Application of peanut AhNF-YC9 gene in improving oil content and salt tolerance of plants

Abstract

The invention discloses application of a peanut AhNF-YC9 gene in improving the oil content and salt tolerance of plants, and belongs to the technical field of gene engineering. The nucleotide sequence of the peanut AhNF-YC9 gene is as shown in SEQ ID NO: 1, and the coded amino acid sequence of the peanut AhNF-YC9 gene is as shown in SEQ ID NO: 2. The AhNF-YC9 gene is used for constructing a plant expression vector to transform peanuts, so that the AhNF-YC9 gene is excessively expressed in the peanuts, and the oil content of obtained transgenic peanut seeds can be increased by 4.0% at most compared with that of non-transgenic seeds; after stress treatment with 350mM NaCl, the salt tolerance of the transgenic peanut is obviously higher than that of a non-transgenic plant. Therefore, the peanut AhNF-YC9 gene has an important application prospect in improving the oil content and the salt tolerance of the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to the application of peanut AhNF-YC9 gene in improving plant oil content and salt tolerance. Background Art

[0002] Salt damage is one of the most serious adverse environmental hazards in agricultural production. Long-term exposure to salt damage can cause slowed plant growth or even death, leading to a significant decrease in yield and quality. As global temperatures gradually rise, salt damage will pose a long-term threat to crop production and safety. Studies have shown that existing crop varieties can experience yield reductions of over 30% compared to normal conditions after long-term exposure to salt damage. Due to the low efficiency and long production cycles of conventional breeding techniques, it is difficult to breed salt-tolerant, high-yielding varieties in the short term. Therefore, identifying salt-tolerance-related genes, establishing salt-tolerance molecular marker-assisted breeding techniques, and cultivating new salt-tolerant varieties are important approaches to effectively utilize salinized soils.

[0003] Peanuts are a widely cultivated oilseed and cash crop worldwide, playing a vital role in agricultural production. Peanuts are also a major source of edible vegetable oil, and increasing their oil content has become one of the most important goals of peanut quality breeding. Currently, the average oil content of major peanut varieties is only 51.4%, with some large varieties boasting less than 50%. As a key oilseed crop, every 1 percentage point increase in oil content can increase net revenue for peanut oil processing companies 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 food crops. 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 the peanut AhNF-YC9 gene in improving the oil content and salt tolerance of plants.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The invention discloses an application of the peanut AhNF-YC9 gene in improving plant oil content and salt tolerance. The amino acid sequence encoded by the peanut AhNF-YC9 gene is shown in SEQ ID NO: 2.

[0007] Based on the above scheme, the nucleic acid sequence of the peanut AhNF-YC9 gene is shown in SEQ ID NO: 1.

[0008] A method for improving plant oil content and salt tolerance, comprising increasing the expression of a peanut AhNF-YC9 gene in the plant to improve the oil content and salt tolerance of the plant, wherein the amino acid sequence encoded by the peanut AhNF-YC9 gene is shown in SEQ ID NO: 2.

[0009] Based on the above scheme, the nucleic acid sequence of the peanut AhNF-YC9 gene is shown in SEQ ID NO: 1.

[0010] On the basis of the above scheme, a plant expression vector of the peanut AhNF-YC9 gene was constructed and transformed into plants to express the gene in the plants, thereby increasing the expression level of the AhNF-YC9 gene in the plants.

[0011] On the basis of the above scheme, the method of transforming plants is one of the following methods: Agrobacterium-mediated method, gene gun method, electric shock method, PEG method, and liposome method.

[0012] Based on the above scheme, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

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

[0014] Advantages of the technical solution of the present invention The present invention cloned the AhNF-YC9 gene from peanut; constructed a plant expression vector for the AhNF-YC9 gene, then transformed it into peanuts, overexpressing it in peanuts and significantly increasing their oil content. The oil content of transgenic peanut kernels can be up to 4.0 percentage points higher than that of non-transgenic kernels. Furthermore, overexpressing the AhNF-YC9 gene in peanuts significantly improves their salt tolerance. After treatment with 350 mM NaCl, the transgenic peanuts exhibited significantly greater salt tolerance than non-transgenic plants. Therefore, the AhNF-YC9 gene has significant potential for applications in increasing plant oil content and salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The relative expression levels of peanut AhNF-YC9 gene at different time periods after 350 mM NaCl stress treatment; Figure 2 is the oil content of the kernels of AhNF-YC9-overexpressing transgenic peanut (OE) and non-transgenic control (WT); Figure 3 The growth of transgenic peanut (OE-1, OE-2) and non-transgenic control (WT) plants under normal growth and salt stress treatment; Figure 4 Relative electrical conductivity of transgenic peanuts (OE-1, OE-2) and non-transgenic control (WT) after normal growth and salt stress treatment; Figure 5 NBT and DAB staining of leaves of transgenic peanut (OE-1, OE-2) and non-transgenic control (WT) after normal growth and salt stress treatment; Figure 6 MDA content, POD content, Pro content and SOD content of transgenic peanuts (OE-1, OE-2) and non-transgenic controls (WT) after normal growth and salt stress treatment. DETAILED DESCRIPTION

[0016] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. Below, in conjunction with specific examples, the present invention will be further described in detail with reference to data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0017] 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 this field or according to the product instructions. The experimental materials, reagents, and drugs used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0018] The sources of the experimental materials used in the following examples are as follows: Escherichia coli DH5α was maintained by the Laboratory of Genetics Research, Qingdao Agricultural University; Agrobacterium tumefaciens strain EHA105 (purchased from Beijing Tianenze Gene Technology Co., Ltd.); The transgenic peanut recipient material was Huayu 23 (provided by the Peanut Molecular Breeding Laboratory of Qingdao Agricultural University); Plant expression vector pCAMBIAsuper1300 (provided by the Peanut Molecular Breeding Laboratory of Qingdao Agricultural University); The Yuhua No. 9 material is preserved by Qingdao Agricultural University.

[0019] Example 1 1. Cloning of the peanut AhNF-YC9 gene RNA was extracted from Yuhua No. 9 (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 primer pair was used to clone the peanut AhNF-YC9 gene. The cDNA sequence of the gene is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2.

[0020] SEQ ID NO:1 (5'→3') ATGGATCATCAAGGGCATGGCCAAAACCCATCCATGGGGGTTGTCAGTAGTGGGGCTCAA 60 CTAACGTATGGTTCCAATCCGTACCAGCCAAACCAAATGACTGGGGCACCAGGGTCAGTT 120 GTTACATCGGTTGGGAACATGCAAAGCGGTCAACCTGCTGGAGCTCAACTGGGACAACAT 180 CAACTTGCTTATCAGCATATTCATCAGCAACAACAGCAGCAACTTCAGCAACAACTACAG 240 GCTTTTTGGGCAAATCAATACCAAGAAATTGAGAAGGTAACTGATTTCAAGAACCACAGT 300 CTCCCCTTGGCAAGGATCAAGAAGATTATGAAGGCTGATGAGGATGTTAGAATGATATCG 360 GCTGAGGCACCTGTCATATTTGCAAGGGCATGCGAAATGTTCATTTTAGAGTTAACCCTG 420 CGTTCTTGGAATCACACTGAAGAGAACAAAAGAAGAACCCTTCAGAAAAATGATATTGCT 480 GCTGCAATCACGAGGACCGATATCTTTGATTTCTTGGTTGATATTGTGCCTCGTGAGGAC 540 TTGAAAGATGAAGTGCTTGCATCGATCCCAAGAGGAACAATGCCTGTTGCAGGGCCAGCT 600 GATGCAATGCCTTACTGTTATATGCCGCCTCAACATGCACCCCAAGTTGGACCTGCAGGT 660 GTCATAATGGGTAAGCCTGTGATGGACCCAAATATGTACGCTCAGCAATCTCATCCCTAC 720 ATGGCTCCACAAATGTGGCCGCAGCCGCCAGACCAACGACAATCATCTCCGGATCACTAG 780 SEQ ID NO:2 MDHQGHGQNPSMGVVSSGAQLTYGSNPYQPNQMTGAPGSVVTSVGNMQSGQPAGAQLGQH 60 QLAYQHIHQQQQQQLQQQLQAFWANQYQEIEKVTDFKNHSLPLARIKKIMKADEDVRMIS 120 AEAPVIFARACEMFILELTLRSWNHTEENKRRTLQKNDIAAAITRTDIFDFLVDIVPRED 180 LKDEVLASIPRGTMPVAGPADAMPYCYMPPQHAPQVGPAGVIMGKPVMDPNMYAQQSHPY 240 MAPQMWPQPPDQRQSSPDH 259 The amplification primer sequences are as follows: P1: 5'-ATGGATCATCAAGGGCATGGCCA-3' (SEQ ID NO: 3); P2: 5'-CTAGTGATCCGGAGATGATTGTC-3' (SEQ ID NO: 4); 2. Effects of salt stress on peanut AhNF-YC9 gene expression a. Treat 'Yuhua 9' seedlings with 350 mM NaCl. Leaves were collected at different time points (0, 6, 12, 24, and 48 hours after treatment) and immediately frozen in liquid nitrogen for later use. 0.05 g of peanut leaves treated at different time points were collected, quickly frozen in liquid nitrogen, and ground into a powder. RNA was extracted using an RNA extraction kit. The extracted total RNA was treated with DNase I and purified.

[0021] b. The samples were reacted on an ABI 7500 FAST fluorescence quantitative PCR instrument.

[0022] The 20 µL reaction system includes: 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.

[0023] The amplification procedure was as follows: pre-denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s. After each cycle, the plate was slowly raised to 94°C for melting curve preparation. Each reaction was performed in triplicate.

[0024] c. Primers for AhNF-YC9 gene quantitative PCR are: Forward primer sequence: 5′- GGAATCACACTGAAGAGAACAAAAG - 3′ (SEQ ID NO: 5); Reverse primer sequence: 5′- CACGAGGCACAATATCAACCA - 3′ (SEQ ID NO: 6).

[0025] The peanut Actin gene was used as the internal standard, and the primers for the internal standard gene were: Forward primer sequence: 5′- GTGGCCGTACAACTGGTATCGT -3′ (SEQ ID NO: 7); Reverse primer sequence: 5′- ATGGATGGCTGGAAGAGAACT 3′ (SEQ ID NO: 8).

[0026] The expression changes of AhNF-YC9 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-YC9 gene changed significantly after 24 h of salt stress treatment, indicating that it was induced by salt stress.

[0027] Example 2 The steps for constructing the AhNF-YC9 gene plant expression vector are as follows: (1) Using the cDNA of Yuhua No. 9 as a template, the coding region of the AhNF-YC9 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: Forward primer: 5′-ctgcaggggcccggggtcgacATGGATCATCAAGGGCATGG-3′ (SEQ ID NO: 9); Reverse primer: 5′-gcccttgctcaccatggtaccCTAGTGATCCGGAGATGATTGTCG-3′ (SEQ ID NO: 10); (2) The PCR product was recovered and ligated with the cloning vector pMD18-T (purchased from TaKaRa) using T4 DNA ligase. The ligation product was transformed into Escherichia coli DH5α. The recombinant plasmid was extracted and double-digested with SalI and KpnI. The enzyme-digested fragment containing the AhNF-YC9 gene was recovered and cloned into the corresponding enzyme cleavage site of the plant expression vector pCAMBIAsuper1300 to obtain the plant expression vector pCAMBIAsuper1300-AhNF-YC9 for the gene.

[0028] Example 3 Application of AhNF-YC9 gene in increasing plant oil content A method for increasing the oil content of peanuts, comprising the following steps: The plant expression vector pCAMBIAsuper1300-AhNF-YC9 prepared in Example 2 was transformed into peanut, comprising the following steps: a. Preparation, activation, and culture preparation of recombinant Agrobacterium strains: The pCAMBIAsuper1300-AhNF-YC9 recombinant plasmid was transformed into competent Agrobacterium strain EHA105 cells using the liquid nitrogen freeze-thaw method. Recombinant strains harboring the recombinant plasmid were screened. Single colonies of the recombinant strains were selected and inoculated into YEB liquid medium (50 mg / L rifampicin, 50 mg / L kanamycin). Culture was performed at 28°C and 180 rpm until the OD600 reached 0.5-0.8. Then, 2 mL of the culture medium was transferred to 50 mL of YEB medium (50 mg / L rifampicin, 50 mg / L kanamycin) and cultured to an OD600 of 0.6. The culture was centrifuged at 5000 rpm for 10 minutes and resuspended in an equal volume of liquid MSB5 for later use.

[0029] b. Isolation of peanut explants: Select plump peanut seeds (Huayu No. 23), soak them in 70% alcohol for 1 minute, soak them in 0.1% mercuric chloride for 20 minutes, rinse them with sterile water 3-5 times, and cut each cotyledon into two halves longitudinally.

[0030] c. Agrobacterium-mediated genetic transformation: Immerse the explants in the prepared Agrobacterium solution and infect them with gentle shaking at 28°C and 90 rpm for 10 minutes. Remove any remaining solution with sterile filter paper and inoculate onto SIM induction medium in the dark for 3 days. Transfer the explants to SIM induction medium supplemented with 250 mg / L cephalosporin, insert the cut end of the explant into the medium, and incubate for approximately 2 weeks to induce shoot formation. Culture conditions: light intensity of 1500-2000 lx, photoperiod of 12 h, and temperature of 26°C ± 1°C.

[0031] The explants that formed clustered buds were transferred to SEM medium containing 250 mg / L cephalosporin and 100 mg / L kanamycin to select resistant buds and cultured for 2 weeks. The culture conditions were: light intensity of 1500-2000lx, light for 12h, and temperature of 26℃±1℃.

[0032] 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 induction of bud elongation. The culture was continued for about 4 weeks, during which time the buds were subcultured 2-3 times.

[0033] d. PCR detection of transgenic plants Genomic DNA was extracted from the regenerated plants, and primers were designed using the vector sequence and the AhNF-YC9 gene sequence for PCR amplification. The PCR reaction program was: 95°C for 5 minutes, 95°C for 50 seconds, 58°C for 50 seconds, 72°C for 1 minute, 30 cycles, and 72°C for 10 minutes.

[0034] e. Grafting and transplanting of transgenic positive plants Use sterile seedlings of about 15 days old as rootstocks, remove 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, and cut a V-shaped wound about 1 cm long at the lower end, with the incision smooth. 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 sterilized seedling medium for acclimatization for 2 weeks, and then transplant them into the matrix soil until the pods are harvested.

[0035] f. Determination of oil content in transgenic peanut kernels Peanut kernel oil content was determined according to the Determination of Oil Content in Oilseeds (Residual Method) (NY / T 1285-2007). Weigh at least 10 g of T3 transgenic and non-transgenic peanut kernels and dry them in an oven at 100 ± 2°C for approximately 2 hours. Use a tissue crusher to crush the kernels into a uniform powder. Weigh at least 1.5 g of the peanut sample and place it in a dried filter paper tube. Dry the filter paper tube and sample in an oven at 105 ± 2°C for 3 hours, then cool to room temperature in a desiccator. Soak the filter paper tube in an extractant overnight. Extract the sample for 8 hours. Calculate the oil content by weight. Repeat the measurement three times for each sample. The results showed that compared with the oil content of non-transgenic peanut kernels (52.31%), the oil content of peanut kernels of the six transgenic lines reached 56.34%, 55.98%, 55.64%, 55.79%, 56.05% and 56.07%, respectively, and the oil content could be increased by 3.3-4.0 percentage points ( Figure 2 ).

[0036] Example 4 Application of AhNF-YC9 gene in improving plant salt tolerance A method for improving salt tolerance of peanuts, comprising the following steps: The plant expression vector pCAMBIAsuper1300-AhNF-YC9 prepared in Example 2 was transformed into peanut (Huayu 23) using the same steps as described in Example 3, ae.e., to obtain transgenic peanut seedlings, i.e., overexpressing plants.

[0037] Two-week-old peanut seedlings were watered with 350 mM NaCl solution for 15 days. Phenotypic changes in overexpressing plants and non-transgenic controls were observed, and the relative electrical conductivity of their leaves was measured. The results showed that after NaCl treatment, plants overexpressing the AhNF-YC9 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 29.41% and 39.71%, respectively ( Figure 4 ).

[0038] In order to detect the accumulation of reactive oxygen species in the leaves of transgenic positive plants and non-transgenic controls after salt stress treatment, NBT and DAB staining were used to measure the accumulation of reactive oxygen species ( Figure 5 ),in, Figure 5 In the NBT staining results, the more blue the leaves are, the more O2 − The more the accumulation, the less blue part of the leaves of transgenic plants (OE-1 and OE-2) than the non-transgenic control (WT), indicating that O2 − The accumulation amount is small. In the DAB staining results, the more brown part of the leaves, the more H2O2 accumulation; the transgenic plants (OE-1 and OE-2) have less brown part than the non-transgenic control (WT), indicating that the accumulation amount of H2O2 is small. The H2O2 and O2 − 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 63.32% and 62.37%, respectively; the POD content increased by 62.33% and 60.34%, respectively; the Pro content increased by 92.67% and 99.71%, respectively; the SOD content increased by 111.61.38% and 121.96%, respectively ( Figure 6 ).

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Application of peanut AhNF-YC9 gene in improving plant oil content and salt tolerance, characterized in that: The amino acid sequence encoded by the peanut AhNF-YC9 gene is shown in SEQ ID NO:

2.

2. The use of the peanut AhNF-YC9 gene in improving plant oil content and salt tolerance according to claim 1, characterized in that: The nucleic acid sequence of the peanut AhNF-YC9 gene is shown in SEQ ID NO:

1.

3. A method for increasing oil content and salt tolerance of plants, characterized in that: By increasing the expression level of the peanut AhNF-YC9 gene in the plant, the oil content and salt tolerance of the plant are improved. The amino acid sequence encoded by the peanut AhNF-YC9 gene is shown in SEQ ID NO:

2.

4. The method for improving plant oil content and salt tolerance according to claim 3, wherein: The nucleic acid sequence of the peanut AhNF-YC9 gene is shown in SEQ ID NO:

1.

5. The method for improving plant oil content and salt tolerance according to claim 4, characterized in that: By constructing a plant expression vector of the peanut AhNF-YC9 gene and transforming the plant, the gene is expressed in the plant, thereby increasing the expression level of the AhNF-YC9 gene in the plant.

6. The method for improving plant oil content and salt tolerance according to claim 5, characterized in that: The method of transforming plants is one of Agrobacterium-mediated method, gene gun method, electric shock method, PEG method and liposome method.

7. The method for improving plant oil content and salt tolerance according to claim 6, characterized in that: The Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

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

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