A gene LrF3'H, a flavonoid 3'-hydroxylase from Lilium minjiangense, and its application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- KUNMING UNIV OF SCI & TECH
- Filing Date
- 2024-08-21
- Publication Date
- 2026-05-26
Smart Images
Figure CN118703538B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of molecular biology and genetic engineering, specifically to a 3'-hydroxylase gene for flavonoids in *Lilium minjiangense* that enhances plant resistance to fungal infection. LrF3'H And its applications. Background Technology
[0002] Viruses, bacteria, and fungi are pathogenic microorganisms that harm plants year-round, causing huge losses to agricultural production. Among them, fungal diseases account for more than 70% (Gao Youhui, Zheng Zehui, Zhang Yue, et al. Research progress on the mechanism of rhizosphere microecology in controlling soil-borne fungal diseases of crops. Journal of Agricultural University, 2021, 26(6): 100-113.). Pathogenic fungi infect the roots, stems, leaves, fruits, and other parts of plants, and can cause disease throughout the entire growth cycle of the plant. Symptoms include tissue necrosis, rot, and wilting. The occurrence of fungal diseases affects the growth and development of plants, leading to reduced yield and quality. Chemical control is the main method for controlling plant fungal diseases in agriculture. However, with the large-scale and repeated use of pesticides, not only has drug resistance been induced in pathogens, but the problems of food safety and environmental pollution caused by pesticide residues have also become increasingly serious. Therefore, the use of green, efficient, and safe control methods is an inevitable trend. As an advanced biotechnology, genetic engineering can be used in agricultural production to cultivate resistant varieties, significantly reduce the use of pesticides, and improve crop growth traits, which is of great benefit to the sustainable development of agriculture.
[0003] Plant secondary metabolites are important substances produced in response to environmental changes. There are approximately 100,000 different plant secondary metabolites. Flavonoid metabolites generally possess defensive functions, allelopathic effects, resistance to pests and diseases, UV protection, and antioxidant properties (Ge Shibei, Zhang Xuening, Han Wenyan, et al. Research progress on the biosynthesis and stress resistance mechanisms of plant flavonoids. Journal of Horticulture, 2023, 50(01): 209-224.). Based on structural differences, flavonoids are generally divided into seven subclasses: flavonols, flavones, isoflavones, anthocyanins, flavanones, flavanols, and chalcones. Flavonoids, as a class of antimicrobial compounds in plants, inhibit fungal growth and reproduction through mechanisms such as causing microbial membrane lysis or rupture, inhibiting biofilm formation, inhibiting cell membrane synthesis, inhibiting nucleic acid synthesis, and interfering with electron transport chains and ATP synthesis (Shen N, Wang T, Gan Q, et al. Plant flavonoids:classification, distribution, biosynthesis, and antioxidant activity. FoodChemistry, 2022, 383: 132531.). Cherry blossom extract is a flavonoid metabolite and a phenolic phytoalexin, effective against rice blast fungus (…). Magnaporthe oryzae It has antibacterial activity and can inhibit the endocytosis of the effector protein of rice blast fungus and enhance the resistance of rice to rice blast (Jiang L, Zhang X, Zhao Y, et al. Phytoalexin sakuranetinattenuates endocytosis and enhances resistance to rice blast. Nature Communications, 2024, 15(1): 3437.).
[0004] Flavonoid 3'-hydroxylase (F3'H) is a member of the cytochrome P450 family. In the flavonoid biosynthesis pathway, the B-ring hydroxylation pattern is determined by two monooxygenases belonging to the P450 family: flavonoid 3'-hydroxylase (F3'H) and flavonoid 3'5'-hydroxylase (F3'5'H). The key enzyme F3'H in flavonoid biosynthesis catalyzes the hydroxylation of the B-ring 3' position of naringenin and dihydrokaempferol, generating eriodictyol and dihydroquercetin. These two products are important precursors in the biosynthesis of anthocyanins and proanthocyanidins (Liu X, Gong Q, Zhao C, et al. Genome-wide analysis of cytochrome P450 genes in...). Citrus clementina and characterization of a CYP gene encoding flavonoid 3'-hydroxylase. Horticulture Research, 2023, 10(2):uhac283.). Anthocyanin content and color state of plants are related to... F3'H Related to gene expression levels, broccoli F3'H Decreased expression levels reduced the accumulation of anthocyanins and delphinidin (Gu H, Yu H, Wang J, et al. A 43bp-deletion in the F3'H Gene reducing anthocyanins is responsible for keeping buds green at low temperatures in Broccoli. (International journal of molecular sciences, 2023, 24(14): 11391). Rhododendron regulation of anthocyanins at different flowering stages and in different tissues. F3'H Gene expression regulates anthocyanin levels; overexpression F3'H The gene leads to a significant increase in anthocyanin content in petals (Wu Zehang et al. Cloning and functional analysis of the flavonoid 3'-hydroxylase (F3'H) gene in Belgian rhododendron. Biotechnology Bulletin, 2024, 40(06): 251-259).
[0005] Lilies belong to the genus *Lilium* in the family Liliaceae. LiliumLilies are a collective term for all species, subspecies, varieties, forms, and cultivars of perennial bulbous flowers. Lilies have a long history of cultivation in China and are widely distributed. In recent years, their planting area has expanded rapidly year by year. However, in the process of lily production, the threat of diseases has become a major factor restricting their yield. Among them, fungal diseases are particularly prominent, mainly including wilt, gray mold, and anthracnose. In particular, lily wilt is frequent and causes serious damage, attracting widespread attention. Wilt is mainly caused by the genus *Fusarium* (*Fusarium*). Fusarium spp . This disease is caused by fungi and can lead to rot of lily leaves, roots, and stems. Lily plants infected with wilt are stunted. In the early stages of the disease, the lower leaves die first, and as the disease progresses upwards, the upper leaves also die. After infection, the underground parts, such as the roots, turn light brown and rot. In severe cases, the entire bulb rots, leading to stem and root rot and ultimately plant death (Deng Jieling, Huang Fengling, Lu Jiesi, et al. Integrated pest management of major diseases and pests of ornamental lilies. Agricultural Research and Application, 2016, (06): 69-71). Minjiang lily ( Lilium regale *Lilium oxypetalum*, native to China, is found only in rock crevices in river valleys and mountainsides at altitudes of 800–2700 m in the Minjiang River basin. It exhibits strong resistance to wilt disease and is an important germplasm resource for modern lily breeding. The flavonoid synthesis pathway is closely related to plant defense mechanisms. Flavonoid 3'-hydroxylase, as a key enzyme in the biosynthesis of flavonoid secondary metabolites, plays a crucial role in the synthesis of flavonoids in *Lilium oxypetalum*. LrF3'H The discovery and functional analysis of genes have significant research implications and application value. Summary of the Invention
[0006] This invention provides a 3'-hydroxylase gene for flavonoids in Lilium minjiangense. LrF3'H And its effect on improving the resistance of tobacco to mold growth on grass stems ( Phoma herbarum ) and Alternaria solanacearum ( Alternaria solani Applications in resistance.
[0007] This invention cloned the flavonoid 3'-hydroxylase gene from Lilium minjiangense. LrF3'H , LrF3'H The nucleotide sequence is shown in SEQ ID NO:1. The full-length cDNA sequence of this gene is 740 bp, containing a 426 bp open reading frame, a 284 bp 5' untranslated region, and a 30 bp 3' untranslated region, encoding a protein with the amino acid sequence shown in SEQ ID NO:2.
[0008] In this invention LrF3'H The coding region of the gene is the nucleotide sequence shown in positions 285-710 of SEQ ID NO:1.
[0009] This invention isolates and clones a flavonoid 3'-hydroxylase gene from Lilium minjiangense.LrF3'H The complete cDNA fragment was obtained using Agrobacterium tumefaciens (Gastrointestinal rust) Agrobacterium tumefaciens The inventors mediated the transfer of the target gene into recipient plants and overexpressed it. Further experiments were conducted to verify whether this gene could enhance the plant's antifungal activity, laying the foundation for future applications in improving the resistance of tobacco and other plants to fungal diseases. The gene was named... LrF3'H .
[0010] The above LrF3'H Genes are applied to enhance the antifungal properties of tobacco, and the specific operation is as follows:
[0011] (1) Amplification LrF3'H Specific primers were used to extract total RNA from the roots of *Lilium minjiangense*, and the RNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). LrF3'H The full-length coding region was extracted, then ligated into the pGEM-T vector, and clones containing the target gene were obtained by sequencing.
[0012] (2) Using restriction endonucleases Eco RI and Bam HI enzyme digestion of pGEM-T- LrF3'H The target gene fragment was obtained by gel extraction from the vector. The plant expression vector pCAMBIA2300s was digested with the same restriction enzyme, and the desired large vector fragment was obtained by gel extraction. The obtained fragment was then... LrF3'H The gene fragment was linked with the pCAMBIA2300s fragment to construct a plant overexpression vector, and then the constructed recombinant vector was transformed into tobacco for expression via Agrobacterium tumefaciens.
[0013] (3) Transformants were screened using the resistance markers on the recombinant vector T-DNA, and real transgenic plants were obtained by PCR detection. The ability of transgenic plants to resist fungal infection was analyzed, and finally transgenic plants with significantly enhanced resistance to fungi were screened.
[0014] This invention, through genetic engineering, cultivates disease-resistant plants, overcoming the shortcomings of traditional breeding methods. It not only shortens the breeding cycle but also simplifies the process and makes it easier to obtain highly resistant materials. This invention originates from the Minjiang lily. LrF3'HGenes can enhance plant resistance to fungi. Introducing this gene into tobacco can produce new varieties and materials with fungal resistance. Utilizing genetic engineering technology to cultivate resistant plant varieties and materials has significant advantages and irreplaceable importance; it not only facilitates large-scale production of crops, medicinal herbs, and horticultural plants, significantly reducing the use of chemical pesticides, but also saves costs in agricultural production, reduces environmental pollution, and provides a new method for improving plant resistance to fungal diseases. Therefore, this invention has broad market application prospects. Attached Figure Description
[0015] Figure 1 This invention contains pCAMBIA2300s- LrF3'H The image shows the PCR detection results of Agrobacterium LBA4404 plasmid. In the image, the marker is the DL2000 DNA Marker (Takara Bio Engineering Dalian Co., Ltd., China), composed of six DNA fragments of 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. The positive control is pCAMBIA2300s- LrF3'H PCR products using plasmid as template; negative control PCR products using sterile water as template; serial numbers 1-20 are PCR products using the plant expression vector pCAMBIA2300s- LrF3'H The bacterial culture after being transformed into Agrobacterium tumefaciens LBA4404 was used as a template for PCR products;
[0016] Figure 2 This is the present invention. LrF3'H The image shows the PCR detection results of genomic DNA from transgenic tobacco. In the image, the marker is the DL2000 DNA Marker (Takara Bio Engineering Dalian Co., Ltd., China), composed of six DNA fragments: 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. The positive control is pCAMBIA2300s- LrF3'H The plasmid-based PCR product; WT is a PCR product using total DNA from non-transgenic tobacco (wild-type) as a template; the negative control is a PCR product using sterile water as a template; numbers 1-20 are partial samples of plasmid-based PCR products. LrF3'H PCR products using transgenic tobacco genomic DNA as a template;
[0017] Figure 3 This is the present invention. LrF3'H The antibacterial effect of 60% methanol extract of transgenic tobacco is shown in the figures. Figures a and b show *Pseudomonas aeruginosa* and *Alternaria solani*, respectively. WT represents 60% methanol extract of wild-type tobacco, and Buffer represents the blank control (60% methanol solution). H11, H13, and H18 represent... LrF3'H60% methanol extracts from different strains of genetically modified tobacco;
[0018] Figure 4 This is the present invention. LrF3'H The diagram shows the resistance identification of transgenic tobacco to pathogenic fungi. Figures a and b show wild-type tobacco and transgenic tobacco after inoculation with *Pseudomonas stolonifera* and *Alternaria alternata*, respectively. LrF3'H The resistance of transgenic tobacco to two fungi is shown in the figure. WT represents wild-type tobacco; H11, H13, and H18 represent... LrF3'H Transgenic tobacco strains. Detailed Implementation
[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the methods in the embodiments are conventional methods, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.
[0020] Example 1: LrF3'H Gene cloning and sequence analysis
[0021] Total RNA was extracted from the roots of *Lilium minjiangense*. The roots, inoculated with *Fusarium oxysporum*, were ground into powder using liquid nitrogen and then transferred to centrifuge tubes. Total RNA was extracted using the guanidine isothiocyanate method. The first strand of cDNA was synthesized using reverse transcriptase M-MLV (Promega, USA) as a template. The reaction system and procedure were as follows: 5 μg of total RNA was added, followed by 50 ngoligo (dT), 2 μL of dNTP (2.5 mM), and DEPC water, until the reaction volume reached 14.5 μL. After mixing, the mixture was denatured at 70°C for 5 min, then rapidly cooled on ice for 5 min. Next, 4 μL of 5× First-stand buffer, 0.5 μL of RNasin (200 U), and 1 μL of M-MLV (200 U) were added sequentially. The mixture was mixed and briefly centrifuged, then incubated at 42°C for 1.5 h. The reaction was terminated by heating at 70°C for 10 min. The synthesized first strand of cDNA was stored at -20°C for later use.
[0022] Using the synthesized first-strand cDNA as a template, the target gene is amplified. LrF3'H The upstream and downstream primer sequences used were 5' ATGGCCTCCAAATCATG 3' and 5' CGCTAGAGTTTCTCATGCT 3', respectively. Advantage was employed. TM2. The target gene was amplified using PCR Enzyme (Clontech, USA). PCR reaction conditions: 94℃ for 5 min; 94℃ for 30 s, 52℃ for 30 s, 72℃ for 30 s, 32 cycles; 72℃ for 7 min. The reaction system (20 μL) consisted of 0.5 μL cDNA, 2 μL 10×Advantage 2 PCR Buffer, 0.4 μL 50×dNTP Mix (10 mM each), 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 0.4 μL Advantage 2 PCR Polymerase Mix, and 15.9 μL PCR-Grade water. After PCR, 5 μL was used for agarose gel electrophoresis to detect the specificity and size of the amplified products.
[0023] The PCR product was TA cloned using the pGEM-T easy Vector System I (Promega, USA). The reaction system and procedure were as follows: 1.5 μL of PCR product was added to 1 μL of pGEM-T Vector (50 ng / μL) and 2.5 μL of 2×Ligation solution I, mixed well, and incubated overnight at 16°C. The ligation product was then transformed into E. coli DH5 using the heat shock transformation method. α In the middle stage, positive clones were screened using LB solid medium containing ampicillin (Amp). Several single colonies were selected, and after shaking, they were amplified. LrF3'H Specific primers identified the multiple cloning site insertion. LrF3'H Clones. The identified clones were sequenced, and the final results were obtained. LrF3'H The full-length cDNA is 740 bp. Analysis using the NCBI ORF finder (http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) revealed that it contains a 426 bp open reading frame. LrF3'H The protein encodes 141 amino acids with a molecular weight of approximately 15.41 kDa and an isoelectric point of approximately 8.98. Analysis was performed using SignalP 6.0 (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ). LrF3'H The encoded protein sequence is analyzed to determine if it possesses an N-terminal signal peptide. Protein prediction is displayed... LrF3'H The absence of a signal peptide suggests that the protein is an intracellular protein.
[0024] Example 2: Construction of plant overexpression vectors
[0025] Insertion plasmid DNA was extracted using the SanPrep column-based plasmid DNA mini-extraction kit (Sangon Biotech Shanghai Co., Ltd., China). LrF3'H pGEM-T-E. coli plasmid LrF3'H The plasmid of the plant expression vector pCAMBIA2300s was also analyzed; 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. Restriction endonucleases were then used to... Eco RI (TaKaRa, Japan) and Bam HI (TaKaRa, Japan) separately tested plasmid pGEM-T- LrF3'H Double digestion with pCAMBIA2300s (100 μL system) was performed. The reaction system and operation procedure were as follows: Take 20 μL of pGEM-T- LrF3'H Alternatively, add 10 μL of 10×K buffer and 4 μL of pCAMBIA2300s plasmid sequentially. Eco RI, 6μL Bam HI, 60 μL ddH2O, mix well, centrifuge briefly, and incubate overnight at 37°C; spot all enzyme digestion products onto an agarose gel for electrophoresis, then... LrF3'H The fragments and the large fragment of the pCAMBIA2300s vector were recovered by gel electrophoresis. The entire process was carried out using the SanPrep column DNA gel recovery kit (Sangon Biotech Shanghai Co., Ltd., China). 1 μL of the recovered product was taken and the size and concentration of the recovered fragments were detected by agarose gel electrophoresis and stored at -20℃ for later use.
[0026] Using T4 DNA Ligase (TaKaRa, Japan), the recovered DNA was... LrF3'H The DNA fragment and the pCAMBIA2300s vector fragment were ligated. The reaction system (20 μL) and the operation procedure were as follows: Take 10 μL... LrF3'H The DNA fragment was added sequentially with 2 μL pCAMBIA2300s vector DNA, 2 μL 10×T4 DNA Ligase Buffer, 1 μL T4 DNA Ligase, and 5 μL ddH2O. After mixing, the mixture was briefly centrifuged and then incubated overnight at 16°C. The ligation product was then transformed into *E. coli* DH5α using a heat shock transformation method. Positive clones were screened using solid medium containing 50 mg / L kanamycin (Km). Single colonies were selected and cultured, and the bacterial culture was used as a template for amplification. LrF3'H PCR was performed using specific primers to select... LrF3'H Clones that were successfully ligated with pCAMBIA2300s were added to glycerol and stored at -80°C for later use.
[0027] Extract and purify pCAMBIA2300s- from the above-mentioned Escherichia coli. LrF3'H Plasmid. The constructed plant expression vector pCAMBIA2300s- was then frozen and thawed in liquid nitrogen. LrF3'H Transfected into Agrobacterium tumefaciens LBA4404 competent cells. The procedure was as follows: Take 2 μg of pCAMBIA2300s- LrF3'H The plasmid was added to a centrifuge tube containing 200 μL of competent cells, gently mixed, and incubated on ice for 5 min. Then, it was transferred to liquid nitrogen and frozen for 1 min, followed by immediate incubation at 37°C for 5 min, then immediately incubated on ice for 2 min. 800 μL of LB broth was added, and the cells were incubated at 28°C with shaking for 4 h. The activated Agrobacterium was plated on LB solid medium containing 50 mg / L Km and incubated statically at 28°C. Single colonies were selected and shaken for further amplification. LrF3'H PCR was performed using specific primers to detect pCAMBIA2300s- LrF3'H Whether it is transferred into Agrobacterium, the result is as follows Figure 1 As shown, multiple positive clones were successfully obtained, and after adding glycerol, they were stored at -80℃ for later use.
[0028] Example 3: Agrobacterium-mediated plant genetic transformation and screening of transgenic plants
[0029] The transgenic recipient in this experiment was tobacco. Tobacco seeds were soaked in 75% alcohol for 30 seconds, washed with sterile water, soaked in 0.1% HgCl2 for 8 minutes, washed several times with sterile water, sown on 1 / 2 MS medium, and cultured in the dark at 28℃ for 6 days. After germination, they were transferred to a light incubator (25℃, 16 h / d light). Subcultured monthly with 1 / 2 MS medium thereafter.
[0030] Removed from a -80℃ freezer containing pCAMBIA2300s- LrF3'H Agrobacterium LBA4404 strain containing plasmids was inoculated into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampin, and cultured at 28°C until the medium became turbid. 1 mL of the turbid bacterial culture was transferred to LB solid medium containing 50 mg / L Km and cultured at 28°C for 48 h. Subsequently, an appropriate amount of Agrobacterium was scraped from the LB solid medium and inoculated into MGL liquid medium supplemented with 20 mg / L acetylsyleugenol, and cultured at 28°C with shaking for 2-3 h to activate the Agrobacterium.
[0031] Take leaves from sterile tobacco seedlings and cut them into 1 cm pieces. 2The leaf discs on both sides were completely immersed in the MGL liquid medium containing activated Agrobacterium for 15 minutes. The bacterial solution on the leaf surface was then blotted dry with sterile filter paper. The leaf discs were then placed on a co-culture medium for room temperature incubation. The co-culture medium for tobacco transformation was MS medium + 0.02 mg / L 6-BA + 2.1 mg / L NAA + 30 g / L sucrose + 6 g / L agar. The mixture was co-cultured at 22°C in the dark for 2 days.
[0032] After co-culture, the leaf discs were transferred to MS selection medium supplemented with antibiotics for differentiation into seedlings, and transgenic plants were screened simultaneously. The tobacco selection medium consisted of MS medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 6 g / L agar + 50 mg / L Km + 200 mg / L cefotaxime sodium salt (Cef). During selection culture, the culture flasks were transferred to a light incubator (25℃, 16 h / d light, 8 h / d darkness). After the tobacco plants sprouted, they were subcultured on MS medium containing 50 mg / L Km and 200 mg / L Cef. The regenerated tobacco seedlings were then transferred to MS medium containing 50 mg / L Km to allow rooting, and the best-rooted seedlings were selected for further testing.
[0033] Genomic DNA was extracted from the leaves of transgenic tobacco plants using the CTAB method. 1 μL of the extracted genomic DNA was analyzed for integrity and concentration by agarose gel electrophoresis. Using the genomic DNA from the transgenic plants as a template, amplification was performed... LrF3'H PCR was performed using specific primers. After PCR, 8 μL of the product was used for agarose gel electrophoresis to detect positive transgenic plants. The amplification results of some tobacco transgenic plants are shown below. Figure 2 As shown, LrF3'H A total of 28 positive transgenic tobacco plants were screened.
[0034] Example 4: LrF3'H Antibacterial activity analysis of 60% methanol extract of transgenic plants
[0035] Laboratory-preserved pathogenic fungi were inoculated onto PDA solid medium (200 g / L potato, 15 g / L agar, 20 g / L glucose) and incubated in the dark at 28°C. When colonies grew to approximately 2-3 cm in diameter, methanol extract was added, and the in vitro antifungal activity of the transgenic plants was analyzed. To extract total flavonoids from tobacco, 5 g of transgenic tobacco plants (numbered H11, H13, and H18) and wild-type leaves were placed in a mortar, ground into powder with liquid nitrogen, and then 20 mL of 60% methanol was added. Extraction was performed using an ultrasonic shaker for 45 min. After extraction, the mixture was centrifuged at 10000 g for 10 min, and the supernatant was collected for further experiments. 20 μL of 60% methanol solution, 60% methanol extract of wild-type tobacco, and 60% methanol extract of H11 / H13 / H18 transgenic tobacco were dropped onto sterile filter paper discs of fungal culture medium and incubated at 28°C for 3-5 days. The growth of the fungi was observed and evaluated accordingly. LrF3'H The in vitro antifungal activity of genetically modified tobacco was as follows: Figure 3 As shown, LrF3'H The 60% methanol extract of genetically modified tobacco (H11, H13, H18) showed a significant inhibitory effect on the growth of *Pseudomonas stolonifera* and *Alternaria alternata*.
[0036] Example 5: LrF3'H Analysis of resistance of transgenic tobacco leaves to pathogenic fungi
[0037] The pathogenic fungi preserved in the laboratory were inoculated onto PDA solid medium (200 g / L potato, 15 g / L agar, 20 g / L glucose) and incubated in the dark at 28°C for 7 days to prepare 10 5 A spore suspension of spores / mL produced uniformly sized wounds on the upper left side of both wild-type tobacco and H11 / H13 / H18 transgenic tobacco. 20 μL of the spore suspension was then applied to the wounds. Leaves were placed on sterile filter paper moistened with water for humidification and incubated in the dark at 28°C for 5-7 days. The leaves were then removed and the disease development was observed. Results are as follows... Figure 4 As shown, LrF3'H Genetically modified tobacco exhibits high levels of resistance to *Alternaria alternata* and *Alternaria solani*.
Claims
1. A gene for 3'-hydroxylase of flavonoids from Lilium minjiangense. LrF3'H In improving the resistance of tobacco to mold growth on stems ( Food herbs ) and Alternaria solanacearum ( Alternaria solani Application in resistance, Minjiang lily flavonoid 3'-hydroxylase gene LrF3'H The nucleotide sequence is shown in SEQ ID NO:1.