Use of maize gene zmgg1 in controlling maize yield

Abstract

The present invention relates to the technical field of plant genetic engineering. Provided is use of a maize gene ZmGG1 in controlling a maize yield. A protein encoded by ZmGG1 is as shown in SEQ ID NO. 2, and the gene is located in the 9th chromosome of maize and controls the kernel row number and the ear weight. CRISPR / Cas9 technology is used to knock out the gene to inhibit gene expression, such that the kernel row number and the ear weight can be increased.

Description

Application of the maize gene ZmGG1 in controlling maize yield Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. Specifically, it relates to the application of the maize gene ZmGG1 in controlling maize yield. The gene of this invention is located on chromosome 9 of maize and controls important yield traits such as the number of rows in the female ear and / or ear weight. Background Technology

[0002] Maize yield is a complex quantitative trait controlled by multiple genes. Ear length, number of kernels per row, number of rows per ear, ear weight, and cob weight are important components of maize yield. Analyzing maize yield traits by breaking them down into different yield factors helps to elucidate the genetic basis of yield trait formation, enabling breeders to more effectively utilize genetic resources to design breeding strategies and achieve high-efficiency breeding. At a specific planting density, maize yield per unit area is determined by the kernel yield per ear and the number of ears; the kernel yield per ear is determined by the number of kernels per ear and the weight of 100 kernels; the number of kernels per ear is determined by the number of rows per ear and the number of kernels per row; ear length and ear diameter are significantly correlated with the number of kernels per row and the number of rows per ear, respectively. Referring to yield and related trait data of 32 different maize varieties published in Argentina from 1965 to 2016, a consistent maximum possible planting density was maintained in the experimental design, with three replicates in a randomized block design. The results showed that over the past 50 years of breeding, maize yield has increased at an average rate of 113 kg / ha / year. This increase in yield was positively correlated with the increase in the number of kernels per ear, but unrelated to changes in individual kernel weight. Ear biomass accumulation increased year by year, while the tasseling and silking interval shortened, and flowering time became more consistent. However, kernel formation efficiency remained unchanged. The gradual trends of all traits were as expected, indicating that the increase in the number of kernels per ear is a key reason for the year-on-year increase in yield. Understanding the genetic basis of ear length and row kernel number is crucial for comprehending the mechanism of maize yield formation and provides a theoretical basis for breeding practices.

[0003] Therefore, this study used genetic methods to isolate a gene, ZmGG1, located on chromosome 9 of maize, that controls ear row number and ear weight. This gene encodes a G protein γ subunit protein involved in the plant's sugar signaling pathway. Based on the genetic phenotype and related molecular biological analysis of transgenic materials, the biological function of this gene in controlling traits such as ear row number and ear weight was confirmed. Genetic transformation research on ZmGG1 can provide genetic resources and theoretical support for maize breeding. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the maize gene ZmGG1 in controlling maize yield, wherein the protein encoded by the gene is shown in SEQ ID NO.2.

[0005] To achieve the above objectives, the present invention adopts the following technical measures:

[0006] Application of the maize gene ZmGG1 in controlling maize yield, wherein the protein encoded by the gene is shown in SEQ ID NO.2 (XP_008670224.1);

[0007] The applications described above, specifically:

[0008] Application of reducing the expression of the maize gene ZmGG1 in increasing maize yield;

[0009] Application of knocking out, suppressing or silencing the expression of the maize gene ZmGG1 in improving maize yield;

[0010] In the above applications, preferably, the knockout is performed using the CRISPR / Cas9 system. The knocked-out gene translates into a protein that has no original function or cannot be translated into a protein, thus achieving the effect of increasing maize yield.

[0011] Preferably, the target sites of gRNA in the CRISPR / Cas9 system are TGATGGTGGTATAAGTCGG and GAGGAATTCGATCTCGG.

[0012] Application of increasing the expression level of the maize gene ZmGG1 in reducing maize yield;

[0013] The above-described application involves introducing substances that enhance the expression of the ZmGG1 gene in maize into maize.

[0014] In the above-described applications, preferably, the substance is a nucleic acid molecule containing the ZmGG1 gene, or its expression cassette, recombinant vector, or recombinant microorganism;

[0015] The ZmGG1 gene is shown in SEQ ID NO.1.

[0016] In the above-described applications, the control of maize yield is achieved by controlling the number of rows per ear and / or ear weight.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This invention cloned and confirmed the gene ZmGG1, which controls ear row number and ear weight, in maize. The relationship between ear row number and ear weight and the expression level of ZmGG1 was also confirmed; reducing its expression level increased ear row number and ear weight. The difference in ear grain number between transgenic and wild-type materials is attributed to a decrease in the protein level encoded by this gene due to editing of its coding region. Mechanistically, this gene is believed to participate in the plant's sugar signaling response, increasing the sugar signaling response, thereby regulating the expression of downstream genes and ultimately controlling the differentiation activity of maize inflorescence meristems, affecting yield traits such as ear row number and ear weight. Therefore, this invention provides a new genetic resource for maize yield improvement. Attached Figure Description

[0019] Figure 1 is a schematic diagram of maize ZmGG1 gene knockout material;

[0020] Where: A is a schematic diagram of the editing of the maize ZmGG1 gene, in which zmgg1-1 is missing the ATCGACT base and zmgg1-2 is missing the G base. The insertion and deletion of these bases both lead to premature termination of protein translation;

[0021] B is a schematic diagram of the protein structure of the maize ZmGG1 gene knockout material. From top to bottom, they are: wild-type family (WT) isolated from transgenic heterozygous plants, and two ZmGG1 gene editing families, zmgg1-1 and zmgg1-2.

[0022] C is a schematic diagram of the female ear of maize ZmGG1 gene knockout material. From top to bottom, they are the wild-type family (WT), and two ZmGG1 gene-edited families, zmgg1-1 and zmgg1-2.

[0023] D is a schematic diagram of the number of ear rows in maize ZmGG1 gene knockout materials. The bars from left to right are the wild-type family (WT), the two ZmGG1 gene editing families zmgg1-1 and zmgg1-2, respectively.

[0024] E is a schematic diagram of the ear width of maize ZmGG1 gene knockout materials. The bars from left to right are the wild-type family (WT), the two ZmGG1 gene editing families zmgg1-1 and zmgg1-2, respectively.

[0025] F is a schematic diagram of ear weight of maize ZmGG1 gene knockout materials. The bars from left to right are the wild-type family (WT), and two ZmGG1 gene-edited families, zmgg1-1 and zmgg1-2.

[0026] Figure 2 is a schematic diagram of the expression pattern of the maize ZmGG1 gene;

[0027] Among them: A: ZmGG1 is highly expressed in maize meristems; BC: In situ hybridization results show that ZmGG1 is specifically expressed in IM (inflorescence meristem), SPM (spikelet pair meristem) and SM (spikelet meristem) of 2mm young ears. Detailed Implementation

[0028] The following embodiments further define the present invention. Based on the following description and examples, those skilled in the art can determine the basic features of the present invention, and can make appropriate improvements and modifications to the present invention without departing from its spirit and scope, so as to make it suitable for various uses and conditions. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all derived from commercial channels or publicly disclosed materials.

[0029] Example 1: ZmGG1 Cloning

[0030] Total DNA was extracted from leaves of inbred line KN5585 (Liu, et al. High-throughput CRISPR / Cas9 mutagenesis streamlin es trait gene identification in maize. The Plant Cell, 2020, 32: 1397–1413). Primers G1-F and G1-R were designed based on the genome reference sequence of maize B73 (National Crop Germplasm Center). The ZmGG1 gene was amplified by PCR and resequencing in KN5585 material, and the complete nucleotide sequence of the ZmGG1 gene was obtained (SEQ ID NO.1). The protein encoded by this gene is shown in SEQ ID NO.2.

[0031] Total DNA was extracted from plant leaves using the CTAB method. The PCR amplification program was as follows: pre-denaturation at 94℃ for 5 min, followed by 34 cycles of denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 60 s, and finally extension at 72℃ for 5 min.

[0032] Table 1. Primers and their sequences used in this invention

[0033] Example 2: Genetic transformation of ZmGG1 in maize

[0034] The genetic transformation for ZmGG1 gene knockout utilized ZmGG1 from the transgenic recipient material KN5585 as the applied gene, with the sequence shown in SEQ ID NO.1. Gene targets were designed using the CRISPR-P website (http: / / cbi.hzau.edu.cn / crispr / ), ultimately yielding guide RNAs (Target: TGATGGTGGTATAAGTCGG and GAGGAATTCGATC TCGG). Based on these guide RNAs, two ZmU6-Target-sgRNA fragments (sequences shown in SEQ ID NO.3 and SEQ ID NO.4) were synthesized using gene synthesis and constructed into the commercially available pEASY-T1 vector.

[0035] The fragment was amplified by PCR using primers pU6F1 and gRR1 (primer sequences are shown in Table 1, primer ID 2). The CPB-ZmUbi-hspCas9 vector was linearized by HindIII digestion, and the fragments were recovered by electrophoresis gel extraction and detected. Guide RNA was ligated into the target vector CPB-ZmUbi-hspCas9 (CN113004383A) via homologous recombination. Finally, the clone was sequenced using CRI SPR vector detection primers (primer sequences are shown in Table 1, primer ID 3) to confirm that the target fragment had been ligated into the vector.

[0036] The correctly cloned plasmid was transformed into the maize inbred line KN5585 via Agrobacterium-mediated transformation (genetic transformation was performed by the Life Science and Technology Center of China Seed Group Co., Ltd.). Using specific ZmGG1 gene detection primers (primer sequences are shown in Table 1, primer ID 4), two maize transformation events with KN5585 as the background were screened and obtained (Figure 1, A), zmgg1-1 and zmgg1-2. Among them, ZmGG1-1 lacked the GATCGACT base (ZmGG1-1 contains the sequence shown in SEQ ID NO. 5), and ZmGG1-2 lacked the G base (ZmGG1-2 contains the sequence shown in SEQ ID NO. 6). The deletion of these bases led to premature termination of protein translation (Figure 1, B). Furthermore, in 2023, the phenotypic values ​​of ear length and number of kernels per row in maize knockout families were investigated in Gansu (Figure 1, C). The results showed that, compared with wild type, after the loss of function of the ZmGG1 gene, the number of rows per ear in zmgg1-1 and zmgg1-2 maize increased by 11.1% and 9.4% respectively (Figure 1, D), and the ear width increased by 19.9% ​​and 14.9% respectively (Figure 1, E). Based on the above results, it is demonstrated that reducing the expression of the ZmGG1 gene can increase the ear length and number of kernels per row in maize, and the average weight per ear is increased by about 11.2% compared with wild type (Figure 1, F).

[0037] Example 3: Expression analysis of ZmGG1

[0038] Based on the maize B73 expression database, the expression pattern of the ZmGG1 gene was analyzed. The ZmGG1 gene was highly expressed in maize meristems (FPKM) (Figure 2A). At the same time, we used RNA in situ hybridization to verify the specific expression pattern of ZmGG1 in the ~2mm young ears (Figure 2BC, primer sequences are shown in Table 1, primer ID5). This gene is mainly highly expressed in the early IM, SPM and SM of maize young ears. Therefore, ZmGG1 affects traits such as ear length and number of kernels per row in maize.

Claims

1. Application of the maize gene ZmGG1 in controlling maize yield, wherein the protein encoded by the gene is shown in SEQ ID NO.2, and the application process is to increase maize yield by reducing the expression of the maize gene ZmGG1.

2. The application according to claim 1, characterized in that: The application process described above increases maize yield by knocking out, suppressing, or silencing the expression of the maize gene ZmGG1.

3. The application according to claim 2, characterized in that: The knockout process uses the CRISPR / Cas9 system, and the knocked-out gene translates into a protein that has no original function or cannot be translated into a protein.

4. The application according to claim 3, characterized in that: The target sites of gRNA in the CRISPR / Cas9 system are TGATGGTGGTATAAGTCGG and GAGGAATTCGATCTCGG.

5. The application according to claim 1, characterized in that: The ZmGG1 gene is shown in SEQ ID NO.

1.

6. The application according to claim 1, characterized in that: The control of maize yield is achieved by controlling the number of rows per ear and / or ear weight.

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