Application of osSMO1-2 gene in regulating content of cholesterol in rice

Abstract

The present application relates to plant cholesterol synthesis, and particularly relates to application of OsSMO1-2 gene in regulating rice cholesterol content. A method for regulating rice cholesterol content is provided, which comprises: increasing content or activity of OsSMO1-2 protein in rice to increase cholesterol content in rice, or reducing content or activity of OsSMO1-2 protein in rice to reduce cholesterol content in rice; the amino acid sequence of the OsSMO1-2 protein is shown as SEQ ID NO:2. The rice with increased or reduced cholesterol content obtained by the method has important application value in cultivating new rice varieties, and the rice with increased cholesterol content can also be used as a plant chassis for mass production of downstream metabolites of cholesterol.

Description

Technical Field

[0001] This invention relates to plant cholesterol synthesis, and more particularly to the application of the OsSMO1-2 gene in regulating cholesterol content in rice. Background Technology

[0002] Sterols are a unique class of lipid metabolites found in all eukaryotes, playing a crucial role in regulating membrane fluidity and transmembrane transport. The main sterols differ significantly among different biological groups. In animals, the most typical sterol is cholesterol, which regulates membrane fluidity and is a precursor to bile acids, vitamin D, and various hormones. Fungi primarily produce ergosterol, which functions similarly to cholesterol. Plants synthesize a variety of sterols, with phytosterols being the most abundant, mainly including campesterol, β-sitosterol, and stigmasterol, which play similar roles in plant cell membranes. Plants also synthesize cholesterol, but in extremely low amounts.

[0003] The biosynthesis of sterols is a complex metabolic process involving multiple enzymatic reactions. The biosynthesis of phytosterols can be divided into three stages. The first stage begins with the synthesis of acetyl-CoA, ultimately producing cycloartenol. The second stage converts cycloartenol to 24-methylenecholest-7-enol. The third stage converts 24-methylenecholest-7-enol to campesterol, β-sitosterol, and stigmasterol. The biosynthesis of phytosterols and cholesterol shares some common precursors and multiple biosynthetic enzymes. Cycloartenol is a key metabolic node, directing some metabolic flux into the phytosterol synthesis pathway and others into the cholesterol synthesis pathway. Plants use cholesterol as a precursor to synthesize brassinolide, steroidal saponins, steroidal alkaloids, or other sterol derivatives. Therefore, plants with high cholesterol content have significant application value in the biosynthesis of downstream cholesterol metabolites. Summary of the Invention

[0004] The purpose of this invention is to provide a method for regulating the cholesterol content in rice, and to use this method to create rice with increased or decreased cholesterol content.

[0005] This invention provides a method for regulating cholesterol content in rice, comprising: increasing the content or activity of OsSMO1-2 protein in rice to increase cholesterol content in rice, or decreasing the content or activity of OsSMO1-2 protein in rice to decrease cholesterol content in rice; the amino acid sequence of the OsSMO1-2 protein is shown in SEQ ID NO:2.

[0006] In some embodiments, the content or activity of the OsSMO1-2 protein in rice is increased by overexpressing the OsSMO1-2 gene in rice, or the content or activity of the OsSMO1-2 protein in rice is decreased by silencing or knocking out the OsSMO1-2 gene in rice; the OsSMO1-2 gene encodes the OsSMO1-2 protein.

[0007] In some embodiments, the nucleotide sequence of the OsSMO1-2 gene is shown in SEQ ID NO:1.

[0008] In some embodiments, the OsSMO1-2 gene is integrated into the rice genome using transgenic technology to achieve overexpression of the OsSMO1-2 gene.

[0009] In some embodiments, the OsSMO1-2 gene is cloned into the plant binary expression vector pCAMBIA1301, and the obtained recombinant vector is introduced into rice recipient material through Agrobacterium-mediated rice genetic transformation technology, and OsSMO1-2 overexpression lines are screened.

[0010] In some embodiments, the OsSMO1-2 gene is edited in rice using the CRISPR / Cas9 system to achieve silencing or knockout of the OsSMO1-2 gene.

[0011] In some embodiments, the target sequence of the OsSMO1-2 gene used in the CRISPR / Cas9 system is shown in SEQ ID NO:3.

[0012] In some embodiments, the OsSMO1-2 gene is knocked out by introducing into rice the gene encoding an sgRNA (single-stranded guide RNA) that targets the target sequence and the gene encoding the Cas9 protein.

[0013] In some embodiments, the gene encoding the sgRNA targeting the target sequence is cloned into the plant gene editing vector pYLCRISPR / Cas9Pubi-H, and the obtained recombinant vector is introduced into rice recipient material through Agrobacterium-mediated rice genetic transformation technology, and OsSMO1-2 gene knockout lines are obtained by screening.

[0014] Rice with increased or decreased cholesterol content obtained by using the above-described method for regulating cholesterol content in rice also falls within the scope of this invention.

[0015] The application of rice with increased cholesterol content obtained by the above-mentioned method of regulating cholesterol content in rice in the biosynthesis of downstream cholesterol metabolites also falls within the scope of this invention.

[0016] The application of the OsSMO1-2 protein or the gene encoding the OsSMO1-2 protein in the preparation of rice with increased or decreased cholesterol content is also within the scope of this invention.

[0017] The rice can be a rice variety (such as Nipponbare or Longjing 11) or an intermediate material in the rice breeding process. The rice recipient material can be embryogenic callus induced by mature embryo, callus induced by young panicle, callus induced by anther, immature embryo, or meristem.

[0018] The inventors of this application have discovered a gene related to cholesterol synthesis in rice—the OsSMO1-2 gene. In the rice varieties Nipponbare and Ryūjō 11 (LJ11), the coding sequence (CDS) of the OsSMO1-2 gene is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2 for the OsSMO1-2 protein. Experiments have shown that OsSMO1-2 gene knockout lines do not accumulate cholesterol at all (Example 1), while OsSMO1-2 overexpression lines have approximately 10 times higher cholesterol content compared to the wild type (Example 2). OsSMO1-2 gene knockout and overexpression lines can be used to study rice growth and development, stress resistance, and nutritional value, and have important application value in breeding new rice varieties. OsSMO1-2 overexpression lines can accumulate large amounts of cholesterol, which can be used as a plant chassis for the large-scale production of downstream cholesterol metabolites. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the OsSMO1-2 gene, as well as the structural diagrams of the sgRNA expression cassette and Cas9 expression cassette of the gene editing vector; gT1 is the target site, UTR is the untranslated region, OsU3 and maize Pubi are promoters, sgRNA represents the coding sequence of a single-stranded guide RNA, NLS is the nuclear localization signal peptide, and Noster is the terminator.

[0020] Figure 2 This demonstrates the OsSMO1-2 gene knockout line (ossmo1-2) of rice. CR #1, ossmo1-2 CR #2 and ossmo1-2 CR #3) OsSMO1-2 gene mutation type; gT1 is the target site, and the PAM motif is CGG.

[0021] Figure 3 For the wild-type (LJ11-WT) and OsSMO1-2 gene knockout lines of rice. CR #1, ossmo1-2 CR#2 and ossmo1-2 CR #3) Statistical analysis results of the relative expression level of OsSMO1-2 gene in leaves; compared with LJ11-WT, p<0.05 (1 asterisk).

[0022] Figure 4 For the wild-type (LJ11-wt) and OsSMO1-2 gene knockout lines of rice (ossmo1-2) CR #1, ossmo1-2 CR #2 and ossmo1-2 CR Statistical analysis results of bud sterol components of #3), where DW represents dry weight; compared with LJ11-wt, p<0.05 (1 asterisk), p<0.01 (2 asterisks), p<0.001 (3 asterisks), P>0.05 (ns).

[0023] Figure 5 For the wild-type (LJ11-WT) and OsSMO1-2 gene knockout lines of rice. CR #1, ossmo1-2 CR #2 and ossmo1-2 CR #3) Gas chromatogram of bud sterol components, with the horizontal axis representing time (min) and the vertical axis representing signal intensity.

[0024] Figure 6 The results of statistical analysis of the relative expression levels of the OsSMO1-2 gene in leaves of wild-type Longjing 11 rice (LJ11-WT) and OsSMO1-2 overexpression lines (UBI:OsSMO1-2#1, UBI:OsSMO1-2#2 and UBI:OsSMO1-2#3); compared with LJ11-WT, p<0.001 (3 asterisks).

[0025] Figure 7 Gas chromatograms (a) and statistical analysis results (b) of sterol components in the leaves of seedlings of wild-type rice Longjing 11 (LJ11-WT) and OsSMO1-2 overexpression lines (UBI:OsSMO1-2#1, UBI:OsSMO1-2#2 and UBI:OsSMO1-2#3); in (a), the horizontal axis represents time (min) and the vertical axis represents signal intensity; in (b), DW represents dry weight, and compared with LJ11-WT, p<0.05 (1 asterisk), p<0.01 (2 asterisks), p<0.001 (3 asterisks), P>0.05 (ns).

[0026] Figure 8 The structural formulas of various sterol intermediates are shown. Detailed Implementation

[0027] The present invention is further described below with reference to embodiments. The following embodiments are merely illustrative and explanatory of the present invention and do not limit the scope of the present invention in any way.

[0028] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and are of laboratory purity. Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] The plasmid vectors used in the following examples:

[0030] pYLCRISPR / Cas9Pubi-H is a plant gene editing vector, with the Addgene plasmid number #66187. pCAMBIA1301 is a plant binary expression vector, with the GenBank accession number AF234297.

[0031] The Longjing 11 used in the following examples is a conventional japonica rice variety, described in the article "Xu Xide, Sun Haizheng, Sun Shuhong, et al. Breeding of Longjing 11, an early-maturing, high-quality and high-yielding rice variety [J]. Heilongjiang Agricultural Sciences, 2002(6):2. DOI:10.3969 / j.issn.1002-2767.2002.06.017." This variety is available for purchase.

[0032] The reagents used in the following examples:

[0033] The betulinol (CAS No.: 473-98-3) standard was purchased from Sigma-Aldrich. Standards for cholesterol (CAS No.: 57-88-5), campesterol (CAS No.: 474-62-4), β-sitosterol (CAS No.: 83-46-5), stigmasterol (CAS No.: 83-48-7), cycloatinol (CAS No.: 469-38-5), 24-methylenecycloatinol (CAS No.: 1449-09-8), cycloeucalyptol (CAS No.: 469-39-6), and 24-methylenecholest-7-enol (CAS No.: 1176-52-9) were purchased from MCE (MedChemExpress), a US company. The episterol standard was purchased from TRA (Toronto Research Chemicals), a Canadian company. 4-Methyl-24-ethylidene-7-cholestenol, Δ-acidosterol, and isoflavone are phytosterol intermediates prepared in our laboratory, and their structural formulas are as follows: Figure 8 As shown.

[0034] In the following examples, GraphPad Prism statistical software was used to process the experimental data, and the results are expressed as mean ± standard deviation. Significant differences were analyzed using Student's t-test, with p < 0.05 indicating significance, p < 0.01 indicating highly significant, p < 0.001 indicating extremely significant, and p > 0.05 indicating no significance (ns).

[0035] Example 1: Obtaining rice materials with reduced cholesterol content

[0036] Using the rice variety Longjing 11 (LJ11) as the original material, a rice OsSMO1-2 gene knockout line was created using CRISPR / Cas9 gene editing technology. The coding sequence (CDS) of the OsSMO1-2 gene is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2 for the OsSMO1-2 protein.

[0037] 1. Construction of gene editing vectors

[0038] Based on the nucleotide sequence of the OsSMO1-2 gene, a CRISPR / Cas9 target site gT1 was designed. Figure 1 The target sequence is 5'-GGCGGCCTGGTTCCGCTACTCGG-3' (SEQ ID NO:3). 5'-GGCGGCCTGGTTCCGCTACT-3' is the sgRNA target recognition sequence, and the 3' CGG is the PAM sequence required for SpCas9 recognition. According to the sgRNA construction requirements of the pYLCRISPR / Cas9 vector system, BsaI enzyme (Thermo Fisher Scientific, FD0293) was added to both ends of the target sequence to digest compatible sticky ends, artificially synthesizing oligonucleotides Os-SMO3-U3T1F: 5'-ggcaGGCGGCCTGGTTCCGCTACT-3' (SEQ ID NO:4) and Os-SMO3-U3T1R: 5'-aaacAGTAGCGGAACCAGGCCGCC-3' (SEQ ID NO:5).

[0039] The two oligonucleotides (Os-SMO3-U3T1F and Os-SMO3-U3T1R) were annealed to form a double-stranded target sequence adapter. Following the pYLCRISPR / Cas9 vector construction method reported in "Ma, Qunyu Zhang, Qinlong Zhu, et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants[J]. Molecular Plant, 2015, 8(8):1274-1284. DOI:10.1016 / j.molp.2015.04.007," the double-stranded target sequence adapter was first ligated into a BsaI-digested sgRNA intermediate vector to obtain an sgRNA expression cassette containing the OsU3 promoter and the OsSMO1-2-gT1 target sequence; subsequently, BsaI-mediated Golden... The Gate cloning method was used to assemble the sgRNA expression cassette into the pYLCRISPR / Cas9Pubi-H binary vector (RRID: Addgene_66187), replacing the ccdB negative selection fragment in the vector to obtain the OsSMO1-2 gene editing vector. Figure 1 The vector, named OsSMO1-2-CRISPR, contains Cas9 driven by the maize ubiquitin promoter Pubi and carries a hygromycin resistance selection marker, making it suitable for genetic transformation and genome editing in monocotyledonous plants such as rice.

[0040] 2. Agrobacterium-mediated transformation

[0041] The obtained OsSMO1-2-CRISPR vector was transformed into *Agrobacterium tumefaciens* strain EHA105 using a freeze-thaw method. The transformed bacterial culture was plated on LB solid medium containing kanamycin (50 mg / L) and rifampin (25 mg / L) and incubated at 28°C with inverted incubation for 2-3 days. Single colonies were picked and inoculated into LB liquid medium containing kanamycin (50 mg / L) and rifampin (25 mg / L) and cultured at 28°C with shaking at 200 rpm until the culture became turbid. Using the bacterial culture as a template, PCR was performed using the vector backbone primers SP-L1 and SP-R to detect whether the target vector had been successfully transformed into *Agrobacterium tumefaciens* EHA105. The bacterial culture PCR test confirmed the presence of recombinant *Agrobacterium* carrying the OsSMO1-2-CRISPR vector, and this strain was named EHA105-OsSMO1-2-CRISPR.

[0042] The nucleotide sequences of the primers are as follows:

[0043] SP-L1: 5'-GCGGTGTCATCTATGTTACTAG-3' (SEQ ID NO: 6)

[0044] SP-R: 5'-TGCAATAACTTCGTATAGGCT-3' (SEQ ID NO:7)

[0045] 3. Genetic transformation of rice

[0046] Rice genetic transformation was performed following the method published by Hiei et al. in *The Plant Journal* in 1994 (Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. *The Plant Journal*, 1994, 6: 271-282). The specific procedure is as follows:

[0047] Preparation of Agrobacterium infection medium: Recombinant Agrobacterium EHA105-OsSMO1-2-CRISPR was inoculated into LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and cultured at 28°C with shaking at 200 rpm until OD. 600 The OD value was 0.6–1.0. The bacterial cells were collected by centrifugation at 5000 rpm for 10 min. The cells were washed once with infection buffer (containing 68.5 g / L sucrose, 36 g / L glucose, 500 mg / L acid-hydrolyzed casein, 500 mg / L L-proline, and 100 μmol / L acetylsylgenone, pH adjusted to 5.2–5.4), and then resuspended in fresh infection buffer and the OD value was adjusted. 600 The concentration was 0.2, and the recombinant Agrobacterium EHA105-OsSMO1-2-CRISPR infection solution was obtained.

[0048] Agrobacterium infection and co-culture: Callus tissue from wild-type rice variety LJ11 was placed in freshly prepared recombinant Agrobacterium EHA105-OsSMO1-2-CRISPR infection solution, gently shaken to mix, and then infected for 10–20 min. After infection, excess bacterial solution remaining on the surface of the callus tissue was removed with sterile filter paper to avoid excessive proliferation of Agrobacterium during the co-culture stage. Subsequently, the callus tissue was transferred to co-culture medium supplemented with acetylsyleugenone (N6D solid medium, containing 30 g / L sucrose, 10 g / L glucose, 2 mg / L 2,4-D, 300 mg / L acid-hydrolyzed casein, 500 mg / L L-proline, 100 μmol / L acetylsyleugenone and 3–4 g / L Gelrite, pH adjusted to 5.2–5.4) and co-cultured in the dark at 25–28℃ for 2–3 days.

[0049] Callus screening: After co-culture, the callus tissue was transferred to a sterile liquid culture medium supplemented with antibacterial antibiotics and washed repeatedly to remove Agrobacterium bacteria attached to the surface of the callus tissue. After washing, the surface moisture of the callus tissue was blotted dry with sterile filter paper, and then transferred to a screening medium (N6D solid medium supplemented with 50 mg / L hygromycin B and 250 mg / L cefotaxime) that simultaneously added plant screening agent and antibacterial antibiotics, and cultured in the dark at 28°C. During the screening process, the callus tissue was transferred to fresh screening medium every 10–14 days. After 2–3 rounds of screening, resistant callus tissue with good growth status was selected for subsequent differentiation culture.

[0050] Differentiation and rooting culture: The selected resistant callus was transferred to differentiation medium (MS solid medium, containing MS basal salts and vitamins, 30 g / L sucrose, 30 g / L sorbitol, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 30 mg / L hygromycin B, 250 mg / L cefotaxime, and 3–4 g / L Gelrite, pH adjusted to 5.8) and cultured under light conditions to induce adventitious shoot differentiation. The culture conditions were set at 26–28℃ and a photoperiod of 16 h light / 8 h dark. After the callus differentiated into green buds and developed into seedlings, the healthy regenerated buds were transferred to rooting medium (1 / 2 MS solid medium, containing 15 g / L sucrose, 0.1 mg / L NAA, 20 mg / L hygromycin B, 100 mg / L cefotaxime, and 3–4 g / L Gelrite, pH adjusted to 5.8). Continue culturing with g / L ELrite and pH adjusted to 5.8; maintain suitable light and temperature conditions during the rooting culture stage, and carry out hardening treatment after the seedlings have formed a complete root system and reached the transplanting standard.

[0051] Hardening off and transplanting: Remove well-rooted, resistant regenerated seedlings from the culture container, gently remove the culture medium attached to the roots, and then transplant them into sterilized substrate or nutrient soil. In the early stage of hardening off, it is necessary to maintain a high ambient humidity, which can be achieved by covering with a transparent cover or plastic wrap to retain moisture and reduce plant water transpiration. During the hardening off process, gradually extend the ventilation time and reduce the ambient humidity to help the plants gradually adapt to the external environment. After 5-7 days of hardening off, select surviving and normally growing plants to be transplanted to a greenhouse or artificial climate chamber for cultivation, and finally obtain T0 generation transgenic rice plants.

[0052] Identification of positive plants: When T0 generation transgenic rice plants reached a suitable growth stage, young leaves were collected and genomic DNA was extracted. The hygromycin resistance gene was amplified by PCR using primers hptII-F and hptII-R to screen for positive transgenic plants. Using the genomic DNA of the positive transgenic plants as a template, the target region was amplified using primers OsSMO1-2-TLJ11-F and OsSMO1-2-TLJ11-R and Sanger sequencing was performed. Sequence alignment analysis was conducted to determine if any editing events such as insertion, deletion, or base substitution had occurred at the target site.

[0053] The nucleotide sequences of the primers are as follows:

[0054] hptII-F: 5'-ATGAAAAAGCCTGAACTCACCGC-3' (SEQ ID NO: 8)

[0055] hptII-R: 5'-CTATTCCTTTGCCCTCGGACGAG-3' (SEQ ID NO:9)

[0056] OsSMO1-2-TLJ11-F: 5'-GCCGGAGATCGATCCTCTGT-3' (SEQ ID NO: 10)

[0057] OsSMO1-2-TLJ11-R: 5'-CCAGAAGGTGGTCATGTGGC-3' (SEQ ID NO: 11)

[0058] PCR reaction mixture: 25 μL 2×Phanta Max Master Mix, 2 μL each of forward and reverse primers, 5 μL template, and ddH2O to a final volume of 50 μL. Reaction program: 95℃ for 5 min; 30 cycles (95℃ for 30 sec, 55℃ for 30 sec, 72℃ for 30 sec / Kb); 72℃ for 5 min.

[0059] Sequencing verification revealed OsSMO1-2 gene knockout lines with different mutation types, which were named ossmo1-2. CR#1, ossmo1-2 CR #2, ossmo1-2 CR #3. For example... Figure 2 As shown, relative to the coding sequence of the OsSMO1-2 gene in wild-type rice (LJ11-WT), ossmo1-2 CR #1 inserts 1 bp (T) between the 83rd and 84th bases, ossmo1-2 CR #2 inserts 1 bp (C) between the 83rd and 84th bases, ossmo1-2 CR #3 contains the deletion of bases 84 and 85 (CT). These mutation types all lead to premature termination of OsSMO1-2 protein translation.

[0060] The aforementioned OsSMO1-2 gene knockout lines were continuously planted for two seasons in transgenic fields in Fuyang, Hangzhou and Sanya, Hainan. After two generations of propagation, ossmo1-2 was obtained. CR #1, ossmo1-2 CR #2, ossmo1-2 CR #3 homozygous mutant line.

[0061] 4. Detection of OsSMO1-2 gene expression levels

[0062] The RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN, DP441) was used to extract total RNA from wild-type rice LJ11 and ossmo1-2. CR #1, ossmo1-2 CR #2, ossmo1-2 CRRNA was extracted from leaf tissues of the #3 homozygous mutant line. The extracted RNA was reverse transcribed into cDNA using a HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R212). Using the cDNA as a template, real-time quantitative PCR (qPCR) was performed on a Roche LightCycler® 480 II instrument (Vazyme, Q711) using a ChamQ Universal SYBR qPCRMaster Mix to detect the expression level of the OsSMO1-2 gene. The OsACTIN1 gene (Os03g0718100) was selected as an internal control gene. The reaction mixture consisted of 10 μL of 2×SYBR qPCRMaster Mix, 0.4 μL each of forward and reverse primers, 2 μL of cDNA template, and 7.2 μL of ddH2O. Reaction program: (96℃ 30 sec) 1 cycle; (96℃ 10 sec, 60℃ 30 sec) 40 cycles; (95℃ 15 sec, 60℃ 60 sec, 95℃ 15 sec) 1 cycle. Relative gene expression levels were measured using 2... -ΔΔCt The calculations were performed using the method. Each treatment included three biological replicates and three technical replicates.

[0063] The qPCR primers for the OsSMO1-2 gene are as follows:

[0064] SMO1-2-qPCR-F: 5'-GAGTACCATGACTACCACCATTTC-3' (SEQ ID NO: 12)

[0065] SMO1-2-qPCR-R: 5'-TATCTGTAGCCTCTGTCAGTCC-3' (SEQ ID NO: 13)

[0066] The qPCR primers for the internal reference gene are as follows:

[0067] qPCR-ACTIN1-F: 5'-TGCTATGTACGTCGCCATCCAG-3' (SEQ ID NO: 14)

[0068] qPCR-ACTIN1-R: 5'-AATGAGTAACCACGCTCCGTCA-3' (SEQ ID NO: 15)

[0069] Test results as follows Figure 3As shown. Compared to the wild-type LJ11 (LJ11-WT), ossmo1-2 CR #1, ossmo1-2 CR #2, ossmo1-2 CR The expression level of the OsSMO1-2 gene in the leaves of #3 was significantly reduced.

[0070] 5. Sterol extraction and analysis of OsSMO1-2 gene knockout lines

[0071] Take ossmo1-2 respectively CR #1, ossmo1-2 CR #2, ossmo1-2 CR Seeds of the #3 homozygous mutant line were soaked in ultrapure water at 37°C for 48 h. After sprouting, they were germinated at 28°C for 5 days. Five days after germination, the sprouts were cut, freeze-dried, and 10 mg of the freeze-dried sprouts were weighed into a 2 mL tube. 1 mL of saponification solution (containing 10% KOH (w / v), 10% H2O (v / v), 90% ethanol (v / v), and 10 μL / mL betulin was added, and the mixture was heated at 75°C for 1 h. The tube was then opened and heated again to completely evaporate the ethanol. After the ethanol had evaporated, 500 μL of ethyl acetate and an equal volume of water were added to the sample. The mixture was vortexed thoroughly and then centrifuged at 12,000 rpm for 10 minutes to promote phase separation. 20 μL of the sample from the upper ethyl acetate layer was carefully transferred to a 2 mL glass vial compatible with an automated sampling system. The solvent was evaporated under a nitrogen stream, and then 50 μL of a 1-(trimethylsilyl)imidazolium-pyridine mixture (Sigma-Aldrich) was added to the residue. The sample was incubated at 70 °C for 30 minutes.

[0072] The samples were analyzed using a Thermo ISQ-LT GC-MS system. A Thermo TG-5HT column (30 m × 0.25 mm × 0.10 μm) was used. The mass spectrometer detector was set to scan mode, with a scan range of 60–800 m / z and a solvent delay time of 10 min. The gas chromatography conditions were as follows: 1 μL of sample was injected at 250 °C in split mode (10:1), with an ultrahelium flow rate of 1.2 mL / min. The temperature program was as follows: initial injection temperature of 170 °C held for 2 min, then increased to 290 °C at 6 °C / min, held for 4 min, and then increased to 340 °C at 25 °C / min.

[0073] Each strain was configured with three biological replicates, and each biological replicate was performed three times technically. Statistical analysis was conducted on the types and accumulation amounts of sterol intermediates in each strain. Figure 4 and Figure 5The germination cholesterol content of wild-type rice LJ11 (LJ11-wt) is approximately 0.09 mg / g dry weight (DW), ossmo1-2 CR #1, ossmo1-2 CR #2, ossmo1-2 CR The #3 homozygous mutant line had a bud cholesterol content of 0, meaning it did not accumulate cholesterol at all. Figure 4 Compared to wild-type LJ11 rice (LJ11-wt), ossmo1-2 CR #1, ossmo1-2 CR #2, ossmo1-2 CR The accumulation of campesterol, sitosterol, stigmasterol, cycloartenol, episterol, and isoflavone was decreased in the #3 homozygous mutant line, while the accumulation of other sterol intermediates did not differ significantly. Figure 4 This indicates that OsSMO1-2 actively participates in the cholesterol biosynthesis pathway. Loss of function of the OsSMO1-2 gene results in rice not accumulating cholesterol at all.

[0074] Example 2: Obtaining rice materials with increased cholesterol content

[0075] Using the rice variety Longjing 11 (LJ11) as the original material, an OsSMO1-2 gene overexpression line was created by Agrobacterium-mediated genetic transformation. The coding sequence (CDS) of the OsSMO1-2 gene is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2 for the OsSMO1-2 protein.

[0076] 1. Construction of OsSMO1-2 overexpression vector

[0077] Using pCAMBIA1301 as the base vector, an OsSMO1-2 overexpression vector driven by the maize ubiquitin promoter was constructed. The specific procedure is as follows:

[0078] Total RNA was extracted from leaves of wild-type rice variety LJ11 using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN, DP441). cDNA was synthesized from the total RNA using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R212). Using the cDNA as a template, the full-length coding sequence (CDS) of the OsSMO1-2 gene was amplified by PCR using primers OsSMO1-2-CDS-F and OsSMO1-2-CDS-R. After agarose gel electrophoresis, the PCR product was recovered and purified using the Omega Agarose Gel Recovery Kit (D2500-02) to obtain the OsSMO1-2 gene CDS fragment.

[0079] The nucleotide sequences of the primers are as follows:

[0080] OsSMO1-2-CDS-F: 5'-ATGCTCCCGTACGCGACG-3' (SEQ ID NO: 16)

[0081] OsSMO1-2-CDS-R: 5'-TTAATCTTGTTTCCCGTTGCCGAAT-3' (SEQ ID NO: 17)

[0082] Using the pYLCRISPR / Cas9Pubi-H vector as a template, the maize ubiquitin promoter fragment was amplified by PCR using primers Pubi-F and Pubi-R. After the PCR product was detected by agarose gel electrophoresis, it was recovered and purified using the Omega Agarose Gel Recovery Kit (D2500-02) to obtain the ubiquitin promoter fragment.

[0083] The nucleotide sequences of the primers are as follows:

[0084] Pubi-F: 5'-GTCGTGCCCCTCTAGAGAT-3' (SEQ ID NO: 18)

[0085] Pubi-R: 5'-CTGCAGAAGTAACACCAAACAACAGG-3' (SEQ ID NO: 19)

[0086] Using the purified ubiquitin promoter fragment as a template, PCR was performed using primers Pubi-BamHI-F and Pubi-SMO1-2-F1-R to obtain the first amplification product. Using the purified OsSMO1-2 gene CDS fragment as a template, PCR was performed using primers Pubi-SMO1-2-F2-F and SMO1-2-Eco91I-R to obtain the second amplification product. Subsequently, using the first and second amplification products as templates, the two fragments were ligated into a long fragment Pubi-OsSMO1-2 using primers Pubi-BamHI-F and SMO1-2-Eco91I-R via overlap PCR.

[0087] The nucleotide sequences of the primers are as follows:

[0088] Pubi-BamHI-F: 5'-cgagctcggtacccggggatccGTCGTGCCCCTCTCTAGAGAT-3' (SEQ IDNO: 20)

[0089] Pubi-SMO1-2-F1-R: 5'-GTACGGAGCATCTGCAGAAGTAACACCAAACAACAGG-3' (SEQ IDNO: 21)

[0090] Pubi-SMO1-2-F2-F: 5'-TTACTTCTGCAGATGCTCCCGTACGCGAC-3' (SEQ ID NO: 22)

[0091] SMO1-2-Eco91I-R: 5'-ggggaaattcgagctggtcaccTTAATCTTGTTTCCCGTTGCCGAAT-3' (SEQ ID NO: 23)

[0092] The pCAMBIA1301 vector (GenBank accession number AF234297) was double-digested with restriction endonucleases BamHI (Thermo Fisher, FD0054) and Eco91I (Thermo Fisher, FD0394) to obtain a linearized pCAMBIA1301 vector. The digestion mixture consisted of 2 μL BamHI enzyme, 2 μL Eco91I enzyme, 3 μL pCAMBIA1301 plasmid (300 ng / μL), 5 μL digestion buffer, and 38 μL ddH2O. The digestion conditions were 37°C for 1 hour.

[0093] The amplified long fragment Pubi-OsSMO1-2 was ligated into the linearized pCAMBIA1301 vector using the ClonExpress II OneStep Cloning Kit (C112), a non-ligase-dependent one-step cloning method developed by Novizan, to obtain the ligation product. The reaction mixture consisted of: 1 μL Exnase II homologous recombinase, 2 μL 5×CE II buffer, 2 μL Pubi-OsSMO1-2 fragment, 3 μL linearized pCAMBIA1301 vector, and 2 μL ddH2O. The reaction conditions were: incubation at 37°C for 30 minutes.

[0094] The ligation product was transformed into *E. coli* DH5α competent cells using a heat shock method. The transformation product was plated on LB agar plates containing 50 mg / L kanamycin and incubated at 37°C for 12–16 h. Single colonies were picked and inoculated into LB liquid medium containing 50 mg / L kanamycin, and cultured at 37°C with shaking. Colony identification was then performed using primers ppubi-1890-F1 and nos-57-R1.

[0095] The nucleotide sequences of the primers are as follows:

[0096] pubi-1890-F1:5'-GCCTGCCTTCATACGCTAT-3' (SEQ ID NO:24)

[0097] nos-57-R1:5'-AAGACCGGCAACAGGATTCA-3' (SEQ ID NO:25)

[0098] Plasmids from PCR-identified positive clones were extracted and double-digested with BamHI and Eco91I enzymes to verify the size of the insert fragment. The double-digested recombinant plasmids were sequenced to confirm that the ubiquitin promoter, the coding region of the OsSMO1-2 gene, and the linker sequence were correct, the reading frame was intact, and there were no mutations. The correctly sequenced recombinant plasmid was named pCAMBIA1301-Ubi-OsSMO1-2.

[0099] 2. Obtaining OsSMO1-2 overexpression lines in rice

[0100] The recombinant plasmid pCAMBIA1301-Ubi-OsSMO1-2 was transformed into *Agrobacterium tumefaciens* EHA105 competent cells using a freeze-thaw method. The transformed cells were then plated on LB agar plates containing kanamycin (50 mg / L) and rifampin (25 mg / L) and incubated at 28°C for 2–3 days. Single colonies were picked and inoculated into LB liquid medium containing kanamycin (50 mg / L) and rifampin (25 mg / L), and cultured with shaking at 28°C. Colony-specific PCR was performed using primers ppubi-1890-F1 and nos-57-R1. The correctly identified recombinant *Agrobacterium* was named EHA105-pCAMBIA1301-Ubi-OsSMO1-2. This recombinant *Agrobacterium* was then expanded and used for genetic transformation in rice.

[0101] Using wild-type rice callus (LJ11) as recipient material, rice genetic transformation was performed using the aforementioned recombinant Agrobacterium EHA105-pCAMBIA1301-Ubi-OsSMO1-2, following the Agrobacterium-mediated rice transformation method described in the literature "Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 1994, 6: 271-282". Specific steps are detailed in Example 1. T0 generation OsSMO1-2 overexpressing lines were obtained, and three of these lines were named UBI:OsSMO1-2#1, UBI:OsSMO1-2#2, and UBI:OsSMO1-2#3, respectively.

[0102] Total RNA was extracted from the leaves of wild-type rice LJ11, UBI:OsSMO1-2#1, UBI:OsSMO1-2#2, and UBI:OsSMO1-2#3 seedlings using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN, DP441). The total RNA was then reverse transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R212). Real-time quantitative PCR (qPCR) was performed using the cDNA as a template to detect the expression level of the OsSMO1-2 gene. The specific procedures for real-time quantitative PCR are described in Example 1.

[0103] The results showed that, compared with wild-type rice LJ11 (LJ11-WT), the expression level of the OsSMO1-2 gene in the OsSMO1-2 overexpression lines UBI:OsSMO1-2#1, UBI:OsSMO1-2#2, and UBI:OsSMO1-2#3 was significantly increased. Figure 6 ).

[0104] 3. Sterol extraction and analysis of OsSMO1-2 overexpression lines

[0105] OsSMO1-2 overexpression lines UBI:OsSMO1-2#1, UBI:OsSMO1-2#2, and UBI:OsSMO1-2#3 were cultured. Leaves from 15-day-old seedlings were freeze-dried, and 10 mg of the freeze-dried leaves were weighed into a 2 mL tube. 1 mL of saponification solution (containing 10% KOH (w / v), 10% H2O (v / v), 90% ethanol (v / v), and 10 μL / mL betulin was added, and the mixture was heated at 75 °C for 1 h. The tube was then opened and heated again to allow complete ethanol evaporation. 500 μL of ethyl acetate and an equal volume of water were added to the sample. The mixture was thoroughly vortexed and then centrifuged at 12,000 rpm for 10 min to promote phase separation. 20 μL of the sample from the upper ethyl acetate layer was carefully transferred to a 2 mL glass vial compatible with an automated sampling system. The solvent was evaporated under a nitrogen stream, and then 50 μL of a 1-(trimethylsilyl)imidazolium-pyridine mixture (Sigma-Aldrich) was added to the residue. The sample was incubated at 70 °C for 30 minutes.

[0106] The samples were analyzed using a Thermo ISQ-LT GC-MS system. A Thermo TG-5HT column (30 m × 0.25 mm × 0.10 μm) was used. The mass spectrometer detector was set to scan mode, with a scan range of 60–800 m / z and a solvent delay time of 10 min. The gas chromatography conditions were as follows: 1 μL of sample was injected at 250 °C in split mode (10:1), with an ultrahelium flow rate of 1.2 mL / min. The temperature program was as follows: initial injection temperature of 170 °C held for 2 min, then increased to 290 °C at 6 °C / min, held for 4 min, and then increased to 340 °C at 25 °C / min.

[0107] The contents of cholesterol, stigmasterol, sitosterol, and campesterol were statistically analyzed, and the results are as follows: Figure 7As shown. In the leaves of wild-type rice LJ11 (LJ11-WT) at the 15-day seedling stage, the cholesterol content was approximately 0.02 mg / g dry weight (DW), the stigmasterol content was approximately 0.32 mg / g dry weight, the sitosterol content was approximately 1.2 mg / g dry weight, and the campesterol content was approximately 0.38 mg / g dry weight (…). Figure 7 In the 15-day-old leaves of OsSMO1-2 overexpression lines UBI:OsSMO1-2#1, UBI:OsSMO1-2#2, and UBI:OsSMO1-2#3, the cholesterol content was approximately 0.2 mg / g dry weight, about 10 times higher than that of the wild type; the campesterol content was approximately 0.7 mg / g, about 1.8 times higher than that of the wild type; however, the accumulation of sitosterol and stigmasterol did not change significantly, with only a slight decrease in stigmasterol accumulation observed in the two overexpression lines. Figure 7 These results indicate that the OsSMO1-2 gene plays a crucial role in the biosynthesis of cholesterol in rice, suggesting the existence of a novel and previously unexplored cholesterol synthesis pathway in rice. OsSMO1-2 overexpression lines can produce large amounts of cholesterol, which could serve as a plant chassis for the large-scale production of downstream cholesterol metabolites.

Claims

1. A method for regulating cholesterol content in rice, comprising: The method involves increasing the content or activity of OsSMO1-2 protein in rice to increase the cholesterol content in rice, or decreasing the content or activity of OsSMO1-2 protein in rice to decrease the cholesterol content in rice; the amino acid sequence of the OsSMO1-2 protein is shown in SEQ ID NO:

2.

2. The method as described in claim 1, characterized in that, The content or activity of the OsSMO1-2 protein in rice is increased by overexpressing the OsSMO1-2 gene in rice, or the content or activity of the OsSMO1-2 protein in rice is decreased by silencing or knocking out the OsSMO1-2 gene in rice; the OsSMO1-2 gene encodes the OsSMO1-2 protein.

3. The method as described in claim 2, characterized in that, The nucleotide sequence of the OsSMO1-2 gene is shown in SEQ ID NO:

1.

4. The method as described in claim 2 or 3, characterized in that, The OsSMO1-2 gene was integrated into the rice genome using transgenic technology to achieve overexpression of the OsSMO1-2 gene.

5. The method as described in claim 2 or 3, characterized in that, The OsSMO1-2 gene was edited in rice using the CRISPR / Cas9 system to achieve silencing or knockout of the OsSMO1-2 gene.

6. The method as described in claim 5, characterized in that, The target sequence of the OsSMO1-2 gene used in the CRISPR / Cas9 system is shown in SEQ ID NO:

3.

7. The method as described in claim 6, characterized in that, The OsSMO1-2 gene was knocked out by introducing the gene encoding the sgRNA that targets the target sequence and the gene encoding the Cas9 protein into rice.

8. Rice with increased or decreased cholesterol content obtained by the method according to any one of claims 1-7.

9. The application of rice with increased cholesterol content obtained by the method according to any one of claims 1-7 in the biosynthesis of downstream cholesterol metabolites.

10. The use of the OsSMO1-2 protein as described in claim 1 or the gene encoding the OsSMO1-2 protein in the preparation of rice with increased or decreased cholesterol content.

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