Application of maize ZmArf1a gene in maize variety selection

Abstract

The application discloses application of a maize ZmArf1a gene in maize variety breeding and belongs to the technical field of molecular breeding. The CDS sequence of the maize ZmArf1a gene is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4. The application first finds that the plant with the knocked-out maize ZmArf1a gene grows normally, the tassel is fertile, the female spike axis and the grain are normal and full, but the leaf length, the leaf width and the plant height are significantly reduced. The provided maize ZmArf1a gene can be used as a candidate gene for breeding a dwarf and dense planting maize variety, and is expected to reduce the workload of breeding and accelerate the breeding process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular breeding, and particularly relates to application of a maize ZmArf1a gene in maize variety breeding. BACKGROUND

[0002] Maize is one of the largest grain crops in China, which not only provides a large amount of food, but also plays an important role in many 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 corn, corn starch, corn oil, etc., which not only provides rich raw materials for the food industry, but also meets people's diverse dietary needs), animal husbandry (the high nutritional value of maize makes it an indispensable part of livestock feeding, and is an important component of animal feed), and stabilizes agricultural product prices and ensures national food security.

[0003] Developing higher-yield cultivation and management techniques and cultivating high-yield new varieties is an eternal goal of Chinese scientific researchers. Traditional maize cultivation has tall plants and spread leaves, which is not conducive to high-density planting production. Maize dwarfing and high-density planting can improve land utilization rate, water and fertilizer utilization rate, and save resources. In addition, since the maize plants of dwarfing and high-density planting are shorter and the leaves are straight, the crown structure of the maize population can be effectively improved, the ventilation and light transmission are enhanced, the lodging resistance of the plants is provided, the occurrence of diseases and pests is reduced, and thus the yield per unit area and economic benefits are significantly improved.

[0004] Through cultivation of new varieties and improvement of crop plant type structure, the main way to improve the tolerance of crops to dense planting has important practical significance for improving crop yield. Traditional breeding methods are time-consuming, long-cycle, and have unpredictable results, and the rapid development of transgenic technology provides an important way for improving crop yield, stress resistance and disease resistance. Using genetic engineering technology to obtain a variety with improved maize plant type in genetics and then to improve the yield of maize has become an important way of modern genetic breeding. SUMMARY

[0005] The purpose of the present application is to provide application of a maize ZmArf1a gene in maize variety breeding, and to provide a candidate gene related to maize plant height and plant type for breeding of new dwarf and high-density maize varieties.

[0006] To achieve the above purpose, the present application provides application of a maize ZmArf1a gene in maize variety breeding, 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 corn variety breeding refers to breeding a dwarf and high-density planting corn variety.

[0008] Preferably, after knocking out or inhibiting the expression of the corn ZmArf1a gene, the corn plant height is reduced, the leaf length is shortened, and the leaf width is narrowed.

[0009] Preferably, the knocking out is performed by homologous recombination or CRISPR-Cas9 technology, and after knocking out the corn ZmArf1a gene, the translated protein has no original function or cannot be translated into a protein.

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

[0011] The application of the vector and / or strain containing the corn ZmArf1a gene in the breeding of a dwarf and high-density planting corn variety, characterized in that the vector is a knockout vector of the corn ZmArf1a gene, and the strain is an Escherichia coli and / or Agrobacterium containing the corn ZmArf1a gene.

[0012] Therefore, the application provides the application of the corn ZmArf1a gene in the breeding of a corn variety, and the specific technical effects are as follows:

[0013] (1) The application first discovers that the plant with the knocked-out corn ZmArf1a gene has normal growth and development, the male spike is fertile, the female spike axis and the kernel are normal and full, but the leaf length, the leaf width and the plant height are significantly reduced.

[0014] (2) The corn ZmArf1a gene provided by the application can be used as a candidate gene for breeding a dwarf and high-density planting corn variety, and is expected to reduce the workload of breeding and accelerate the breeding process.

[0015] The technical solutions of the application will be further described in detail below with the aid of the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the description of the embodiments of the application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0017] Figure 1 is the gRNA sequencing result of the T1 generation transgenic strain in Example 2 of the application; wherein arfla ko1 and arfla ko2 is the knockout strain, Arfla NTFigure 1 shows the results of the analysis of the Arf1a gene in the control group; the blue line and blue text in the figure represent target point 1, the green line and green text represent target point 2, the black dotted line represents the omitted normal sequence, the black font with a blue background represents PAM (Proto-spacer adjacent motif), the red font represents the mutation site, and the blue and green "-" represents the deletion site, and the orange rectangle represents the Arf domain;

[0018] Figure 2 Arf1a in Example 3 of the present application NT1 and arfla ko1 Family plant phenotype photos and statistical results; wherein a is a corn plant photo at the milk stage, Bar = 10 cm; b is a photo of the ear node and the two nodes of the stem below it; c is a photo of the ear leaf; d is a column chart of the corn plant height; e is a column chart of the corn plant ear height; f is a column chart of the corn plant stem thickness; g is a column chart of the corn plant leaf width; h is a column chart of the corn plant leaf length; i is a column chart of the number of corn plant leaves; wherein *** indicates a significant difference;

[0019] Figure 3 Arf1a in Example 3 of the present application NT1 and arfla ko1 Family ear phenotype chart; wherein a is a mature female ear photo, Bar = 2 cm; b is a male ear photo at the mature stage, bar = 2 cm; c is a corn earlet photo, Bar = 1 mm; d is a column chart of the corn male ear length; e is a column chart of the number of corn male ear branches; f is a column chart of the corn female ear length; g is a column chart of the corn ear thickness; h is a column chart of the row kernel number; i is a column chart of the ear row number;

[0020] Figure 4 Arf1a in Example 3 of the present application NT2 and arfla ko2 Family plant and ear phenotype chart; a is a corn plant photo, Bar = 10 cm; b is a mature female ear photo, Bar = 2 cm; c is a corn leaf photo, Bar = 5 cm; d is a photo of the plant ear node and the two nodes of the stem below it, bar = 5 cm; e is a male ear photo, bar = 2 cm; f is a column chart of the corn female ear length; g is a column chart of the corn ear thickness; h is a column chart of the corn row kernel number; i is a column chart of the corn ear row number; j is a column chart of the corn male ear length; k is a column chart of the corn plant height; l is a column chart of the corn plant ear height; m is a column chart of the corn plant stem thickness; n is a column chart of the corn plant leaf length; o is a column chart of the corn plant leaf width; p is a column chart of the number of corn plant leaves. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are further described below through the drawings and examples.

[0022] In order to make the purposes, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application are described clearly and completely below through the drawings and examples. The following detailed description is the description of examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meanings as generally understood by those skilled in the art to which the present application belongs.

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

[0024] Example 1

[0025] The ZmArf1a gene was cloned as follows:

[0026] The total DNA of the corn inbred line KN5585 plant leaves was extracted by the CTAB method, the primer Arf1a-F (the sequence is shown as SEQ ID NO. 1) and Arf1a-R (the sequence is shown as SEQ ID NO. 2) were designed according to the corn B73 (National Crop Germplasm Center) genome reference sequence, and the extracted KN5585 plant DNA was used as a template, and the Arf1a-F and Arf1a-R primers were used for PCR, and the PCR system was 15 μL, which was configured according to the instruction manual of Taq enzyme. The PCR amplification program was: 94°C pre-denaturation for 5 min; then 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 60 s, for 34 cycles; finally 72°C extension for 5 min. The PCR product was sent to the company for Sanger sequencing, and the complete nucleotide sequence of the ZmArf1a gene in the KN5585 material was obtained. The CDS sequence of the ZmArf1a gene in the KN5585 material is shown as SEQ ID NO. 3, and the amino acid sequence is shown as 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 ZmArfla gene was genetically transformed in maize as follows:

[0048] S21, genetic transformation of ZmArfla gene knockout uses the full-length CDS sequence of ZmArfla in transgenic receptor material KN5585 (the sequence is shown as SEQ ID NO. 3) as the application gene sequence, and the gene target is designed according to the website http: / / cbi.hzau.edu.cn / crispr / , and finally two Guide RNA are obtained: Target-1 (the sequence is shown as SEQ ID NO. 5) 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 are designed using two Guide RNA sequences, respectively, Target-1F (the sequence is shown as SEQ ID NO. 7) and Target-2F (the sequence is shown as SEQ ID NO. 8). The first and second ZmU6 promoters are 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, using CPB-ZmUbi-hspCas9 vector as a template; the first and second Target+SgRNA fragments are 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. ZmU6-Target1-sgRNA, ZmU6-Target2-sgRNA fragments are amplified using pU6F1 and gRR0, pU6F2 and gRR1 for overlap, and are multi-homologous recombined into the CPB-ZmUbi-hspCas9 vector (the construction method is shown in Chinese invention patent CN116574754A) digested by HindIII enzyme.

[0052] The constructed vector was transformed into *E. coli* and cultured using standard methods. Once colonies appeared, single clones were picked and sequenced using CRISPR-F (sequence shown in SEQ ID NO. 14) and CRISPR-R (sequence shown in SEQ ID NO. 15) primers. Clones whose genes were correctly ligated into the vector were identified as positive clones. The plasmids of positive clones were then genetically transformed into the maize inbred line KN5585 using Agrobacterium-mediated transformation.

[0053] The genetically transformed seedlings were planted in the field, and gRNA was extracted from each plant using a kit. The extracted gRNA was sent to the company for sequencing, yielding two maize transformation events with KN5585 as the background. The harvested seeds were from the T1 generation, designated as the stable-editing transgenic event ZmArf1a-KO, and named arf1a. ko1 and arf1a ko2 Edited materials from the same event that did not occur are used as a reference (ZmArf1a-NT).

[0054] In the spring of 2023, stable-edited transgenic plants were planted, and genotyping was performed on the T1 generation of the ZmArf1a-KO transgenic strain. The results are as follows... Figure 1 As shown, arf1a ko1 The CDS sequence of the strain is shown in SEQ ID NO.16, arf1a ko2 The CDS sequence of the strain is shown in SEQ ID NO.17. arf1a ko1 The edit type is a 1bp deletion at target 1 and a 2bp deletion at target 2, arf1a ko1 The deletion results in a mutation at amino acids 50-64, GFNVETVEYKNISFT (sequence shown in SEQ ID NO.18), into DSMLKLLSTRTLAS (sequence shown in SEQ ID NO.19), and a deletion at amino acid 63. This leads to structural changes in the G2 and G3 loops of the conserved domain of the Arf1a protein, potentially resulting in the loss of function of the ZmArf1a gene. The G2 loop connects the A1 helix and B2 chain of the Arf1a protein and contains a conserved Thr residue responsible for Mg... 2+ The combination; the G3 ring is Mg 2+ It provides residues by binding with γ-phosphate, located at the N-terminus of the A2 helix.

[0055] arf1a ko2 The editing type is a 1bp deletion at target site 1 and a 3bp deletion at target site 2, resulting in a frameshift mutation that causes premature termination of translation, forming a truncated protein 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] The phenotype of the two ZmArfla gene knockout plants obtained in Example 2 was investigated, 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 and milk ripening stages of maize. These mainly included 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 Tables 2 and 3. Data 3sigma rule: Normal values are within the range of mean plus or minus three standard deviations, and others are outliers. After removing the outliers, GraphPad Prism software (GraphPadPrism8.0.2.263) was used to statistically analyze the data, and the results are as Figures 2 to 4 shown. Among them, leaf length is the length of the ear leaf from the leaf sheath to the leaf tip; 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 total number of leaves of the whole plant 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 that 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 lengths and numbers of stem 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 stem 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 stem internode lengths, 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) were significantly different from the control (P < 0.01). Plant height and ear height were shorter, and stem diameter, leaf length, and leaf width were thinner, shorter, and narrower. Figure 2 (Table 2).

[0084] Table 2. Key phenotypic statistics of ZmArf1a CRISPR / Cas9 families.

[0085] Trait Arf1a NT1 ]]> 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 Arf1a NT2 ]]> 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 the number of tassel branches, KNR represents the number of kernels per row, KRN represents the number of rows per 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-positive plants / number of positive-positive plants. Bold text in the table indicates P < 0.05.

[0087] The length and number of branches of the tassel were measured and statistically analyzed. An indoor seed evaluation was conducted after the maize female ears were harvested, specifically including measurements of ear length, ear diameter, number of kernels per row, and number of rows per ear. arf1a ko1 The length of the male spike was 24.96±5.02 cm (n=12), the number of male spike branches was 4.42±1.44 (n=12), the length of the female spike was 9.54±1.19 cm (n=8), the spike diameter was 3.75±0.51 cm (n=8), the number of kernels per row was 17.45±2.73 (n=10), and the number of kernels per spike row was 14.45±1.24 (n=10). Arf1a NT1 The length of the male spike was 30.02±2.98 cm (n=16), the number of male spike branches was 6.74±1.57 (n=16), the length of the female spike was 12.02±1.49 cm (n=14), the spike diameter was 4.23±0.41 cm (n=14), the number of kernels per row was 21.11±3.72 (n=12), and the number of kernels per spike row was 15.39±1.58 (n=16). The results showed that arf1a ko1 With Arf1a NT1 There were highly significant differences (P<0.01) in tassel length (P=8.193E-04), number of tassel branches (P=3.621E-04), ear length (P=9.5E-06), and ear diameter (P=3.28E-05), and significant differences (P<0.05) in number of kernels per row (P=0.014) and number of rows per ear (P=0.049). In other words, the female ear was shorter and had fewer branches, while the female ear was shorter and narrower, with fewer kernels per row and fewer rows per ear. Figure 3 (Table 3).

[0088] Statistics of the stem internode length and the number of internodes in the ZmArf1a CRISPR / Cas9 family

[0089]

[0090] ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The difference in plant height between plants was caused by the difference in length of the 7th, 8th, 10th and 11th internodes from the base (Table 3).

[0093] arf1a ko2 The tassel length of arf1a was 228.16±4.78 cm (n=10), the tassel branch number was 5.8±1.55 (n=10), the ear length was 10.84±2.15 cm (n=7), the ear thickness was 3.89±0.38 cm (n=7), the grain number per row was 17.57±4.93 (n=7), and the ear row number was 14±2 (n=7). NT2 The tassel length of arf1a was 30.53±2.02 cm (n=14), the tassel branch number was 6.11±1.59 (n=28), the ear length was 12.55±1.53 cm (n=14), the ear thickness was 4.14±0.49 cm (n=15), the grain number per row was 21±3.27 (n=19), and the ear row number was 16±2.63 (n=19). ko2 The tassel length (P=0.478) and the tassel branch number (P=0.731) of arf1a NT2 had no significant difference (P>0.05) with those of Arf1a ko2 The ear length (P=0.020), the ear thickness (P=0.029), the grain number per row (P=0.039) and the ear row number (P=0.044) of arf1a NT2 had significant difference (P<0.05) with those of Arf1a Figure 4 , and the ear length and the ear thickness were shorter and narrower, and the grain number per row and the ear row number were less.

[0094] The above results show that the change of G2 and G3 structure in arf1a ko1 significantly affects the vegetative and reproductive growth of mutant plants. ko2 The frame shift mutation caused by the base deletion of arf1a ko significantly affects the vegetative and reproductive growth of mutant plants.

[0095] Therefore, the present application first discovers that the plant with the knocked-out ZmArf1a gene of corn grows normally, the tassel is fertile, the ear axis and the kernel are normal and full, but the leaf length, the leaf width and the plant height are significantly reduced, and the corn ZmArf1a gene provided can be used as a candidate gene for breeding a dwarf and dense corn variety, which is expected to reduce the workload of breeding and accelerate the breeding process.

[0096] It should be pointed out finally that the above examples are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. Application of the maize ZmArfla gene in maize variety selection, characterized in that: The CDS sequence of the corn ZmArf1a gene is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4; The corn variety breeding refers to breeding a dwarf and dense planting corn variety; Knocking out the corn ZmArf1a gene can reduce the plant height, shorten the leaf length and narrow the leaf width of corn; Knocking out the corn ZmArf1a gene by using the CRISPR-Cas9 technology can make the translated protein of the gene lose the original function or be unable to be translated into protein.

2. Use of the vector and / or strain comprising the maize ZmArfla gene as claimed in claim 1 in the selection of a maize compact variety, characterized in that: The vector is a knock-out vector of the corn ZmArf1a gene, and the strain is Escherichia coli and / or Agrobacterium containing the knock-out vector of the corn ZmArf1a gene.

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