Lily MYB family member LrMYB1 gene and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a lily MYB family member LrMYB1 gene and application thereof, the nucleotide sequence of the gene LrMYB1 is as shown in SEQ ID NO.1, and the coded amino acid sequence of the gene LrMYB1 is as shown in SEQ ID NO.2. The lily gene LrMYB1 is used for promoting lignin synthesis of plants; the lily gene LrMYB1 is used for increasing the resistance of plants to botrytis cinerea. Through construction of a 35S overexpression vector and overexpression analysis of the gene in arabidopsis thaliana, it is found that the lignin content in stems of LrMYB1 transgenic plants is significantly increased compared with that of wild plants, leaves slightly roll up, meanwhile, the resistance to botrytis cinerea is enhanced, and it is further found that the LrMYB1 positively regulates and controls the resistance to botrytis cinerea through instantaneous overexpression of LrMYB1 in lily and tobacco leaves. Therefore, a foundation is laid for analyzing lily lignin synthesis regulation and gray mold resistance molecular mechanisms, a new target is provided for improving gray mold resistance of lilies or other plants by using a transgenic technology in future, and important theoretical and application values are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a lily MYB family member LrMYB1 gene and application thereof. Background Art

[0002] In nature, the evolution of various organisms has always followed the principle of natural selection. Therefore, under the influence of the survival of the fittest, existing plants have often developed a series of defense mechanisms in order to avoid various biological and non-biological damage.

[0003] Among the transcription factor families of plants, there is a MYB transcription factor family, which 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. Zou Hongzhu et al. studied the important role of the MYB transcription factor family in the yellowing process of Yunnan peony flowers, providing candidate genes for peony flower color improvement and molecular breeding, and found that PdMYB2 in the MYB transcription factor family has a positive regulatory effect in the flavonoid synthesis pathway, which can promote the accumulation of anthocyanins and increase the expression of structural genes in the flavonoid pathway. （邹红竹，韩璐璐，周琳，吕纪杭，贾莹华，王雁，滇牡丹 MYB 家族成员鉴定及PdMYB2 的功能验证，林业科学研究，DOI:10.13275 / j.cnki.lykxyj.2022.005. 001） Zhang Yining et al. found that the wheat MYB transcription factor gene TaPHR1 is involved in regulating the number of spikelets per ear ( 张逸宁，张艳菲，汪敏，王景一，李龙，李超男，杨德龙，毛新国，景蕊莲．小麦 MYB 转录因子基因 TaPHR1 参与调控每穗小穗数[J / OL]．作物学报.https: / / kns.cnk i.net / kcms2 / detail / 11.1809.S.20230705.1720.002.html） In addition, MYB also affects the plant's disease resistance. Wang Haotian et al. summarized the basic characteristics of R2R3-MYB transcription factors and focused on the transcriptional regulation mechanism of R2R3-MYB transcription factors in the response process of plant drought, low temperature, high salt stress, etc. （王浩田，蒋景龙，王倩，魏丽娜，胡佳．R2R3-MYB 转录因子响应植物抗逆机制研究进展[J / OL]．分子植物育种） .

[0004] Lily is one of the famous flowers at home and abroad, and is also an important vegetable, occupying an important share in the domestic and foreign markets. The growth and development of lily plants are regulated by many factors, such as environmental factors and genetic factors. Wang Rui et al. cloned a LhTTG1 gene using Oriental lily as material. The gene is mainly expressed in petals, and the expression is highest in flower buds during the coloring period. (王瑞, 刘思 泱,何祥凤,赵祥云,王文和, 2022,百合 TTG1 基因的克隆、表达及其与 MYB 和 bHLH 的互作分析,分子植物育种, 20(16): 5258-5266.) Fan Wenguang et al. studied lily of Lanzhou and found that by constructing evolutionary tree analysis, conservative domain prediction, gene expression analysis and correlation analysis between transcription factors and structural genes with MYB and bHLH transcription factors of the model plant Arabidopsis, they preliminarily screened out three MYB genes and one bHLH gene that can promote the synthesis of anthocyanins in the bulbs of lily of Lanzhou ( 范文广，李保豫，田辉，李欣，任海伟，曹莹莹，姜欣彤，柴佳靖，陈少青．兰州百合鳞茎花青素合成相关 MYB 与 bHLH 转录因子的筛选分析[J / OL]．食品与发酵工业.http s: / / doi.org / 10.13995 / j.cnki.11-1802 / ts.033270）Wang Xueqian and others studied the identification of the MYB transcription factor family of Asian lilies and the screening of MYB genes that regulate pollen abortion, and found that the LaMYB gene plays an important regulatory role in the process of pollen abortion. This study provides basic data for in-depth exploration of the function of the LaMYB gene in the process of pollen abortion （王雪倩， 袁国振， 吴泽，李茜，滕年军，亚洲百合MYB转录因子家族的鉴定及调控花粉败育MYB基因的筛选，农业生物技术学报，2019, 27(11): 1951~1961 DOI: 10.3969 / j.issn.1674-7968.2019.11.006） .

[0005] Botrytis cinerea is one of the most common diseases that harms 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 production, and quality to deteriorate. 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 disease-resistant lily varieties is the most effective, economical and environmentally friendly measure to solve the problem of lily gray mold disease. Therefore, the excavation of lily gray mold resistance genes and the analysis of their important physiological functions are of great theoretical and practical value for the cultivation of disease-resistant lily varieties.

[0006] 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 is used in lily disease-resistant breeding. Sun et al. (2016) isolated the ATP-binding transporter gene ( LrABCF1 ), which was introduced into petunia ( Petunia × hybrida ) found that transgenic plants were resistant to Botrytis cinerea ( B. cinerea ) has significantly improved resistance （Sun D, Zhang X, Li S,et al. (2016)LrABCF1, a GCN-type ATP-binding cassette transporter fromLilium regale, is involved in defense responses against viral and fungal pathogens. Planta, 244:1185-1199.） In addition, it was found that LrWRKY genes with different three-dimensional structures had different B. cinerea ) show different functions in the regulation of resistance （Fu Y, Li J, Wu H, et al. (2022) Analyses ofBotrytis cinerea-responsiveLrWRKY genes fromLilium regale reveal d istinct roles of two LrWRKY transcription factors in mediating responses toB. cinerea. Plant Cell Reports , 41(4): 995-1012.） The MYB family plays an important regulatory role in plant development and morphological construction and adverse stress. Currently, there are few reports on the functions of lily MYB family genes, and the mechanism by which MYB family genes regulate lignin synthesis and improve gray mold resistance is still unclear.

[0007] The present invention cloned a MYB gene member responsive to Botrytis cinerea from Minjiang lily, and elucidated its biological function through reverse genetics methods, providing a new target for analyzing its mechanism of action and subsequent lily genetic breeding improvement. Summary of the invention

[0008] The purpose of the present invention is to provide a lily MYB family member LrMYB1 gene and its application, to provide a new target for the regulation of lignin synthesis in lily plants, and to provide more gene options for improving the gray mold resistance of lily, and ultimately to provide strong support for obtaining excellent traits of lily through molecular breeding.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention also provides the application of the LrMYB1 gene in the regulation of lignin synthesis in plants, characterized in that the nucleotide sequence of the LrMYB1 gene is as shown in SEQ ID NO.1 or has 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.

[0010] Furthermore, the amino acid sequence of the LrMYB1 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.

[0011] Furthermore, the LrMYB1 gene regulates the lignin synthesis in plants to promote the synthesis of lignin in plants.

[0012] The present invention also provides the application of the LrMYB1 gene in regulating the resistance of plants to Botrytis cinerea. The nucleotide sequence of the LrMYB1 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.

[0013] Furthermore, the amino acid sequence of the LrMYB1 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.

[0014] Furthermore, the regulation of the LrMYB1 gene in the plant's resistance to Botrytis cinerea is to enhance the plant's resistance to Botrytis cinerea.

[0015] Furthermore, the LrMYB1 gene mediates the plant's immune response to Botrytis cinerea by regulating the lignin synthesis pathway.

[0016] The present invention also provides a method for regulating lignin synthesis, which increases the expression level of the LrMYB1 gene in the plant by introducing a recombinant vector, an expression cassette, a cell or a recombinant bacterium containing the LrMYB1 gene into the plant cell.

[0017] The present invention also provides a method for regulating the resistance of a plant to Botrytis cinerea, by introducing a recombinant vector, an expression cassette, a cell or a recombinant bacterium containing the LrMYB1 gene into plant cells to increase the expression level of the LrMYB1 gene in the plant.

[0018] The present invention has at least the following beneficial effects: 1. The present invention cloned the full-length gene sequence of LrMYB1, a member of the MYB family gene, from Lilium chinense for the first time, and obtained the protein sequence encoded by it. Quantitative PCR analysis showed that the gene was highly expressed in the stems of adult plants. Overexpression analysis of the gene in Arabidopsis thaliana revealed that the rosette leaves of the transgenic plants were slightly curled, and the lignin content in the stems was significantly higher than that of the wild-type control group, providing a key transcription factor for the regulation of lignin synthesis in Lilium plants in the future.

[0019] 2. The present invention shows through molecular genetics and biochemical analysis that overexpression or transient expression of LrMYB1 gene in Arabidopsis, lily or tobacco increases the resistance of transformed plants to Botrytis cinerea ( Botrytis cinerea ) has enriched the disease-resistant gene pool of plants, provided new molecular targets for improving the gray mold resistance of lily and other plants, and laid a theoretical foundation for analyzing the regulatory mechanism of lily's resistance to gray mold. 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 Minjiang lily LrMYB1 with four similar MYB 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 analysis of LrMYB1 gene in Lilium chinense. The horizontal axis is root, mature stem, young stem, leaf, flower and external bulb. The vertical axis is the relative expression level. Figure 3This is the electrophoresis diagram of the restriction enzyme digestion verification analysis of the vector plgne-35S::LrMYB1, where M is a 12kb DNA marker; lane 1 is a plasmid control; lane 2 is a picture after Kpn I-Sal I digestion; Figure 4 This is the electrophoresis diagram of PCR amplified transgenic Arabidopsis plants. Lanes 1-15 are transgenic lines, lane 16 is a positive control (plasmid as template), lane 17 is a 1kb DNA marker, and lanes 18 and 19 are negative controls. Figure 5 GUS staining analysis of transgenic Arabidopsis plant leaves. A is the wild-type plant (WT) control, B, C and D are three independent transgenic lines OX-L3, OX-L8, and OX-L15. The scale bar represents 1 mm. Figure 6 To detect the phenotype and lignin content of transgenic plants, A and B are the phenotypes of wild-type (WT) and LrMYB1-overexpressing plants grown in greenhouse soil for about 5 weeks; C and D are the observations of phloroglucinol staining of the cross-section of the stem at the base of the wild-type and transgenic plants; E is the lignin quantification of the wild-type (WT) and transgenic plants (OX-L3, OX-L8, OX-L15) at about 7 weeks using the AcBr method.

[0022] Figure 7 Figure 2 Analysis of resistance of transgenic Arabidopsis thaliana leaves to Botrytis cinerea. A, B, C, and D are the phenotypes of leaves of WT, OX-L3, OX-L8, and OX-L15 plants inoculated with Botrytis cinerea for 2 days, respectively. E is a quantitative analysis of the corrosion area of ​​Arabidopsis leaves inoculated with Botrytis cinerea for 2 days. The error bars represent the ±SE values ​​of three repeated experiments. Different letters represent significant differences. Duncan test: p value < 0.05.

[0023] Figure 8 Figure 3 is the effect of transient transformation of LrMYB1 gene in lily leaves on resistance to Botrytis cinerea; A and B are the effect pictures of transformed leaves 5 days after infection with Botrytis cinerea; A is the control group, B is the experimental group (injected with 35S::LrMYB1 overexpression vector); C and D are GUS staining analysis of transformed lily leaves, D is the staining of lily leaves injected with control solution, E is the staining of lily leaves injected with Agrobacterium containing 35S::LrMYB1 vector, the scale bar represents 1 mm; E is the quantitative analysis of the corrosion area of ​​lily leaves inoculated with Botrytis cinerea for 5 days, the error bars represent 3 repeated experiments ± SE values, different letters represent significant differences, Duncan test: p value <0.05.

[0024] Fig. 9Figure 3 is the effect of transient transformation of LrMYB1 gene in tobacco leaves on resistance to Botrytis cinerea; AD is the effect of Botrytis cinerea infection on transformed leaves 5 days later, A and B are control groups (Control), C and D are experimental groups (injected with 35S::LrMYB1 overexpression vector); E is the quantitative analysis of the corrosion area of ​​tobacco leaves inoculated with Botrytis cinerea for 5 days, the error bars represent 3 repeated experiments ± SE values, different letters represent significant differences, Duncan test: p value <0.05.

[0025] Fig.10 Quantitative PCR analysis of the effect of LrMYB1 overexpression on the expression of genes in the lignin biosynthesis pathway (AtC4H, At4CL1, AtC3H, AtCCR1, AtF5H, AtCOMT); AtActin gene was used as the internal reference, error bars represent 3 repeated experiments ± SE values, asterisks represent Student's T test: ** P < 0.01. Implementation

[0026] The present invention is further described in detail below in conjunction with specific examples and drawings, but the scope of protection of the present invention is not limited to the above contents. The raw materials described in the examples are all common commercially available 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.

[0027] Through comparative transcriptome sequencing analysis, we previously discovered a unigene gene that was upregulated in response to Botrytis cinerea, which belongs to the MYB gene family and was named LrMYB1.

[0028] Example 1 Cloning and sequence analysis of the LrMYB1 gene of Lilium chinense 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.

[0029] 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).

[0030] The PCR amplification system was high-fidelity amplification enzyme Prime STAR HS (R010A, TaKaRa) 0.25 μL, 5× PrimeSTAR Buffer (Mg 2+ Plus) 5μL, forward primer (LrMYB1-F, 10μM) 0.5μL, reverse primer (LrMYB1-R, 10μM) 0.5μL, template (DNA) 1μL, dNTP (2.5mM) 2μL, sterile ddH 2 Add 0.1% HO to make up to 25 μL.

[0031] The forward and reverse primers are: LrMYB1-F:5'-ATGGGAAGGCACTCCTGT-3' LrMYB1-R: 5'-CTAATAGTGTATCACTCCCTGAGC-3' PCR reaction program: pre-denaturation at 95°C, 5 min; 95°C, 30 s; 55°C, 40 s; 72°C, 1 min 10 s, 38 cycles; 72°C, 5 min.

[0032] The PCR product was analyzed by agarose gel electrophoresis, and a specific amplification band was observed between 1000-1500 bp under ultraviolet light. It was purified according to the gel recovery kit (9672, Takara) and used for later use.

[0033] The purified DNA fragment was tagged with A using a blunt end A reagent and connected to pMD20-T vector (6019, Takara) through TA cloning. The ligation product was transformed into Escherichia coli DH5a, and 1-2 positive clones were picked from the LB culture plate containing ampicillin (100 mg / L) for sequencing analysis. The results showed that the full-length sequence of the Minjiang lily LrMYB1 gene was shown in SEQ ID NO.1, including an open reading frame of 1101 bp (including a stop codon).

[0034] According to SEQ ID NO.1, the protein sequence of LrMYB1 of Lilium chinense was obtained by DNAman software translation as shown in SEQ ID NO.2, which contains 366 amino acids (* indicates the termination signal). The nucleotide sequence of LrMYB1 was compared with NCBI database by BLAST, and no highly similar sequence was found, but it had high sequence identity with Iris pallida hypotheticalprotein M6B38_114215, Elaeis guineensis transcription factor MYB61, Asparagusofficinalis transcription factor MYB86-like and Cinnamomum micranthum f.kanehirae R2R3-MYB transcription factor MYB2, which were 218 / 385 (57%), 222 / 403 (55%), 212 / 365 (58%) and 219 / 417 (53%), respectively.

[0035] Protein sequence analysis was performed by comparing the protein sequence at http: / / multalin.toulouse.inra.fr / multalin / Figure 1 ), and found that LrMYB1 belongs to the R2R3-MYB family and is a new member of the lily MYB family.

[0036] Example 2 Tissue expression specificity of LrMYB1 gene Using Lilium chinense as the material, different tissues including roots, adult stems (flowering stage), young stems (seedling stage), leaves, flowers and outer 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 LrMYB1 gene in different tissues of lily.

[0037] Detection 18S The gene primers are: 18S-qF:5'-CGCAAGGCTGAAACTTAAAGG -3' 18S -qR:5'- CAGACAAATCGCTCCACCAAC -3' The primers for detecting the LrMYB1 gene are: LrMYB1-QF: 5'- TCCCGTTCTGGGAAGCTGGT-3' LrMYB1-QR: 5'- GCAGCGTCAACTCCGACCAT-3' The relative expression level analysis was performed using 2 −ΔΔCt Method (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 LrMYB1 gene was the highest in adult stem tissue, followed by young stem, flower and outer scale, and the lowest in leaves and roots, indicating that this gene is likely to be involved in the growth and development of secondary vascular tissue.

[0038] Example 3 Construction of LrMYB1 gene overexpression vector and genetic transformation of Arabidopsis (1) Construction of Plgne-35S::LrMYB1 overexpression vector According to the plgne vector sequence and LrMYB1 gene sequence (SEQ ID NO.1), primers LrMYB1-inf-F (forward primer) and LrMYB1-inf-R (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 to perform specific amplification of the LrMYB1 gene fragment.

[0039] The primer sequences are as follows: LrMYB1-inf-F: 5'-acagggtacccgg ggatcc ATGGGAAGGCACTCCTGT-3' LrMYB1-inf-R: 5'-ggaattcctgcag gtcgac CTAATAGTGTATCACTCCCTGAGC-3' The bold and underlined sequence of the LrMYB1-inf-F primer is the BamHI restriction site, and the bold and underlined sequence of the LrMYB1-inf-R primer is the Sal I restriction site. Lowercase letters indicate that this is the In-fusion cloning vector linker sequence.

[0040] 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, sterile ddH 2 Add HO to make up to 50 μL.

[0041] PCR reaction conditions: pre-denaturation at 95°C, 5 min; 95°C, 30 s; 55°C, 40 s; 72°C, 1 min 10 s, 38 cycles; 72°C, 5 min.

[0042] 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.

[0043] 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 ddH 2 O to 20 μL; react at 37°C for 3 h. After the enzyme digestion, the plgne vector large fragment was recovered using the Takara agarose gel recovery kit.

[0044] The plgne-35S::LrMYB1 recombinant expression vector was constructed using seamless cloning technology (In-fusion HD Cloning Kit, Takara).

[0045] The recombination reaction system is: Purified PCR fragment (recovery of LrMYB1 target fragment) 50ng; Linearized vector (plgne) 100ng; 5X In-fusion HD Enzyme Premix 2μL; sterile ddH 2O was added to 10 μL. Then, the above recombinant reaction system was transformed into Escherichia coli DH5a according to the molecular cloning experiment guide and spread on a screening culture plate containing kanamycin (100 mg / L). The correct recombinant expression vector plgne-35S::LrMYB1 containing the LrMYB1 gene fragment was obtained through positive clone screening and sequencing. After KpnI and SalI restriction enzyme digestion verification, Figure 3 As shown in (lane 2), a band of about 1.1 kb was cut out, indicating that the LrMYB1 gene (1101 bp) was successfully connected. It was further sent to BGI for sequencing verification. The 5' end of the target gene LrMYB1 in the recombinant expression vector is located downstream of the constitutive promoter 35S, which can overexpress the LrMYB1 gene; LrMYB1 The 3' end is assembled with a 35S poly A terminator, which can effectively terminate the transcription of the fusion gene. The NPTII gene and the GUS reporter gene are assembled on the recombinant expression vector as screening markers for transgenic plants. Kanamycin can be used to screen transgenic plants and detect GUS staining. The LB and RB sequences are assembled on the recombinant expression vector to promote the integration of the expression frame and the screening marker gene NPTII assembled therein into the plant receptor chromosome.

[0046] (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::LrMYB1 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.

[0047] 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+) 2 μL, 2.5 mM / L dNTP 2 μL, 10 uM / L forward and reverse primers 0.5 μL each, 5 U / μL TaqDNA polymerase 0.2 μL, 13.8 μL ddH 2 O; amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, a total of 35 cycles; 72℃ extension for 7 min; 16℃ storage.

[0048] 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 18, 19) and transgenic negative plants (lanes 12, 13, 14). Npt II marker gene was amplified only in lanes 1-11, 15 and 16 (positive control), and a total of 12 positive transgenic lines were obtained, indicating that the recombinant expression frame had been introduced into the genome of the Arabidopsis transgenic lines.

[0049] Furthermore, the transgenic positive plants were analyzed and detected by GUS staining. The seedlings of Plgne-35S::LrMYB1 transgenic lines OX-L1, OX-L2, OX-L3 and wild-type WT plants were taken as materials for GUS chemical staining analysis. The specific method is referred to (Jefferson, RA (1987). Assaying chimeric genes in plants: the GUSgene fusion 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), OX-L2 (C), and OX-L3 (D) 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 LrMYB1 transgenic have been successfully obtained.

[0050] Example 4 Morphological observation of transgenic LrMYB1 plants and wild type Transgenic LrMYB1 and wild-type seeds were sown on sterile plates. After about 2-3 weeks, when 4 leaflets grew, they were transplanted into sterilized soil and cultured normally in a light culture room (photoperiod of 14h light / 10h dark, temperature of about 23±2℃). The growth and development of wild-type and transgenic LrMYB1 Arabidopsis were observed and compared. About 5 weeks after transplanting, it was found that ( Figure 6A, B), compared with the wild-type control, the rosette leaves of the transgenic plants were slightly curled, suggesting that overexpression of LrMYB1 may affect the synthesis of lignin. Furthermore, the cross-sections of the stems at the base of the wild-type and transgenic plants were stained with phloroglucinol (Li C., et al . Plant Cell Physiol. 2015, 56(12): 2436-2446.) analysis, the results showed that ( Figure 6 C, D) The stem vascular tissue of the transgenic plants was stained darker. When the plants were grown for about 7 weeks, the AcBr method (Fu Y., et al. Plant Physiol Biochem, 2020, 157: 379-389) quantitative analysis showed that the lignin content in transgenic plants (OX-L3, OX-L8 and OX-L15) was significantly higher than that in wild-type (WT) controls, and the transgenic line OX-L15 had the highest lignin content. Therefore, overexpression of LrMYB1 in Arabidopsis promoted the synthesis of lignin.

[0051] Example 6 Response of transgenic LrMYB1 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 48 hours. The results are shown in ( Figure 7 AD), the extent of corrosion of leaves of transgenic Arabidopsis plants (OX-L3, OX-L8 and OX-L15) infected with Botrytis cinerea was lower than that of wild type (WT) control; quantitative determination of corrosion area showed that ( Figure 7 E) The average damaged area of ​​leaves transgenic for LrMYB1 (OX-L3, OX-L8 and OX-L15) was 0.97, 0.69 and 0.79 cm 2 , while the wild type had an injury area of ​​approximately 1.31 cm 2 Therefore, overexpression of the LrMYB1 gene increased the resistance of transformed Arabidopsis to Botrytis cinerea and showed disease resistance characteristics.

[0052] Example 7: Functional verification of LrMYB1 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::LrMYB1 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.

[0053] After 4 days of culture, the leaves of lilies transformed with 35S::LrMYB1 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::LrMYB1 could be stained light blue ( Figure 8 D), while no blue was detected in the control transformation ( Figure 8 (C) shows that LrMYB1 was successfully expressed in lily leaves after transient transformation.

[0054] Inoculation with Botrytis cinerea ( B. cinerea ) for 5 days, and observe the corrosion of lily leaves by gray mold. Figure 8 A and B show the growth of Botrytis cinerea on leaves of lily plants overexpressing LrMYB1. B. cinerea ) The infection area of ​​the control group was smaller than that of the gray mold. Figure 8 As shown in E, the area of ​​gray mold corrosion on the leaves of lily overexpressing LrMYB1 (35S::LrMYB1) is about 2.11 cm 2 , which was significantly smaller than the control group (3.08 cm 2 ), which is consistent with the qualitative observation results. The above shows that overexpression of LrMYB1 gene in lily leaves enhances the resistance to Botrytis cinerea ( B. cinerea ) resistance, providing new targets for lily molecular genetic breeding.

[0055] Example 8: Functional analysis of LrMYB1 gene in tobacco leaves Tobacco is a model plant commonly used in plant immunology research and has good representativeness. To this end, according to the method described in Example 7, the Agrobacterium suspension containing the recombinant plasmid Plgne-35S::LrMYB1 or the control transformation liquid was transformed into tobacco leaves, and then inoculated with Botrytis cinerea ( B. cinerea ) After 5 days, the disease condition of petunia leaves was observed. Fig. 9 As shown, the number of Botrytis cinerea on leaves transformed with LrMYB1 ( B. cinerea ) Infection area ( Fig. 9The infection area of ​​pathogens in C and D was significantly smaller than that in the control group ( Fig. 9 Compared with the control group ( Fig. 9 Middle E), the area of ​​the damaged lesions on the LrMYB1 transformed leaves increased from 5.99 cm 2 Reduced to 2.30cm 2 The results showed that expressing LrMYB1 in the model plant tobacco also improved resistance to Botrytis cinerea, providing an important reference for disease-resistant breeding of other plants.

[0056] Example 9: Effect of overexpression of LrMYB1 on the expression of key genes in the lignin synthesis pathway Quantitative PCR was used to analyze the expression of LrMYB1 gene and lignin biosynthesis pathway genes in transgenic Arabidopsis. Quantitative PCR primers for lignin pathway genes (AtC4H, At4CL1, AtC3H, AtCCR1, AtF5H and AtCOMT) can be found in (Fu et al., 2020, Plant Physiology and Biochemistry, 157: 379-389.) Total RNA from wild-type and transgenic Arabidopsis thaliana (OX-L3 and OX-L15) plants was extracted using Trizol reagent (Invitrogen™), residual DNA was removed using DNase I (Invitrogen™), and first-strand cDNA was synthesized using cDNA reverse transcription reagent (6210A, Takara) according to the instructions. TM II (PR820A, TaKaRa) reagent for quantitative detection. AtActin The gene was used as the internal reference, and 2 -△△CT The relative expression of genes was analyzed by Fig.10 shown.

[0057] from Fig.10 As can be seen from the results, the LrMYB1 gene was significantly upregulated in the transgenic lines (OX-L3 and OX-L15). The key genes for lignin synthesis, AtC4H, At4CL1, and AtCCR1, were upregulated in two independent transgenic lines. The results showed that LrMYB1 may mediate the immune response of Arabidopsis plants to Botrytis cinerea by regulating the lignin synthesis pathway.

[0058] In addition, the sequence listing of the above-mentioned SEQ ID NO.1-SEQ ID NO.12 is attached: Sequence 1: SEQ ID NO.1 LrMYB1 nucleotide sequence, length 1101 bp Sequence 2: SEQ ID NO.2 LrMYB1 amino acid sequence, 366 amino acids long MGRHSCCYKQKLRKGLWSPEEDEKLLNHITRYGHGCWSSVPKQAGLQRCGKSCRLRWINYLRPDLKRGTFSPQEESLIIELHAVLGNRWSQIAAQLPGRTDNEIKNLWNSCIKKKLKQQGIDPNTHKPLIGAPERAPSESDKISGSSDQKLLAASGSTNPIIPDPSPSLIPVFDSSSSFNCRP SSTTGYYPLQQLNCQSGLVGLSSSSNPLLWLNQYSRLNNNNMSTDFICNNSSTIVPSINLLPDNSLPGCAGINSVPFWEAGNTSNSTGSSGSNTSGIELQSNSSIFDGSILSWSELTLHKDSQIHLDSEPADIKWLEYLQVGYPISAAIQSQTQTSYSGIKAESHGHPENTSNVWTDLAQGVIHY* Sequence 3: SEQ ID NO.3 LrMYB1-F nucleotide sequence, length 18 bp ATGGGAAGGCACTCCTGT Sequence 4: SEQ ID NO.4 LrMYB1-R nucleotide sequence, length 24 bp CTAATAGTGTATCACTCCCTGAGC Sequence 5: SEQ ID NO.5 18S-qF nucleotide sequence, length 21 bp CGCAAGGCTGAAACTTAAAGG Sequence 6: SEQ ID NO.6 18S-qR nucleotide sequence, length 21 bp CAGACAAATCGCTCCACCAAC Sequence 7: SEQ ID NO.7 LrMYB1-QF nucleotide sequence, length 20 bp TCCCGTTCTGGGAAGCTGGT Sequence 8: SEQ ID NO.8 LrMYB1-QR nucleotide sequence, length 20 bp GCAGCGTCAACTCCGACCAT Sequence 9: SEQ ID NO.9 LrMYB1-inf-F nucleotide sequence, length 37 bp acagggtacccggggatccATGGGAAGGCACTCCTGT Sequence 10: SEQ ID NO.10 LrMYB1-inf-P nucleotide sequence, length 43 bp ggaattcctgcaggtcgacCTAATAGTGTATCACTCCCTGAGC Sequence 11: SEQ ID NO.11 Npt II-F nucleotide sequence, length 27 bp CGGCTATGACTGGGCACAACAGACAAT Sequence 12: SEQ ID NO.12 Npt II-R nucleotide sequence, length 25 bp CTCGGCAGGAGCAAGGTGAGATGAC The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of LrMYB1 gene in regulating lignin synthesis in plants, It is characterized in that The nucleotide sequence of the LrMYB1 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, It is characterized in that The amino acid sequence of the LrMYB1 gene is expressed 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, It is characterized in that The LrMYB1 gene regulates the lignin synthesis in plants to promote the synthesis of plant lignin.

4. Application of LrMYB1 gene in regulating plant resistance to Botrytis cinerea, It is characterized in that The nucleotide sequence of the LrMYB1 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.

5. The use according to claim 4, It is characterized in that The amino acid sequence of the LrMYB1 gene is expressed 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.

6. The use according to claim 4, It is characterized in that The regulation of the LrMYB1 gene on the plant's resistance to Botrytis cinerea is to enhance the plant's resistance to gray mold.

7. The use according to claim 4, It is characterized in that The LrMYB1 gene mediates the plant's immune response to Botrytis cinerea by regulating the lignin synthesis pathway.

8. A method for regulating lignin synthesis, It is characterized in that The expression level of the LrMYB1 gene in the plant is increased by introducing a recombinant vector, an expression cassette, a cell or a recombinant bacterium containing the LrMYB1 gene into the plant cell.

9. A method for regulating the resistance of a plant to Botrytis cinerea, It is characterized in that The expression level of the LrMYB1 gene in the plant is increased by introducing a recombinant vector, an expression cassette, a cell or a recombinant bacterium containing the LrMYB1 gene into the plant cell.

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