CN116970625A

CN116970625A - Functional identification and application of garlic AsFMO1 gene

Info

Publication number: CN116970625A
Application number: CN202311160993.4A
Authority: CN
Inventors: 杨彩霞
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-09-11
Filing date: 2023-09-11
Publication date: 2023-10-31

Abstract

The application discloses a key gene AsFMO1 participating in alliin biosynthesis and functional identification and application thereof, belonging to the technical field of plant biology. The application provides a garlic genetic transformation method, and verifies that the AsFMO1 gene is a key gene for alliin biosynthesis through the overexpression of the AsFMO1 gene and CRISPR/Cas9 gene editing technology. The obtained transgenic garlic with high alliin makes a certain contribution to garlic breeding work and application of alliin as a medical intermediate, and has a certain practical application value.

Description

Functional identification and application of garlic AsFMO1 gene

Technical Field

The application relates to functional identification and application of alliin biosynthesis key gene AsFMO 1. Belongs to the fields of molecular biology and biotechnology.

Background

Garlic is an important seasoning and medicinal plant, and alliin is only sulfur-containing amino acid in garlic in nature, and is one of main bioactive substances in garlic. Alliin has unique pharmacological actions, including anti-inflammatory, blood sugar reducing, anti-tumor, blood lipid reducing, liver protecting and the like. Studies have shown that alliin synthesized de novo is preferentially produced in chloroplast-containing cells, so that garlic leaves are the main site of alliin synthesis, and alliin is transported to bulbs and the like by the vascular system with the shift of growth cycle.

Since allinase only uses S-allyl-L-cysteine sulfoxide, and does not use the biosynthesized intermediate sulfide as a substrate to hydrolyze to generate various sulfur-containing compounds with biological activity, the S-oxidation of the intermediate sulfide is one of the important steps in the biosynthesis of S-allyl-L-cysteine sulfoxide. Flavin-containing monooxygenases (FMO; EC 1.14.13.8) are responsible for catalyzing stereoselective S-oxidation reactions, relying on flavin adenine dinucleotide (FAD, flavin adenine dinucleotide) and reduced nicotinamide adenine dinucleotide phosphate (NADPH, nicotinamide adenine dinucleotide phosphate).

At present, little is known about the molecular mechanism of alliin biosynthesis, and related enzymes and complex molecular regulatory mechanisms in the synthesis process still need to be further explored and studied. The research proves that the AsFMO1 is a key gene for alliin biosynthesis through cloning the AsFMO1, performing qRT-PCR analysis, subcellular localization, constructing an AsFMO1 over-expression strain and a knockout mutant by using an agrobacterium-mediated garlic genetic transformation method, performing functional verification and application on the AsFMO1 by using an alliin content measurement method and the like.

Disclosure of Invention

The application aims to disclose a key gene AsFMO1 participating in alliin biosynthesis and functional identification and application thereof.

The application provides an agrobacterium-mediated garlic genetic transformation method, which is used for transferring an AsFMO1 gene into garlic to cause the alliin content to change obviously.

The first aim of the application is to provide a key gene AsFMO1 for alliin biosynthesis, the nucleotide sequence of the AsFMO1 gene is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.

Further, the gene CDS sequence in the Laiwu white garlic is amplified to construct an over-expression vector, and the AsFMO1 protein is found to be positioned in cytoplasm and nucleus through transient infection of tobacco leaf cells by agrobacterium.

Furthermore, the AsFMO1 gene is shown by qRT-PCR to have higher expression level mainly in leaves in the bulb expansion period.

Further, the gene codes for a Flavin-containing monooxygenase monooxygenase, regulating the synthesis of alliin in Allium plants; regulating and controlling alliin content.

The second object of the application is to provide an application of alliin biosynthesis key gene AsFMO 1.

Further, the gene CDS sequence in the Laiwu white garlic is amplified to construct an over-expression vector, and the transgenic plant obtained by an agrobacterium-mediated genetic transformation method can greatly improve the gene expression level and alliin content.

Further, by designing a double-target CRISPR/Cas9 gene editing vector, the obtained frameshift mutant has the characteristics of reduced gene expression level and obviously reduced alliin content.

Furthermore, plants with high alliin content are created by editing AsFMO1 genes, and the material can be used for producing and processing alliin serving as a medical intermediate.

The application also provides a recombinant expression vector containing the AsFMO1 gene sequence, a primer sequence and recombinant bacteria, and any recombinant expression vector containing the gene sequence also belongs to the protection scope of the application.

The beneficial effects are that:

the application clones a gene which can regulate the biosynthesis of alliin. The application provides application of AsFMO1 in garlic breeding and application of alliin as a medical intermediate. The alliin content of the gene mutant is obviously reduced; meanwhile, the alliin content of the gene overexpression strain is obviously up-regulated, and the gene can be used as a material for producing and processing alliin medical intermediates.

(IV) description of the drawings

FIG. 1 is a diagram showing the expression of AsFMO1 gene in different garlic tissues

A represents germination period, B represents seedling stage, C represents bulb expansion period

FIG. 2 is a map of subcellular localization of AsFMO1 protein

FIG. 3 is a construction diagram of AsFMO1 gene overexpression vector

A represents an AsFMO1 gene amplification diagram, and M represents a molecular weight marker; b represents an AsFMO1 gene overexpression vector bacterial liquid PCR identification agarose gel electrophoresis diagram, 1-6 represents an AsFMO1 gene band amplified in bacterial liquid PCR, and 7 represents a negative control; c represents AsFMO1 gene over-expression vector schematic diagram

FIG. 4 is a construction diagram of the AsFMO1 gene CRISPR/Cas9 vector

A represents a target position schematic diagram; b represents a agarose gel electrophoresis diagram of a PCR amplification target spot, and M represents a molecular weight marker; c represents agarose gel electrophoresis diagram of PCR amplification target; d represents AsFMO1 gene CRISPR/Cas9 carrier bacterial liquid PCR identification agarose gel electrophoresis diagram, 1-8 represents bacterial liquid PCR positive clone, 9 represents negative control

FIG. 5 is a diagram showing the regeneration process of transformed plants

A represents a garlic bulb germination map, B represents a callus map formed on a callus induction medium, C represents a callus subculture map, D represents a co-culture map after infection, E represents a resistance callus screening map, F represents an adventitious bud development map on a differentiation medium, G represents a sterile seedling rooting map on a rooting medium, and H represents a sterile seedling transplanting map

FIG. 6 is a PCR detection of positive transgenic plants

M represents molecular weight marker, 1 represents recombinant plasmid SV-Cas9-AsFMO1-Bar positive control, 2-3 represents different knockout resistant strains, 4 represents negative control, 5 represents untransformed plant, 6 represents recombinant plasmid pCOMBIA3300-AsFMO1-eGFP positive control, 7-9 represents different over-expression resistant strain, 10 represents negative control, 11 represents untransformed plant

FIG. 7 is an analytical view of T0 generation mutant plants

A represents PCR amplification of AsFMO1 fragment containing target site, M represents molecular weight marker, 1 represents AsFMO1-1 mutant genome, and 2 represents AsFMO1-2 mutant genome; b represents an AsFMO1 target sequence peak diagram; c represents an AsFMO1 target sequence alignment chart; d represents the amino acid sequence of AsFMO1 mutant protein

FIG. 8 is a diagram showing the expression of AsFMO1 gene in transgenic plants

FIG. 9 is a chart showing alliin content measurement

A represents an alliin chromatogram of an untransformed plant, B represents an alliin chromatogram of an asfmo1 mutant strain, C represents an alliin chromatogram of an OE over-expression strain, and D represents the content of alliin.

(fifth) detailed description of the application

The application will be further described with reference to specific examples. It is clear that these embodiments are merely illustrative and do not limit the scope of the application in any way, including but not limited to the following examples, modifications or optimizations of the details of the application are within the scope of the application.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Unless explicitly stated otherwise, the experimental methods mentioned in the examples below are conventional.

Unless explicitly stated otherwise, the biochemical reagents mentioned in the examples below are conventional reagents.

Example 1: expression profiling of the garlic AsFMO1 Gene

1. Analysis of expression of AsFMO1 Gene in different tissues of Garlic

qRT-PCR was performed with the primers AsFMO1-F-qPCR and AsFMO1-R-qPCR using the Laiwa garlic cDNA in germination, seedling and bulb expansion phases as templates (FIG. 1). The AsFMO1 gene was found to be expressed in all 3 stages, and the expression level of AsFMO1 was higher in leaves at the early, late and mature stages of bulb expansion, especially in leaves at the mature stage of bulb expansion, whereas the expression level was very low in white leaves in bulbs at the sprouting and seedling stages.

2. AsFMO1 protein subcellular localization analysis

A fusion expression vector 35S of AsFMO1 gene and 35S of GFP gene are used for constructing the fusion expression vector 35S of AsFMO1-GFP, GV3101 agrobacterium is transformed, tobacco leaf cells are transiently infected by the agrobacterium, and after illumination culture for 2-3 d, LMS880 laser confocal microscopy is used for observation (FIG. 2). GFP signals in tobacco epidermal cells are observed through a confocal laser fluorescence microscope, and the subcellular localization of the AsFMO1-GFP fusion protein is found that the AsFMO1 protein is mainly localized in cytoplasm and nucleus.

The expression profiling method described above is as follows:

1. real-time fluorescent quantitative PCR

(1) Extraction of garlic total RNA

(1) Wrapping the cleaned and wiped mortar, grinding rod and medicine spoon with tinfoil, and placing into oven at 180deg.C for 4 hr. After a 1.5mL centrifuge tube and a gun head used in the RNA extraction process are wrapped by newspapers, sterilizing by high-pressure steam at 121 ℃ for 50min, and drying for later use after sterilization;

(2) pre-cooling a mortar and a grinding rod by liquid nitrogen in advance, adding 100-200 mg of garlic tissue samples, quickly grinding the garlic tissue samples into powder by liquid nitrogen, and quickly transferring the powder samples into a pre-cooled centrifuge tube by using a pre-cooled medicine spoon;

(3) immediately adding 1mL of precooled Trizol and 10 mu L of beta-mercaptoethanol, vortex shaking for 1-2 min, mixing uniformly, standing for 15min, reversing a few centrifuge tubes every 1min, and fully mixing the sample and the Trizol;

(4) centrifuging 12000g at 4deg.C for 15min, transferring supernatant to a new centrifuge tube, adding 300 μl chloroform, shaking vigorously for 15s (inhibiting vortex), and standing for 15min;

(5) centrifuging 12000g at 4deg.C for 15min, transferring supernatant to a new centrifuge tube, adding 450 μl isopropanol to precipitate nucleic acid, mixing upside down, and standing at-20deg.C for 20min;

(6) centrifuging at 4deg.C for 15min at 12000g, discarding supernatant, adding 1mL 75% ethanol (prepared with DEPC water), repeatedly reversing, washing precipitate, and sucking ethanol;

(7) repeating washing the precipitate once, sucking out the ethanol, centrifuging at 4 ℃ for 5min, carefully sucking out the residual ethanol, and drying the precipitate in an ultra-clean workbench for 5min;

(8) adding 20-100 mu L DEPC water for re-dissolving according to the amount of precipitation, split charging 10 mu L after dissolving, detecting the quality and concentration of the subsequent RNA, and storing the residual RNA sample at-80 ℃.

(2) RNA concentration and quality detection

(1) Concentration detection

OD determination with Nanodrop 2000 using DEPC water as a blank ₂₆₀ /OD ₂₃₀ 、OD ₂₆₀ /OD ₂₈₀ Values were recorded for RNA concentration. OD of RNA ₂₆₀ /OD ₂₈₀ The ratio should be between 1.8 and 2.0;

(2) integrity detection

1000ng of RNA was placed in a pretreated PCR tube, and 2. Mu. L RNA Loading Buffer was added to the tube for agarose gel electrophoresis, and the electrophoresis band was observed. At least 2 bands of 28sRNA and 18sRNA, respectively, should be visible in the RNA solution of good quality. The 28sRNA band intensity of the completely undegraded RNA sample should be twice that of the 18sRNA band intensity. If the 5sRNA band appears at the bottom end and the brightness exceeds the first two bands, or the tail of the bands is serious, the degradation of RNA is indicated.

(3) cDNA Synthesis

And selecting RNA with qualified extraction quality for subsequent reverse transcription experiments. cDNA synthesis experiments were performed using a reverse transcription kit, the reaction system was as follows:

(1) removal of genomic DNA from total RNA of plants:

the reaction system was prepared according to the following table, gently pipetted and stirred uniformly and centrifuged briefly. Reacting at 42 ℃ for 2min;

(2) synthesis of first strand of cDNA:

reverse transcriptase was added to the reaction liquid of step 1 according to the following table, gently pipetted and stirred uniformly and centrifuged briefly. Reacting at 50 ℃ for 15min, and then reacting at 85 ℃ for 5s;

(4) Real-time fluorescent quantitative PCR

(1) The reaction system was prepared using the cDNA obtained by reverse transcription as a template according to the following table:

(2) qRT-PCR procedure denaturation to extension cycle 40 times, PCR amplification according to the following table:

2. subcellular localization

(1) PCR reaction system and reaction conditions

Specific primer sequences (AsFMO 1-F and AsFMO 1-R) are designed according to the CDS sequence of the AsFMO1 gene, and PCR amplification is carried out by taking the Litsea lycra cDNA as a template and adopting Phanta Max Super-Fidelity DNA Polymerase, wherein the specific reaction system is as follows:

the PCR amplification reaction was performed as follows:

(2) PCR product band detection and sequencing

At the same time as PCR amplification, a 4% agarose gel was prepared. The size of the band was measured by 4% agarose gel, 5. Mu.L of standard DNA Marker was added to compare the DNA size of the amplified product, DNA was recovered, ligated into expression vectors, the vectors were transformed into Escherichia coli DH. Alpha. E.coli competent cells, and positive clones were screened for sequencing.

3. Transient transformation of tobacco epidermal cells

(1) Thawing strain preserved at-80deg.C on ice, sucking 100 μl of strain solution, inoculating to 1mL of YEP liquid culture medium containing Kan and Rif, and shaking culturing in shaking table at 28deg.C overnight;

(2) 1mL of the bacterial liquid is inoculated into 10mL of YEP liquid culture medium containing Kan and Rif, and shake culture is carried out in a shaking table at 28 ℃ for overnight;

(3) transferring 1-2 mL of the bacterial liquid into 50mL of YEP liquid culture medium containing Kan and Rif, shaking and culturing overnight in a shaking table at 28 ℃ until OD is reached ₆₀₀ ＝1.0；

(4) Centrifuging at 5000rpm for 5min, discarding supernatant, and collecting thallus;

(5) adding 3mL MMA (AS) into the tube for sucking and resuspending, and standing for 3h at room temperature in a dark environment;

(6) after the completion of standing, the mixture was centrifuged at 5000rpm for 5 minutes, the supernatant was discarded, the cells were resuspended in 1mL of MMA, 30mL of MMA was added and mixed upside down, and the OD was obtained ₆₀₀ The value is adjusted to 0.8;

(7) selecting tobacco leaves with less veins and growing days reaching about 30d, injecting bacterial liquid with the adjusted light absorption value from the back of the leaves by using a small injector (a needle is removed), and enabling the injection port to be attached to the leaves as much as possible so that the bacterial liquid can fully enter;

(8) culturing for 2-3 d under illumination, tearing the tobacco epidermis in distilled water to prepare slices, observing fluorescent signals by using a laser confocal microscope, and photographing and recording.

Amplification and sequencing of candidate genes

Primer sequences involved in the above experiments:

example 2: acquisition and identification of transgenic plants

1. Identification of overexpressing plants

The protein coding region (CDS) of the AsFMO1 gene was amplified using specific primers and ligated into the vector transformed with pCOMBIA3300-eGFP (fig. 3) and transformed with lauca white garlic by agrobacterium-mediated genetic transformation technique (fig. 5). Screening positive plants were identified by resistance screening and PCR (fig. 6), and the expression level of AsFMO1 gene in T0 generation overexpressing lines was significantly up-regulated relative to the expression level of untransformed plant genes (fig. 8).

2. Knock-out strain identification

Meanwhile, a CRISPR/Cas9 knockout vector was constructed (fig. 4), two targets containing the targeted AsFMO1 gene were introduced into the SV-Cas9 vector, and the lauchi garlic was transformed by agrobacterium-mediated genetic transformation technique (fig. 5). 2 mutant lines (AsFMO 1-1, asFMO 1-2) (FIG. 6) were screened by PCR identification and amplification sequencing (FIG. 7), and the expression level of the asFMO1 gene in the T0 generation mutant line was significantly down-regulated relative to the expression level of the untransformed plant gene (FIG. 8).

The experimental methods described above are as follows:

1. recombinant expression vector construction

(1) Construction of overexpression vector

(1) Primer design: the CDS sequence of the gene is downloaded at NCBI (https:// www.ncbi.nlm.nih.gov /) site, and appropriate cleavage sites are selected from the expression vector plasmid multiple cloning sites according to the cleavage sites in the CDS sequence and added to the 5' ends of the forward and reverse amplification primers. In the experiment, two enzyme cutting sites of BamHI and SmaI are selected, and a primer pair is used: OE-AsFMO1-F; OE-AsFMO1-R;

(2) carrying out PCR amplification by using Phanta Max Super-Fidelity DNA Polymerase and taking cDNA of Laiwu white garlic as a template, carrying out PCR electrophoresis detection and product recovery after the amplification is completed, connecting the mixture into a cloning vector, and sequentially carrying out competent cell conversion of escherichia coli, screening positive colonies by escherichia coli colony PCR, sequencing a fungus sample and plasmid extraction;

(3) after the plasmid extraction is completed, respectively carrying out enzyme digestion on the expression vector plasmid and the cloning vector plasmid by using restriction enzymes of the selected enzyme digestion sites, and then carrying out electrophoresis detection and product recovery;

(4) and (3) connecting the target DNA fragment into an expression vector, and sequentially carrying out escherichia coli competent cell transformation, escherichia coli colony PCR screening positive colonies, bacterial sample sequencing and plasmid extraction.

(2) CRISPR/Cas9 gene editing vector construction

(1) Primer design: designing a target point by utilizing an online website SSC, selecting a sequence which is high in predicted editing efficiency and located in a coding region as the target point, and further verifying the specificity of the target point through BLAST comparison of garlic whole genome and using the target point for primer synthesis;

(2) the SV-Cas9 vector is used as a template, primers MT1T2-F and MT1T2-R are used for PCR amplification of target fragments, PCR products are checked by gel electrophoresis, and target band gel is cut off and recovered according to the band position. And then, taking the gel recovery product as a template, carrying out PCR (polymerase chain reaction) secondary amplification on the target fragment by using primers CAP-F and CAP-R, checking the PCR product by gel electrophoresis, and cutting off the target gel and recovering according to the position of the gel. Reacting the recovered PCR product with the digested SV-Cas9 carrier in a PCR instrument at 50 ℃ for 50min;

the system is as follows:

(3) after the enzyme digestion connection reaction is completed, E.coli competent cell transformation, E.coli colony PCR screening positive colony, bacterial sample sequencing and plasmid extraction are sequentially carried out.

2. Acquisition of recombinant Agrobacterium

The LBA4404 Agrobacterium competent cells (20 mu L) were put on ice to melt, after complete melting, 10 mu L of the recombinant vector was added, gently sucked and stirred with a pipette to mix well, and then placed on ice for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min, and ice bath for 5min. mu.L of YEP liquid medium was added thereto, and the mixture was cultured at 28℃for 2 to 3 hours at 200 rpm. 6000rpm, centrifuging for 1min, discarding the supernatant in an ultra-clean workbench, lightly sucking and beating the supernatant remained in the centrifuge tube by using a liquid-transfering device, uniformly smearing the supernatant on a YEP solid culture medium with antibiotics containing kanamycin and rifampicin, and culturing for 2-3 d in a culture box at 28 ℃ in an inverted manner. Colony PCR is used for screening positive strains.

3. Agrobacterium-mediated garlic genetic transformation

(1) Placing the healthy and undamaged garlic cloves serving as explants in a refrigerator at 4 ℃ for one month for dormancy breaking, peeling off outer protective leaves of the dormancy breaking explants and removing brown garlic heels at the base, soaking the outer protective leaves in a super clean workbench for 15min, pouring out 75% of ethanol, washing the outer protective leaves with sterile water, sterilizing the outer protective leaves for 30min with 4% of sodium hypochlorite, discarding the 4% of sodium hypochlorite, washing the outer protective leaves with sterile water for 3-5 times, placing the sterilized outer protective leaves in a gun head box containing a proper amount of sterile water, culturing the outer protective leaves in dark at 25 ℃ for 7d until root tips grow out, cutting the root tips of the outer protective leaves into a callus induction culture medium by using a sterilized scalpel, and culturing the outer protective leaves in dark at 25 ℃ for 4 weeks to obtain callus with the diameter of 5-10 mm; the callus induction medium comprises the following raw materials with the following concentration: MS,1 g/L2, 4-D,1 mg/L6-BA, 30g/L sucrose, 7g/L agar;

(2) Soaking the callus in an infectious microbe liquid to obtain infected callus; the infection bacterial liquid is an activated agrobacterium bacterial liquid carrying recombinant plasmids;

(3) Sucking the liquid on the surface of the infected callus, spreading on a co-culture medium, performing dark culture for 3d, transferring to a first-sieve culture medium, performing dark culture for 15-20 d, and transferring to a second-sieve culture medium, performing dark culture for 15-20 d to obtain the resistant callus; the co-culture medium comprises the following raw materials in concentration: MS,1 mg/L2, 4-D,1 mg/L6-BA, 30g/L sucrose, 7g/L agar, 100. Mu.M AS; the first-sieve culture medium comprises the following raw materials in concentration: MS,1 mg/L2, 4-D,1 mg/L6-BA, 30g/L sucrose, 7g/L agar, 400mg/L Cef,15mg/L Basta; the two-sieve culture medium comprises the following raw materials in concentration: MS,1 mg/L2, 4-D,1 mg/L6-BA, 30g/L sucrose, 7g/L agar, 400mg/L Cef,30mg/L Basta;

(4) Inoculating the resistant callus on a differentiation medium for induction culture to obtain adventitious buds; the differentiation medium comprises the following raw materials in concentration: MS/SH,3 mg/L6-BA, 30g/L sucrose, 7g/L agar;

(5) Inoculating the adventitious buds on a rooting culture medium for rooting culture to obtain transgenic seedlings; the rooting culture medium comprises the following raw materials in concentration: MS,30g/L sucrose, 7g/L agar;

(6) Transplanting the transgenic seedlings into nutrient soil (perlite: vermiculite: peat soil=1:3:6) for culture.

Preferably, the pH of the callus induction medium in step (1) is 5.8-6.0.

Preferably, the soaking time in the step (2) is 10min, and the soaking period needs to be continuously oscillated.

Preferably, the preparation method of the infectious microbe liquid in the step (2) comprises the following steps:

(1) Thawing agrobacterium strain stored at-80deg.C and carrying recombinant plasmid on ice, sucking 100 μL of bacterial liquid, inoculating to 1mL of YEP liquid culture medium, placing in shaking table at 28deg.C for shake culture overnight, sucking 1mL of bacterial liquid, inoculating to 10mL of YEP liquid culture medium, and shake culturing in shaking table at 28deg.C for overnight to obtain enlarged bacterial liquid; the YEP liquid medium comprises 50 mug/mL kanamycin and 100 mug/mL rifampicin;

(2) Transferring 1-2 mL of the expanded bacterial liquid into 50mL of YEP liquid culture medium, shaking and culturing overnight in a shaking table at 28 ℃ until the bacterial liquid reaches OD ₆₀₀ Obtaining activated bacterial liquid with the concentration of 0.6-0.8; the YEP liquid medium comprises 50 mug/mL kanamycin and 100 mug/mL rifampicin;

(3) Centrifuging the activated bacterial liquid, and collecting bacterial bodies;

(4) Re-suspending the thalli by using an infection liquid, centrifuging, collecting thalli, and diluting to OD by using the infection liquid ₆₀₀ =0.4 to 0.7. The dyeing liquid comprises the following raw materials in concentration: 4.42g/L MS, 10g/L sucrose, 10g/L glucose, 0.04g/L L-cysteine, 100. Mu.M AS.

Preferably, the dark culture temperature in the step (3) is 25 ℃, and the pH of the co-culture medium, the first-sieve culture medium and the second-sieve culture medium is 5.8-6.0.

Preferably, the temperature of the adventitious bud development culture in the step (4) is 25 ℃, the photoperiod is 12 hours of illumination, the photoperiod is 12 hours of light shielding, and the illumination intensity is 200 mu mol.m ^-2 ·s ^-1 。

Preferably, the rooting culture in the step (5) has a temperature of 25 ℃, a photoperiod of 12 hours of illumination and 12 hours of light shielding, and an illumination intensity of 200 mu mol.m ^-2 ·s ^-1 。

4. Identification of transgenic plants

Extracting the obtained T ₀ The generation transgenic strain DNA is used for identifying positive plants through specific primer sequences (specific primers used by the over-expression strain are eGFP-F and eGFP-R, and specific primers used by the knockout strain are BlpR-F and BlpR-R), and simultaneously amplifying DNA fragments containing two targets of the knockout strainThe gene editing situation was analyzed by sequencing. And determining the relative expression condition of the T0 generation transgenic plant by a qRT-PCR method.

Primer sequences involved in the above experiments:

example 3: determination of alliin content

The untransformed plants with the same growth period are selected as a control group and transgenic plants (asfmo 1 mutant line and OE over-expression line) as experimental groups to measure the alliin content (figure 9), which shows that the alliin content of the mutant line is reduced by one third compared with the control plant, and the alliin content in the OE over-expression line is about twice that of the control plant. The AsFMO1 gene is shown to regulate the synthesis of alliin, and is a key gene for the biosynthesis of alliin.

The experimental procedure described above is as follows:

1. preparation of alliin standard solution

Accurately weighing alliin standard substances, dissolving with water, and diluting into 6 standard solutions with different mass concentrations of 0.4 mug/mL, 1.0 mug/mL, 4.0 mug/mL, 10.0 mug/mL, 40.0 mug/mL and 100.0 mug/mL respectively.

2. Sample pretreatment

About 0.4g of sample is weighed, 0.5mL of water is added for boiling for 15min for enzyme deactivation, the mixture is ground into slurry after cooling, 20% ethanol solution is added for constant volume to 5mL, ultrasonic leaching is carried out for 2h,8000g of centrifugation is carried out for 10min, and the supernatant is taken out. The volume was fixed to 5mL with 20% ethanol and the membrane was filtered to be derivatised.

3. Conditions for liquid chromatography of alliin

(1) Chromatograph: agilent 1100 high performance liquid chromatograph, detection wavelength is 214nm;

(2) Chromatographic column: compass C18 (2) reverse phase chromatography column (250 mm. Times.4.6 mm,5 μm);

(3) Column temperature: 30 ℃;

(4) Flow rate: 0.8mL/min;

(5) Sample injection volume: 5. Mu.L;

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

4. Determination of a Standard Curve

Detecting peak areas of the standard solutions in sequence according to the chromatographic conditions, wherein the peak areas are taken as ordinate, and the concentrations are taken as abscissa; and calculating to obtain the standard curve of alliin, the linear range and the correlation coefficient.

Sequence listing

Functional identification and application of garlic AsFMO1 gene

CDS sequence of SEQ ID No.1 Garlic AsFMO1 Gene

DNA

1374bp

ATGGTTTCCTCATCTTGCTCGTCCATTCCCAAGATGCCCGTCACCCCCCTGTCACTCGTCACCCG

CCATGTAGCAATTATTGGCGCCGGCGCAGCTGGTCTCGTCACCGCCCGTGAGCTCCGCCGCGAA

GGCCATACAACCACTATTTTCGAACGCGGATCTTCAATCGGTGGAACATGGATCTACACGCCTG

ATACAGAACCCGACCCGATGAGCCAAGATTCGTCCCGACCTATAGTACACTCCAGCCTGTACAA

GTCGTTGCGAACGAACTTACCGCGCGAAGTGATGGGCTTTCTCGATTATCCTTTTGTGGAGAAA

ACGAACGGCGGAGATCGGAGGAGGTTTCCAGGACACGAAGAGGTGTTAGATTATTTGGAGAGG

TTCGGGAGGGAATTTGGGGTATCCAGAGAAGTGGGTATGGAAAAAGAGGTGGTTAGGGTTGAT

ATGGAGCAGGGCGGGAAATGGACGGTCAAATGGAAGGGAAAAGATGGAGGCGGCGGCGAGG

AGGGTTTTGATGCGGTGGTTGTTTGCAATGGGCATTATACTGAGCCTAGGTTTGCGGAGATTCC

TGGAATTGATGTATGGCCTGGGAAGCAAATGCATAGCCATAACTATCGTATCCCCGAACCGTTT

CATGACCAAGTTGTGGTTATCATTGGGAGTTCCGCTAGTGCCGTTGATATCTCAAGAGATGTTG

CCAGATTTGCCAAAGAAGTTCACATTGCAAATAGGTCAATAACTGAGGGCACGCCAGCAAAGC

AACCTGGGTATGATAATATGTGGCTTCATTCAATGATAAAAATCACTCATAATGATGGCTCTGT

GGTATTCCATGATGGATGTTCCGTTCATGTTGATGTCATCATGCACTGCACCGGGTATGTTTATA

ACTTCCCATTCCTTAATACGAATGGAATCGTCACTGTGGACGACAACCGAGTGGGGCCACTGTA

TAAACATGTGTTCCCTCCATTACTAGCTCCATCTCTCTCTTTCGTTGGAATACCATGGAAGATTG

TTCCCTTCCCCTTATGCGAACTACAAAGCAAGTGGATAGCTGCAGTTTTATCTGGTCGAATTTCA

CTCCCAACGAAAAAGGAGATGATGGAAGATGTGGAAGCATACTACAAACAAATGGAAGCTGCT

GGAATTCCAAAAAGATACACACATAATATCGGGCATAACCAGTTTGACTACGATGACTGGCTT

GCTAATGAATGTGGATATAGTTGCATTGAAGAGTGGAGAAGGCTGATGTACAAGGAGGTGAGC

AAAAACAGGAAAGAGCGGCCTGAAAGCTATCGCGATGAATGGGACGATGATCACTTGGTTGCA

CAAGCAAGAGAAACTTTCAGCAAATTTCTTTCATAASEQ ID NO.2AsFMO1 gene coding protein sequence

Protein amino acid sequence

457aa

MVSSSCSSIPKMPVTPLSLVTRHVAIIGAGAAGLVTARELRREGHTTTIFERGSSIGGTWIYTPDTEPD

PMSQDSSRPIVHSSLYKSLRTNLPREVMGFLDYPFVEKTNGGDRRRFPGHEEVLDYLERFGREFGVS

REVGMEKEVVRVDMEQGGKWTVKWKGKDGGGGEEGFDAVVVCNGHYTEPRFAEIPGIDVWPGK

QMHSHNYRIPEPFHDQVVVIIGSSASAVDISRDVARFAKEVHIANRSITEGTPAKQPGYDNMWLHSM

IKITHNDGSVVFHDGCSVHVDVIMHCTGYVYNFPFLNTNGIVTVDDNRVGPLYKHVFPPLLAPSLSF

VGIPWKIVPFPLCELQSKWIAAVLSGRISLPTKKEMMEDVEAYYKQMEAAGIPKRYTHNIGHNQFD

YDDWLANECGYSCIEEWRRLMYKEVSKNRKERPESYRDEWDDDHLVAQARETFSKFLS。

Claims

1. A key gene AsFMO1 involved in alliin biosynthesis is characterized by a nucleotide sequence shown as SEQ ID NO. 1.

2. The protein amino acid sequence encoded by the gene of claim 1, which is represented by the sequence shown in SEQ ID NO. 2.

3. A vector comprising the entire nucleotide sequence of claim 1.

4. A recombinant protein comprising the amino acid sequence of claim 2.

5. An agrobacterium-mediated garlic genetic transformation method, which comprises the following steps:

(1) Placing the explant in a refrigerator at 4 ℃ for one month to break dormancy, peeling off outer protective leaves of the explant which breaks dormancy and removing brown garlic heels at the base, soaking in a super clean workbench for 15min with 75% ethanol, pouring out 75% ethanol, washing cleanly with sterile water, sterilizing with 4% sodium hypochlorite for 30min, discarding 4% sodium hypochlorite, washing with sterile water for 3-5 times, placing the sterilized explant in a gun head box containing a proper amount of sterile water, culturing in dark at 25 ℃ for 7d until root tips grow out, cutting the root tips of the explant with a sterilized scalpel for about 1cm, inoculating in a callus induction culture medium, culturing in dark at 25 ℃ for about 4 weeks to obtain callus; the callus induction medium comprises the following raw materials with the following concentration: MS,1 g/L2, 4-D,1 mg/L6-BA, 30g/L sucrose, 7g/L agar;

(2) Soaking the callus in an infectious microbe liquid for 10min, and continuously oscillating during the soaking period to obtain the infected callus; the infection bacterial liquid is an activated agrobacterium bacterial liquid carrying recombinant plasmids;

(3) Sucking the liquid on the surface of the infected callus, spreading on a co-culture medium, performing dark culture for 3d, transferring to a first-sieve culture medium, performing dark culture for 15-20 d, and transferring to a second-sieve culture medium, performing dark culture for 15-20 d to obtain the resistant callus; the co-culture medium comprises the following raw materials in concentration: MS,1 mg/L2, 4-D,1 mg/L6-BA, 30g/L sucrose, 7g/L agar, 100. Mu.M AS; the first-sieve culture medium comprises the following raw materials in concentration: MS,1 mg/L2, 4-D,1 mg/L6-BA, 30g/L sucrose, 7g/L agar, 400mg/L Cef,15mg/L Basta; the two-sieve culture medium comprises the following raw materials in concentration: MS,1 mg/L2, 4-D,1 mg/L6-BA, 30g/L sucrose, 7g/L agar, 400mg/L Cef,30mg/LBasta;

(4) Inoculating the resistant callus on a differentiation medium for induction culture to obtain adventitious buds; the differentiation medium comprises the following raw materials in concentration: MS,3 mg/L6-BA, 30g/L sucrose, 7g/L agar;

6. The preferred preparation method of the infectious microbe liquid in the step (2) in claim 5 comprises the following steps:

(2) Transferring 1-2 mL of the expanded bacterial liquid into 50mL of YEP liquid culture medium, and culturing overnight in a shaking table at 28 ℃ in a shaking way until the OD600 = 0.6-0.8 to obtain activated bacterial liquid; the YEP liquid medium comprises 50 mug/mL kanamycin and 100 mug/mL rifampicin;

(4) And (3) re-suspending the thalli by using an infection liquid, centrifuging, collecting thalli, and diluting the thalli to the OD600 = 0.4-0.7 by using the infection liquid. The dyeing liquid comprises the following raw materials in concentration: 4.42g/L MS, 10g/L sucrose, 10g/L glucose, 0.04g/L L-cysteine, 100. Mu.M AS.

7. The method according to claim 5, wherein the temperature of the adventitious bud development culture in the step (4) is 25 ℃, the photoperiod is 12 hours of illumination, the photoperiod is 12 hours of light shielding, and the illumination intensity is 200 mu mol m & lt-2 & gts & lt-1 & gt.

8. The rooting culture in the step (5) is carried out at 25 ℃ for 12 hours under light, 12 hours under dark conditions and 200 mu mol m-2 s-1 under light intensity.

9. A method for changing the alliin content is characterized in that,

(1) Altering the nucleotide sequence of claim 1 as a target;

(2) Manipulating the expression level of the gene of claim 1 by biotechnology means (including but not limited to gene editing, RNAi, T-DNA insertion, overexpression, etc.);

(3) The protein of claim 3 having its activity altered by biotechnological means.

10. Use of the gene of claim 1 in garlic breeding and alliin as a pharmaceutical intermediate.