CeNF-YA1 gene, expression vector and its application in plant oil regulation

Abstract

This invention belongs to the field of molecular biology technology, specifically involving CeNF‑YA1 Genes, expression vectors, and their applications in plant oil regulation. This invention provides for the first time... CeNF‑YA1 Genes, the ones mentioned CeNF‑YA1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, with a coding region of 792 bp. This is the first time a gene containing [a specific gene structure] has been constructed. CeNF‑ YA1 A series of vectors, including subcellular localization of the gene, yeast hybridization, and plant overexpression, were used to demonstrate that the CeNF-YA1 protein possesses transcriptional activation function and is located in the cell nucleus, consistent with the basic characteristics of a transcription factor. This is the first discovery of its kind. CeNF‑YA1 Overexpression of the gene can significantly increase the oil content of Arabidopsis seeds and leaves; therefore, the technical solution of the present invention can be used to increase the oil content of plant seeds and vegetative tissues, and shows its application prospects in increasing the oil content of seeds and vegetative tissues.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically involving CeNF-YA1 Genes, expression vectors and their applications in the regulation of plant oils. Background Technology

[0002] Tiger nuts ( Cyperus esculentus (L.) belongs to the Cyperaceae family of the Poales order. Also known as oil sedge or tiger nut, it is a root crop with high nutritional value. It is characterized by its wide adaptability, great development potential, and high oil yield per acre, making it a highly sought-after new food ingredient in the fields of health food and sustainable agriculture.

[0003] Dietary lipid synthesis and accumulation are controlled by a multi-gene regulatory network, in which transcription factors play a crucial role, such as AP2 / EREBP, B3, NF-Y, MYB, WRKY, DOF, and bZIP. WRI1 ( WRINKLED1 This gene belongs to the AP2 / ERF transcription factor family and is a key gene confirmed to regulate lipid accumulation during oilseed seed development. Currently, information regarding tiger nuts... WRI Gene-like studies have been reported, for example, in Chinese patent CN 118531058 A, on a tiger nut. CeWRI1b The application of genes in the regulation of plant oils provides CeWRI1b Genes, and confirmed CeWRI1b Overexpression can be complementary atwri1-1 The wrinkled phenotype and oil content of mutant seeds can significantly increase the oil content of leaves; another example is Chinese patent CN118064452 A, a tiger nut. CeWRI2 Genes, expression vectors, and their applications in plant oil regulation have led to the discovery of... CeWRI2 Genes can be used to increase the oil content of plant seeds and vegetative tissues; Chinese Patent CN 118064455 A: A Kind of Tiger Nut CeWRI3 Genes, expression vectors, and their applications in plant oil regulation have led to the discovery of... CeWRI3 Genes can be used to increase the oil content of plant seeds and vegetative tissues. However, no such gene has been observed to date. CeNF-YA1 Reports on genes and their role in increasing the oil content of plant seeds and vegetative tissues. Summary of the Invention

[0004] The purpose of this invention is to provide CeNF-YA1 Genes, expression vectors and their applications in the regulation of plant oils.

[0005] This invention provides CeNF-YA1 Genes, the ones mentioned CeNF-YA1The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] The present invention provides the technical solution described above. CeNF-YA1 The CeNF-YA1 protein encoded by the gene, the amino acid sequence of which is shown in SEQ ID NO.11.

[0007] This invention provides a solution containing the above-described technical solution. CeNF-YA1 The gene expression vector includes one or more of the following: cell localization vector pNC-Cam1304-CeNF-YA1, yeast hybridization vector, and plant overexpression vector pCAMBIA1301-CeNF-YA1;

[0008] The yeast hybridization vector is one or more of the following: pNC-GBKT7-CeNF-YA1F, pNC-GBKT7-CeNF-YA1N1, pNC-GBKT7-CeNF-YA1N2, and pNC-GBKT7-CeNF-YA1C.

[0009] Preferably, pNC-Cam1304-CeNF-YA1 is constructed using CeNF-YA1F2 and CeNF-YA1R2;

[0010] Alternatively, pNC-GBKT7-CeNF-YA1F can be constructed using CeNF-YA1F2 and CeNF-YA1R2;

[0011] pNC-GBKT7-CeNF-YA1N1 was constructed using CeNF-YA1F2 and CeNF-YA1R3;

[0012] pNC-GBKT7-CeNF-YA1N2 was constructed using CeNF-YA1F2 and CeNF-YA1R4;

[0013] pNC-GBKT7-CeNF-YA1C was constructed using CeNF-YA1F3 and CeNF-YA1R2;

[0014] pCAMBIA1301-CeNF-YA1 was constructed using CeNF-YA1F4 and CeNF-YA1R5;

[0015] The nucleotide sequence of CeNF-YA1F2 is shown in SEQ ID NO.4;

[0016] The nucleotide sequence of CeNF-YA1R2 is shown in SEQ ID NO.5;

[0017] The nucleotide sequence of CeNF-YA1F3 is shown in SEQ ID NO.7;

[0018] The nucleotide sequence of CeNF-YA1R3 is shown in SEQ ID NO.6;

[0019] The nucleotide sequence of CeNF-YA1F4 is shown in SEQ ID NO.9;

[0020] The nucleotide sequence of CeNF-YA1R4 is shown in SEQ ID NO.8;

[0021] The nucleotide sequence of CeNF-YA1R5 is shown in SEQ ID NO.10.

[0022] This invention provides a method for amplifying the above-described technical solution. CeNF-YA1 Primer pairs for the gene, said primer pairs including CeNF-YA1F1 and CeNF-YA1R1;

[0023] The nucleotide sequence of CeNF-YA1F1 is shown in SEQ ID NO.2;

[0024] The nucleotide sequence of CeNF-YA1R1 is shown in SEQ ID NO.3.

[0025] The present invention provides the technical solution described above. CeNF-YA1 The application of the gene, the CeNF-YA1 protein, the expression vector, or the primer pair in the regulation of plant oils;

[0026] The plants mentioned include Arabidopsis thaliana and / or tobacco.

[0027] Preferably, the regulation of plant oils includes: increasing the concentration of plant oils in the plant oil content of oils. CeNF-YA1 The level of gene expression promotes the accumulation of oil in plant seeds or plant tissues, thereby increasing the oil content.

[0028] Preferably, the plant tissue includes one or more of leaves, tubers, and rhizomes.

[0029] This invention provides a method for increasing the oil content of plants or promoting the accumulation of plant oils, comprising the following steps:

[0030] The above technical solution is described CeNF-YA1 Genes are introduced into target plants to increase the oil content or promote oil accumulation in the target plants.

[0031] Preferably, the target plant includes: tobacco and / or Arabidopsis thaliana.

[0032] Beneficial effects:

[0033] This invention provides for the first time CeNF-YA1 Genes, the ones mentioned CeNF-YA1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and its coding region is 792 bp in length.

[0034] This invention is the first to construct a system containing CeNF-YA1 A series of vectors, including subcellular localization of the gene, yeast hybridization, and plant overexpression, were developed, and for the first time it was demonstrated that the CeNF-YA1 protein has transcriptional activation function and is located in the cell nucleus, which conforms to the basic characteristics of transcription factors.

[0035] This invention demonstrates the effectiveness of transforming Arabidopsis thaliana using an inflorescence-based dyeing method. CeNF-YA1 Overexpression of the gene can significantly increase the oil content of Arabidopsis seeds and leaves; therefore, it can be used to increase the oil content of plant seeds and vegetative tissues, showing its application prospects in increasing the oil content of seeds and vegetative tissues. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0037] Figure 1 For the present invention CeNF-YA1 PCR amplification results of the gene: M: DNA marker III; CK: blank control; 1: CeNF-YA1 ;

[0038] Figure 2 For the present invention CeNF-YA1 BLASTN alignment of genes in the NCBI GenBank database;

[0039] Figure 3 For the present invention CeNF-YA1 Schematic diagram of sequence characteristics and evolutionary analysis of encoded proteins: A: Conserved domain analysis of CeNF-YA1 protein; B: Multiple sequence alignment of CeNF-YA1 and EgNF-YA3; C: Evolutionary analysis of CeNF-YA1, EgNF-YA3 and NF-YA protein in Arabidopsis thaliana;

[0040] Figure 4 This is a diagram showing the subcellular localization of the CeNF-YA1 protein in tobacco leaf cells according to the present invention.

[0041] Figure 5 Figure showing the identification results of the transcriptional activation function of CeNF-YA1 protein in yeast according to the present invention: pNC-GBKT7: empty vector; F: full-length coding region; N1: N-terminal sequence 1; N2: N-terminal sequence 2, which contains the CBFB-NFYA domain; C: C-terminal sequence, which is the downstream sequence of the CBFB-NFYA domain.

[0042] Figure 6 The graph shows the regulatory effect of transient overexpression of the CeNF-YA1 gene in tobacco leaves on lipid accumulation according to the present invention: the horizontal axis represents different days after transformation, and the vertical axis represents the fold increase in triglycerides (TAG) in the treatment relative to the empty vector control. The graph shows the average of three biological replicates, and uppercase letters indicate the results based on... P The difference was significant (<0.01).

[0043] Figure 7 For the present invention CeNF-YA1 Figure 1 shows the regulatory effect of gene overexpression on seed and leaf lipid accumulation in Arabidopsis thaliana: A: TAG content in transgenic and empty-transplant control T3 generation seeds; B: fold increase in TAG content in leaves of transgenic plants relative to empty-transplant T3 generation plants, showing the average of three biological replicates. "**" indicates based on... P The difference was significant (<0.01). Detailed Implementation

[0044] The method for constructing the expression vector described in this invention has no special requirements and can be constructed using methods known in the art.

[0045] The NC kit described in this invention was purchased from Hainan Nixing Biotechnology Co., Ltd.; total RNA was extracted using the Tiangen plant polysaccharide and polyphenol RNA extraction kit (catalog number DP441); reverse transcription was performed using the Takara PrimeScript™ RTreagent Kit with gDNA Eraser kit (catalog number RR047A).

[0046] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0047] In this invention, the primers used are shown in Table 1.

[0048] Table 1 Primer Information

[0049]

[0050] The present invention CeNF-YA1The nucleotide sequence of the gene is shown in SEQ ID NO.1, specifically: 5′-ATGGCCAAGCCTGTCTCCTCCTCAACTCAGTCCTCAAGTAGATCCCATCAAGATGATTCAGCTTCAGCTTTGAGTGATACTTCTGTCTATTCTGGTAGTTATGATAAGAGAAATGATCAGGTGAAATCAGCTTTATCCCTTGGCAATGGTGATTCCGCTTTTACTGCTCCAAAGCTTGATTATTTGCAGCCCTTTACATGCGTGCCGTACCCATATCCAGATGCCTGTTATGGCAGCGACATGACTGCATATGCTTCACCTCCGATGGTAAATGCACAGATGATGGGGATGGGGTCCAATTCGCGGGTGCCACTACCTCTCGAGCCTGCAGCAGAGGAACCAATATTTGTGAATGCAAAGCAGTACCATGCAATTCTTAGAAGGAGACAACTGCGTGCAAAGTTGGAGGCTGAAAATAAACTTGTGAAAGTCCGGAAGCCATATCTCCATGAATCACGGCATCTTCATGCGATGAAGCGGGCTCGGGGATCTGGTGGTCGGTTTCTTAATACCAAGCAGCAGTCTCAGCAGGGCCAGCTGTCAGACAGCCCAACACATGGCCTAAAAGTCTTAGGCACTAACATCGGACCTCAAGCTGAAGCCACTAATCAAACTATAGCAGGTGCTTCTGTGGCAGTAACTCCATCTAACTCCAGAAATGCACCAACCGAGCATAAGATCAACTATGGATCCCTGGATTTTAGAGGAGGGTTTCAAGCTGTAAATGGGTTTGGGAACAGAATTGGGATCAACAGTAGCTCCTCTCAGTGCGGTATCTCTATCATGCCGTGA-3′。

[0051] The amino acid sequence of the CeNF-YA1 protein described in this invention is shown in SEQ ID NO.11, specifically: MAKPVSSSTQSSSRSHQDDSASALSDTSVYSGSYDKRNDQVKSALSLGNGDSAFTAPKLDYLQPFTCVPYPYPDACYGSDMTAYASPPMVNAQMMGMGSNSRVPLPLEPAAEEPIFVNAKQYHAILRRRQLRAKLEAENKLVKVRKPYLHESRHLHAMKRARGSGGRFLNTKQQSQQGQLSDSPTHGLKVLGTNIGPQAEATNQTIAGASVAVTPSNSRNAPTEHKINYGSLDFRGGFQAVNGFGNRIGINSSSSQCGISIMP*.

[0052] Example 1 CeNF-YA1 Gene cloning and sequence analysis

[0053] (1) Total RNA was extracted from tubers of 10 different developmental stages of Tiger nut No. 3. The mRNA was then reverse transcribed into cDNA and used as a template for PCR amplification. Based on the full-length cDNA obtained from the transcriptome, primer pairs CeNF-YA1F1 (SEQ ID NO. 2) and CeNF-YA1R1 (SEQ ID NO. 3) were designed using Primer Premier 5.0.

[0054] (2) After optimizing the PCR conditions based on the Tm values ​​of the primers, PCR amplification was performed using the upstream primer CeNF-YA1F1, the downstream primer CeNF-YA1R1, and the reverse transcribed cDNA as templates. The PCR amplification reaction system is shown in Table 2. The PCR reaction program was as follows: 98℃ for 3 min; 98℃ for 30 sec, 60℃ for 30 sec, 72℃ for 50 sec (35 cycles); 72℃ for 5 min; and stored at 4℃.

[0055] Table 2 PCR amplification reaction system

[0056]

[0057] (3) PCR amplification yielded a specific band of approximately 800 bp. Figure 1 After gel extraction using the OMEGA gel extraction kit (catalog number D2500-02), the cells were cloned into the Takara cloning vector pMD19-T (catalog number 6013). The cells were then sequenced after blue-white screening and colony PCR verification.

[0058] (4) Sequence analysis showed that, CeNF-YA1The coding region (CDS) is 792 bp in length, with a GC content of 47.47%, and is predicted to encode 263 amino acids. Its theoretical molecular weight is 28.31 kDa, isoelectric point (pI) is 9.51, total average hydrophobicity index (GRAVY) is -0.573, instability coefficient (II) is 57.22, and aliphatic index (AI) is 63.88.

[0059] (5) BLASTN was used to compare the sequence against the NCBI GenBank database, and no similar sequences were found. Figure 2 This confirms that the cloned gene is a new gene.

[0060] (6) CDD (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?) analysis and sequence alignment showed that the CeNF-YA1 protein contains one conserved CBFB_NFYA domain ( Figure 3 A and 3B). To further reveal the evolutionary characteristics of CeNF-YA1, a phylogenetic tree was constructed using MEGA6 (https: / / www.megasoftware.net / ) along with EgNF-YA3 and NF-YA proteins in Arabidopsis thaliana. The results are as follows. Figure 3 As shown in C.

[0061] Combination Figure 3 As shown in C, CeNF-YA1 clustered with EgNF-YA3 (Egu032172), AtNF-YA5 (AT1G54160), AtNF-YA6 (AT3G14020), AtNF-YA3 (AT1G72830), and AtNF-YA8 (AT1G17590), with sequence similarities of 50.54%, 44.70%, 39.46%, 42.70%, and 38.07%, respectively.

[0062] Example 2 Subcellular localization analysis of CeNF-YA1 protein

[0063] (1) WoLF PSORT (https: / / www.genscript.com / wolf-psort.html) analysis showed that CeNF-YA1 may be located in the cell nucleus.

[0064] (2) To confirm the above prediction results, primer pairs CeNF-YA1F2 (SEQ ID NO.4) and CeNF-YA1R2 (SEQ ID NO.5) as shown in Table 1 were designed near the start and stop codons of the sequencing gene. The PCR product in Example 1 was diluted 100 times as a template for PCR amplification (the PCR amplification system and procedure were the same as those in Example 1). After PCR amplification, the target fragment was gel-cleaved and cloned into pNC-Cam1304-SubN using the NC kit (using Hainan Nixing Biotechnology Co., Ltd.) to construct the fusion expression vector pNC-Cam1304-CeNF-YA1 with EGFP. The NC ligation system is shown in Table 3.

[0065] Table 3 NC Connection System

[0066]

[0067] (4) Transform the recombinant plasmid pNC-Cam1304-CeNF-YA1 into Agrobacterium tumefaciens strain GV3101 (containing pSoup-P19), the specific procedure is as follows:

[0068] (4-1) Add 1 μg of nuclear localization positive plasmids pNC-Cam1304-H2A and pNC-Cam1304-CeNF-YA1 to 100 μL of competent cells GV3101 and mix well by pipetting.

[0069] (4-2) Ice bath for 5 min, freeze in liquid nitrogen for 5 min, then water bath at 37℃ for 5 min, and stand on ice for 5 min;

[0070] (4-3) Add 700 μL of antibiotic-free LB medium and incubate at 28℃ and 200 rpm for 2 h with shaking.

[0071] (4-4) Spread the bacterial cells on solid LB medium supplemented with 50 mg / L kanamycin and 100 mg / L rifampin, and incubate at 28°C for 2-3 days;

[0072] (4-5) Select single clones for colony PCR detection, and select positive clones for genetic transformation experiments.

[0073] (5) The micro-injection method was used to transform 4-week-old tobacco leaves. The specific process is as follows:

[0074] (5-1) Inoculate the above-mentioned positive bacteria into 2 mL of LB liquid medium (50 mg / L rifampin) and incubate overnight at 28°C and 210 rpm;

[0075] (5-2) Take 1 mL of bacterial culture into 30 mL of LB liquid medium and incubate at 28°C and 210 rpm until the bacterial concentration OD600 is 0.8;

[0076] (5-3) Centrifuge at 5000 rpm for 3 min to collect bacterial cells, remove the supernatant and add resuspension buffer (10 mmol / L MgCl2, 0.2 mmol / L acetylsalicylic acid (AS) and 10 mmol / L MES, adjust the pH to 5.6 with KOH) to resuspend the bacterial cells. Repeat twice, and adjust the concentration of the resuspension to OD600 of 0.6.

[0077] (5-4) After resuspending the bacterial solution, incubate it at 28°C for 3-5 h. Then, inject the bacterial solution into the lower epidermis of the tobacco plant using a 1 mL syringe with the needle removed (one gene is injected into multiple leaves of one tobacco plant). You can use the needle to slightly puncture the lower epidermis of the leaf before injection.

[0078] (5-5) After the tobacco leaves were cultured in the dark for 2-4 days after injection, the injected tobacco leaves were used to prepare slides for fluorescence signal detection.

[0079] (6) Laser confocal microscopy revealed that the fluorescence signals of the experimental group (green fluorescence) and the positive control (red fluorescence) were found in the cell nucleus and highly overlapped, indicating that CeNF-YA1 plays a role in the cell nucleus. Figure 4 ).

[0080] Example 3 Identification of the transcriptional activation function of CeNF-YA1 protein

[0081] (1) To identify the transcriptional activation function of CeNF-YA1 and the location of the activation domain, primers as shown in Table 1 were designed near the CBFB_NFYA domain. The PCR product in Example 1 was diluted 100 times as a template, and CeNF-YA1F2 (SEQ ID NO.4) and CeNF-YA1R2 (SEQ ID NO.5) were used as primers to amplify the full length of the coding region of CeNF-YA1 (SEQ ID NO.1). After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 to construct the yeast two-hybrid bait vector pNC-GBKT7-CeNF-YA1F.

[0082] Using the PCR product diluted 100-fold in Example 1 as a template, and CeNF-YA1F2 (SEQ ID NO.4) and CeNF-YA1R3 (SEQ ID NO.6) as primers, the N1 end of CeNF-YA1 (i.e., nucleotide sequence 1-327 bp shown in SEQ ID NO.1) was amplified. After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 to construct the yeast two-hybrid bait vector pNC-GBKT7-CeNF-YA1N1.

[0083] Using the PCR product diluted 100-fold in Example 1 as a template, and CeNF-YA1F2 (SEQ ID NO.4) and CeNF-YA1R4 (SEQ ID NO.8) as primers, the N2 end of CeNF-YA1 (i.e., nucleotide sequence 1-510 bp as shown in SEQ ID NO.1, containing the CBFB_NFYA domain) was amplified. After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 to construct the yeast two-hybrid bait vector pNC-GBKT7-CeNF-YA1N2.

[0084] Using the PCR product diluted 100-fold in Example 1 as a template, and CeNF-YA1F3 (SEQ ID NO.7) and CeNF-YA1R2 (SEQ ID NO.5) as primers, the C-terminus of CeNF-YA1 (i.e., nucleotide sequence 511-792 bp shown in SEQ ID NO.1, downstream of the CBFB_NFYA domain) was amplified. After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 to construct the yeast two-hybrid bait vector pNC-GBKT7-CeNF-YA1C.

[0085] The PCR amplification method and NC ligation method described above are the same as those in Example 2.

[0086] (2) The empty vector (pNC-GBKT7) and four recombinant plasmids (pNC-GBKT7-CeNF-YA1F, pNC-GBKT7-CeNF-YA1N1, pNC-GBKT7-CeNF-YA1N2 and pNC-GBKT7-CeNF-YA1C) were transferred into Y2H-Gold yeast cells. The transformed yeast cells were then selected on tryptophan-deficient medium. Positive bacteria were selected and screened on tryptophan and histidine-deficient medium. Subsequently, the positive bacteria were subjected to X-α-gal colorimetric reaction.

[0087] (3) The results showed that the engineered bacteria transformed with pNC-GBKT7-CeNF-YA1F, pNC-GBKT7-CeNF-YA1N1, and pNC-GBKT7-CeNF-YA1N2 could survive on selective media and showed a blue color, while yeast cells transformed with empty vector and pNC-GBKT7-CeNF-YA1C failed to show β-galactosidase activity. This indicates that CeNF-YA1 has transcriptional activation function, and the activation domain is located upstream of the CBFB_NFYA domain ( Figure 5 ).

[0088] Example 4 CeNF-YA1 Identification of lipid regulation function

[0089] (1) For identification CeNF-YA1 To investigate its function in regulating plant oils, the above PCR product was diluted 100-fold as a template and CeNF-YA1F4 (SEQ ID NO. 9) and CeNF-YA1R5 (SEQ ID NO. 10) were used as primers for PCR amplification. The target fragment was then recovered from the gel and cloned into the plant overexpression vector pCAMBIA1301 using homologous recombination to construct the recombinant vector pCAMBIA1301-CeNF-YA1.

[0090] (2) The recombinant vector pCAMBIA1301-CeNF-YA1 and the empty pCAMBIA1301 were separately transformed into GV3101 (containing pSoup-P19), and 4-week-old tobacco leaves were transformed by micro-injection method; samples were collected 1 day, 3 days and 5 days after transformation for the determination of oil content.

[0091] (3) Oil content determination results based on Agilent 7890A (HP-FFAP, 30 9 0.25 mm ID, 0.25 lm, Santa Clara, CA, USA) showed that, compared with the control group converted to empty loading, the TAG content in the experimental group increased by 1.52, 1.57 and 2.05 times after 1 day, 3 days and 5 days of conversion, respectively. Figure 6 ).

[0092] Example 5 CeNF-YA1 Application in the regulation of lipids in seeds and vegetative tissues

[0093] (1) For evaluation CeNF-YA1To explore its potential application in regulating plant oils, the engineered bacteria containing the recombinant vector pCAMBIA1301-CeNF-YA1 or the empty vector pCAMBIA1301 were used to transform wild-type Arabidopsis thaliana using the inflorescence immersion method. The transformed plants were then cultured under the same conditions until maturity and seed harvesting. T1 seeds were screened for resistance on MS solid plates containing 25 µg / mL hygromycin to obtain T2 generation positive transgenic plants. After maturation, these plants underwent further resistance screening to obtain stably inherited T3 generation positive transgenic plants.

[0094] (2) To analyze the effect of gene overexpression on seed oil regulation, oil content was measured in T3 generation transgenic Arabidopsis seeds. The results showed that the oil content of the transgenic seeds was 32.51%, which was significantly higher than that of the empty-vectored seeds (27.50%). Figure 7 A);

[0095] (3) For evaluation CeNF-YA1 The application value of this study in regulating oil content in plant vegetative tissues was investigated. Leaves from the aforementioned T3 generation transgenic Arabidopsis thaliana were collected for oil content determination. The results showed that, compared to the control group (transformed into an empty vector), oil content was significantly higher. CeNF-YA1 The oil content of the leaves increased by 3.57 times due to overexpression. Figure 7 B).

[0096] (4) The above results indicate that, CeNF-YA1 It has the function of promoting oil accumulation and shows its application prospect in increasing the oil content of seeds and nutrient tissues.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. CeNF-YA1 Genes, characterized by, The CeNF-YA1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The claim 1 CeNF-YA1 The CeNF-YA1 protein encoded by the gene is characterized by, The amino acid sequence of the CeNF-YA1 protein is shown in SEQ ID NO.

11.

3. Containing the contents of claim 1 CeNF-YA1 Gene expression vectors, characterized in that, The expression vector includes one or more of the following: cell localization vector pNC-Cam1304-CeNF-YA1, yeast hybridization vector, and plant overexpression vector pCAMBIA1301-CeNF-YA1; The yeast hybridization vector is one or more of the following: pNC-GBKT7-CeNF-YA1F, pNC-GBKT7-CeNF-YA1N1, pNC-GBKT7-CeNF-YA1N2, and pNC-GBKT7-CeNF-YA1C.

4. The expression vector according to claim 3, characterized in that, pNC-Cam1304-CeNF-YA1 was constructed using CeNF-YA1F2 and CeNF-YA1R2; Alternatively, pNC-GBKT7-CeNF-YA1F can be constructed using CeNF-YA1F2 and CeNF-YA1R2; Alternatively, pNC-GBKT7-CeNF-YA1N1 can be constructed using CeNF-YA1F2 and CeNF-YA1R3; Alternatively, pNC-GBKT7-CeNF-YA1N2 can be constructed using CeNF-YA1F2 and CeNF-YA1R4; Alternatively, pNC-GBKT7-CeNF-YA1C can be constructed using CeNF-YA1F3 and CeNF-YA1R2; Alternatively, pCAMBIA1301-CeNF-YA1 can be constructed using CeNF-YA1F4 and CeNF-YA1R5; The nucleotide sequence of CeNF-YA1F2 is shown in SEQ ID NO.4; The nucleotide sequence of CeNF-YA1R2 is shown in SEQ ID NO.5; The nucleotide sequence of CeNF-YA1F3 is shown in SEQ ID NO.7; The nucleotide sequence of CeNF-YA1R3 is shown in SEQ ID NO.6; The nucleotide sequence of CeNF-YA1F4 is shown in SEQ ID NO.9; The nucleotide sequence of CeNF-YA1R4 is shown in SEQ ID NO.8; The nucleotide sequence of CeNF-YA1R5 is shown in SEQ ID NO.

10.

5. For amplifying the device described in claim 1 CeNF-YA1 A primer pair for a gene, characterized in that, The primer pair includes CeNF-YA1F1 and CeNF-YA1R1; The nucleotide sequence of CeNF-YA1F1 is shown in SEQ ID NO.2; The nucleotide sequence of CeNF-YA1R1 is shown in SEQ ID NO.

3.

6. The claim 1 CeNF-YA1 The application of the gene or the expression vector as described in claim 3 or 4 in the regulation of plant oils. The plants mentioned are: Arabidopsis thaliana and / or tobacco; The regulation of plant oils is achieved by increasing the levels of oils in plants. CeNF-YA1 The level of gene expression promotes the accumulation of oil in plant seeds or plant tissues, thereby increasing the oil content.

7. The application according to claim 6, characterized in that, The plant tissues include: leaves and / or seeds.

8. A method for increasing the oil content of plants or promoting the accumulation of plant oils, characterized in that, Includes the following steps: The claim 1 CeNF-YA1 Genes are introduced into target plants to increase the oil content or promote oil accumulation in the target plants. The target plants are: tobacco and / or Arabidopsis thaliana.

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