ZmDi19-7 protein and application in regulating corn kernel and plant height

Abstract

The application discloses a kind of ZmDi19-7 proteins, the amino acid sequence of the ZmDi19-7 protein is as shown in SEQ ID NO.1, the substance expressed by the ZmDi19-7 gene of the application can regulate plant kernel length, kernel width, kernel hundred-grain weight, plant starch granule size, has important guiding significance to the development of corn kernel, corn kernel improvement and high product variety selection.Gene expression substance can regulate plant height, regulate plant stem number, regulate plant stem length, regulate plant cell length, has important guiding significance to the development of corn plant height, corn plant height improvement and dense variety selection breeding.Using biotechnology for corn breeding work, can accelerate the variation speed of genetic gene, greatly shorten the breeding period, accelerate new variety cultivation.

Description

TECHNICAL FIELD

[0001] The present application relates to a ZmDi19-7 protein and its application in regulating maize kernel and plant height, relates to C12N, and particularly relates to the field of microorganisms and compositions thereof. BACKGROUND

[0002] Maize is one of the crops with the highest total yield, is an important food crop and forage crop, has great production potential and high economic benefits. The existing method for increasing grain yield is to increase the planting density of maize, but under the condition of dense planting, the maize internode elongation becomes thin, the plant height increases, the resistance to lodging is poor, and lodging disaster is prone to occur in the maize maturing season, resulting in yield reduction. Therefore, it is crucial to reasonably reduce the plant height, improve the lodging resistance of maize, improve the group photosynthetic efficiency, and then promote the yield increase of dense planting. In addition, the yield increase of maize can also be improved by improving the size of maize kernel, the weight of 100 kernels, and the starch content of kernel, which has important guiding significance for cultivating new maize varieties with high yield.

[0003] Chinese patent CN112941087A discloses an application of maize ZmBES1 / BZR1-2 gene in improving plant drought resistance. By isolating and cloning the ZmBES1 / BZR1-2 gene, the drought resistance of plants can be improved, which provides a train of thought for cultivating drought-resistant and salt-resistant transgenic plants, and has important significance for crop drought-resistant breeding and production application. However, it does not involve the yield-increasing effect of maize. Chinese patent CN110157713A discloses a maize drought-resistant gene ZmDi19-7 and its application. The maize drought-resistant gene ZmDi19-7 is integrated into the cells, tissues and organs of plants and overexpressed, which can improve the drought resistance of plants. However, it does not involve the yield-increasing effect of ZmDi19-7 gene on maize. SUMMARY

[0004] In order to improve the kernel performance of maize and optimize the plant height effect of maize, the first aspect of the present application provides a ZmDi19-7 protein, wherein the amino acid sequence of the ZmDi19-7 protein is shown as 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 the 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] TCTGTCGTCGTTCGGCCTTGGCTTCCCTACCTCAGACGCGGAGCAAGC

[0022] ATCCAAGTCGACTGTTTCCATTCCTGATGATGCAACGACGGTGAAAGAAGC

[0023] GCCCGCTCAGGCACGGAAGCTAAGTATCGATTCGTCGCTCACAAGTGAAG

[0024] AAAGGGAGCTGAAGCGGAAGCAAGCCCGCGTCAGAGCCACGTTCGTGCAGGACCTGCTGCTCTCTACTCTATTCGGCGACTGA.

[0025] As a preferred embodiment, the genomic sequence of the ZmDi19-7 nucleic acid is shown as 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] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0035] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0036] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0037] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0038] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0039] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0040] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0041] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0042] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0043] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0044] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0045] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[0046] TGAAAAGCAGCTTATGCTTGTTGGCTGCTTTTAGTGTATTCTGAGAAGCAG

[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 extraction method of the ZmDi19-7 nucleic acid comprises the following steps:

[0076] S1 obtaining plant material;

[0077] S2 extracting RNA;

[0078] S3 reverse transcription;

[0079] S4 PCR reaction.

[0080] As a preferred embodiment, the specific steps of obtaining plant material in step S1 are as follows: selecting selfing line B73 corn plant material, selecting corn seeds with uniform kernel size, sowing in nutrient soil, and taking corn leaves for RNA extraction when growing to three to four leaf stage.

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

[0082] M1 total RNA is extracted by Trizol method, high-temperature sterilization and inactivation of mortar and grinding rod, pre-cooling, liquid nitrogen grinding of corn leaf sample to powder, picking powder and transferring to 2mL RNA free EP tube, adding 1mL Trizol extraction solution, fully shaking and mixing in vortex shaker, and standing on ice in dark for 10min;

[0083] M2 after adding 500μL RNA nucleic acid extraction solution (the mass ratio of RNA nucleic acid extraction solution to sample powder is 24:1), vortex shaking for 10s, mixing, standing on ice for 10min to extract impurities;

[0084] M3 using high-speed benchtop centrifuge 12000xg, 4℃ centrifugation for 10min;

[0085] M4 transferring 600μL supernatant to a new 1.5mL RNA free EP tube, adding equal volume of isopropanol, repeatedly inverting and mixing, standing on ice for 10min;

[0086] M5 centrifuge at 12000 x g, 4°C for 10 min using high speed bench top refrigerated centrifuge;

[0087] M6 discard supernatant, add 1 mL 75 vol% ethanol solution (DEPC water preparation), mix well;

[0088] M7 centrifuge at 12000 x g, 4°C for 3 min using high speed bench top refrigerated centrifuge;

[0089] M8 repeat S6, S7 steps, centrifuge at 12000 x g, 4°C for 3 min using high speed bench top refrigerated centrifuge;

[0090] M9 dissolve RNA precipitate with 50 μL DEPC water;

[0091] M10 measure the concentration of RNA ng / uL and OD 260 / 280 value, and at the same time use nucleic acid gel electrophoresis to detect the quality of RNA.

[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] Component Volume 5x Evo M-MLV RT Master Mix 2 μL Total RNA 1 μL RNase free water 7 μL Component Volume Primer star Mix 12.5 μL Upstream primer (10 μM) 1 μL Downstream primer (10 μM) 1 μL

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

[0095] As a preferred embodiment, the procedure of reverse transcription in step S3 is as follows: mix the reagents at 35-37°C for 15-20 min, cycle 1-3 times; then at 80-85°C for 3-8 s, cycle 1-3 times; finally at 4°C, cycle 1-2 times, and save for standby.

[0096] As a preferred embodiment, the procedure of reverse transcription in step S3 is as follows: mix the reagents at 37°C for 15 min, cycle 1 time; then at 85°C for 5 s, cycle 1 time; finally at 4°C, cycle 1 time, after the reaction is completed, the product is stored at -20°C or in an ice box for standby.

[0097] As a preferred embodiment, the specific primers of 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] Template (cDNA) 1 μL RNA free water 9.5 μL Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10

[0102] As a preferred embodiment, the program of the PCR reaction in step S4 comprises: pre-denaturation 95-98℃, 10-15min, 1-3 cycles; denaturation 95-98℃, 10-15s; annealing 55-58℃, 3-8s, 35-40 cycles; extension 70-75℃, 1-3s; total extension 70-75℃, 10-15min, 1-3 cycles; preservation 3-5℃, 1-2 cycles, standby.

[0103] As a preferred embodiment, the program of the PCR reaction in step S4 comprises: pre-denaturation 98℃, 10min, 1 cycle; denaturation 98℃, 10s; annealing 58℃, 5s, 35 cycles; extension 72℃, 1s; total extension 72℃, 10min, 1 cycle; preservation 4℃, 1 cycle, standby.

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

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

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

[0107] (1) The ZmDi19-7 protein can regulate the grain length, grain width, 100-grain weight, starch granule size of plants, and has important guiding significance for regulating the development of corn kernels, improving corn kernels and high-yield variety breeding.

[0108] (2) The ZmDi19-7 protein can regulate the plant height, regulate the number of stem nodes, regulate the length of stem nodes, regulate the length of plant cells, and has important guiding significance for regulating the development of corn plant height, improving corn plant height and dense-tolerance variety breeding.

[0109] (3) The ZmDi19-7 protein can accelerate the variation speed of genetic genes, greatly shorten the breeding period, and accelerate the breeding of new varieties by using biotechnology in corn breeding. BRIEF DESCRIPTION OF DRAWINGS

[0110] Figure 11Figure 1 is a diagram of the structure of the corn ZmDi19-7 gene (A); RT-qPCR of the knockout of the corn ZmDi19-7 gene (B) and semi-RT-qPCR results of the knockout of the corn ZmDi19-7 gene (C).

[0111] Figure 12 Figure 4 is a comparison of wild-type KN5585 and ZmDi19-7 gene knockout mutant plants in a breeding base after the grains matured, a comparison of grain shape (A); a comparison of grain length and width [actual (B) length (D); width (E)]; a comparison of grain thickness [actual (C) thickness (F)]; a comparison of grain weight per 100 grains (G).

[0112] Component Volume Figure 5 is an optical microscope observation of the grains, embryos and endosperms of 10 days, 15 days and 20 days after pollination; actual (A) and weight change value line graph (B) of the embryos; weight change value line graph (C) of the endosperms; weight change value line graph (D) of the grains.

[0113] 5x Evo M-MLV RT Master Mix 2 μL Figure 6 is a paraffin section observation of the grains, embryos and endosperms of 10 days, 15 days and 20 days after pollination.

[0114] Total RNA 1 μL Figure 7 is a longitudinal section scanning electron microscope observation of wild-type KN5585 and ZmDi19-7 gene knockout mutant plants in a breeding base (actual A, electron microscope B, C); starch grain size statistics bar graph (D) in the unit area field of view; starch grain number statistics bar graph (E) in the unit area field of view.

[0115] RNase free water 7 μL Figure 8 is the expression of 12 key genes related to starch synthesis detected by RT-qPCR experiments.

[0116] Component Volume Figure 9 is the expression of the gene ZmDi19-7 in various tissues detected by real-time fluorescence quantitative PCR (RT-qPCR).

[0117] Primer star Mix 12.5 μL Figure 10 is the expression of the corn ZmDi19-7 gene in stem nodes (C, D); stem tip meristems (E); embryos of 25 days, 30 days, 35 days and 40 days after pollination (A) (the red arrow points to the embryo of the grain, and the part other than the grain epidermis is the endosperm of the grain); leaves (B) after in situ hybridization.

[0118] Upstream primer (10 μM) 1 μL Figure 11 is a comparison of the plant height and stem traits of different transgenic materials and wild types.

[0119] Downstream primer (10 μM) 1 μLThe numerical comparison chart of the plant height, stem node number and stem node length of different transgenic materials and wild type; plant height (A); stem node number (B); stem node length (C).

[0120] Template (cDNA) 1 μL The paraffin section of the second stem node on the ear position of different transgenic materials and wild type plants was observed by Leica optical microscope, and the physical map (A), cell area (B) and cell length (C) were observed.

[0121] RNA free water 9.5 μL The effect of ZmDi19-7 gene on the 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 which is shown as SEQ ID NO. 1.

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

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

[0126] The extraction method of the ZmDi19-7 nucleic acid comprises the following steps:

[0127] S1 obtaining plant material;

[0128] S2 extracting RNA;

[0129] S3 reverse transcription;

[0130] S4 PCR reaction.

[0131] The specific steps of obtaining plant material in step S1 are as follows: selecting selfing line B73 corn plant material, selecting corn seeds with uniform kernel size, sowing in nutrient soil, and taking corn leaves for RNA extraction when growing to three to four leaf stage.

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

[0133] M1 total RNA is extracted by Trizol method, high temperature is used for sterilization and inactivation of mortar and grinding rod, and corn leaf sample is ground into powder after pre-cooling, the powder is transferred to 2mL RNA free EP tube, 1mL Trizol extraction solution is added, and the mixture is fully shaken and mixed in vortex shaker, and then it is placed on ice in dark for 10min;

[0134] M2 Add 500 μL RNA nucleic acid extraction solution (mass ratio of RNA nucleic acid extraction solution to sample powder is 24:1) after vortexing for 10 s, and extract impurities on ice for 10 min;

[0135] M3 Use a high-speed benchtop centrifuge to centrifuge at 12000 x g, 4°C for 10 min;

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

[0137] M5 Use a high-speed benchtop centrifuge to centrifuge at 12000 x g, 4°C for 10 min;

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

[0139] M7 Use a high-speed benchtop centrifuge to centrifuge at 12000 x g, 4°C for 3 min;

[0140] M8 Repeat the S6, S7 steps, use a high-speed benchtop centrifuge to centrifuge at 12000 x g, 4°C for 3 min;

[0141] M9 Dissolve the RNA precipitate with 50 μL of DEPC water;

[0142] M10 Measure the concentration ng / uL and OD 260 / 280 value of the RNA, and simultaneously detect the quality of the RNA using nucleic acid gel electrophoresis.

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

[0144] Figure 7 Figure 1 Figure 1 Figure 1 Figure 2 Figure 2 Figure 2 Figure 2

[0145] After all the reagents are added, use a low-speed centrifuge to centrifuge at 200 rpm for 10-15 s to fully mix the reagents.

[0146] The procedure for reverse transcription in the step S3 is as follows: mix the reagents at 37°C for 15 min, cycle 1 time; then at 85°C for 5 s, cycle 1 time; finally at 4°C, cycle 1 time. After the reaction is completed, the product is stored at -20°C or in an ice box for later use.

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

[0148] ZmDi19-7-R: TCAGTCGCCGAATAGAGTAGAG.

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

[0150] Figure 3 Figure 4 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 9 Figure 9 Figure 10 Figure 11

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

[0152] A ZmDi19-7 protein in the application of regulating corn kernels.

[0153] The ZmDi19-7 gene editing knockout mutant of corn is created, and the specific implementation is as follows:

[0154] 1.1 Target point design of the ZmDi19-7 gene editing knockout mutant of corn

[0155] The CRISPR / Cas9 basic vector (pCXB-053-CCDB vector) used in the application is donated by Unami Biotechnology (Jiangsu) Co., Ltd. Two target points are selected on the ZmDi19-7 protein domain sequence, and an expression cassette of double-target sgRNA and Cas9 protein is 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 point is 5'-AGTGGGCTTCCCGTAGATCT-3', and targets the 1643-1662th position of sequence 3 in the sequence table of the ZmDi19-7 gene; the nucleotide sequence of the sgRNA2 target point is 5'-TGGCTTCCCTACCTCAGACG-3', and targets the 1766-1785th nucleotide of sequence 3 in the sequence table.

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

[0157] The CCDB sequence in the pCXB-053-CCDB vector is cut off by BSAI enzyme digestion, two independent single target vectors are constructed by T4 ligase respectively, and then sgRNA2 is connected by recombinase to form a double target knockout vector of ZmDi19-7-sgRNA1-srRNA2; the specific steps are: 1.2.1 Synthesis of primers, and dissolution in ultrapure water and mixing uniformly; 1.2.2 Configuration of annealing buffer (Tris-HCl 10.0 mM, pH 8.0, EDTA 0.1 mM, NaCl: 50.0 mM); 1.2.3 Annealing and ligation, annealing buffer 50.0 μL, primer mixture 5.0 μL, (95℃, 3min, cooling to 16℃).

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

[0159] Select ZmDi19-7 #KO1 and ZmDi19-7 #KO2 homozygous lines, respectively, when the plants grow to six or seven leaves, respectively, take the leaves to extract RNA, the method is referred to the above RNA extraction method, and then the above reverse transcription method is used to obtain a certain concentration of cDNA.

[0160] According to the sequence information of the gene ZmDi19-7 (Zm00001d038999), specific quantitative primers are designed, and the following two pairs of primers are selected according to the sequence of the knockout line:

[0161] ZmDi19-7-RT: AGATCTACGGGAAGCCCACT

[0162] ZmDi19-7-RT: AGTCGACTTGGATGCTTGCT

[0163] Then the performance test is carried out.

[0164] Example 2

[0165] A ZmDi19-7 protein, the amino acid sequence, nucleic acid sequence, and genomic sequence of the ZmDi19-7 protein are the same as in Example 1, and the extraction method of the ZmDi19-7 nucleic acid is the same as in Example 1. The difference lies in that:

[0166] Application of a ZmDi19-7 protein in regulating the height of a maize plant.

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

[0168] According to the sequence information of the gene ZmDi19-7, specific quantitative primers were designed, the length of the primers was set at 20 bp, the Tm value was at 57.0-60.0 °C, and the GC% content was at 40-60%. The sequences were as follows: forward primer: GACTGTTTCCATTCCTGATG; reverse primer: TCGCCGAATAGAGTAGAGAG. The materials of Root-V4, Stem-V4, Leaf-V4, Bract-V17, Ear-V17, Silk-V17, Pollen-R1, Embryo-30 DAP and Endosperm-30 DAP at the 10 periods were taken, and the expression of the gene ZmDi19-7 in each tissue was detected by real-time fluorescent quantitative PCR (RT-qPCR) Figure 12

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

[0170] To further analyze the distribution of the ZmDi19-7 gene in different tissues, the stem nodes of the maize B73 inbred line at the V4 period, the stem tip meristem at the jointing period, and the leaf tissue were obtained. In addition, when the pollination period arrived, the maize ears at 25 days, 30 days, 35 days, and 40 days after pollination were selected, and the kernels were carefully taken out and fixed in the FAA fixing solution (50wt% ethanol, 5wt% acetic acid, and 3.7wt% methanol) for the in situ hybridization experiment.

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

[0172] The experimental materials were taken from the B73 inbred line at different growth periods or different growth tissues, including the shoot apical meristem (SAM) at 17 days after sowing, the stem at the four-leaf stage, the leaf after pollen scattering, and the kernels at 25 days, 30 days, 35 days, and 40 days after pollination, which were fixed in the FAA fixing solution containing 50wt% ethanol, 5wt% acetic acid, and 37wt% formaldehyde. Referring to the full-length CDS sequence of the ZmDi19-7 gene, the sense strand (GCAACATGGATATTTATTCAAGAATCGCCGCCG) and the antisense strand (CGGCGGCGATTCTTGAATAAATATCCATGTTGC) RNA probes were designed. The in situ hybridization experiment was done by the Wuhan Seville Biological Company. The in situ hybridization of ZmDi19-7 used the sense and antisense probes, with the sense probe as the control.

[0173] ​2.3 The creation of ZmDi19-7 transgenic plants overexpressing, the specific implementation is as follows:

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

[0175] 2.3.2 Genetic transformation of the vector overexpressing ZmDi19-7 gene (completed by Yumi Biotechnology (Jiangsu) Co., Ltd.).

[0176] 2.4 The creation of maize ZmDi19-7 gene editing knockout mutants, the specific implementation is as follows:

[0177] 2.4.1 Target site design of maize ZmDi19-7 gene editing knockout mutants

[0178] The CRISPR / Cas9 basic vector (pCXB-053-CCDB vector) used in the present application is donated by Yumi Biotechnology (Jiangsu) Co., Ltd. Two targets are selected on the ZmDi19-7 protein domain sequence to construct an expression cassette of double-target sgRNA and Cas9 protein, 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 nucleotide of sequence 3 in the sequence table of ZmDi19-7 gene; the nucleotide sequence of the sgRNA2 target is 5'-TGGCTTCCCTACCTCAGACG-3', targeting the 1766-1785th nucleotide of sequence 3 in the sequence table.

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

[0180] By BSAI enzyme digestion, the CCDB sequence in the pCXB-053-CCDB vector is cut off, two independent single-target vectors are constructed by T4 ligase, and then the sgRNA2 is connected by recombinase to form a double-target knockout vector of ZmDi19-7-sgRNA1-srRNA2; the specific steps are as follows: 1) Synthesize primers and dissolve in ultrapure water, then mix uniformly; 2) Configure annealing buffer (Tris-HCl 10.0mM, PH8.0, EDTA 0.1mM, NaCl: 50.0mM); 3) Annealing and ligation, annealing buffer 50.0μL, primer mixture 5.0μL, (95℃, 3min, cooling to 16℃).

[0181] 2.4.3 Genetic transformation of maize ZmDi19-7 gene editing knockout mutants (completed by Unicarve Biotech (Jiangsu) Co., Ltd.)

[0182] 2.4.4 Expression level detection of maize ZmDi19-7 gene in transgenic overexpression lines and knockout lines

[0183] Select ZmDi19-7 from transgenic overexpression lines of gene ZmDi19-7 #OE3 Homozygous positive plant lines, and select ZmDi19-7 from knockout lines #KO1 and ZmDi19-7 #KO2 Homozygous lines, respectively, when the plants grow to six, seven leaves, respectively, take the leaves to extract RNA, the method is referred to the above RNA extraction method, and then through the above reverse transcription method, get a certain concentration of cDNA.

[0184] According to the sequence information of gene ZmDi19-7 (Zm00001d038999), design specific quantitative primers, and select the following two pairs of primers according to the sequence of knockout lines:

[0185] ZmDi19-7-RT: AGATCTACGGGAAGCCCACT

[0186] ZmDi19-7-RT: AGTCGACTTGGATGCTTGCT

[0187] Then perform performance test.

[0188] Performance test

[0189] Performance test for example 1:

[0190] 1.1 Perform real-time fluorescent quantitative PCR technology on wild type maize and ZmDi19-7 gene editing knockout mutant maize, knockout maize ZmDi19-7 gene structure (such as ​ A), the test results show that: compared with the wild type, the expression of gene ZmDi19-7 in knockout lines is below 0.5, even lower (such as ​ B), and we again verified our knockout lines and wild type by semi-quantitative method, the results are consistent with the results of real-time fluorescent quantitative PCR (such as ​ C), indicating that the knockout mutant can be used for biological function verification.

[0191] 1.2 Kernel phenotype analysis of ZmDi19-7 gene knockout mutant:

[0192] 1.2.1 Wild type KN5585 and ZmDi19-7 gene knockout mutant plants were planted in spring in the corn molecular breeding base of the Hefei Anhui Agricultural University Farm, and after the seeds were mature, the corn ears of each line were harvested, dried to constant weight, and the corn seeds were observed and statistically analyzed (as shown in ​ ) Compared with the wild type, the ZmDi19-7 #KO1 and ZmDi19-7 #KO2 grain shape was significantly smaller than the wild type (as shown in ​ A); the ZmDi19-7 #KO1 and ZmDi19-7 #KO2 grain length and width were also significantly smaller than the wild type (as shown in ​ B-E), although the grain thickness was not significantly different from the wild type, the ZmDi19-7 #KO1 and ZmDi19-7 #KO2 grain weight per 100 grains was significantly smaller than the wild type, and the weight per 100 grains was reduced by 18.92% and 14.94%, respectively (as shown in ​ D). The results showed 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 Effect of ZmDi19-7 gene knockout mutant on grain development:

[0194] 1.3.1 Effect of ZmDi19-7 gene knockout mutant on grain development: Corn kernel size is a key factor in determining corn yield. In order to clearly understand the development of corn kernel, the present study observed the kernel, embryo, and endosperm at 10 days, 15 days, and 20 days after pollination under an optical microscope and by paraffin sectioning, and measured different tissues at different times. As shown in ​ , ​ , at the early stage of kernel development (10DAP), compared with the wild type KN5585, the ZmDi19-7 #KO1 and ZmDi19-7 #KO2 mutants had faster embryo tissue development and larger embryo volume, and the endosperm weight and kernel weight were significantly smaller than the wild type; at the middle stage of kernel development (15DAP and 20DAP), the ZmDi19-7 gene knockout mutant had significantly slower kernel development, and at 15DAP, there was no significant difference in embryo weight between the wild type and the ZmDi19-7 gene knockout mutant, but at 15DAP and 20DAP, the embryo size and kernel size of the ZmDi19-7 gene knockout mutant were significantly smaller than the wild type. The results showed that reducing the expression of ZmDi19-7 gene or inhibiting the activity of ZmDi19-7 protein significantly inhibited the development of endosperm and affected the development of kernel size.

[0195] 1.3.2 Grain paraffin section observation: Specific experimental method of paraffin section:

[0196] Select wild type corn kernels and ZmDi19-7 gene knockout mutant corn kernels at 10 days, 15 days, and 20 days after pollination, respectively, and put them into prepared fixing liquid FAA (50wt% ethanol, 5wt% acetic acid, 3.7wt% formaldehyde) for room temperature storage for later use. The specific steps of paraffin section are as follows:

[0197] 1.3.2.1 Tissue fixation: Put the vial containing the fixing liquid and the material into a vacuum drying box, slowly vacuumize until the material sinks to the bottom of the vial, replace the fresh fixing liquid, and store at 4°C overnight.

[0198] 1.3.2.2 Dehydration: Dehydrate the fixed material by sequentially immersing it in gradient concentration alcohol (85wt%, 95wt%, 100wt%, 100wt%) for 1h each time.

[0199] 1.3.2.3 Transparency: After dehydration, sequentially immerse the material in different concentration mixtures of xylene and alcohol (25wt% xylene: 75wt% ethanol, 50wt% xylene: 50wt% ethanol, 100wt% xylene) for 1h each time.

[0200] 1.3.2.4 Wax immersion: Immers the material in the solution of xylene: wax with a volume ratio of 1:1, and store in a 37°C oven overnight.

[0201] 1.3.2.5 Wax replacement: Replace the wax twice, 2h each time.

[0202] 1.3.2.6 Wax block trimming, sectioning, and drying: Trim the wax block for easy sectioning, use tweezers to place the cut wax band on a water-coated glass slide, and store in an oven overnight to make the section stick.

[0203] 1.3.2.7 Dewaxing and rehydration: First, dewax with xylene for 30min, then rehydrate with alcohol of different concentrations (100wt%, 95wt%, 85wt%, 70wt%, 50wt%, 30wt%) for 10min each time.

[0204] 1.3.2.8 Staining and observation: After the above step, place the glass slide in a staining tank containing 1wt% toluidine blue dye solution for 10s, separate the color with distilled water for 10s, and observe and take pictures under a LEICA (LEICA DM5000B) optical microscope. The test results are shown in ​ .

[0205] 1.4 Effects of the ZmDi19-7 gene knockout mutant on kernel starch development: Starch is the most important storage substance in maize kernels, and it mainly originates from the endosperm. Starch development is closely related to kernel size. To clearly observe the morphology of starch granules in maize kernels, this study used scanning electron microscopy to observe longitudinal sections of maize kernels from different genetic materials. Specific methods for scanning electron microscopy observation:

[0206] KN5585 and ZmDi19-7 #KO1 ZmDi19-7 #KO2 After harvesting, the corn seeds were dried to a constant weight, and the kernels basically met the drying requirements for scanning electron microscopy. The corn kernels were longitudinally cut with a sharp blade, and relatively intact kernel samples were selected, placed in a sample basket, and put into a critical point dryer for thorough drying. Afterward, the magnification was adjusted, and the state of the starch granules in the corn kernels was observed, photographed, and recorded for subsequent comparative analysis.

[0207] like ​ As shown, the starch morphology of wild-type KN5585 is irregular polygonal and tightly packed; while that of the ZmDi19-7 gene knockout mutant is spherical and loosely packed. We statistically analyzed the size and number of starch granules per unit area of ​​field for both wild-type and knockout mutants, finding that the starch granules of the ZmDi19-7 gene knockout mutant were significantly smaller than those of the wild-type, and the number of starch granules in the ZmDi19-7 gene knockout mutant was significantly greater than that in the wild-type. These results indicate that reducing the expression of the ZmDi19-7 gene or inhibiting the activity of the ZmDi19-7 protein significantly inhibits starch granule development, thereby affecting grain size development.

[0208] 1.5 The ZmDi19-7 gene influences the expression of key genes in the starch synthesis pathway:

[0209] This study found that the starch granules in the ZmDi19-7 gene knockout mutant were significantly smaller, suggesting that the ZmDi19-7 gene may be involved in the starch synthesis pathway in grains. Twelve key genes related to starch synthesis were detected using RT-qPCR. ​Zm00001d045042 (ZmShl), Zm00001d050032 (ZmBt2), Zm00001d044129 (ZmSh2), Zm00001d045462 (ZmWxl), Zm00001d045261 (ZmSsl), Zm00001d037234 (ZmSSIIa), Zm00001d000002 (ZmDul), Zm00001d016684 (ZmAel), Zm00001d049753 (ZmSul), Zm00001d019479 (ZmGBSSIIa), these genes were significantly down-regulated in ZmDi19-7 #KO1 Zm00001d004438 (ZmZpul) was significantly up-regulated in ZmDi19-7 #KO1 The results showed that reducing the expression of ZmDi19-7 gene or inhibiting the activity of ZmDi19-7 protein could directly or indirectly regulate the expression of key genes in the starch synthesis pathway to affect the development of endosperm, and then regulate the size of the kernel.

[0210] Performance test for Example 2:

[0211] 2.1 The expression of ZmDi19-7 gene in different tissues was detected by RT-qPCR. ​ The results showed that ZmDi19-7 gene was widely expressed in corn tissues, with the highest expression in stem nodes, suggesting that the gene might have an important function in regulating the development of corn height.

[0212] 2.2 The results of detecting the specific expression of ZmDi19-7 gene in different tissues. ​ The results showed that the ZmDi19-7 gene in corn was specifically expressed in stem nodes, stem tip meristem, 25-day, 30-day, 35-day, and 40-day embryos after pollination, while the expression in leaves and endosperm was low.

[0213] 2.3 Real-time fluorescent quantitative PCR was performed on wild-type corn and ZmDi19-7 overexpression corn plants, and the structure of ZmDi19-7 gene was knocked out (such as ​ The test results showed that the overexpression was 9-10 times that of the wild type, and the expression of ZmDi19-7 gene in the knockout lines was below 0.5, even lower (such as ​ left 3, 4), and we again verified our overexpression lines (such as ​ left 2) and knockout lines by semi-quantitative method, and the results were consistent with those of real-time fluorescent quantitative PCR.

[0214] 2.4 Phenotype analysis of transgenic plants overexpressing ZmDi19-7 gene and knock-out mutant plants:

[0215] The wild type KN5585, transgenic plants overexpressing ZmDi19-7 gene and transgenic plants with ZmDi19-7 gene knock-out were planted in the corn molecular breeding base of Anhui Agricultural University in Hefei, with a planting density of 4000 plants per mu. The plants were managed in the same way as general corn field management. After the plant type was fixed (30 days after pollination), the plant height, stem node number and stem node length of the corn were measured and counted. ​ The results showed that, compared with the wild type control, the stem node number of the transgenic corn overexpressing ZmDi19-7 gene was significantly increased, while the stem node number of the transgenic corn with ZmDi19-7 gene knock-out was significantly reduced. At the same time, the stem node length of the transgenic corn overexpressing ZmDi19-7 gene was increased, while the stem node length of the transgenic corn with ZmDi19-7 gene knock-out was significantly shortened. This result indicated that reducing the expression of ZmDi19-7 gene or inhibiting the activity of ZmDi19-7 protein was helpful to reduce the plant height of corn and improve the resistance to lodging.

[0216] 2.5 Observation of stem node sections of transgenic plants overexpressing ZmDi19-7 gene and knock-out mutant plants:

[0217] The second stem node at the ear position of the wild type (KN5585), transgenic plants overexpressing ZmDi19-7 gene and gene editing knock-out mutant plants at the jointing stage were taken for paraffin section, and after staining, the sections were observed by Leica optical microscope. It was found that the cell area and cell length of the gene editing knock-out mutant plants were significantly smaller than those of the wild type KN5585, while the cell length of the transgenic plants overexpressing ZmDi19-7 gene was significantly larger than that of the wild type KN5585, indicating that after reducing the expression of ZmDi19-7 or inhibiting the activity of ZmDi19-7, the cell elongation was significantly inhibited (as shown in ​ ).

[0218] 2.6 Stem phenotype of transgenic plants overexpressing ZmDi19-7 gene and knock-out mutant plants: the test results are shown in ​ .

Claims

1. An application of the ZmDi19-7 gene or ZmDi19-7 protein in reducing maize plant height, characterized in that, Maize plant height is reduced by decreasing the expression of the ZmDi19-7 gene or inhibiting the activity of the ZmDi19-7 protein; the amino acid sequence of the ZmDi19-7 protein is shown in SEQ ID NO.1; the nucleic acid sequence of the ZmDi19-7 gene is shown in SEQ ID NO.

2.

2. The application of the ZmDi19-7 gene or ZmDi19-7 protein according to claim 1 in reducing maize plant height, characterized in that, The genomic sequence of the ZmDi19-7 gene is shown in SEQ ID NO.

3.

3. The application of the ZmDi19-7 gene or ZmDi19-7 protein according to claim 1 in reducing maize plant height, characterized in that, The cloning method for the ZmDi19-7 gene includes the following steps: S1 obtains plant materials; S2 RNA extraction; S3 reverse transcription; S4PCR reaction.

4. The application of the ZmDi19-7 gene or ZmDi19-7 protein according to claim 3 in reducing maize plant height, characterized in that, The specific steps for obtaining plant materials in step S1 are as follows: select inbred maize plant materials, select maize seeds of uniform size, sow them in nutrient soil, and when they grow to the three- to four-leaf stage, take maize leaves for RNA extraction.

5. The application of the ZmDi19-7 gene or ZmDi19-7 protein according to claim 3 in reducing maize plant height, characterized in that, The specific primers for the ZmDi19-7 gene used in the PCR reaction in step S4 include: ZmDi19-7-F: ATGGACTCCGAGCACTGGATCT and ZmDi19-7-R: TCAGTCGCCGAATAGAGTAGAG.

Images