A ZmHLH2 gene that regulates maize leaf angle and plant height and its application

Abstract

This invention discloses a ZmHLH2 gene that regulates maize leaf angle and plant height, and its applications, belonging to the field of plant genetic engineering technology. The genomic sequence of the ZmHLH2 gene is shown in SEQ ID NO:1, the CDS sequence is shown in SEQ ID NO:3, and the amino acid sequence of its expressed protein is shown in SEQ ID NO:2. This invention utilizes CRISPR / Cas9 technology to knock out the maize ZmHLH2 gene. The mutant exhibits significantly reduced leaf angle and plant height, which is of great significance for maize high-density breeding. Upright leaves allow densely planted crops to capture more light by reducing mutual shading, thereby contributing to increased crop yield per unit area.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular biology, specifically to the ZmHLH2 gene that regulates maize leaf angle and plant height and its applications. Background Technology

[0002] Maize is an important food crop in my country and a significant source of feed and bioenergy. Due to continuous population growth and the resulting economic expansion, the demand for maize is constantly increasing. How to improve maize yield per unit area is a crucial issue. Increasing evidence suggests that increasing planting density is an effective strategy for increasing crop yield per unit area. However, increasing maize planting density leads to a significant decrease in the red light (R):far-red light (FR) ratio, causing Shade Avoidance Responses (SAR). This signal can serve as a warning of impending competition from neighboring plants and triggers several physiological changes in maize plants, leading to a series of adverse consequences, including reduced branching, stem elongation, increased lodging susceptibility, increased auricle height, premature flowering and senescence, reduced light utilization, reduced grain filling, and active suppression of defense responses against pathogens and herbivores, ultimately resulting in reduced maize biomass and yield (Martinez-Garcia and Rodriguez-Concepcion, 2023; Wang et al., 2016). A key factor determining the optimal planting density for field maize is the maize's response to shading. Therefore, in order to maximize crop yield, there is an urgent need to develop new varieties with traits that mitigate the harmful effects of SAR.

[0003] Leaf angle and plant height (LA) are the angles between the vertical stem and the midrib of the leaf in a plant, and are key agronomic traits regulating the structure of densely planted plants. Upright leaves enhance light perception, photosynthetic efficiency, ventilation, and stress resistance in densely planted canopies, thereby achieving dense planting and increasing cereal crop yields. For example, the rice mutant OsDWARF4 exhibits upright leaves and higher biomass and yield than the wild type under dense planting conditions (Sakamoto et al., 2006). In maize, two classic genes, ZmLG1 (Liguleless1) and ZmLG2 (Liguleless2), play a crucial role in the formation of the tongue-shaped region that alters leaf angle (Moreno et al., 1997; Walshe et al., 1998). In the lg1 and lg2 mutants, the absence of ligules and auricles leads to excessively upright leaves, reduced leaf angle, and smaller plant height (Moreno et al., 1997; Walshe et al., 1998; Mantilla-Perez and Salas Fernandez, 2017). Introducing the lg2 mutant allele into maize inbred lines increases maize grain yield (Pendletone et al., 1968; Lambert and Johnson, 1978). Recent studies have demonstrated that ZmRAVL1 directly activates ZmBRD1 expression, leading to increased BR levels and greater leaf angle and plant height (Tian et al., 2019). ZmLG1 can directly bind to the upstream regulatory sequence of ZmRAVL1. Furthermore, ZmDRL1 (a YABBY protein with a C2C2 zinc finger DNA-binding domain, a negative regulator of LA) can physically interact with ZmLG1, inhibiting the transcriptional activation of ZmRAVL1 by ZmLG1, thereby reducing maize leaf angle and plant height (Strable et al., 2017). Over the past 30 years, several genes related to leaf angle and plant height formation have been identified in maize based on genetic, molecular, and transcriptomic analyses; however, the molecular mechanisms regulating these parameters require further investigation (Caoe et al., 2022). This study involves ZmHLH2, an atypical bHLH transcription factor composed of 261 amino acids with a conserved HLH functional domain, whose biological function has not yet been reported in maize. This invention investigates the molecular biological function of this gene in regulating maize leaf angle and plant height, and provides gene resources for breeding superior high-yielding maize varieties with a dense plant type. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a ZmHLH2 gene that regulates maize leaf angle and plant height and its application.

[0005] This invention is achieved using the following technical solution:

[0006] The first objective of this invention is to provide a ZmHLH2 gene that regulates the leaf angle and plant height of maize, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] As a further improvement to the present invention, the protein encoded by the ZmHLH2 gene has the amino acid sequence shown in SEQ ID NO.2.

[0008] The second objective of this invention is to provide an application of the above-mentioned ZmHLH2 gene in regulating maize leaf angle and plant height.

[0009] As a further improvement to the present invention, the expression of the ZmHLH2 gene regulates the leaf angle and plant height of maize to become smaller.

[0010] A third objective of this invention is to provide a knockout vector, wherein the knockout vector is a CRISPR knockout vector containing an sgRNA target site sequence for knocking out the ZmHLH2 gene described above.

[0011] As a further improvement to the present invention, the sgRNA target site sequence comprises the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5:

[0012] SEQ ID NO.4: Targel1: 5'-TGTCGTCGGGGCGACGTGGC-3';

[0013] SEQ ID NO. 5: Targel2: 5'-GGCGGCGTGAGGACTCAGGG-3'. .

[0014] The fourth objective of this invention is to provide a genetically engineered bacterium, wherein the host cell is Agrobacterium containing the above-mentioned knockout vector.

[0015] The fifth objective of this invention is to provide a method for cultivating maize with small leaf angles and short plant height by knocking out the maize ZmHLH2 gene to obtain maize plants with smaller leaf angles and shorter plant height.

[0016] As a further improvement to the present invention, the following steps are included:

[0017] (1) Construct a recombinant gene CRISPR / Cas9 knockout vector containing the target site sequence of the maize ZmHLH2 gene sgRNA.

[0018] (2) The CRISPR / Cas9 knockout vector was transferred into Agrobacterium competent cells, and the maize callus tissue was infected by Agrobacterium-mediated transformation. Maize seedlings were obtained by culturing and the seedlings were obtained after the sequencing was correct. The seedlings were cultured and propagated to obtain maize plants with small leaf angles and short plant height.

[0019] This invention utilizes CRISPR / Cas9 gene knockout technology to knock out the ZmHLH2 gene in the KN5585 inbred line, obtaining a ZmHLH2 loss-of-function mutant. Sequencing analysis detected two mutation types: the first mutant has a 60-base deletion, resulting in a large fragment deletion of the ZmHLH2-encoded protein sequence; the second mutant has a 59-base deletion, causing premature termination of ZmHLH2-encoded protein expression. Both knockout mutation types resulted in a decrease in the angle between maize leaves.

[0020] The beneficial effects of this invention are: the ZmHLH2 gene provided by this invention can regulate the leaf angle and plant height of maize, which is of great significance for maize high-density breeding; maize varieties with upright leaves have better canopy structure and higher light energy utilization, thus achieving higher yields. Attached Figure Description

[0021] Figure 1 It is a ZmHLH2 mutant phenotype; Figure 1 In the middle: A, the target site of the ZmHLH2 gene; B, the mutant (zmbhlh2-1) has 60 bases deleted at the first exon, resulting in a large deletion in the protein sequence; the mutant (zmbhlh2-2) has 59 bases deleted at the first exon, resulting in disordered protein sequence and premature termination of protein expression.

[0022] Figure 2 In the middle: A, leaf angle and plant height phenotypes of wild-type KN5585 and mutant; B, C, plant height, leaf angle and plant height angle statistics of wild-type KN5585 and mutant; ns indicates no significant difference, ** indicates significant difference (P<0.001, Student's t-test);

[0023] Figure 3 This shows the expression of ZmHLH2 in different maize tissues;

[0024] Figure 4 This shows the expression of ZmHLH2 after Shade processing. Detailed Implementation

[0025] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention will be further described below with reference to specific illustrations.

[0026] 1. Materials

[0027] Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products. All primers used are indicated upon their first appearance, and subsequent use of the same primers follows the same indication.

[0028] 2. Method

[0029] 2.1 Obtaining and identifying ZmHLH2 knockout mutants

[0030] This invention utilizes CRISPR / Cas9 technology to design and construct a knockout vector for ZmHLH2. The design principles for the sgRNA target site sequence are as follows: 1) The knockout site is located in the coding sequence (CDS) region and preferably at the protein's front end or in an important functional domain; 2) It should cover a higher proportion of the transcript; 3) There should be no off-target effects or off-target effects should be located in intergenic regions; 4) Target sites with high editing efficiency are preferred; 5) The sequence should have a relatively balanced GC content and be less prone to secondary structure formation. This invention uses the Huazhong Agricultural University CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ) for design, selecting targets with high target scores, low off-target rates, and suitable locations as sgRNA target site sequences. The following sequences are the targets used when constructing the CRISPR knockout vector for the ZmHLH2 gene:

[0031] SEQ ID NO.4: Targel1:5'-TGTCGTCGGGGCGACGTGGC-3'

[0032] SEQ ID NO. 5: Targel2: 5'-GGCGGCGTGAGGACTCAGGG-3'.

[0033] The two target sequences were constructed into a CRISPR knockout vector to obtain the ZmHLH2 knockout vector. This knockout vector was then transformed into Agrobacterium, and the target gene was transferred into mature callus tissue of KN5585 maize using Agrobacterium-mediated maize transgenic technology. After co-culture, selection, and differentiation / rooting, transgenic T0 generation maize seedlings were obtained. Primers were designed to amplify a 400-500 bp DNA segment containing the initial CRISPR target site, and the amplified DNA was sent to a sequencing company for sequencing. The sequencing primer sequences are as follows:

[0034] SEQ ID NO.6: 5'-TTCACTCCTATCTCCGTTTG-3'

[0035] SEQ ID NO.7: 5'-GCAGGCTTTAATTTGCTG-3'.

[0036] The sequencing results were compared with the ZmHLH2 genome sequence (DNA sequence as shown in SEQ ID NO.1, CDs sequence as shown in SEQ ID NO.3). It was found that the first mutation type resulted in a deletion of 60 bases, and the second mutation type resulted in a deletion of 59 bases, leading to premature termination of expression of the protein ZmHLH2 (SEQ ID NO.2) encoded by ZmHLH2. Figure 1 Transgenic plants (AC) were named zmbhlh2-1 and zmbhlh2-2, respectively. After normal culture and propagation, T1 generation transgenic plants were obtained. After normal growth, it was found that compared to the wild-type KN5585, both zmbhlh2-1 and zmbhlh2-2 exhibited varying degrees of reduced leaf angle and plant height, resulting in shorter plant heights. The results are as follows: Figure 2 As shown. Upright leaves allow densely planted crops to capture more light by reducing mutual shading. Therefore, the significance of smaller leaf angles and plant height in agricultural applications lies primarily in enabling more dense crop planting, thereby helping to increase crop yield per unit area.

[0037] 2.4 Expression Pattern Analysis of ZmHLH2 under Different Shade Treatments

[0038] Maize was treated with shade for different time periods (0, 1, 2, 4 h), and the expression of ZmHLH2 at different shade treatment times was detected using real-time quantitative PCR. The steps included:

[0039] 2.4.1 Total RNA extraction from maize

[0040] Total RNA was extracted from maize using the HiPure Total RNA Midi Kit (dual-column method) from MGI Genomics.

[0041] Plant leaf tissue was ground into powder using liquid nitrogen. Using a pre-cooled liquid nitrogen spatula, the powder was placed into a 1.5 mL RNase-free EP tube. 800 μL of lysis buffer (RL) was added, and the mixture was vortexed for 15 seconds and incubated at room temperature for 3 minutes. The lysate was centrifuged at 14000 rpm for 5 minutes to precipitate the fragments. A gDNA Filter Mini Column was placed in a 2 mL collection tube. The tissue supernatant was transferred to a gDNA filter column. Centrifuged at 14000 rpm for 2 minutes. The gDNA filter column was discarded. An equal volume of 70% ethanol was added to the filtrate, and the mixture was pipetted 3-5 times. A HiPure RNA Mini Column was placed in a 2 mL collection tube. 700 μL of the mixture was transferred to the RNA column. The column was centrifuged at 12000 rpm for 1 minute. The filtrate was discarded, and the column was returned to the collection tube. The remaining mixture was transferred to the column. The column was centrifuged at 12000 rpm for 1 minute. After discarding the filtrate, reassemble the column into the collection tube. Add 500 μL of Buffer RW1 to the column. Centrifuge at 12000 rpm for 1 min. Discard the filtrate, add 500 μL of Buffer RW2 (diluted with ethanol) to the column, centrifuge at 12000 rpm for 1 min, and repeat this step once. Discard the eluent and reassemble the column into the collection tube. Centrifuge the empty column at 12000 rpm for 2 min. This step thoroughly removes any residual ethanol from the column. Transfer the column to a 1.5 mL centrifuge tube. Add 50 μL of N-Nase Free Water to the center of the column membrane. Incubate at room temperature for 2 min. Centrifuge at 12000 rpm for 1 min. Discard the column and store the RNA at -80℃. First, use a nucleic acid analyzer to determine the RNA concentration and whether there are any residual impurities. Then, run a gel electrophoresis (50 mL, 1% gel) to verify the integrity of the RNA, thus obtaining total corn RNA.

[0042] 2.4.2 Reverse transcription into cDNA

[0043] Reverse transcription experiment utilizes III. Obtain cDNA by reverse transcription using the 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit (YEASEN). Taking a 20 μL system as an example, first, add 1 μg total RNA, 3 μL 5× gDNA digester MiX enzyme, and 11 μL diluted water. Pre-denature at 42℃ for 2 min, then add 5 μL 4× ⅢsuperMixplus. Reaction conditions: 25℃ for 5 min, 55℃ for 15 min, 85℃ for 5 min.

[0044] 2.4.3 qRT-PCR experiment

[0045] qRT-PCR experiment based on The primer sequences used for qRT-PCR are as follows, as per the GreenMasterMix instructions:

[0046] SEQ ID NO: 8: 5'-GACGATGAAGAGGTGGTGGT-3'

[0047] SEQ ID NO: 9: 5'-GGACAGCAGGCAGGTGAG-3'.

[0048] The results showed that the expression level of ZmHLH2 was significantly increased after 4 hours of Shade treatment. Figure 4 ).

[0049] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A kind ZmHLH2 The application of genes in regulating maize leaf angle and plant height is characterized by, The ZmHLH2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. ZmHLH2 Gene knockout reduces the leaf angle and plant height in maize.

2. A method for cultivating maize with small leaf angles and short plant height, characterized in that, For corn ZmHLH2 Gene knockout mutations resulted in smaller leaf angles and shorter plant height. ZmHLH2 Mutant maize plants, the ZmHLH2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The method for cultivating maize with small leaf angles and short plant height according to claim 2, characterized in that, Includes the following steps: (1) Constructing a structure containing corn ZmHLH2 Recombinant gene CRISPR / Cas9 knockout vector containing the gene sgRNA target site sequence; (2) The CRISPR / Cas9 knockout vector was transferred into Agrobacterium competent cells, and the maize callus tissue was infected by Agrobacterium-mediated transformation. Maize seedlings were obtained by culturing and the seedlings were obtained after the sequencing was correct. After that, the seedlings were cultured and propagated to obtain maize plants with small leaf angles and short plant height.

4. The method for cultivating maize with small leaf angles and short plant height according to claim 3, characterized in that, The sgRNA target site sequence is shown in the nucleotide sequences of SEQ ID NO.4 and SEQ ID NO.5: SEQ ID NO.4: Targel1:5'-TGTCGTCGGGGCGACGTGGC-3'; SEQ ID NO. 5: Targel2: 5'-GGCGGCGTGAGGACTCAGGG-3'.

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