Corn kernel size control gene ZmILR1 and molecular identification method and application thereof

Abstract

The application relates to a gene for controlling corn kernel size ZmILR1 and a molecular identification method and application thereof, and belongs to the technical field of plant genetic breeding and functional gene research. ZmILR1 The gene encodes an IAA-amino acid hydrolase, mutation of the gene leads to small corn kernels, and overexpression of the gene can create corn materials with increased kernel length, kernel width and hundred-kernel weight. According to the sequence difference between the mutant gene and the wild-type gene at the DNA level, a functional molecular marker A1A4 is developed, which can be used for rapid identification and screening of small-kernel corn materials. The application provides a theoretical basis and application tool for analyzing the molecular mechanism of corn kernel size, carrying out molecular marker-assisted breeding and hybrid selection, and provides important gene resources and technical support for corn high-yield and high-quality molecular breeding.

Description

Technical Field

[0001] This invention relates to genes that control the size of maize kernels. ZmILR1 Its molecular identification methods and applications belong to the field of plant genetics and breeding and functional gene research technology. Background Technology

[0002] corn( Zea mays Maize (L.) is one of the world's most important food and industrial raw material crops, widely used in food, feed, and bioenergy (Ranum et al., 2014). Kernel size, as a key trait for maize yield and quality, directly affects yield per unit area and commercial value, and is also an important basis for screening high-quality breeding materials. In-depth analysis of the genetic regulatory mechanisms of kernel size will help to discover key functional genes and develop molecular markers (Palacios-Rojas et al., 2020), improving the efficiency and accuracy of high-yield and high-quality maize breeding, and has significant theoretical and practical application value.

[0003] Maize kernel size is regulated by multiple factors, including genetics, development, physiology, and environment. Kernel formation begins with double fertilization (Russell, 1992), subsequently developing into a structure containing the endosperm, embryo, and seed coat. The endosperm further differentiates into various cell types, such as the starch endosperm, the BETL (beta-transport layer), and the aleurone layer (AL) (Leroux et al., 2014). The determination of kernel size involves cell division, expansion, and material accumulation, and its regulatory mechanisms are highly complex. Previous studies have identified a number of key regulatory genes covering multiple signaling pathways, including the ubiquitin-proteasome pathway (e.g., ZmGW2 ), G protein signaling pathways (such as ZmGS3MAPK signaling pathway and photosynthetic product transport network, etc. (Li et al., 2021, Zhang et al., 2019, Feng et al., 2018, Myers et al., 2011, Sosso et al., 2012). In addition, different types of transcription factors (such as MADS-box, bZIP, bHLH, etc.) have also been shown to play a significant role in grain development (Feng et al., 2018, Schmidt et al., 1987, Montag et al., 1995). miRNA and RNA splicing also play important roles in post-transcriptional regulation of grain development (Zheng et al., 2019). In terms of physiological regulation, plant hormones such as auxin, gibberellin, cytokinin, ethylene, and brassinosteroids synergistically regulate grain length, width, and weight, forming a complex hormonal interaction network (Sun et al., 2021, Wang et al., 2020, Myers et al., 2011, Mimura et al., 2018). Although these studies have made significant progress in rice, wheat, and maize, many key pathways remain undeciphered, and further research is needed to uncover new regulatory factors and mechanisms of action.

[0004] In the plant hormone regulatory network, auxin (IAA) has a particularly significant impact on grain development. Previous studies have shown that IAA synthesis genes, such as... ZmYUC1 Transport-related genes, such as ZmPIN1 Signal transduction genes, such as ZmARF and ZmAux / IAA All of these are closely related to the size of maize kernels (Bernardi et al., 2012, Chen et al., 2020). For example, ZmSK2 Through with ZmIAA28 Interactions jointly regulate grain development (Wang et al., 2022). ZmVPS29 This positively regulates grain length through the IAA signaling pathway (Chen et al., 2020). However, the application of IAA hydrolysis-related genes in grain development regulation has not yet been reported.

[0005] This invention is the first to identify a factor significantly correlated with corn kernel size. ZmILR1This invention elucidates the role of a gene in the IAA degradation pathway, demonstrating that its mutation leads to smaller maize kernels. Overexpression of this gene can create maize materials with increased kernel length, width, and 100-kernel weight. The invention also develops functional molecular markers that can be used for rapid screening and molecular identification of mutant materials, providing important genetic resources and technical support for high-yield and high-quality molecular breeding of maize. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a key regulatory gene for maize kernel size. ZmILR1 Furthermore, the gene and its functional molecular markers were developed, providing insights into their application in breeding large-grain maize germplasm resources. This research falls under the fields of plant functional gene research and molecular breeding technology. The maize mentioned... ZmILR1 The full-length DNA, cDNA, and amino acid sequences of the gene are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0007] Specifically, this invention uses the laboratory core maize inbred lines B404 and B406 as parental materials, constructs an F2 segregating population through hybridization, and identifies a key gene that significantly regulates maize kernel size using map-based cloning and other methods. ZmILR1 Further analysis revealed that in small-kernel maize, the first exon of this allele underwent a 3-base insertion and a 33 bp deletion, resulting in inactivation of the encoded protein and manifesting as a distinctly small-kernel phenotype. This mutant gene... Zmilr1 The nucleotide sequence is shown in SEQ ID NO.4.

[0008] Furthermore, based on the sequence differences at the DNA level between small-grained maize and wild-type maize, this invention has developed and designed the functionally specific InDel molecular marker A1A4, which can be used for rapid screening and molecular identification of mutant materials. This molecular marker can be widely applied to the precise identification of large and small-grained maize germplasm, providing a simple and effective tool for the efficient utilization of superior germplasm resources related to grains.

[0009] This invention utilizes a genetic transformation platform for gene... ZmILR1 Overexpression resulted in increased grain length, width, and 100-grain weight in the plants. This invention not only enriches the genetic basis of molecular regulation of grain size but also provides important genetic resources and technical support for molecular breeding of high-yield and high-quality maize.

[0010] The present invention has the following beneficial technical effects:

[0011] For the first time, corn was clearly defined. ZmILR1 The genetic link between genes and seed size.

[0012] Provided ZmILR1Small-grained maize materials with allelic variations can be used for research on the mechanism of maize grain size regulation and germplasm innovation.

[0013] Identified Zmilr1 A key functional mutation site in the gene was identified, which significantly reduced the IAA-amino acid hydrolase activity of the ZmILR1 protein, revealing its functional basis in the regulation of IAA metabolism.

[0014] based on Zmilr1 A functional molecular marker, A1A4, was designed and developed for the mutation site. The primer sequences are AI-F (SEQ ID NO.5) and A4-R (SEQ ID NO.6), which can be used for rapid and accurate identification of small-grained materials and improve the efficiency of marker-assisted selection in breeding.

[0015] The applicability of the molecular marker A1A4 in distinguishing between large and small grains in the F2 segregating population was verified, demonstrating its good resolution and practicality, and providing an effective tool for high-throughput screening of maize kernel size-related resources.

[0016] Using genetic transformation platforms ZmILR1 Genetic transformation using overexpression vectors yielded transgenic overexpression materials with positive plants exhibiting increased grain length, width, and 100-grain weight, providing important genetic resources and technical support for obtaining large-grained maize. Attached Figure Description

[0017] Figure 1 Phenotypic analysis of wild-type and small-seed materials in the genetic mapping population parents and F2 segregating population.

[0018] Figure A shows the grain phenotypes of the two parents, B404 and B406, in the F2 genetic mapping population, as well as the ear and grain phenotypes of the F2 segregating population; Figure B is a statistical illustration of the grain length, grain width, and 100-grain weight of the wild type and small grains in the F2 segregating population.

[0019] Figure 2 Grain development process at different stages for wild-type and small-grained materials

[0020] We continuously observed the phenotypic characteristics of wild-type and small-seeded F2 populations, taking samples and photographs based on the pollination date to determine the period of phenotypic differences and morphological changes in small seeds.

[0021] Figure 3 For corn ZmILR1 Fine mapping and map-based cloning of genes

[0022] Figure 4 For the two parents B404 and B406 Zm00001eb304520 Gene sequence and amino acid variation analysis

[0023] against Zm00001eb304520 Sequencing revealed a base difference in the first exon of the gene between the two parents, resulting in a change in the amino acid sequence.

[0024] Figure 5 Functional molecular markers were designed and developed based on differentially expressed sites in wild-type and small-seed gene sequences.

[0025] For both parents Zm00001eb304520 We designed specific markers to verify the base sequence differences present in the first exon of the gene.

[0026] Figure 6 A comparison of the grain phenotypes of overexpressing plants and wild-type plants.

[0027] Build a Ubi strong starter driver Zm00001eb304520 Genetic transformation was performed using the overexpression vector to obtain positive plant seeds, which were then compared with wild-type plant seeds. Figure A shows the morphological phenotypes of wild-type and overexpression plant seeds; Figure B shows the statistical distribution of grain length, grain width, and 100-seed weight between wild-type and overexpression seeds. Detailed Implementation

[0028] The following embodiments are used to illustrate the present invention, but do not limit the scope of the invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are all within the scope of the invention. Unless otherwise specified, the synthesis and sequencing of primers and genes used in the embodiments were performed by Beijing Qingke Biotechnology Co., Ltd. Other biochemical reagents, unless otherwise specified, are conventional commercially available reagents, and the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0029] Example 1: Obtaining and describing the F2 population and small-seed phenotypic materials of maize genetic mapping

[0030] The F2 population with different kernel sizes was obtained by the patent applicant through hybridization of two inbred lines, B404 and B406, from the germplasm bank of the Institute of Biological Agriculture, University of Science and Technology Beijing, followed by self-pollination of the F1 generation. B404 is the parent with the large kernel phenotype, and B406 is the parent with the small kernel phenotype. Figure 1 A). The F2 ears of self-pollinated offspring from the F1 generation showed significant differences in kernel size phenotype (large and small kernels). Figure 1 A), there were also significant differences in grain length, grain width, and 100-grain weight. Figure 1 B). Stereoscopic photography was performed on samples of different pollination stages, showing that the small grain phenotype was observable with the naked eye 12 days after pollination. Simultaneously, the development of the endosperm and embryo in these samples was found to be delayed compared to the wild type. Figure 2 ).

[0031] The segregation of large and small grains in the F2 population conformed to a single-gene segregation ratio of 3:1. The chi-square test and significance test results showed that the observed data did not differ significantly from the theoretical ratio of 3:1, which was consistent with the 3:1 ratio hypothesis. That is, the grain size phenotype of this population showed obvious single-gene recessive inheritance (Table 1).

[0032] Table 1. Genetic analysis of grain size phenotypic segregation in the F2 segregating population.

[0033]

[0034] Three hundred small-grained individual plants were selected from the F2 population for the localization and cloning of maize kernel size genes.

[0035] The method for extracting DNA from corn leaves is as follows: 1) Cut an appropriate amount of leaves and chop them into small pieces. Place them into 2.0ml centrifuge tubes that have been pre-labeled with numbers, and add a steel ball. 2) Arrange the centrifuge tubes containing the leaves and steel ball in order on a centrifuge tube rack (8×5) for the sampler, and immerse the entire tube in a container filled with liquid nitrogen for 1-2 minutes (Note: the liquid nitrogen should just cover the centrifuge tube rack slightly). 3) Place the frozen centrifuge tube rack into the slot of the sampler (Thmorgan Cell Killer CK-1000), tighten it, close the cap, and sample at 1200 rpm for 20 seconds. 4) Use a magnet to remove the steel ball from the centrifuge tube. 5) Add 700μL LCTAB extraction buffer (preheated to 65℃), and incubate in a 65℃ water bath for 30 minutes, inverting the tube 1-2 times during the incubation period. 6) Add 700 μL of chloroform:isoamyl alcohol (24:1) extraction buffer, tighten the cap, invert and mix well, being careful not to erase the label (Note: Chloroform is a corrosive reagent; wear PE disposable gloves and handle in a fume hood). 7) Centrifuge at 12000 rpm for 5 min until clear phase separation. Transfer 400 μL of the supernatant to a new 1.5 mL microcentrifuge tube (pre-fill with 800 μL of pre-chilled anhydrous ethanol, discard the pipette tip, tighten the cap, label and check for accuracy, invert and mix well. Place in a -20℃ refrigerator for 30 min). Centrifuge at 12000 rpm for 10 min until the precipitate adheres to the bottom of the tube, discard the supernatant. 8) Wash the precipitate twice with 70% ethanol, invert the 1.5 mL microcentrifuge tube onto a piece of paper laid flat on the table, and allow it to air dry. 9) Add 100-200 μL of 1×TE buffer or ddH2O to dissolve the precipitate. 10) Store the sample in a -20℃ refrigerator for later use.

[0036] The SSR marker primer sequences used are shown in Table 2, and the PCR amplification system and amplification program are shown in Tables 3 and 4.

[0037] Table 2 SSR markers used for gene mapping

[0038]

[0039] Table 3. PCR reaction system

[0040]

[0041] Table 4. SSR marker amplification procedure

[0042]

[0043] Gene mapping information, such as Figure 3 As shown. Using BSA-seq analysis and specific markers for map-based cloning, candidate genes were located in a region of approximately 600 kb on chromosome 7, from 26.43 Mb to 27.05 Mb, between markers M2 and M6. Fine mapping results showed that the candidate genes were located in the 26.738 Mb and 26.918 Mb regions, between markers M4 and M5. There are four annotated genes within this region: Zm00001eb304500 , Zm00001eb304510 , Zm00001eb304520 and Zm00001eb304530 Cloning comparisons of these four candidate gene coding sequences in both parents revealed that, except for... Zm00001eb304520 Apart from the two genes, the other three genes showed no base sequence differences between the two parents. Comparative analysis of the two parents... Zm00001eb304520 Sequence differences revealed a base change in the first exon sequence of this gene in the small-seeded parent B406 compared to the large-seeded parent B404. Specifically, there was a 3-base TCT insertion at position 4 and a 33-base deletion at position 17. The differences in DNA and amino acid sequences of the Zm00001eb304520 gene between the two parents are shown below. Figure 4 A and Figure 4 As shown in B. Query the MaizeGDB database. Zm00001eb304520 Functional annotation revealed that this gene is an amino acid conjugate that hydrolyzes part of the plant growth regulator indole-3-acetic acid (IAA). Based on its functional annotation, the inventors named this gene... ZmILR1 .

[0044] Example 3: Development of the molecular marker A1A4

[0045] In this invention, the parental parent is targeted. ZmILR1 The differentially expressed site (30bp deletion in the first exon) was identified, and primers were designed using Primer 5.0 software. Figure 5 A) A functional molecular marker, A1A4, was developed. The obtained DNA was amplified by PCR using designed primers; the methods and procedures are shown in Table 5.

[0046] Table 5. PCR amplification conditions

[0047]

[0048] In the large parent B404, the amplified fragment size was 171 bp. Due to a 3 bp insertion and a 33 bp deletion in the small parent B406, the amplified fragment size became 141 bp. Electrophoresis using a 3% agarose gel showed the following amplified band patterns for both parents: Figure 5 As shown in B, this marker can specifically distinguish between the two parents. ZmILR1 difference.

[0049] Example 4: Practicality Analysis of Molecular Marker A1A4

[0050] The functional markers developed in Example 3 were validated using the F2 segregating population. Experimental results showed that homozygous large-grained materials from the large-grained parent B404 and the F2 segregating population only amplified a 171bp product; small-grained materials from the small-grained parent B406 and the F2 segregating population only amplified a 141bp product; heterozygous wild-type materials amplified two bands, one at 171bp and the other at 141bp. The band sizes determined by the markers were correct and met expectations. Figure 5 (B) This indicates that the primer pair can specifically detect small-seed phenotypic materials in the F2 segregating population and can simultaneously distinguish wild-type. ZmILR1 Genes and small seeds Zmilr1 Gene.

[0051] Example 5: Obtaining overexpression plants and grain phenotype

[0052] To further determine ZmILR1 Differences in grain morphology-related traits were analyzed based on the PCUB vector framework. ZmILR1 Overexpression genetic transformation vector. This vector uses the Ubiquitin promoter (maize polyubiquitin gene), enabling it to drive... ZmILR1 It exhibits strong expression ability in plants. It is obtained through genetic transformation. ZmILR1 Seeds from positive overexpression plants were analyzed, and their morphological phenotypes were identified. The results showed that overexpression plants had increased seed length and width, and a higher weight per 100 seeds. Figure 6 ).

[0053] This invention confirms ZmILR1 It plays an important role in regulating maize kernel size and has developed small kernel-specific functional markers, providing important genetic resources and technical support for improving maize kernel traits and molecular breeding for high-yield and high-quality maize.

[0054] References

[0055] BERNARDI J, LANUBILE A, LI Q B, et al. 2012. Impaired auxinbiosynthesis in the defective endosperm18 mutant is due to mutational loss ofexpression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein inmaize. Plant Physiol [J], 160: 1318-1328.

[0056] CHEN L, LI Y X, LI C, et al. 2020. The retromer protein ZmVPS29regulates maize kernel morphology likely through an auxin-dependent process(es). Plant Biotechnol J [J], 18: 1004-1014.

[0057] FENG F, QI W, LV Y, et al. 2018. OPAQUE11 Is a Central Hub of theRegulatory Network for Maize Endosperm Development and Nutrient Metabolism.Plant Cell [J], 30: 375-396.

[0058] LEROUX B M, GOODYKE A J, SCHUMACHER K I, et al. 2014. Maize earlyendosperm growth and development: from fertilization through cell typedifferentiation. Am J Bot [J], 101: 1259-1274.

[0059] LI Q, ZHOU S, LIU W, et al. 2021. A chlorophyll a oxygenase 1 geneZmCAO1 contributes to grain yield and waterlogging tolerance in maize.Journal of Experimental Botany [J], 72: 3155-3167.

[0060] MIMURA M, KUDO T, WU S, et al. 2018. Autonomous and non-autonomousfunctions of the maize Shohai1 gene, encoding a RWP-RK putative transcriptionfactor, in regulation of embryo and endosperm development. Plant J [J].

[0061] MONTAG K, SALAMINI F, THOMPSON R D 1995. ZEMa, a member of a novelgroup of MADS box genes, is alternatively spliced in maize endosperm. NucleicAcids Res [J], 23: 2168-2177.

[0062] MYERS A M, JAMES M G, LIN Q, et al. 2011. Maize opaque5 encodesmonogalactosyldiacylglycerol synthase and specifically affects galactolipidsnecessary for amyloplast and chloroplast function. Plant Cell [J], 23: 2331-2347.

[0063] PALACIOS-ROJAS N, MCCULLEY L, KAEPPLER M, et al. 2020. Mining maizediversity and improving its nutritional aspects within agro-food systems.Compr Rev Food Sci Food Saf [J], 19: 1809-1834.

[0064] RANUM P, PEñA-ROSAS J P, GARCIA-CASAL M N 2014. Global maizeproduction, utilization, and consumption. Ann N Y Acad Sci [J], 1312: 105-112.

[0065] RUSSELL S D. Double Fertilization[C] / / S. D. RUSSELL, C. DUMAS.International Review of Cytology.Academic Press,1992:357-388.

[0066] SCHMIDT R J, BURR F A, BURR B 1987. Transposon tagging and molecularanalysis of the maize regulatory locus opaque-2. Science [J], 238: 960-963.

[0067] SOSSO D, CANUT M, GENDROT G, et al. 2012. PPR8522 encodes achloroplast-targeted pentatricopeptide repeat protein necessary for maizeembryogenesis and vegetative development. J Exp Bot [J], 63: 5843-5857.

[0068] SUN H, XU H, LI B, et al. 2021. The brassinosteroid biosynthesisgene, ZmD11, increases seed size and quality in rice and maize. PlantPhysiology and Biochemistry [J], 160: 281-293.

[0069] WANG Y, LIU W, WANG H, et al. 2020. ZmEHD1 Is Required for KernelDevelopment and Vegetative Growth through Regulating Auxin Homeostasis. PlantPhysiol [J], 182: 1467-1480.

[0070] WANG Y, XU J, YU J, et al. 2022. Maize GSK3-like kinase ZmSK2 isinvolved in embryonic development. Plant Sci [J], 318: 111221.

[0071] ZHANG J, WU S, BOEHLEIN S K, et al. 2019. Maize defective kernel5 isa bacterial TamB homologue required for chloroplast envelope biogenesis. JCell Biol [J], 218: 2638-2658.

[0072] ZHENG L, ZHANG X, ZHANG H, et al. 2019. The miR164-dependentregulatory pathway in developing maize seed. Mol Genet Genomics [J], 294:501-517。

Claims

1. Application of the maize ZmILR1 gene in regulating maize kernel size; characterized in that, Overexpression of the ZmILR1 gene in maize causes maize kernel enlargement, while knocking out the ZmILR1 gene in maize yields small-kernel maize material; the full-length DNA sequence of the maize ZmILR1 gene is shown in SEQ ID NO.1, and the cDNA sequence of the maize ZmILR1 gene is shown in SEQ ID NO.

2.

2. A small-seed maize gene Zmilr1, characterized in that, The gene is obtained by inserting 3 TCT bases at position 4 of the first exon of the ZmILR1 gene as described in claim 1, and simultaneously deleting 33 bases at positions 17-49; the DNA sequence of the small seed gene Zmilr1 is shown in SEQ ID NO.

4.

3. A method for creating large-kernel corn material, characterized in that, By using transgenic technology to increase the expression of the maize ZmILR1 gene as described in claim 1, or by using ZmILR1 transgenic lines to introduce ZmILR1 into maize materials with different genetic backgrounds through hybridization, backcrossing, or other means, maize materials with increased grain length, grain width, and 100-grain weight can be obtained.

4. A method for creating small-kernel corn material, characterized in that, The ZmILR1 gene described in claim 1 can be knocked out using CRISPR / Cas9 gene editing technology, RNA interference technology, or other technologies to obtain maize material with smaller kernels.

5. A method for creating small-grained maize materials with different genetic backgrounds, characterized in that, The small-grained maize material obtained by the method described in claim 4 is hybridized and backcrossed with the target material, thereby enabling the target material to acquire the Zmilr1 mutant gene and the small-grained trait.

6. The functional marker A1A4 developed for the maize small kernel gene Zmilr1 as described in claim 2, characterized in that, The functional marker A1A4 includes a first primer A1-F and a second primer A4-R, whose sequences are SEQ ID NO.5 and SEQ ID NO.6, respectively.

7. The application of the functional marker A1A4 of the maize small grain gene Zmilr1 as described in claim 6 in maize breeding, characterized in that, This functional marker can simultaneously distinguish between the maize ZmILR1 gene of claim 1 and the maize small kernel gene Zmilr1 of claim 2.

8. The method for creating large-grain maize material according to claim 3, wherein the overexpression vector used in the transgenic method is an overexpression vector of the ZmILR1 gene driven by the Ubi strong promoter.

Images