Genes controlling maize kernel dehydration rate and kernel moisture content and uses thereof

Abstract

The present application relates to genes for controlling maize kernel dehydration rate and kernel moisture content and application thereof, and belongs to the field of molecular genetics. The present application provides genes ZmMYBST1 and ZmMYBR43 for controlling maize kernel dehydration rate and kernel moisture content, and further discloses a method for changing maize kernel moisture content or dehydration rate by operating ZmMYBST1 and ZmMYBR43 by means of genetic engineering.

Description

Technical Field

[0001] This invention relates to genes that control the dehydration rate and moisture content of maize kernels and their applications, belonging to the field of molecular genetics. Background Technology

[0002] Grain moisture content is a key factor affecting the quality of mechanized corn harvesting, safe storage, and economic benefits. The moisture content of the grains at harvest has a significant impact on corn harvesting, drying, storage, transportation, and processing. Excessive moisture content often causes economic losses for corn growers and operators, reduces economic benefits, and easily leads to grain mold, affecting corn quality. In addition, mechanized grain harvesting has become one of the main factors limiting corn production in my country, and the most critical link in mechanized corn grain harvesting is that the moisture content of corn grains at harvest cannot reach the standard moisture content of ≤25% for mechanized grain harvesting (Wang Z, Wang X, Zhang L, Liu X, Di H, Li T, Jin X. QTL underlying field grain drying rate after physiological maturity in maize (Zea Mays L.) [J]. Euphaitica, 2012, 185(3): 521-528.). Therefore, the breeding of corn varieties with low grain moisture content at harvest is very important. In addition, low grain moisture content can shorten the growth cycle of corn, which is of great significance for pre-frost harvesting in high-latitude regions of my country and for not affecting wheat planting in the Huang-Huai-Hai region.

[0003] On the other hand, the appropriate harvest moisture content varies depending on the harvesting purpose. For example, corn kernels intended for silage require a high moisture content during harvest, generally controlled above 30%; while for fresh corn, to maintain better taste and nutritional value, the moisture content can be as high as 75%. Therefore, QTLs or functional genes that can control the moisture content and dehydration rate of corn kernels have significant industrial value.

[0004] Since grain dehydration and moisture content traits are controlled by multiple genes, clarifying the regulatory network of these traits will help to discover more genes that play a role in grain dehydration and moisture content traits, and enrich the technical means to artificially regulate grain dehydration and moisture content traits. Summary of the Invention

[0005] To address the aforementioned issues, this invention analyzes and screens transcription factors bound to the qKDR1 region, obtaining the genes ZmMYBST1 and ZmMYBR43 that control the dehydration rate and moisture content of maize kernels. Furthermore, it discloses a method for manipulating ZmMYBST1 and ZmMYBR43 using genetic engineering techniques to alter the moisture content or dehydration rate of maize kernels.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides an application of a maize gene in improving the moisture content or dehydration rate of maize kernels, characterized in that the LOC number of the maize gene in the B73 reference genome is Zm00001d000184 or Zm00001d029875.

[0008] In some implementations, the nucleotide sequence of the maize gene described above is shown in any one of SEQ ID NO.1 to SEQ ID NO.4.

[0009] The present invention also provides the application of a protein in improving the moisture content or dehydration rate of corn kernels, characterized in that the amino acid sequence of the protein is as shown in either SEQ ID NO.5 or SEQ ID NO.6.

[0010] The present invention also provides a method for increasing the moisture content of maize kernels or reducing the dehydration rate, characterized in that: the expression of the gene shown in claim 1 of maize is inhibited and / or the gene is knocked out, and plants with increased moisture content of maize kernels or reduced dehydration rate are selected;

[0011] In some implementations, the methods for inhibiting gene expression and / or knocking out the gene include gene editing or RNA interference;

[0012] In some implementations, the target sequences for the gene editing described above are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0013] This invention also provides a kit for increasing the moisture content of corn kernels or reducing the dehydration rate, characterized in that it comprises Cas9 protein and any one of the following RNA molecules:

[0014] (1) An RNA molecule capable of recognizing the target sequence of claim 3; in some embodiments, the sequence of the RNA molecule is shown in SEQ ID NO. 9 and SEQ ID NO. 10;

[0015] (2) A DNA molecule encoding the RNA described in (1);

[0016] (3) Vectors that express the RNA described in (1).

[0017] The present invention also provides a mutant gene, characterized in that: the nucleic acid sequence of the mutant gene is shown in any one of SEQ ID NO.11 to SEQ ID NO.16.

[0018] The present invention also provides the above-described method, or kit, or application of the mutant gene in improving the traits of corn kernel moisture content or dehydration rate.

[0019] Compared with the prior art, the beneficial effects of the present invention are: the ZmMYBST1 and ZmMYBR43 genes provided by the present invention affect the dehydration rate and moisture content of maize kernels, and can be used to improve the kernel dehydration rate and moisture content traits, and to breed new maize varieties with different kernel dehydration rates. Attached Figure Description

[0020] Figure 1 Expression patterns of ZmMYBST1, ZmMYBR43, and RPG genes in different tissues at different growth stages of maize. The horizontal axis represents different tissue samples, and the vertical axis represents the expression level.

[0021] Figure 2 A schematic diagram of the effector and reporter structures in the LUC experiment.

[0022] Figure 3 The inhibitory effect of ZmMYBST1 or ZmMYBR43 on LUC reporter genes.

[0023] Figure 4 Dehydration rate of maize kernels in ZmMYBST1 and ZmMYBR43 knockout lines. KO1, KO2, and KO3 represent different knockout lines. WT: recipient control; n: sample size. Kernel moisture content was measured using the AUDDC (Area Under the Dry Down Curve, AUDDC): a method to evaluate rate of dry down in maize[J]. Crop Sci., 2010, 50(6): 2347-2354.

[0024] Figure 5 Grain moisture content at harvest of ZmMYBST1 and ZmMYBR43 knockout lines. KO1, KO2, and KO3 represent different knockout lines. WT: recipient control; n: sample size. Detailed Implementation

[0025] The following definitions and methods are provided to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terms are to be understood in accordance with their conventional usage by those skilled in the art. All patent literature, academic papers, industry standards, and other publicly available publications cited herein are incorporated herein by reference in their entirety.

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance thereof are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0027] Example 1: Screening of qKDR1 binding proteins

[0028] Patent 2024101031551 discloses a QTL—qKDR1—that controls the rate of dehydration in maize kernels. This invention aims to further elucidate the mechanism by which qKDR1 functions, screen for transcription factors that bind to it, and thereby identify new genes related to kernel dehydration and water content.

[0029] The inventors first analyzed publicly available chromatin immunoprecipitation sequencing data (see Tu X, Mejía-Guerra MK, Valdes Franco JA, et al. Reconstructing the maize leaf regulatory network using ChIP-seq data of 104 transcription factors. Nat Commun, 2020, 11:5089) and found that 43 transcription factors can bind to the qKDR1 fragment site. Further analysis of the expression levels of these transcription factors in B73 and in knockout materials of another gene, RPG, regulated by the qKDR1 fragment (see patent 202410103198X), resulted in the removal of 26 genes with low or no expression in the seeds. Further analysis of the expression levels of the remaining 17 genes in tissues throughout the entire growth period of B73 revealed that the expression patterns of the genes Zm00001d000184 and Zm00001d029875 in maize kernels, embryos, and endosperm were similar to those of RPG. Figure 1 The inventors named the Zm00001d000184 and Zm00001d029875 genes ZmMYBST1 and ZmMYBR43, respectively (the genome sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), to further verify their functions.

[0030] Table 1 shows transcription factors that may bind to qKDR1.

[0031]

[0032]

[0033] KO-1 to KO-3 represent three expression data points from RPG gene knockout materials, while KO-1 to KO-3 represent three expression data points from wild-type B73 materials.

[0034] Example 2: Functional Verification of the Combination of ZmMYBST1 and ZmMYBR43

[0035] The inventors designed a transient transcriptional activity assay based on the LUC (Firefly luciferase) system to verify the binding of ZmMYBST1 and ZmMYBR43 proteins to the qKDR1 fragment. Specifically:

[0036] The protein-coding sequences of ZmMYBST1 and ZmMYBR43 driven by the CaMV 35S promoter (as shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively) were used as effectors, and pRPG3D and pRPG3K containing the qKDR1 fragment were used as reporters. The structures of the effectors and reporters are shown in (…). Figure 2 The LUC promoter in the reporter uses the promoter of the RPG gene (sequence shown in SEQ ID NO. 14 of patent 2024101028173). ZmMYBST1 and ZmMYBR43 were co-expressed with pRPG3D or pRPG3K as experimental groups, while the control group (containing qKDR1) contained no coding gene. 3D (fragment) or pRPG3K (including qKDR1) 3K (segment), qKDR1 3D Fragments and qKDR1 3K The fragment sequence is shown in Example 3 of patent 2024101028173. Co-expression was used as a control group. Protoplasts were extracted from leaves of etiolated seedlings of maize inbred line B73, transformed with plasmids, and after transformation and culture, cellular proteins were extracted and collected using the dual-luciferase reporter assay system (Promega, catalog number E1980) according to the kit instructions. Finally, the relative LUC activity, i.e., the LUC / REN ratio, was calculated.

[0037] The experimental results showed that, compared with the control group, the experimental group co-expressed with ZmMYBST1 or ZmMYBR43 resulted in a strong inhibition of the LUC activity of the reporter genes of both reporters. Figure 3 This experimental result indicates that both transcription factors ZmMYBST1 and ZmMYBR43 bind to the qKDR1 region and inhibit the expression of the RPG gene.

[0038] Example 3 Gene Knockout Experiment

[0039] The inventors further used the CRISPR-Cas9 tool to knock out the ZmMYBST1 and ZmMYBR43 genes in maize to verify their impact on the dehydration traits of maize kernels.

[0040] Maize B104 was selected as the receptor, and the designed target sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, while the gRNA sequences binding to the target are shown in SEQ ID NO.9 and SEQ ID NO.10. The remaining maize gene editing procedures can be found in Example 2 of CN112646013A.

[0041] The results showed that knocking out the ZmMYBST1 and ZmMYBR43 genes using CRISPR-Cas9 technology yielded three lines (named KO1, KO2, and KO3, respectively). Field tests indicated that, compared to the non-knockout recipient control plants, the dehydration rate of maize kernels in KO1, KO2, and KO3 was significantly slower. Figure 4 Further assessment of the moisture content at harvest revealed that KO1 increased the moisture content by 1.78%, KO2 by 1.05%, and KO3 by 3.31%. Figure 5 These results indicate that the ZmMYBST1 and ZmMYBR43 genes and their encoded proteins promote the dehydration of maize kernels, and inhibiting or knocking out these two genes can reduce the dehydration rate of kernels and increase kernel moisture content.

[0042] Analysis of the edited gene sequences of ZmMYBST1 and ZmMYBR43 in KO1, KO2, and KO3 showed that the ZmMYBST1 and ZmMYBR43 genes in KO1 were mutated to (SEQ ID NO.11 and SEQ ID NO.12), in KO2 to (SEQ ID NO.13 and SEQ ID NO.14), and in KO3 to (SEQ ID NO.15 and SEQ ID NO.16). Introducing these mutated genes into other maize varieties using backcrossing can improve maize kernel dehydration and moisture content traits, resulting in maize varieties with slower dehydration rates and higher moisture content.

[0043] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of inhibiting or knocking out maize genes in increasing maize kernel moisture content or reducing dehydration rate, characterized by: The LOC numbers of the maize genes in the B73 reference genome are Zm00001d000184 and Zm00001d029875; The nucleotide sequence of the maize gene with LOC number Zm00001d000184 is shown in either SEQ ID NO. 1 or SEQ ID NO.

3. The nucleotide sequence of the maize gene with LOC number Zm00001d029875 is shown in either SEQ ID NO. 2 or SEQ ID NO.

4.

2. The application of inhibiting or knocking out a protein in increasing the water content of corn kernels or reducing the dehydration rate, characterized in that: The protein in question is encoded by maize with LOC numbers Zm00001d000184 and Zm00001d029875 in the B73 reference genome. The amino acid sequence of the protein encoded by the maize gene with LOC number Zm00001d000184 is shown in SEQ ID NO. 5; The amino acid sequence of the protein encoded by the maize gene with LOC number Zm00001d029875 is shown in SEQ ID NO.

6.

3. A method for increasing the moisture content of corn kernels or reducing the dehydration rate, characterized in that: Suppress the expression of the gene described in claim 1 and / or knock out the gene, and select plants with increased corn kernel moisture content or decreased dehydration rate.

4. The method according to claim 3, characterized in that: The methods for suppressing gene expression and / or knocking out the gene include gene editing or RNA interference.

5. The method according to claim 4, characterized in that: The target sequence for gene editing is shown in SEQ ID NO. 7 or SEQ ID NO.

8.

6. A kit for increasing the moisture content of corn kernels or reducing the dehydration rate, characterized in that: Including Cas9 protein and any of the following: (1) RNA molecules with sequences as shown in SEQ ID NO. 9 and SEQ ID NO. 10; (2) The DNA molecule encoding the RNA described in (1); (3) A vector for expressing the RNA described in (1).

7. A mutant gene, characterized in that: The nucleic acid sequence of the mutated gene is shown in any one of SEQ ID NO. 11 to SEQ ID NO.

16.

8. The method according to any one of claims 3-5, or the kit according to claim 6, or the use of the mutant gene according to claim 7 in increasing the moisture content of maize kernels or reducing the dehydration rate.

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