AU2021106992A4 - Peanut adversity stress gene ahmyb30 and application thereof - Google Patents

Abstract

OF THE DISCLOSURE The invention relates to the field of genetic engineering, and in particular to a peanut adversity stress gene AhMYB30 and application thereof. The gene AhMYB30 can be used for improving the resistance of plants to low temperature stress and salt stress. 1/1 DRAWINGS: WT OE1 OE2 OE3 c 0 0 Fig. 1 WT OE1 OE2 OE3 11a(l Rille , .6 Iiti SF Fig1

Description

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PEANUT ADVERSITY STRESS GENE AHMYB30 AND APPLICATION THEREOF

BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The invention belongs to the field of genetic engineering, and particularly relates to a peanut adversity stress gene AhMYB30 and application thereof.

[0003] 2. Background

[0004] The yield and quality of peanuts are seriously influenced by the stress of drought, salt and alkali, and the yield reduction rate of the peanuts caused by drought in China is more than 20 percent every year. However, high-resistance varieties are difficult to be bred by using the conventional breeding method due to narrow genetic basis and absence of gene sources with high drought resistance, cold resistance and salt tolerance in existing peanut varieties. With the rapid development of molecular biology, the researches for improving the stress tolerance of plants by using a transgenic technology have indicated remarkable achievements in recent years, and stress-related genes and signal transduction pathways have been further understood.

[0005] MYB-related MYB transcription factors are also a relatively large subfamily in plants, of which the previous researches have mainly focus on their regulation to circadian rhythms and cell morphogenesis, but less on functional regulation to plant salt stress.

BRIEF SUMMARY OF THE INVENTION

[0006] The invention provides a peanut adversity stress gene AhMYB30 with nucleotide sequence shown as SEQ ID No. 1.

[0007] Meanwhile, the invention provides a cloning method of the peanut adversity stress gene AhMYB30, which comprises the following specific steps:

[0008] (1) Preparation and treatment of materials: Germinating peanut seeds in mixed soil containing nutrient soil and vermiculite in ratio of 2: 1, growing seedlings under 16h light/8h ) dark at 28°C/22°C after germination, placing the peanut seedlings in an illumination incubator at 4 °C when the peanut seedlings grow to trefoil stage, taking peanut leaves and roots as materials at Oh, lh, 3h, 6h, 12h, 24h, 48h and 72h respectively, and storing all materials in an ultra-low temperature refrigerator at -80 °C for later use;

[0009] (2) Extraction of RNA and Synthesis of cDNA

[0010] Separating and extracting peanut seedling RNA, removing DNA pollution from the obtained RNA followed by synthesis of cDNA; synthesizing cDNA by using an M-MLV reverse transcription kit (Takara) in which the 25 L of reaction system contains 2 tg of RNA, placing a reverse transcription product on ice for 5 min after reverse transcription reaction for 60 min at 42 °C, and then storing the reverse transcription product in a low-temperature refrigerator at 20 °C for later use;

[0011] (3) Cloning of genes

[0012] Cloning with RT-PCR in which polymerase used for PCR amplification is LA TaqTM DNA polymerase (TaKaRa), and the following components are added into 25 L of system: 2.5 L of Ox PCR buffer (containing MgCl2); 2.5 L of 10 mM dNTPs; 1 L cDNA template; 0.5 pL of LA polymerase and 17.5 L of ddH 20;

[0013] Under PCR reaction conditions as follows: (a) at 94 °C for 5 min; (b) at 94 °C for 45 s; 54 °C for 60 s; 72 °C for 90 s; up to 35 cycles; (c) 72 °C for 10 min;

[0014] The primer is AhMYB30-S: 5'-ATGAAGTGGGAAGTAGAAGTA-3'and AhMYB30 A: 5'-TCTCTATAATATAAGTGAAG-3';

[0015] Purifying PCR products with gel extraction kit (Omega) after separating the PCR products by 1% agarose gel electrophoresis, ligating the purified products to pMD18-T Easy vector (Takara) and sequencing (Sangon, Shanghai).

[0016] The gene AhMYB30 can be used for improving the resistance of plants to low temperature stress and salt stress.

BRIEF DESCRIPTION OF DRAWINGS

[0017] Fig. 1 shows the phenotypes of arabidopsis with overexpressed AhMYB30 under low temperature stress and wild type Arabidopsis

[0018] Fig. 2 shows the phenotypes of arabidopsis with overexpressed AhMYB30 under salt stress and wild type arabidopsis

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0020] Example 1

[0021] A cloning method of a peanut adversity stress gene AhMYB30 comprises the following specific steps:

[0022] (1) Preparation and treatment of materials: Germinating seeds of Arachis hypogaea L. cultivar Huayu33 in mixed soil containing nutrient soil and vermiculite in ratio of 2: 1, growing seedlings under 16h light/8h dark at 28 °C/22 °C after germination, placing the peanut seedlings in an illumination incubator at 4 °C when the peanut seedlings grow to trefoil stage, taking peanut leaves and roots as materials at Oh, 1h, 3h, 6h, 12h, 24h, 48h and 72h respectively, and storing all materials in an ultra-low temperature refrigerator at -80 °C for later use;

[0023] (2) Extraction of RNA and Synthesis of cDNA

[0024] Separating and extracting peanut seedling RNA with RNeasy Mini Kit (Qiagen), removing DNA pollution from the obtained RNA by RQ1 RNase-free DNaseI and the performing synthesis of cDNA; synthesizing cDNA by using an M-MLV reverse transcription kit (Takara) in which the 25 L of reaction system contains 2 g of RNA, placing a reverse transcription product on ice for 5 min after reverse transcription reaction for 60 min at 42 °C, and then storing the reverse transcription product in a low-temperature refrigerator at -20 °C for later use

[0025] (3) Cloning of genes

[0026] Cloning with RT-PCR in which polymerase used for PCR amplification is LA TaqTM DNA polymerase (TaKaRa), and the following components are added into 25 L of system: 2.5 L of 1Ox PCR buffer (containing MgCl2); 2.5 L of 10 mM dNTPs; 1 L cDNA template; 0.5 pL of LA polymerase and 17.5 L of ddH 20

[0027] under PCR reaction conditions as follows: (a) at 94 °C for 5 min; (b) at 94 °C for 45 s; 54 °C for 60 s; 72 °C for 90 s; up to 35 cycles; (c) 72 °C for 10 min;

[0028] The primers are as follows: AhMYB30-S: 5'-ATGAAGTGGGAAGTAGAAGTA-3' (SEQ ID No. 2) and AhMYB30-A: 5'-TCTCTATAATATAAGTGAAG-3'(SEQ ID No. 3);

[0029] Purifying PCR products with gel extraction kit (Omega) after separating the PCR products by 1% agarose gel electrophoresis, ligating the purified products to pMD18-T Easy vector (Takara) and sequencing (Sangon, Shanghai).

[0030] The sequencing result shows that the gene AhMYB30 has full length of 1553bp, sequence as shown in SEQ ID No.1, open reading frame of 1104bp, and codes 368 amino acids.

[0031] Example 2

[0032] Vector construction and genetic transformation

[0033] AhMYB30 is constructed on an expression vector (pCAMBIA1301 is used in the example) by the following primers:

[0034] AhMYB30-S: 5'-GGATCCATGAAGTGGGAAGTAGAAGTA-3'(SEQ ID No. 4) and Ah MYB30-A:5'-CTGCAGCTATAATATAAGTGAAGAAAA-3'(SEQ ID No. 5).The enzyme cleavage sites are BamHI and PstI. The constructed vector is transformed into agrobacterium LBA4404 after the correctness of the ligated gene sequence is determined by sequencing, and the gene is transferred into arabidopsis through the mediation of agrobacterium. Genetic transformation of arabidopsis is performed by the flower-dipper method. Transgenic positive plants are screened with 50 mg/L hygromycin.

[0035] (5) Stress treatment of transgenic plants

[0036] Low-temperature treatment: arabidopsis seeds are germinated in mixed soil containing vermiculite and nutrient soil in the ratio of 1: 2, and then grown under long-day conditions (16 h light/8 h dark) at 22 °C.After growing for about 20 days, the plants are transferred to a biochemical incubator and treated for 3 h at -8 °C. The treated material is then transferred to 22 °C under long day conditions to restore growth and observe the phenotypes. All treatments are repeated at least 3 times.

[0037] The low-temperature treatment test shows that the phenotype of the arabidopsis with overexpressed AhMYB30 (OEl-OE3) is not obviously different from that of wild type arabidopsis (WT) in absence of low-temperature treatment (Normal conditioning). During growth restoration after treatment at -8 °C (Cold treated) for 3 h, the arabidopsis with overexpressed AhMYB30 (OEl-OE3) mostly can restore growth with more than 80% of survival rate while wild type arabidopsis (WT) has less than 50% of survival rate (Fig. 1).

[0038] Salt treatment: wild type arabidopsis seeds and seeds of three lines of transgenic Arabidopsis with overexpressed AhMYB30 are placed at 4 °C for 3 d to break dormancy, and then sown into 1/2MS culture medium together. After the seeds germinate for 3 d, the wild type arabidopsis seedlings and the transgenic arabidopsis seedlings are transferred into saline 1/2MS culture medium with NaCl concentration of 0 and 200 mM respectively, followed by observing the root elongation condition and the leaf color change of the arabidopsis after culturing in an illumination incubator for 7 d. All treatments are repeated at least 3 times.

[0039] The Salt treatment results show that the phenotype of arabidopsis with overexpressed AhMYB30 (OEl-OE3) is not obviously different from that of wild arabidopsis (WT) in a solid medium without NaCl (Normal conditioning), and partial leaves of the transgenic arabidopsis (OEl-OE3) still keep green in a solid medium with 200 mM NaCl (Salt treated) while the leaves of the wild Arabidopsis (WT) are all yellowed, and the root length of the transgenic arabidopsis in 200 mM NaCl is obviously higher than that of the wild arabidopsis (Fig. 2).

[0040] The results show that the overexpression of AhMYB30 in arabidopsis improves the resistance of transgenic plants to low temperature and salt stress.

[0041] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any skilled in the art may make changes of variations on the technical content disclosed above to become equivalent embodiments with the same changes. However, any simple modification, equivalent change and variation made on the above embodiments by using the contents without departing from the technical solution of the present invention based on the technical essence of the present invention are all still within the protection scope of the present invention.

Claims (3)

What is claimed is:

1. A peanut adversity stress gene AhMYB30 has a nucleotide sequence shown as SEQID No. 1.

2. A primer pair for cloning the gene of claim 1, with sequences as follows:AhMYB30-S: 5'-ATGAAGTGGGAAGTAGAAGTA-3' AhMYB30-A: 5'- TCTCTATAATATAAGTGAAG-3'

3. The application of the peanut adversity stress gene AhMYB30 of claim 1 inimproving cold and salt tolerance of plants.