A corn dwarfing control gene ZmEXO84C and its application

Abstract

The invention discloses a corn dwarfing control gene ZmEXO84C and its application, and relates to the technical field of functional genes and plant gene breeding. The nucleotide sequence of the ZmEXO84C gene is shown in SEQ ID NO.1. The ZmEXO84C gene is highly expressed in the leaves, tassels and embryos of corn, and the amino acid sequence of the protein encoded by the ZmEXO84C gene is shown in SEQ ID NO.2. The invention performs phenotypic identification on the mutant induced by EMS of corn, separates the obtained dwarfing mutant d2201, constructs a population, and combines genetic analysis and Mutmap positioning to determine that ZmEXO84C is an effect gene that causes the dwarfing of the mutant d2201. The gene has a nonsense mutation in the mutant d2201, which can significantly reduce the plant height. The use of the functional gene is helpful to improve the plant height and dense breeding of corn, and has important breeding application potential.

Description

Technical Field

[0001] The invention relates to the technical field of functional genes and plant gene breeding, and in particular to a corn dwarfing control gene ZmEXO84C and an application thereof. Background Art

[0002] As my country's largest food crop, the increase in corn yield is of great significance in ensuring national food security. Corn yield is closely related to plant height, and a moderate reduction in plant height is conducive to increasing planting density and yield. Therefore, identifying excellent corn dwarf mutants, exploring their related genes and analyzing their molecular mechanisms of function are of great significance to modern corn breeding.

[0003] The height of corn is closely related to hormone levels in the body. Studies have shown that the reported mutants and their dwarfing genes are mainly related to hormones such as gibberellin (GA), brassinosteroids (BR) and auxin (IAA). GA, BR and IAA mainly affect plant stem elongation through anabolism and signal transduction. In addition, plant height is also closely related to factors such as cell division, cell elongation and cell wall biosynthesis.

[0004] The material exchange between organelles inside eukaryotic cells depends on vesicle transport, which plays an important role in the performance of cell functions and life activities. The Exocyst complex is an evolutionarily conservative multi-subunit tethering complex that is widely present in eukaryotes and is mainly responsible for the tethering and fusion of vesicles and plasma membranes at polarized exocytosis sites. The Exocyst complex is mainly composed of eight subunits, namely SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70 and EXO84. In recent years, genes encoding each subunit of the exocyst complex have been identified in a variety of plants and their functions have been studied. Evidence has shown that the exocyst complex plays an important role in plant polar growth and cell morphology, including cell wall formation, polar auxin transport, fertilization, and response to pathogens. At present, the research on the EXO84 subunit is mainly concentrated in species such as Arabidopsis, but there is little research on its function and molecular regulation mechanism in corn. Summary of the invention

[0005] The purpose of the present invention is to provide a corn dwarfing control gene ZmEXO84C and application thereof.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The first aspect of the present invention provides a maize dwarfing control gene ZmEXO84C, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The second aspect of the present invention also provides a maize dwarfing control gene ZmEXO84C encoding protein, the amino acid sequence of the ZmEXO84C gene encoding protein is shown in SEQ ID NO.2.

[0009] The third aspect of the present invention also provides a use of the ZmEXO84C gene mutant as described above in regulating corn plant height.

[0010] A further improvement is that the method for regulating corn plant height is: by backcrossing or gene editing technology, the ZmEXO84C gene function of the corn plant is lost, thereby reducing the corn plant height.

[0011] The present invention has the following beneficial effects:

[0012] The present invention performs phenotypic identification on mutants induced by EMS of corn, separates the obtained dwarf mutant d2201, constructs a population, and combines genetic analysis and Mutmap positioning to determine that the ZmEXO84C gene is the effect gene that causes the dwarfing of the mutant d2201. The gene undergoes a nonsense mutation in the mutant d2201, which can significantly reduce the plant height of corn plants. The application of this functional gene is helpful to improve the plant height of corn and density-resistant breeding, and has important breeding application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Comparison of plant height and ear position between B73 and d2201 mutants (in the figure, A: dwarf phenotype of d2201 mutant; B: plant height statistics; C: ear height statistics (n=25));

[0014] Figure 2 Comparison of grain trait development between B73 and d2201 mutants (in the figure, A: grain length; B: grain width; C: comparison of 100-grain weight; D: comparison of grain length; E: comparison of grain width (n=25));

[0015] Figure 3 Comparison of cell morphology of the third internode in the aerial part of B73 and d2201 mutants (in the figure, A: internode longitudinal section; B: internode cross section; C: comparison of cell length in longitudinal section (n=300); D: comparison of cell width in longitudinal section (n=300); E: cell length in cross section (n=300); F: comparison of cell width in cross section (n=300));

[0016] Figure 4 Comparison of cell morphology of the internodes in the upper part of the ear between B73 and d2201 mutants (in the figure, A: longitudinal section of S+1 internode; B: comparison of cell length of S+1 longitudinal section (n=300); C: comparison of cell width of S+1 longitudinal section (n=300));

[0017] Figure 5 Comparison of endogenous hormone content between B73 and d2201 mutants (in the figure, A: IAA content in nodes; B GA content in nodes; C: IAA content in internodes; D: GA content in internodes);

[0018] Figure 6 Genetic positioning and mutation site analysis of the maize d2201 mutant (in the figure, A: Mutmap positioning analysis of the d2201 mutant; B: analysis of the mutation site of the effector gene; C: sequencing analysis of the mutation site);

[0019] Figure 7 Phenotypic comparison of B73, zmexo84c-1(d2201), zmexo84c-2 and zmexo84c-1×zmexo84c-2 (in the figure, A: plant comparison; B: plant height statistics (B73, zmexo84c-1, zmexo84c-1×zmexo84c-2n=25, zmexo84c-2n=20, the same below); C: ear height statistics; D: leaf length statistics (S: ear leaf, S+1: the first leaf above the ear leaf, S-1: the first leaf below the ear leaf); E: leaf width statistics; F: leaf angle statistics (S+5: the fifth leaf above the ear leaf, S+1: the first leaf above the ear leaf, S-1: the first leaf below the ear leaf));

[0020] Figure 8 Comparison of internode phenotypes between B73 and zmexo84c-1 (in the figure, A: comparison of each internode (the left side of each internode is B73, and the right side is zmexo84c-1); B: comparison of male spikes; C: statistics of the height of the last node (n=25); D: statistics of internode length (S: internode where the main spike is attached, S-1: the first internode below the spike internode, S+1: the first internode above the spike internode, and so on, n=25));

[0021] Fig. 9 Tissue expression pattern analysis of ZmEXO84C (in the figure, V3-R: root at 3-leaf stage; V3-L: leaf at 3-leaf stage; V8-R: root at 8-leaf stage; V8-S: stem at 8-leaf stage; V8-L: leaf at 8-leaf stage; V11-S: stem at 11-leaf stage; V11-L: leaf at 11-leaf stage; R1-S: stem at silking stage; R1-L: leaf at silking stage; SK: silk at silking stage; E: ear; T: tassel; EM: embryo 20 days after pollination);

[0022] Note: In the above figures, * indicates that the data are significantly different, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0023] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0024] The maize B73 inbred line used in this experiment was maintained by the National and Local Joint Engineering Laboratory for Crop Stress Breeding and Disaster Reduction, College of Life Sciences, Anhui Agricultural University; the d2201 and its allelic mutants used were screened from the maize EMS mutant library (http: / / maizeems.qlnu.edu.cn / ).

[0025] Corn sampling and breeding materials were planted in the experimental fields of the breeding base.

[0026] Unless otherwise specified, the methods used in this test are conventional methods known to those skilled in the art, and the reagents not otherwise specified are commercially available products.

[0027] The content of the present invention is described in detail below in conjunction with specific implementation methods.

[0028] 1. Identification of mutant d2201

[0029] 1.1. Agronomic traits analysis

[0030] The B73 and d2201 mutants were planted in the experimental field of the breeding base at the same time, with a row length of 5m and a plant spacing of 25cm. 25 plants were selected during the pollination period and numbered and labeled. Agronomic traits such as plant height and ear height were measured and counted, and open-pollinated ears were harvested to examine grain-related traits.

[0031] The statistical results of plant height and ear height are as follows Figure 1 As shown in Figure 2, it can be seen that the plant height and ear height of the d2201 mutant are significantly lower than those of the B73 inbred line. Figure 2 As shown, it can be seen that the dwarfing of the d2201 mutant has a certain effect on the development of grain traits.

[0032] 1.2 Analysis of internode cell morphology of d2201 mutant

[0033] The internode tissues of B73 inbred line and d2201 mutant were sliced ​​and observed. The third internode of the aerial part of B73 inbred line and d2201 mutant was sampled and sliced ​​to analyze the cell size. Figure 3 As shown, the cell volume of the aerial internodes of the d2201 mutant was reduced, and the dwarf phenotype of the mutant was closely related to the shortening of the longitudinal length of the cells.

[0034] The first internode (S+1) on the ear of B73 inbred line and d2201 mutant was further sliced ​​and its cell size was counted. Figure 4 The size of the internode cells in the upper part of the ear of the d2201 mutant was also significantly reduced, which was similar to the statistical results of the cytological observation of the third internode in the aboveground part, indicating that the dwarf phenotype of the d2201 mutant may be closely related to the shortening of the longitudinal length of the internode cells.

[0035] 1.3. Determination of endogenous hormone content

[0036] B73 and d2201 mutants were sown at the same time. When the growth period was about 40 days, the third node and internode of the aboveground part were taken, cut into pieces and frozen in liquid nitrogen. Three biological replicates were set for each group of samples, and the mass of each biological replicate sample was 1g. Hormone extraction and detection were completed by Shandong Guocangjian Biotechnology Co., Ltd.

[0037] The results are as follows Figure 5 As shown, in the internodes, the auxin content of the d2201 mutant was significantly higher than that of the B73 inbred line. 3 , GA 4 and GA 7 The content was slightly higher than that of B73 inbred line. In the nodes, the auxin content of d2201 mutant was significantly higher than that of B73 inbred line. 1 The content is slightly lower than that of B73 inbred line, GA 3 , GA 4 and GA 7 The above results indicate that the dwarfing of d2201 mutant may be closely related to the change of IAA content in stems.

[0038] 1.4 Analysis of the inheritance of dwarfing traits in the d2201 mutant

[0039] The maize d2201 mutant was crossed with the B73 inbred line to obtain the F 1 Generation, then F 1 F 2 Generation group, F 2 The first generation of population was planted in the experimental field for field observation and it was found that F 2 The plant height phenotypes were separated in the population. 2 The wild-type and mutant phenotype plants in the F generation population were counted, and a total of 156 maize plants were obtained. 2 In the first generation population, 120 plants showed wild-type phenotype and 36 plants showed d2201 mutant phenotype, with a segregation ratio of 3.3:1. The chi-square test χ 2 =0.307<χ 2 0.05(1) = 3.84, which is consistent with Mendel's theoretical segregation ratio of 3:1, indicating that the dwarfing trait of d2201 is controlled by a single recessive gene.

[0040] 2. Gene localization of the d2201 mutant

[0041] 2.1 Mutmap Positioning Analysis

[0042] From B73×d2201F 2 Leaves of 18 mutant plants were taken from the first generation segregation population, and the genome was extracted for equal mixed pool BSA sequencing. The Mutmap method was used to locate the gene, and SNPs with mutations from C to T or G to A and SNP-index = 1, as well as mutation types including termination mutations and splicing site mutations, were screened. Finally, 9 SNPs were obtained, and the relationship between the chromosome where the SNP was located and the SNP-index position was analyzed.

[0043] like Figure 6 As shown, an obvious signal peak appeared on chromosome 7. Combined with the BSA sequencing results and functional annotation, it was found that there was a gene Zm00001d021828 on the chromosome, which encodes an exocyst complex EXO84 subunit. In the mutant d2201, the fifth exon region of the gene mutated from C to T, resulting in a nonsense mutation (CAG-TAG) in the gene. Therefore, it was speculated that the dwarf phenotype of the d2201 mutant was caused by the Zm00001d021828 mutation, and the gene was named ZmEXO84C, and the mutant was named zmexo84c-1.

[0044] Primers were designed on both sides of the mutation site of the ZmEXO84C gene. The primer information is shown in Table 1. DNA was extracted from the zmexo84c-1 mutant and sequenced. The sequence was compared with that of the B73 inbred line. The results showed that the 22nd base C in the 5th exon of the ZmEXO84C gene in the zmexo84c-1 mutant genome mutated to T, which was a homozygous mutation, confirming that the site was mutated ( Figure 6 ).

[0045] Table 1 Primer information for mutation site verification

[0046]

[0047] 2.2. Mutant gene allele verification and agronomic trait analysis

[0048] To further confirm that the ZmEXO84C mutation is the main cause of the dwarf phenotype of the d2201 mutant, another mutant of this gene was obtained from the maize EMS mutant library and named zmexo84c-2. Primers were designed according to its mutation site and sequenced for verification. The primer information is shown in Table 1. The 138th base G in the 5th exon of the ZmEXO84C gene in the zmexo84c-2 mutant genome mutated to A ( Figure 6 ).

[0049] The zmexo84c-1 mutant was crossed with the zmexo84c-2 homozygous plants to obtain F 1 Generation plants, B73, zmexo84c-1, zmexo84c-2 and F 1 The field phenotypes of the next generation plants were statistically analyzed. Figure 7 As shown, the zmexo84c-1 mutant, zmexo84c-2 mutant and F 1 The plant height and ear height of the first generation plants were lower than those of the B73 inbred line, and the plant height and ear height of the zmexo84c-2 mutant were slightly higher than those of the zmexo84c-1 mutant.

[0050] The results are as follows Figure 7 As shown in Figure 2, compared with the B73 inbred line, the average plant height and ear height of the zmexo84c-1 mutant decreased by about 35.1% and 43.3%, respectively, and the average plant height and ear height of the zmexo84c-2 mutant decreased by about 23.9% and 21.1%, respectively. 1 The average plant height and ear height of the plants decreased by about 24.8% and 39.9%, respectively. 1 The plant height and ear height of the first generation plants were significantly lower than those of the B73 inbred line. In addition, the statistical investigation of the leaf morphology of the three-leafed rods in different lines found that compared with the B73 inbred line, the two mutants and F 1 The leaf length and width of the first generation plants were shortened to varying degrees. The length of the three-leafed leaf of zmexo84c-1 was shortened by about 35%, and the width was shortened by about 13%. The length of the three-leafed leaf of zmexo84c-2 was shortened by about 22%, and the width was shortened by about 10%. 1 The length of the three-leafed leaves of the first generation plants was shortened by about 27%, and the leaf width was shortened by about 7%. The angle between the first leaf on the ear and the first leaf below the ear of the zmexo84c-1 mutant was higher than that of the B73 inbred line, and the angle between the fifth leaf on the ear was lower than that of the B73 inbred line. The angle between the first leaf on the ear and the first leaf below the ear of the zmexo84c-2 mutant was higher than that of the B73 inbred line, and the angle between the fifth leaf on the ear and the B73 inbred line was not significantly different. 1The angle of the first leaf above the ear of the F1 generation plants was higher than that of the B73 inbred line, and the angles of the first leaf below the ear and the fifth leaf above the ear were not significantly different from those of the B73 inbred line. The above results showed that the dwarf phenotype of the F1 generation plants was similar to that of the zmexo84c-2 mutant, which fully proved that the dwarf phenotype of the d2201 mutant was caused by the mutation of the ZmEXO84C gene.

[0051] In order to further analyze the reasons for the reduction of plant height and ear height in the zmexo84c-1 mutant, the internode length and the height of the last node were counted. Figure 8 As shown in the figure, compared with the B73 inbred line, the height of the last node and the length of each internode of the zmexo84c-1 mutant were shortened to varying degrees, among which the height of the last node was shortened by about 32.1%, the second internode under the tassel was shortened by about 19.2%, and the fifth internode under the main ear was shortened by about 48.1%. The above results indicate that the shortening of the internode length of the zmexo84c-1 mutant may be the main reason for the dwarfing of the plant, and the shortening of the internodes under the ear is greater than that of the internodes above the ear.

[0052] 3. Analysis of tissue expression pattern of ZmEXO84C

[0053] 3.1 RNA extraction from corn tissue

[0054] Take the roots and leaves of the wild-type strain at V3 stage, roots, stems and leaves at V8 stage, stems and leaves at V11 stage, stems and leaves at the silking stage, filaments, tassels, female spikes, and embryos 20 days after pollination under normal growth, freeze them in liquid nitrogen, store at -80℃, and extract total RNA respectively. The extracted RNA is reverse transcribed, and the sample is added according to the system in Table 2. The PCR reaction conditions are: 37℃, 15min; 85℃, 5sec; 4℃ insulation. After the reaction, the cDNA product obtained by reverse transcription is diluted with 190μL DEPC water and stored at -20℃.

[0055] Table 2 Reverse transcription loading system

[0056]

[0057] 3.2 Fluorescence quantitative PCR reaction

[0058] Using cDNA as a template, Primer3Plus was used to design primers, and the product size was 100-150bp. Primer3Plus (https: / / www.bioinformatics.nl / cgi-bin / primer3plus / primer3plus.cgi). qRT-PCR used a fluorescent quantitative PCR kit, with the GAPDH gene as an internal reference. Each template was guaranteed to have 3 biological replicates and 3 technical replicates. The primer sequences are shown in Table 3. The qRT-PCR reaction system was loaded according to Table 4, and the reaction procedure was shown in Table 5. After the reaction was completed, the CT value was exported and the relative expression of the gene was calculated.

[0059] Table 3 Gene quantification primers

[0060]

[0061] Table 4 qRT-PCR sample loading system

[0062]

[0063] Table 5 qPCR reaction procedure

[0064]

[0065] The results are as follows Fig. 9 As shown, ZmEXO84C is expressed in different tissues of maize, and the expression trend is basically consistent with the gene tissue expression level published in the MaizeGDB database, among which the expression level in leaves, tassels and embryos is higher.

[0066] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for reducing corn plant height, characterized in that: Using backcrossing or gene editing to make corn plants ZmEXO84C Gene function mutation, thereby reducing corn plant height, the mutation is ZmEXO84C The 22nd base in the 5th exon region of the gene mutated from C to T. ZmEXO84C The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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