Tagetes gene, cloning method, expression vector and application

Abstract

The application provides a marigold gene, a cloning method, an expression vector and application. Compared with the prior art, the TeLFI1 gene is cloned from marigold as follows: high-abundance expression sequences in leaves are assembled by using marigold transcriptome sequencing; then, specific PCR primers are designed by using primer design software Primer Premier, total RNA is extracted from marigold leaves, reverse transcription PCR is carried out, and the TeLFI1 gene is cloned. When the TeLFI1 is expressed in tomatoes, the leaf color can be changed, and mottled leaves or gold-edged leaves are formed. The expression of the TeLFI1 gene in tomatoes changes volatile flavor substances in the fruits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and in particular to a Tagetes erecta L. gene, a cloning method, an expression vector and application, a gene cloned from Tagetes erecta L., and application of the gene in changing plant leaf color and fruit flavor. BACKGROUND

[0002] Tagetes erecta L. is an annual dicotyledonous herb of the Asteraceae family with ornamental and medicinal and agronomic values. Due to its bright color and long flowering period, it is easy to cultivate and adapt to various soil environments, and has become an ornamental plant and is widely planted around the world. The flowers of Tagetes erecta L. account for more than half of the world's loose flower yield due to their bright colors. In addition, the flowers, leaves and roots of Tagetes erecta L. are often used to treat acute upper respiratory tract infection, whooping cough and the like, and are also a traditional plant medicine. At present, Tagetes erecta L. has been introduced as a characteristic and dominant crop in some areas of China, and large-scale planting of Tagetes erecta L. has been developed, and through the construction of a complete industry chain, economic benefits have been improved, so that Tagetes erecta L. has formed a certain industrialization scale.

[0003] Tagetes erecta L. is rich in carotenoids, polyphenols, thiophenes, alkaloids and flavonoids and other secondary metabolites, and these secondary metabolites have activities of preventing and treating degenerative diseases, anti-tumor, antioxidant and the like, have high nutritional value and medicinal value, and are widely used in the medical, health care and food industries. Therefore, Tagetes erecta L. has also become an important plant resource for extracting these natural secondary metabolites.

[0004] Plant secondary metabolites are small-molecule organic compounds produced by genetic genes under the action of environmental conditions during the growth and development of plants, and they play a crucial role in the life activities of plants and are important bioactive substances utilized by humans. The synthesis of secondary metabolites in plants is catalyzed by enzymes related to secondary metabolic pathways, and in order to further isolate and identify functional genes regulating the biosynthesis of secondary metabolites, using different germplasm materials, tissue types and developmental stages of Tagetes erecta L. for differential expression gene analysis has become an important method. Through transcriptome, proteomics and other means, differential expression sequences are obtained, from which candidate genes catalyzing and regulating the synthesis and accumulation of specific secondary metabolites are screened, and through further genetic transformation and phenotype analysis, the functions of the candidate genes are determined, which provides a possibility for obtaining new gene resources and for plant genetic trait improvement. SUMMARY

[0005] The present application aims to provide a marigold gene, a cloning method, an expression vector, clone a gene sequence from marigold, and name it Tagetes erecta Leaf and Flavor Improvement 1 (TeLFI1 for short); clone the TeLFI1 gene from marigold: assemble high-abundance expression sequences in leaves by using marigold transcriptome sequencing; then design PCR specific primers by using primer design software Primer Premier, extract total RNA from marigold leaves, perform reverse transcription-PCR (RT-PCR), and clone to obtain the TeLFI1 gene.

[0006] The present application also aims to provide an application of the marigold gene, an application in changing leaf color, or an application in improving fruit flavor.

[0007] The specific technical scheme of the present application is as follows:

[0008] A marigold gene, referred to as TeLFI1, has a nucleotide sequence as shown in SEQ ID NO. 1;

[0009] The present application provides a cloning method of a marigold gene, comprising the following steps:

[0010] 1) Extract RNA from marigold leaf tissue;

[0011] 2) Obtain cDNA by reverse transcription;

[0012] 3) Design primer sequences, and clone the TeLFI1 gene by using the obtained cDNA.

[0013] In step 3), the primer sequences are designed as follows: TeLFI1F, with a sequence as shown in SEQ ID NO. 3; and TeLFI1R, with a sequence as shown in SEQ ID NO. 4.

[0014] The reagents used in step 3) are as follows: place 200 μL EP tubes on ice, and add the reagents: high-fidelity DNA polymerase 0.5 μL; 5×Buffer 10 μL; dNTP 4 μL; cDNA 1 μL; TeLFI1F 1 μL; TeLFI1R 1 μL; and ddH2O 32.5 μL;

[0015] In step 3), the amplification program is as follows: pre-denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 s, recombination at 56℃ for 20 s, and extension at 72℃ for 70 s, with 30 cycles of denaturation-recombination-extension; and total extension at 72℃ for 5 min.

[0016] The application provides a tagetes gene expression vector, wherein the tagetes gene expression vector comprises the nucleotide sequence as shown in SEQ ID NO. 1.

[0017] The tagetes gene expression vector is inserted into a plant expression vector pBI121 by using a tagetes gene TeLFI1 to obtain a recombinant expression vector.

[0018] The amino acid sequence coded by the tagetes gene is shown in SEQ ID NO. 2.

[0019] MSVALIWVVSPNSELCNRLETTKFADLSKSRNCLRASKIKNFEKKDKCKSFCYMNADFSGFSGSNYAKNPGLISRVVANPGGELAVSSEQL

[0020] VYDVVLKQAALVKEQMRNKEDIEVKPDIVLPGSLGLLSEAYDRCGEVCAEYAKTFYLGTLLMTPERRKAIWAIYVWCRRTDELVDGPNASH

[0021] ITPKALDRWESRLEDLYNGRPFDMLDAALSDTVSKFPVDIQPFKDMIDGMRMDLRKSRYENFDELYLYCYYVAGTVGLMSVPIMGIAPESN

[0022] APTESVYNAALALGIANQLTNILRDVGEDARRGRVYLPQDELAQAGLSDEDIFAMKVTDKWRFFMKKQIKRARTFFDQAEEGVTQLSSASRWPVWASLLLYRQILDEIEANDYNNFTKRAYVSKPKKIVALPIAYAKSLVPPSSRNLVSN.

[0023] The application further provides an application of the tagetes gene, which is used in changing leaf color or improving fruit flavor.

[0024] The application clones TeLFI1 gene from Tagetes erecta, and the expression amount of TeLFI1 gene in Tagetes erecta leaves is high. When TeLFI1 is expressed in tomato through genetic transformation, the chlorophyll content in leaves is obviously reduced, the leaf color is changed, and marbled leaves or gold-edged leaves are formed. Tagetes erecta does not have obvious fruit structure, and when TeLFI1 is expressed in tomato, the volatile substance composition in tomato fruits is changed, and compared with the wild type, the contents of flavor substances 3-hexenol, 2-hexenal, 1-penten-3-ketone and 2-isobutylthiazole are obviously increased. The gene with the nucleotide sequence shown in SEQ ID NO. 1 plays a role in the formation of leaf pigment substances and fruit volatile components, and the cloned TeLFI1 gene from Tagetes erecta can be used to change the leaf color and fruit volatile flavor components of plants.

[0025] Compared with the prior art, the Tagetes erecta gene TeLFI1 of the application provides a new gene resource for plant trait improvement, and can be used to change the leaf color and fruit flavor of plants. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 TeLFI1 gene expression changes the color of tomato leaves, A: wild type tomato Micro-Tom and its TeLFI1 overexpression plant; B: wild type tomato Ailsa Craig and its TeLFI1 overexpression plant;

[0027] Figure 2 TeLFI1 gene expression changes the flavor of tomato fruits, the upper chromatogram is the purge and trap chromatogram of wild type tomato fruits, and the lower chromatogram is the purge and trap chromatogram of TeLFI1 overexpression tomato fruits. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the embodiments of the application clearer, the technical scheme in the embodiments of the application will be described clearly and completely below in combination with the embodiments of the application. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0029] In the following examples, if specific experimental conditions are not indicated, they are according to conventional conditions well known to those skilled in the art, such as the conditions described in Sambrook J. and Russell, D.W. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or the conditions recommended by the manufacturer.

[0030] Example 1: Marigold Transcriptome Sequencing

[0031] To obtain the functional gene expression sequences, the leaves and flower tissues of mature marigold 'Super Chrysanthemum King' plants were used for transcriptome sequencing and assembly annotation.

[0032] 1. Reagents

[0033] Plant RNA extraction kit was purchased from Beijing TransGen Biotech Co., Ltd., DNase I was purchased from Sangon Biotech (Shanghai) Co., Ltd., and RNA library preparation kit (Illumina Truseq) was purchased from Sangon Biotech (Shanghai) Co., Ltd. TM The RNA sample prep kit was from Meiji Biotechnology Co., Ltd., and the rest of the reagents were either imported and repackaged or domestically produced analytical grade products.

[0034] 2. Plant materials

[0035] The 'Super Chrysanthemum King' (Tagetes erecta L.) was provided by Chifeng Xinhui Horticulture Company.

[0036] 3. Methods

[0037] 3.1 RNA Extraction

[0038] RNA was extracted from marigold leaf tissue, and the experimental procedures were performed according to the kit manufacturer's instructions.

[0039] 1) Grind the marigold tissue into powder rapidly in liquid nitrogen. Take 100 mg of tissue and add it to 500 μL of BB6 solution (add 10 μL of β-mercaptoethanol to every 1 mL of BB6, prepare fresh each time, and vortex vigorously to mix). Incubate at room temperature for 3 min;

[0040] 2) Centrifuge at 12000g for 2-5 minutes, and transfer the supernatant from the centrifuge tube to an RNase-free centrifuge tube;

[0041] 3) Add 0.5 times the volume of anhydrous ethanol to the supernatant and mix well;

[0042] 4) Vortex thoroughly mix and disperse the sediment;

[0043] 5) Add the obtained solution and precipitate together into a centrifuge column, centrifuge at 12000g for 30s, and discard the eluent;

[0044] 6) Add 500 μL of CB6 solution, centrifuge at 12000g for 30 seconds at room temperature, and discard the effluent;

[0045] 7) Add 80 μL of DNase I working solution to the center of the centrifuge column (to prepare DNase I working solution: take 70 μL of Reaction Buffer and put it into an RNase-free tube, then add 30 U of DNase I and mix well), let it stand at room temperature for 15 min, and repeat step 6).

[0046] 8) Add 500 μL of WB6 solution (add anhydrous ethanol before use), centrifuge at 12000g for 30s, and discard the effluent;

[0047] 9) Repeat step 8);

[0048] 10) Centrifuge at 12000g for 2 minutes at room temperature to completely remove residual ethanol;

[0049] 11) Add 50 μL of RNase-free water to the center of the centrifuge column and let it stand at room temperature for 1 min;

[0050] 12) Centrifuge at 12000g for 2 minutes at room temperature to elute RNA;

[0051] 13) Detect the purity and concentration of the RNA sample and store the RNA at -80℃.

[0052] 3.2 Transcriptome Sequencing and Assembly Annotation

[0053] Transcriptome sequencing using Illumina Truseq TM The RNA sample prep kit was used with the Illumina Novaseq 6000 sequencing platform, following these steps:

[0054] 1) Enrich mRNA with magnetic beads containing Oligo dT and randomly fragment the mRNA;

[0055] 2) Synthesize cDNA strands using mRNA as a template and purify cDNA using microbeads;

[0056] 3) The purified double-stranded cDNA is then repaired at the ends and ligated with sequencing adapters. Fragment size selection is performed using microbeads, and cDNA libraries are obtained by PCR enrichment.

[0057] 4) Detect the concentration of the library and the size of the inserted fragments;

[0058] 5) The cDNA library was sequenced using the Illumina Novaseq 6000 high-throughput sequencing platform;

[0059] 6) Remove adapter sequences, non-A / C / T / G bases at the 5' end, and low-quality sequences from the original sequencing reads to obtain high-quality sequencing data;

[0060] 7) Use Trinity (https: / / github.com / trinityrnaseq / trinityrnaseq / wiki) to concatenate the filtered original sequence data;

[0061] 8) Using BLAST+ software, all transcripts obtained from transcriptome sequencing assembly were compared with the NR (NCBI Non-redundant Protein Sequences Database, https: / / www.ncbi.nlm.nih.gov / publish / ), Swiss-Prot (Swiss-Prot Protein Sequences Database, http: / / www.geneontology.org), Pfam (Protein Family, http: / / pfam.xfam.org / ), eggNOG (Evolutionary Genetics of Genes, Non-supervised Orthologous Groups, http: / / eggnogdb.embl.de / # / app / home), GO (Gene Ontology, http: / / www.geneontology.org), and KEGG (Kyoto Encycolpedia of Genes and Genomes, http: / / www.genome.jp / kegg / ) databases to obtain annotation information for the expressed sequences;

[0062] 9) Estimate gene expression levels using FPKM (Fragments Per Kilobase of exon mode per Mileon mapped fragments) values.

[0063] 4. Results

[0064] Following the steps outlined above, RNA was extracted from marigold leaves and flower tissues, libraries were constructed, and transcriptome sequencing was performed. The sequenced sequences were then assembled. Quantitative analysis of the expression levels of the assembled genes revealed that the TeLFI1 gene exhibited high expression levels in all three marigold leaf libraries, with an average expression level of 132, which is 8.3 times higher than that in mature flowers. The high abundance of TeLFI1 expression in mature plant leaves suggests that it may play a functional role in leaves.

[0065] Example 2: Cloning of the Marigold TeLFI1 Gene

[0066] Based on the assembled sequence of the marigold TeLFI1 gene in Example 1, PCR-specific primers were designed using Primer Premier software. Total RNA was extracted from mature marigold leaves, and the TeLFI1 gene was cloned by reverse transcription PCR (RT-PCR).

[0067] 1. Reagents

[0068] Plant RNA extraction kit, reverse transcriptase, dNTPs, and high-fidelity DNA polymerase were purchased from Beijing TransGen Biotech Co., Ltd. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the remaining reagents were either imported and repackaged or domestically produced analytical grade products.

[0069] 2. Vectors and strains

[0070] The cloning vector pEASY-Blundt Simple Cloning Vector and the Escherichia coli (Escherichiacoli) strain DH5α were purchased from Beijing TransGen Biotechnology Co., Ltd.

[0071] 3. Culture medium and reagents

[0072] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L. Adjust pH to 7.0 with NaOH and autoclave.

[0073] LB solid medium: Add 1% agar powder to the above LB liquid medium, autoclave, and prepare LB solid medium.

[0074] 100×Mg 2+ Solution: Weigh 20.33g MgCl2·6H2O and 24.65g MgSO4·7H2O and dilute to 100mL deionized water, then autoclave.

[0075] SOC medium: tryptone 20 g / L, yeast extract 5 g / L, NaCl 0.58 g / L, KCl 0.19 g / L, 100×Mg2+ 10 mL of NaOH was added to adjust the pH to 7.0 and then autoclaved. Then 2 mL of filtered, sterilized 1 mol / L glucose was added.

[0076] 1000× kanamycin: 50 mg / mL, dissolved in sterile deionized water, stored at -20℃.

[0077] 4. Methods

[0078] 4.1 RNA extraction from marigold leaf tissue

[0079] Perform the operation steps as described in 3.1 of Example 1.

[0080] 4.2 RT-PCR

[0081] 4.2.1RT

[0082] 1) Take 1 μg of total RNA and 1 μL of polyT 18 Mix the (10 μM) primers, add RNase-free ddH2O to a final volume of 12.75 μL, and mix gently.

[0083] 2) Keep warm at 65℃ for 5 minutes, then immediately transfer to an ice bath and let stand for 2 minutes;

[0084] 3) Add 4 μL of 5× reaction buffer, 2 μL of 10 mM dNTP, 0.25 μL of RNA inhibitor (40 U / μL), and 1 μL of reverse transcriptase (100 U / μL), and react at 42℃ for 1 h to synthesize first-strand cDNA;

[0085] 4) Heat at 95℃ for 5 minutes to inactivate the reverse transcriptase and terminate the reaction.

[0086] 4.2.2 PCR

[0087] Based on the deduced TeLFI1 gene sequence obtained in Example 1, primer sequences were designed using Primer Premier software as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The specific sequences are as follows:

[0088] TeLFI 1F:5'atgtctgttgctctgatttgggt 3', SEQ ID NO.3;

[0089] TeLFI 1R:5'gtttgaaactaagtttcttgatgat 3', SEQ ID NO.4;

[0090] The TeLFI1 gene was cloned using the cDNA from marigold leaf tissue obtained in step 4.2.1 of this embodiment.

[0091] Place the 200 μL EP tube on ice and add the reagent:

[0092] High-fidelity DNA polymerase, 0.5 μL;

[0093] 5×Buffer, 10μL;

[0094] dNTP, 4 μL;

[0095] cDNA, 1 μL;

[0096] TeLFI 1F, 1μL;

[0097] TeLFI 1R, 1μL;

[0098] ddH2O, 32.5 μL;

[0099] Amplification was performed according to the following procedure: 98℃ for 2 min (pre-denaturation); 98℃ for 10 s (denaturation), 56℃ for 20 s (renaturation), 72℃ for 70 s (extension), for 30 cycles of denaturation-renaturation-extension; 72℃ for 5 min (total extension).

[0100] The above operations yielded the PCR amplification product of the TeLFI1 gene.

[0101] 4.3 Ligation of PCR amplification products with pEASY-BLUNT vector

[0102] The TeLFI1 gene PCR amplification product obtained in step 4.2 of this embodiment was ligated with the pEASY-Blunt Simple Cloning Vector at a molar ratio of 1:4 (25℃, 15min). The ligation system is as follows:

[0103] pEASY-Blunt Simple Cloning Vector (50μg / μL), 4μL;

[0104] PCR product (~150 μg / μL), 1 μL;

[0105] 4.4 Escherichia coli transformation

[0106] 1) Take out the frozen Escherichia coli strain DH5α competent cells and thaw them in an ice bath;

[0107] 2) Gently mix the ligation product described in 4.3 with competent E. coli cells and incubate on ice for 30 minutes;

[0108] 3) Heat shock at 42℃ for 90 seconds, then immediately ice bath for 1-2 minutes;

[0109] 4) Add 0.8 mL of SOC, mix well, and incubate at 37°C with gentle shaking for 1 h;

[0110] 5) Centrifuge at 13000 rpm for 1 min at room temperature, discard a portion of the supernatant, leaving about 200 μL of supernatant. Mix the supernatant with the cells using a pipette tip, spread it on LB solid medium containing kanamycin (50 μg / mL), and incubate at 37°C for 12 h.

[0111] 4.5 Colony PCR Identification

[0112] The *E. coli* described in step 4.4 of this embodiment were then subjected to colony PCR identification to confirm that the inserted fragment was the target gene. The reaction system and amplification procedure were as described in 4.2.2 above.

[0113]

[0114] Example 3: Construction of a plant expression vector for the TeLFI1 gene

[0115] PCR amplification was performed using the pEASY-TeLF I 1 plasmid obtained in Example 2 as a template, and Kpn I and Sal I restriction sites were introduced. The PCR product and the plant expression vector pBI 121 were digested with Kpn I and Sal I, respectively. The digested products were recovered from the gel, ligated, and transformed into Escherichia coli and Agrobacterium.

[0116] 1. Reagents

[0117] Plasmid extraction kits and gel extraction kits were purchased from Tiangen Biotech Co., Ltd.; high-fidelity DNA polymerase and T4 DNA ligase were purchased from TransGen Biotech Co., Ltd.; restriction endonucleases Kpn I and Sal I were purchased from New Engl and Biolabs. All other reagents were either imported and repackaged or domestically produced analytical grade products.

[0118] 2. Escherichia coli strains and Agrobacterium strains

[0119] Escherichia coli strain DH5α was purchased from Beijing TransGen Biotechnology Co., Ltd., and Agrobacterium tumefaciens strain GV2260 was preserved in the laboratory.

[0120] 3. Culture medium and antibiotics

[0121] The preparation methods for LB liquid medium, LB solid medium, and SOC medium are as described in Example 2.

[0122] 1000× kanamycin: The preparation method is as described in Example 2.

[0123] 1000×rifampicin: 20 mg / mL, dissolved in sterile deionized water, stored at -20℃.

[0124] 4. Methods

[0125] 4.1 Plasmid Extraction

[0126] Plasmids were extracted from the pEASY-TeLF I1 vector and the plant expression vector pBI 121 obtained in Example 2, and the experimental steps were performed according to the instructions of the kit manufacturer.

[0127] 1) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and set aside for later use;

[0128] 2) Centrifuge at 12000 rpm for 1 min to collect the bacterial precipitate and discard the supernatant; add 250 μL of P1 solution (with RNase A added), and mix by pipetting until the bacterial precipitate is completely resuspended;

[0129] 3) Add 250 μL of P2 solution, gently invert the centrifuge tube to allow the cells to fully lyse;

[0130] 4) Add 350 μL of P3 solution, immediately and gently invert the centrifuge tube, and then centrifuge at 12000 rpm for 10 min;

[0131] 5) Transfer the supernatant to a new centrifuge tube and centrifuge at 12,000 rpm for 5 minutes;

[0132] 6) Carefully transfer the supernatant to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0133] 7) Add 500 μL of PD solution, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0134] 8) Add 600 μL of PW solution (with anhydrous ethanol added), centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat the operation once;

[0135] 9) Centrifuge at 12000 rpm for 2 min to remove any remaining PW solution;

[0136] 10) Transfer the adsorption column to a new centrifuge tube, add 50 μL of sterile deionized water to the center of the column, incubate at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to elute the DNA.

[0137] 4.2 PCR

[0138] PCR amplification was performed using the obtained pEASY-TeLF I 1 plasmid as a template, and Kpn I and Sal I restriction sites were introduced to amplify the coding sequence. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively, and the specific sequences are as follows:

[0139] TeLFI F2:5'ggggtaccatgtctgttgctctgatttgggt 3', SEQ ID NO.5;

[0140] TeLFI R2:5'gcgtcgacgtttgaaactaagtttcttgatgat 3', SEQ ID NO.6;

[0141] 4.3 Enzyme digestion

[0142] The PCR amplification products of the expected size were purified, and then the pBI 121 plasmid and TeLFI1 gene amplification products obtained in 4.1 and 4.2 were digested with restriction endonucleases Kpn I and Sal I.

[0143] 4.4 Glue Recycling

[0144] The plasmids and TeLFI1 gene amplification products after enzyme digestion as described in 4.3 were recovered by gel extraction, and the experimental procedures were performed according to the instructions of the kit manufacturer.

[0145] 1) Add 500 μL of equilibration solution BL to the gel recovery adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid at the bottom of the column;

[0146] 2) Wearing plastic gloves, collect the electrophoretically separated fragments on the UV gel cutting instrument and put the cut fragments into the pre-prepared EP tubes;

[0147] 3) Determine the volume of PC solvent to add based on the quality of the adhesive, and add it at a 1:1 volume ratio. Place the EP tube in the heater and dissolve at 50℃ for 10-15 minutes, until the adhesive is completely dissolved;

[0148] 4) After the sol is completely dissolved, let it cool to room temperature, then transfer the dissolved liquid into the sol recovery adsorption column and let it stand for 3 minutes.

[0149] 5) Centrifuge at 12000 rpm for 1 min, then discard the waste liquid at the bottom of the collection tube. Add 600 μL of washing buffer PW to the adsorption tube;

[0150] 6) Centrifuge at 12000 rpm for 1 min, then discard the waste liquid at the bottom of the collection tube. Repeat the washing process once.

[0151] 7) Place the adsorption column in a centrifuge and centrifuge at 12,000 rpm for 3 minutes, then let it stand for 15 minutes;

[0152] 8) Add 30 μL of eluent EB to the adsorption membrane at the center of the adsorption column, let stand for 3 min, and centrifuge at 12000 rpm for 3 min to obtain the gel recovery product.

[0153] 4.5 Ligation of enzyme-digested fragments

[0154] The TeLFI1 gene fragment and pBI 121 plasmid fragment recovered from the enzyme digestion in section 4.4 were ligated using T4 ligase. The ligation reaction system was as follows: 25℃, 3h:

[0155] TeLFI1 gene fragment, 4 μL;

[0156] pBI 121, 3 μL;

[0157] 5×buffer, 2μL;

[0158] T4 ligase, 1 μL;

[0159] 4.6 Escherichia coli transformation

[0160] The experimental procedure is as described in section 4.4 of Example 2.

[0161] 4.7 Colony PCR Identification of Recombinant Plasmids

[0162] The method steps are as described in 4.5 of Example 2.

[0163] The recombinant vector (named pBI 121-TeLFI 1) was identified by colony PCR and then sequenced. Sequencing results showed that the TeLFI1 coding sequence was ligated into the pBI 121 vector. The correctly sequenced recombinant E. coli were stored at -80°C.

[0164] 4.8 Agrobacterium-mediated transformation

[0165] 1) Extract the pBI 121-TeLFI 1 plasmid according to the steps described in 4.1 of this embodiment;

[0166] 2) Add pBI 121-TeLFI 1 plasmid to 50 μL of Agrobacterium strain GV2260 competent cells, gently stir to mix, and incubate on ice for 30 min;

[0167] 3) Place in liquid nitrogen for 1 minute for cooling shock;

[0168] 4) Place the EP tube on a 37℃ constant temperature heater and heat for 5 minutes;

[0169] 5) Add 800 μL of SOC culture medium and incubate in a shaker at 28°C and 200 rpm for 4-5 hours;

[0170] 6) Centrifuge the bacterial culture at 4000 rpm for 5 min;

[0171] 7) In the clean bench, aspirate the supernatant, leaving approximately 100 μL. Gently pipette the remaining bacterial cells to suspend and mix them.

[0172] 8) Spread the bacterial solution evenly on LB solid medium containing kanamycin (50 μg / mL) and rifampin (20 μg / mL) using sterilized glass beads, and incubate at 28℃ for 48 h.

[0173] 9) Perform colony PCR identification according to the same method steps as in Example 2, 4.5, and store the identified positive Agrobacterium in the recombinant plasmid at -80℃.

[0174] Example 4: Genetic transformation of the TeLFI1 gene in tomato

[0175] The TeLFI1 gene was genetically transformed into tomato using Agrobacterium-mediated transformation, and the TeLFI1 gene was quantitatively identified in the genetically transformed tomato lines.

[0176] 1. Reagents

[0177] Acetyleugenol, 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (6-BA), indoleacetic acid (IAA), and kinetin (KT) were purchased from Sigma-Aldrich, USA; RNA extraction kit, reverse transcriptase, and TransStart Green qPCR SuperMix real-time quantitative PCR reagent were purchased from TransGen Biotech, Ltd., Beijing; primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and all other reagents were either imported and repackaged or domestically produced analytical grade products.

[0178] 2. Plant materials

[0179] Tomatoes (Solanumlycopersicum variety Micro-Tom, Solanumlycopersicum variety Alsa Craig) are grown in artificial climate cultivation chambers.

[0180] 3. Solutions and culture media

[0181] 20×AB salt solution: Weigh 2g NaCl, 0.3g KCl, 0.005g FeSO4, 0.06g MgSO4·7H2O, and 0.3g CaCl2·2H2O respectively, add deionized water to dissolve completely, and bring the volume to 200mL. Sterilize at 121℃ for 20min, and store at -20℃.

[0182] 0.5M 2-morpholinoethanesulfonic acid (MES): Weigh 4.88g of MES, add deionized water to dissolve it completely, then adjust the pH to 5.6-6.0 with 0.5M NaOH solution, and finally bring the volume to 50mL. Sterilize by filtration through a 0.22μm filter membrane and store at -20℃.

[0183] 20% glucose solution: Weigh 20g of glucose, dissolve and dilute to 100mL of deionized water, filter through a 0.22μm filter membrane for sterilization, and store at -20℃.

[0184] 50× phosphate buffer: Prepare 0.078 g / mL NaH2PO4 solution and 0.087 g / mL K2HPO4 solution respectively, then mix them evenly at a ratio of 5:1 (v / v), filter through a 0.22 μm filter membrane for sterilization, and store at -20℃.

[0185] 250× Acetyleugenone (ACE): Weigh 0.5g of ACE, dissolve and dilute to 50mL of 70% ethanol, filter through a 0.22μm filter membrane for sterilization, dispense into 1.5mL sterile EP tubes, and store at -20℃.

[0186] 500×6-Benzylaminopurine (6-BA): Weigh 50 mg of 6-BA, dissolve it completely in 5 mL of 1 M KOH solution, then bring the volume to 50 mL with deionized water, sterilize by filtration through a 0.22 μm filter membrane, and store at -20 °C.

[0187] 500× Indoleacetic Acid (IAA): Weigh 50 mg of IAA, dissolve it completely in anhydrous ethanol, then bring the volume to 50 mL with deionized water, sterilize by filtration through a 0.22 μm filter membrane, and store at -20 °C.

[0188] 5000×2,4-Dichlorophenoxyacetic acid (2,4-D): Weigh 50 mg of 2,4-D, dissolve it completely in 1 M KOH solution, then bring the volume to 50 mL with deionized water, filter through a 0.22 μm filter membrane for sterilization, and store at -20 °C.

[0189] 10000× Kinetin (KT): Weigh 50 mg of kinetin, dissolve it completely in 1 M HCl solution, then bring the volume to 50 mL with deionized water, sterilize by filtration through a 0.22 μm filter membrane, and store at -20 °C.

[0190] 500×Timetin: Weigh 5g of Timetin, dissolve and dilute to 50mL of deionized water, sterilize by filtration through a 0.22μm filter membrane, and store at -20℃.

[0191] Tissue culture infection medium (50mL): In a clean bench, add 2.5mL of 20×AB salt solution, 1mL of 0.5M MES, 2.5mL of 20% glucose solution, 1mL of 50× phosphate buffer, and 200μL of 250×ACE to the sterilized vessel in sequence, mix well and set aside.

[0192] 1 / 2MS medium: Weigh 10g sucrose, 1.5g MS medium powder, and 3.5g agar powder. First, add deionized water and stir to dissolve it completely. Then, adjust the pH to 5.6-6.0 with 1M KOH solution. Finally, bring the volume to 500mL with deionized water and sterilize at 121℃ for 20min.

[0193] Pre-medium: After sterilization, cool 1 / 2 MS medium (500 mL) to about 60°C, add 500 μL of 500×6-BA and 20 μL of 500×IAA, mix thoroughly, and dispense into sterile petri dishes for later use.

[0194] Co-culture medium: After sterilizing MS medium (500mL) and cooling it to about 60℃, add 250×ACE 735μL, 5000×2,4-D 100μL and 10000×KT 50μL to the medium, mix thoroughly, and dispense into sterile petri dishes for later use.

[0195] Screening media: After sterilization and cooling, MS medium (500 mL) is mixed with 1 mL of 500×6-BA, 100 μL of 500×IAA, 1 mL of 500×Timentin, and 1000×kanamycin. The mixture is then dispensed into sterile petri dishes or tissue culture flasks for later use. Based on the final kanamycin concentration, three screening media are prepared: 60 μg / mL, 65 μg / mL, and 70 μg / mL.

[0196] Rooting medium: After sterilization, cool MS medium (500mL) to about 60℃, add 1mL of 500×IAA, 1mL of 500×Timentin, and 700μL of 1000×Kanamycin to the medium and mix thoroughly. Dispense into sterile tissue culture bottles for later use.

[0197] 4. Methods

[0198] 4.1 Genetic transformation of tomatoes

[0199] 1) Aseptic seedling culture of tomato: Take an appropriate amount of wild-type tomato seeds and place them in a 50mL centrifuge tube. Treat the seeds with 75% ethanol for 5 minutes in a clean bench, and then wash them with sterile water. Then treat the seeds with 15% sodium hypochlorite solution for 15 minutes, and then wash them with sterile water. Spread the seeds evenly on sterile filter paper, and then use sterile forceps to evenly spot them on the surface of 1 / 2MS medium. Incubate at 25℃ in the dark for about one week until the seeds show white sprouts, and then transfer them to light conditions to grow aseptic seedlings.

[0200] 2) Pre-culture of tomato explants: Sterile seedlings were picked up with sterile forceps in a clean bench, the roots were cut off, and the cotyledons and stems were cut into callus tissue segments of about 5 mm in length. They were placed in pre-culture medium and cultured for 2 days under 25°C light conditions.

[0201] 3) Agrobacterium infection: Agrobacterium strains transformed with the TeLFI1 gene plant genetic transformation vector were removed from a -80℃ freezer and streaked onto LB medium containing kanamycin (50 μg / mL) and rifampin (20 μg / mL), and cultured at 28℃ for 2 days. Single colonies were picked and placed in 3 mL of liquid medium containing the same antibiotics, and cultured at 28℃ for 12 h. 2 mL of the bacterial culture was added to LB medium containing the same antibiotics to expand the culture to OD. 600 When the concentration is approximately 0.5, centrifuge at 5000 rpm for 5 min. Resuspend the bacterial cells in the induction solution to achieve a final concentration of OD0.5. 600=0.1. The tomato cotyledons and stem segments, pre-cultured for 2 days, were transferred to the induction solution and soaked for 15 minutes. Then, the explants were removed, evenly placed on sterile filter paper, the bacterial solution was blotted dry, and transferred to a co-culture medium, incubated at 25°C for 2 days.

[0202] 4) Callus screening and culture: Leaves and stem segments infected with Agrobacterium in the co-culture medium were placed in a selection medium with a kanamycin concentration of 60 mg / mL and cultured at 25°C for about 30 days. Then, they were successively transferred to selection media with antibiotic concentrations of 65 mg / mL and 70 mg / mL for subculture.

[0203] 5) Rooting culture: Take out the differentiated seedlings that have grown to a height of about 5 cm from the screening medium and place them in the rooting medium. Incubate at 25℃ for about 30 days until roots are formed. Then remove the differentiated seedlings, rinse the roots with agar, and transfer them to soil culture pots for growth in an artificial culture room.

[0204] 4.2 Identification of transgenic plants

[0205] 1) Based on the full-length TeLFI1 gene sequence cloned in Example 2, quantitative PCR primers were designed using the primer design software PrimerPremier. UBI, which is stably expressed in tomato tissues, was used as an internal control gene for quantitative PCR analysis of TeLFI1 expression levels. The TeLFI1 primers were TeLFI RTF and TeLFI RTR, and the UBI primers were UBI F and UBI 6R. The primer sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively. The specific sequences are as follows:

[0206] TeLFI RTF:5'tcagagcaattggtttatgatgtgg 3', SEQ ID NO.7;

[0207] TeLFI RTR:5'ccaagtgacccaggaagcacaat 3', SEQ ID NO.8;

[0208] UBI F:5'aggttgatgacactggaaaggt 3', SEQ ID NO.9;

[0209] UBI R: 5'aatcgcctccagccttgttgta 3', SEQ ID NO. 10.

[0210] 2) Collect leaves from wild-type and genetically transformed tomato plants respectively, and extract RNA as described in step 3.1 of Example 1. The extracted total RNA is then reverse transcribed to obtain cDNA as described in step 4.2 of Example 2.

[0211] 3) Using the cDNA obtained above as a template and UBI as an internal reference gene, quantitative PCR analysis of TeLFI1 expression level was performed.

[0212] The quantitative PCR reaction system is as follows:

[0213] Forward primer (10 μM), 0.5 μL;

[0214] Reverse primer (10 μM), 0.5 μL;

[0215] 2×TransStart Green qPCR SuperMix, 10 μL;

[0216] 9 μL of cDNA;

[0217] Reaction conditions: 95℃ for 30s; 95℃ for 5s, 60℃ for 15s, 72℃ for 10s, 40 cycles. The cDNA used was the cDNA template described in Example 4.2 diluted 30-fold for quantitative PCR. After amplification, the temperature was increased by 0.5℃ per cycle for 60 cycles, followed by melting curve analysis. Each sample was tested three times.

[0218] 5. Results

[0219] Quantitative PCR results showed that, compared with untransformed wild-type tomato plants, the expression level of the TeLFI1 gene was significantly increased in positively transformed plants of two tomato varieties, Micro-Tom and Ai l sa Cra ig. Genetically transformed tomato lines expressing the TeLFI1 gene were obtained by Agrobacterium-mediated transformation.

[0220] Example 5: TeLFI 1 gene overexpression alters tomato leaf color

[0221] The tomato lines transfected with the TeLFI1 gene obtained in Example 4 were planted in parallel with wild-type plants for phenotypic observation and analysis.

[0222] 1. Reagents and materials

[0223] Acetone was domestically produced and of analytical grade. Tomato plants were grown in an artificial climate incubator.

[0224] 2. Method

[0225] Wild-type tomato Micro-Tom and its TeLFI1 overexpression line, and wild-type tomato Ailsa Craig and its TeLFI1 overexpression line were grown in parallel in a culture room, and their phenotypes were observed and photographed.

[0226] Leaves of wild-type and TeLFI1-overexpressing tomato plants of equal weight and growth location were collected, ground with liquid nitrogen, and then extracted by soaking in 80% acetone at 4°C for 14 hours in the dark. The absorbance of the extract at 645 nm and 663 nm was measured using a spectrophotometer to determine the chlorophyll content in the leaves. The calculation formula was: Chlorophyll content = (8.02A) / (663nm) * ... 663 +20.21A 645 )×V / W (where V is the volume of the sample solution and W is the mass of the sample).

[0227] 4. Results

[0228] Compared to the wild type, tomato plants inoculated with the TeLFI1 gene showed a significant change in leaf color, forming a pattern similar to... Figure 1 The leaves shown are variegated or golden-edged. Chlorophyll content measurements showed that the average chlorophyll content in wild-type Micro-Tom leaves was 1.2 mg / g FW, while in TeLFI1-overexpressing lines it was 0.7 mg / g FW; the average chlorophyll content in wild-type Ailsa Craig leaves was 2.7 mg / g FW, while in TeLFI1-overexpressing lines it was 1.5 mg / g FW. In both tomato materials, TeLFI1 gene expression significantly reduced the chlorophyll content in the leaves. Changes in chlorophyll content in leaves can cause changes in leaf color. The expression of the TeLFI1 gene in this invention does not cause a uniform change in leaf color, but rather creates unpredictable, non-uniform, and ornamental leaf color changes. Furthermore, variegated and golden-edged leaves can appear simultaneously in the same tomato material, indicating the pleiotropic function of the TeLFI1 gene in changing leaf color. Simultaneously, since similar results were produced in different tomato materials, it demonstrates the universality of the TeLFI1 gene in changing leaf color. Based on the results of this embodiment, the gene with the nucleotide sequence shown in SEQ ID NO.1 plays a role in leaf pigment synthesis, and the TeLFI1 gene cloned from marigold in this embodiment can be used to change the leaf color of plants.

[0229] Example 6: TeLFI1 gene overexpression alters the synthesis of flavor components in tomato fruit

[0230] The tomato lines transfected with the TeLFI1 gene obtained in Example 4 were planted in parallel with wild-type plants, and their fruit flavor phenotypes were detected and analyzed.

[0231] 1. Materials

[0232] The tomato plants were grown in an artificial climate incubator.

[0233] 2. Method

[0234] 1) Sample preparation: Fresh fruits of wild-type tomatoes with TeLFI1 gene overexpression from the same growth stage were taken, quickly ground into a paste, and 0.4g was placed in a 40mL purge bottle, sealed, and injected for testing.

[0235] 2) Purge and trap conditions

[0236] The prepared sample was injected using an autosampler. High-purity nitrogen was used for purging: pre-purging time 0.2 min, purging time 20 min, purging temperature 35℃, and purging flow rate 40 mL / min. Once the volatile components were adsorbed onto the adsorbent, the collector was heated to 200℃, and the collected components were desorbed by helium for 0.5 min, then directly transferred to a gas chromatography system.

[0237] 3) Chromatographic conditions

[0238] Chromatographic column: HP-5MS capillary column (30m×250μm, 0.24μm); injection port temperature 250℃, carrier gas (He) flow rate 1mL / min, split ratio 15:1; temperature program 40℃ held for 3min, increased to 100℃ at 8℃ / min, then increased to 280℃ at 10℃ / min and held for 10min.

[0239] 4) Mass spectrometry conditions

[0240] Electron impact ion source, electron energy 70eV, ion source temperature 230℃, transmission line temperature 280℃, quadrupole temperature 150℃; scanning range 33-500m / z, full scan mode.

[0241] 5) Use the NIST standard mass spectrometry database to search the mass spectrometry results and identify the chemical composition of each sample.

[0242] 4. Results

[0243] The volatile components of tomato fruit were detected and analyzed using a purge-trap-gas chromatography-mass spectrometry method. Figure 2 The results showed that, compared with the wild type, the fruits of tomatoes transformed with the TeLFI1 gene contained the same proportion of hexanal, but also contained more 2-hexenal, hexanol and 1-penten-3-one, as well as 3-hexenol and a small amount of 2-isobutylthiazole, which were not detected in the wild type. Transplantation of the TeLFI1 gene enabled tomatoes to synthesize more tomato flavor compounds.

[0244] The main volatile components in tomato fruit are aldehydes, alcohols, ketones, esters, phenols, and heterocyclic compounds. Aldehydes primarily produce a grassy aroma, enhancing the freshness of tomatoes; alcohols contribute sweetness and play a crucial role in flavor enhancement; ketones produce floral, fruity, and sweet aromas. These flavor compounds can be categorized according to their metabolic pathways: fatty acid-derived volatiles, branched-chain amino acid derivatives, cucurbitacin-like derivatives, and phenylalanine derivatives. Volatile components in these pathways, such as 3-hexenal, β-ionone, hexanal, 1-penten-3-one, β-damascone, 2-hexenal, 2-isobutylthiazole, and phenylacetaldehyde, significantly contribute to the flavor of tomato fruit. 3-Hexenol, in particular, possesses a strong, fresh, green leaf aroma and is a permitted edible flavoring under GB2760-96, primarily used in the formulation of various fruit and vegetable flavorings. Mixing 3-hexenol, hexanal, 1-penten-3-one, 2-hexenol, 2-isobutylthiazole, and β-ionone in appropriate proportions can yield the aromatic flavor of ripe tomatoes. Expression of the TeLFI1 gene increases the content of fatty acid-derived volatiles 3-hexenol, 2-hexenol, hexanol, and 1-penten-3-one, as well as the branched-chain amino acid derivative volatile 2-isobutylthiazole in the fruit. This increase is mainly achieved by influencing the synthesis of volatiles in the fatty acid metabolism pathway, thereby enhancing the flavor compounds and their content. Therefore, based on the results of this embodiment, the gene with the nucleotide sequence shown in SEQ ID NO. 1 plays a function in the formation of volatile components in fruits. The TeLFI1 gene cloned from marigolds in this embodiment can be used to improve the flavor of plant fruits.

[0245] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A marigold gene, characterized in that, The nucleotide sequence of the marigold gene is shown in SEQ ID NO.

1.

2. The protein encoded by the marigold gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A method for cloning the marigold gene as described in claim 1, characterized in that, The cloning method includes the following steps: 1) RNA extraction from marigold leaf tissue; 2) Obtain cDNA through reverse transcription; 3) Design primer sequences and use the obtained cDNA for... TeLFI1 Gene cloning.

4. The cloning method according to claim 3, characterized in that, In step 3), the primer sequences are designed as follows: TeLFI1F, as shown in SEQ ID NO.3; and TeLFI1R, as shown in SEQ ID NO.

4.

5. The cloning method according to claim 4, characterized in that, In step 3), place the 200 μL EP tube on ice and add the following reagents: 0.5 μL high-fidelity DNA polymerase; 10 μL 5×Buffer; 4 μL dNTP; 1 μL cDNA; 1 μL TeLFI1F; 1 μL TeLFI1R; 32.5 μL ddH2O.

6. The cloning method according to claim 3 or 5, characterized in that, In step 3), the gene clone amplification program is as follows: 98℃ for 2 min pre-denaturation; 98℃ for 10 s denaturation, 56℃ for 20 s annealing, 72℃ for 70 s extension, with 30 cycles of denaturation-annealing-extension; 72℃ for 5 min total extension.

7. A marigold gene expression vector, characterized in that, The marigold gene expression vector comprises the marigold gene as described in claim 1.

8. The marigold gene expression vector according to claim 7, characterized in that, The marigold gene was inserted into the plant expression vector pBI121 to construct a recombinant expression vector.

9. An application of the marigold gene according to claim 1, characterized in that, The application of the marigold gene in altering tomato leaf color.

10. An application of the marigold gene according to claim 1, characterized in that, The application of the marigold gene in improving the flavor of tomato fruit.

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