Application of TaMYB55 protein and its encoding gene in regulating plant heat tolerance

Abstract

The present invention discloses the application of TaMYB55 protein and its encoding gene in regulating plant heat resistance. TaMYB55 protein is derived from wheat ( Summer wheat L.). The gene encoding TaMYB55 protein also belongs to the protection scope of the present invention. The gene encoding TaMYB55 protein is named TaMYB55 The present invention also protects the application of TaMYB55 protein in regulating plant heat resistance. The present invention is of great significance for the research and application of plant heat resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to TaMYB55 protein, encoding gene and application in regulating plant heat resistance. Background Art

[0002] Wheat is the world's second largest food crop, providing staple food for nearly 40% of the world's population and contributing about 20% of the world's dietary calories. Its cultivated area exceeds 220 million hectares, and it plays a vital role in agricultural production.

[0003] As a crop suitable for cool climates, wheat is extremely sensitive to high temperature stress. Data show that global high temperature stress causes about 15%-20% loss of wheat yield each year, among which the losses in the flowering and filling stages are the most significant. The influence of dry hot winds often shortens the wheat filling period by about 5-7 days, ultimately resulting in a 10%-15% reduction in yield and quality deterioration. Studies have shown that when the temperature rises by 1°C, the average yield of wheat decreases by 6%, and the decrease in some high-temperature sensitive areas can even reach 10%. Therefore, improving the heat tolerance of wheat is one of the key tasks in responding to climate change.

[0004] Through the selection and breeding of heat-resistant varieties, the discovery of key heat-resistant genes and the analysis of heat-resistant molecular mechanisms, it is hoped that new ideas will be provided for coping with high temperature stress and contribute to ensuring food security. Summary of the invention

[0005] In view of this, the object of the present invention is to provide TaMYB55 protein, encoding gene and application in regulating plant heat tolerance.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. TaMYB55 protein, selected from any of the following:

[0008] (a1) a protein having an amino acid sequence as shown in SEQ ID NO.1;

[0009] (a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (a1).

[0010] As one of the preferred technical solutions, the tag described in (a2) is a Flag tag, a His tag, a MBP tag, a HA tag, a myc tag, a GST tag and / or a SUMO tag.

[0011] 2. Genes encoding the aforementioned proteins TaMYB55 , selected from any of the following:

[0012] (b1) a DNA molecule whose coding region is shown in SEQ ID NO.2;

[0013] (b2) a DNA molecule having a nucleotide sequence as shown in SEQ ID NO.3;

[0014] (b3) A DNA molecule that hybridizes with the DNA molecule defined in (b1) or (b2) under stringent conditions and encodes the protein.

[0015] As one of the preferred technical solutions, the stringent conditions described in (b3) are: hybridization and membrane washing twice at 68°C in a solution of 2×SSC (nucleic acid hybridization washing solution) with a mass fraction of 0.1% SDS (sodium dodecyl sulfate), each time for 5 minutes, and hybridization and membrane washing twice at 68°C in a solution of 0.5×SSC with a mass fraction of 0.1% SDS, each time for 15 minutes.

[0016] 3. Related biological materials, selected from any of the following:

[0017] (c1) containing the above TaMYB55 Gene sgRNA expression cassette;

[0018] (c2) containing the above TaMYB55 A recombinant vector or recombinant microorganism containing the gene or the expression cassette as described in (c1).

[0019] As one of the preferred technical solutions, the expression cassette in (c1) refers to a DNA capable of expressing the sgRNA protein in a host cell, and any targeting TaMYB55 Single-stranded guide RNA of the gene, i.e., sgRNA, is used to construct a knockout plant cell containing the sgRNA. TaMYB55 Plant binary expression vector PUbi414- TaMYB55 .

[0020] As one of the preferred technical solutions, the recombinant vector in (c2) is constructed from a plant expression vector, and the plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment.

[0021] 4. The aforementioned TaMYB55 protein, TaMYB55 Application of genes or related biological materials in regulating plant heat tolerance.

[0022] As one of the preferred technical solutions, by promoting the expression of TaMYB55 protein or increasing TaMYB55 Gene expression improves plant heat tolerance.

[0023] 5. The aforementioned TaMYB55 protein, TaMYB55 Application of genes or related biological materials in breeding of heat-tolerant transgenic plants.

[0024] 6. A method for cultivating a heat-resistant transgenic plant, comprising: TaMYB55 The recombinant vector of the gene sgRNA is introduced into the recipient plant, and through induction, subculture, pre-culture, co-culture, resistance screening, differentiation, rooting culture, seedling hardening and transplanting, a transgenic plant with improved heat resistance is obtained.

[0025] As one of the preferred technical solutions, the recipient plant is a monocotyledonous plant or a dicotyledonous plant, more preferably a plant of the Poaceae family, more preferably a plant of the genus Triticum, more preferably a hexaploid wheat, and more preferably a wheat Fielder.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides TaMYB55 protein, encoding gene and application in regulating plant heat resistance, wherein TaMYB55 protein is derived from wheat ( Triticum aestivum L.). The gene encoding TaMYB55 protein also belongs to the protection scope of the present invention. The gene encoding TaMYB55 protein is named TaMYB55 The present invention also protects the application of TaMYB55 protein in regulating plant heat resistance. The present invention is of great significance for the research and application of plant heat resistance.

[0028] The present invention uses genetic engineering technology to inhibit TaMYB55 The expression of the gene can significantly reduce the heat resistance of wheat. The invention has important significance for the research and application of plant heat resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0030] Picture 1 This is the electrophoresis diagram of PCR identification of transgenic plants.

[0031] Picture 2 Schematic diagram of editing types of transgenic plants.

[0032] Picture 3 The phenotypic photos of the heat-treated transgenic plants are shown in Figure 1. From left to right, the order is Fielder control wheat, TaMYB55 Knockout strains Tamyb55 -9 and Tamyb55 -10.

[0033] Picture 4 The survival rate of the heat treatment group is shown in the figure below. The vertical axis is the survival rate. **** represents p≤0.0001. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0036] 1. Discovery of TaMYB55 protein and its encoding gene

[0037] A new protein was found in the roots of wheat Fielder cultured in water for 8 days. Its amino acid sequence is shown in SEQ ID NO.1 and named TaMYB55 protein. The gene encoding TaMYB55 protein was named TaMYB55 Gene. In the cDNA of wheat Fielder, TaMYB55 The coding sequence of the gene is shown in SEQ ID NO.2.

[0038] 2. Construction of recombinant plasmid

[0039] Reference "Optimization of Agrobacterium-mediated transformation inspring bread wheat using mature and immature embryos" (Kumar R, Mamrutha HM, Kaur A, Venkatesh K, Sharma D, Singh GP. 2019, Molecular Biology Reports 46:1845–1853), will TaMYB55 The sgRNA of the gene was connected to the wheat U6 promoter to form an sgRNA expression cassette; the sgRNA expression cassette was connected to the PUbi414 plasmid (Shanghai Kelei Biotechnology Co., Ltd.) to obtain the recombinant plasmid PUbi414- TaMYB55 . Recombinant plasmid PUbi414- TaMYB55 Sequencing verification has been performed.

[0040] TaMYB55 The sgRNA primer pairs for the genes are as follows:

[0041] TaMYB55 -414F: 5′-GATGGACCAGCGGCTGCCGATGG- 3′;

[0042] TaMYB55 -414R: 5′-GCAGAGGCCCTGGCGGCGGCTTGG- 3′.

[0043] 3. Preparation of transgenic plants

[0044] 1. Recombinant plasmid PUbi414- TaMYB55 Introduce into Agrobacterium tumefaciens EHA105 (WEIDI, CAT#: AC1013) to obtain recombinant Agrobacterium.

[0045] 2. The recombinant Agrobacterium obtained in step 1 was used to infect the embryonic callus of wheat Fielder, and then differentiation culture, rooting culture, and herbicide resistance screening (screening concentration was 250 mg / L) were carried out in sequence to obtain 11 T0 generation regenerated plants.

[0046] 3. The 11 T0 generation regenerated plants obtained in step 2 were identified by PCR.

[0047] PCR identification method: Take plant leaves, extract genomic DNA, and use a primer pair consisting of cas9-F and cas9-R for PCR amplification. If an amplification product is obtained, the identification result is positive and the plant is a transgenic plant.

[0048] cas9-F: 5′-CTAAGCGGAACAGCGACAAG- 3′;

[0049] cas9-R: 5′-GGCCAGGTAGAGGAAGTTCAC-3′.

[0050] Among the 11 T0 generation regenerated plants, 11 were transgenic plants.

[0051] The electrophoresis diagram of transgenic plants identified by PCR is shown in Picture 1 . Picture 1 In the figure, M is a molecular weight marker, #1 to #11 represent different transgenic plants, H2O represents sterile water (negative control), and WT represents wheat Fielder plants (negative control).

[0052] The 11 transgenic plants screened in step 3 were identified by sequencing the sgRNA knockout target sites TaMYB55 The situation of gene editing.

[0053] (1) Take plant leaves and extract DNA.

[0054] (2) Using DNA as a template, the amplified product is sequenced and verified.

[0055] For identification TaMYB55A Gene coding region such as SEQ ID NO.4 is used to identify TaMYB55 The primer pairs for genes (homologous genes in the A genome) are as follows:

[0056] TaMYB55A -F: 5′-AAACATCCATCACACGACGA-3′;

[0057] TaMYB55A -R: 5′-AGCAAACCACATCCACTGCT-3′.

[0058] TaMYB55B The primer pairs for the genes (homologous genes in the B genome) are as follows:

[0059] TaMYB55B -F: 5′-TGGTCGGAACTAGCTTGGAG-3′;

[0060] TaMYB55B -R: 5′-GTGTCAATCGATCGGCCTAA - 3′.

[0061] TaMYB55D Gene coding region such as SEQ ID NO.5, used to identify TaMYB55 The primer pairs for genes (homologous genes in the D genome) are as follows:

[0062] TaMYB55D -F: 5′-CCAGTTAATGCGGGAAACAT- 3′;

[0063] TaMYB55D -R: 5′-GTCCGATCGGTCTAAGCAAA-3′.

[0064] PCR reaction system (10μl): 2×Mix 5μl, primer F (2μM) 1μl, primer R (2μM) 1μl, DNA template 1μl, ddH2O 2μl.

[0065] PCR reaction program: 94 o C pre-denaturation 5min; 94 o C 20s, 58 o C 20s, 72 o C for 30 s, 35 cycles; 72°C for 5 min; 12°C for 5 min.

[0066] The amplified products were sequenced and analyzed using SnapGene software.

[0067] Some results can be found in Picture 2 . Picture 2 2 are shown in TaMYB55 Transgenic lines ( Tamyb55 -9 and Tamyb55 -10), Tamyb55 -9A inserts 1 bp (T), Tamyb55 -10 A Insert 1 bp (T); Tamyb55 -9B inserts 1 bp (A), Tamyb55 -10 B Insertion 1 bp (A); Tamyb55 -9D inserts 1 bp (A), Tamyb55 -10 D Insert 1 bp (T).

[0068] 5. Obtain offspring through self-pollination.

[0069] Transgenic plants are self-pollinated and seeds are obtained, which are T1 generation seeds. T1 generation seeds are cultivated into plants, which are T1 generation plants. T1 generation plants are self-pollinated and seeds are obtained, which are T2 generation seeds. T2 generation seeds are cultivated into plants, which are T2 generation plants. Take leaves of T2 generation plants, extract DNA for PCR identification (the method is the same as step 4). For a certain T2 generation plant, if the PCR sequencing result of the plant is identified as a positive transgenic plant, the T2 generation plant and its offspring are a homozygous transgenic strain.

[0070] Two transgenic lines were randomly selected ( Tamyb55 -9 and Tamyb55 -10) Carry out the identification in step five.

[0071] 4. Identification of resistance to (high temperature) heat stress

[0072] Seeds for testing: Tamyb55 -9 series T3 seeds, Tamyb55 -10 series T3 seeds, wheat Fielder seeds.

[0073] Culture conditions: 22°C, 16h light / 8h dark.

[0074] (1) Take the test seeds, soak them in a 1% sodium hypochlorite aqueous solution for 15 min, and then wash them with distilled water for 6 times.

[0075] (2) Take the seeds obtained in step (1) and place them in a dark place at 4°C for 3 days.

[0076] (3) Take the seeds obtained in step (2) and culture them until the seedlings germinate for 2 days.

[0077] (4) Take the seedlings with the same growth and transfer them to rectangular culture pots (size 30cm*24cm*15cm), sow 120 seeds in each culture pot. After 7 days of culture, transfer them to a 42℃ incubator and culture them for 7-14 days. After that, transfer them to a 20℃ incubator to recover for one week, and then calculate the survival rate. The results showed that after high temperature treatment at 42℃, TaMYB55Compared with the wild-type Fielder, the knockout materials had more wilting, the leaves of the plants were more wilted and yellowed than those of Fielder, and the stems were more wilted than those of the wild-type Fielder ( Picture 3 ), the plant survival rate is lower ( Picture 4 ), showed a poorer heat stress tolerance phenotype. Therefore, the results showed that the knockout TaMYB55 The gene can significantly reduce wheat's resistance to heat (high temperature) stress.

[0078] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Inhibition TaMYB55 Application of gene expression in reducing heat tolerance in wheat; characterized in that, TaMYB55 Gene, selected from any of the following: (b1) a DNA molecule whose coding region is shown in SEQ ID NO.2; (b2) a DNA molecule having a nucleotide sequence as shown in SEQ ID NO.3; TaMYB55 The amino acid sequence of the TaMYB55 protein encoded by the gene is shown in SEQ ID NO.

1.

2. Inhibition TaMYB55 The application of gene expression related biological materials in reducing the heat tolerance of wheat is characterized by: Related biological materials, selected from any of the following: (c1) contains TaMYB55 Gene sgRNA expression cassette; (c2) A recombinant vector or recombinant microorganism containing the expression cassette described in (c1).

3. The use according to claim 2, characterized in that: The recombinant vector in (c2) is constructed from a plant expression vector, and the plant expression vector includes a binary Agrobacterium vector.

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