Application of peanut AhNF-YA3 gene in improving oil content and / or salt tolerance of plants

Abstract

The invention discloses application of a peanut AhNF-YA3 gene in improving the oil content and / or salt tolerance of plants, and belongs to the technical field of gene engineering. The nucleotide sequence of the peanut AhNF-YA3 gene for improving the oil content and / or the salt tolerance of the plant is as shown in SEQ ID NO: 1. After the gene is used for constructing a plant expression vector and transforming peanuts, the oil content of peanut seed kernels can be remarkably improved, and the oil content is at least improved by 3.9 percent compared with that of wild types; the salt tolerance can be obviously improved, at least 350mM NaCl can be tolerated, and the method has a relatively good application prospect in the field of improving the oil content and / or the salt tolerance of the plants.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of peanut AhNF-YA3 gene in improving plant oil content and / or salt tolerance. Background Art

[0002] Studies have shown that existing crop varieties will reduce their yields by more than 30% compared to normal conditions after long-term exposure to salt damage. Since conventional breeding technology is inefficient and has a long cycle, it is difficult to breed salt-tolerant and high-yield varieties in the short term. Therefore, exploring salt-tolerant genes, establishing salt-tolerant molecular marker-assisted breeding technology, and breeding new salt-tolerant varieties are important ways to effectively utilize salinized soil.

[0003] Peanuts are widely planted oil and cash crops around the world, and they play an important role in agricultural production. Peanuts are also the main source of edible vegetable oil, and increasing the oil content has become one of the most important goals of peanut quality breeding. The average oil content of the main peanut varieties promoted for planting in the past 20 years is only 51.4%, and the oil content of some large peanut varieties planted in the main production areas in the north is less than 50%. About 50% of peanuts are used for oil extraction. For every 1 percentage point increase in oil content, peanut oil processing companies can increase their net income by 7%.

[0004] Therefore, exploring the genes related to peanut salt tolerance and oil synthesis, creating new salt-tolerant and high-oil materials, and then breeding new salt-tolerant and high-oil varieties are expected to make full use of the vast saline land without competing with grain crops for land. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide an application of peanut AhNF-YA3 gene in improving plant oil content and / or salt tolerance.

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

[0007] Application of peanut AhNF-YA3 gene in improving plant oil content and / or salt tolerance, wherein the amino acid sequence encoded by the peanut AhNF-YA3 gene is as shown in SEQ ID NO: 2.

[0008] Based on the above scheme, the nucleic acid sequence of the peanut AhNF-YA3 gene is shown in SEQ ID NO:1.

[0009] A product for improving the oil content and / or salt tolerance of plants, the product being a recombinant vector, a recombinant bacterium, an expression cassette or a transgenic cell line containing a peanut AhNF-YA3 gene coding region sequence, wherein the nucleic acid sequence of the peanut AhNF-YA3 gene is shown in SEQ ID NO: 1.

[0010] A method for improving plant oil content and / or salt tolerance, comprising constructing a coding region sequence of a peanut AhNF-YA3 gene into a plant expression vector, transforming plant cells, and expressing the vector in the plant to improve the plant oil content and / or salt tolerance; the nucleic acid sequence of the peanut AhNF-YA3 gene is shown in SEQ ID NO: 1.

[0011] Based on the above scheme, the oil content of the plant is increased by at least 3.9 percentage points relative to the wild type.

[0012] Based on the above scheme, the improved plant salt tolerance is tolerating at least 350 mM NaCl.

[0013] Based on the above scheme, the plant is peanut.

[0014] Advantages of the technical solution of the present invention

[0015] The invention clones AhNF-YA3 gene from peanuts; overexpressing AhNF-YA3 gene in peanuts can significantly increase its oil content. The oil content of transgenic peanut kernels can be increased by up to 5.0 percentage points compared with non-transgenic kernels.

[0016] The invention overexpresses AhNF-YA3 gene in peanuts, and can also significantly improve its salt tolerance. After being treated with 350mM NaCl stress, the salt tolerance of transgenic peanuts is significantly stronger than that of non-transgenic plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The relative expression levels of peanut AhNF-YA3 gene at different time periods after 300 mM NaCl stress treatment;

[0018] Figure 2 The oil content of the kernels of AhNF-YA3-overexpressing transgenic peanut (OE) and non-transgenic control (WT) (different lowercase letters indicate the significance level when p=0.05);

[0019] Figure 3 The growth of transgenic peanut (OE-1, OE-2) and non-transgenic control (WT) plants under normal growth and salt stress treatment;

[0020] Figure 4 Relative conductivity of transgenic peanut (OE-1, OE-2) and non-transgenic control (WT) after normal growth and salt stress treatment (different lowercase letters indicate the significance level when p=0.05);

[0021] Figure 5NBT and DAB staining of leaves of transgenic peanut (OE-1, OE-2) and non-transgenic control (WT) after normal growth and salt stress treatment;

[0022] Figure 6 MDA content, POD content, Pro content and SOD content of transgenic peanut (OE-1, OE-2) and non-transgenic control (WT) after normal growth and salt stress treatment (different lowercase letters indicate the significance level when p=0.05). DETAILED DESCRIPTION

[0023] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0024] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0025] The sources of the experimental materials used in the following examples are as follows:

[0026] Escherichia coli DH5α was maintained by the Laboratory of Genetics Research, Qingdao Agricultural University;

[0027] Agrobacterium tumefaciens strain EHA105 (purchased from Beijing Tianenze Gene Technology Co., Ltd.);

[0028] The transgenic peanut receptor material was Huayu 23 (provided by the Peanut Molecular Breeding Laboratory of Qingdao Agricultural University).

[0029] The expression vector pCAMBIAsuper1300 was maintained by the Laboratory of Genetics Research, Qingdao Agricultural University;

[0030] Yuhua No. 9 peanut material is a high-oil peanut variety bred by Professor Wang Jingshan of Qingdao Agricultural University.

[0031] Example 1

[0032] Cloning and Expression Analysis of Peanut AhNF-YA3 Gene

[0033] 1. Cloning of peanut AhNF-YA3 gene

[0034] RNA was extracted from Yuhua No. 9 material (a high-oil peanut variety bred by Qingdao Agricultural University) and reverse transcribed into cDNA. The cDNA was used as a template and the following primers were used to clone the peanut AhNF-YA3 gene. The cDNA sequence of the gene was shown in SEQ ID NO: 1, and the amino acid sequence encoded was shown in SEQ ID NO: 2.

[0035] SEQ ID NO:1(5'→3')

[0036]

[0037] SEQ ID NO:2

[0038]

[0039] The amplification primer sequences are as follows:

[0040] P1: 5′-ATGAAGCCATTCCTCTTACTG-3′ (SEQ ID NO: 3);

[0041] P2: 5′-TCAACAACCAAAGGTGTGTCCC-3′ (SEQ ID NO: 4);

[0042] 2. Effects of salt stress on the expression of peanut AhNF-YA3 gene

[0043] a. Treat the seedlings of 'Yuhua No. 9' with 300mM NaCl, take the seedling leaves at different time periods (0h, 6h, 12h, 24h and 48h after treatment), and immediately freeze them in liquid nitrogen for later use. Take 0.05g of peanut young leaves treated with stress at different time periods, freeze them in liquid nitrogen and grind them into powder, and extract RNA using RNA extraction kit. The extracted total RNA is treated with DNase I and purified.

[0044] b. The samples were reacted on an ABI 7500FAST fluorescent quantitative PCR instrument.

[0045] The 20 μL reaction system included: 10 μL 2×SybrGreen qPCR Master Mix, 0.25 μL each of 20 μmol / L forward and reverse primers, and 20 ng of reverse transcription product.

[0046] The amplification procedure is: first pre-denaturation at 94°C for 2 minutes; then enter 40 cycles of reaction, with denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds in each cycle; after the cycle is completed, slowly increase the temperature to 94°C to prepare the melting curve. Each reaction is set up with 3 replicate wells.

[0047] c. Primers for AhNF-YA3 gene quantitative PCR are:

[0048] Forward primer sequence: 5'-TGGCACATGCTCCTTTTCC-3' (SEQ ID NO: 5);

[0049] Reverse primer sequence: 5'-TGCTCGTGACTGTCTCCTCCT-3' (SEQ ID NO: 6).

[0050] The peanut Actin gene was used as the internal standard, and the primers for the internal standard gene were:

[0051] Forward primer sequence: 5′-GTGGCCGTACAACTGGTATCGT-3′ (SEQ ID NO: 7);

[0052] Reverse primer sequence: 5'-ATGGATGGCTGGAAGAGAACT 3' (SEQ ID NO: 8).

[0053] The expression changes of AhNF-YA3 gene before and after salt stress treatment were measured. Figure 1 As shown by Figure 1 It can be seen that the expression level of AhNF-YA3 gene changed significantly after salt stress treatment, indicating that it was induced by salt stress.

[0054] Example 2

[0055] Construction of plant expression vector of AhNF-YA3 gene

[0056] (1) Using the cDNA of Yuhua No. 9 as a template, the coding region of the AhNF-YA3 gene was cloned, and Sal I and Kpn I restriction sites were introduced into the upstream and downstream primers respectively; the primer sequences used were:

[0057] Forward primer: 5′-ctgcaggggcccggggtcgacATGAAGCCATTCCTCTTACTGAATC-3′ (SEQ IDNO: 9);

[0058] Reverse primer: 5′-gcccttgctcaccatggtaccACAACCAAAGGTGTGTCCCCC-3′ (SEQ ID NO: 10);

[0059] (2) The PCR product was recovered and ligated with the cloning vector pMD18-T (purchased from TaKaRa) under the action of T4 DNA ligase. The ligation product was transformed into Escherichia coli DH5α. The recombinant plasmid was extracted and double-digested with Sal I and Kpn I to recover the restriction fragment containing the AhNF-YA3 gene and cloned into the corresponding restriction site of the plant expression vector pCAMBIAsuper1300 to obtain the plant expression vector pCAMBIAsuper1300-AhNF-YA3 of the gene.

[0060] Example 3

[0061] Application of AhNF-YA3 gene in increasing oil content in plants

[0062] A method for increasing the oil content of peanuts, comprising the following steps:

[0063] The plant expression vector pCAMBIAsuper1300-AhNF-YA3 prepared in Example 2 was transformed into peanut, comprising the following steps:

[0064] a. Preparation, activation and bacterial liquid preparation of Agrobacterium recombinant strains: The pCAMBIAsuper1300-AhNF-YA3 recombinant plasmid was transformed into Agrobacterium strain EHA105 competent cells using liquid nitrogen freeze-thaw method, and the recombinant strain containing the recombinant plasmid was screened out. Pick a single colony of the recombinant strain and inoculate it into YEB (rifampicin 50 mg / L, kanamycin 50 mg / L) liquid culture medium, and culture it at 28°C and 180rpm until OD600 = 0.5-0.8, then take 2mL of bacterial liquid and transfer it to 50mL YEB (rifampicin 50 mg / L, kanamycin 50 mg / L) culture medium, and culture it to OD600 = 0.6. After centrifuging the bacterial liquid at 5000rpm for 10min, suspend it with the same volume of liquid MSB5 for later use.

[0065] b. Separation of peanut explants: Select full peanut seeds (Huayu No. 23), soak them in 70% alcohol for 1 min, soak them in 0.1% mercuric chloride for 20 min, rinse them with sterile water for 3-5 times, and cut each cotyledon into two halves longitudinally.

[0066] c. Agrobacterium-mediated genetic transformation: The explants were immersed in the prepared Agrobacterium solution in step a, and infected for 10 minutes at 28°C and 90 rpm with gentle shaking. The residual solution was dried with sterile filter paper, and then inoculated into SIM induction medium (MS macronutrient medium + B5 micronutrient medium + 2mg / L BAP + 1.5mg / L 2,4-D) and co-cultured in the dark for 3 days. Transferred to SIM induction medium supplemented with 250mg / L cephalosporin, the cut end of the explant was embedded in the medium, and cultured for about 2 weeks to induce clustered buds. The culture conditions were: light intensity of 1500-2000lx, light for 12h, and temperature of 26°C±1°C.

[0067] The explants that formed clustered buds were transferred to SEM medium (MS macronutrient medium + B5 micronutrient medium + 2mg / L BAP) containing 250mg / L cephalosporin and 100mg / L kanamycin to select resistant buds and cultured for 2 weeks. The culture conditions were: light intensity of 1500-2000lx, light intensity of 12h, and temperature of 26℃±1℃.

[0068] After culturing for 2 weeks, the adventitious buds were cut off and transferred to SEM medium containing 250 mg / L cephalosporin and 150 mg / L kanamycin for screening of resistant buds and inducing bud elongation. The culture was carried out for about 4 weeks, during which time the buds were subcultured 2-3 times.

[0069] d. PCR detection of transgenic plants

[0070] The genomic DNA of the regenerated plants was extracted, and primers were designed using the pCAMBIAsuper1300 vector sequence and the AhNF-YA3 gene sequence for PCR amplification. The PCR reaction program was: 95°C, 5 min; 95°C, 50 s, 58°C, 50 s, 72°C, 1 min, 30 cycles; 72°C, 10 min.

[0071] e. Grafting and transplanting of transgenic positive plants

[0072] Use sterile seedlings of about 15 days old as rootstocks, cut off the main stem part more than 2 cm away from the cotyledons, and split the upper end of the rootstock vertically with a scalpel, with a depth of about 1 cm. When the transgenic plant seedlings grow to about 3 cm, cut the regenerated seedlings from the base of the bud cluster as scions, cut the lower end into a V-shaped wound about 1 cm long, and the incision is flat. Insert the scion into the rootstock so that the cambium of the rootstock and scion are in close contact, and then wrap the interface with sealing film with appropriate tightness. Place the grafted seedlings in MSB5 culture medium for aseptic culture for 3-4 days; then transplant them into the sterilized seedling medium for acclimatization for 2 weeks, and then transplant them into the matrix soil until the pods are harvested.

[0073] f. Determination of oil content in transgenic peanut kernels

[0074] The oil content of peanut kernels was determined by referring to the determination of oil content in oilseeds (residual method) (NY / T 1285-2007). Weigh more than 10g of T3 transgenic peanut kernels and non-transgenic kernels, put them in an oven at 100±2℃ and dry them for about 2h, and use a tissue crusher to crush the peanut kernels into uniform powder. Weigh more than 1.5g of peanut sample and put them into a dried filter paper tube, put the filter paper tube and the sample in an oven at 105℃±2℃ and dry them for 3h, then put them in a desiccator and cool them to room temperature. Soak the filter paper tube in the extractant overnight, take it out and extract the sample for 8h. Weigh and calculate the oil content, and repeat the determination 3 times for each sample. The results showed that compared with the oil content of non-transgenic peanut kernels (51.75%), the peanut kernel contents of the six transgenic lines reached 56.75%, 55.84%, 55.69%, 55.97%, 56.48%, and 56.12%, respectively, and the oil content could be increased by 3.9-5.0 percentage points ( Figure 2 ).

[0075] Example 4

[0076] Application of AhNF-YA3 gene in improving plant salt tolerance

[0077] A method for improving the salt tolerance of peanuts, comprising the following steps:

[0078] The plant expression vector pCAMBIAsuper1300-AhNF-YA3 prepared in Example 2 was transformed into peanut (Huayu 23), and the specific steps were as described in ae of Example 3. Finally, transgenic peanut seedlings, i.e., overexpression plants, were obtained.

[0079] Peanut seedlings grown for 2 weeks were watered with 300mM NaCl solution, and then 3 days later, the seedlings were watered with 350mM NaCl solution for 15 days, and the phenotypic changes of overexpressed plants and non-transgenic controls were observed. The results showed that after NaCl treatment, plants overexpressing the AhNF-YA3 gene (OE) (OE-1 and OE-2) showed strong salt tolerance, while non-transgenic peanut plants (WT) wilted severely ( Figure 3 Compared with the non-transgenic control, the relative conductivity of the two overexpression lines (OE-1 and OE-2) decreased by 50.74% and 51.44%, respectively ( Figure 4 ).

[0080] In order to detect the accumulation of reactive oxygen species in the leaves of transgenic positive plants and non-transgenic controls after salt stress, NBT and DAB were used to stain them ( Figure 5 ),in, Figure 5 In the NBT staining results, the more blue the leaves are, the more 2-The more the accumulation, the less blue part of the leaves of transgenic plants (OE-1 and OE-2) than those of the non-transgenic control (WT), indicating that O 2- The accumulation amount is small. In the DAB staining results, the more brown parts of the leaves, the more H2O2 accumulated; the transgenic plants (OE-1 and OE-2) had less brown parts than the non-transgenic control (WT), indicating that the H2O2 accumulation amount was small. The MDA content, POD content, Pro content and SOD content of the leaves of the transgenic positive plants and the non-transgenic control were further determined. The results showed that compared with the non-transgenic control, the MDA content of the two overexpression lines (OE-1 and OE-2) decreased by 53.79% and 49.82%, respectively; the POD content increased by 59.06% and 63.98%, respectively; the Pro content increased by 68.87% and 128.33%, respectively; the SOD content increased by 139.38% and 146.36%, respectively ( Figure 6 ).

[0081] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. Application of peanut AhNF-YA3 gene in improving plant oil content and / or salt tolerance, characterized in that: The amino acid sequence encoded by the peanut AhNF-YA3 gene is as shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The nucleic acid sequence of the peanut AhNF-YA3 gene is shown in SEQ ID NO:

1.

3. A product for increasing the oil content and / or salt tolerance of plants, characterized in that: The product is a recombinant vector, recombinant bacteria, expression cassette or transgenic cell line containing the peanut AhNF-YA3 gene coding region sequence, and the nucleic acid sequence of the peanut AhNF-YA3 gene is shown in SEQ ID NO:

1.

4. A method for increasing the oil content and / or salt tolerance of plants, characterized in that: The coding region sequence of the peanut AhNF-YA3 gene is constructed into a plant expression vector, and plant cells are transformed to express it in the plant to improve the oil content and / or salt tolerance of the plant; the nucleic acid sequence of the peanut AhNF-YA3 gene is shown in SEQ ID NO:

1.

5. The method for improving plant oil content and / or salt tolerance according to claim 4, characterized in that: The increased plant oil content is at least 3.9 percentage points higher than that of the wild type.

6. The method for improving plant oil content and / or salt tolerance according to claim 4, characterized in that: The improved salt tolerance of plants is tolerating at least 350 mM NaCl.

7. The method for improving plant oil content and / or salt tolerance according to any one of claims 4 to 6, characterized in that: The plant is peanut.

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