Application of ZmbHLH156 protein or coding gene thereof in regulation and control of corn plant height

Abstract

The invention discloses an application of a ZmbHLH156 protein or an encoding gene thereof in regulating and controlling the height of a corn plant. It is found for the first time that the corn bHLH family transcription factor ZmbHLH156 protein or the coding gene thereof can regulate and control the plant height, a recombinant vector p3301-35S containing a ZmbHLH156 gene CDS sequence shown in SEQ ID NO: 2 in a sequence table is converted into corn, the plant heights of obtained T2 and T3 generation transgenic lines are statistically shown to be extremely remarkably lower than that of wild type corn, and the corn bHLH family transcription factor ZmbHLH156 protein or the coding gene of the corn bHLH family transcription factor ZmbHLH156 protein or the coding gene thereof can regulate and control the plant height. The method has high practical application value in plant variety improvement or breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of a ZmbHLH156 protein or its encoding gene in regulating the growth of maize plants. Background Technology

[0002] Maize (Zea mays L.) belongs to the genus Zea in the family Poaceae. It is an important economic, feed, and food crop, playing a vital role in production and daily life. Plant height is an important agronomical trait of maize. Studying the gene function that regulates plant height and constructing an ideal plant structure is of great significance for improving maize's lodging resistance and increasing its yield.

[0003] bHLH family transcription factors are key regulatory proteins widely distributed in eukaryotes, regulating target gene expression through specific binding to DNA. In plants, they primarily participate in core life processes such as plant growth and development, and organ morphogenesis by forming complexes.

[0004] While there are reports on the function of bHLH family proteins in plants under stress, there are no studies on their application in maize plant height. Therefore, the application of a ZmbHLH156 protein or its encoding gene in regulating maize plant height is urgently needed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an application of the ZmbHLH156 protein or its encoding gene in regulating the growth rate of maize plants.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide an application of the ZmbHLH156 protein or its encoding gene, or biological materials containing its encoding gene, in regulating the growth rate of maize plants.

[0008] Preferably, the application reduces plant height by promoting the expression of the ZmbHLH156 gene; and / or increases plant height by inhibiting the expression of the ZmbHLH156 gene.

[0009] Preferably, the application promotes or inhibits the expression of the ZmbHLH156 gene through genetic engineering or gene editing technology.

[0010] Preferably, the ZmbHLH156 protein has any one of the following amino acid sequences:

[0011] 1) The amino acid sequence shown in SEQ ID NO. 1; or

[0012] 2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO 1.

[0013] Preferably, the gene encoding the ZmbHLH156 protein has any one of the following nucleotide sequences:

[0014] 1) The nucleotide sequence shown in SEQ ID NO. 2, or

[0015] 2) The nucleotide sequence encoding a protein with the same function obtained by substituting, deleting, or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 2;

[0016] 3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO .2 under stringent conditions.

[0017] Preferably, the biomaterial is an expression cassette, vector, host cell, or recombinant bacteria.

[0018] The second objective of this invention is to provide a method for preparing transgenic plants of dwarf maize by introducing or overexpressing a gene encoding the ZmbHLH156 protein into the maize genome.

[0019] Preferably, the nucleotide sequence shown in SEQ ID NO. 2 is introduced or overexpressed in the plant genome.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention is the first to discover that the maize bHLH family transcription factor ZmbHLH156 protein or its encoding gene can regulate plant height. When maize is transformed with the recombinant vector p3301-35S containing the CDS sequence of the ZmbHLH156 gene shown in SEQ ID NO:2 in the sequence listing, the plant height of the T2 and T3 generation transgenic lines is statistically significantly lower than that of wild-type maize, which has high practical application value in plant variety improvement or breeding. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of the plant expression vector p3301-35S in Example 1 of the present invention.

[0023] Figure 2 This refers to the transcriptional level determination results of the ZmbHLH156 gene in transgenic maize overexpressing the maize bHLH family transcription factor ZmbHLH156 in Example 3 of this invention; where WT is wild-type B104, and OE#1, OE#2, and OE#4 are maize bHLH family transcription factor ZmbHLH156 overexpression lines. "" indicates significance at the P<0.0001 level.

[0024] Figure 3 This is the result of the phenotypic analysis of plant height in maize overexpression lines investigated in Example 4 of the present invention. Among them, WT is wild-type B104, and OE#1, OE#2 and OE#4 are maize bHLH family transcription factor ZmbHLH156 overexpression lines. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0026] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0027] Example 1: Cloning of the bHLH family transcription factor ZmbHLH156.

[0028] The forward primer is shown in SEQ ID NO. 3, and the reverse primer is shown in SEQ ID NO. 4;

[0029] The ZmbHLH156 gene was cloned and sequenced from By4944 using the forward primer 5'-GGGGACTCTTGACATGCAGCTCTTCCAAGGAGAG-3' and the reverse primer 5'-GGAAATTCGAGCTGTCAGTTTGAGCTGGAGATCTTGC-3', and its gene sequence is shown in SEQ ID NO. 2; the amino acid sequence of the protein encoded by it is shown in SEQ ID NO. 1.

[0030] The PCR reaction program was as follows: 95℃ for 3 min pre-denaturation, 95℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, 34 cycles, and 72℃ for 5 min extension.

[0031] Example 2: Construction of a plant expression vector for the bHLH family transcription factor ZmbHLH156.

[0032] The PCR product obtained in Example 1 was directly cloned into a clone using the enzyme digestion and ligation method. Figure 1The plant expression vector p3001-35S was used. The p3001-35S vector and PCR product were first recovered by digestion with NcoI and BstEII. The recovered product was then incubated at 50°C for 25 min. The ligation product was transformed into *E. coli* Top10 and cultured overnight at 37°C. Positive clones were screened and sequenced the following day. The plant transformation method used was Agrobacterium-mediated transformation. The selection marker in the plant was Bar.

[0033] Example 3: Determination of expression level of bHLH family transcription factor ZmbHLH156.

[0034] The expression level of the ZmbHLH156 gene in wild-type maize and the transgenic maize obtained in Example 2 was determined using quantitative real-time PCR (RT-PCR). RT-PCR was performed on an ABI StepOnePlus instrument, and the fluorescence signal was detected using StepOne Software. The reaction system was as follows:

[0035]

[0036] The reaction parameters were a two-step method: 95℃, 30s, hot start; 95℃, 10s, 60℃, 30s, 40 cycles. Gene expression was standardized and plotted using Excel. In the above transgenic maize, ZmbHLH156 was upregulated to varying degrees, such as... Figure 2 As shown. The primer sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6. Among them, SEQ ID NO. 5 is the forward primer; SEQ ID NO. 6 is the reverse primer.

[0037] Example 4: bHLH family transcription factor ZmbHLH156 reduces maize plant height.

[0038] The Agrobacterium tumefaciens culture obtained in Example 2 was sent to the company for transformation, resulting in three transgenic maize lines overexpressing ZmbHLH156. The results of plant height phenotypic analysis are as follows: Figure 3 The results showed that transformation of maize with the bHLH family transcription factor ZmbHLH156 resulted in a significant reduction in plant height.

[0039] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of ZmbHLH156 protein or its encoding gene, or biological materials containing its encoding gene, in regulating maize plant height.

2. The application according to claim 1, characterized in that, The application reduces plant height by promoting the expression of the ZmbHLH156 gene; and / or increases plant height by inhibiting the expression of the ZmbHLH156 gene.

3. The application according to claim 2, characterized in that, The application promotes or inhibits the expression of the ZmbHLH156 gene through genetic engineering or gene editing technology.

4. The application according to any one of claims 1-3, characterized in that, The ZmbHLH156 protein has any of the following amino acid sequences: 1) The amino acid sequence shown in SEQ ID NO. 1; or 2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO 1.

5. The application according to any one of claims 1-3, characterized in that, The gene encoding the ZmbHLH156 protein has any of the following nucleotide sequences: 1) The nucleotide sequence shown in SEQ ID NO. 2, or 2) The nucleotide sequence encoding a protein with the same function obtained by substituting, deleting, or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 2; 3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO .2 under stringent conditions.

6. The application according to claim 4 or 5, characterized in that, The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

7. A method for preparing transgenic plants of dwarf maize, characterized in that, According to the application described in claim 6, a gene encoding the ZmbHLH156 protein is introduced or overexpressed in the maize genome.

8. The method for preparing transgenic dwarf maize plants according to claim 1, characterized in that, Introducing or overexpressing the nucleotide sequence shown in SEQ ID NO. 2 into the plant genome.

Images