ZmDi19-7 protein and application of ZmDi19-7 protein in regulation and control of corn kernel and plant height

Abstract

The invention discloses a ZmDi19-7 protein, the amino acid sequence of the ZmDi19-7 protein is shown as SEQ ID NO.1. A substance expressed by the ZmDi19-7 gene can regulate and control the length, the width, the hundred-grain weight and the size of plant starch grains, and the ZmDi19-7 gene has important guiding significance on regulation and control of development of corn grains, improvement of the corn grains and breeding of high-yield varieties. The gene expressed substance can regulate and control the plant height, the plant stem node number, the plant stem node length and the plant cell length, and has important guiding significance on regulation and control of corn plant height development, corn plant height improvement and density-tolerant variety breeding. The corn breeding work is carried out by applying a biotechnology, the variation speed of genetic genes can be accelerated, the breeding period is shortened to a great extent, and the breeding of new varieties is accelerated.

Description

Technical Field

[0001] The invention relates to a ZmDi19-7 protein and application thereof in regulating corn grains and plant height, relates to C12N, and specifically relates to the field of microorganisms and compositions thereof. Background Art

[0002] Corn is one of the crops with the highest total yield. It is an important food crop and feed crop with great production potential and high economic benefits. The existing method to increase grain yield is to increase the planting density of corn. However, under dense planting conditions, the internodes of corn become elongated and thinner, the plant height increases, and the lodging resistance is poor. It is easy to suffer from lodging disasters during the corn maturity season, resulting in reduced yield. Therefore, it is very important to reasonably reduce plant height, improve the lodging resistance of corn, and improve the photosynthetic efficiency of the group, thereby promoting dense planting and increased yield. In addition, the yield of corn can be increased by improving the size of corn kernels, the weight of 100 kernels, and the starch content of the kernels, which has important guiding significance for the cultivation of new high-yield corn varieties.

[0003] Chinese invention patent CN112941087A discloses an application of corn ZmBES1 / BZR1-2 gene in improving plant drought tolerance. By isolating and cloning ZmBES1 / BZR1-2 gene, plant drought tolerance can be improved, providing ideas for cultivating drought-resistant and salt-resistant transgenic plants, which is of great significance to crop drought-resistant breeding and production applications. However, it does not involve the yield-increasing effect on corn. Chinese invention patent CN110157713A discloses a corn drought-resistant related gene ZmDi19-7 and its application. The corn drought-resistant related gene ZmDi19-7 is integrated into the cells, tissues and organs of the plant and overexpressed, which can improve the drought resistance of the plant, but it does not involve the yield-increasing effect of the ZmDi19-7 gene on corn. Summary of the invention

[0004] In order to improve the kernel performance of corn and optimize the plant height effect of corn, the first aspect of the present invention provides a ZmDi19-7 protein, the amino acid sequence of the ZmDi19-7 protein is shown in SEQ ID NO.1.

[0005] ZmDi19-7 amino acid sequence SEQ ID NO.1:

[0006] MDSEHWISRLAAAKRFYAAQLGHSDRAGMDELEMDEEVRPEFPCPYCYEDH

[0007] DVGSLCAHLEEEHPFEPQAAACPVCSEMVTKDMVNHITTQHGYLFKNRRRL

[0008] RRFIIPGSQALSLLSRDLREAHLQVLLGGGGQRSSDNSSSSSATNISADPLLSSF

[0009] GLGFPTSDAEQASKSTVSIPDDATTVKEAPAQARKLSIDSSLTSEERELKRKQARVRATFVQDLLLSTLFGD*.

[0010] As a preferred embodiment, the nucleic acid sequence of the ZmDi19-7 amino acid is shown as SEQ ID NO.2.

[0011] The nucleic acid sequence of ZmDi19-7 amino acid is SEQ ID NO.2:

[0012] ATGGACTCCGAGCACTGGATCTCGCGCCTGGCCGCCGCGAAGCGCTTCTAC

[0013] GCGGCGCAGCTCGGCCACAGCGATCGGGCCGGGATGGATGAGCTGGAGAT

[0014] GGACGAGGAGGTCAGGCCCGAGTTCCCCTGCCCCTACTGCTACGAGGACC

[0015] ACGACGTCGGATCCCTCTGCGCGCACCTGGAGGAGGAGCACCCGTTCGAA

[0016] CCCCAAGCCGCGGCCTGCCCTGTCTGCTCAGAGATGGTTACGAAGGATATG

[0017] GTTAACCATATTACTACGCAACATGGATATTTATTCAAGAATCGCCGCCGGC

[0018] TGCGCAGATTCATCATTCCAGGCAGCCAGGCCCTCTCTTTGCTGAGCCGAG

[0019] ATCTACGGGAAGCCCACTTGCAGGTGCTCCTCGGAGGAGGCGGACAGAGA

[0020] TCCAGCGACAACAGCAGCAGCAGCAGCGCCACGAACATTTCGGCTGATCC

[0021] TCTTCTGTCGTCGTTCGGCCTTGGCTTCCCTACCTCAGACGCGGAGCAAGC

[0022] ATCCAAGTCGACTGTTTCCATTCCTGATGATGCAACGACGGTGAAAGAAGC

[0023] GCCCGCTCAGGCACGGAAGCTAAGTATCGATTCGTCGCTCACAAGTGAAG

[0024] AAAGGGAGCTGAAGCGGAAGCAAGCCCGCGTCAGAGCCACGTTCGTGCAGGACCTGCTGCTCTCTACTCTATTCGGCGACTGA。

[0025] As a preferred embodiment, the genomic sequence of the ZmDi19-7 nucleic acid is as shown in SEQ ID NO.3.

[0026] Genomic sequence of ZmDi19-7 nucleic acid SEQ ID NO.3:

[0027] ATGGACTCCGAGCACTGGATCTCGCGCCTGGCCGCCGCGAAGCGCTTCTAC

[0028] GCGGCGCAGCTCGGCCACAGCGGTGCGGCTACCGCGCTCCCTCCAGCCCC

[0029] GTGTTCCGTTCCGCTGGGTCTGCCTTCGTGGCGGCGTGGTTTCTGACTGCT

[0030] TCACCCTGGGCTTGCTTTTGCGGGCGCAGATCGGGCCGGGATGGATGAGCT

[0031] GGAGATGGACGAGGAGGTCAGGCCCGAGTTCCCCTGCCCCTACTGCTACG

[0032] AGGACCACGACGTCGGATCCCTCTGCGCGCACCTGGAGGAGGAGCACCCG

[0033] TTCGAACCCCAAGCCGCGGTGAGTGCCCCCCCACCGGACGCCTTAGGCCC

[0034] TGTTTGGCACAGCTGCTGCTGGTTGAAAAAGCAGCTTATCTGATAAGCTGG

[0035] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0036] CTGAACTAATAAGCTGCTGGAGAAGCTAGCTGTTTGGCAAAACTTCTGCTT

[0037] AAATTTGTGAAGAAGCTGAAAATAAGCTGTGCCAAACAGGGCCTTAGTCT

[0038] GGTGTGTTTTGCCCTCCTATGAATTGCTAGAGAGGATTTAGCGAATTTGTGC

[0039] TTGGTTGACCTGGGAACTGACCACATGATGCTAGTCGTGTCGGCGCGGGTG

[0040] CTTGATCATATGGGTTATATACCTTGTTCTGATTGGAATTTGGAACTAGCAGC

[0041] TCGTTGGCGTTGACTTTTGAAGAACGAGATTGTTCTTCTTTTACACTTTTCG

[0042] GGATCTATTGGACAGGAGAAGTATGGGAAAGATGGTACAATTTGATTACTT

[0043] GAACTATCCTTTTAGTCTGCCTAAATGAAACGAGAGTTTTGGGAAATGAATA

[0044] TATGACAACTTGGAGCTTCTCGGAGATGCAAAGTGAAAGCTAATCTATCAT

[0045] ACGTCAGTGTTATAAGCCTGTGGCTGGGGTTTTTGTCAATATACAACCATGG

[0046] ACTGAAGTCTGCTACAAGAAAGCTTACAAATAGGTATCTATGAGAAGAAAC

[0047] GATTCAAGTTTGATCCGAAGAATGAACATCGCTGCCTTCGGACTAGTGAAC

[0048] TGGAACATCAGCTTTGGAAGGAACCCAGTAACTCAGTATCAGGGGTAGAA

[0049] CTTCTAGCTTATCCTAAGTTGTAGATTTAAGAGCTTAGTTGTCTCCAATGTTT

[0050] ATGGTATCTGCTGCTGCAAGTTGATTTGAGGACCCTTTCTCTTCGCCTCAGT

[0051] TGAAACGATCTTTTGCATAAGCTATAAGCCTATAGCATGGACGAAACTCACC

[0052] TTAACGATGTCGTTGCATTTATGCCCATCTTGCTGGTTACTTTATTCCTGCCC

[0053] TGATTTTTTGCACTGCCCATTCCAGGCCTGCCCTGTCTGCTCAGAGATGGTT

[0054] ACGAAGGATATGGTTAACCATATTACTACGCAACATGGATATTTATTCAAGGT

[0055] ATTCTCACACCGTGGACATGTGAATTCGTGGTTCTTGACCTCGCTCACCAA

[0056] GTCACCATTTGATGCATACGAGTGTAGGCTTACAGTTTATGAAGTGCTTTTT

[0057] TCTCTCCCTTGAAACTCATCCGCCTTCTGCCCTCCCGCAGAATCGCCGCCG

[0058] GCTGCGCAGATTCATCATTCCAGGCAGCCAGGCCCTCTCTTTGCTGAGCCG

[0059] AGATCTACGGGAAGCCCACTTGCAGGTGCTCCTCGGAGGAGGCGGACAGA

[0060] GATCCAGCGACAACAGCAGCAGCAGCAGCGCCACGAACATTTCGGCTGAT

[0061] CCTCTTCTGTCGTCGTTCGGCCTTGGCTTCCCTACCTCAGACGCGGAGCAA

[0062] GCATCCAAGTCGACTGTTTCCATTCCTGATGATGCAACGACGGTGAAAGAA

[0063] GCGCCCGCTCAGGCACGGAAGCTAAGGTATATTTTATACGCATCCATGCTAA

[0064] AATGAAACGCTAGTGAGTCGACAGGCTTAACAACCACATCGTTTTTTCGCC

[0065] CAGTCGTGGCGGACACCATAGTCTTCATGTTACTTTCTGGACTACTAGTTTA

[0066] GAGTCGGGCATCTGAACTGTCTCGTGTGACAGGGAAATCTTATATGTGGCC

[0067] ACCATGCAAATCTGAGCCAGTAGTTCTCGTAGTCTTGTAGACTAGATAGCTC

[0068] GTTTTGTTTACCCGTTCGGAAAGATGCCTCCTGTTGAATAATAAAGTCTTCC

[0069] AACCAGCAACTTGGTTTCATTGAAAAAATGATGAGCAGTGTAGTATCAAAC

[0070] TTCCTCTTCGCCCTTCTGCAACTTGCTGTTTTGTAAAACGCATAGATGAAGC

[0071] AGGCTGTCCACGCGACCATTAAACCCTGCTCATCTTACCAATTGATTGTACA

[0072] CACAACACAGTTCAGTCATCAGATGTGTTCGTTTTTTTTTGTGGCACAGTAT

[0073] CGATTCGTCGCTCACAAGTGAAGAAAGGGAGCTGAAGCGGAAGCAAGCC

[0074] CGCGTCAGAGCCACGTTCGTGCAGGACCTGCTGCTCTCTACTCTATTCGGCGACTGA.

[0075] As a preferred embodiment, the method for extracting ZmDi19-7 nucleic acid comprises the following steps:

[0076] S1 Obtain plant material;

[0077] S2 extracts RNA;

[0078] S3 reverse transcription;

[0079] S4PCR reaction.

[0080] As a preferred embodiment, the specific steps of obtaining plant materials in step S1 are as follows: select inbred line B73 corn plant materials, select corn seeds with uniform grain size, sow them in nutrient soil, and when they grow to the three-leaf to four-leaf stage, take corn leaves for RNA extraction.

[0081] As a preferred embodiment, the method for extracting RNA in step S2 comprises the following steps:

[0082] M1 used the Trizol method to extract total RNA. The mortar and grinding rod were sterilized and inactivated by high temperature heating. The corn leaf samples were ground into powder by precooling with liquid nitrogen. The powder was transferred to a 2 mL RNAfree EP tube, and 1 mL of Trizol extraction solution was added. The tube was placed on a vortex shaker and thoroughly shaken and mixed. The tube was placed on ice in the dark for 10 min.

[0083] M2 was added with 500 μL RNA nucleic acid extract (the mass ratio of RNA nucleic acid extract to sample powder was 24:1), vortexed for 10 seconds to mix, and placed on ice for 10 minutes to extract impurities;

[0084] M3 was centrifuged in a high-speed tabletop refrigerated centrifuge at 12,000 × g at 4°C for 10 min;

[0085] M4: Transfer 600 μL of the supernatant to a new 1.5 mL RNA free EP tube, add an equal volume of isopropanol, invert repeatedly to mix, and place on ice for 10 min;

[0086] M5 was centrifuged in a high-speed tabletop refrigerated centrifuge at 12,000 × g at 4°C for 10 min;

[0087] Discard the supernatant of M6, add 1 mL of 75 vol% ethanol solution (prepared with DEPC water), and mix well;

[0088] M7 was centrifuged at 12,000 × g for 3 min at 4°C using a high-speed desktop refrigerated centrifuge;

[0089] Repeat steps S6 and S7 for M8, centrifuge at 12,000 × g and 4°C for 3 min using a high-speed desktop refrigerated centrifuge;

[0090] M9 used 50 μL DEPC water to dissolve the RNA precipitate;

[0091] M10 measures RNA concentration in ng / uL and OD 260 / 280 values, and uses nucleic acid gel electrophoresis to detect RNA quality.

[0092] As a preferred embodiment, the reverse transcription in step S3 is performed using a kit. The reverse transcription reaction system is as follows:

[0093] Components volume 5×Evo M-MLV RT Master Mix 2μL Total RNA 1μL RNase free water 7μL

[0094] After adding all reagents, use a low-speed centrifuge at 200 rpm for 10-15 seconds to fully mix the reagent components.

[0095] As a preferred embodiment, the reverse transcription procedure in step S3 is as follows: react the mixed reagent at 35-37°C for 15-20 min, and cycle 1-3 times; then react at 80-85°C for 3-8s, and cycle 1-3 times; finally, react at 4°C for 1-2 cycles and store for later use.

[0096] As a preferred embodiment, the reverse transcription procedure in step S3 is as follows: react the mixed reagent at 37°C for 15 minutes, and cycle once; then react at 85°C for 5 seconds, and cycle once; finally, react at 4°C, and cycle once. After the reaction, the product is stored at -20°C or in an ice box for use.

[0097] As a preferred embodiment, the specific primers for ZmDi19-7 nucleic acid used in the PCR reaction in step S4 include:

[0098] ZmDi19-7-F: ATGGACTCCGAGCACTGGATCT and

[0099] ZmDi19-7-R: TCAGTCGCCGAATAGAGTAGAG.

[0100] As a preferred embodiment, the PCR reaction system in step S4 is as follows:

[0101] Components volume Primer star Mix 12.5μL Upstream primer (10 μM) 1μL Downstream primer (10 μM) 1μL Template (cDNA) 1μL RNA free water 9.5μL

[0102] As a preferred embodiment, the procedure of the PCR reaction in step S4 includes: pre-denaturation at 95-98°C, 10-15min, 1-3 cycles; denaturation at 95-98°C, 10-15s; annealing at 55-58°C, 3-8s, 35-40 cycles; extension at 70-75°C, 1-3s; total extension at 70-75°C, 10-15min, 1-3 cycles; storage at 3-5°C, 1-2 cycles, for standby use.

[0103] As a preferred embodiment, the procedure of the PCR reaction in step S4 includes: pre-denaturation at 98°C, 10 min, 1 cycle; denaturation at 98°C, 10 s; annealing at 58°C, 5 s, 35 cycles; extension at 72°C, 1 s; total extension at 72°C, 10 min, 1 cycle; storage at 4°C, 1 cycle, and standby.

[0104] The second aspect of the present invention provides an application of ZmDi19-7 protein in regulating corn grains.

[0105] The third aspect of the present invention provides an application of ZmDi19-7 protein in regulating the growth of corn plants.

[0106] Compared with the prior art, the present invention has the following beneficial effects:

[0107] (1) The ZmDi19-7 protein and the gene expressed by the present invention can regulate plant grain length, grain width, 100-grain weight, and plant starch granule size, and have important guiding significance for regulating the development of corn grains, corn grain improvement, and high-yield variety breeding.

[0108] (2) The ZmDi19-7 protein and the gene expressed by the present invention can regulate plant height, the number of plant nodes, the length of plant nodes, and the length of plant cells, and have important guiding significance for regulating the development of corn plant height, improving corn plant height, and breeding dense-resistant varieties.

[0109] (3) The ZmDi19-7 protein of the present invention can accelerate the mutation rate of genetic genes by applying biotechnology to corn breeding, shorten the breeding cycle to a great extent, and accelerate the cultivation of new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1The structure diagram of the knockout maize ZmDi19-7 gene (A); RT-qPCR of the knockout maize ZmDi19-7 gene (B) and semi-RT-qPCR results of the knockout maize ZmDi19-7 gene (C).

[0111] Figure 2 Comparison of grains of wild-type KN5585 and ZmDi19-7 knockout mutant plants after grain maturity in the breeding base: grain shape comparison (A); grain length and width comparison [actual (B) length value (D); width value (E)]; grain thickness comparison [actual (C) thickness value (F)]; 100-grain weight comparison value (G).

[0112] Figure 3 These are optical microscope observations of the grains, embryos, and endosperms 10, 15, and 20 days after pollination; actual pictures (A) numerical line graph of grain weight changes (B) numerical line graph of embryo weight changes (C) numerical line graph of endosperm weight changes (D).

[0113] Figure 4 These are paraffin section observations of the kernels, embryos, and endosperms 10, 15, and 20 days after pollination.

[0114] Figure 5 Scanning electron microscopy observations of the longitudinal sections of grains of wild-type KN5585 and ZmDi19-7 gene knockout mutant plants in the breeding base (A, electron microscopy images B and C); statistical bar graph of starch granule size per unit area of ​​view (D); statistical bar graph of starch granule number per unit area of ​​view (E).

[0115] Figure 6 RT-qPCR experiments were used to detect 12 key genes related to starch synthesis and their regulatory expression.

[0116] Figure 7 Real-time fluorescence quantitative PCR technology (RT-qPCR) was used to detect the expression level of gene ZmDi19-7 in various tissues.

[0117] Figure 8 The expression of maize ZmDi19-7 gene in stem nodes (C, D); stem apical meristem (E); embryos 25, 30, 35, and 40 days after pollination (A) (the red arrow refers to the embryo of the grain, and the part outside the arrow refers to the endosperm of the grain except the grain epidermis); leaves (B); after in situ hybridization.

[0118] Fig. 9 This is a comparison chart of plant height and stem traits between different transgenic materials and the wild type.

[0119] Fig.10The figures are a comparison of the plant height, number of nodes, and length of nodes of different transgenic materials and the wild type; plant height (A); number of nodes (B), and node length (C).

[0120] Fig.11 Paraffin sections of cells in cross section and longitudinal section of the second stem node at the ear position of different transgenic materials and wild-type plants were observed under a Leica optical microscope. Actual image (A), cell area (B), cell length (C).

[0121] Fig.12 This is the effect of ZmDi19-7 gene on stem length of different transgenic materials and wild-type plants. DETAILED DESCRIPTION

[0122] Example 1

[0123] A ZmDi19-7 protein, the amino acid sequence of the ZmDi19-7 protein is shown in SEQ ID NO.1.

[0124] The nucleic acid sequence of the amino acid ZmDi19-7 is shown in SEQ ID NO.2.

[0125] The genomic sequence of the ZmDi19-7 nucleic acid is shown in SEQ ID NO.3.

[0126] The method for extracting the ZmDi19-7 nucleic acid comprises the following steps:

[0127] S1 Obtain plant material;

[0128] S2 extracts RNA;

[0129] S3 reverse transcription;

[0130] S4PCR reaction.

[0131] The specific steps of obtaining plant materials in step S1 are as follows: select inbred line B73 corn plant materials, select corn seeds with uniform grain size, sow them in nutrient soil, and when they grow to the three-leaf to four-leaf stage, take corn leaves for RNA extraction.

[0132] The method for extracting RNA in step S2 comprises the following steps:

[0133] M1 used the Trizol method to extract total RNA. The mortar and grinding rod were sterilized and inactivated by high temperature heating. The corn leaf samples were ground into powder by precooling with liquid nitrogen. The powder was transferred to a 2mL RNA free EP tube, and 1mL Trizol extraction solution was added. The tube was placed in a vortex shaker and fully shaken and mixed. The tube was placed on ice in the dark for 10 minutes.

[0134] M2 was added with 500 μL RNA nucleic acid extract (the mass ratio of RNA nucleic acid extract to sample powder was 24:1), vortexed for 10 seconds to mix, and placed on ice for 10 minutes to extract impurities;

[0135] M3 was centrifuged in a high-speed tabletop refrigerated centrifuge at 12,000 × g at 4°C for 10 min;

[0136] M4: Transfer 600 μL of the supernatant to a new 1.5 mL RNA free EP tube, add an equal volume of isopropanol, invert repeatedly to mix, and place on ice for 10 min;

[0137] M5 was centrifuged in a high-speed tabletop refrigerated centrifuge at 12,000 × g at 4°C for 10 min;

[0138] Discard the supernatant of M6, add 1 mL of 75 vol% ethanol solution (prepared with DEPC water), and mix well;

[0139] M7 was centrifuged at 12,000 × g for 3 min at 4°C using a high-speed desktop refrigerated centrifuge;

[0140] Repeat steps S6 and S7 for M8, centrifuge at 12,000 × g and 4°C for 3 min using a high-speed desktop refrigerated centrifuge;

[0141] M9 used 50 μL DEPC water to dissolve RNA precipitate;

[0142] M10 measures RNA concentration in ng / uL and OD 260 / 280 values, and uses nucleic acid gel electrophoresis to detect RNA quality.

[0143] In step S3, the reverse transcription is performed using a kit. The reverse transcription reaction system is as follows:

[0144] Components volume 5×Evo M-MLV RT Master Mix 2μL Total RNA 1μL RNase free water 7μL

[0145] After adding all reagents, use a low-speed centrifuge at 200 rpm for 10-15 seconds to fully mix the reagent components.

[0146] The reverse transcription procedure in step S3 is as follows: react the mixed reagent at 37°C for 15 minutes, for one cycle; then react at 85°C for 5 seconds, for one cycle; finally, react at 4°C for one cycle. After the reaction is completed, the product is stored at -20°C or in an ice box for later use.

[0147] The specific primers for the ZmDi19-7 nucleic acid used in the PCR reaction in step S4 include: ZmDi19-7-F: ATGGACTCCGAGCACTGGATCT and

[0148] ZmDi19-7-R: TCAGTCGCCGAATAGAGTAGAG.

[0149] The PCR reaction system in step S4 is as follows:

[0150] Components volume Primer star Mix 12.5μL Upstream primer (10 μM) 1μL Downstream primer (10 μM) 1μL Template (cDNA) 1μL RNA free water 9.5μL

[0151] The procedure of the PCR reaction in step S4 includes: pre-denaturation at 98°C, 10 min, 1 cycle; denaturation at 98°C, 10 s; annealing at 58°C, 5 s, 35 cycles; extension at 72°C, 1 s; total extension at 72°C, 10 min, 1 cycle; storage at 4°C, 1 cycle, and standby.

[0152] Application of ZmDi19-7 protein in regulating corn grains.

[0153] The specific implementation plan for creating a ZmDi19-7 gene editing knockout mutant of maize is as follows:

[0154] 1.1 Design of target site for gene editing knockout mutant of maize ZmDi19-7:

[0155] The CRISPR / Cas9 basic vector (pCXB-053-CCDB vector) used in the present invention was donated by Weimi Biotechnology (Jiangsu) Co., Ltd. Two targets were selected on the ZmDi19-7 protein domain sequence, and an expression cassette of dual-target sgRNA and Cas9 protein was constructed, and inserted into the binary expression vector pCXB-053-CCDB to complete the construction of the CRISPR / Cas9 knockout vector. The two sgRNAs targeting ZmDi19-7 are sgRNA1 and sgRNA2. The nucleotide sequence of the sgRNA1 target is 5'-AGTGGGCTTCCCGTAGATCT-3', targeting the 1643-1662th position of sequence 3 in the sequence table of the ZmDi19-7 gene; the nucleotide sequence of the sgRNA2 target is 5'-TGGCTTCCCTACCTCAGACG-3', targeting the 1766-1785th nucleotides of sequence 3 in the sequence table.

[0156] 1.2 Construction of maize ZmDi19-7 gene editing knockout mutant vector

[0157] The CCDB sequence in the pCXB-053-CCDB vector was cut off by BSAI digestion, and two independent single-target vectors were first constructed by T4 ligase, and then sgRNA2 was connected by recombinase to form a dual-target knockout vector of ZmDi19-7-sgRNA1-srRNA2; specific steps: 1.2.1 Synthesize primers, dissolve them in ultrapure water and mix them evenly; 1.2.2 Prepare annealing buffer (Tris-HCl 10.0mM, pH8.0, EDTA 0.1mM, NaCl: 50.0mM); 1.2.3 Annealing and connection, annealing buffer 50.0μL, primer mixture 5.0μL, (95℃, 3min, cool to 16℃).

[0158] 1.3 Genetic transformation of maize ZmDi19-7 gene editing knockout mutant (completed by Weimi Biotechnology (Jiangsu) Co., Ltd.)

[0159] Select ZmDi19-7 from the ZmDi19-7 knockout strain #KO1 and ZmDi19-7 #KO2 For homozygous strains, when the plants grow to six or seven leaves, RNA is extracted from leaves respectively, using the above-mentioned RNA extraction method, and then a certain concentration of cDNA is obtained through the above-mentioned reverse transcription method.

[0160] Then, based on the sequence information of gene ZmDi19-7 (Zm00001d038999), specific quantitative primers were designed, and the following two pairs of primers were selected with reference to the sequence of the knockout strain:

[0161] ZmDi19-7-RT:AGATCTACGGGAAGCCCACT

[0162] ZmDi19-7-RT:AGTCGACTTGGATGCTTGCT

[0163] Then conduct performance testing.

[0164] Example 2

[0165] A ZmDi19-7 protein, wherein the amino acid sequence, nucleic acid sequence and genome sequence of the ZmDi19-7 protein are the same as those in Example 1, and the method for extracting the ZmDi19-7 nucleic acid is the same as that in Example 1. The difference is:

[0166] Application of ZmDi19-7 protein in regulating the growth of corn plants.

[0167] 2.1. Analysis of tissue expression pattern of maize ZmDi19-7 gene:

[0168] According to the sequence information of gene ZmDi19-7, specific quantitative primers were designed, with a primer length of 20 bp, a Tm value of 57.0℃-60℃, and a GC% content of 40%-60%, and the sequence was obtained: forward primer: GACTGTTTCCATTCCTGATG; reverse primer: TCGCCGAATAGAGTAGAGAG. Roots (Root-V4), stems (Stem-V4), leaves (Leaf-V4) in the V4 period, bracts (Bract-V17), ear (Ear-V17), silk (Silk-V17) in the V17 period, pollens (Pollen-R1) in the R1 period, and embryos (Embryo-30 DAP) and endosperms (Endosperm-30 DAP) 30 days after pollination were collected, and the expression level of gene ZmDi19-7 in various tissues was detected by real-time fluorescence quantitative PCR (RT-qPCR). Figure 7 ).

[0169] 2.2. In situ hybridization of maize ZmDi19-7 gene

[0170] To further analyze the distribution of ZmDi19-7 gene in different tissues, we obtained the stem nodes, stem apical meristems and leaf tissues of maize B73 inbred line V4. In addition, when it came to the pollination period, we selected corn ears 25, 30, 35 and 40 days after pollination, and carefully took out the kernels and put them into FAA fixative (50wt% ethanol, 5wt% acetic acid, 3.7wt% methanol) for in situ hybridization experiments.

[0171] 2.2.1 The in situ hybridization experimental method is as follows:

[0172] The experimental materials were taken from B73 inbred lines at different growth stages or different growth tissues, including shoot apical meristem (SAM) 17 days after corn sowing, stalks at the four-leaf stage, leaves after pollination, and grains 25 days, 30 days, 35 days, and 40 days after pollination. The above samples were fixed in FAA fixative containing 50wt% ethanol, 5%wt acetic acid, and 37wt% formaldehyde. With reference to the full-length CDS sequence of the ZmDi19-7 gene, (GCAACATGGATATTTATTCAAGAATCGCCGCCG) sense strand and (CGGCGGCGATTCTTGAATAAATATCCATGTTGC) antisense strand RNA probes were designed. The in situ hybridization experiment was performed by Wuhan Seville Biotechnology Co., Ltd. The in situ hybridization of ZmDi19-7 used sense and antisense probes, with sense probes as controls.

[0173] 2.3 Creation of transgenic plants overexpressing ZmDi19-7, the specific implementation plan is as follows:

[0174] 2.3.1 Construction of a vector for overexpressing the ZmDi19-7 gene;

[0175] 2.3.2 The vector overexpressing the ZmDi19-7 gene was used for genetic transformation of corn (completed by Weimi Biotechnology (Jiangsu) Co., Ltd.).

[0176] 2.4 Creation of gene editing knockout mutant of maize ZmDi19-7, the specific implementation plan is as follows:

[0177] 2.4.1 Target design of ZmDi19-7 gene editing knockout mutant in maize

[0178] The CRISPR / Cas9 basic vector (pCXB-053-CCDB vector) used in the present invention was donated by Weimi Biotechnology (Jiangsu) Co., Ltd. Two targets were selected on the ZmDi19-7 protein domain sequence, and an expression cassette of dual-target sgRNA and Cas9 protein was constructed, and inserted into the binary expression vector pCXB-053-CCDB to complete the construction of the CRISPR / Cas9 knockout vector. The two sgRNAs targeting ZmDi19-7 are sgRNA1 and sgRNA2. The nucleotide sequence of the sgRNA1 target is 5'-AGTGGGCTTCCCGTAGATCT-3', targeting the 1643-1662th position of sequence 3 in the sequence table of the ZmDi19-7 gene; the nucleotide sequence of the sgRNA2 target is 5'-TGGCTTCCCTACCTCAGACG-3', targeting the 1766-1785th nucleotides of sequence 3 in the sequence table.

[0179] 2.4.2. Construction of gene editing knockout mutant vector for maize ZmDi19-7

[0180] The CCDB sequence in the pCXB-053-CCDB vector was cut off by BSAI digestion, and two independent single-target vectors were first constructed by T4 ligase, and then sgRNA2 was connected by recombinase to form a dual-target knockout vector of ZmDi19-7-sgRNA1-srRNA2; specific steps: 1) synthesize primers, dissolve them in ultrapure water and mix them evenly; 2) prepare annealing buffer (Tris-HCl 10.0mM, PH8.0, EDTA 0.1mM, NaCl: 50.0mM); 3) annealing and connection, annealing buffer 50.0μL, primer mixture 5.0μL, (95℃, 3min, cool to 16℃).

[0181] 2.4.3 Genetic transformation of maize ZmDi19-7 gene editing knockout mutant (completed by Weimi Biotechnology (Jiangsu) Co., Ltd.)

[0182] 2.4.4 Detection of the expression level of the maize ZmDi19-7 gene in transgenic overexpression lines and knockout lines

[0183] Select ZmDi19-7 from the transgenic overexpression strain of gene ZmDi19-7 #OE3 Homozygous positive plant lines, and then select ZmDi19-7 from the knockout lines #KO1 and ZmDi19-7 #KO2 For homozygous strains, when the plants grow to six or seven leaves, RNA is extracted from leaves respectively, using the above-mentioned RNA extraction method, and then a certain concentration of cDNA is obtained through the above-mentioned reverse transcription method.

[0184] Then, based on the sequence information of gene ZmDi19-7 (Zm00001d038999), specific quantitative primers were designed, and the following two pairs of primers were selected with reference to the sequence of the knockout strain:

[0185] ZmDi19-7-RT:AGATCTACGGGAAGCCCACT

[0186] ZmDi19-7-RT:AGTCGACTTGGATGCTTGCT

[0187] Then conduct performance testing.

[0188] Performance Testing

[0189] For the performance test of Example 1:

[0190] 1.1 Wild-type corn and ZmDi19-7 gene-edited knockout mutant corn were subjected to real-time fluorescence quantitative PCR technology to knock out the corn ZmDi19-7 gene structure (such as Figure 1 A), the test results show that compared with the wild type, the expression level of gene ZmDi19-7 in the knockout strain is below 0.5, or even lower (such as Figure 1 B), and we again verified our knockout strains and wild type by semi-quantitative methods, and the results were consistent with those of real-time fluorescence quantitative PCR (e.g. Figure 1 C), indicating that knockout mutants can be used to verify biological functions.

[0191] 1.2 Analysis of grain phenotype of ZmDi19-7 gene knockout mutant:

[0192] 1.2.1 The wild-type KN5585 and ZmDi19-7 knockout mutant plants were planted in the Nongcuiyuan Maize Molecular Breeding Base of Anhui Agricultural University in Hefei in spring. After the kernels matured, the corn ears of each strain were harvested and dried to constant weight. The kernels were observed and statistically analyzed (e.g. Figure 2 ). Compared with the wild type, ZmDi19-7 #KO1 and ZmDi19-7 #KO2 The grain shape is significantly smaller than that of the wild type (such as Figure 2 A); ZmDi19-7 #KO1 and ZmDi19-7 #KO2 The length and width of the grains were also significantly smaller than those of the wild type (e.g. Figure 2 BE), although the grain thickness was not significantly different from that of the wild type, ZmDi19-7 #KO1 and ZmDi19-7 #KO2 The 100-grain weight of the seeds was significantly smaller than that of the wild type, and the 100-grain weight was reduced by 18.92% and 14.94%, respectively ( Figure 2 D). The results indicate that reducing the expression of ZmDi19-7 gene or inhibiting the activity of ZmDi19-7 protein significantly inhibited the development of grain size and greatly reduced crop yield.

[0193] 1.3 Effects of ZmDi19-7 knockout mutant on grain development:

[0194] 1.3.1 Effects of ZmDi19-7 knockout mutant on grain development: Corn grain size is a key factor in determining corn yield. In order to clearly understand the development of corn grains, this study conducted optical microscopic observations and paraffin section observations on grains, embryos, and endosperms 10, 15, and 20 days after pollination, and measured different tissues at different times. Figure 3 , Figure 4 As shown, at the early stage of grain development (10 DAP), ZmDi19-7 #KO1 and ZmDi19-7 #KO2 The mutant embryonic tissue developed faster, the embryo volume was larger, and the endosperm weight and grain weight were significantly smaller than those of the wild type; however, in the middle stage of grain development (15DAP and 20DAP), the grain development speed of the ZmDi19-7 knockout mutant was significantly slowed down. There was no significant difference in embryo weight between the wild type and the ZmDi19-7 knockout mutant at 15DAP, but the endosperm and grain size of the ZmDi19-7 knockout mutant were significantly smaller than those of the wild type at 15DAP and 20DAP. This result indicates that reducing the expression of the ZmDi19-7 gene or inhibiting the activity of the ZmDi19-7 protein significantly inhibited the development of the endosperm and affected the development of grain size.

[0195] 1.3.2 Observation of paraffin sections of seeds: Specific experimental methods of paraffin sections:

[0196] Wild-type corn kernels and ZmDi19-7 knockout mutant corn kernels 10, 15, and 20 days after pollination were selected and placed in the prepared fixative FAA (50 wt% ethanol, 5% wt acetic acid, 3.7 wt% formaldehyde) and stored at room temperature for later use. The specific steps for paraffin sectioning are as follows:

[0197] 1.3.2.1 Tissue fixation: Place the vial containing the material and the fixative into a vacuum drying box, slowly draw the vacuum until the material sinks to the bottom of the bottle, replace with fresh fixative, and incubate at 4°C overnight.

[0198] 1.3.2.2 Dehydration: The fixed materials were immersed in gradient concentrations of alcohol (85wt%, 95wt%, 100wt%, 100wt%) for dehydration, each time for 1 hour.

[0199] 1.3.2.3 Transparency: The dehydrated material was immersed in mixed solutions of different concentrations of xylene and alcohol (25wt% xylene: 75wt% ethanol, 50wt% xylene: 50wt% ethanol, 100wt% xylene) for 1 hour each time.

[0200] 1.3.2.4 Wax dipping: Immerse the material from the previous step in a solution with a volume ratio of xylene to wax of 1:1 and bake in an oven at 37°C overnight.

[0201] 1.3.2.5 Waxing: Waxing twice, each time for 2 hours.

[0202] 1.3.2.6 Trimming wax blocks, slicing, and drying: Trim the wax blocks to facilitate slice handling. Use tweezers to place the cut wax strips on a water-added slide and place them on a slide baking machine overnight to ensure that the slices stick firmly.

[0203] 1.3.2.7 Dewaxing and rehydration: first dewax with xylene for 30 min, then rehydrate with different concentrations of alcohol (100wt%, 95wt%, 85wt%, 70wt%, 50wt%, 30wt%), each time for 10 min. 1.3.2.8 Staining and observation: After the above step, put the slide into a dye vat containing 1wt% toluidine blue dye for 10s, color it with distilled water for 10s, and observe and take pictures with a Leica (LEICA DM5000B) optical microscope. The test results are shown in Figure 4 .

[0204] 1.4 Effect of ZmDi19-7 knockout mutant on grain starch development: The main storage material in corn kernels is starch, which is mainly developed from the endosperm. The development of starch is closely related to the size of the kernels. In order to clearly observe the morphology of starch granules in corn kernels, this study observed the longitudinal sections of corn kernels of different genetic materials using a scanning electron microscope. Specific methods for scanning electron microscope observation:

[0205] KN5585 and ZmDi19-7 #KO1 、ZmDi19-7 #KO2 After the corn seeds are harvested, they are dried to constant weight and the kernels basically meet the drying requirements of the scanning electron microscope. The corn kernels are cut longitudinally with a sharp blade, and relatively intact kernel samples are selected and placed in a sample basket, which is then placed in a critical point dryer for full drying. After that, the magnification is adjusted, the state of the starch granules in the corn kernels is observed, and photos are taken for record and subsequent comparative analysis.

[0206] like Figure 5 As shown in the figure, the starch morphology of wild-type KN5585 is irregular polygonal and closely arranged, while the starch granules of ZmDi19-7 knockout mutant are spherical and loosely arranged. We counted and analyzed the starch granule size and number of starch granules per unit area of ​​the wild-type and knockout mutant, respectively, and found that the starch granule size of ZmDi19-7 knockout mutant was significantly smaller than that of wild-type, and the number of starch granules of ZmDi19-7 knockout mutant was significantly greater than that of wild-type. This result shows that reducing the expression of ZmDi19-7 gene or inhibiting the activity of ZmDi19-7 protein significantly inhibited the development of starch granules, thereby affecting the development of grain size.

[0207] 1.5ZmDi19-7 gene affects the expression of key genes in the starch synthesis pathway:

[0208] This study found that the size of starch granules in the ZmDi19-7 gene knockout mutant was significantly reduced, and it was speculated that the ZmDi19-7 gene may be involved in the starch synthesis pathway of grains. RT-qPCR experiments were used to detect 12 key genes related to starch synthesis. Figure 6As shown, Zm00001d045042 (ZmSh1), Zm00001d050032 (ZmBt2), Zm00001d044129 (ZmSh2), Zm00001d045462 (ZmWx1), Zm00001d045261 (ZmSs1), Zm00001d037234 (ZmSSIIа), Zm00001d000002 (ZmDu1), Zm00001d016684 (ZmAe1), Zm00001d049753 (ZmSu1), Zm00001d019479 (ZmGBSSIIa), and ZmDi1-7 were found in these genes. #KO1 The expression of Zm00001d004438 (ZmZpu1) was significantly downregulated in ZmDi19-7 #KO1 The results showed that reducing the expression of ZmDi19-7 gene or inhibiting the activity of ZmDi19-7 protein may directly or indirectly regulate the expression of key genes in the starch synthesis pathway to affect the development of endosperm and thus regulate the size of grains.

[0209] For the performance test of Example 2:

[0210] 2.1 RT-qPCR was used to detect the expression of gene ZmDi19-7 in various tissues ( Figure 7 The results showed that the ZmDi19-7 gene was widely expressed in maize tissues, with the highest expression in the stem nodes. It is speculated that this gene may play an important role in regulating the development of maize plant height.

[0211] 2.2 Detection of specific expression of ZmDi19-7 gene in different tissues ( Figure 8 The results showed that the maize ZmDi19-7 gene was specifically expressed in the stem nodes, stem apical meristem, and embryos at 25, 30, 35, and 40 days after pollination, while its expression was low in leaves and endosperm.

[0212] 2.3 Wild-type maize and maize plants overexpressing gene ZmDi19-7 were subjected to real-time fluorescence quantitative PCR technology to knock out the maize ZmDi19-7 gene structure (such as Fig. 9 ), the test results showed that the overexpression was 9 to 10 times that of the wild type. Compared with the wild type, the expression level of gene ZmDi19-7 in the knockout strain was below 0.5, or even lower (such as Fig. 9 3, 4 on the left), and we again verified our overexpression lines by semi-quantitative methods (e.g. Fig. 9 Left 2) and knockout strains, the results were consistent with the results of real-time fluorescence quantitative PCR.

[0213] 2.4 Analysis of plant height phenotypes of ZmDi19-7 gene overexpression transgenic plants and knockout mutants:

[0214] At the Nongcuiyuan Maize Molecular Breeding Base of Anhui Agricultural University in Hefei, wild-type KN5585, overexpression transgenic plants, and gene knockout transgenic plants were planted at a planting density of 4,000 plants / mu. The management was the same as that of ordinary maize fields. After the plant type was fixed (30 days after pollination), phenotypic measurements were performed to count the maize plant height, number of stem nodes, and length of stem nodes. Fig.10 The results showed that compared with the wild-type control, the number of stem nodes of transgenic corn overexpressing ZmDi19-7 increased significantly, while the number of stem nodes of gene knockout transgenic corn decreased significantly; at the same time, the length of stem nodes of transgenic corn overexpressing ZmDi19-7 increased, while the length of stem nodes of gene knockout transgenic corn was significantly shortened. These results indicate that reducing the expression of the ZmDi19-7 gene or inhibiting the activity of the ZmDi19-7 protein can help reduce the plant height of corn and improve its lodging resistance.

[0215] 2.5 Observation of stem node sections of transgenic plants with overexpression of ZmDi19-7 gene and knockout mutants:

[0216] The second stem node at the ear position of the wild type (KN5585), transgenic plants overexpressing ZmDi19-7, and gene-edited knockout mutant plants at the jointing stage were taken, and cross-sectioned and longitudinally cut into paraffin sections. After staining, they were observed under a Leica optical microscope. It was found that the cell area and cell length of the gene-edited knockout mutant plants were significantly smaller than those of the wild type KN5585, while the cell length of the transgenic plants overexpressing ZmDi19-7 was significantly greater than that of the wild type KN5585, indicating that after reducing the expression of ZmDi19-7 or inhibiting the activity of ZmDi19-7, cell elongation was significantly inhibited (such as Fig.11 ).

[0217] 2.6 Stem phenotypes of ZmDi19-7 gene overexpression transgenic plants and knockout mutants: Test results are shown in Fig.12 .

Claims

1. A ZmDi19-7 protein, characterized in that The amino acid sequence of the ZmDi19-7 protein is shown in SEQ ID NO.

1.

2. The ZmDi19-7 protein according to claim 1, characterized in that The nucleic acid sequence of the amino acid ZmDi19-7 is shown in SEQ ID NO.

2.

3. The ZmDi19-7 protein according to claim 2, characterized in that The genomic sequence of the ZmDi19-7 nucleic acid is shown in SEQ ID NO.

3.

4. The ZmDi19-7 protein according to claim 2, characterized in that The cloning method of the ZmDi19-7 nucleic acid comprises the following steps: S1 Obtain plant material; S2 extracts RNA; S3 reverse transcription; S4PCR reaction.

5. The ZmDi19-7 protein according to claim 4, characterized in that The specific steps of obtaining plant materials in step S1 are as follows: select inbred corn plant materials, select corn seeds with uniform grain size, sow them in nutrient soil, and when they grow to the three-leaf to four-leaf stage, take corn leaves for RNA extraction.

6. The ZmDi19-7 protein according to claim 4, characterized in that The reverse transcription procedure in step S3 is as follows: react the mixed reagent at 35-37°C for 15-20 minutes, and cycle 1-3 times; then react at 80-85°C for 3-8 seconds, and cycle 1-3 times; finally, react at 4°C for 1-2 cycles and store for later use.

7. The ZmDi19-7 protein according to claim 4, characterized in that The specific primers for the ZmDi19-7 nucleic acid used in the PCR reaction in step S4 include: ZmDi19-7-F: ATGGACTCCGAGCACTGGATCT and ZmDi19-7-R: TCAGTCGCCGAATAGAGTAGAG.

8. The ZmDi19-7 protein according to claim 4, characterized in that The procedure of the PCR reaction in step S4 includes: pre-denaturation at 95-98°C, 10-15min, 1-3 cycles; denaturation at 95-98°C, 10-15s; annealing at 55-58°C, 3-8s, 35-40 cycles; extension at 70-75°C, 1-3s; total extension at 70-75°C, 10-15min, 1-3 cycles; storage at 3-5°C, 1-2 cycles, and standby.

9. Use of the ZmDi19-7 protein according to any one of claims 1 to 8 in regulating corn grains.

10. Use of the ZmDi19-7 protein according to any one of claims 1 to 8 in regulating the growth of corn plants.

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