Corn compactness tolerance gene Zmhlh4 and application thereof in regulating plant height and leaf angle

Abstract

The application discloses a maize compactness tolerance gene ZmHLH4 and application thereof in regulating plant height and leaf angle, and belongs to the technical field of plant genetic engineering.The nucleotide sequence of the ZmHLH4 gene is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein is shown as SEQ ID NO.2.The application detects the expression of the ZmHLH4-HA fusion protein by Western Blot, and analyzes the phenotype of the ZmHLH4 gene overexpression plant, and finds that the plant height and the leaf angle of the overexpression plant are obviously reduced, the lower plant height and the upright leaf can make the compactly planted crops reduce mutual shielding and capture more light, thereby helping to improve the yield per unit area of the crops, and the research on the ZmHLH4 gene has important significance for cultivating maize compactness tolerance varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering and molecular biology, and particularly relates to a maize density-tolerant gene ZmHLH4 and application thereof in regulating plant height and leaf angle. BACKGROUND

[0002] How to improve the grain yield per unit area of maize is a very important problem, and increasing planting density is an effective strategy to improve the yield per unit area of crops. A key factor determining the optimal planting density of maize in the field is the response of maize to shading. Therefore, in order to maximize the yield of crops, it is urgent to breed new varieties with the trait of reducing the harmful effects of SARS. Plant height and leaf angle are the angle between the vertical stem and the midrib of the leaf, which are key agronomic traits for regulating the structure of densely planted plants.

[0003] The ZmHLH4 gene is an atypical bHLH transcription factor composed of 81 amino acids with a HLH conserved functional domain, and its biological function has not been reported in maize. The prior art disclosed in CN118667836A discloses the application of gene ZmHLH2 in regulating plant height and leaf angle, specifically "knocking out the ZmHLH2 gene of maize, the leaf angle and plant height of the mutant are significantly reduced", which is the only public report on the maize HLH gene family capable of regulating plant height. The development and mining of the functions of other members of the HLH gene family have positive significance for studying the role of the HLH gene family in plant phenotype regulation. At the same time, by mining more genes related to plant height and leaf angle regulation, more excellent gene resources can be provided for breeding new varieties of high-yield maize with dense tolerance. SUMMARY

[0004] The purpose of the present application is to provide a maize density-tolerant gene ZmHLH4 and its application in regulating plant height and leaf angle, so as to provide a new HLH family gene related to the regulation of maize plant height and leaf angle, and provide more excellent gene resources for the breeding of new maize varieties.

[0005] The present application adopts the following technical solutions to achieve the above purposes:

[0006] The first purpose of the present application is to provide a maize density-tolerant gene ZmHLH4, wherein the nucleotide sequence of the ZmHLH4 gene is shown as SEQ ID NO. 1.

[0007] As a further improvement of the present application, the encoded protein of the ZmHLH4 gene has an amino acid sequence as shown in SEQ ID NO. 2.

[0008] The second purpose of the present application is to provide the application of the above-mentioned ZmHLH4 gene in regulating the leaf angle and plant height of maize.

[0009] As a further improvement of the present application, the overexpression of the ZmHLH4 gene regulates the smaller leaf angle and the shorter plant height of the corn.

[0010] A third object of the present application is to provide an overexpression vector, which is obtained by introducing the above-mentioned corn compactness tolerance gene ZmHLH4 into a plasmid.

[0011] As a further improvement of the present application, the nucleotide sequence of the specific primer required when constructing the overexpression vector is shown in SEQ ID NO. 3 and SEQ ID NO. 4:

[0012] SEQ ID NO. 3: ZmHLH4-FP: 5' CGCGACGTGCCTCTAG 3';

[0013] SEQ ID NO. 4: ZmHLH4-RP: 5' TATGTTCCTGTCGACGG 3'.

[0014] As a further improvement of the present application, the plasmid is NEWMOL.

[0015] A fourth object of the present application is to provide a method for cultivating corn with small leaf angle and short plant height, which overexpresses the corn ZmHLH4 gene to obtain a ZmHLH4 gene overexpression corn plant with short plant height and small leaf angle.

[0016] As a further improvement of the present application, the method comprises the following steps:

[0017] (1) constructing a recombinant plasmid NEWMOL-ZmHLH4 containing the corn ZmHLH4 gene;

[0018] (2) introducing the recombinant plasmid NEWMOL-ZmHLH4 into an Agrobacterium competent cell, using Agrobacterium-mediated infection to invade corn callus, culturing to obtain corn seedlings, and culturing and breeding to obtain corn plants with short plant height and small leaf angle after sequencing.

[0019] The present application has the following beneficial effects: the present application introduces the ZmHLH4 gene into the B73 inbred line to obtain a plant with overexpression of the ZmHLH4 function, and through sequencing analysis and phenotype identification, it is found that the overexpression of the ZmHLH4 gene can lead to a shorter plant height and a smaller leaf angle of the corn. The present application first discovers that the ZmHLH4 gene can regulate the plant height and the leaf angle of the corn, which is of great significance for cultivating corn compactness tolerance varieties; corn varieties with upright leaves have a good canopy structure and a high light energy utilization rate, thereby obtaining a high yield. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a vector map for constructing ZmHLH4 overexpression line NEWMOL-ZmHLH4;

[0021] Figure 2 Figure 2 is a phenotype of wild type B73 and overexpression plant height and leaf angle; Figure 3 is a phenotype of wild type B73 and overexpression plant height and ear height and upper leaf angle and lower leaf angle and SPAD value statistics;

[0022] Figure 3 Figure 4 is a ZmHLH4 overexpression phenotype; Figure 1 Figure 5 is a ZmHLH4 overexpression plant protein identification;

[0023] Figure 4 Figure 6 is an expression of ZmHLH4 overexpression plant after white light and shade treatment;

[0024] Figure 5 Figure 7 is an evolutionary tree constructed using MEGA11;

[0025] Figure 6 Figure 8 is a protein sequence alignment using DNAMAN;

[0026] Figure 7 Figure 9 is a comparison of plant height ratio and leaf angle ratio of ZmHLH4 / ZmHLH2 overexpression plant and wild type plant with the same genetic background; wherein: A, comparison of plant height ratio of ZmHLH4 and B73 and plant height ratio of Zmhlh2 and Kn5585; B, comparison of leaf angle ratio of ZmHLH4 and B73 and leaf angle ratio of Zmhlh2 and Kn5585. DETAILED DESCRIPTION

[0027] In order to make the technical means, creative features, purposes and effects realized by the present application easy to understand, the present application is further described below in combination with specific drawings,

[0028] 1. Materials

[0029] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified. The reagents and other materials used are commercially available unless otherwise specified. The primers used are indicated at the first occurrence, and the same primers used thereafter are the same as the first indicated content.

[0030] 2. Methods

[0031] 2.1 Obtaining of ZmHLH4 expression vector and phenotype identification thereof

[0032] The application uses wild type corn (B73) as material, extracts total RNA, and synthesizes cDNA first strand by reverse transcription of the extracted total RNA, uses the obtained cDNA first strand as a template for PCR amplification, performs amplification to obtain a 240bp gene fragment, connects the gene fragment to a cloning vector NEWMOL to obtain NEWMOL-ZmHLH4 which is transformed into E. coli, picks positive clones and performs sequencing, compares the sequencing result with the ZmHLH4 genomic sequence (the DNA sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2), and the sequencing result is consistent with the predicted result, and the specific primers for PCR amplification are as follows:

[0033] SEQ ID NO. 3: ZmHLH4-FP: 5' CGCGACGTGCCTCTAG 3';

[0034] SEQ ID NO. 4: ZmHLH4-RP: 5' TATGTTCCTGTCGACGG 3'.

[0035] As shown in Figure 1 , the plasmid NEWMOL-ZmHLH4 is extracted, that is, the corn ZmHLH4 gene overexpression recombinant plasmid NEWMOL-ZmHLH4 is obtained, and the recombinant plasmid has a HA fusion protein.

[0036] The constructed recombinant vector is transformed into Agrobacterium, and the target gene is transformed into the mature callus of corn B73 by using the Agrobacterium infection corn transgenic technology, and through co-culture, screening and differentiation rooting process, the transgenic T0 generation corn seedlings are obtained.

[0037] The leaves are taken for qRT-PCR experiment to detect the RNA expression level and Western Blotting to detect the expression of ZmHLH4-3HA fusion protein in NEWMOL-ZmHLH4. The plants with successful protein identification are named as ZmHLH4OE1 and ZmHLH4OE2, respectively, and the transgenic T1 generation plants are obtained by normal culture and propagation, and after normal growth, it is found that compared with the wild type B73, ZmHLH4OE1 and ZmHLHOE2 have different degrees of dwarfing and leaf angle reduction, and the results are shown in Figure 2 . The upright leaves can reduce the mutual shading of the densely planted crops and capture more light. Therefore, the reduction of plant height and leaf angle is mainly significant for the agricultural application in that it can improve the planting density of crops, thereby helping to improve the yield per unit area of crops.

[0038] 2.2 Analysis of expression mode of ZmHLH4 transgenic plants

[0039] The expression of ZmHLH4 is detected by real-time fluorescent quantitative PCR, and the steps include:

[0040] 2.2.1 Total RNA extraction from corn

[0041] Total RNA extraction from corn was performed using HiPure Total RNA Midi Kit (double column method) from Omega Bio-Tek.

[0042] Plant leaf tissue was ground to powder with liquid nitrogen. The powder was transferred to a 1.5 mL RNase-free EP tube with a pre-chilled spatula in liquid nitrogen. 800 μL of lysis solution RL was added. After vortexing for 15 s, the sample was incubated at room temperature for 3 min. The lysate was centrifuged at 14,000 r / min for 5 min, and the pellet was discarded. The gDNA Filter Mini Column was assembled in a 2 mL collection tube. The supernatant was transferred to the gDNA filter column. The column was centrifuged at 14,000 r / min for 2 min. 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. The HiPure RNA Mini Column was assembled in a 2 mL collection tube. 700 μL of the mixture was transferred to the RNA column. The column was centrifuged at 12,000 r / min for 1 min. 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 12,000 r / min for 1 min. The filtrate was discarded, and the column was returned to the collection tube. 500 μL of Buffer RW1 was added to the column. The column was centrifuged at 12,000 r / min for 1 min. 500 μL of Buffer RW2 (diluted with ethanol) was added to the column, and the column was centrifuged at 12,000 r / min for 1 min. The procedure was repeated once. The filtrate was discarded, and the column was returned to the collection tube. The column was centrifuged at 12,000 r / min for 2 min. The column was transferred to a 1.5 mL centrifuge tube. 50 μL of RNase-Free Water was added to the center of the membrane. The sample was incubated at room temperature for 2 min. The column was centrifuged at 12,000 r / min for 1 min. The column was discarded, and the RNA was stored at -80°C. The concentration and purity of the RNA were determined using a nucleic acid detector. The integrity of the RNA was verified by running a gel (50 mL, 1% gel). The total RNA from corn was obtained.

[0043] 2.2.2 Reverse transcription to cDNA

[0044] The reverse transcription experiment was performed using III 1 stStrand cDNA Synthesis Super Mix for qPCR(gDNA digester plus) kit (YEASEN) Reverse transcription to obtain cDNA, for example, 20 μL system, first take 1 μg total RNA, 3 μL 5x gDNA digester Mix enzyme, 11 μL distilled water, 42°C pre-denaturation 2 min, then add Ⅲsuper Mix plus. Reaction conditions: 25°C for 5 min, 55°C for 15 min, 85°C for 5 min.

[0045] 2.2.2 qRT-PCR experiment

[0046] qRT-PCR experiment according to qPCR Green Master Mix instruction, the primer sequence used for qRT-PCR is:

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

[0048] SEQ ID NO. 6: 5'-AGTCTAGGGTCCTCCACCAT-3'.

[0049] It was found that the expression amount of ZmHLH4OE1 and ZmHLH4OE2 was significantly increased Figure 3 A).

[0050] 2.3 ZmHLH4 transgenic plant protein content detection analysis

[0051] The protein expression was detected by extracting protein from the leaves of the transgenic plants, and the steps included:

[0052] The leaves of mature ZmHLH4 transgenic plants were taken, then placed in the middle of a 1.5 ml centrifuge tube with tweezers, quickly wrapped the tube opening with tin foil, put into liquid nitrogen for quick freezing, then ground into powder with a drill and electric drill, quickly added 200 ul SDS Loading buffer to the powder, mixed and boiled for 5 min, after cooling, centrifuged at 14000 rpm for a short time, and then took an appropriate amount of sample for detection.

[0053] SDS-PAGE electrophoresis: After the SDS-PAGE gel was prepared, it was placed in the electrophoresis tank, and the electrophoresis liquid was added. After electrophoresis at 80 V for 30 min (after the sample was run into the separation gel), 120 V electrophoresis was performed until the blue band ran to the bottom of the glass plate. A PVDF membrane with the same size as the gel was prepared and immersed in methanol for 30 s to activate it. The gel was placed at the negative electrode, and the PVDF membrane was placed at the positive electrode. The electrophoresis tank was placed in an ice water mixture, and the voltage was set at 20 V for about 1 h. After the transfer was completed, it was placed in a blocking solution (5% skim milk powder, 10 ml TBST) and sealed in a shaking bed at room temperature for 1 h. After sealing, the primary antibody was added to the 10 ml TBST blocking solution (5% skim milk powder) at a ratio of 1:1000, and the membrane was immersed and sealed overnight at 4°C with slow shaking. Then the PVDF membrane was washed with TBST (120 rpm) for 3 times, each for 10 min. The corresponding secondary antibody was added to the blocking solution (5% skim milk powder) at a ratio of 1:10,000, and the membrane was transferred to a shaking bed at room temperature for 1 h. TBST was shaken (120 rpm) for 3 times, each for 10 min. The washed PVDF membrane could be developed by chemiluminescence (ECL chemiluminescence super-sensitive color reagent kit provided by Shanghai Yisen Biological Technology Co., Ltd.).

[0054] It was found that the expression of ZmHLH4 OE1 and ZmHLH4 OE2 proteins was as shown in Figure 3

[0055] 2.4 Corn shade phenotype system

[0056] After the corn was grown for 3 days (V1) after germination in a 28°C incubator under white light (WL, B=15 μmol m-2s-1, R=96 μmol m-2s-1, FR=21 μmol m-2s-1; R / FR=4.57; 16h WL / 8h), the seedlings in the shade treatment group were grown for 10 days under shade conditions (Shade, B=15 μmol m-2s-1, R=12 μmol m-2s-1, FR=105 μmol m-2s-1; R / FR=0.11; 16h WL / 8h), and the control group was grown for 10 days under white light. Photographs were taken, and the plant height, first leaf sheath, hypocotyl length, root length, leaf angle (first leaf), and new leaf chlorophyll content were measured as phenotype indicators. In order to more accurately determine the shade avoidance ability of corn, the change rate of phenotype under shade and white light [(SH-WL) / WL, Figure 4 midline] was introduced as an additional phenotype indicator.

[0057] 2.5 Comparison with ZmHLH2 mutants

[0058] Figure 5 ​The phylogenetic tree was constructed using MEGA11, which was based on protein sequences, constructed by maximum likelihood method, Bootstrap analysis, and the branch length was proportional to the estimated evolutionary distance. Figure 6 The protein sequence alignment was performed using DNAMAN, which included the amino acid sequences of ZmHLH2 and ZmHLH4, global alignment was used, and the default parameters were used for analysis. The alignment results showed the homology and variation sites between the sequences, and the homology of 100% was marked with red, and the homology of >=50% was marked with green. It can be seen from Figures 5-6 that the homology of ZmHLH2 and ZmHLH4 is low, which are two different genes.

[0059] According to the patent document CN118667836A, the deletion of ZmHLH2 gene leads to the decrease of plant height and leaf angle of wild type plants. In order to further compare the regulation function of ZmHLH4 gene of the present application, the mutant strain of ZmHLH2 gene constructed in the patent document CN118667836A was self-crossed with the Kn5585 wild type inbred line with the same genetic background to analyze the ratio of plant height and leaf angle, and the overexpression plant of ZmHLH4 gene constructed in the present application was self-crossed with the B73 wild type inbred line with the same genetic background to analyze the ratio of plant height and leaf angle, and the results are shown in Figure 7 It can be seen from the figure that compared with the reported ZmHLH2, the regulation effect of ZmHLH4 on leaf angle is more significant.

[0060] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application, and various changes and improvements can be made without departing from the spirit and scope of the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A maize compactness tolerance gene ZmHLH4 In the use of regulating maize leaf angle and plant height, characterized in that, The nucleotide sequence of the gene ZmHLH4 is shown as SEQ ID NO. 1, the gene ZmHLH4 overexpression regulates the corn leaf angle to be smaller and the plant height to be shorter.

2. A method of growing corn with small leaf angle and short plant height, characterized by, Genes for density tolerance in maize ZmHLH4 Overexpression resulted in shorter plant height and smaller leaf angle. ZmHLH4 In maize plants overexpressing the gene, wherein the gene ZmHLH4 The nucleotide sequence is shown in SEQ ID NO.

1.

3. The method of breeding corn plants having reduced leaf angle and reduced plant height according to claim 2, wherein, comprising the steps of: (1) Constructing a recombinant plasmid containing a corn ZmHLH4 gene NEWMOL-ZmHLH4 ; (2) The recombinant plasmid is NEWMOL-ZmHLH4 The cells were transferred into competent Agrobacterium cells and infected with maize callus tissue using Agrobacterium-mediated inoculation. Maize seedlings were obtained by culturing and, after correct sequencing, were cultured and propagated to obtain short-statured maize plants with small leaf angles.

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