Application of Maize Gene ZmGG1 in Controlling Maize Yield

Abstract

This invention, belonging to the field of plant genetic engineering technology, discloses the use of the maize gene ZmGG1 in controlling maize yield. The protein encoded by ZmGG1 is shown in SEQ ID NO. 2. This gene is located on maize chromosome 9 and controls the number of rows and weight of maize ears. Knocking out this gene and inhibiting its expression using CRISPR / Cas9 technology can increase the number of rows and weight of maize ears.

Description

Technical Field

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

[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. Dissecting maize yield traits into distinct yield factors facilitates understanding the genetic basis underlying yield traits, helping breeders more effectively utilize genetic resources to design breeding strategies and achieve efficient breeding. At a specific planting density, maize yield per unit area is determined by kernel yield per ear and number of ears; kernel yield per ear is determined by kernels per ear and 100-kernel weight, and kernels per ear are determined by number of rows per ear and kernels per row. Maize ear length and ear diameter are significantly correlated with kernels per row and number of rows per ear, respectively. Using yield and related trait data published by Argentina between 1965 and 2016 for 32 different maize varieties, the experimental design maintained a consistent maximum possible planting density and randomized blocks with three replicates. Results show that over the past 50 years of corn breeding, maize yield has increased at an average rate of 113 kg / ha / year. This yield increase is positively correlated with an increase in kernel number per ear and is unrelated to changes in individual kernel weight. Biomass accumulation per ear has increased annually, while the tassel-silking interval has shortened, and flowering has become more consistent. However, kernel formation efficiency has remained constant, and the gradual trends of all traits are consistent with expectations, indicating that increasing kernel number per ear is a key driver of the annual yield increase. Unraveling the genetic basis of ear length and kernel number per row is crucial for understanding the mechanisms of corn yield formation and provides a theoretical basis for breeding practice.

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

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

[0005] In order to achieve the above object, 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 mentioned above are:

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

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

[0010] In the above-mentioned applications, preferably, the knockout is carried out using the CRISPR / Cas9 system, and the protein translated from the knocked-out gene has no original function or cannot be translated into protein, thereby achieving the effect of increasing corn yield.

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

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

[0013] The application described above is to introduce substances that increase the expression of the maize ZmGG1 gene into maize;

[0014] In the above application, 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 as SEQ ID NO.1.

[0016] In the above application, the control of corn yield is achieved by controlling the number of ear rows and / or ear weight.

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

[0018] The present invention cloned and confirmed the gene ZmGG1, which controls the number of rows and weight of ears in maize, and confirmed the relationship between the number of rows and weight of ears and the expression level of ZmGG1. By reducing its expression level, the number of rows and weight of ears in maize can be increased. The difference in kernel number per ear between transgenic and wild-type materials is due to editing of the gene's coding region, which causes a decrease in the level of the protein encoded by the gene. In terms of its mechanism of action, it is believed that this gene is involved in the sugar signal response in the plant body, increasing the response to sugar signals, thereby regulating the expression of downstream genes, ultimately controlling the differentiation activity of the maize inflorescence meristem, affecting maize yield traits such as the number of rows and weight of ears. Therefore, the present invention provides a new genetic resource for improving maize yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of maize ZmGG1 gene knockout material;

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

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

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

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

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

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

[0026] Figure 2 Schematic diagram of the expression pattern of the maize ZmGG1 gene;

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

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

[0029] Example 1: ZmGG1 cloning

[0030] Total DNA was extracted from the leaves of the 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 PCR amplified and resequenced in the 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 from plant leaves was extracted using the CTAB method, and the PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, followed by 34 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 60 s, and finally extension at 72°C for 5 min.

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

[0033]

[0034]

[0035] Example 2: Genetic transformation of ZmGG1 in maize

[0036] The genetic transformation of ZmGG1 gene knockout was carried out by using ZmGG1 in the transgenic recipient material KN5585 as the applied gene, the sequence of which is shown in SEQ ID NO.1. http: / / cbi.hzau.edu.cn / crispr / ) for gene target design, ultimately obtaining Guide RNA (Target: TGATGGTGGTATAAGTCGG and GAGGAATTCGATC TCGG). Based on the Guide RNA, two ZmU6-Target-sgRNA fragments (sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4) were synthesized by gene synthesis and constructed into the commercial pEASY-T1 vector.

[0037] 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 using HindIII single enzyme digestion, recovered and detected by electrophoresis, and the Guide RNA was connected to the target vector CPB-ZmUbi-hspCas9 (CN113004383A) by homologous recombination. Finally, the resulting clone was sequenced using CRISPR vector detection primers (primer sequences are shown in Table 1, Primer ID 3) to confirm that the target fragment was connected to the vector.

[0038] The correctly cloned plasmid was transformed into the maize inbred line KN5585 via Agrobacterium-mediated transformation (genetic transformation was completed by the Life Science Technology Center of China National Seed Group Co., Ltd.). Using specific ZmGG1 gene detection primers (primer sequences are shown in Table 1, primer ID4), two maize transformation events were screened in the KN5585 background ( Figure 1 A), zmgg1-1 and zm gg1-2, among which ZmGG1-1 lacks the GATCGACT base (ZmGG1-1 contains the sequence shown in SEQ ID NO.5), and ZmGG1-2 lacks the G base (ZmGG1-2 contains the sequence shown in SEQ ID NO.6). The deletion of these bases leads to the premature termination of protein translation ( Figure 1 Furthermore, in 2023, the phenotypic values ​​of maize ear length and kernel number per row in ZmGG1 gene knockout families were investigated in Gansu ( Figure 1 The results showed that compared with WT, the number of ears and rows of maize in zmgg1-1 and zmgg1-2 increased by 11.1% and 9.4%, respectively, after the ZmGG1 gene function was lost. Figure 1 D), the ear width increased by 19.9% ​​and 14.9% ( Figure 1Based on the above results, it was shown that reducing the expression of ZmGG1 gene can increase the ear length and the number of kernels per row of corn. Compared with the wild type, the average weight per ear increased by about 11.2% ( Figure 1 Middle F).

[0039] Example 3: Expression analysis of ZmGG2

[0040] Based on the maize B73 expression database, the expression pattern of the ZmGG1 gene was analyzed. The expression level of the ZmGG1 gene in maize meristem was very high (FPKM) ( Figure 2 At the same time, we used RNA in situ hybridization to verify the specific expression pattern of ZmGG1 in ~2mm young ears ( Figure 2 The primer sequences are shown in Table 1 , primer ID 5). This gene is highly expressed in the early IM, SPM, and SM of the maize ear; therefore, ZmGG1 affects maize traits such as ear length and kernel number per row.

Claims

1. Corn Genes ZmGG1 The application in controlling corn yield, the protein encoded by the gene is shown in SEQ ID NO.2, and the application process is to reduce the corn gene ZmGG1 expression to increase maize yield.

2. The use according to claim 1, characterized in that: The application process is to knock out, inhibit or silence corn genes ZmGG1 expression to increase maize yield.

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

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

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

1.

6. The use according to claim 1, characterized in that: The corn yield is controlled by controlling the number of ear rows and / or ear weight.

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