Application of corn ZmArf1a gene in corn variety breeding

Abstract

The invention discloses application of a corn ZmArf1a gene in corn variety breeding, and belongs to the technical field of molecular breeding. The CDS sequence of the corn ZmArf1a gene is as shown in SEQ ID NO.3, and the amino acid sequence of the corn ZmArf1a gene is as shown in SEQ ID NO.4. It is found for the first time that a plant with the corn ZmArf1a gene knocked out grows and develops normally, tassels are fertile, female spike stalks and grains are normal and full, but the leaf length, the leaf width and the plant height are remarkably reduced; the corn ZmArf1a gene provided by the invention can be used as a candidate gene for breeding dwarf and close-planting corn varieties, and is expected to reduce the breeding workload and accelerate the breeding process.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular breeding, and particularly to the application of the maize ZmArf1a gene in the breeding of maize varieties. Background Art

[0002] Maize is one of the food crops with the largest planting area in China. It not only provides a large amount of food, but also occupies an important position in multiple industries such as industry (through deep processing, maize can be converted into various products, such as starch, alcohol, citric acid, L-lactic acid, food-grade erythritol, etc.), food processing (fresh maize, maize starch, maize oil, etc., which not only provides rich raw materials for the food industry, but also meets the diverse dietary needs of people), and animal husbandry (the high nutritional value of maize makes it an indispensable part of livestock feeding and an important part of livestock feed). It also plays a crucial role in stabilizing agricultural product prices and ensuring national food security.

[0003] Continuously developing higher-yield cultivation and management techniques and cultivating high-yield new varieties are the eternal goals of Chinese scientific researchers. Traditional cultivated maize has tall plants and spreading leaves, which is not conducive to high-density planting and production. The dwarfing and close planting of maize can improve land utilization rate and water and fertilizer utilization rate, and save resources by optimizing the crop planting structure. In addition, due to the shorter plants and upright leaves of dwarfing and close planting maize, it can effectively improve the canopy structure of the maize population, enhance ventilation and light transmission, thereby improving the lodging resistance of the plants, reducing the occurrence of pests and diseases, and significantly increasing the yield and economic benefits per unit area.

[0004] By cultivating new varieties and improving the plant type structure of crops, it is the main way to improve the density tolerance of crops and has important practical significance for increasing crop yields. Traditional breeding methods are time-consuming and have a long cycle, with unpredictable results. The rapid development of transgenic technology provides an important way to improve crop yields, stress resistance, and disease resistance. Obtaining varieties with improved maize plant type genetically by using genetic engineering technology and then increasing the yield of maize has become an important way of modern genetic breeding. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of the maize ZmArf1a gene in the breeding of maize varieties, and to provide a candidate gene related to maize plant height and plant type for the cultivation of new dwarfing and close planting maize varieties.

[0006] To achieve the above purpose, the present invention provides the application of the maize ZmArf1a gene in the breeding of maize varieties. The CDS sequence of the maize ZmArf1a gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0007] Preferably, the breeding of maize varieties refers to the breeding of dwarf and high-density maize varieties.

[0008] Preferably, knocking out the maize ZmArf1a gene or inhibiting the expression of the maize ZmArf1a gene results in a decrease in maize plant height, a shortening of leaf length, and a narrowing of leaf width.

[0009] Preferably, homologous recombination or CRISPR-Cas9 technology is used for knocking out. After knocking out the maize ZmArf1a gene, the protein translated by the gene has no original function or cannot be translated into a protein.

[0010] Preferably, the inhibition of maize ZmArf1a gene expression is achieved by antisense RNA technology or interfering RNA technology.

[0011] The application of the vector and / or strain containing the above maize ZmArf1a gene in the breeding of dwarf and high-density maize varieties is characterized in that: the vector is a knockout vector of the maize ZmArf1a gene, and the strain is Escherichia coli and / or Agrobacterium tumefaciens containing the maize ZmArf1a gene.

[0012] Therefore, the application of the maize ZmArf1a gene provided by the present invention in the breeding of maize varieties has the following specific technical effects:

[0013] (1) The present invention first discovers that the plants with the maize ZmArf1a gene knocked out grow and develop normally, the tassels are fertile, the ear axes and grains of the female ears are normal and plump, but the leaf length, leaf width and plant height are significantly reduced;

[0014] (2) The maize ZmArf1a gene provided by the present invention can be used as a candidate gene for the breeding of dwarf and high-density maize varieties, which is expected to reduce the breeding workload and accelerate the breeding process.

[0015] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1 It is the gRNA sequencing result of the T1 generation transgenic lines in Example 2 of the present invention; where arf1a ko1 and arf1a ko2 are knockout lines, Arfla NTwas the control group; in the figure, the blue lines and blue text represent target 1, the green lines and green text represent target 2, the black dashed lines represent the omitted normal sequences, the text in black with a blue background represents PAM (Proto-spacer adjacent motif), the red text represents the mutation sites, the blue and green "-" represent the deletion sites, and the orange rectangle represents the Arf domain;

[0018] Figure 2 is Arf1a in Example 3 of the present invention NT1 and arf1a ko1 Phenotype photos and statistical results of the Arf1a and arf1a family plants; where a is a photo of a maize plant at the milk-ripe stage, Bar = 10 cm; b is a photo of the stem of the ear node and the two lower nodes; c is a photo of the ear leaf; d is a histogram of the plant height of the maize plant; e is a histogram of the ear height of the maize plant; f is a histogram of the stem diameter of the maize plant; g is a histogram of the leaf width of the maize plant; h is a histogram of the leaf length of the maize plant; i is a histogram of the number of leaves of the maize plant; where *** indicates extremely significant differences;

[0019] Figure 3 is Arf1a in Example 3 of the present invention NT1 and arf1a ko1 Phenotype diagrams of the Arf1a and arf1a family ears; where a is a photo of a mature female ear, Bar = 2 cm; b is a photo of a mature male ear, bar = 2 cm; c is a photo of a young ear of maize, Bar = 1 mm; d is a histogram of the length of the maize male ear; e is a histogram of the number of branches of the maize male ear; f is a histogram of the length of the maize female ear; g is a histogram of the ear diameter of the maize; h is a histogram of the number of grains per row; i is a histogram of the number of rows of ears;

[0020] Figure 4 is Arf1a in Example 3 of the present invention NT2 and arf1a ko2 Phenotype diagrams of the Arf1a and arf1a family plants and ears; a is a photo of a maize plant, Bar = 10 cm; b is a photo of a mature female ear, Bar = 2 cm; c is a photo of a maize leaf, Bar = 5 cm; d is a photo of the stem of the ear node and the two lower nodes of the plant, bar = 5 cm; e is a photo of a male ear, bar = 2 cm; f is a histogram of the length of the maize female ear; g is a histogram of the ear diameter of the maize; h is a histogram of the number of grains per row of the maize; i is a histogram of the number of rows of ears of the maize; j is a histogram of the length of the maize male ear; k is a histogram of the plant height of the maize plant; l is a histogram of the ear height of the maize plant; m is a histogram of the stem diameter of the maize plant; n is a histogram of the leaf length of the maize plant; o is a histogram of the leaf width of the maize plant; p is a histogram of the number of leaves of the maize plant. Detailed implementation manners

[0021] The technical solutions of the present invention will be further described below with reference to the drawings and examples.

[0022] To make the objectives, technical solutions and advantages of this application more clear, thorough and complete, the technical solutions of the present invention will be clearly and completely described below through the accompanying drawings and embodiments. The following detailed descriptions are all descriptions of embodiments, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0023] The instrument equipment and reagent materials used in the embodiments are all obtained through commercial channels; the method steps not described in detail in the embodiments are all conventional technical means in this field.

[0024] Example 1

[0025] Clone the ZmArf1a gene, specifically as follows:

[0026] Extract the total DNA of the leaves of the maize inbred line KN5585 plants by the CTAB method. Design primers Arf1a-F (the sequence is shown in SEQ ID NO.1) and Arf1a-R (the sequence is shown in SEQ ID NO.2) according to the genomic reference sequence of maize B73 (National Crop Germplasm Center). Using the extracted DNA of KN5585 plants as a template and Arf1a-F and Arf1a-R as primers for PCR, the PCR system is 15 μL, and it is configured according to the instructions attached to the Taq enzyme. The PCR amplification program is: pre-denaturation at 94 °C for 5 min; then denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 60 s, for 34 cycles; finally, extension at 72 °C for 5 min. Send the PCR product to the company for Sanger sequencing to obtain the complete nucleotide sequence of the ZmArf1a gene in the KN5585 material. The CDS sequence of the ZmArf1a gene in the KN5585 material is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0027] SEQ ID NO.3:

[0028] ATGGGGCTCTCCTTTGGGAAGCTGTTCAGCCGCCTCTTCGCCAAGAAGGA

[0029] GATGAGGATTCTCATGGTCGGGCTCGATGCCGCCGGTAAGACCACCATCC

[0030] TCTATAAGCTCAAGCTCGGCGAGATCGTCACCACCATCCCCACCATTGGA

[0031] TTCAATGTTGAAACTGTTGAGTACAAGAACATTAGCTTCACCGTTTGGGAT

[0032] GTTGGTGGCCAGGACAAGATCAGGCCCCTGTGGAGGCACTACTTTCAGAA

[0033] CACGCAGGGACTTATTTTTGTTGTAGACAGCAATGATAGGGAACGTGTTG

[0034] TTGAGGCTAGAGATGAGCTCCACAGGATGCTGAATGAGGATGAGCTGCGT

[0035] GATGCCGTGCTGCTTGTATTTGCAAACAAACAAGATCTTCCCAATGCTATG

[0036] AATGCTGCTGAAATTACTGACAAGCTTGGTCTGCATTCTCTTCGCCAGCGG

[0037] CACTGGTACATCCAGAGCACTTGTGCTACATCTGGTGAAGGGTTGTATGA

[0038] GGGGCTTGATTGGCTTTCCAACAACATCGCCAACAAGTCTTGA

[0039] SEQ ID NO.4:

[0040] MGLSFGKLFSRLFAKKEMRILMVGLDAAGKTTILYKLKLGEIVTTIPTIGFNVE

[0041] TVEYKNISFTVWDVGGQDKIRPLWRHYFQNTQGLIFVVDSNDRERVVEARDE

[0042] LHRMLNEDELRDAVLLVFANKQDLPNAMNAAEITDKLGLHSLRQRHWYIQS

[0043] TCATSGEGLYEGLDWLSNNIANKS

[0044] Table 1 Primers and Their Sequences

[0045]

[0046] Example 2

[0047] The ZmArf1a gene was genetically transformed in maize as follows:

[0048] S21. For the genetic transformation of ZmArf1a gene knockout, the full-length CDS sequence of ZmArf1a (the sequence is shown as SEQ ID NO.3) in the transgenic receptor material KN5585 was used as the applied gene sequence. According to the website http: / / cbi.hzau.edu.cn / crispr / , gene target design was carried out, and finally two Guide RNAs were obtained: Target-1 (the sequence is shown as SEQ ID NO.5 respectively) and Target-2 (the sequence is shown as SEQ ID NO.6).

[0049] SEQ ID NO.5:CCCCACCATTGGTAAGCCTTTTCCC

[0050] SEQ ID NO.6:AGAACATTAGCTTCACCGTTTGGG

[0051] S22. Two Target primers were designed using the two Guide RNA sequences respectively, namely Target-1F (the sequence is shown as SEQ ID NO.7) and Target-2F (the sequence is shown as SEQ ID NO.8). Using the CPB-ZmUbi-hspCas9 vector as a template, the first and second ZmU6 promoters were amplified using pU6F1 (the sequence is shown as SEQ ID NO.9) and pU6R (the sequence is shown as SEQ ID NO.10), pU6F2 (the sequence is shown as SEQ ID NO.11) and pU6R; the first and second Target+SgRNA fragments were amplified using Target-1F and gRR0 (the sequence is shown as SEQ ID NO.12), Target-2F and gRR1 (the sequence is shown as SEQ ID NO.13) respectively. The ZmU6-Target1-sgRNA and ZmU6-Target2-sgRNA fragments were amplified by overlap using pU6F1 and gRR0, pU6F2 and gRR1, and were multi-homologously recombined onto the CPB-ZmUbi-hspCas9 vector digested with HindIII (the construction method is shown in Chinese invention patent CN116574754A).

[0052] The constructed vector was transferred into Escherichia coli for conventional culture. After colonies grew out, monoclonal colonies were picked. The obtained clones were sequenced using the CRISPR vector detection primers CRISPR-F (the sequence is shown in SEQ ID NO.14) and CRISPR-R (the sequence is shown in SEQ ID NO.15), and the clones with the gene correctly ligated to the vector were determined as positive clones. The plasmids of the positive clones were genetically transformed into the maize inbred line KN5585 by the Agrobacterium-mediated method.

[0053] The obtained genetically transformed seedlings were planted in the field. The gRNA of each plant was extracted using a kit and sent to the company for sequencing detection. Two maize transformation events with KN5585 as the background were obtained. The harvested seeds were the T1 generation, denoted as the stably edited transgenic event ZmArf1a-KO, and were named arf1a ko1 and arf1a ko2 . The materials without editing in the same event were used as the control ZmArf1a-NT.

[0054] In the spring of 2023, the stably edited transgenic events were planted, and the genotypes of the transgenic T1 generation of ZmArf1a-KO were detected. The results are as Figure 1 shown. The CDS sequence of the arf1a ko1 line is shown in SEQ ID NO.16, and the CDS sequence of the arf1a ko2 line is shown in SEQ ID NO.17. The editing type of arf1a ko1 is a 1-bp base deletion at target site 1 and a 2-bp base deletion at target site 2. The deletion causes the amino acids GFNVETVEYKNISFT (the sequence is shown in SEQ ID NO.18) at positions 50-64 to mutate to DSMLKLLSTRTLAS (the sequence is shown in SEQ ID NO.19), and the 63rd amino acid is deleted, resulting in changes in the G2 loop and G3 loop structures of the conserved domain of the Arf1a protein, which may lead to the loss of the function of the ZmArf1a gene. The G2 loop connects the A1 helix and B2 strand of the Arf1a protein and contains a conserved Thr residue responsible for the binding of Mg ko1 ; the G3 loop provides residues for the binding of Mg 2+ and γ-phosphate and is located at the N-terminus of the A2 helix. 2+

[0055] The editing type of arf1a ko2 is a 1-bp base deletion at target site 1 and a 3-bp deletion at target site 2, resulting in a frameshift mutation that causes premature termination of translation and forms a truncated protein composed of 90 amino acids.

[0056] SEQ ID NO.16: ​

[0057] ATGGGGCTCTCCTTTGGGAAGCTGTTCAGCCGCCTCTTCGCCAAGAAGGA

[0058] GATGAGGATTCTCATGGTCGGGCTCGATGCCGCCGGTAAGACCACCATCCT

[0059] CTATAAGCTCAAGCTCGGCGAGATCGTCACCACCATCCCCACATTGGATTC

[0060] AATGTTGAAACTGTTGAGTACAAGAACATTAGCTTCCGTTTGGGATGTTGG

[0061] TGGCCAGGACAAGATCAGGCCCCTGTGGAGGCACTACTTTCAGAACACGC

[0062] AGGGACTTATTTTTGTTGTAGACAGCAATGATAGGGAACGTGTTGTTGAGG

[0063] CTAGAGATGAGCTCCACAGGATGCTGAATGAGGATGAGCTGCGTGATGCCG

[0064] TGCTGCTTGTATTTGCAAACAAACAAGATCTTCCCAATGCTATGAATGCTGC

[0065] TGAAATTACTGACAAGCTTGGTCTGCATTCTCTTCGCCAGCGGCACTGGTA

[0066] CATCCAGAGCACTTGTGCTACATCTGGTGAAGGGTTGTATGAGGGGCTTGA

[0067] TTGGCTTTCCAACAACATCGCCAACAAGTCTTGA

[0068] SEQ ID NO.17:

[0069] ATGGGGCTCTCCTTTGGGAAGCTGTTCAGCCGCCTCTTCGCCAAGAAGGA

[0070] GATGAGGATTCTCATGGTCGGGCTCGATGCCGCCGGTAAGACCACCATCCT

[0071] CTATAAGCTCAAGCTCGGCGAGATCGTCACCACCATCCCCACATTGGATTC

[0072] AATGTTGAAACTGTTGAGTACAAGAACATTAGCTTCGTTTGGGATGTTGGT

[0073] GGCCAGGACAAGATCAGGCCCCTGTGGAGGCACTACTTTCAGAACACGCA

[0074] GGGACTTATTTTTGTTGTAGACAGCAATGATAGGGAACGTGTTGTTGAGGC

[0075] TAGAGATGAGCTCCACAGGATGCTGAATGAGGATGAGCTGCGTGATGCCGT

[0076] GCTGCTTGTATTTGCAAACAAACAAGATCTTCCCAATGCTATGAATGCTGCT

[0077] GAAATTACTGACAAGCTTGGTCTGCATTCTCTTCGCCAGCGGCACTGGTAC

[0078] ATCCAGAGCACTTGTGCTACATCTGGTGAAGGGTTGTATGAGGGGCTTGAT

[0079] TGGCTTTCCAACAACATCGCCAACAAGTCTTGA

[0080] Example 3

[0081] Examine the phenotypes of the 2 ZmArf1a gene knockout plants obtained in Example 2, which are specifically as follows:

[0082] Phenotypic investigations were carried out on two stable editing events of ZmArf1a CRISPR / Cas9 T1 generation. Important agronomic traits were investigated during the grain formation stage and milk-ripe stage of maize. Mainly including plant height, ear height, leaf length of the ear leaf, leaf width of the ear leaf, number of leaves, etc. The statistical results are shown in Table 2 and Table 3. Data 3sigma rule: The values within the range of mean plus or minus three standard deviations are normal values, and others are abnormal values. After removing the abnormal values, GraphPad Prism software (GraphPad Prism 8.0.2.263) was used to statistically analyze the data, and the results are as Figures 2 to 4 shown. Among them, leaf length is the length from the leaf sheath to the leaf tip of the ear leaf; plant height is the length from the base stem node of the plant to the top of the tassel; ear height is the length from the base stem node of the plant to the ear stem node; stem diameter is the average of the diameters at the middle of the first stem node above the ground and the middle of the stem node where the ear is located; leaf width is the length at the widest part of the ear leaf; number of leaves is the number of whole-plant leaves at the silking stage; ear length is the length of the female ear at the first ear position harvested; ear diameter is the diameter at the thickest part of the female ear at the first ear position harvested; number of ear rows is the number of grains in the cross-section at the middle of the female ear at the first ear position harvested; number of grains per row is the number of grains in the longitudinal section of the female ear at the first ear position harvested; tassel length is the length from the base to the top of the tassel; maize young ear photo is a photo of the female ear at the 8-leaf stage.

[0083] The results showed that the plant height of arf1a ko1 was 137.49±12.37 cm (n = 19), the ear height was 60.79±9.03 cm (n = 19), the stem diameter was 12.08±2.65 mm (n = 14), the leaf length was 58.37±5.81 cm (n = 14), the leaf width was 7.41±0.75 cm (n = 14), the number of leaves was 11.58±0.9 (n = 15), while the plant height of Arf1a NT1 was 162.46±12.12 cm (n = 23), the ear height was 69.17±5.02 cm (n = 23), the stem diameter was 15.21±2.16 mm (n = 14), the leaf length was 71.41±2.78 cm (n = 20), the leaf width was 8.86±0.98 cm (n = 20), and the number of leaves was 12.52±0.73 (n = 23). The internode length and number of internodes of Arf1a NT1 and arf1a ko1 plants were statistically analyzed, and it was found that there was a significant difference in the number of internodes between the arf1a ko1 mutant and the control (0.01 < P < 0.05), and there were extremely significant and significant differences in the 4th, 8th, 9th, 11th, and 12th internodes of the internode length, and there was no significant difference between the remaining internodes (P > 0.05) (Table 3). The above results indicate that arf1a ko1The plant height (P = 1.03E-08), ear height (P = 1.75E-06), stem diameter (P = 1.5E-05), leaf length (P = 8.75E-13) and leaf width (P = 6.84E-06) of the plants showed extremely significant differences (P<0.01) compared with the control. The plant height and ear height became shorter, while the stem diameter, leaf length and leaf width became thinner, shorter and narrower ( Figure 2 , Table 2).

[0084] Table 2 Statistical analysis of important phenotypes of ZmArf1a CRISPR / Cas9 families

[0085] Trait <![CDATA[Arf1a NT1 > <![CDATA[arf1a ko1 > KO-NT P-value N PH (cm) 162.46±12.12 137.49±12.37 -24.97 1.03E-08 23 / 19 EH (cm) 69.17±5.02 60.79±9.03 -8.38 1.75E-06 23 / 19 EL (cm) 12.02±1.49 9.54±1.19 -2.48 9.5E-06 14 / 8 EW (cm) 4.23±0.41 3.75±0.51 -0.475 3.28E-05 14 / 8 TL (cm) 30.02±2.98 24.96±5.02 -5.06 8.19E-04 16 / 12 TBN 6.74±1.57 4.42±1.44 -2.52 3.62E-04 16 / 12 KNR 21.11±3.72 17.45±2.73 -3.66 0.014 12 / 10 KRN 15.39±1.58 14.45±1.24 -0.95 0.049 16 / 9 LL (cm) 71.41±2.78 58.37±5.81 -13.04 8.57E-13 20 / 14 LW (cm) 8.86±0.98 7.41±0.75 -1.45 6.84E-06 20 / 14 Trait <![CDATA[Arf1a NT2 > <![CDATA[arf1a ko2 > KO-NT P-value N PH (cm) 168.06±11.53 150.08±22.13 -17.99 0.002 32 / 10 EH (cm) 74.65±7.27 63.93±12.74 -10.73 0.013 32 / 10 EL (cm) 12.55±1.53 10.84±2.15 -1.71 0.020 16 / 7 EW (cm) 4.14±0.49 3.89±0.38 -0.45 0.029 16 / 7 TL (cm) 30.53±2.02 28.16±4.78 -1.93 0.478 28 / 10 TBN 6.11±1.59 5.8±1.55 -0.31 0.731 28 / 10 KNR 21±3.27 17.57±4.93 -3.43 0.039 16 / 7 KRN 16±2.63 14±2 -2.00 0.044 16 / 7 LL (cm) 65.05±6.86 62.78±5.89 -2.29 0.006 7 / 26 LW (cm) 8.93±1.32 8.09±0.40 -0.84 0.062 7 / 26

[0086] Note: PH represents plant height, EH represents ear height, EL represents ear length, EW represents ear diameter, TL represents tassel length, TBN represents tassel branch number, KNR represents kernel number per row, KRN represents row number of ear, LL represents leaf length, LW represents leaf width, KO-NT represents the difference between the average values of KO and NT, and N represents the number of negative single plants / positive single plants. Bold fonts in the table indicate P<0.05.

[0087] The tassel length and tassel branch number were measured and statistically analyzed, and indoor ear evaluation was carried out after harvesting the maize ears, including measuring the ear length, ear diameter, kernel number per row and row number of ear. The tassel length of arf1a ko1 was 24.96±5.02 cm (n = 12), the tassel branch number was 4.42±1.44 (n = 12), the ear length of the female ear was 9.54±1.19 cm (n = 8), the ear diameter was 3.75±0.51 cm (n = 8), the kernel number per row was 17.45±2.73 grains (n = 10), and the row number of ear was 14.45±1.24 grains (n = 10). The tassel length of Arf1a NT1 was 30.02±2.98 cm (n = 16), the tassel branch number was 6.74±1.57 (n = 16), the ear length of the female ear was 12.02±1.49 cm (n = 14), the ear diameter was 4.23±0.41 cm (n = 14), the kernel number per row was 21.11±3.72 grains (n = 12), and the row number of ear was 15.39±1.58 grains (n = 16). The results showed that there were extremely significant differences in the tassel length (P = 8.193E-04), tassel branch number (P = 3.621E-04), ear length of the female ear (P = 9.5E-06), and ear diameter (P = 3.28E-05) between arf1a ko1 and Arf1a NT1 (P<0.01), and significant differences in the kernel number per row (P = 0.014) and row number of ear (P = 0.049) (P<0.05). That is, the tassel length was shorter, the tassel branch number was fewer, the ear length and ear diameter of the female ear were shorter and narrower, and the kernel number per row and row number of ear were fewer ( Figure 3 , Table 3).

[0088] Statistics of Stem Internode Length and Number of Internodes in the ZmArf1a CRISPR / Cas9 Family

[0089]

[0090]

[0091] Note: The first internode is the internode connecting to the tassel, i.e., the top of the stem. Since the number of stem internodes in the control and mutants is inconsistent, only the internode values that both have are listed. The bold font in the table indicates the value of P < 0.05.

[0092] arf1a ko2 The plant height of arf1a is 150.08 ± 22.13 cm (n = 10), the ear height is 63.93 ± 12.74 cm (n = 10), the stem diameter is 15.02 ± 3.58 mm (n = 9), the leaf length is 62.78 ± 5.89 cm (n = 9), the leaf width is 8.93 ± 1.32 cm (n = 9), and the number of leaves is 12.11 ± 1.17 (n = 9). Arf1a NT2 The plant height of Arf1a is 168.06 ± 11.53 cm (n = 32), the ear height is 74.65 ± 7.27 cm (n = 32), the stem diameter is 14.38 ± 2.02 mm (n = 32), the leaf length is 65.05 ± 6.86 cm (n = 26), the leaf width is 8.09 ± 0.40 cm (n = 26), and the number of leaves is 12.35 ± 0.61 (n = 26). The above results show that arf1a ko2 has significant differences in plant height (P = 0.002), ear height (P = 0.013), and leaf length (P = 0.006) compared with Arf1a NT2 (p < 0.05); there is no significant difference in leaf width (P = 0.062) between arf1a ko2 and Arf1a NT2 (P > 0.05). The stem internode length and number of stem internodes of Arf1a NT2 and arf1a ko2 plants were statistically analyzed, and it was found that there was no significant difference in the total number of internodes between arf1a ko2 and Arf1a NT2 (P > 0.05), and there were extremely significant differences between the 7th and 8th internodes of arf1a ko2 and Arf1a NT2 (P < 0.001), and significant differences between the 10th and 11th internodes (0.01 < P < 0.05). Therefore, arf1a ko2 and Arf1a NT2The difference in plant height among plants is caused by the difference in the length of the 7th, 8th, 10th, and 11th internodes counted from the base (Table 3).

[0093] arf1a ko2 has a tassel length of 228.16 ± 4.78 cm (n = 10), a tassel branch number of 5.8 ± 1.55 (n = 10), an ear length of 10.84 ± 2.15 cm (n = 7), an ear diameter of 3.89 ± 0.38 cm (n = 7), a grain number per row of 17.57 ± 4.93 grains (n = 7), and an ear row number of 14 ± 2 grains (n = 7). Arf1a NT2 has a tassel length of 30.53 ± 2.02 cm (n = 14), a tassel branch number of 6.11 ± 1.59 (n = 28), an ear length of 12.55 ± 1.53 cm (n = 14), an ear diameter of 4.14 ± 0.49 cm (n = 15), a grain number per row of 21 ± 3.27 grains (n = 19), and an ear row number of 16 ± 2.63 grains (n = 19). The results show that arf1a ko2 and Arf1a NT2 have no significant difference in the phenotypes of tassel length (P = 0.478) and tassel branch number (P = 0.731) (P > 0.05), and arf1a ko2 and Arf1a NT2 have significant differences in ear length (P = 0.020), ear diameter (P = 0.029), grain number per row (P = 0.039), and ear row number (P = 0.044) (P < 0.05) ( Figure 4 , Table 2), and also show that the ear length and ear diameter are shorter and narrower, and the grain number per row and ear row number are fewer.

[0094] The above results show that the changes in the G2 and G3 structures in arf1a ko1 significantly affect the vegetative and reproductive growth of mutant plants. The frameshift mutation caused by the base deletion in arf1a ko2 makes the mutant significantly different from the control plants and ear phenotypes. The difference in plant height causes differences in nutrient absorption and utilization by plants. Therefore, the mutation in arf1a ko affects the vegetative and reproductive growth of mutant plants.

[0095] Therefore, the present invention for the first time discovers that plants with the maize ZmArf1a gene knocked out grow and develop normally, have fertile tassels, and the ear axes and grains are normal and plump, but the leaf length, leaf width, and plant height are significantly reduced; the provided maize ZmArf1a gene can be used as a candidate gene for breeding dwarf and dense planting maize varieties, and is expected to reduce the breeding workload and accelerate the breeding process.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of the maize ZmArf1a gene in maize variety breeding, characterized in that: The CDS sequence of the maize ZmArf1a gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

2. Use of the maize ZmArf1a gene in maize variety breeding according to claim 1, characterized in that: Maize variety breeding refers to breeding dwarf and high-density maize varieties.

3. Use of the maize ZmArf1a gene according to claim 1 in maize variety breeding, characterized in that: Knocking out the maize ZmArf1a gene or inhibiting the expression of the maize ZmArf1a gene results in a decrease in maize plant height, a shortening of leaf length, and a narrowing of leaf width.

4. Use of the maize ZmArf1a gene according to claim 3 in maize variety breeding, characterized in that: Knocking out is carried out using homologous recombination or CRISPR-Cas9 technology. After knocking out the maize ZmArf1a gene, the protein translated by the gene has no original function or cannot be translated into a protein.

5. Use of the maize ZmArf1a gene according to claim 3 in maize variety breeding, characterized in that: Inhibiting the expression of the maize ZmArf1a gene is achieved through antisense RNA technology or interfering RNA technology.

6. Use of the vector and / or strain containing the maize ZmArf1a gene described in claim 1 in the breeding of maize varieties with dwarfing and high-density planting, characterized in that: The vector is a knockout vector for the maize ZmArf1a gene, and the strains are Escherichia coli and / or Agrobacterium containing the maize ZmArf1a gene.

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