A Dactylis glomerata gene related to flavonoid synthesis and its application

Abstract

The present invention discloses a Duckgrass gene related to flavonoid synthesis and its application. The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.4. Studies have shown that overexpressing the gene in Arabidopsis thaliana can cause albinism in the Arabidopsis thaliana, and the expression levels of its flavonoid synthesis-related genes all decrease, indicating that the gene is a negative regulatory gene for flavonoid synthesis and can be used for the synthesis of flavonoid compounds, which is beneficial to the industrial production of flavonoid compounds. At the same time, by regulating the expression of the gene, it can be used to improve Duckgrass strains, promote the development and utilization of high-quality grass forage Duckgrass, and make a significant contribution to the improvement of traditional forage.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to a orchardgrass gene related to flavonoid synthesis and an application thereof. Background Art

[0002] Dactylis, a genus of the Poaceae subfamily Festucoideae, is a perennial, cool-season forage grass cultivated worldwide. It boasts numerous leaves, high yield, shade tolerance, strong adaptability, good palatability, and high nutritional value. It can be used for green fodder, hay preparation, or silage, making it one of the four most widely distributed grasses in the world. Approximately 14,000 tons of Dactylis seeds are produced annually worldwide, accounting for 3.3% of the world's temperate forage seeds. Currently, Dactylis is cultivated in Qinghai, Gansu, Shaanxi, Shanxi, Henan, Jilin, Jiangsu, Hubei, Sichuan, and Xinjiang provinces for both mowing and grazing. It is a popular food for various poultry species and has achieved excellent economic and ecological benefits, demonstrating broad prospects for its utilization. Flowering period is a key agronomic trait of Dactylis, closely linked to its quality and yield. Flowering period marks the transition from vegetative to reproductive growth, and forage quality and yield also change accordingly. Duckgrass is suitable for mixed sowing with legume forages. Targeted cultivation of duckgrass varieties with different flowering periods and combination with legume forage varieties with the same maturity period can extend the grazing time of mixed grasslands and improve overall production performance and utilization efficiency.

[0003] Flavonoids are a large class of polyphenolic compounds produced by plants that play an important role in maintaining plant health, development, and growth in various organs. At the same time, increasing evidence also shows that flavonoids are beneficial to livestock health. For example, adding flavonoids to ruminant diets can improve ruminant productivity. Flavonoids can be divided into flavonoids, flavonols, isoflavones, anthocyanins, chalcones, neuroketones, flavans, etc. Flavonoids are widely present in the biosynthetic pathways of plants and produce extremely rich secondary metabolites. They are one of the largest groups of secondary metabolites and the main source of plant pigments. They accumulate in various parts of the plant. However, genes related to flavonoid synthesis in Dactylis gloeospermum have not yet been developed or reported. Summary of the Invention

[0004] The purpose of the present invention is to provide a Duckgrass gene related to flavonoid synthesis. Overexpression of this gene in Arabidopsis thaliana can cause albinism in Arabidopsis thaliana, and the expression levels of its flavonoid synthesis-related genes all decrease, indicating that this gene is a negative regulatory gene for flavonoid synthesis and can be used for the industrial production of flavonoid compounds; at the same time, by regulating the expression of this gene, it can be used to improve Duckgrass varieties.

[0005] In order to achieve the above object, the present invention provides a Dactylis gracilis gene DgCMO-like related to flavonoid synthesis, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0006] The present invention also provides a protein encoded by the above-mentioned Dactylis glomerata gene DgCMO-like, the amino acid sequence of which is shown in SEQ ID NO.4.

[0007] The present invention also provides a recombinant vector containing the Dactylis gracilis gene DgCMO-like nucleotide sequence.

[0008] The present invention also provides a recombinant engineering bacterium containing the recombinant vector.

[0009] The orchardgrass gene DgCMO-like provided by the present invention can be used in forage improvement and breeding.

[0010] The orchid grass gene DgCMO-like provided by the present invention can be used in the industrial production of flavonoid compounds.

[0011] The Dactylis gracilis gene DgCMO-like provided by the present invention can be used to create ornamental plants, especially to create albino ornamental plants.

[0012] The present invention has the following advantages:

[0013] The present invention discloses for the first time a Dactylis grassi gene DgCMO-like related to flavonoid synthesis, which promotes the annotation of Dactylis grassi gene function.

[0014] Since overexpression of the gene DgCMO-like leads to a decrease in flavonoid synthesis, gene knockout technology can be used to reduce the expression level of the gene DgCMO-like in Dactylis grassi to increase the synthesis of flavonoids. This can address the shortcomings of traditional forage improvement technology, which has slow effects and long cycles, and can be used to develop Dactylis grassi varieties with high flavonoid synthesis efficiency, helping to shorten breeding time, improve breeding efficiency, and promote the development and utilization of high-quality Poaceae forage Dactylis grassi. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the subcellular localization of the protein encoded by the gene DgCMO-like in the present invention.

[0016] Figure 2 This is the resistance screening result of the T1 generation of Arabidopsis thaliana that overexpressed the gene DgCMO-like in the present invention.

[0017] Figure 3 This is the expression level of the DgCMO-like gene in the positive seedlings of the overexpression homozygous strain in the present invention.

[0018] Figure 4 The figure shows the phenotypic comparison between the wild type and overexpression strains in the present invention.

[0019] Figure 5 It is the expression level of the gene related to flavonoid synthesis in the present invention.

[0020] Figure 6 These are the results of anthocyanin content detection in the wild type and overexpression strains of the present invention. DETAILED DESCRIPTION

[0021] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0022] Note: Methods not specifically described in this application are all conventional methods in the art, and reagents and consumables not specifically given are all conventional reagents and consumables in the art.

[0023] Experimental Example 1 Gene Acquisition

[0024] 1. Selection of experimental materials

[0025] Total RNA was extracted from young leaves of the Duckgrass cultivar '2006-1', grown at the Wenjiang campus of Sichuan Agricultural University. RNA was extracted using the Tiangen (Beijing) Biochemical Technology Co., Ltd. Plant Total RNA Extraction Kit, following the included instructions. RNA integrity was assessed by 1% agarose gel electrophoresis, and RNA concentration and purity were determined using an ultra-micro spectrophotometer. Reverse transcription was performed using the TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit, following the included instructions.

[0026] 2. Amplification of target gene

[0027] (1) Cloning of gene fragments:

[0028] Using the Dactylis glomerata reference genome (sequencing data in the reference article Huang L. Genome assembly provides insights into the genome evolution and flowering regulation of orchardgrass. Plant Biotechnol J) as a template, primers were designed based on the full-length sequence. The specific primer sequences are as follows (5'→3'):

[0029] F(SEQ ID NO.1):ATGGCGATAGCGCGATCCCTG;

[0030] R (SEQ ID NO. 2): CCGCGGCCGGTGAGGTCGGC.

[0031] The extracted RNA was reversed to obtain cDNA, and PCR amplification was performed using the cDNA as a template using the primers designed above. The amplification was performed using the 2×PhantaMax MasterMix (Dye Plus) kit from Vazyme. The operation procedure was referred to the enclosed instruction manual.

[0032] The PCR amplification reaction system was 50 μL, including 25 μL of 2×PhantaMax Master Mix (DyePlus), 2 μL of F primer (10 μM), 2 μL of R primer (10 μM), 4 μL of template DNA, and 17 μL of ddH2O.

[0033] The PCR amplification reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, 35 cycles; and extension at 72°C for 5 min.

[0034] The amplified product was recovered and sequenced, and the amplified nucleotide sequence was obtained as shown in SEQ ID NO.3, which was named DgCMO-like. At the same time, the amino acid sequence of the protein encoded by it was shown in SEQ ID NO.4.

[0035] DgCMO-like full gene sequence (SEQ ID NO.3):

[0036]

[0037] DgCMO-like amino acid sequence (SEQ ID NO.4):

[0038] MAIARSLTPSSTASAARAARVRSRRAATSRVAATGAAAAAAEPARRLVAEFDPAIPLASAVTTPSGWYTDPEFLRLELDRVFFRGWQAVGHIGQVKNPNDFFTGRLGNVEFVICRDANGKLHAFHNVCRHHASLLACGSGQKVCFQCPYHGWTYGLDGTLLKATRISGIKNFNKNDFGLKPIKVATWGPFVLAKFDDFTQDTGDD VVGDEWLGSASDLLSRSGIDTSLPHICRREYIIECNWKVFCDNYLDGGYHVPYAHGALASGLQLQSYETLAYERVSVQRCESAAAEQEDIDRLGTKATYAFVYPNFMINRYGPWMDTNLAVPLDATRCKVVFDYFLDESLLDDQSFIDRSLEDSEPVQMEDIALCEGVQRGLGSPAYGVGRYAPSVEMAMHHFHCLLHADLTGRA.

[0039] Experimental Example 2: Functional Analysis of DgCMO-like Gene

[0040] 1. Determine the expression location of DgCMO-like genes using subcellular localization

[0041] (1) Construction of clones:

[0042] The pAN580 plasmid was used as an empty vector, double-digested with XbaI and BamHI, and the DgCMO-like gene sequence was ligated to the digested pAN580 plasmid through cloning recombination technology. The ligation product was sent for sequencing to identify the positive clones. The positive clones were recorded as pAN580-DgCMO-like and transformed into Escherichia coli DH5α competent cells for preservation.

[0043] (2) Transformation of rice protoplasts

[0044] The positive clone plasmid obtained above was transformed into protoplasts as follows:

[0045] 1) Cultivate rice seedlings in the dark at approximately 30°C for 7-15 days, remove stems and leaves, rinse surface dirt with water, and remove the outermost leaf sheaths;

[0046] 2) Place the stems and leaves of the seedlings on a clean plastic board and cut them into small pieces using a sharp blade;

[0047] 3) Add 5-10 mL of enzymatic solution to completely immerse the tissue and shake slowly at 28°C for 4-5 hours.

[0048] 4) Filter the protoplasts through a 40 μm filter, transfer to a 2 mL centrifuge tube, and centrifuge at 600 rpm for 5 min until a turbid precipitate is visible.

[0049] 5) Directly aspirate the supernatant, wash twice with 10 mL of pre-cooled W5 solution, and centrifuge at 600 rpm for 5 minutes at 25°C;

[0050] 6) Add appropriate amount of MMG solution as needed to suspend the mixture to a concentration of 2*10 5 / mL, microscopic examination showed that the protoplasts were round and had few ruptures;

[0051] 7) Take 100 μL of the obtained protoplast suspension, 10 μL of plasmid DNA, and an equal volume of PEG4000 solution, mix gently and evenly, and let it stand at room temperature for 10-15 minutes;

[0052] 8) Dilute the protoplasts with 1 mL of W5 and mix well to terminate the reaction;

[0053] 9) Centrifuge at 600 rpm for 5 min to collect the protoplasts and remove the supernatant;

[0054] 10) Add 1 mL of W5 solution for washing.

[0055] After transient expression in rice protoplasts, DgCMO-like fluorescence was observed under a laser confocal microscope to obtain the subcellular localization of DgCMO-like protein. Figure 1 As shown, it can be seen that the fluorescence signal of DgCMO-like protein is obvious in the cytoplasm, which can be determined that the protein is localized in the granular aggregates in the cytoplasm.

[0056] 2. Functional analysis of genes

[0057] (1) Construction of overexpression recombinant vector

[0058] The plasmid pHG-35S was double-digested with BamHI and PstⅠ, and the DgCMO-like gene sequence was ligated to the double-digested pHG-35S plasmid by cloning recombination technology. The ligation product was sent for sequencing to identify the positive clones. The positive clones were recorded as pHG-35S-DgCMO-like and transformed into Escherichia coli DH5α competent cells for preservation.

[0059] (2) Agrobacterium transformation of Arabidopsis thaliana

[0060] 1) Extract the recombinant vector pHG-35S-DgCMO-like constructed above and transform it into Agrobacterium by the following steps:

[0061] a. Take the competent Agrobacterium GV3101 stored at -80℃ and place it in the palm of your hand for a while until it partially melts. When it is in an ice-water mixture, insert it into ice;

[0062] b. Add 0.1 μg (no more than 10 μL) of plasmid DNA per 100 μL of competent medium, mix thoroughly by hand at the bottom of the tube, and incubate on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0063] c. Add 700 μL of LB liquid medium without antibiotics and culture at 28°C, 200 rpm, shaking for 2 to 3 hours;

[0064] d. Centrifuge at 6000 rpm for one minute to harvest the bacteria. Collect approximately 100 μL of the supernatant and gently pipette to resuspend the bacteria. Spread the suspension onto an LB plate containing the appropriate antibiotic and incubate the plate upside down at 28°C for 2-3 days. Randomly select a single colony and perform colony PCR to identify the correct Agrobacterium clone and label it for future use.

[0065] e. Use a sterile pipette tip to pick up a single colony of the labeled Agrobacterium and inoculate it into 1.5 mL of LB liquid medium containing the corresponding antibiotic (in a 50 mL blue-capped centrifuge tube) and culture at 30°C, 200 rpm, and shake for 24 hours.

[0066] f. The Agrobacterium culture was inoculated with a small shaker at a ratio of 1% and added to 100 mL of LB liquid medium containing antibiotics at 30°C and shaken until OD600 = 1.0;

[0067] g. Centrifuge at 20°C and 4,000 rpm for 15 min to collect the cells; pipette the cells evenly with transformation buffer and resuspend them to an OD600 of about 1.0 to obtain recombinant engineered bacteria.

[0068] 2) The obtained recombinant engineered bacterial suspension was transformed into Arabidopsis thaliana in the following steps:

[0069] a. Water the Arabidopsis plants that are bolting and flowering one day in advance;

[0070] b. Turn the small pot upside down and place all the inflorescences into the bacterial solution pre-suspended in transformation buffer for about 30 seconds;

[0071] c. Repeat the above transformation steps once after 7 days. After 2-3 weeks, water as little nutrient solution as possible to accelerate aging. Collect the mature seeds in paper bags and place them in a desiccator for 7 days.

[0072] The conversion buffer is prepared as follows:

[0073]

[0074] 3) Screening of transgenic Arabidopsis

[0075] a. Preparation of culture medium: Arabidopsis culture medium was 1 / 2MS (0.8% agar powder, without sucrose, pH = 5.8);

[0076] b. Seed disinfection: 70% ethanol for 1 minute, add 1 mL of 7% sodium hypochlorite solution (containing 1 drop of Tween) for 10 minutes, mix by inversion for 5 minutes, and rinse 5 times with sterile water;

[0077] c. Resuspend the sterilized seeds in 100 μL of sterile water, pipette with a 1 mL pipette tip, and spot onto a 1 / 2 MS medium plate (with selection antibiotics added: 50 μg / mL KAN, 30 μg / mL HYG, or 50 μM Glufosinate-ammonium).

[0078] d. Seal the plate and place in a refrigerator at 4°C for 48 hours for vernalization. Then place in an artificial climate chamber to begin germination and growth. The plant growth environment is 60% relative humidity, a constant temperature of 20-22°C, and a photoperiod of 16 hours light and 8 hours dark, with a light intensity of 80-200 μmol / M 2 / S;

[0079] e. Observe after 8-15 days, identify the positive ones and transplant them into planting soil;

[0080] f. Preparation of planting soil: Mix peat soil and vermiculite in a ratio of 2:1 and set aside;

[0081] g. Soil soaking: Fill the soil into the planting pot to about 1 cm from the pot mouth, and use Huawuque compound fertilizer (N, P, K = 20%, 20%, 20%) to completely soak it;

[0082] h. Transplanting: 20 days after germination, select healthy and uniform seedlings and transplant them into a culture soil soaked with flowers beforehand, cover with plastic wrap, and remove it after the seedlings are alive;

[0083] i. Samples were taken at the seedling stage for PCR identification to obtain Arabidopsis T1 generation resistance-positive plants.

[0084] 20-day-old seedlings were sampled and DNA was extracted using the Plant Genomic DNA Extraction Kit (DP305) according to the included instructions. PCR was performed on the extracted DNA. PCR primers were designed based on the DgCMO-like gene sequence. The specific sequences are as follows (5'→3'). The amplification product of this primer pair is 574 bp:

[0085] DgCMO-like-F (SEQ ID NO.5): ATGAGGTGGCCGGAGCAG;

[0086] DgCMO-like-R (SEQ ID NO. 6): CTGTTCGGAAGATAAAGCG.

[0087] 16 seedlings were randomly selected for identification. After PCR, the electrophoresis results of their products were as follows: Figure 2 As shown in the figure, lane M is the DL2000 marker, lanes 1 to 16 are transgenic Arabidopsis seedlings numbered OE1, OE2, ..., OE16, and lane con is wild-type Arabidopsis. The electrophoresis results show that positive Arabidopsis plants overexpressing the DgCMO-like gene were successfully obtained.

[0088] 4) Verification of DgCMO-like expression in T3 transgenic lines

[0089] Four T3 generation homozygous transgenic seedlings were randomly selected and RNA was extracted after they grew for 3 weeks. The expression levels of DgCMO-like genes in 7 homozygous positive seedlings were verified. The expression levels are shown in Figure 3 shown

[0090] 5) Phenotypic differences in transgenic Arabidopsis seedlings

[0091] a. Phenotypic determination of overexpression lines:

[0092] The phenotypes of Arabidopsis plants overexpressing the DgCMO-like gene were compared with those of the wild type. It was found that the seedlings of plants overexpressing the gene showed albinism. The phenotypes of the wild type and the DgCMO-like overexpressing lines OE6 and OE8 were compared as follows: Figure 4 This suggests that the expression of this gene may be related to the synthesis of flavonoids, and overexpression of this gene will lead to a decrease in flavonoid synthesis.

[0093] b. Fluorescence quantitative determination of genes related to flavonoid synthesis:

[0094] Three weeks after germination, samples were collected from the positive overexpression lines (OE6 and OE10) and wild-type Arabidopsis thaliana (WT) and subsequently frozen in liquid nitrogen. Three replicates were obtained for each sample. RNA was extracted and reverse transcribed from the obtained samples using the same experimental methods as above.

[0095] Conventional qPCR was used to perform fluorescence quantification of seven genes in each sample for Arabidopsis thaliana related to flavonoid biosynthesis (AtBAN, AtTT4, AtTT5, AtTT7, AtCHIL, AtF3H, and AtDFR). Figure 5 As shown in the results, except for the AtBAN gene, the other genes related to flavonoid synthesis showed a downward trend in the overexpression strains, such as AtTT4, AtTT5, AtTT7, AtCHIL, AtF3H and AtDFR. Anthocyanin is an important member of the flavonoid family. At the same time, the anthocyanin content was detected, and the quantitative results were as follows. Figure 6 As shown in the figure, it can be seen that the anthocyanin content of the gene overexpression strain is also lower than that of the wild type. Figure 5-6 It can be seen that the Dactylis gracilis DgCMO-like gene may be a negative regulatory gene for flavonoid synthesis, and overexpression of this gene will lead to a decrease in flavonoid synthesis.

[0096] In summary, DgCMO-like is a Dactylis grassi gene involved in flavonoid biosynthesis. Overexpression of this gene significantly reduces flavonoid biosynthesis, suggesting that gene knockout technology could be used to reduce DgCMO-like expression in Dactylis grassi and increase flavonoid biosynthesis. By constructing an overexpression vector for this gene and introducing it into plants, albino seedlings can be produced, potentially enabling the creation of ornamental plants, such as the albino Arabidopsis thaliana described above, as well as other albino ornamental plants.

[0097] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A Dactylis glomerata gene DgCMO-like related to flavonoid synthesis, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The protein encoded by the Dactylis glomerata gene DgCMO-like as claimed in claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

4.

3. A recombinant vector comprising the nucleotide sequence of the Dactylis gracilis gene DgCMO-like as claimed in claim 1.

4. A recombinant engineered bacterium containing the recombinant vector as claimed in claim 3.

5. Use of the Dactylis gracilis gene DgCMO-like as claimed in claim 1 in the creation of albino Arabidopsis thaliana.

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