Lilium lancifolium LlMYC2 gene and application thereof

Abstract

The invention discloses a lilium lancifolium LlMYC2 gene. The nucleotide sequence of the lilium lancifolium LlMYC2 gene is as shown in SEQ ID NO: 1; the amino acid sequence of the expression protein of the lilium lancifolium LlMYC2 gene is as shown in SEQ ID NO: 2; a specific primer pair for cloning the lilium lancifolium LlMYC2 gene, namely LlMYC2-F and LlMYC2-R, has nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4. The invention also discloses an application of the lilium lancifolium LlMYC2 gene in lilium lancifolium breeding, and the lilium lancifolium LlMYC2 gene can promote the development of lilium lancifolium microbulbs. The method is beneficial to expanding development mechanisms of different organs of plants and analyzing a lily bulb development regulation and control mechanism.

Description

Technical Field

[0001] The invention relates to the field of garden plant biotechnology, in particular to a Lilium lily L1MYC2 gene and application thereof. Background Art

[0002] Lily (Lilium spp.) is a plant of the genus Lilium in the family Liliaceae, and is a variant of wild lily. Lily has extremely high ornamental value, and can also be used as a health food and commonly used Chinese medicine for both medicinal and edible purposes, so it is deeply loved by Chinese consumers. Its flowers contain aromatic oils and can be used as spices; its bulbs are rich in starch and are a precious food. They are also used as medicines, with the effects of moistening the lungs and relieving coughs, clearing away heat, calming the nerves and promoting diuresis. Lily bulbs are the bulbs of lilies. Lilies are planted with vegetative bulbs, and the bulbs are divided into large, medium and small. In production, it is generally best to select medium bulbs of 50-60g for planting. However, the current lily bulb production technology is still insufficient, facing problems such as long production cycle and bulb expansion. It usually takes several years for small bulbs to grow into bulbs that meet commercial standards in the field, resulting in increased production costs. In addition, domestic bulbs are generally small, which affects the growth and development of lilies and the quality of flowers, resulting in about 90% of bulbs relying on imports. MYC transcription factor is an important member of the bHLH family. In recent years, studies have shown that MYC2, a key transcription factor in the jasmonic acid signaling pathway, is involved in plant growth, development and defense. Currently, research on the regulation of microbulb development by MYC2 gene has not been conducted in lily. In summary, in-depth research on the development of lily bulbs is of great significance for promoting the industrialization of lily bulbs in my country.

[0003] With the rapid development of molecular biotechnology, molecular breeding has gradually become one of the important means of horticultural plant breeding. Exploring the developmental regulation mechanism of lily microbulbs has important theoretical value for expanding the developmental mechanism of different plant organs and analyzing the mechanism of lily microbulb growth regulation. Summary of the invention

[0004] The present invention discloses the expression pattern of MYC2, a key transcription factor of the jasmonic acid signaling pathway, in the microbulb development process of Lilium lancifolium and its positive regulatory role. Specifically, the present invention analyzes the expression pattern of the LlMYC2 gene in the microbulb development process of Lilium lancifolium, confirms that the LlMYC2 gene is involved in the microbulb development process, clones the complete amino acid coding segment of the LlMYC2 gene, constructs a transient silencing vector, and verifies its regulatory role on the microbulb development in Lilium lancifolium microbulbs.

[0005] The nucleotide sequence of the Lilium lily L1MYC2 gene of the present invention is shown in SEQ ID NO:1.

[0006] The amino acid sequence of the expressed protein of the Lilium lily L1MYC2 gene of the present invention is shown in SEQ ID NO:2.

[0007] The specific primer pair used to clone the Lilium lily LlMYC2 gene of the present invention, LlMYC2-F and LlMYC2-R, have nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0008] The invention discloses an application of the Lilium lily L1MYC2 gene in Lilium lily breeding.

[0009] In the application of the present invention, the Lilium lily LlMYC2 gene can promote the development of Lilium lily microbulbs.

[0010] The method for analyzing the expression pattern of the Lilium lily L1MYC2 gene of the present invention comprises the following steps:

[0011] (1) Collection of materials during the development of Lilium lily microbulbs: Collection of materials during the development of Lilium lily microbulbs: Collection of materials at different developmental stages of Lilium lily bulbils: There are 4 stages in total, S0 (stage before bulbils), S1 (stage with small white spots on bulbils), S2 (stage with green bulbils) and S3 (stage with mature bulbils). Collection of materials at different developmental stages of Lilium lily bulblets: Select the middle scales of Lilium lily bulbs with similar growth and size and no diseases, infiltrate them with carbendazim, and then culture them on 0.7% water agar medium in a completely dark incubator (23±2°C). According to the diameter size of the bulblets of Lilium lily during the development process, they are divided into S1 (diameter < 0.3 cm), S2 (diameter 0.3-0.45 cm), S3 (diameter 0.45-0.6 cm), S4 (diameter 0.6-0.75 cm), S5 (diameter 0.75-0.9 cm), and S6 (diameter > 0.9 cm).

[0012] (2) Extraction of total RNA during microbulb development: Use a total RNA extraction kit and a reverse transcription kit to extract total RNA and synthesize cDNA from the material taken in step (1);

[0013] (3) Detection and analysis of the relative expression of the LlMYC2 gene during microbulb development The cDNA obtained in step (2) was used as a template and amplified using a real-time fluorescence quantitative PCR kit;

[0014] In the method of the present invention, the quantitative primer pair used in step (3), LlMYC2-qPCR-F and LlMYC2-qPCR-R, have nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6.

[0015] The method for constructing a transient silencing vector of the Lilium lily L1MYC2 gene of the present invention comprises the following steps:

[0016] (1) Obtaining the target fragment: a 306 bp specific fragment was selected in the non-conserved domain of the LlMYC2 sequence for silencing, and the upstream primer LlMYC2-TRV2-EcoRI-F and the downstream primer LlMYC2-TRV2-XhoI-R were designed. The nucleotide sequences thereof are shown in SEQ ID NO:7 and SEQ ID NO:8, and restriction sites were added to both ends of the primers. PCR amplification was performed using the LlMYC2 full-length plasmid as a template. A PCR amplification reaction was performed to obtain an amplified product of the LlMYC2 coding region with restriction sites, i.e., the target fragment; the restriction sites were EcoRI and XhoI;

[0017] (2) Recovering the target fragment, digesting the LlMYC2 gene PCR product fragment with restriction sites and the vector pTRV2 in step (1), and recovering the restriction products respectively, and then connecting the recovered LlMYC2 gene PCR product fragment and the vector pTRV2;

[0018] (3) connecting the target fragment digested and recovered in step (2) to the pTRV2 vector digested and recovered;

[0019] (4) The ligation product of step (3) was transformed into Escherichia coli competent cells DH5α, and positive single clones were identified by colony PCR;

[0020] (5) The expression vector constructed in step (4) is transformed into Agrobacterium competent EHA105, and the obtained engineered bacteria EHA105 / pTRV2-LlMYC2 is used to transform Lilium lily microbulbs.

[0021] The present invention is different from the prior art in that:

[0022] The present invention obtains candidate genes related to the development process of Lilium lily microbulbs through transcriptome analysis, and confirms that the candidate genes are involved in regulating the development of microbulbs by analyzing the expression patterns of the candidate genes during the development of microbulbs. The complete amino acid coding segment of the gene is cloned, a silencing vector is constructed, and the function of the gene is verified; the present invention is conducive to expanding the development mechanism of different plant organs and analyzing the regulation mechanism of lily bulb development.

[0023] The present invention transforms Lilium lily microbulbs with a pTRV2 silencing vector that controls the microbulb development gene LlMYC2, and finds that the development of the silencing strain is significantly slower than that of the pTRV2 empty vector.

[0024] The cloning and application of the Lilium lily LlMYC2 gene of the present invention will be further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The electrophoresis result of the cloning of the LlMYC2 gene of Lilium lily in the present invention;

[0026] Figure 2 This is an expression diagram of the LIMYC2 gene of the present invention during the development of bulbils of Lilium lily;

[0027] Figure 3 This is an expression diagram of the LIMYC2 gene of the present invention during the development of Lilium lily bulblets;

[0028] Figure 4 The phenotype, relative diameter increase rate and LIMYC2 gene expression level of the LlMYC2 gene silenced strain of Lilium lily bulblets in the present invention;

[0029] Figure 5 The phenotype, diameter and LIMYC2 gene expression level of the bud LlMYC2 gene silenced strain of Lilium lily in the present invention;

[0030] Figure 6 This is a diagram of the microbulb development pattern regulated by the LlMYC2 gene in the present invention. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are intended to provide the reader with a clear and specific understanding, but the content does not limit the overall scope of the present invention. The experimental methods in the following embodiments, unless otherwise specified, all adopt conventional experimental operations. The test materials used in the embodiments of the present invention are all from conventional biochemical reagent stores to ensure the quality and purity of the reagents. All quantitative tests were repeated three times, and the results were averaged. The index for judging the development of microbulbs is the relative bulb increase rate / diameter.

[0032] 1. LlMYC2 gene cloning

[0033] The LlMYC2 gene was obtained by screening the transcriptome data of bulbil development of Lilium lily, and its transcript sequence was put into ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) to obtain its open reading frame (ORF) sequence. The longest ORF sequence obtained was compared in NCBI protein BLAST (https: / / blast.ncbi.nlm.nih.gov / -Blast.cgi), and the comparison was successful if it matched the transcriptome annotation result. According to the ORF sequence obtained in the previous stage, the specific cloning primers were designed using Premier 5 software, as shown in Table 1, and PCR amplification was performed using Lilium lily bulbil cDNA as a template. The total amplification system was 50 μL, the specific reaction system was shown in Table 2, and the reaction procedure was shown in Table 3. The PCR amplification product was detected by 1.2% agarose gel electrophoresis and the target fragment was recovered using a DNA recovery kit (Novozyme, DC301); the target fragment was connected to the TA / Blunt-Zero intermediate vector (Novozyme, Nanjing) and transformed into Escherichia coli competent DH5α (Qingke, Beijing), cultured in LB solid medium (containing 50 mg / L kanamycin) at 37°C for 12-18 hours, and single clones were randomly picked and subjected to colony PCR detection. The colony PCR reaction system is shown in Table 4, and the reaction procedure is shown in Table 5. Sequencing analysis was completed by Beijing Qingke Biotechnology Co., Ltd., and the sequencing results were compared in DNAMAN software.

[0034] Table 1 Names and sequences of primers for cloning LlMYC2 gene

[0035]

[0036] Table 2 High-fidelity enzyme PCR reaction system

[0037]

[0038] Table 3 High-fidelity enzyme PCR reaction program

[0039]

[0040] Table 4 Colony PCR reaction system

[0041]

[0042] Table 5 Colony PCR reaction procedure

[0043]

[0044] Summary: The present invention screened a candidate differentially expressed gene LIMYC2 from the transcriptome of bulbils of Lilium lily, and BLASTed a 2148 bp CDS series through the NCBI ORF Finder online website. Primers were designed based on the CDS sequence, and a specific band of 2148 bp was obtained by PCR amplification using bulbils of Lilium lily as the test material ( Figure 1 ).

[0045] LlMYC2 gene sequence:

[0046]

[0047] Amino acid sequence encoded by LlMYC2 gene:

[0048] MNLWTDDNASMMEAFMASTPDLHSFPWAAPQTPALTPPPPFTASSASTPPPPPPVYFNQDTLQQRLQTLIEGARESWTYAIFWQSSVDAAAGGSLLGWGDGYYKGCEQDKRIQRRAVTPAEQEHRKRVLRELNSIISGGAGSPDEAVDEEVTDTEWFFLISMTHSFINGDGLPGQAFF SGAASWFAGANYLASAPCERAQQAQTFGMQTMVCVPVGFGVLELGSTELIFQSSEIMAQIKLLFDFQNPGGGVAAIGGTTAAGFWPPAEASPAPPHDQGEDDPAAIWISDPSVIEIKDSVSPPPATTEFPIPKPQIQFDNPSSNSKAPVQFDNPSINSKAPIGNPNPRPIPVQQQYNGS SSKQPQPQPQHQNAGIPNPPTQPFFTRDLNFSEEFGSSSAPPQPYKPETGEILNFGEMKRNSSPVTGSSLFSQHHRVGSSSAAIDERKNKATSRPNNDEGMLSFSSAPARQASVSQVMSGGGIISAAGGGGIDSEHSDLDASVREVESSRVVEPEKPRPRKRGRKPANGREEPLNHVEAE RQRREKLNQRFYALRAVVPNVSKMDKASLLGDAIAYINEIKSKMQTMEEEKEGLEAQIEELRKDRVSRPAPPPDRAHLNTMMNGGMGNGRSHGVEVDVRPLGPEVGADVMLRIQTPNRNHPAARLMAALRELELEVFWASVSIVKELMVQQLTVKMSKTRLYTQEQLTAVLFNMISEGA

[0049] 2. Analysis of LlMYC2 expression pattern during microbulb development

[0050] 1. Collection of Lilium bulbs during microbulb development

[0051] Bulbs of Lilium lily at different developmental stages (S0, S1, S2, S3) and bulblets of Lilium lily at different developmental stages (S1, S2, S3, S4, S5, S6) were taken for total RNA extraction and candidate gene fluorescence quantitative PCR detection.

[0052] 2. Total RNA extraction and cDNA synthesis from Lilium lily bulbs during microbulb development

[0053] The FastPure polysaccharide and polyphenol plant RNA rapid extraction kit (Novozyme, R401) was used to extract bulbils and bulblets of Lilium lily at different developmental stages, and the reverse transcription (+gDNA wiper) kit (Novozyme, R312) was used to synthesize 1 μg RNA into the first chain of cDNA to obtain bulbils and bulblets of Lilium lily at different developmental stages. The reverse transcription system is as follows:

[0054] Table 6 Reverse transcription system composition

[0055]

[0056] 3. Real-time fluorescence quantitative PCR (qPCR) quantitative detection of gene expression

[0057] Real-time fluorescence quantitative PCR primers were designed according to the candidate gene CDS. The specific primers are shown in Table 7. PremixEx Taq TM II (TAKARA, RR820A) was used to detect the expression level of the target gene. The fluorescence quantitative reaction system and reaction procedure are shown in Tables 8 and 9. LlFP was used as the internal reference gene, and the measurement instrument was Bio-Rad CFX96TM Real-Time System real-time fluorescence quantitative PCR instrument. Each sample was repeated three times.

[0058] Table 7 Primer names and sequences used in the present invention

[0059]

[0060] Table 8 Real-time fluorescence quantitative PCR reaction system

[0061]

[0062] Table 9 Real-time fluorescence quantitative PCR reaction adopts a three-step method, and the specific procedures are as follows:

[0063]

[0064] 4. Analysis of LlMYC2 expression pattern during microbulb development

[0065] According to the above fluorescence quantitative PCR results, Graph Pad Prism8 and DPS were used for significant difference analysis, and two-sided Student's t-test was used. P<0.05(*) indicated significant difference, and P<0.01(**) indicated extremely significant difference. The experimental results showed that LlMYC2 was highly expressed in S1 and S3 of bulbils of Lilium lily and showed an upward trend. It was speculated that there were two expansion processes in the development of bulbils of Lilium lily, and LlMYC2 may play a promoting role in these two expansion processes. At the same time, the same situation also existed in the development of bulblets of Lilium lily, and it was speculated that LlMYC2 played a promoting role in the two expansion processes of bulblets of Lilium lily. It showed that LlMYC2 was a key gene regulating the development of microbulbs.

[0066] Summary: The present invention detected the expression level of LlMYC2 during the development period of Lilium lily microbulbs by qRT-PCR method. Figure 2 and Figure 3 As shown in the results, the expression level of LlMYC2 in the bulbils S1 and S3 of Lilium lily was high and showed an upward trend. It is speculated that there are two expansion processes in the development of bulbils of Lilium lily, and LlMYC2 may play a promoting role in these two expansion processes. The same situation also exists in the development of the bulblets of Lilium lily, and it is speculated that LlMYC2 plays a promoting role in the two expansion processes of the bulblets of Lilium lily.

[0067] 3. Application of microbulb development regulatory gene LlMYC2

[0068] 1. Construction of gene silencing vector:

[0069] (1) Ligation and transformation of target fragment and vector

[0070] A 306bp specific fragment was selected in the non-conserved domain of the LlMYC2 sequence for silencing, and the upstream primer LlMYC2-TRV2-F and the downstream primer LlMYC2-TRV2-R were designed. Restriction sites were added to both ends of the primers, and PCR amplification was performed using the LlMYC2 full-length plasmid as a template. The PCR amplification reaction was performed to obtain the LlMYC2 coding region amplification product with restriction sites, that is, the target fragment. The LlMYC2 gene PCR product fragment with restriction sites and the vector pTRV2 were digested, and the restriction products were recovered respectively, and then the recovered LlMYC2 gene PCR product fragment and the vector pTRV2 were connected; the target fragment after restriction digestion and recovery was connected to the pTRV2 vector after restriction digestion and recovery; the connection product was transformed into Escherichia coli competent cells DH5α, coated on the corresponding resistance screening medium, and inverted in a 37°C constant temperature incubator for 12-16h. Colony PCR identified positive single clones, and sequencing analysis was completed by Beijing Qingke Biotechnology Co., Ltd.

[0071] (2) Gene function verification

[0072] The recombinant plasmid TRV2-LIMYC2 with correct recombinant sequencing results, as well as the empty plasmids TRV1 and TRV2 were transferred into the competent EHA105 Agrobacterium. The Agrobacterium strains were streaked on LB solid medium containing antibiotics (50 mg / L kanamycin and 25 mg / L rifampicin), and after inverted culture for 48 hours, each Agrobacterium strain was placed in 100 ml of LB liquid containing kanamycin and rifampicin antibiotics, and shaken overnight. The bacteria were collected in a 50 mL centrifuge tube at 5000 rpm for 10 min, and the bacteria at the bottom of the centrifuge tube were resuspended with a resuspension solution (10 mM MgCl2, 10 mM MES, 200 μM AS; pH 5.8), and the concentration of the Agrobacterium solution was adjusted to OD600 = 0.8-1.0 using a spectrophotometer. TRV1 was mixed with TRV2 and recombinant TRV2 in equal volumes of 1:1, and allowed to stand for 3-5 hours in the dark. Add the resuspension solution to cover the microbulbs of Lilium lily, use a vacuum pump to pump the air pressure in the pump to 0.8MPa, maintain it for 5min, then slowly release the air to slowly restore the air pressure in the pump to the standard atmospheric pressure (repeat twice), and dry the excess Agrobacterium liquid on the scales. The bulbils were cultured in the greenhouse (60d), and the scales were cut on 0.7% water agar (30d), placed in a dark environment, and the microbulbs were morphologically observed. Image J and Excel 2007 software were used to count and calculate the increase rate of the diameter of the bulbils and the relative diameter of the small bulbs. The silencing bulbils and small bulbs were quickly frozen with liquid nitrogen and stored in a -80℃ refrigerator for subsequent analysis of the expression of LlMYC2.

[0073] Summary: The present invention silenced the gene in the scales and bulbils of Lilium lily, and selected 30 days and 60 days as sampling time to count and sample the size of the bulblets and bulbils of Lilium lily. Figure 4 and Figure 5 As shown in the results, the expression of LlMYC2 gene decreased by about 1.44, 1.76 and 3.37 times in the three lines with bulbil silencing, and decreased by about 1.71, 1.92 and 2.54 times in the three lines with bulbil silencing. After 30 days of LlMYC2 silencing in scales, the size of the bulbil of Lilium lily was significantly smaller than that of the control group, and the increase rate of its bulb diameter was reduced by about 2.25 times compared with the control group; similarly, after 60 days of LlMYC2 silencing in bulbils, the size of the bulbil of Lilium lily was significantly smaller than that of the control group, and its bulb diameter was reduced by about 1.15 times compared with the control group. This indicates that LlMYC2 plays a positive regulatory role in the expansion of Lilium lily microbulbs. The primers, PCR system and program used in the detection of LlMYC2 gene expression are the same as those in Tables 7 to 9.

[0074] The present invention is to explore the genes related to the regulation of the microbulb development of Lilium lily. The invention obtains the candidate gene LlMYC2 related to the microbulb development by analyzing the transcriptome of the bulbil development process of Lilium lily. The data of the S0 to S3 period are analyzed, and a differentially expressed gene annotated as LlMYC2 is screened. The gene has a high expression in the S2 and S3 periods. By analyzing the expression pattern of LlMYC2 during the microbulb development process, it is determined that LlMYC2 is involved in the microbulb development process, and a corresponding silencing vector is constructed. The LlMYC2 silencing vector is transferred into the plant to verify its function.

[0075] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Lilium lily L1MYC2 gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The expressed protein of the Lilium lily L1MYC2 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO:

2.

3. A specific primer pair for cloning the LlMYC2 gene of Lilium lily according to claim 1, LlMYC2-F and LlMYC2-R, whose nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

4. Use of the Lilium lily L1MYC2 gene according to claim 1 in Lilium lily breeding.

5. The use according to claim 4, characterized in that: The Lilium lily LlMYC2 gene can promote the development of Lilium lily microbulbs.

6. The method for analyzing the expression pattern of the Lilium lily L1MYC2 gene according to claim 1, characterized in that: The steps include: (1) The development process of Lilium lily microbulbs: The bulbils of Lilium lily were collected at different developmental stages: there were 4 stages in total, including S0, the stage of no bulbils, S1, the stage of small white spots on the bulbils, S2, the stage of green bulbils, and S3, the stage of mature bulbils. The materials of Lilium lily bulblets at different developmental stages were collected: the middle scales of Lilium lily bulblets with similar growth and size and no diseases were selected, and after being infiltrated with carbendazim, the scales were placed on 0.7% water agar medium and cultured in a completely dark incubator at a temperature of 23±2°C; according to the diameter size of the Lilium lily bulblets during the development process, they were divided into S1: diameter <0.3cm, S2: diameter 0.3-0.45cm, S3: diameter 0.45-0.6cm, S4: diameter 0.6-0.75cm, S5: diameter 0.75-0.9cm, S6: diameter >0.9cm; (2) Extraction of total RNA during microbulb development: Use a total RNA extraction kit and a reverse transcription kit to extract total RNA and synthesize cDNA from the material taken in step (1); (3) Detection and analysis of relative expression of LlMYC2 gene during microbulb development The cDNA obtained in step (2) was used as a template and amplified using a real-time fluorescence quantitative PCR kit.

7. The method according to claim 6, characterized in that The quantitative primer pair used in step (3), LlMYC2-qPCR-F and LlMYC2-qPCR-R, have nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:

6.

8. The method for constructing a transient silencing vector of the Lilium lily L1MYC2 gene according to claim 1, characterized in that: The steps include: (1) Obtaining the target fragment: a 306 bp specific fragment was selected in the non-conserved domain of the LlMYC2 sequence for silencing, and the upstream primer LlMYC2-TRV2-EcoRI-F and the downstream primer LlMYC2-TRV2-XhoI-R were designed. The nucleotide sequences thereof are shown in SEQ ID NO:7 and SEQ ID NO:8, and restriction sites were added to both ends of the primers. PCR amplification was performed using the LlMYC2 full-length plasmid as a template. A PCR amplification reaction was performed to obtain an amplified product of the LlMYC2 coding region with restriction sites, i.e., the target fragment; the restriction sites were EcoRI and XhoI; (2) Recovering the target fragment, digesting the LlMYC2 gene PCR product fragment with restriction sites and the vector pTRV2 in step (1), and recovering the restriction products respectively, and then connecting the recovered LlMYC2 gene PCR product fragment and the vector pTRV2; (3) connecting the target fragment digested and recovered in step (2) to the pTRV2 vector digested and recovered; (4) The ligation product of step (3) was transformed into Escherichia coli competent cells DH5α, and positive single clones were identified by colony PCR; (5) The expression vector constructed in step (4) is transformed into Agrobacterium competent EHA105, and the obtained engineered bacteria EHA105 / pTRV2-LlMYC2 is used to transform Lilium lily microbulbs.

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