Flavin monooxygenase genes, products and their applications in alliin synthesis

Abstract

The present invention discloses flavin monooxygenase genes, products and their application in alliin synthesis, belonging to the technical field of plant genetic engineering. The present invention screened multiple flavin monooxygenase genes AsFMO1, AsFMO2, AsFMO3, AsFMO4 and AsFMO5 (SEQ ID NO.1-5) in alliin biosynthesis from "Jinxiang Siliuban", and identified two particularly excellent genes AsFMO1 and AsFMO2 (SEQ ID NO.1-2). This study revealed the key genes for garlic alliin biosynthesis, and used garlic genetic transformation methods to obtain transgenic garlic with high and low alliin content, which contributed to the molecular breeding of garlic and had practical application value.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a flavin monooxygenase gene, a product and application thereof in alliin synthesis. Background Art

[0002] Garlic (Allium sativum L.) is an important vegetable, condiment and medicinal plant. Alliin is a sulfur-containing amino acid that only exists in garlic in nature and is also one of the main bioactive substances in garlic. As one of the important bulb vegetables, garlic has a spicy and pungent taste and is a common spice and food additive. Sulfur-containing compounds, such as allicin and its metabolites, are the main reasons for the spicy effect and spicy smell of garlic. Traditionally, garlic and its related compounds are considered to have a variety of biological activities, including anticancer, antioxidant, antidiabetic, renal protection, antifungal and antihypertensive activities. In addition, garlic is also used in traditional medicine for the treatment of indigestion, respiratory and urinary tract infections and heart disease. It has anthelmintic, antipyretic, sedative and diuretic effects. Although alliin has unique nutritional and medicinal value, the molecular regulatory pathways of alliin biosynthesis remain unclear, so it is crucial to study the molecular regulatory mechanism of alliin biosynthesis.

[0003] S-allylcysteine ​​is stereospecifically oxidized to (+)-S-allyl-L-cysteine ​​sulfoxide by garlic extract, indicating the presence of an oxidase in garlic that can catalyze the synthesis of (+)-S-allyl-L-cysteine ​​sulfoxide. In the article “Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic”, Yoshimoto et al. first discovered a gene encoding a flavin-containing monooxygenase (FMO) in Fukuchi-howaito (a garlic variety from Hokkaido, Japan) based on ESTs, and named it AsFMO1 (NCBI No.AB924383.1). Through heterologous expression in yeast, it was shown that AsFMO1 was involved in alliin biosynthesis in vitro. AsFMO1 is consistent with class B flavin monooxygenases, has NADP, FAD and FMO conserved motifs, and the S-oxidation reaction catalyzed by it depends on the presence of NADPH and FAD. AsFMO1 functions as an S-allyl-l-cysteine-S-oxygenase and may convert stored γ-glutamyl-S-allyl-l-cysteine ​​into alliin during germination or synthesize alliin de novo in leaves. However, no functional reports on AsFMO1 regulating the alliin biosynthesis process in garlic have been found, and its molecular regulatory mechanism and application remain unclear. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide flavin monooxygenase genes, products and their application in alliin synthesis. In view of the progress of the prior art, the present invention, in combination with long-term research and exploration, screened multiple flavin monooxygenase genes AsFMO1, AsFMO2, AsFMO3, AsFMO4 and AsFMO5 (SEQ ID NO.1-5) in alliin biosynthesis from "Jinxiang Siliuban", and identified two particularly excellent genes AsFMO1 and AsFMO2 (SEQ ID NO.1-2). This study further revealed the garlic alliin biosynthesis gene, and used the garlic genetic transformation method to obtain transgenic garlic with high alliin content and low alliin content, which contributed to the molecular breeding of garlic and has practical application value.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a flavin-containing monooxygenase gene is provided, wherein the nucleotide sequence of the flavin-containing monooxygenase gene is shown in any one of SEQ ID NO.1 to SEQ ID NO.5.

[0007] The second aspect of the present invention provides the use of the flavin monooxygenase gene in regulating the synthesis of alliin in garlic.

[0008] The third aspect of the present invention provides a protein encoded by a flavin monooxygenase gene, wherein the amino acid sequence of the protein encoded by the flavin monooxygenase gene is shown in any one of SEQ ID NO.50 to SEQ ID NO.54.

[0009] The fourth aspect of the present invention provides the use of the protein encoded by the flavin monooxygenase gene in regulating the synthesis of alliin in garlic.

[0010] The fifth aspect of the present invention provides an expression cassette containing the flavin monooxygenase gene.

[0011] The sixth aspect of the present invention provides a recombinant expression vector containing the flavin monooxygenase gene.

[0012] The seventh aspect of the present invention provides a recombinant bacterium containing the flavin monooxygenase gene.

[0013] The eighth aspect of the present invention provides the use of the expression cassette, the recombinant expression vector or the recombinant bacteria in regulating the synthesis of alliin in garlic.

[0014] A ninth aspect of the present invention provides a method for regulating plant alliin synthesis, comprising:

[0015] Promoting the expression of flavin monooxygenase genes in target plants to obtain plants with higher alliin content than the target plants;

[0016] Alternatively, the flavin monooxygenase gene in the target plant is silenced or knocked out to obtain a plant with a lower alliin content than the target plant;

[0017] The flavin monooxygenase gene is transferred into the target plant via an expression vector;

[0018] The flavin monooxygenase gene is a nucleic acid molecule as shown in any one of the following i) to ii):

[0019] i) the nucleotide sequence is a nucleic acid molecule shown in any one of SEQ ID NO.1 to SEQ ID NO.5;

[0020] ii) A nucleic acid molecule encoding the amino acid sequence shown in any one of SEQ ID NO.50 to SEQ ID NO.54 except i).

[0021] A tenth aspect of the present invention provides application of the method for regulating the alliin content in plants in plant breeding, wherein the plant is garlic.

[0022] The garlic reference genome used in this study was from Dishuizao (NCBIGEO: GSE145455), and AsFMO1 (NCBINo.AB924383.1) was used as the reference sequence to identify AsFMO genes in the whole genome. To identify all members of the garlic FMO gene family, a preliminary screening was first performed based on the garlic genome annotation, and then the candidate genes were submitted to the protein domain prediction website Pfam for protein domain prediction, and then further verified using the online websites Interpro and SMART. Finally, the FMO members were identified in the garlic genome and finally determined to be AsFMO. Class B flavin monooxygenases have two typical dinucleotide binding domains, FAD and NAD(P)H, and always bind to NADP+ in the catalytic process of class B flavin monooxygenases. Studies have shown that binding to NADP+ may have the effect of stabilizing flavin peroxide intermediates. Class B flavin monooxygenase can oxidize carbon atoms, sulfur atoms and other (hetero) atoms on substrate molecules, so it is also called multifunctional flavin-containing monooxygenase. The oxidation reaction in which it participates is characterized by high stereo, chemical and regional specificity.

[0023] Based on the two typical dinucleotide binding domains of FAD and NAD(P)H in plant FMOs, the inventors finally identified 5 FMOs members (Asa3G05097.1, Asa4G04959.1, Asa4G04963.1, Asa5G05156.1, Asa6G04803.1). Asa3G05097.1 has 11 fewer amino acids at the N-terminus than the protein encoded by NCBI No.AB924383.1, and they were named AsFMO1, AsFMO2, AsFMO3, AsFMO4 and AsFMO5 (the nucleotide sequences of AsFMO1, AsFMO2, AsFMO3, AsFMO4 and AsFMO5 genes are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively). IDNO.5, the amino acid sequences encoded by the AsFMO1, AsFMO2, AsFMO3, AsFMO4 and AsFMO5 genes are SEQ IDNO.50, SEQ ID NO.51, SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO.54, respectively, see the nucleotide sequence table for details). See the nucleotide sequence table for details). In addition, all FMOs members have four conserved motifs (FAD-binding, NADP-binding, TGY and FMO-identifying). Further sequence alignment revealed that all five FMOs members contain FAD-binding, NADP-binding, TGY and FMO-identifying characteristic sequences, which are consistent with the four conserved motifs of class B flavin monooxygenases, among which the FAD binding motif and TGY motif are extremely conserved. In addition, the AsFMO1 protein has an additional NADP binding motif (GGGEEG) at positions 155-160 of the amino acid sequence compared to the other four members. The brief process is as follows:

[0024] 1. Construction of AsFMO1 and AsFMO2 gene overexpression vectors

[0025] In order to identify the key roles of AsFMO1 and AsFMO2 in alliin biosynthesis, we used cDNA reverse transcribed from RNA of Jinxiang four six petals as template.

[0026] AsFMO1 Primer Pair:

[0027] Forward primer-1 (SEQ ID NO. 6): 5′-CGGATCCGATGGTTTCCTCATCT-3′;

[0028] Reverse primer-1 (SEQ ID NO. 7): 5′-CCCGGGTGAAAGAAATTTGCTGAA-3′;

[0029] AsFMO2 Primer Pair:

[0030] Forward primer-2 (SEQ ID NO. 8): 5′-CGGGATCCATGGTCTCTGAATATATCAACTCC-3′;

[0031] Reverse primer-2 (SEQ ID NO.9): 5'-TCCCCCGGGTATTTTAATAACATCTGGATCTAGCTG

[0032] -3';

[0033] PCR amplified a band of about 1300 bp, and the sequencing lengths were 1374 bp and 1371 bp respectively, obtaining the CDS sequences of the AsFMO1 gene and the AsFMO2 gene.

[0034] After the amplified target band was recovered from the gel, it was connected to the pCOMBIA3300-eGFP vector after double restriction digestion using T4 ligase, transformed into Escherichia coli, and positive clones were screened for bacterial liquid PCR and sequencing.

[0035] 2. Construction of CRISPR / Cas9 for AsFMO1 and AsFMO2 genes

[0036] The online website SSC was used to predict suitable high-scoring targets, and the target sequences were compared to select targets with good specificity. According to this principle, two targets were finally selected in the coding regions of the AsFMO1 and AsFMO2 genes. The sequence of target 1 of AsFMO1 was 5′-TTTGTGGAGAAAACGAACGG-3′ (SEQ ID NO.10), and the sequence of target 2 was 5′-TGGAAGGGAAAAGATGGAGG-3′ (SEQ ID NO.11); the sequence of target 1 of AsFMO2 was 5′-AATTCTCCGAGGGAGATCAT-3″ (SEQ ID NO.12), and the sequence of target 2 was 5′-TGTAGGCATGACGAATACCA-3′ (SEQ ID NO.13). The vector construction process includes two reactions: the first reaction is to use the SV-Cas9 vector (CRISPR / Cas9 vector) as a template and amplify with primers containing the target site; the second reaction is to use the amplified product of the first step as a template for amplification, and then purify the PCR product, connect it with the single enzyme-cut SV-Cas9 vector using seamless cloning technology, and transform E. coli. After preliminary screening of bacterial liquid PCR, multiple positive clones were obtained, and the sequencing results were compared using DNAMAN to confirm the correct single clone and obtain the SV-Cas9 vector containing the correct target site.

[0037] 3. Obtaining transgenic lines

[0038] After obtaining the above-mentioned target vector, embryonic callus induced from garlic root tip was selected as the receptor material, and the recombinant plasmids SV-Cas9-AsFMO1-Bar, SV-Cas9-AsFMO2-Bar, pCOMBIA3300-AsFMO1-eGFP, and pCOMBIA3300-AsFMO2-eGFP were transferred into garlic by Agrobacterium-mediated method. After activating LBA4404 Agrobacterium containing recombinant plasmids SV-Cas9-AsFMO1-Bar, SV-Cas9-AsFMO2-Bar, pCOMBIA3300-AsFMO1-eGFP, and pCOMBIA3300-AsFMO2-eGFP, embryonic callus with less than 2 subculture times was infected with infection solution, AsFMO1 and AsFMO2 transgenic overexpression strains and transgenic knockout strains were obtained. The genomes of transformed and untransformed plants were extracted, and the recombinant plasmids SV-Cas9-AsFMO1-Bar, SV-Cas9-AsFMO2-Bar, pCOMBIA3300-AsFMO1-eGFP, and pCOMBIA3300-AsFMO2-eGFP were used as positive controls, the untransformed garlic leaf genome was used as negative control, and water was used as blank control. PCR was performed on them using specific primers to obtain 5 T0 generation plants. The knockout mutant strains amplified the herbicide gene Bar fragment by PCR, indicating that the exogenous gene had been integrated into the genome, and the target fragment had undergone gene editing. The AsFMO1 mutant strains were named asfmo1-1 and asfmo1-2 in sequence; the AsFMO2 mutant strains were named asfmo2-1 and asfmo2-2 in sequence. The eGFP gene was amplified by PCR in the overexpression lines, indicating that the exogenous gene had been integrated into the genome. The AsFMO1 transgenic lines were named OE-1, OE-2, and OE-3 in sequence, and the AsFMO2 transgenic lines were named OE-4, OE-5, and OE-6 in sequence.

[0039] RNA was extracted from young leaves of T0 transgenic lines and untransformed plants and reverse transcribed, and the expression of AsFMO1 and AsFMO2 genes was detected by fluorescence quantitative PCR. In terms of the expression of AsFMO1 and AsFMO2 genes, compared with untransformed plants, the expression of AsFMO1 gene in asfmo1-1 and asfmo1-2 mutant lines was significantly downregulated, and the expression of AsFMO2 gene in asfmo2-1 and asfmo2-2 mutant lines was significantly downregulated. The expression of AsFMO1 overexpression lines was upregulated by 3 times, and the expression of AsFMO2 overexpression lines was upregulated by 2.5 times. In terms of alliin content, compared with untransformed plants, the AsFMO1 overexpression lines increased by 82%, and the mutant lines decreased by 37%; the AsFMO2 overexpression lines increased by 50%, and the mutant lines decreased by 27%.

[0040] According to the final determination of alliin content, combined with bioinformatics analysis and expression characteristics analysis of the AsFMOs gene family, the flavin-containing monooxygenases in alliin biosynthesis are AsFMO1, AsFMO2, AsFMO3, AsFMO4 and AsFMO5. As AsFMO1 and AsFMO2 protein structures are extremely similar, and both are expressed at the highest levels in leaves during the bulb expansion period, AsFMO1 and AsFMO2 are key genes for alliin synthesis.

[0041] Beneficial effects of the present invention:

[0042] The present invention preliminarily screened out 49 FMOs candidate sequences based on the genome annotation information of Dishui Zao garlic, and finally identified 5 FMOs members: AsFMO1, AsFMO2, AsFMO3, AsFMO4 and AsFMO5 (SEQ ID NO.1-5) based on the two typical dinucleotide binding domains of FAD and NAD(P)H in plant FMOs, and identified two particularly excellent genes AsFMO1 and AsFMO2 (SEQ ID NO.1-2) from them. This study revealed the key genes of garlic alliin biosynthesis, and used garlic genetic transformation methods to obtain transgenic garlic with high and low alliin content, which contributed to garlic breeding and had practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 For the structural domain analysis of AsFMOs gene family members; Figure 1 A in the middle is the candidate sequence domain analysis; Figure 1 B in the figure is the structural domain analysis of AsFMOs.

[0044] Figure 2 To analyze the conserved motifs and gene structures of AsFMOs gene family members; Figure 2A in the middle is the conservative motif analysis of AsFMOs family members. Figure 2 Figure B shows the gene structure analysis of AsFMOs members.

[0045] Figure 3 Schematic diagram of the three-dimensional structure of AsFMOs protein; Figure 3 A in the figure is the three-dimensional structure of AsFMO1 protein. Figure 3 B in the figure is the three-dimensional structure of AsFMO2 protein. Figure 3 C in the figure is the three-dimensional structure of AsFMO3 protein. Figure 3 D in the figure is the three-dimensional structure of AsFMO4 protein. Figure 3 (E) is the three-dimensional structure of AsFMO5 protein.

[0046] Figure 4 Phylogenetic tree analysis of garlic AsFMOs, Arabidopsis AtFMOs and japonica rice OsFMOs.

[0047] Figure 5 Subcellular localization of AsFMOs protein.

[0048] Figure 6 is the expression of AsFMOs genes in different tissues of garlic; among them, GFLG is the outer green leaves of the bulb at the germination stage, WFLG is the inner white leaves of the bulb at the germination stage, SLG is the storage leaves at the germination stage, RG is the root at the germination stage, GFLS is the outer green leaves of the bulb at the seedling stage, WFLS is the inner white leaves of the bulb at the seedling stage, SLS is the storage leaves at the seedling stage, RS is the root at the seedling stage, EBBE is the bulb at the early stage of bulb expansion, MBBE is the bulb at the middle stage of bulb expansion, LBBE is the bulb at the late stage of bulb expansion, MaBBE is the bulb at the mature stage of bulb expansion, ELBE is the leaf at the early stage of bulb expansion, MLBE is the leaf at the middle stage of bulb expansion, LLBE is the leaf at the late stage of bulb expansion, and MaLBE is the leaf at the mature stage of bulb expansion.

[0049] Figure 7 For the regeneration process of transformed plants; Figure 7 A in the middle is the root apex. Figure 7 B in the middle is callus tissue, Figure 7 Middle C is subculture, Figure 7 D in the middle is co-culture, Figure 7 E in the middle is for screening culture, Figure 7 F in the middle is differentiation culture, Figure 7 Middle G is rooting culture, Figure 7 Middle H is the mature seedling.

[0050] Figure 8is the expression level of AsFMO1 and AsFMO2 genes in transgenic plants; Control-1 is the expression level of AsFMO1 gene in untransformed plants, Control-2 is the expression level of AsFMO2 gene in untransformed plants, asfmo1-1 and asfmo1-2 are the expression levels of AsFMO1 gene in mutant strains, asfmo2-1 and asfmo2-2 are the expression levels of AsFMO2 gene in mutant strains, OE-1, OE-2 and OE-3 are the expression levels of AsFMO1 gene in overexpression strains, and OE-4, OE-5 and OE-6 are the expression levels of AsFMO2 gene in overexpression strains.

[0051] Fig. 9 is the alliin content of transgenic plants and wild-type plants; Fig. 9 A in the middle is the alliin chromatogram of the untransformed plant. Fig. 9 B in the figure is the alliin chromatogram of the asfmo1 mutant strain. Fig. 9 Middle C is the alliin chromatogram of the OE overexpression strain. Fig. 9 D is the standard curve of alliin, Fig. 9 E is the content of alliin. DETAILED DESCRIPTION

[0052] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0053] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0054] The experimental materials used in the examples of the present invention but not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels.

[0055] Example 1: Identification of garlic FMO genes

[0056] The garlic genome used in this study was the Dishuizao reference gene, and AsFMO1 (NCBINo.AB924383.1) was used as the reference sequence to identify AsFMO genes in the whole genome. To identify all members of the garlic FMO gene family, e.g. Figure 1As shown, the preliminary screening was first performed according to the garlic genome annotation, and then the candidate genes were submitted to the protein domain prediction website Pfam for protein domain prediction, and then further verified using the online websites Interpro and SMART. Finally, the FMO members were identified in the garlic genome and finally determined to be AsFMO. AsFMO was named according to the location of the chromosome and visualized using TBtools. The basic physicochemical properties of all members were analyzed using the ProtParam tool in the Expasy online website, including the number of amino acids, protein relative molecular weight (Mw), theoretical isoelectric point (pI), lipid solubility coefficient, instability coefficient and overall average hydrophilicity (GRAVY).

[0057] (1) Analysis of the conserved motif and gene structure of AsFMO

[0058] The conservative motif analysis of AsFMO was performed using the MEME website, where the number of occurrences of each sequence motif site was set to 0 or 1, the number of predicted motifs was 10, the minimum width of the motif was 6, and the maximum width was 50. To analyze the gene structure of AsFMO, the number, length, and position of introns and exons of the AsFMO gene were submitted from the GFF3 file in the garlic genome annotation and visualized using TBtools.

[0059] Results Figure 2 , analyzing the exon-intron structure of all members of the AsFMOs family, AsFMO1, AsFMO2 and AsFMO3 all contain 7 exons, and AsFMO4 and AsFMO5 all contain 4 exons. AsFMO1, AsFMO2 and AsFMO3 are very similar in terms of conserved motifs and gene structure.

[0060] (2) Analysis of the three-dimensional structure of AsFMO protein

[0061] In order to analyze the protein three-dimensional structure of AsFMO, the amino acid sequence was imported into the protein three-dimensional structure analysis website Phyre2 for data analysis. The protein three-dimensional structure was predicted and its function was analyzed based on the confidence of the prediction.

[0062] Results Figure 3 , AsFMO1, AsFMO2 and AsFMO3 have similar structures, and AsFMO4 and AsFMO5 have similar structures.

[0063] (3) AsFMO system evolution analysis

[0064] NCBI was used to perform BLAST to obtain amino acid sequences with high homology to AsFMO, and then UniProt was used to convert the gene names of the sequences. Finally, MEGA11.0 software was used for evolutionary analysis.

[0065] Results Figure 4 , AsFMO1 is most closely related to Os10g0553800, AsFMO4 and AsFMO5 are most closely related to Os11g0207700, and AsFMO2 and AsFMO3 are most closely related to Arabidopsis (At5g07800, At5g61290). AsFMO1, AsFMO2, and AsFMO3 belong to clade III, which is the same clade as FMOGS-OX (such as At5g61290) found in Arabidopsis; AsFMO4 and AsFMO5 belong to clade II, which is the same clade as the YUCCA family;

[0066] (4) Subcellular localization analysis of AsFMOs gene family members

[0067] WoLF PSORT was used to speculate the subcellular localization of AsFMOs family proteins. AsFMOs gene was used as the research object, the CDS of AsFMOs gene and 35S::GFP gene were used to construct the fusion expression vector 35S::AsFMOs-GFP, transformed into GV3101 Agrobacterium, and Agrobacterium was used to transiently infect tobacco leaf cells. After 2-3 days of light culture, LMS880 laser confocal microscope was used to observe. GFP signal in tobacco epidermal cells was observed by laser confocal fluorescence microscopy.

[0068] Results Figure 5 , it can be seen that the AsFMOs:GFP fusion protein is localized in the subcellular space, and the AsFMOs protein is localized in the cytoplasm.

[0069] Example 2: AsFMO1 and AsFMO2 gene cloning

[0070] (1) Extraction of total RNA from garlic (Trizol method)

[0071] ① Pre-cool the mortar and grinding rod with liquid nitrogen, add 100-200 mg of garlic tissue sample, quickly grind it into powder with liquid nitrogen, and quickly transfer the powdered sample to a pre-cooled centrifuge tube with a pre-cooled medicine spoon.

[0072] ②Immediately add 1 mL of pre-cooled Trizol and 10 μL of β-mercaptoethanol, vortex for 1-2 minutes to mix, let stand for 15 minutes, and invert the centrifuge tube several times every 1 minute to fully mix the sample and Trizol.

[0073] ③ Centrifuge at 12000g and 4℃ for 15min, transfer the supernatant to a new centrifuge tube, add 300μL chloroform to the tube, shake vigorously for 15s, and let stand for 15min.

[0074] ④ Centrifuge at 12000g and 4℃ for 15min, transfer the supernatant to a new centrifuge tube, add 450μL of isopropanol to the tube to precipitate the nucleic acid, mix by inversion, and let stand at -20℃ for 20min.

[0075] ⑤ Centrifuge at 12000g and 4℃ for 15min, discard the supernatant, add 1mL 75% ethanol (prepared with DEPC water), repeatedly invert to wash the precipitate, and aspirate the ethanol.

[0076] ⑥ Repeat the washing of the precipitate. After aspirating the ethanol, centrifuge at 7500g and 4℃ for 5 minutes. Carefully aspirate the remaining ethanol and blow dry the precipitate in an ultra-clean workbench for 5 minutes.

[0077] ⑦ Add 20-100 μL DEPC water to dissolve the precipitate according to the amount of precipitation. After dissolution, aliquot 10 μL for subsequent RNA quality and concentration testing. The remaining RNA sample is stored at -80°C.

[0078] (2) RNA concentration and quality testing

[0079] ①Concentration detection

[0080] Using DEPC water as blank control, OD was measured by Nanodrop 2000. 260 / OD 230 OD 260 / OD 280 The RNA concentration is recorded. 260 / OD 280 The ratio should be between 1.8-2.0.

[0081] ②Integrity detection

[0082] Take 1000ng RNA in a pretreated PCR tube, add 2μL RNA Loading Buffer for agarose gel electrophoresis, and observe the electrophoresis bands. A good quality RNA solution should have at least 2 bands, 28sRNA and 18sRNA. The brightness of the 28sRNA band of a completely undegraded RNA sample should be twice that of the 18sRNA band. If a 5sRNA band appears at the bottom and its brightness exceeds that of the first two bands, or the band tails severely, it means that the RNA is degraded.

[0083] (3) Synthesis of cloned cDNA

[0084] Select RNA with qualified extraction quality for subsequent reverse transcription experiments. Use the Novozymes reverse transcription kit HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) for cDNA synthesis experiments. The process is as follows:

[0085] ① RNA template denaturation

[0086] ② Genomic DNA removal

[0087] ③Prepare the first-strand cDNA synthesis reaction solution

[0088] Example 3: Analysis of AsFMOs gene expression patterns

[0089] In order to study the function of AsFMOs gene, the expression pattern of AsFMOs gene in garlic germination, seedling and bulb expansion stages was analyzed.

[0090] (1) cDNA synthesis

[0091] Select RNA with qualified extraction quality for subsequent reverse transcription experiments. Use the Novozymes reverse transcription kit HiScript IIQ RT SuperMix for qPCR (+gDNA wiper) for cDNA synthesis experiments. The process is as follows:

[0092] ① Removal of genomic DNA from plant total RNA:

[0093] ② Synthesis of the first strand of cDNA:

[0094] (2) Real-time fluorescence quantitative PCR

[0095] ①Use the cDNA obtained by reverse transcription as a template and prepare the reaction system according to Table 1:

[0096] Table 1 qRT-PCR reaction system preparation

[0097]

[0098] ②qRT-PCR reaction program, according to the reaction program in Table 2, denaturation to extension cycle 40 times, PCR amplification:

[0099] Table 2qRT-PCR reaction procedure

[0100]

[0101] The qRT-PCR primer sequences involved in the above experiments are:

[0102]

[0103] Results Figure 6 After analysis, AsFMO1 and AsFMO2 were expressed at higher levels in leaves at the early, late and mature stages of bulb expansion, especially in leaves at the mature stage of bulb expansion; AsFMO3 and AsFMO4 were expressed at lower levels in garlic tissues; AsFMO5 was expressed at higher levels in storage leaves at the germination and seedling stages than in other tissues. AsFMO1 and AsFMO2 protein structures are extremely similar, and both are expressed at the highest levels in leaves at the bulb expansion stage, it is predicted that AsFMO1 and AsFMO2 are key genes for alliin synthesis, and AsFMO1 and AsFMO2 were selected for subsequent experiments.

[0104] Example 4: Construction of AsFMO1 and AsFMO2 gene overexpression vectors

[0105] (1) Expansion of AsFMO1 and AsFMO2 gene fragments

[0106] Using the reverse transcribed cDNA as a template, the PCR amplification system (25 μL) was prepared according to Table 3. The AsFMO1 and AsFMO2 genes used their corresponding primer pairs (SEQ ID NO. 6-7 and SEQ ID NO. 8-9), and other conditions were the same:

[0107] Table 3 PCR reaction system preparation

[0108]

[0109] According to the reaction program in Table 4, the amplification was performed with 38 cycles of denaturation and extension:

[0110] Table 4 PCR reaction program

[0111]

[0112] After the reaction, add 2 μL 10× Loading Buffer to each sample, mix gently with a pipette, and electrophoresed in 1% agarose gel for 15 min. Observe the target bands with a gel imager and cut out the correct size for gel recovery.

[0113] (2) Connection and transformation of target fragment and expression vector

[0114] The AsFMO1 and AsFMO2 gene overexpression vectors were constructed with pCOMBIA3300-eGFP as the vector backbone, and transformed into Agrobacterium (the present invention adopts the freeze-thaw method to transform Agrobacterium). The DNA fragments of the AsFMO1 and AsFMO2 genes obtained by amplification were digested with restriction endonucleases BamHI and SmaI, and the fragments after digestion were ligated with T4 ligase. The operation steps were carried out according to the instructions of T4 DNA Ligase, a product of Thermo Fisher Scientific-CN, and then the ligation products were transformed into Escherichia coli DH5α competent cells, and cultured overnight on LB solid medium containing kanamycin (50 mg / L). White colonies were picked and cultured overnight in LB liquid medium containing kanamycin (50 mg / L). Plasmid DNA was extracted using the GoldHiEndoFree Plasmid Maxi Kit and sequenced. The correctly identified AsFMO1 gene overexpression vector was transformed into Agrobacterium LBA4404 competent cells to obtain Agrobacterium strains that could be used for transformation. The positive strains were preserved with glycerol for subsequent genetic transformation.

[0115] Example 5: Construction of CRISPR / Cas9 knockout vectors for AsFMO1 and AsFMO2 genes

[0116] (1) PCR amplification of target band

[0117] ① Using the SV-Cas9 vector as a template, amplify the target fragment by PCR, and test the PCR product by gel electrophoresis. According to the position of the band, cut out the target band gel for recovery.

[0118] ② Using the above gel recovery product as a template, amplify the target fragment by PCR. The PCR product is tested by gel electrophoresis. According to the position of the band, cut out the target band and recover it by gel.

[0119] (2) Single enzyme digestion and dephosphorylation of SV-Cas9 vector

[0120] ①In a PCR centrifuge tube, add reagents according to Table 5, mix well and react at 37℃ for 30 minutes.

[0121] Table 5 Enzyme digestion reaction system preparation

[0122]

[0123] ②After the above reaction is completed, add 1 μL FastAP, centrifuge briefly, react at 37°C for 15 min, and then react at 85°C for 5 s.

[0124] ③ After the reaction is completed, use the undigested plasmid as a control and run electrophoresis in 1% agarose gel for 15 minutes. Observe the enzyme cleavage status in a gel imager and cut the gel for recovery if the size is correct.

[0125] (3) Connection and transformation of target fragment and expression vector

[0126] The digested vector was connected to the fragment according to the LightNing product of Yugong Biotech. TM DNA Assembly Mix Plus instructions, then the ligation product was transformed into Escherichia coli DH5α competent cells, and cultured overnight on LB solid medium containing kanamycin (50 mg / L). Pick white colonies and culture them overnight in LB liquid medium containing kanamycin (50 mg / L). Use the plasmid DNA extraction kit GoldHiEndoFree Plasmid Maxi Kit to extract plasmid DNA, perform sequence determination, and transform the correctly identified AsFMO1 gene and AsFMO2 gene CRISPR / Cas9 knockout vectors into Agrobacterium LBA4404 competent cells, and obtain Agrobacterium strains that can be used for transformation, and use glycerol to preserve the positive strains for subsequent genetic transformation (the present invention adopts the freeze-thaw method to transform Agrobacterium).

[0127] Primer sequences involved in the above experiments:

[0128]

[0129] Example 6: Agrobacterium-mediated transformation of garlic root tips and acquisition of resistant plants

[0130] (1) Infection and co-cultivation

[0131] The day before infection, inoculate the preserved bacteria into 10 mL LB medium containing 50 mg / L kanamycin + 50 mg / L rifampicin, shake the bacteria at 28°C and 220 rpm for 12 h in the morning, transfer to a 50 mL system and shake overnight, collect the bacteria after 12 hours of culture, resuspend the bacteria in the pre-infection solution and set aside.

[0132] In the clean bench, take the garlic callus in good condition and place it in a sterile culture dish. Use a sterilized scalpel to cut the callus. Avoid cutting the callus too small. Use sterilized tweezers to place the cut garlic callus in the infection solution. Oscillate for 10 minutes to make the callus fully contact with the infection solution. Dry the filter paper and transfer the infected callus to the co-culture medium. Culture it in the dark at 25℃ for 3 days.

[0133] (2) Screening of resistant callus

[0134] Take the uncontaminated garlic callus and place it in a sterile centrifuge tube, rinse it with 400mg / L sterile water for 4-5 times, then rinse it with sterile water for 4-5 times, pour it into a sterile culture dish covered with dry filter paper, and absorb the residual sterile water. Transfer the above-treated callus to a screening medium containing 400mg / L cephalexin and 15mg / L herbicide Basta, and culture it in the dark at 25℃ for 15-20 days. After the culture is completed, transfer it to a screening medium containing 400mg / L cephalexin and 30mg / L herbicide Basta, and culture it in the dark at 25℃ for 15-20d.

[0135] (3) Differentiation of resistant callus and transplanting

[0136] In the clean bench, discard the browned callus tissue, transfer the uninfected callus tissue to the differentiation medium, culture it under light for 15-30 days, transfer it to the rooting medium after the adventitious buds grow, culture it under light until 3-4 roots grow, and then transplant it to the nutrient soil (in the nutrient soil, the mass ratio of perlite, vermiculite and peat soil is 1:3:6) to obtain the resistant plants.

[0137] The regeneration process of transformed plants is shown in Figure 7 .

[0138] Example 7: Identification of transgenic plants

[0139] (1) Extraction of garlic genomic DNA (CTAB method)

[0140] ① Preheat 2% CTAB in a water bath to 65°C and place the garlic material in a centrifuge tube.

[0141] ② After grinding and crushing the garlic material with liquid nitrogen, add 600 μL of 2% CTAB and mix well. Place in a 65°C water bath for 30 min, inverting every 5-8 min.

[0142] ③ Add 500 μL of the mixed solution in the fume hood, in which the volume ratio of chloroform to isoamyl alcohol is 24:1, and mix well.

[0143] ④ Centrifuge at 13000rpm for 10min and transfer the supernatant to a new 1.5mL centrifuge tube.

[0144] ⑤Repeat steps ③ and ④.

[0145] ⑥ Add 400 μL of isopropanol, invert to mix, and place at -20°C for 20 min.

[0146] ⑦ Centrifuge at 13000rpm for 10min and discard the supernatant.

[0147] ⑧ Add 1 mL of 75% ethanol, mix thoroughly and wash the precipitate, aspirate the ethanol, centrifuge at 7500 rpm for 5 min, and discard the remaining ethanol.

[0148] ⑨Repeat step ⑧ to blow dry the precipitate.

[0149] ⑩ Add 50-100 μL ddH2O according to the amount of precipitate, dissolve and store at 4°C overnight.

[0150] (2) Identification of asfmo1 and asfmo2 mutants

[0151] ① After obtaining T0 generation resistant plants, the garlic genome was extracted using the CTAB method, and PCR molecular detection was performed using the unique Bar marker gene on the vector. The detection primers were:

[0152] BlpR-F: 5'-ATGAGCCCAGAACGACGCC-3' (SEQ ID NO.42)

[0153] BlpR-R: 5'-AATCTCGGTGACGGGCAGGA-3' (SEQ ID NO.43)

[0154] ② Design specific primers upstream and downstream of the target site of the AsFMO1 genomic sequence:

[0155] Target-F: 5'-ATGTAGCAATTATTGGCGCCG-3'(SEQ ID NO.44)

[0156] Target-R: 5'-CCTAGGCTCAGTATAATGCCCAT-3' (SEQ ID NO.45)

[0157] ③ Design specific primers upstream and downstream of the target site of the AsFMO2 genomic sequence:

[0158] Target-2F: 5'-TGGGCAATGGCTATATGAA-3' (SEQ ID NO.46)

[0159] Target-2R: 5'-TCGAGTAATGTCCATTGGCCA-3' (SEQ ID NO.47)

[0160] Then, PCR amplification was performed to obtain the genomic fragments containing AsFMO1 and AsFMO2 (PCR reaction system and procedure are shown in Tables 8 and 9). The PCR product was directly sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After the results were obtained, the sequencing results were compared with the wild-type sequence. Analysis of the mutation type was performed using DNAMAN to compare the sequence with the sequence in the NCBI database and screen for homozygous mutants.

[0161] ③The relative expression of T0 generation transgenic plants was determined by qRT-PCR method.

[0162] (3) Identification of overexpressing transgenic plants

[0163] ① After obtaining T0 generation resistant plants, the garlic genome was extracted using the CTAB method, and PCR molecular detection was performed using the unique eGFP marker gene on the vector. The detection primers were:

[0164] eGFP-F: 5'-ATGGTGAGCAAGGGCGAGG-3' (SEQ ID NO.48)

[0165] eGFP-R: 5'-CTTGTACAGCTCGTCCATGC-3' (SEQ ID NO.49)

[0166] Prepare PCR amplification system (15 μL) according to Table 6:

[0167] Table 6 Identification PCR reaction system preparation

[0168]

[0169]

[0170] According to the reaction program in Table 7, perform 30 cycles of denaturation and extension for amplification:

[0171] Table 7 Identification PCR reaction program

[0172]

[0173] After the reaction, electrophoresis was performed in 1% agarose gel for 15 min, and the size of the target band was observed on a gel imager to see if it was correct.

[0174] ②The relative expression of T0 generation overexpression transgenic plants was determined by qRT-PCR method.

[0175] Example 8: Analysis of the expression levels of AsFMO1 and AsFMO2 genes in positive transgenic plants

[0176] RNA was extracted from young leaves of T0 transgenic lines and untransformed plants and reverse transcribed, and the expression levels of AsFMO1 and AsFMO2 genes were detected by fluorescence quantitative PCR.

[0177] The results are as follows Figure 8As shown, in terms of the expression levels of AsFMO1 and AsFMO2 genes, compared with the untransformed plants, the expression of AsFMO1 gene in asfmo1-1 and asfmo1-2 mutant strains was significantly downregulated, and the expression of AsFMO2 gene in asfmo2-1 and asfmo2-2 mutant strains was significantly downregulated. The expression of AsFMO1 overexpression strain was upregulated by 3 times, and the expression of AsFMO2 overexpression strain was upregulated by 2.5 times.

[0178] Example 9: Analysis of alliin content in positive transgenic plants

[0179] Untransformed plants with the same growth period were selected as the control group and transgenic plants (asfmo mutant strain, OE overexpression strain) were selected as the experimental group to determine the alliin content (three biological replicates).

[0180] (1) Preparation of alliin standard solution

[0181] Accurately weigh the alliin standard, dissolve it in water, and dilute it into 6 standard solutions with different mass concentrations, namely 0.4 μg / mL, 1.0 μg / mL, 4.0 μg / mL, 10.0 μg / mL, 40.0 μg / mL, and 100.0 μg / mL.

[0182] (2) Sample pretreatment

[0183] Weigh about 0.4g of sample, add 0.5mL of water and boil for 15min to inactivate enzyme, grind into slurry after cooling, add 20% ethanol solution to 5mL, ultrasonically extract for 2h, centrifuge at 8000g for 10min, remove supernatant, dilute to 5mL with 20% ethanol, filter membrane for derivatization.

[0184] (3) Liquid chromatography analysis conditions of alliin

[0185] ①Chromatograph: Agilent 1100 high performance liquid chromatograph, detection wavelength is 214 nm.

[0186] ②Chromatographic column: Compass C18; reversed phase chromatography column (250mm*4.6mm, 5μm).

[0187] ③Column temperature: 30℃.

[0188] ④Flow rate: 0.8mL / min.

[0189] ⑤Injection volume: 5μL.

[0190] ⑥Mobile phase: water: methanol = 70:30 (V / V).

[0191] (4) Determination of standard curve

[0192] The peak area of ​​each standard solution was detected in turn according to the above chromatographic conditions, with the peak area as the ordinate and the concentration as the abscissa; the standard curve, linear range and correlation coefficient of alliin were calculated.

[0193] The results are as follows Fig. 9 As shown in the results, the liquid chromatography and alliin content analysis of the control group and the experimental group showed that the alliin content of the asfmo1 mutant line was 37% lower than that of the control plant, and the alliin content of the asfmo2 mutant line was 27% lower than that of the control plant; the alliin content of the AsFMO1 overexpression line was 82% higher than that of the control plant, and the alliin content of the AsFMO2 overexpression line was 50% higher than that of the control plant. The results showed that the AsFMO1 and AsFMO2 genes are key genes for alliin biosynthesis.

[0194] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A flavin monooxygenase gene, characterized in that: The nucleotide sequence of the flavin monooxygenase gene is shown as SEQ ID NO.

2.

2. Use of overexpression of the flavin-containing monooxygenase gene of claim 1 in promoting alliin synthesis in garlic; or, use of silencing or knocking out the flavin-containing monooxygenase gene of claim 1 in inhibiting alliin synthesis in garlic.

3. A protein encoded by a flavin monooxygenase gene, characterized in that: The amino acid sequence of the protein encoded by the flavin monooxygenase gene is shown in SEQ ID NO.

51.

4. Application of enhancing the expression of the protein encoded by the flavin monooxygenase gene according to claim 3 in promoting the synthesis of alliin in garlic; or, application of weakening the expression of the protein encoded by the flavin monooxygenase gene according to claim 3 in inhibiting the synthesis of alliin in garlic.

5. An expression cassette comprising the flavin monooxygenase gene according to claim 1.

6. A recombinant expression vector containing the flavin monooxygenase gene according to claim 1.

7. A recombinant bacterium containing the flavin monooxygenase gene according to claim 1.

8. Use of the expression cassette of claim 5, the recombinant expression vector of claim 6 or the recombinant bacterium of claim 7 in promoting or inhibiting the synthesis of alliin in garlic, characterized in that: Overexpressing the flavin monooxygenase gene of claim 1 to promote alliin synthesis in garlic; or silencing or knocking out the flavin monooxygenase gene of claim 1 to inhibit alliin synthesis in garlic.

9. A method for regulating the synthesis of alliin in garlic, characterized in that: include: Promoting the expression of flavin monooxygenase genes in garlic to obtain garlic plants with increased alliin content; Alternatively, the flavin monooxygenase gene in garlic can be silenced or knocked out to obtain garlic plants with reduced alliin content; The flavin monooxygenase gene is transferred into garlic via an expression vector; The flavin monooxygenase gene is a nucleic acid molecule as shown in the following i) or ii): i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.51 other than i).

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