VJC0483, Changsha Abiwei Biotechnology Co., Ltd.) and pB7WG2.0 (Dual-LUC) (Cat. No.: VT13019, Qingdao Cresbo Biotechnology Co., Ltd.) to obtain the recombinant vectors OsEPR1-pGADT7 (yeast single hybrid) and OsEPR1-pB7WG2.0 (Dual-LUC). proOsLOX1 The sequences were constructed into vectors pHIS2 (Cat. No.: HG-VJC0481, Changsha Abiwei Biotechnology Co., Ltd.) and pGEXT4-1 (Cat. No.: HG-VYA0221, Changsha Abiwei Biotechnology Co., Ltd.) to obtain proOsLOX1-pHIS2 and proOsLOX1-pGEXT4-1; the upstream 2000 bp proOsLOX12 The sequences were constructed into vectors pHIS2 and pGEXT4-1, respectively, to obtain proOsLOX12-pHIS2 and proOsLOX12-pGEXT4-1. The construction of the vectors refers to the Gateway system of Example 2. The interaction was verified using the mature Y1H and Dual-LUC experimental systems. The Y1H system uses the yeast competent system of Y187. The pGADT7 and pHIS2 containing the target gene can normally grow plaques on the three-deficient plates with 3-AT, which can determine the interaction between DNA and protein. Dual-LUC was verified in tobacco leaves, and the binding of DNA and protein was determined by fluorescent signals. The vector and primer sequences are shown in SEQ ID NO.36, SEQ ID NO.37, SEQID NO.38 and SEQ ID NO.39, see Table 9.

[0068] Table 9. Vector and primer information

[0069]

[0070] The results are as follows Figure 4 and Figure 5As shown, OsEPR1 can bind to OsLOX1 , OsLOX12 The promoter region of the gene significantly inhibits its expression.

[0071] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for increasing the content of volatile metabolites in rice, characterized in that: include: The expression of the transcription factor OsEPR1 in rice is reduced or knocked out, wherein the amino acid sequence of the transcription factor OsEPR1 is shown in SEQ ID NO.41, and the volatile metabolites are one or more of hexanal, 1-octen-3-ol, 1-octen-3-one, n-octanol, and cis-3-hexenal.

2. The method for increasing the content of volatile metabolites in rice according to claim 1, characterized in that: The nucleotide sequence of the transcription factor OsEPR1 is shown in SEQ ID NO.

40.

3. A method for increasing the content of volatile metabolites in rice according to claim 1 or 2, characterized in that: The transcription factor OsEPR1 was knocked out using the CRISPR / Cas9 gene editing system.

4. The method for increasing the content of volatile metabolites in rice according to claim 3, characterized in that: The sgRNA target sequences of the CRISPR / Cas9 gene editing system are shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.

11.

5. The method for increasing the content of volatile metabolites in rice according to claim 4, characterized in that: The amplification primers for amplifying the OsEPR1 editing sequence by the CRISPR / Cas9 gene editing system are shown as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.

19.

6. The method for increasing the content of volatile metabolites in rice according to claim 5, characterized in that: Using the CRISPR / Cas9 gene editing system to knock out the transcription factor OsEPR1 also includes: connecting the amplified product into pHUE411 to obtain a plant transformation plasmid containing the OsEPR1 gene editing function of CRISPR / Cas9, and obtaining an OsEPR1 gene knockout vector.

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