CN110484647B - Molecular markers associated with maize cell wall digestibility and their identification primers and applications - Google Patents

Abstract

The invention relates to a molecular marker related to maize cell wall digestibility, identification primer and application thereof, and belongs to the technical field of plant genetic engineering. The mutation resulted in loss of CAD enzymatic activity, which in turn affected lignin levels in the cell wall and resulted in a 29.5% increase in cell wall digestibility. The invention provides a detection method for the single base mutation and the influence on the digestibility of the maize cell wall after the single base mutation, and provides a new target for molecular breeding in the future.

Description

Molecular markers associated with maize cell wall digestibility and their identification primers and applications

technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a single-base mutation in the coding region of a CAD gene related to the maize reddish-brown vein mutant bm1 and a marker thereof. The altered gene contributes to the phenotype of maize lignin composition change, and then Improve corn cell wall digestibility.

Background technique

Maize (Zea mays L.) is an annual C4 herbaceous plant belonging to the family Poaceae, and is a multi-purpose crop for "grain-energy-feeding". Plant cell walls are mainly composed of cellulose, hemicellulose and lignin, which are important factors in determining biomass energy conversion efficiency and forage quality. Analyzing the regulatory mechanism related to the lignin metabolic pathway of maize, and changing the lignin composition to increase the digestibility of the cell wall has important practical guiding significance for the genetics and breeding of maize.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a molecular marker related to maize cell wall digestibility, the marker is a mutation of wild-type thymidine (T) from wild-type thymidine deoxyribonucleotide (T) at the 307th position of the marker. Adenine deoxyribonucleotide (A); the mutation can result in loss of CAD enzymatic activity, which in turn affects lignin levels in the cell wall.

The second object of the present invention is to provide a primer for identifying the above molecular marker for rapid identification of the site mutation, and the primer sequences are ZmCAD2F: AAATCATGGCTTGGTTTGA; ZmCAD2R: CCTTATCTCGTCCACTTCTCG.

Further, the PCR reaction system used for identification was 50 μL, including 2 μL of forward/reverse primers, 4 μL of DNA template, 0.5 μL of PrimeSATR high-fidelity DNA amplification enzyme, 25 μL of reaction buffer, 4 μL of dNTP, and ddH ₂ O supplemented to 50 μL. The reaction program was: 98°C, 4 minutes pre-denaturation; 98°C denaturation for 10 seconds, 61°C annealing for 5 seconds, 72°C extension for 2 minutes, 34 cycles of reaction; 72°C chain extension for 7 minutes, 12°C termination reaction, amplified DNA band 457bp.

The present invention also provides the application of the above-mentioned molecular markers in the cultivation of new maize products.

1. The present invention utilizes molecular biology means to obtain the mutation site of the maize CAD gene through PCR amplification, and develops molecular markers at this site, which can be used for rapid identification of cad mutants;

2. The present invention starts from the CAD gene of the key enzyme of lignin synthesis, analyzes the influence mechanism of maize lignin synthesis on cell wall digestibility, and can provide a new direction for the breeding of maize with high cell wall digestibility.

The beneficial effects of the present invention compared with the prior art:

1. The identification of maize cad mutants in the present invention is to analyze the influence of lignin content and composition changes on cell wall digestibility from the synthesis pathway of lignin itself. Through the analysis of cell wall components in the mutants and the test of lignocellulose digestibility, a germplasm resource with constant biomass and constant lignin content, but reduced lignin monomer components, thereby improving cell wall digestibility, is provided for maize And the directed design breeding of other monocotyledonous grasses provides a new breakthrough direction.

2. The molecular marker for single-base mutation of CAD gene in the cad mutant of the present invention can provide a rapid identification method, provide a new target for molecular marker-assisted selection breeding, and greatly save the breeding cycle of crops.

Description of drawings

Figure 1. Phenotype and phloroglucinol staining of maize cad mutants. A and B are the vein phenotypes of the wild-type and cad mutants, respectively; C and D are the phloroglucinol staining images of the wild-type and cad mutants, respectively.

Figure 2. Mutation sites of CAD genes in maize cad mutants. A and B are the sequencing results of CAD genes in wild-type and cad mutants (positions 297-317), of which position 307 is the mutation site.

Figure 3. Enzymatic activity detection of single-base mutations in CAD. A and B phenotype mutated and unmutated enzyme activity chromatograms, respectively.

Figure 4. Total amount and monomer content of lignin in maize cad mutants. A and B represent the total amount of lignin and the monomer content, respectively.

Figure 5. Total cell wall cellulose, enzymatic sugar content and saccharification efficiency of the veins of maize cad mutants. A, B and C represent the total amount of cellulose, enzymatic sugar content and cell wall saccharification efficiency, respectively.

Detailed ways

The specific data of a molecular marker related to cell wall digestibility is described below, and the examples are only used to explain the present invention, and are not intended to limit the scope of the invention. The materials, reagents, molecularly labeled probes, etc. used in the following examples can be purchased from the company through commercial channels unless otherwise specified.

Example 1: Mutant phenotype and molecular identification

Seeds of maize cad mutants were obtained by chemical mutagenesis of pollen by EMS. Using ordinary nutrient soil, seeding and cultivation, the phenotype was continuously observed, and when the seedlings grew to the six-leaf stage, the leaf veins appeared reddish-brown phenotype (A and B in Figure 1). Fresh reddish-brown vein tissue was taken, and non-red (B73) vein tissue was taken as a control, and phloroglucinol was used to stain the cross-section of the leaf vein. stain at room temperature for 3 minutes; add an equal volume of 50% hydrochloric acid dropwise, stain at room temperature for 3 minutes; add 40% glycerol dropwise. Observation records were completed within 15 minutes. The results showed darkening of the color of the mutants (C and D in Figure 1), indicating accumulation of phenylpropanal in the mutants.

Subsequently, we extracted genomic DNA by CTAB method. The specific operations were as follows: (1) Take fresh leaf vein tissue, grind it in liquid nitrogen, add 600 μL of 2% CTAB (add 2‰β-mercaptoethanol at the same time), and take a water bath at 65°C for 30 minutes; (2) ) Add an equal volume of chloroform:isoamyl alcohol (24:1), shake vigorously for 15 seconds, mix well, and place at room temperature for 3 minutes; (3) Centrifuge at 12,000 rpm for 10 minutes, take the supernatant into a new centrifuge tube, add an equal volume of pre-cooling (4) Centrifuge at 12,000 rpm for 15 minutes, discard the supernatant, add 75% ethanol to wash the precipitate 1-2 times, air dry naturally, and add 50 μL ddH ₂ O dissolves the precipitate to obtain an aqueous solution of genomic DNA. The primers ZmCAD2-ORFF and ZmCAD2-ORFR were used as probes for PCR detection. The primer sequences are as follows:

ZmCAD2-ORFF: CGGCTTTCTTTCCCAACTCC

ZmCAD2-ORFR: CATTAAAAACTGACCATCCATCGT

The PCR reaction system was 50 μL, including 2 μL of forward/reverse primers, 4 μL of DNA template, 0.5 μL of PrimeSATR high-fidelity DNA amplification enzyme, 25 μL of reaction buffer, 4 μL of dNTP, and ddH ₂ O supplemented to 50 μL. The reaction program was as follows: 98°C, 4 minutes pre-denaturation; 98°C denaturation for 10 seconds, 61°C annealing for 5 seconds, 72°C extension for 2 minutes, 34 cycles of reaction; 72°C chain extension for 7 minutes, termination of the reaction at 12°C. After detection, the coding region of ZmCAD2 did not change, and the sequencing results showed that the 307-position nucleic acid was mutated from wild-type thymidine deoxyribonucleotide (T) to adenine deoxyribonucleotide (Figure 2).

To further verify the mutation of this site, we designed molecular probes that can be used to rapidly identify this site. The probe sequences are: ZmCAD2F: AAATCATGGCTTTGGTTTGA; ZmCAD2R: CCTTATCTCGTCCACTTCTCG. The PCR reaction system was 50 μL, including 2 μL of forward/reverse primers, 4 μL of DNA template, 0.5 μL of PrimeSATR high-fidelity DNA amplification enzyme, 25 μL of reaction buffer, 4 μL of dNTP, and ddH ₂ O supplemented to 50 μL. The reaction program was: 98°C, 4 minutes pre-denaturation; 98°C denaturation for 10 seconds, 61°C annealing for 5 seconds, 72°C extension for 1 minute, 34 cycles of reaction; 72°C chain extension for 7 minutes, 12°C termination reaction. Using this probe, a DNA band of 457 bp can be easily and rapidly detected for identification of mutation sites.

Example 2: Detection of CAD enzyme activity of mutants

In order to verify the effect of the above single-base mutation on CAD enzyme activity, we performed artificial site-directed mutagenesis (CADm) at this site of wild type, and expressed wild type CAD and mutant CADm in vitro respectively. Overlapping PCR was used for mutation method, and E. coli system was used for expression of recombinant protein in vitro. All methods used conventional molecular biology techniques.具体操作为：(1)CAD原CDS片段及定点突变片段CADm获得，序列扩增引物：ZmCAD2F1：ATGGGGAGCCTGGCGTCCG；ZmCAD2R1：TCAGTTGCTGGCCGCATCC；ZmCAD2mF1：GCCGCGAGTGCAGCCCCTG；ZmCAD2mR1：CAGGGGCTGCACTCGCGGC；ZmCAD2BamHIF1：CGGGATCCCGATGGGGAGCCTGGCGTCCG；ZmCAD2HindIIIR1:CCA AGCTTGGTCAGTTGCTGGCCGCATCC。 Amplification of CAD original CDS fragments: the first round PCR reaction system is 50 μL, ZmCAD2F1/ZmCAD2R1 each 2 μL, DNA template 4 μL, PrimeSATR high-fidelity DNA amplification enzyme 0.5 μL, reaction buffer 25 μL, dNTP 4 μL, ddH ₂ O supplemented to 50 μL. The reaction program was as follows: 98°C, 4 minutes pre-denaturation; 98°C denaturation for 10 seconds, 58°C annealing for 5 seconds, 72°C extension for 1 minute, 34 cycles of reaction; 72°C chain extension for 7 minutes, termination of the reaction at 12°C. Then use the fragment obtained from the first round of PCR as the template to carry out the second round of PCR, the reaction system is 50 μL, ZmCAD2BamHIF1/ZmCAD2HindIIIR1 each 2 μL, template 4 μL, PrimeSATR high-fidelity DNA amplification enzyme 0.5 μL, reaction buffer 25 μL, dNTP 4 μL, ddH ₂ O Make up to 50 μL. The reaction program was as follows: 98°C, 4 minutes pre-denaturation; 98°C denaturation for 10 seconds, 58°C annealing for 5 seconds, 72°C extension for 1 minute, 34 cycles of reaction; 72°C chain extension for 7 minutes, termination of the reaction at 12°C. Finally, the original CDS fragment of CAD is obtained. Amplification of CAD site-directed mutagenesis fragments: PCR was carried out with ZmCAD2F1/ZmCAD2mR1 and ZmCAD2mF1/ZmCAD2R1 as primers, respectively, PCR reaction system was 50 μL, forward/reverse primers were 2 μL each, DNA template 4 μL, PrimeSATR high-fidelity DNA amplification enzyme 0.5 μL, Reaction buffer 25 μL, dNTP 4 μL, ddH ₂ O supplemented to 50 μL. The reaction program was: 98°C, 4 minutes pre-denaturation; 98°C denaturation for 10 seconds, 58°C annealing for 5 seconds, 72°C extension for 30 seconds, 34 cycles of reaction; 72°C chain extension for 7 minutes, 12°C termination reaction; The fragments obtained from the first PCR were mixed and used as a template. Two rounds of PCR were performed with ZmCAD2F1/ZmCAD2R1 as primers. The PCR reaction system was 50 μL, the forward/reverse primers were 2 μL each, the template was 4 μL, and PrimeSATR high-fidelity DNA amplification enzyme was 0.5 μL. , 25 μL of reaction buffer, 4 μL of dNTP, and 50 μL of ddH ₂ O. The reaction program was as follows: 98°C, 4 minutes pre-denaturation; 98°C denaturation for 10 seconds, 58°C annealing for 5 seconds, 72°C extension for 1 minute, 34 cycles of reaction; 72°C chain extension for 7 minutes, termination of the reaction at 12°C. Finally, the fragment obtained from the second round of PCR was used as the template, and three rounds of PCR were carried out. The reaction system was 50 μL, ZmCAD2BamHIF1/ZmCAD2HindIIIR1 2 μL each, template 4 μL, PrimeSATR high-fidelity DNA amplification enzyme 0.5 μL, reaction buffer 25 μL, dNTP 4 μL, ddH ₂ O make up to 50 μL. The reaction program was as follows: 98°C, 4 minutes pre-denaturation; 98°C denaturation for 10 seconds, 58°C annealing for 5 seconds, 72°C extension for 1 minute, 34 cycles of reaction; 72°C chain extension for 7 minutes, termination of the reaction at 12°C. Finally, the CAD site-directed mutagenesis fragment CADm was obtained. (2) Construction of prokaryotic expression vector: The CAD, CADm fragment and pET32a plasmid were digested with restriction enzymes BamHI and HindIII respectively. The reaction system was 20 μL, CAD fragment/CADm fragment/pET32a plasmid 1 μg, BamHI 1 μL, HindIII 1 μL , the reaction buffer was 2 μL, and ddH ₂ O was supplemented to 20 μL. Incubate at 37°C for 4 hours, then cut the gel for recovery. Then, the digested CAD and CADm fragments were respectively ligated with the digested pET32a plasmid, the reaction system was 10 μL, the digested CAD fragment/CADm fragment 3 μL, the digested pET32a plasmid 1 μL, the reaction buffer 1 μL, T4 ligation Enzyme 1 μL, ddH ₂ O made up to 20 μL. After incubation at 16°C for 2 hours, DH5α was transformed, identified and sequenced. The plasmids with correct sequencing were re-transformed into BL21, identified, and positive single colonies were picked and shaken overnight to be used as seed bacteria solution. (3) To obtain recombinant protein, transfer the inoculated bacterial solution to LB liquid medium at a ratio of 1:100, shake to OD600=0.6-0.8, add 0.1-0.3 mM IPTG, induce at 18°C for about 10-12 hours; collect bacteria by centrifugation The cells were suspended with pre-cooled Lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and the operation was performed on ice; ultrasonically disrupted, ultrasonically on for 5.0 s, and ultrasonically off for 9.0 s, for a total of 30 min; Centrifuge at 10,000 rpm for 30 min; filter the supernatant with a 0.45 μM filter head; load the column: 4°C, add filler, and equilibrate the column filled with Ni-NTA with Lysis buffer; load the filtered protein sample, and pass the sample through the column; Washing buffer (50mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazo0le); eluted with Elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole), and collect the eluted protein, which is the recombinant protein.

Then, we used the recombinant protein obtained above to carry out the in vitro enzyme activity detection test of CAD. The enzymatic reaction system was: 20 μL of phosphoric acid reaction buffer, 1 μL of 2 mM NADPH, 0.4 μL of 5 mM mustardaldehyde, 1 μL of 0.5 mM recombinant protein, and ddH ₂ O was added to 100 μL. Incubate at 30°C for 30 minutes, and add an equal volume of methanol to stop the reaction. After the reaction was terminated, the HPLC method was used for detection. The results showed that the CAD enzyme activity was lost after the mutation of thymidine deoxyribonucleotide at position 307 of the CAD coding region to adenine deoxyribonucleotide (Fig. 3).

Example 3: Detection of mutant lignin content and composition

The lignin content was determined by the acetyl bromide method. The leaf veins of the wild-type and mutant plants at the same leaf position at the heading stage were taken, ground in liquid nitrogen, and dried to extract lignin for analysis. The specific operations are as follows: (1) Treat the sample with methanol: water and chloroform to obtain cell wall residue; (2) Weigh 50 mg of dried cell wall residue powder into a 10 mL test tube, add 5 mL of 25% acetyl bromide, cover with a lid, and take a water bath at 50°C 4 hours; (3) After cooling, centrifuge at 3500rpm for 15 minutes, transfer the supernatant to a 10mL test tube; (4) Prepare a 15mL test tube, first add 2.5mL 2M NaOH and 3mL acetic acid, mix well, then add 1mL of the liquid from the previous step, Mix well; (5) Add 0.25 mL of hydroxylamine, mix well, measure the absorbance at OD ₂₈₀ , and calculate the lignin content. The test results showed that the lignin content in the veins of the cad mutant was not significantly different from that of the wild type (A in Figure 4).

The lignin composition was detected using the Thioacidolysis method. The specific operations are: (1) Prepare a lysis solution, Dioxan: Bron Triflorate Ethyl Etherate: Ethernethiol=35:1:4; (2) Weigh 50 mg of the cell wall residue powder into a sample tube, add 3 mL of the lysis solution, 80°C, 4 hours ; (3) Add 100 μL of 2.5 mg/mL behenate as the internal standard in each sample tube, then add 0.8 mL of saturated NaHCO ₃ and 3 mL of dichloromethane, mix well, centrifuge at 2900 rpm for 10 minutes, extract the lower layer to a glass tube, and repeat Add 3 mL of dichloromethane, centrifuge, and extract the lower layer; (4) Add Na ₂ SO 4 until the solution is clear; (5) Protect from light, vacuum dry with nitrogen, add 40 μL pyridine, 120 μL MSTFA, centrifuge at 2900 rpm for 1 minute, and incubate at 37 °C Half an hour, GC/MS detection. The test results showed that the H-type, G-type and S-type lignin monomers in the leaf veins of the cad mutant were significantly decreased compared with the wild type (B in Figure 4).

Example 4: Assessment of cell wall digestibility of mutants

To evaluate the cell wall digestibility of cad mutants, we examined cell wall cellulose content, enzymatic sugar content, and cell wall saccharification efficiency. First, the veins of maize leaves at the growth and heading stage were taken, dried at 40°C for 96 hours, ground and pulverized for testing. The test method is the phenol sulfate method [Dubois et al, Colorimetric method for determination of sugars and related substances. 1956, Analytical Chemistry, 28: 350-356], and the specific determination method is as follows: using a mixture of cellulase and cellobiase to directly enzymatic Cell wall residues were hydrolyzed for 72 h as a control; pretreated with 1.5% H ₂ SO ₄ at 121 °C for 60 min, and then the same amount of cellulase and cellobiase mixture was used to hydrolyze cell wall residues for 72 h as processing group. The fermentable sugar content of the enzymatic hydrolysate was determined using the phenol sulfate method. Saccharification efficiency is the ratio of the difference between the fermentable sugar content before and after enzymatic hydrolysis to the fermentable sugar content before enzymatic hydrolysis. Therefore, according to the formula: fermentable sugar yield (g/plant)=aerial cell wall carbohydrate yield (g/plant)×saccharification efficiency, the fermentable sugar yield of the transgenic plants was calculated. The assay results showed that there was little difference in cellulose content in the cad mutants, and the saccharification efficiency and enzymatic glycolysis increased significantly by 29.5% and 31.2%, respectively (Fig. 5).

sequence listing

<110> Qilu Teachers College

Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences

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Claims (2)

1. a method for identifying a CAD mutant utilizing a primer of a molecular marker relevant to maize cell wall digestibility, the primer sequence is ZmCAD2F: AAATCATGGCTTTGGTTTGA; ZmCAD2R: CCTTATCTCGTCCACTTCTCG; the molecular marker is the maize wild-type CAD gene encoding A single base mutation in the region, the 307th base was mutated from wild-type T to A, and the cell wall digestibility of the CAD mutant plant was improved; The PCR reaction system of the method is 50 μL, in which forward/reverse primers are 2 μL each, DNA template 4 μL, PrimeSATR high-fidelity DNA amplification enzyme 0.5 μL, reaction buffer 25 μL, dNTP 4 μL, ddH ₂ O supplemented to 50 μL; the reaction program is: 98 ^o C, 4 minutes pre-denaturation; 98 ^o C denaturation for 10 seconds, 61 ^o C annealing for 5 seconds, 72 ^o C extension for 2 minutes, reaction 34 cycles; 72 ^o C chain extension for 7 minutes, The reaction was terminated at 12 ^oC , and the amplified DNA band was 457 bp. 2. the application of the primer of a molecular marker that utilizes the molecular marker relevant to maize cell wall digestibility in the cultivation of corn high cell wall digestibility varieties, the primer sequence is ZmCAD2F: AAATCATGGCTTGGTTTGA; ZmCAD2R: CCTTATCTCGTCCACTTCTCG; the molecular marker is maize wild-type A single base mutation in the coding region of the CAD gene, the base at the 307th position is mutated from wild-type T to A, and the cell wall digestibility of the CAD mutant plant is improved; the high refers to the 307th position of claim 1. The cell wall digestibility of the mutant plant obtained after the base is mutated from wild-type T to A is improved, and the mutant plant is called a corn high cell wall digestibility variety.

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