Application of zmerf13 gene in regulating corn stalk quality

Abstract

The invention discloses an application of the zmerf13 gene in regulating the quality of corn stalks, belongs to the technical field of plant genetic engineering, and provides an application of the zmerf13 gene in regulating the glycolysis efficiency of corn stalks, wherein the nucleotide sequence of the zmerf13 gene is shown in SEQ ID NO.1. And an application of the zmerf13 gene in regulating the synthesis of vascular bundles of corn stalks, regulating the cellulose content of corn stalks, and regulating the lignin content of corn stalks. The invention plants a mutant in which the zmerf13 gene is knocked out and compares it with a wild-type corn plant, and finds that knocking out the zmerf13 gene can promote the improvement of corn glycolysis efficiency, thereby regulating the quality of corn stalks and improving the quality of corn stalks.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of zmerf13 gene in regulating the quality of corn stalks. Background Art

[0002] Corn straw glycolysis can utilize agricultural waste to produce renewable energy and chemicals, reduce dependence on fossil resources, and reduce the risk of environmental pollution. It has broad application prospects in the production of biofuels (such as bioethanol), bio-based plastics (such as polylactic acid), and other renewable products.

[0003] Although corn straw glycolysis is of great significance in the fields of biomass energy and green chemistry, the current technology is not yet fully mature and still faces some key challenges and shortcomings. The hydrolysis products of hemicellulose in corn straw (such as xylose and arabinose) may be metabolized by microorganisms into other byproducts (such as lactic acid, acetic acid, etc.), affecting the yield and purity of the main products (such as ethanol). The ERF (Ethylene Response Factor) family is a class of plant-specific transcription factors, belonging to an important branch of the AP2 / ERF superfamily, and plays an important role in regulating plant response to environmental stress, developmental processes and metabolic regulation. Recent studies have shown that ethylene, as an important plant hormone, is closely related to grain development. Given that ethylene signals often affect plant growth and development processes through downstream ethylene response factors (ERF)-mediated transcriptional regulatory reactions, existing technologies have analyzed the regulatory mechanism of ethylene signal transduction on rice grain shape and grain weight, and also revealed the potential pathway for corn grain size regulation, which is of great significance for the molecular breeding of high-yield and high-quality cereal crop varieties. However, there have been no reports on whether the transcription factor genes related to the ERF family will affect the sudden drop in corn glycolysis. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes the application of the zmerf13 gene in regulating the quality of corn stalks. By planting mutants with the zmerf13 gene knocked out and comparing them with wild-type corn plants, it was found that knocking out the zmerf13 gene can promote the improvement of corn glycolysis efficiency.

[0005] To achieve the above object, the present invention provides the use of the zmerf13 gene in regulating the glycolysis efficiency of corn straw, and the nucleotide sequence of the zmerf13 gene is shown in SEQ ID NO.1.

[0006] Preferably, the glycolysis efficiency of corn stover is improved by knocking down and / or knocking out the zmerf13 gene to reduce its expression level.

[0007] The present invention also provides the use of an expression box, a vector or a recombinant bacterium containing the zmerf13 gene in improving the glycolysis efficiency of corn stalks.

[0008] The present invention also provides a method for improving the glycolysis efficiency of corn straw by utilizing the zmerf13 gene, wherein the corn zmerf13 gene is knocked down and / or knocked out through transgenic, hybridization, backcrossing or selfing methods to reduce its expression level and improve the glycolysis efficiency of corn straw.

[0009] Preferably, the transgenic comprises introducing a recombinant expression vector containing the zmerf gene into corn using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated methods to obtain a transgenic corn line.

[0010] The present invention also provides application of the zmerf13 gene in regulating the synthesis of corn stalk vascular bundles.

[0011] Preferably, the zmerf13 gene is knocked down and / or knocked out to reduce its expression level and inhibit the synthesis of vascular bundles in corn stover.

[0012] The present invention also provides the application of the zmerf13 gene in regulating the cellulose content of corn stalks.

[0013] Preferably, the zmerf13 gene is knocked down and / or knocked out to reduce its expression level and thereby reduce the cellulose content in corn stover.

[0014] The present invention also provides application of the zmerf13 gene in regulating the lignin content of corn stalks.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] The present invention discloses that the ERF family-related gene zmerf13 affects corn, which can improve the quality of corn stalks. By knocking down and / or knocking out the zmerf13 gene, its expression level is reduced, the synthesis of vascular bundles in corn stalks is inhibited, the cellulose content in corn stalks is reduced, and the lignin content in corn stalks is reduced, which can promote the improvement of corn glycolysis efficiency. The specific functions of the ERF family in corn are deeply excavated, which is expected to provide a theoretical basis and practical guidance for the development of efficient glycolysis raw materials and the improvement of biofuel production efficiency, and provide new ideas for the targeted cultivation of high-quality new corn strains using genetic engineering methods. Through these technological breakthroughs, the efficiency and economy of corn stalk glycolysis will be significantly improved, which will help to achieve its wide application in the fields of bioenergy and bio-based materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 For wild type WT and mutant zmerf13 Phenotypic analysis of strains, where A is a mutant zmerf13 The phenotype of wild-type WT corn lines. The left side of the figure is the wild-type WT, and the right side is the mutant zmerf13 , B is the wild type WT and mutant zmerf13 The stems of the strains are thick, C is the wild type WT and the mutant zmerf13 Plant height, D is the cross section of the stem at the base of the wild-type WT plant, the scale bar is 1000 μm, and E is the mutant zmerf13 Cross section of the stem at the base of the plant, scale bar is 1000 μm;

[0019] Figure 2 For wild type WT and mutant zmerf13 Paraffin sections, where A is the wild type WT and B is the mutant zmerf13 ;

[0020] Figure 3 For wild type WT and mutant zmerf13 cellulose content of the strains;

[0021] Figure 4 For wild type WT and mutant zmerf13 lignin content of strains;

[0022] Figure 5 For wild type WT and mutant zmerf13 Glycolytic efficiency of the strains. DETAILED DESCRIPTION

[0023] Example 1

[0024] 1. Mutants zmerf13 The process of obtaining

[0025] Order the MaizeGDB transposon-inserted mutants from the website.

[0026]

[0027] 2. Growth and development of corn stems

[0028] Corn phenotype

[0029] Wild-type maize (WT) and maize mutant ( zmerf13 ) were cultivated in the field for 60 days, and then the phenotype was observed and the plant height and ground diameter were measured. Figure 1 Middle A, Figure 1 Medium B and Figure 1 As shown in C, mutant plants were found zmerf13 The stem diameter was reduced by 6.12% and the plant height was reduced by 2.01% compared with the wild-type plants. zmerf13 The cross section of the stem at the base was observed under a microscope and the mutant zmerf13 The vascular bundles of the wild type are sparsely distributed and less in number, such as Figure 1 Medium D and Figure 1 As shown in E.

[0030] Identification of xylem development

[0031] 1. Paraffin section observation:

[0032] (1) Materials: The fifth internode of the base of wild-type WT and zmerf13 mutant maize grown in the field was collected;

[0033] (2) Fixation of materials: Cut the materials into 5 mm stem segments, place them in a 4% paraformaldehyde solution, vacuum the solution to make the materials sink to the bottom of the tube, and place them in a 4°C refrigerator for 3 days for later use;

[0034] (3) Dehydration: Place the fixed tissue in a 2 mL centrifuge tube and soak the material in 30%, 50%, 75%, 80%, 90%, 95%, 100%, and 100% ethanol, respectively. Place the material on a rotary mixer and rotate for 2.5 h at each concentration.

[0035] (4) Transparency: Use solutions of xylene and anhydrous ethanol at a ratio of 25%, 50%, 75%, 100%, and 100% to immerse the material, place it on a rotary mixer, and treat it for 2.5 hours at each concentration;

[0036] (5) Wax dipping: Melt the paraffin in advance, aspirate half of the liquid in the centrifuge tube, add an equal volume of paraffin, and place in a 75°C oven for dipping. Change the wax every 12 hours until there is no xylene smell.

[0037] (6) Embedding: Open the metal bath (preheated to 95°C), burn the embedding mold with an alcohol lamp, then place it in the metal bath, add a little paraffin, use heated tweezers to take out the material and place it in the embedding mold. When the paraffin at the bottom of the mold is slightly solidified, continue to add paraffin to cover the embedding mold, place the mold cover, and add paraffin to seal it. Place the embedded material on ice to cool. After it is completely cooled, demold the wax block and place it in a 4°C refrigerator for later use;

[0038] (7) Slicing: Set the slicer thickness to 8-10 μm, slice the embedded wax block, and gently pick up the slices with a brush and place them in 42°C ddH 2 After the slide is fully developed, pick it up with an absorbent glass slide and dry it on a 37°C slide dryer. After drying, store it in a 37°C oven for later use;

[0039] (8) Dewaxing and rehydration: Place the prepared sections in a dyeing jar containing 100% xylene for dewaxing for 30 min, repeat once; after dewaxing, wash the sections with 100%, 100%, 95%, 90%, 80%, 70%, 50%, and 30% ethanol in sequence, each time for 5 min; then rinse with ddH 2 O Wash the slides for 5 minutes, repeat once;

[0040] (9) Observation: Use 1% TBO (toluidine blue) for staining. After staining, wash with clean water, cover with a coverslip and observe under a microscope;

[0041] The results are as follows Figure 2 Middle A and Figure 2 As shown in B, the mutant zmerf13 The vascular bundles in the plant are sparsely distributed and less in number than those in the wild type.

[0042] 2 Identification of cell wall components

[0043] The wild-type WT and mutant zmerf13 Changes in cell wall composition of corn stems

[0044] AIR EXTRACTION:

[0045] (1) Take the wild-type WT and mutants grown in the field zmerf13 The fifth internode material of the corn base stem was placed in liquid nitrogen, crushed into powder using a tissue crusher, and packaged in 2 mL centrifuge tubes.

[0046] (2) Take 1 mL of the crushed powder and place it in a 2 mL centrifuge tube. Add 1 mL of 80% ethanol and mix well. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. Repeat once. Add 1 mL of anhydrous ethanol and mix well. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. Repeat once. Add 1 mL of a mixture of chloroform and methanol (volume ratio 1:1) and mix well. Incubate in a 37°C water bath for 40 min. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. Repeat once. Add 1 mL of acetone and mix well. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. Repeat once. Place in a fume hood and blow dry.

[0047] (3) Remove starch with 1U amylase and 1U amyloglucosidase, incubate in a water bath at 37°C overnight; inactivate at 100°C for 10 min; add anhydrous ethanol to a concentration of about 80% for washing, centrifuge at 12,000 rpm for 10 min, discard the supernatant, repeat once; wash with 1mL anhydrous ethanol, centrifuge at 12,000 rpm for 10 min, discard the supernatant, repeat once; mix with 1mL acetone, centrifuge at 12,000 rpm for 10 min, discard the supernatant; place in a fume hood and blow dry, and a pure AIR sample is obtained after complete drying.

[0048] 2.1 Determination of total cellulose content

[0049] (1) Take 2 mg of AIR in a glass tube, add 500 μL of 2M TFA (slowly add along the tube wall), and react in a metal bath at 121°C for 1.5 h;

[0050] (2) Cool at room temperature, centrifuge at 5000g for 5 min, and remove the supernatant;

[0051] (3) Use ddH 2 O Wash the precipitate, centrifuge at 5000g for 5 min, and remove the supernatant;

[0052] (4) Repeat step 3;

[0053] (4) Remove the supernatant, add 200 μL of 72% sulfuric acid, and react for 30 min;

[0054] (5) Add 800 μL ddHO 2 O, 12000rpm, centrifuge for 5min, take 500μL supernatant;

[0055] (6) Preparation of glucose standard curve: weigh 1 mg of glucose standard sample and dilute it into 0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, and 1 mg / mL solutions respectively, and draw 500 μL for the experiment;

[0056] (7) Add 500 μL of 6% phenol and 2.5 mL of 98% sulfuric acid to the supernatant and standard solution, and mix well.

[0057] (8) Take 200 μL of the reaction solution and place it on an ELISA plate, and measure the absorbance at 490 nm.

[0058] The results are as follows Figure 3 As shown, mutants were found zmerf13 The cellulose content in the stem of R. paniculata was 23.34% lower than that of the wild type WT.

[0059] 2.2 Determination of lignin content

[0060] (1) Take 2 mg of AIR and place it in a 2 mL centrifuge tube, add 250 μL of acetone, and evaporate to dryness under air flow;

[0061] (2) Add 100 μL of acetyl bromide solution (25% v / v acetyl bromide / acetic acid) and heat in a 50°C water bath for 2 h;

[0062] (3) Continue heating for 1 hour, shaking every 15 minutes;

[0063] (4) Place the reacted sample on ice to cool down to room temperature, add 2M NaOH to neutralize the acetic acid, and add 70 μL 0.5M hydroxylamine hydrochloride to terminate the reaction;

[0064] (5) Use glacial acetic acid to make the volume of the reaction solution to 2 mL;

[0065] (6) Take 200 μL of the above solution and place it in an ELISA plate, and measure the absorbance at 280 nm.

[0066] Acetyl bromide soluble lignin (% ABSC) = Abs (absorbance value) / coeff (coefficient) * 0.1cm * 2mL * 100% / weight (mg)

[0067] The results are as follows Figure 4 As shown, mutants were found zmerf13 The stem lignin content increased by 21.40% compared with the wild type WT. 2.3

[0069] a. Determination of total sugar and sugar component content

[0070] (1) Accurately weigh 100 mg of the prepared AIR sample and transfer it to a 10 mL quartz centrifuge tube;

[0071] (2) Add 1.5 mL of 72% H 2 SO 4 , incubate in a 30°C water bath for 1 h, shaking every 10 min to mix the sample evenly;

[0072] (3) Transfer all the above materials into a 50 mL glass bottle and add 42 mL of ultrapure water;

[0073] (4) Autoclave at 121°C for 1 h, then cool to room temperature;

[0074] (5) Transfer the above materials into a new 50 mL test tube, wash the glass bottle with 5-10 mL of ultrapure water, and combine them in the 50 mL test tube;

[0075] (6) Centrifuge at 4000 rpm for 30 min. Take the supernatant and store it in a -20℃ refrigerator for phenol-sulfuric acid method detection.

[0076] b. Enzymatic hydrolysis to release sugars and sugar components (with or without acid pretreatment)

[0077] (1) [Without acid pretreatment] Weigh 100 mg of the prepared AIR sample and transfer it to a 15 mL test tube;

[0078] (2) Add 10 mL of enzyme digestion solution to each test tube and mix well (enzyme digestion solution formula: 8.8 mL 0.1 M, pH = 4.8 sodium citrate buffer + 0.2 mL 2% NaN 3 +1mL cellulase);

[0079] (3) After thorough mixing, shake and culture on a shaker at 50°C and 100 rpm for 72 h;

[0080] (4) Centrifuge the test tube at 4000 rpm and take the supernatant for detection by sulfuric acid phenol method;

[0081] (5) [After acid pretreatment] Weigh 100 mg of the prepared AIR sample and transfer it to a 15 mL test tube;

[0082] (6) Add 2.5 mL of 1.5% H 2 SO 4 , sterilize at 121℃ for 1h;

[0083] (7) Centrifuge the glass tube, take the supernatant and put it into a new 15 mL test tube, add 5 mL of ultrapure water to wash the precipitate, centrifuge and take the supernatant and combine it into a 15 mL centrifuge tube;

[0084] (8) The collected supernatant can be stored in a -20°C refrigerator and will be used for phenol-sulfuric acid method detection;

[0085] (9) Resuspend the precipitate with ultrapure water and transfer it to a new 15 mL centrifuge tube. Continue to add water to the old precipitation tube until all the precipitate is transferred to the new 15 mL centrifuge tube. Centrifuge to remove the supernatant, and then wash the residue with citrate solution 2 to 3 times until the pH is 4.8;

[0086] (10) Centrifuge the solution and remove the supernatant;

[0087] (11) Add 10 mL of enzyme digestion solution to 15 mL of sealing solution and follow steps b(2) to b(4).

[0088] c. Phenol sulfuric acid method to detect sugar content

[0089] (1) Standard curve: Prepare glucose solutions with concentrations of 0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively. Add 150 µL of 5% phenol and 750 µL of concentrated sulfuric acid to the solutions and mix them. Then place the mixture in a 30°C water bath for 30 min. Use a spectrophotometer to measure the absorbance at 490 nm, and then prepare a standard curve.

[0090] (2) Take 100 μL of the prepared sample, add 150 μL of 5% phenol and 750 μL of concentrated sulfuric acid to mix, then place the mixture in a 30°C water bath for 30 min, and measure its absorbance at 490 nm using a spectrophotometer;

[0091] (3) The measured total sugar content was recorded as S1, the sugar content without acid treatment was recorded as S2, and the sugar content under acid pretreatment conditions was recorded as S3. The saccharification efficiency with and without acid pretreatment was obtained by calculating S3 / S1 and S2 / S1.

[0092] The results are as follows Figure 5 As shown, mutants were found zmerf13 The glycolysis efficiency of the stem was increased by 8.18% compared with the wild type WT.

[0093] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of zmerf13 gene in regulating corn straw glycolysis efficiency, characterized in that: The nucleotide sequence of the zmerf13 gene is shown in SEQ ID NO.1; by knocking down and / or knocking out the zmerf13 gene, its expression level is reduced and the glycolysis efficiency of corn stover is improved.

2. Use of an expression cassette, vector or recombinant bacterium containing the zmerf13 gene as claimed in claim 1 in improving the glycolysis efficiency of corn stalks, characterized in that: By knocking down and / or knocking out the zmerf13 gene, its expression level is reduced and the glycolysis efficiency of corn straw is improved.

3. A method for improving the glycolysis efficiency of corn straw using the zmerf13 gene as claimed in claim 1, characterized in that: Through transgenic, hybridization, backcrossing or self-pollination methods, the corn zmerf13 gene is knocked down and / or knocked out to reduce its expression level and improve the glycolysis efficiency of corn stover.

4. The use of the zmerf13 gene in regulating corn stalk vascular bundle synthesis as claimed in claim 1, characterized in that: By knocking down and / or knocking out the zmerf13 gene, its expression level is reduced and the synthesis of vascular bundles in corn stover is inhibited.

5. The use of the zmerf13 gene in regulating the cellulose content of corn stalks as claimed in claim 1, characterized in that: By knocking down and / or knocking out the zmerf13 gene, its expression level is reduced and the cellulose content in corn stover is reduced.

6. The use of the zmerf13 gene in regulating the lignin content of corn stover as claimed in claim 1, characterized in that: By knocking down and / or knocking out the zmerf13 gene, its expression level is reduced and the lignin content in corn straw is increased.

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