Lily MYB family member LrMYB2 gene and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a lily MYB family member LrMYB2 gene and application thereof, the nucleotide sequence of the LrMYB2 gene is as shown in SEQ ID NO.1, the coded amino acid sequence of the LrMYB2 gene is as shown in SEQ ID NO.2, and the lily LrMYB2 gene is used for regulating growth and development and flowering phase regulation of lily plants; the lily LrMYB2 gene is used for enhancing the sensitivity of plants to botrytis cinerea; the lily LrMYB2 gene is used for regulating the antioxidant enzyme activity of plants. The gene is subjected to overexpression analysis in arabidopsis thaliana and lilium brownii, the biological function of the gene is discussed, and it is found that the LrMYB2 gene inhibits the growth and development process of arabidopsis thaliana, so that a transgenic plant is more sensitive to botrytis cinerea, meanwhile, the antioxidant enzyme activity is reduced, and therefore, the gene provides a new choice for breeding of a new variety of the plant, and has a good application prospect. The method has important theoretical research significance and practical application value.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, in particular to a lily MYB family member LrMYB2 gene and application thereof. Background Art

[0002] In the natural evolution process of survival of the fittest and natural selection, plants will evolve various characteristics. Among them, the MYB transcription factor family in plants is one of the largest transcription factor families in plants. It plays a very important role in regulating plant growth and development, plant secondary metabolism (including anthocyanin and lignin synthesis), mediating pathogen resistance and responding to hormone signals. MYB transcription factors are named because they contain special domains (1-4 MYB repeat elements). In plants, most MYB factors contain R2R3-MYB domains and belong to the R2R3-MYB family (Huang Qianru, Liu Xiaohui, Zhang Aidong, et al. Research Progress of MYB Gene Family, Journal of Jiangxi Agricultural Sciences, 2022, 34(02):146~153.). At present, the R2R3-MYB transcription factor family has been identified in many plants, including Arabidopsis, rice, wheat, corn and poplar. For example, 126 members have been found in Arabidopsis (Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. CurrOpin Plant Biol, 4: 447-456), and as many as 192 members in poplar (Wilkins O, Nahal H, Foong J, et al. (2009) Expansion and diversification of the Populus R2R3-MYB family of transcription factors. Plant Physiol, 149: 981-993.). MYB affects the plant's disease resistance. In Arabidopsis, Van et al. (2008) used gene knockout technology to construct myb72 Defective mutants were found to have significantly reduced resistance to fungi such as Alternaria and Botrytis cinerea (Van S, Verhagen B, Van R, et al. (2008) MYB72 is required in early signaling steps of rhizobacteria-induced systemic resistance in Arabidopsis. Plant Physiol, 146:1293-1304.).

[0003] Lily is one of the famous flowers at home and abroad, and is also an important vegetable, occupying an important share in both domestic and foreign markets. The growth and development of lily plants are regulated by many factors, such as environmental factors and genetic factors. At present, there are few reports on the research on the development regulation of lily plants. For example, Wang Xueqian et al. proposed the identification of the MYB transcription factor family in Asian lilies and the screening of MYB genes that regulate pollen abortion. The results showed that the LaMYB gene plays an important regulatory role in the process of pollen abortion (Wang Xueqian, Yuan Guozhen, Wu Ze, Li Qian, Teng Nianjun, Identification of the MYB transcription factor family in Asian lilies and the screening of MYB genes that regulate pollen abortion, Journal of Agricultural Biotechnology, 2019, 27(11): 1951~1961). Wang Rui et al. used Oriental lily as the material to study that the lily TTG1 gene is mainly expressed in the petals, with the highest expression in the flower buds during the coloring period. The lily TTG1 gene can act on the expression of anthocyanins (Wang Rui, Liu Siyang, He Xiangfeng, Zhao Xiangyun, Wang Wenhe, Cloning and expression of the lily TTG1 gene and its interaction analysis with MYB and bHLH, Molecular Plant Breeding, 2022, Vol. 20, No. 16, No. 5258 - 5266 pages). Fan Wenguang et al. screened out three MYB genes and one bHLH gene that can promote the synthesis of anthocyanins in the bulbs of Lanzhou lily, providing theoretical support for further in-depth research on anthocyanin regulatory genes in the bulbs of Lanzhou lily (Fan Wenguang, Li Baoyu, Tian Hui et al., Screening and analysis of MYB and bHLH transcription factors related to anthocyanin synthesis in the bulbs of Lanzhou lily, Food and Fermentation Industries, https: / / doi.org / 10.13995 / j.cnki.11-1802 / ts.033270).

[0004] Botrytis cinerea is one of the most common diseases that harm lily field production and storage. Studies have shown that after being infected by Botrytis cinerea, lily plants usually show leaf withering and brown rot of the flowers, which in turn causes the stems to turn black, underground bulbs to reduce yield, and quality to deteriorate (Cao X, Shi S, Zhang Z. (2018) First report of Botrytis leafblight on lily (Lilium longiflorum) caused by Botrytis cinerea in Beijing, China. Plant Dis, 102: 1033). Botrytis cinerea is most likely to occur in humid weather. Once it breaks out, it usually spreads quickly and is difficult to cure. Generally, when Botrytis cinerea occurs, it will cause a 20-40% yield loss, and cause a complete loss of harvest in severely infected areas. Cultivating and applying new lily resistant varieties is the most effective, economical and environmentally friendly measure to solve the problem of lily botrytis cinerea. Therefore, the discovery of lily's resistance or susceptibility genes to gray mold and the analysis of their important physiological functions have important theoretical and practical value for breeding disease-resistant lily varieties.

[0005] Minjiang Lily ( Lilium regale Wilson) is a natural wild germplasm of the genus Lilium in the Liliaceae family, widely distributed in the Minjiang River Basin in Sichuan, my country. Studies have shown that Minjiang lily has high resistance to fungi and viruses, and it is used in disease-resistant lily breeding. Obviously, the MYB family plays an important regulatory role in the developmental morphology and adverse stress of plants, but there are few reports on the functions of lily MYB family genes. The mechanism of MYB family in lily regulating the growth and development of Lilium plants and improving gray mold resistance has not been elucidated. The present invention cloned a MYB gene member that responds to gray mold from Minjiang lily, and clarified its biological function through reverse genetics methods, providing a new target for analyzing its mechanism of action and later lily genetic breeding improvement. Summary of the invention

[0006] The purpose of the present invention is to provide an application of the LrMYB2 gene, to provide a new member of the lily MYB gene LrMYB2 and its application, and to provide a new target for the growth and development regulation and gray mold resistance improvement of lily plants.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention also provides the application of the LrMYB2 gene in the regulation of plant growth and development and flowering period, wherein the nucleotide sequence of the LrMYB2 gene is as shown in SEQ ID NO.1 or a nucleotide sequence with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the LrMYB2 gene expressed is as shown in SEQ ID NO.2 or an amino acid sequence with the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.2.

[0009] Furthermore, the plant is lily or Arabidopsis thaliana.

[0010] Furthermore, the LrMYB2 gene is involved in the growth and development regulation of the plant's stem tissue.

[0011] Furthermore, the regulation of the LrMYB2 gene on the flowering period of the plant is to delay the flowering period of the plant.

[0012] The present invention also provides an application of the LrMYB2 gene in regulating the sensitivity of plants to Botrytis cinerea, wherein the nucleotide sequence of the LrMYB2 gene is as shown in SEQ ID NO.1 or a nucleotide sequence having the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.1; The amino acid sequence of the LrMYB2 gene expressed is as shown in SEQ ID NO.2 or an amino acid sequence having the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.2.

[0013] Furthermore, the LrMYB2 gene regulates the sensitivity of the plant to Botrytis cinerea to enhance the sensitivity of the plant to Botrytis cinerea.

[0014] Furthermore, the LrMYB2 gene affects the sensitivity of plants to Botrytis cinerea by affecting the activity of antioxidant enzymes.

[0015] The present invention also provides a method for regulating the growth and development and flowering period of plants, by introducing a recombinant vector, expression cassette, cell or recombinant bacteria containing the LrWRKY-R2 gene into plant cells to increase the expression level of the LrMYB2 gene in the plant.

[0016] The present invention also provides a method for regulating the sensitivity of plants to Botrytis cinerea, by introducing a recombinant vector, expression cassette, cell or recombinant bacteria containing the LrWRKY-R2 gene into plant cells to increase the expression level of the LrMYB2 gene in the plant.

[0017] The present invention has at least the following beneficial effects: 1. The present invention cloned the full-length sequence of the LrMYB2 gene, a member of the MYB family, from Lilium chinense, and obtained the protein sequence encoded by it. The gene was overexpressed in Arabidopsis thaliana, and it was found that the growth and development process of the transgenic plants was significantly inhibited, and the leaf growth and development, bolting period and flowering period were all delayed compared with the wild-type control. This provides a key negative regulatory factor for the growth and development and flowering period regulation of Lilium plants in the future.

[0018] 2. The present invention shows through molecular genetics and biochemical analysis that the LrMYB2 gene not only affects the growth and development of plants, but also that overexpression of the gene in Arabidopsis and lily enhances the sensitivity of plants to Botrytis cinerea. This study not only enriches the plant disease-susceptibility gene library, but also provides new options for breeding lily disease-resistant varieties through gene knockout or expression inhibition technology in the future. At the same time, it lays an important theoretical foundation for analyzing the molecular regulatory mechanism of lily resistance to gray mold.

[0019] 3. The present invention analyzes the activity of antioxidant enzymes by overexpressing LrMYB2 and finds that LrMYB2 may regulate the growth and development of plants and their sensitivity to Botrytis cinerea by affecting the activity of antioxidant enzymes. Therefore, it provides a theoretical basis for the study of the regulation of antioxidant enzyme activity in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0021] Figure 1 This is the alignment of LrMYB2 from Lilium chinense with two similar MYB4 protein sequences in the NCBI database. The boxes are the conserved R2 and R3-MYB domains analyzed using https: / / smart.embl.de / ; Figure 2 This is the tissue expression specificity analysis of LrMYB2 gene in Lilium chinense. The horizontal axis is root, mature stem, young stem, leaf, flower and external bulb. Figure 3 This is the electrophoresis diagram of the restriction enzyme digestion verification analysis of the vector plgne-35S::LrMYB2, where M is a 12kb DNA marker; lane 1 is a plasmid control; lane 2 is a picture after HindIII-EcoRI digestion; Figure 4 The figure is the electrophoresis diagram of PCR amplified transgenic plants. Lanes 1-6, 9, 10, 14, 15, and 17 are transgenic positive seedling lines, lane 18 is the positive control (plasmid as template), lane 19 is 1kb DNA marker, and lanes 20 and 21 are negative controls. Figure 5 GUS staining analysis of seedlings in transgenic lines, A is the wild-type plant (WT) control, B and C are two independent plgne-35S::LrMYB2 transformed lines OX-L1, OX-L2; Figure 6 Phenotypic observation of transgenic plants. A is the phenotype of transgenic plants grown in greenhouse soil for 2 weeks, from top to bottom are WT, OX-L1, OX-L2; B, C and D are the phenotypes of WT, OX-L1, OX-L2 grown in soil for 4 weeks, E is the phenotype of transgenic plants grown in soil for 7 weeks, from left to right are WT, OX-L1, OX-L2; Figure 7 Analysis of resistance to Botrytis cinerea in transgenic Arabidopsis plants. A, B, and C are the phenotypes of leaves of WT, OX-L1, and OX-L2 plants inoculated with Botrytis cinerea for 3 days, respectively. The scale bar represents 4 mm. D is a quantitative analysis of the corrosion area of ​​Arabidopsis leaves inoculated with Botrytis cinerea for 3 days. The error bars represent the ±SE values ​​of 3 repeated experiments. Different letters represent significant differences. Duncan test: p value < 0.05.

[0022] Figure 8 The effect of transient transformation of LrMYB2 gene on resistance to gray mold in lily leaves; A and B are the effect pictures of transformed leaves infected with Botrytis cinerea 5 days later; A is the control group, B is the experimental group (injected with 35S::LrMYB2 overexpression vector); C is the quantitative analysis of the corrosion area of ​​lily leaves inoculated with Botrytis cinerea for 5 days, the error bars represent the ±SE value of 3 repeated experiments, different letters represent significant differences, Duncan test: p value <0.05. D and E are GUS staining identification of transformed lily leaves, D is the staining of lily leaves injected with control solution, and E is the staining of lily leaves injected with Agrobacterium containing 35S::LrMYB2 vector.

[0023] Fig. 9To quantitatively detect the effect of LrMYB2 overexpression on antioxidant enzyme activity; A is the comparison of peroxidase (POD) activity in the wild-type control (WT) and transgenic plants (OX-L1 and OX-L2), B is the comparison of hydrogen peroxide (CAT) activity in the WT and transgenic plants OX-L1 and OX-L2, and C is the comparison of superoxide dismutase (SOD) activity in the WT and transgenic plants OX-L1 and OX-L2. Error bars represent 3 replicates ± SE values, different letters represent significant differences, Duncan test: p value < 0.05. Implementation

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The raw materials described in the examples are ordinary commercial products unless otherwise specified. The experimental methods described in the examples are not particularly specified, that is, they are operated according to conventional molecular biology experimental methods.

[0025] In the early stage of the present invention, comparative transcriptome sequencing analysis was performed to obtain MYB gene members induced by Botrytis cinerea, and it was found that one of the unigene genes was induced to up-regulate expression, which was named LrMYB2.

[0026] Example 1 Lilium chinense LrMYB2 Gene cloning and sequence analysis Mixed tissues of leaves, stems, roots and flowers of Lilium chinense were used as materials, and total RNA was extracted using TRIzol™ Plus RNA Purification Kit (12183555, Invitrogen™) according to the instructions. Residual trace DNA was removed using DNase I (18047019, Invitrogen™), and the RNA concentration was determined using a spectrophotometer for later use.

[0027] About 2.0 μg of total RNA from Lilium chinense leaves was taken and the first-strand cDNA was synthesized according to the instructions of PrimeScript II first-strand cDNA synthesis kit (6210A, Takara).

[0028] PCR amplification system: high-fidelity amplification enzyme Prime STAR HS (R010A, TaKaRa) 0.25 μL, 5× PrimeSTAR Buffer (Mg 2+Plus) 5μL, forward primer (Myb6864-F, 10μM) 0.5μL, reverse primer (LrMYB2-R, 10μM) 0.5μL, template (DNA) 1μL, dNTP (2.5mM) 2μL, sterile ddH2O to 25μL.

[0029] The forward and reverse primers are: LrMYB2-F: 5'-ATGGTGAGAGCTCCTTGCTGTG-3' LrMYB2-R: 5'-TCAGATTGTGGGCAAGTCCTGC-3' The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; 95°C for 30 s; 60°C for 40 s; 72°C for 1 min, 38 cycles; and 72°C for 7 min.

[0030] The PCR product was analyzed by agarose gel electrophoresis, and a specific amplification band was observed between 750-1000 bp under ultraviolet light. It was purified according to the gel recovery kit (9672, Takara) and used for later use.

[0031] The purified DNA fragment was A-added using a blunt-end A reagent and connected to pMD20-T vector (6019, Takara) by TA cloning. The ligation product was transformed into Escherichia coli DH5a, and 1-2 positive clones were selected from the LB culture plate containing ampicillin (100 mg / L) for sequencing analysis. The results showed that Lilium chinense LrMYB2 The full-length sequence of the gene is shown in SEQ ID NO.1, including an open reading frame of 762 bp (including the stop codon).

[0032] According to SEQ ID NO.1, the protein sequence of LrMYB2 of Lilium edulis was obtained by translation with DNAman software as shown in SEQ ID NO.2, which contains 253 amino acids (* indicates the termination signal). The nucleotide sequence of LrMYB2 was compared with NCBI database by BLAST, and no highly similar sequence was found. The highest Identities were 543 / 679 (80%) with Amana edulis transcription factorMYB4 (MYB4) mRNA. The protein sequence of LrMYB2 was compared with NCBI database by BLAST, and no highly similar sequence was found. The highest Identities were 185 / 258 (72%) with Amana edulis transcription factorMYB4 (QQY00481.1). Lilium regale MYB4 (ASV46333.1) identities are 162 / 262 (62%). Protein sequence analysis was performed by http: / / multalin.toulouse.inra.fr / multalin / ( Figure 1 ), and found that LrMYB2 belongs to the R2R3-MYB family and is a new member of the lily MYB family.

[0033] Example 2 Tissue expression specificity of LrMYB2 gene Using Lilium chinense as the material, different tissues including roots, adult stems (flowering stage), young stems (seedling stage), leaves, flowers and outer bulb scales were collected and ground with liquid nitrogen. Total RNA was extracted using Trizol reagent (Invitrogen™) according to the instructions. PrimeScript TM RT reagent Kit with gDNA Eraser (PR047A, TaKaRa) was used to obtain cDNA by reverse transcription. 18S The gene was used as an internal reference and SYBR Premix Ex Taq TM II (PR820A, TaKaRa) reagents for quantitative RT- P CR detection was used to analyze the expression characteristics of the LrMYB2 gene in different tissues of lily.

[0034] Detection 18S The gene primers are: 18S -qF:5'-CGCAAGGCTGAAACTTAAAGG -3' 18S -qR:5'- CAGACAAATCGCTCCACCAAC -3' The primers for detecting the LrMYB2 gene are: LrMYB2-QF: 5'- ACCAAATGCAAGACCAAGAAAG-3' LrMYB2-QR: 5'-ATTTCTCCCCAGTCACAATAGATG-3' The relative expression level analysis was performed using 2 −ΔΔCtMethod (LivakKJ, Schmittgen TD, 2001. Analysis of relative gene expression data usingrealtime quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25,402–408) Statistical calculation. The results are shown in Figure 2 As shown in the figure, the expression level of LrMYB2 gene is highest in adult stem tissue, followed by outer scales, flowers and young stems, while the expression level is lower in leaves and roots, indicating that this gene is likely to be involved in the growth and development regulation of stem tissue.

[0035] Example 4 Overexpression LrMYB2 Gene vector construction and genetic transformation of Arabidopsis thaliana (1) Construction of Plgne-35S::LrMYB2 overexpression vector According to the plgne vector sequence and LrMYB2 gene sequence (SEQ ID NO.1), primers LrMYB2-inf-F1 (forward primer) and LrMYB2-inf-R1 (reverse primer) were designed, and the seamless cloning (In-fusion) vector linker sequence and restriction site sequence were introduced into the primers. The positive clone plasmid connected by TA in Example 1 was used as a template. LrMYB2 Specific amplification of gene fragments.

[0036] The primer sequences are as follows: LrMYB2-inf-F1: 5'- acagggtacccggggatccATGGTGAGAGCTCCTTGCTGTG-3' LrMYB2-inf-R1: 5'-ggaattcctgcaggtcgacTCAGATTGTGGGCAAGTCCTGC-3' The bold sequence of the LrMYB2-inf-F1 primer is the BamHI restriction site, and the bold sequence of the LrMYB2-inf-R1 primer is the Sal I restriction site. The underlined part is the In-fusion cloning vector linker sequence.

[0037] The PCR reaction system was: 0.5 μL of high-fidelity amplification enzyme PrimeSTAR HS (R010A, TaKaRa), 5x PrimeSTAR Buffer (Mg 2+Plus) 10μL, forward primer (10μM) 1μL, reverse primer (10μM) 1μL, template (50-fold diluted plasmid) 1μL, dNTP (2.5mM) 4μL, and sterile ddH2O to 50μL.

[0038] PCR reaction conditions: pre-denaturation at 95°C, 5 min; 95°C, 30 s; 60°C, 40 s; 72°C, 1 min, 38 cycles; 72°C, 7 min.

[0039] The PCR amplification product was detected by agarose gel electrophoresis. The amplified target fragment had the same size as the expected fragment, and was recovered and purified according to the instructions of the gel recovery kit (9672, Takara) to obtain the target gene fragment.

[0040] The plgne plant binary expression vector was treated with Bam HI and Sal I. The enzyme digestion system was: plgne vector 5 μL; Bam HI 0.5 μL; Sal I 0.5 μL; 10× QuickCut Buffer 2 μL; sterile ddH2O to 20 μL; 37°C for 3 h. After the enzyme digestion, the plgne vector large fragment was recovered using the Takara agarose gel recovery kit.

[0041] The plgne-35S::LrMYB2 recombinant expression vector was constructed using seamless cloning technology (In-fusion HD Cloning Kit, Takara).

[0042] The recombination reaction system is: Purified PCR fragment (recovery of LrMYB2 target fragment) 50ng; Linearized vector (plgne) 100ng; 5X In-fusion HD Enzyme Premix 2μL; Sterile ddH2O to 10μL. Then, according to the molecular cloning experimental guide, the above recombinant reaction system was transformed into Escherichia coli DH5a and spread on a screening culture plate containing kanamycin (100mg / L). The correct clone was obtained through positive clone screening and sequencing. LrMYB2 The recombinant expression vector plgne-35S::LrMYB2 of the gene fragment was verified by HindIII and EcoRI restriction enzyme digestion. Figure 3As shown in (lane 2), it can be seen that a band of about 1.5 kb was cut out, and the distance between HindIII and EcoRI in the plgne empty vector was 576 bp, indicating that the LrMYB2 gene (762 bp) was successfully connected. It was further sent to BGI for sequencing verification. The 5' end of the target LrMYB2 gene in the recombinant expression vector is located downstream of the constitutive promoter 35S, which can overexpress the LrMYB2 gene; LrMYB2 The 3' end is assembled with 35S poly A terminator, which can effectively terminate the transcription of the fusion gene. NPTII Gene and GUS reporter gene, as screening markers for transgenic plants, can be used to screen transgenic plants and detect GUS staining with kanamycin. LB and RB sequences are assembled on the recombinant expression vector to promote the assembly of the expression framework and screening marker gene between them. NPTII Integrates into the plant recipient chromosome.

[0043] (2) Arabidopsis genetic transformation and identification of positive lines Arabidopsis thaliana genetic transformation was carried out using the floral dip method (Zhang X., et al. , Nat Protoc. 2006, 1:641-646). Agrobacterium carrying the Plgne-35S::LrMYB2 vector was introduced into the Columbia Col type Arabidopsis. Resistant Arabidopsis regenerated seedlings were selected with kanamycin (100 mg / L) to obtain transgenic plants. Genomic DNA was extracted using the CTAB method, and the resistance marker gene was amplified by PCR to detect transgenic positive plants.

[0044] The sequences of the forward and reverse primers are as follows, and the amplified fragment size is 270 bp: Npt II-F: 5'-CGGCTATGACTGGGCACAACAGACAAT-3' Npt II-R: 5'-CTCGGCAGGAGCAAGGTGAGATGAC-3' The PCR reaction reagents used were TaKaRa products (RR001A), including: DNA 1 μL, 10× Buffer (containing 20 mM Mg 2+ ) 2μL, 2.5mM / L dNTP 2μL, 10uM / L forward and reverse primers 0.5μL each, 5U / μL TaqDNA polymerase 0.2 μL, 13.8 μL ddH2O; amplification program: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 30s, a total of 35 cycles; 72℃ extension for 7min; 16℃ storage.

[0045] PCR test results Figure 4 As shown, the presence of Npt II marker gene could not be detected in wild-type plants (negative control, lanes 20 and 21) and transgenic negative plants (lanes 7, 8, 11, 12, 13 and 16). Npt II marker gene was amplified only in lanes 1-6, 9, 10, 14, 15 and 17. Lane 18 was a positive control. A total of 11 positive transgenic lines were obtained, indicating that the recombinant expression frame had been introduced into the genome of the Arabidopsis transgenic lines.

[0046] Furthermore, the transgenic positive plants were analyzed and detected by GUS staining. The seedlings of Plgne-35S::LrMYB2 transgenic lines OX-L1, OX-L2 and wild-type WT plants were used as materials for GUS chemical staining analysis. The specific method is referred to (Jefferson, RA (1987). Assaying chimeric genes in plants: the GUS genefusion system. Plant Mol. Biol. Rep. 5, 387–405. doi: 10.1007 / BF02667740). The test results are shown in Figure 5 As shown in the figure, the seedlings of OX-L1 (B) and OX-L2 (C) lines can be stained blue, while no blue can be detected in the WT control (A), indicating that the GUS staining results are consistent with the PCR test, and the positive lines of LrMYB2 transgenic have been successfully obtained.

[0047] Example 5 Observation on the growth and development of LrMYB2 transgenic strains The transgenic LrMYB2 and wild-type seeds were sown on sterile plates. After about 3 weeks, when 4 leaflets grew, they were transplanted into sterilized soil and cultured normally in a light culture room (with a photoperiod of 14 h light / 10 h dark and a temperature of about 23 ± 2 °C). The growth and development of the wild-type and transgenic LrMYB2 Arabidopsis were observed and compared ( Figure 6 ). Two weeks after transplanting into the soil, it was found that the leaves of the wild-type plants were significantly larger than those of the transgenic plants ( Figure 6 After 4 weeks of growth, the wild-type plants were found to have bolted and flowered, while the transgenic plants (OX-L1) had just begun to bolt and OX-L2 was still in the rosette leaf growth and development stage ( Figure 6B, C, D); 7 weeks after transplantation, the rosette leaves of the wild-type plants began to turn yellow, while the leaves of the transgenic plants were in the green leaf stage (6E in the figure). The growth and development process of the transgenic plants was significantly delayed compared with the wild-type plants. The transgenic plants could bloom in about 6 weeks, which was about 2 weeks later than the wild-type. This shows that the LrMYB2 gene can affect the flowering period of plants (Arabidopsis), specifically delaying the flowering period of plants (Arabidopsis).

[0048] Example 6 Response of LrMYB2 transgenic plants to Botrytis cinerea Botrytis cinerea was inoculated into PDA medium (200 g / L potato, 15 g / L agar, 20 g / L glucose) and cultured in the dark at 28°C for about 1 week. Wild-type and transgenic Arabidopsis plants that had grown normally for about 4 weeks in the greenhouse were used as materials. Fresh fungal hyphae blocks of equal size were inoculated onto detached rosette leaves using a cork puncher. The culture dish was kept humid and placed in a light incubator for normal culture. The infection of Botrytis cinerea was observed after 3 days. The experiment was repeated three times. The results are shown in ( Figure 7 A, B, C), the lesion area of ​​transgenic Arabidopsis leaves (OX-L1, OX-L2) infected by Botrytis cinerea is larger than that of wild-type (WT) controls; quantitative analysis of the measured area shows that ( Figure 7 Middle D), the average damaged area of ​​leaves (OX-L1 and OX-L2) with LrMYB2 gene was approximately 134.81 and 127.21 mm 2 , while the wild type has an injury area of ​​about 92.62mm 2 Therefore, it was shown that overexpression of LrMYB2 gene increased the sensitivity of Arabidopsis to Botrytis cinerea and showed disease characteristics.

[0049] Example 7: Functional verification of LrMYB2 gene in lily leaves The transient transformation method of lily leaves was based on the method of Fu et al. (Plant Physiol Bioch, 2020, 157: 380-382). The Agrobacterium suspension carrying the recombinant plasmid Plgne-35S::LrMYB2 vector or the control transformation solution was transformed into lily leaves, and then the leaves were inoculated with Botrytis cinerea ( B. cinerea ) Inoculation was carried out according to the method of Zhang et al. (Mol PlantPathol, 2019, 20: 309-322). A 4 mm bacterial spot was inoculated on the front of the injection leaf and the leaves were kept moist and cultured normally in a light incubator.

[0050] After 4 days of cultivation, the leaves of lilies transformed with 35S::LrMYB2 and the blank control group were taken as materials for GUS chemical staining analysis. The results showed that the leaves of lilies transformed with 35S::LrMYB2 could be stained light blue ( Figure 8 E), while no blue was detected in the control transformation ( Figure 8 Middle D) shows that the lily leaves were transiently transformed and expressed successfully.

[0051] Inoculation with Botrytis cinerea ( B. cinerea After culturing the lilies for 5 days, observe the infection of the lily leaves by gray mold. Figure 8 A and B show the growth of Botrytis cinerea on leaves of lily plants overexpressing LrMYB2 ( B. cinerea ) infection area was significantly larger than that of the control group. Figure 8 As shown in C, the area of ​​gray mold corrosion on the lily leaf overexpressing LrMYB2 (35S::LrMYB2) is about 5.98 cm 2 , which was significantly larger than the control group (3.65 cm 2 ), which is consistent with the qualitative observation results. The above results show that overexpression of LrMYB2 gene in lily leaves enhances the resistance to Botrytis cinerea ( B. cinerea ) provides a theoretical basis for lily molecular genetic breeding.

[0052] Example 8: Effect of overexpression of LrMYB2 on the activity of antioxidant enzymes Antioxidant enzymes such as peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) affect plant growth and development and immunity to pathogens. Arabidopsis plants grown for about 5 weeks were used as materials, and the content of peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) was measured by spectrophotometry (refer to Hui et al., 2012, Plant Cell Rep., 31(6): 1073-1084.). The results showed that ( Fig. 9 ), the POD, SOD and CAT enzyme activities in the transgenic Arabidopsis lines were significantly lower than those in the wild-type control group. This indicates that LrMYB2 may regulate plant growth and development and sensitivity to Botrytis cinerea by affecting the activity of antioxidant enzymes.

[0053] In addition, the sequence table of the above SEQ ID NO.1 to SEQ ID NO.12 is attached as follows: Sequence 1: SEQ ID NO.1 LrMYB2 nucleotide sequence, 762 bp long ATGGTGAGAGCTCCTTGCTGTGAGAAGATGGGGTTGAAGAAGGGGCCATGGACGCAAGAGGAGGACGAGATACTGGTTGCTTACATCCAGAGCTATGGCCATGGAAATTGGAGAGCTCTCCCGAAAGAAGCTGGGTTGCTGAGGTGTGGGAAGAGTTGTCGGCTCCGGTGGACGAACTACTTGAGGCCGGATATCAAGAGAGGGAACTTCAGCAGGGATGAGGAGGAGATGATCATCAGCTTGCATGAGATGCTTGGCAATAGATGGTCTGCGATCGCAGCTAGACTACCCGGAAGAACAGATAACGAAATCAAGAATGTGTGGAACACCCATTTGAAGAAACGGGTTGAACCGACTCAAGCATTTCCTGAATCCAAGAGAAAAACCAAATGCAAGACCAAGAAAGACACAGGAGTCAAATCTGAAGCACCCCACATTGCCCCATCATCACCAGCACAGTCGAACAGCGATATCTGTTCGGCCACCGAATCATCTATTGTGACTGGGGAGAATCATACTCGCATGGTGGATGAGAGAATGGAATCTCTGGAAGAATTGCCAGAGCTCGATGACAGTTTTTGGTCGGAGGCATTGTCGATGGACAACTCCGCCCTATCGAACGATCTTACAACATTCGAAGGCCCACTCGCAACTCCTATCGGTGAACCATTCGGTTCGATCAGTTCTTCCAACAATGAAATGAGCTTCTGGCTAAAGATTTTCATCGAAAATGGTGAGCTGCAGGACTTGCCCACAATCTGA Sequence 2: SEQ ID NO.2 Amino acid sequence of LrMYB2, 253 amino acids in length MVRAPCCEKMGLKKGPWTQEEDEILVAYIQSYGHGNWRALPKEAGLLRCGKSCRLRWTNYLRPDIKRGNFSRDEEEMIISLHEMLGNRWSAIAARLPGRTDNEIKNVWNTHLKKRVEPTQAFPESKR KTKCKTKKDTGVKSEAPHIAPSSPAQSNSDISATESSIVTGENHTRMVDERMESLEELPELDDSFWSEALSMDNSALSNDLTTFEGPLATPIGEPFGSISSSNNEMSFWLKIFIENGELQDLPTI* Sequence 3: SEQ ID NO.3 LrMYB2-F nucleotide sequence, 22 bp long ATGGTGAGAGCTCCTTGCTGTG Sequence 4: SEQ ID NO.4 LrMYB2-R nucleotide sequence, 22 bp long TCAGATTGTGGGCAAGTCCTGC Sequence 5: SEQ ID NO.5 18S-qF nucleotide sequence, 21 bp long CGCAAGGCTGAAACTTAAAGG Sequence 6: SEQ ID NO.6 18S-qR nucleotide sequence, 21 bp long CAGACAAATCGCTCCACCAAC Sequence 7: SEQ ID NO.7 LrMYB2-QF nucleotide sequence, 22 bp long ACCAAATGCAAGACCAAGAAAG Sequence 8: SEQ ID NO.8 LrMYB2-QR nucleotide sequence, 23 bp long ATTCTCCCCAGTCACAATAGATG Sequence 9: SEQ ID NO.9 LrMYB2-inf-F1 nucleotide sequence, 41 bp long ACAGGGTACCCGGGGATCCATGGTGAGAGCTCCTTGCTGTG Sequence 10: SEQ ID NO.10 LrMYB2-inf-R1 nucleotide sequence, 41 bp long GGAATTCCTGCAGGTCGACTCAGATTGTGGGCAAGTCCTGC Sequence 11: SEQ ID NO.11 Npt II-F nucleotide sequence, 27 bp long CGGCTATGACTGGGCACAACAGACAAT Sequence 12: SEQ ID NO.12 Npt II-R nucleotide sequence, 25 bp long CTCGGCAGGAGCAAGGTGAGATGAC Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Application of LrMYB2 gene in regulating plant growth and development and flowering period, characterized in that: The nucleotide sequence of the LrMYB2 gene is as shown in SEQ ID NO.1 or a nucleotide sequence having the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, wherein the amino acid sequence of the LrMYB2 gene expressed is as shown in SEQ ID NO.2 or an amino acid sequence having the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.

2.

3. The use according to claim 1, wherein the plant is lily or Arabidopsis thaliana.

4. The use according to claim 1, wherein the LrMYB2 gene is involved in the growth and development regulation of the plant's stem tissue.

5. The use according to claim 1, wherein the regulation of the LrMYB2 gene in the flowering period of the plant is to delay the flowering period of the plant.

6. Application of LrMYB2 gene in regulating plant sensitivity to Botrytis cinerea, characterized in that: The nucleotide sequence of the LrMYB2 gene is as shown in SEQ ID NO.1 or a nucleotide sequence having the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.1; The amino acid sequence of the LrMYB2 gene expressed is as shown in SEQ ID NO.2 or an amino acid sequence having the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.

2.

7. The use according to claim 6, characterized in that: The LrMYB2 gene regulates the sensitivity of plants to Botrytis cinerea to enhance the sensitivity of plants to Botrytis cinerea.

8. The use according to claim 6, characterized in that: The LrMYB2 gene affects the sensitivity of plants to Botrytis cinerea by affecting the activity of antioxidant enzymes.

9. A method for regulating the growth and flowering period of a plant, characterized in that: The expression level of the LrMYB2 gene in the plant is increased by introducing a recombinant vector, an expression cassette, a cell or a recombinant bacterium containing the LrWRKY-R2 gene into the plant cell.

10. A method for regulating the sensitivity of a plant to Botrytis cinerea, characterized in that: The expression level of the LrMYB2 gene in the plant is increased by introducing a recombinant vector, an expression cassette, a cell or a recombinant bacterium containing the LrWRKY-R2 gene into the plant cell.

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