Use of a microprotein gene ZmHLH1 in regulating elongation of primary roots of plants

Abstract

This invention relates to the application of the microprotein gene ZmHLH1 in regulating primary root elongation in plants, belonging to the field of plant gene breeding technology. The nucleotide sequence of the microprotein gene ZmHLH1 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.2. Overexpression of the ZmHLH1 gene promotes primary root elongation and improves seedling quality. This invention is the first to discover that overexpression of the ZmHLH1 gene can promote primary root growth in maize, providing a new functional element and theoretical basis for breeding superior maize varieties with robust primary roots and uniform seedlings.

Description

Technical Field

[0001] This invention belongs to the field of plant gene breeding technology, specifically involving the application of a microprotein gene ZmHLH1 in regulating the elongation of primary roots in plants. Background Technology

[0002] Seedling establishment is a crucial transitional stage in maize from seed germination to seedling maturity, directly determining the plant's later growth vigor, stress resistance, and final yield. Primary roots, as the earliest established core absorption organs in maize seedlings, directly determine the efficiency of water and nutrient acquisition during the seedling stage, seedling rooting ability, and seedling uniformity through their elongation rate and growth length. They are the core root traits regulating the quality of maize seedling establishment, and improving seedling establishment quality is key to increasing seedling survival rate and overcoming yield bottlenecks. Currently, insufficient elongation of primary roots in the maize seedling stage often leads to phenotypic problems such as shallow rooting, weak stress resistance, and uneven emergence, severely affecting the quality of seedling establishment.

[0003] ZmHLH1 is an HLH-like microprotein composed of 92 amino acids and possessing a conserved HLH functional domain. While existing research has clearly demonstrated the involvement of HLH-like microproteins in the regulation of plant organ development, there is a lack of microprotein resources that can specifically regulate primary root growth. Furthermore, the biological function of ZmHLH1 has not been reported in maize. Therefore, this invention proposes the application of the microprotein gene ZmHLH1 in regulating primary root elongation in plants. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the microprotein gene ZmHLH1 in regulating the elongation of primary roots in plants in order to solve the above-mentioned problems.

[0005] The present invention achieves the above objectives through the following technical solutions: The present invention provides a microprotein gene ZmHLH1, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by which the gene is shown in SEQ ID NO.2.

[0006] This invention also provides an application of the microprotein gene ZmHLH1 in regulating the elongation of primary roots in plants.

[0007] As a further optimization of the present invention, overexpression of the ZmHLH1 gene promotes the elongation of primary roots in plants and improves the quality of seedling establishment.

[0008] As a further optimization of the present invention, the plant is corn.

[0009] As a further optimization of the present invention, in the process of plant breeding, a homozygous crop variety with overexpression of the ZmHLH1 gene is created using genetic engineering technology to obtain a crop variety with elongated primary roots and robust seedlings.

[0010] As a further optimization of the present invention, the specific steps for creating a homozygous maize variety overexpressing the ZmHLH1 gene using genetic engineering technology include: (1) Construct a recombinant plasmid UBI:ZmHLH1-HA containing the ZmHLH1 gene; (2) The recombinant plasmid UBI:ZmHLH1-HA was transferred into Agrobacterium competent cells. The callus tissue of maize was infected by Agrobacterium-mediated transformation, and maize seedlings were obtained by culturing. After the sequence was correct, the seedlings were cultured and propagated to obtain maize plants with primary root elongation.

[0011] The beneficial effects of this invention are as follows: This invention obtained ZmHLH1 overexpressing plants by transforming the ZmHLH1 gene into maize KN5585. Sequencing analysis and phenotypic identification revealed that, compared to the wild type, the primary roots of the ZmHLH1 overexpressing plants were significantly longer. This verifies that ZmHLH1 gene overexpression can promote primary root growth in maize, thereby improving seedling survival rate and seedling quality. This discovery provides a new functional element and theoretical basis for breeding superior maize varieties with robust primary roots and uniform seedlings. Attached Figure Description

[0012] Figure 1 The spectrum of the UBI:HA-Maize vector; Figure 2 Identification of ZmHLH1 overexpressing plants (A: qPCR identification results; B: Western blotting detection results); Figure 3 Primary root phenotype identification of ZmHLH1 overexpressing plants (A: primary root phenotype; B: primary root length statistics). Detailed Implementation

[0013] The present application will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0014] I. Experimental Materials Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products.

[0015] II. Experimental Methods 1. Obtaining ZmHLH1 overexpressing plants This invention utilizes wild-type maize (KN5585) as material, extracts total RNA, and reverse transcribes the extracted total RNA to synthesize cDNA first strand. The obtained cDNA first strand is used as a template for PCR amplification to obtain the corresponding ZmHLH1 gene fragment, which is then ligated into the cloning vector UBI:HA-Maize (see...). Figure 1 UBI:ZmHLH1-HA was obtained from [a company name - likely a company name] and transformed into E. coli. Positive clones were selected and sequenced. The sequencing results were compared with the ZmHLH1 genome sequence (DNA sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2). The sequencing results were consistent with the predicted results. The ZmHLH1 overexpression recombinant plasmid UBI:ZmHLH1-HA was constructed. This recombinant plasmid carries the HA fusion protein (amino acid sequence: YPYDVPDYA).

[0016] The specific primers for PCR amplification are: SEQ ID NO.3: ZmHLH1-FP: 5'-tttggtgttacttctgcagATGTCGAACCGGAGGTCA-3'; SEQ ID NO.4: ZmHLH1-RP: 5'-acctcctccaccCATGAGCAGGCTCCGG-3'.

[0017] The constructed recombinant vector was transferred into Agrobacterium, and the target gene was transferred into the mature callus tissue of maize KN5585 using Agrobacterium infection technology. After co-culture, screening and differentiation and rooting processes, transgenic T0 generation maize seedlings were obtained.

[0018] 2. Analysis of expression patterns in ZmHLH1 overexpressing plants Primary root tissues of T0 generation maize seedlings were collected, and the expression of ZmHLH1 was detected using real-time quantitative PCR (qPCR). The steps are as follows: 2.1 Total RNA extraction from maize Total RNA was extracted from maize primary root tissue using the HiPure Total RNA Midi Kit (dual-column method) from MIG Biotech. The specific procedures are as follows: After thorough grinding with liquid nitrogen, take an appropriate amount of powder into a 1.5 ml RNase-free EP tube, add 800 μL of lysis buffer RL, vortex for 15 s, and let stand at room temperature for 3 min. Centrifuge at 14000 rpm for 5 min to precipitate fragments. Transfer all supernatant to a gDNA filter column (placed in a 2 mL collection tube), centrifuge at 14000 rpm for 2 min, and discard the filter column. Add an equal volume of 70% ethanol to the filtrate and gently mix. Take 700 μL of the mixture each time and pass it through a HiPure RNA Mini column (centrifuge at 12000 rpm for 1 min), discard the filtrate, and repeat until all the column has been passed through. Add 500 μL of Buffer RW1 and 500 μL of Buffer RW2 (already containing ethanol), centrifuge at 12000 rpm for 1 min for each, and discard the filtrate; wash once more with Buffer RW2. Centrifuge an empty column at 12000 rpm for 2 min to completely remove residual ethanol. Transfer the column to a new 1.5 ml centrifuge tube, add 50 μL of RNase-free water to the center of the membrane, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 1 min to elute, discard the column, and store the RNA at -80℃. Assess the concentration and purity using a nucleic acid analyzer, and verify integrity using 1% agarose gel electrophoresis to obtain high-quality maize total RNA.

[0019] 2.2 Reverse transcription into cDNA Reverse transcription was performed using the Hifair® Ⅲ 1st Strand cDNA Synthesis Super Mix for qPCR (gDNA digester plus) kit (YEASEN). For the 20 μL system: Take 1 μg of total RNA, add 3 μL of 5×gDNA digesterMix and RNase-free ddH2O to a total volume of 15 μL, mix gently, and incubate at 42°C for 2 min. After the reaction, add 5 μL of 4×Hifair® Ⅲ Super Mix plus, and set the program as follows: 25°C, 5 min; 55°C, 15 min; 85°C, 5 min, to obtain cDNA.

[0020] 2.3 qPCR experiment The qPCR experiment was performed according to the AceQ® qPCR SYBR® Green Master Mix instructions. The primer sequences used for qPCR were: SEQ ID NO.5: 5'-AGCATCCCATGCAGCAATTC-3'; SEQ ID NO. 6: 5'-TCCTGGTGCAAGTTCCTGAT-3'.

[0021] The results are as follows Figure 2As shown in Figure A, two candidate plants with significantly higher ZmHLH1 gene expression levels than the wild type (KN5585) were screened from transgenic T0 generation maize seedlings by qPCR detection. They were tentatively named ZmHLH1-OE1 and ZmHLH1-OE2.

[0022] 3. Detection and analysis of protein content in ZmHLH1 overexpressing plants Protein extraction and immunoassay: Primary roots of ZmHLH1 transgenic maize grown for 8 days were placed in the middle of a 1.5 ml centrifuge tube, sealed with aluminum foil, and flash-frozen in liquid nitrogen. The roots were ground into powder using an electric drill, and 150 μL of SDS loading buffer, equivalent in volume to the powder, was quickly added. The mixture was vortexed, boiled for 5 min, cooled, and centrifuged at 14000 rpm. The supernatant was used for detection.

[0023] SDS-PAGE and Immunoblot: Prepare SDS-PAGE gel, install the electrophoresis tank, and add an appropriate amount of electrophoresis buffer. After loading the sample, electrophore at 80V for 30 min (to the separating gel interface), then adjust to 120V until bromophenol blue reaches the bottom of the gel. Activate the PVDF membrane with methanol for 30 s, assemble the transfer clip in the order of sponge-filter paper-gel-PVDF membrane-filter paper-sponge (gel negative, membrane positive), place it in the transfer tank, and transfer at a constant voltage of 20V for 30 min. After transfer, block the membrane in TBST containing 5% skim milk powder at room temperature for 2 h. Add primary antibody (1:1000 diluted in blocking buffer), and incubate overnight at 4℃ with gentle shaking. Wash 3 times with TBST (120 rpm, 10 min / wash). Add HRP-labeled secondary antibody (1:10000 diluted in blocking buffer), and incubate at room temperature for 1 h. Wash 3 times with TBST using the same method. Develop using an ECL chemiluminescence ultrasensitive colorimetric kit (Yisheng). Ponceau S staining was used as an internal control.

[0024] The results are as follows Figure 2 As shown in Figure B, the expression levels of the target protein in ZmHLH1-OE1 and ZmHLH1-OE2 were significantly higher than those in the wild type (KN5585), confirming that ZmHLH1-OE1 and ZmHLH1-OE2 are ZmHLH1 overexpressing plants.

[0025] 4. Phenotypic analysis of primary roots in ZmHLH1 overexpressing plants Wild-type seeds KN5585, ZmHLH1-OE1, and ZmHLH1-OE2 were selected and surface-sterilized (immersed in 75% ethanol for 30 seconds, rinsed 3 times with sterile water, immersed in 0.1% sodium hypochlorite for 10 minutes, and rinsed 5 times with sterile water). They were then evenly sown in petri dishes lined with moist filter paper and placed in a plant intelligent incubator (white light, 22℃) for germination culture for 8 days. Ten seedlings of uniform growth were selected from each plant, and the length of the primary root was measured with a ruler. The differences between the plants were statistically analyzed.

[0026] The results are as follows Figure 3As shown, compared with wild-type KN5585, the primary root lengths of ZmHLH1-OE1 and ZmHLH1-OE2 were significantly increased, further verifying that overexpression of the ZmHLH1 gene can significantly promote the elongation of maize primary roots.

[0027] Well-developed primary roots enable maize seedlings to better absorb water and nutrients from the soil, enhancing seedling stability and thus improving seedling survival rate and seedling quality. Therefore, the significance of primary root elongation in agricultural applications lies mainly in its ability to improve maize's stress resistance and growth vigor, thereby helping to increase crop yield per unit area.

[0028] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A microprotein gene ZmHLH1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. The application of the microprotein gene ZmHLH1 as described in claim 1 in regulating the elongation of primary roots in plants.

3. The application according to claim 2, characterized in that: Overexpression of the ZmHLH1 gene promotes the elongation of primary roots in plants and improves the quality of seedling establishment.

4. The application according to claim 2, characterized in that: The plant in question is corn.

5. The application according to claim 2, characterized in that: In the process of plant breeding, genetic engineering technology is used to create homozygous crop varieties with overexpression of the ZmHLH1 gene, resulting in crop varieties with elongated primary roots and robust seedlings.

6. The application according to claim 5, characterized in that: The specific steps for creating homozygous maize varieties overexpressing the ZmHLH1 gene using genetic engineering techniques include: (1) Construct a recombinant plasmid UBI:ZmHLH1-HA containing the ZmHLH1 gene; (2) The recombinant plasmid UBI:ZmHLH1-HA was transferred into Agrobacterium competent cells. The callus tissue of maize was infected by Agrobacterium-mediated transformation, and maize seedlings were obtained by culturing. After the sequence was correct, the seedlings were cultured and propagated to obtain maize plants with primary root elongation.

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