Ceabi3b gene, expression vector and application thereof in plant oil regulation

Abstract

The application belongs to the field of molecular biological technology, and particularly relates to CeABI3B a gene, an expression vector and application thereof in plant oil regulation. CeABI3B The application provides a gene for the first time, a nucleotide sequence of which is shown as SEQ ID NO. 1, and a coding region of which has a full length of 1707 bp. CeABI3B A series of vectors containing the gene are constructed for the first time, including subcellular localization, yeast hybridization and plant overexpression, and it is proved that the CeABI3B protein has a transcription activation function and is located in the cell nucleus, which is in line with the basic characteristics of transcription factors. CeABI3B Not only is it found that transient overexpression of the gene can significantly improve the oil content of tobacco leaves, but also it is proved that overexpression of the gene can significantly improve the oil content of transgenic Arabidopsis thaliana seeds and leaves. CeABI3B Therefore, the cloned and identified gene can be used to improve the oil content of plant seeds and nutrient tissues, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically involving CeABI3B Genes, expression vectors and their applications in the regulation of plant oils. Background Technology

[0002] Tiger nuts (scientific name: Cyperus esculentus Tigernut (Symplocos rubrum) belongs to the genus Cyperus in the family Cyperaceae of the order Poales. It is a newly emerging herbaceous oilseed crop originating in Africa and the Mediterranean coast. Compared to traditional oilseed crops that accumulate oil in their seeds, tigernut is the only known crop to date that accumulates oil at a high level (24%-35%) in its tubers. Discovering and identifying key genes involved in tuber oil accumulation can not only lay the foundation for the genetic improvement of tigernut, but also provide theoretical support and excellent genetic resources for the efficient production of oil from nutrient tissues such as roots, tubers, and leaves.

[0003] Lipid synthesis and accumulation are controlled by a multi-gene regulatory network, in which transcription factors play a key role, such as AP2 / EREBP, B3, NF-Y, MYB, WRKY, DOF, and bZIP. ABI3 belongs to the B3 transcription factor superfamily and was first identified in maize by McCarty et al. VP1 Subsequently, researchers identified homologous genes from Arabidopsis thaliana, rice, and maize. ABI3 / VP1 The ABI3 protein has four domains: an acidic N-terminal domain A and three basic domains B1, B2, and B3, which are conserved in different plants. Domain A is an acidic transcription activator; domain B1 interacts with the bZIP family-specific transcription factor ABI5; domain B2 binds to ABA response elements or G-box elements (CACGTG), thereby participating in abscisic acid response or nuclear localization; and domain B3 is the most conserved domain in the ABI3 protein, capable of binding to various conserved cis-elements in seed-specific gene promoter regions. Transcription factors involved in lipid regulation have been cloned and identified in tiger nuts; for example, Chinese patent CN118531058A reports a tiger nut... CeWRI1b The application of genes in plant oil regulation was demonstrated by transforming Arabidopsis thaliana using the inflorescence staining method. CeWRI1b Overexpression can be complementary wri1-1 The wrinkled phenotype and oil content of mutant seeds; transformation of tobacco using Agrobacterium-mediated microinjection to demonstrate... CeWRI1b Transient overexpression of genes can significantly increase the oil content of leaves; for example, Chinese patent CN118064452A reports a type of tiger nut. CeWRI2Genes, expression vectors, and their applications in regulating plant oils can be used to increase the oil content of plant seeds and vegetative tissues; Chinese patent CN118064455A reports a tiger nut. CeWRI3 Genes, expression vectors, and their applications in regulating plant oils can be used to increase the oil content of plant seeds and vegetative tissues; Chinese patent CN121294462A reports on this. CeNF-YA1 Genes, expression vectors, and their applications in plant oil regulation were discovered for the first time. CeNF-YA1 Overexpression of the gene can significantly increase the oil content of Arabidopsis seeds and leaves; Chinese patent CN121294463A reports a... CeNF-YA3 Genes, expression vectors, and their applications in plant oil regulation have been studied; overexpression of these genes significantly increases the oil content in Arabidopsis seeds and leaves. However, no publicly available research has been conducted to date. CeABI3B 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 CeABI3B This invention is the first to clone genes, expression vectors, and their applications in plant oil regulation. CeABI3B The gene was used to construct a series of vectors for its subcellular localization, yeast hybridization, and plant overexpression; it was demonstrated for the first time that the CeABI3B protein has transcriptional activation function and is located in the cell nucleus, which is consistent with the basic characteristics of transcription factors; tobacco was transformed using Agrobacterium-mediated microinjection, demonstrating... CeABI3B Transient overexpression of the gene significantly increased the oil content in leaves; this was confirmed by transforming Arabidopsis thaliana using the inflorescence immersion method. CeABI3B Gene overexpression significantly increases the oil content of transgenic Arabidopsis seeds and leaves; the cloned and identified gene of this invention... CeABI3B Genes can be used to increase the oil content of plant seeds and vegetative tissues.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides CeABI3B Genes, the ones mentioned CeABI3B The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] This invention provides CeABI3B The gene encodes the CeABI3B protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] This invention provides a product containing the above-mentioned... CeABI3BThe gene expression vector includes one or more of the following: cell localization vector pNC-Cam1304-CeABI3B, yeast hybridization vector, and plant overexpression vector pCAMBIA1301-CeABI3B; The yeast hybridization vector is one or more of the following: pNC-GBKT7-CeABI3BF, pNC-GBKT7-CeABI3BN1, pNC-GBKT7-CeABI3BN2, pNC-GBKT7-CeABI3BC, and pNC-GBKT7-CeABI3BTAD.

[0008] Preferably, pNC-Cam1304-CeABI3B is constructed using primers CeABI3BF2 and CeABI3BR2; Alternatively, pCAMBIA1301-CeABI3B can be constructed using primers CeABI3BF5 and CeABI3BR5; pNC-GBKT7-CeABI3BF was constructed using primers CeABI3BF2 and CeABI3BR2; pNC-GBKT7-CeABI3BN1 was constructed using primers CeABI3BF2 and CeABI3BR3; pNC-GBKT7-CeABI3BN2 was constructed using primers CeABI3BF2 and CeABI3BR4; pNC-GBKT7-CeABI3BC was constructed using primers CeABI3BF3 and CeABI3BR2; pNC-GBKT7-CeABI3BTAD was constructed using primers CeABI3BF4 and CeABI3BR3; The nucleotide sequence of CeABI3BF2 is shown in SEQ ID NO.5; The nucleotide sequence of CeABI3BR2 is shown in SEQ ID NO.6; The nucleotide sequence of CeABI3BF3 is shown in SEQ ID NO.7; The nucleotide sequence of CeABI3BR3 is shown in SEQ ID NO.8; The nucleotide sequence of CeABI3BF4 is shown in SEQ ID NO. 9; The nucleotide sequence of CeABI3BR4 is shown in SEQ ID NO.10; The nucleotide sequence of CeABI3BF5 is shown in SEQ ID NO.11; The nucleotide sequence of CeABI3BR5 is shown in SEQ ID NO.12.

[0009] This invention provides a method for amplifying the above CeABI3B Primer pairs for the gene, said primer pairs including CeABI3BF1 and CeABI3BR1; The nucleotide sequence of CeABI3BF1 is shown in SEQ ID NO.3; The nucleotide sequence of CeABI3BR1 is shown in SEQ ID NO.4.

[0010] This invention provides the aforementioned CeABI3B The application of the gene, the CeABI3B protein, the expression vector, or the primer pair in the regulation of plant oils; The plants include one or more of the following: Arabidopsis thaliana, tobacco, rapeseed, soybean, peanut, sunflower, mustard greens, cabbage, tomato, potato, sweet potato, cassava, beet, corn, wheat, sorghum, sugarcane, oil palm, and coconut.

[0011] Preferably, the regulation of plant oils includes: increasing the concentration of plant oils in the plant oil content of oils. CeABI3B The level of gene expression promotes the accumulation of oil in plant seeds or plant tissues, thereby increasing the oil content.

[0012] Preferably, the plant tissue includes one or more of the following: leaves, stems, tubers, and rhizomes.

[0013] This invention provides a method for increasing the oil content of plants or promoting the accumulation of plant oils, comprising the following steps: The above CeABI3B Genes are introduced into target plants to increase the oil content or promote oil accumulation in the target plants.

[0014] Preferably, the target plant includes one or more of Arabidopsis thaliana and tobacco.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention provides for the first time CeABI3B Genes, the ones mentioned CeABI3B The nucleotide sequence of the gene is shown in SEQ ID NO.1, and its coding region is 1707 bp in length; (2) This invention is the first to construct a system containing CeABI3B 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 CeABI3B protein has transcriptional activation function and is located in the cell nucleus, which is consistent with the basic characteristics of transcription factors. (3) This invention proves CeABI3B Transient overexpression of the gene in tobacco can significantly increase the oil content of leaves. CeABI3BOverexpression of the gene in Arabidopsis thaliana can significantly increase the oil content of transgenic 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

[0016] Figure 1 For the present invention CeABI3B Figures showing the results of the first (A) and second (B) rounds of PCR amplification of the gene; where M: DNA marker III; CK: blank control; 1: CeABI3B ; Figure 2 For the present invention CeABI3B BLASTN alignment of genes in the NCBI GenBank database; Figure 3 This is a schematic diagram of the sequence characteristics and evolutionary analysis of the CeABI3B protein of the present invention; wherein A: analysis of conserved domains of CeABI3B; B: sequence alignment of CeABI3B and AtWRI1; C: evolutionary analysis of CeABI3B and homologous proteins in rice, maize, Arabidopsis thaliana, sunflower, cotton and peanut. Figure 4 This is a diagram showing the subcellular localization of the CeABI3B protein of the present invention in tobacco leaf cells. Figure 5 The diagram shows the identification results of the transcriptional activation function of the CeABI3B protein in yeast according to the present invention: where V: empty vector; F: full-length coding region; N1: N1 end sequence, upstream of the B3 domain; N2: N2 end sequence, upstream of the B3 domain, containing the transcriptional activation domain; C: C-terminal sequence, which is the B3 domain and its downstream; TAD: contains the transcriptional activation domain. Figure 6 For the present invention CeABI3B Figure showing the transient overexpression of the gene in tobacco leaves and its regulatory effect on lipid accumulation: the horizontal axis represents different days after transformation, and the vertical axis represents the experimental group (…). CeABI3B The fold increase in triglycerides (TAG) in the control group (transformed with empty vector WT) and the control group is shown as the mean of three biological replicates. Lowercase letters indicate the basis of measurement. P The difference was statistically significant (<0.05). Figure 7 For the present invention CeABI3B Figure showing the regulatory effect of gene overexpression on seed and leaf lipid accumulation in Arabidopsis thaliana; where A: experimental group ( CeABI3BA: Fold increase in TAG in T3 generation seeds of the experimental and control groups (empty vector WT); B: Fold increase in TAG in leaves of T3 generation plants of the experimental and control groups (empty vector), showing the average of three biological replicates, "**" indicates based on P The difference was statistically significant (<0.01). Detailed Implementation

[0017] 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. 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.

[0018] In this invention, the primers used are shown in Table 1.

[0019] Table 1 Primer Information

[0020] The present invention CeABI3B

[0021] The present invention CeABI3B The amino acid sequence of the CeABI3B protein encoded by the gene is shown in SEQ ID NO.2, and the specific sequence information is as follows: *

[0022] Example 1 CeABI3B Gene cloning and sequence analysis (1) Using tubers of Tiger Pea No. 3 at different developmental stages as material, total RNA was extracted using the Tiangen Plant Polysaccharide Polyphenol RNA Extraction Kit (catalog number DP441): (2) The total RNA was reverse transcribed into cDNA using the Takara PrimeScript™ RT reagent Kit with gDNA Eraser, which was then used as a template for PCR amplification. (3) Based on the full-length cDNA obtained from the transcriptome, primer pairs CeABI3BF1 (SEQ ID NO.3) and CeABI3BR1 (SEQ ID NO.4) were designed using Primer Premier 5.0 as shown in Table 1. (4) After optimizing the PCR conditions according to the Tm values ​​of the primers, PCR amplification was performed using CeABI3BF1 (SEQ ID NO.3) and CeABI3BR1 (SEQ ID NO.4) as primers and the cDNA obtained by reverse transcription as a template. The first round of PCR amplification reaction system is shown in Table 2. The PCR reaction program is as follows: 98℃ for 3 min; 98℃ for 30 sec, 60℃ for 30 sec, 72℃ for 80 sec (35 cycles); 72℃ for 5 min; and stored at 4℃. Table 2 First-round PCR amplification reaction system

[0023] (5) PCR amplification yielded a specific band of approximately 1700 bp. Figure 1 A) was recovered from the gel using the OMEGA gel recovery kit and cloned into the pMD19-T cloning vector from Takara. The sequence was determined after blue-white screening and colony PCR verification. (6) Sequence analysis showed that, CeABI3B The coding region (CDS) is 1707 bp in length, with a GC content of 45.65%, and is predicted to encode 568 amino acids. Its theoretical molecular weight is 63.77 kDa, isoelectric point (pI) is 8.58, total average hydrophobicity index (GRAVY) is -0.572, instability coefficient (II) is 54.45, and aliphatic index (AI) is 70.76. (7) BLASTN alignment using the NCBI GenBank database did not reveal any similar sequences. Figure 2 This confirms that it is a new gene; (8) CDD analysis (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?) and sequence alignment with AtWRI1 showed that CeABI3B contains a conserved B3 domain. Figure 3 The A in the text possesses the transcriptional activation domain (TAD) characteristic unique to the ABI3 family. Figure 3 (B in the middle) (9) To further reveal the evolutionary characteristics of CeABI3B, a phylogenetic tree was constructed using MEGA6 (https: / / www.megasoftware.net / ) with ABI3 proteins from Arabidopsis thaliana (AtABI3), rice (OsABI3), maize (ZmABI3), sunflower (HaABI3), cotton (GhABI3), and peanut (AhABI3). Figure 3 As shown in C, these proteins clearly cluster into three groups, with CeABI3 clustering together with ZmABI3, OsABI3, AtABI3, HaABI3, GhABI3, and AhABI3.

[0024] Example 2: Subcellular localization analysis of CeABI3B protein (1) WoLF PSORT (https: / / www.genscript.com / wolf-psort.html) analysis showed that CeABI3B protein may be located in the cell nucleus.

[0025] (2) To confirm the above prediction results, primer pairs CeABI3BF2 (SEQ ID NO.5) and CeABI3BR2 (SEQ ID NO.6) were designed near the start and stop codons of the sequencing gene, as shown in Table 1.

[0026] (3) Using the PCR product diluted 100-fold in Example 1 as a template, and CeABI3BF2 (SEQ ID NO. 5) and CeABI3BR2 (SEQ ID NO. 6) as primers, PCR amplification was performed. The reaction system for the second round of PCR amplification is shown in Table 3. The PCR reaction program was: 98℃ for 3 min; 98℃ for 30 sec, 60℃ for 30 sec, 72℃ for 60 sec (35 cycles); 72℃ for 5 min; and stored at 4℃. Finally, a clear band of approximately 1700 bp was obtained. Figure 1 After gel extraction and recovery, the B in the sample was cloned into pNC-Cam1304-SubN using an NC kit (purchased from Hainan Nixing Biotechnology Co., Ltd.) to construct the fusion expression vector pNC-Cam1304-CeABI3B with EGFP. The NC ligation system is shown in Table 4.

[0027] Table 3. Second-round PCR amplification reaction system

[0028] Table 4 NC Connection System

[0029] (4) Transform the recombinant plasmid pNC-Cam1304-CeABI3B into Agrobacterium tumefaciens strain GV3101 (containing pSoup-P19), the specific steps are as follows: (4-1) Add 1 μg of empty vector plasmid pNC-Cam1304-SubN and 1 μg of recombinant plasmid pNC-Cam1304-CeABI3B to 100 μL of competent cells GV3101 and mix them by pipetting. (4-2) Ice bath for 30 min, then quickly place in liquid nitrogen for 1 min, and then heat shock in a water bath at 37℃ for 5 min; (4-3) Add 950 μL of antibiotic-free liquid YEP medium and incubate at 28℃ and 200 rpm for 4 h with shaking. (4-4) Centrifuge at 10,000 rpm for 1 min to concentrate the bacterial culture, discard the supernatant and resuspend the bacterial cells in 100 μL of antibiotic-free liquid YEP medium; (4-5) Spread the bacterial cells on solid YEP medium supplemented with 50 mg / L kanamycin and 100 mg / L rifampin, and incubate at 28°C for 2-3 days; (4-6) Select single clones for colony PCR detection and screen positive clones for subsequent genetic transformation experiments.

[0030] (5) The micro-injection method was used to transform 4-week-old tobacco leaves. The specific steps are as follows: (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; (5-2) Take 1 mL of bacterial culture and add it to 30 mL of LB liquid medium. Incubate at 28°C and 210 rpm until the bacterial concentration reaches OD500. 600 It is 0.8; (5-3) Collect bacterial cells by centrifugation at 5000 rpm for 3 min, remove the supernatant, and add resuspension buffer (10 mmol / L MgCl2, 0.2 mmol / L acetylsalicylic acid and 10 mmol / L MES, adjust pH to 5.6 using KOH) to resuspend the bacterial cells. Repeat twice, adjusting the concentration of the resuspension to OD. 600 It is 0.6; (5-4) After resuspending the bacterial solution, incubate it at 28°C for 3-5 hours. Then, inject it into the lower epidermis of the tobacco plant using a 1mL 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. (5-5) After the tobacco leaves were cultured in the dark for 2-4 days after injection, the injected tobacco leaves were taken to prepare slides for fluorescence observation. (6) Laser confocal microscopy showed that the green fluorescence signal in the experimental group highly overlapped with the red fluorescence signal labeled in the cell nucleus, indicating that CeABI3B plays a role in the cell nucleus. Figure 4 ).

[0031] Example 3: Identification of the transcriptional activation function of CeABI3B protein (1) To identify the transcriptional activation function and the location of the activation domain of CeABI3B protein, primers as shown in Table 1 were designed near the B3 domain. (2) Using the PCR product diluted 100 times in Example 1 as a template, CeABI3BF2 (SEQ ID NO.5) and CeABI3BR2 (SEQ ID NO.6) were used as primers to amplify the full length of the coding region of CeABI3B (SEQ ID NO.1). After the PCR amplification was completed, the gel was excised and the product was cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI3BF; Using the PCR product diluted 100-fold in Example 1 as a template, the N1 end (i.e., the first 1-1365 bp of the nucleotide sequence shown in SEQ ID NO.1, upstream of the B3 domain) was amplified using CeABI3BF2 (SEQ ID NO.5) and CeABI3BR3 (SEQ ID NO.8) as primers. After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI3BN1. Using the PCR product diluted 100-fold in Example 1 as a template, the N2 end (i.e., the 1st to 427th bp of the nucleotide sequence shown in SEQ ID NO.1, containing the transcription activation domain) was amplified using CeABI3BF2 (SEQ ID NO.5) and CeABI3BR4 (SEQ ID NO.10) as primers, respectively. After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI3BN2. Using the PCR product diluted 100-fold in Example 1 as a template, the TAD end (i.e., the 45-427 bp of the nucleotide sequence shown in SEQ ID NO.1, containing the transcription activation domain) was amplified using CeABI3BF4 (SEQ ID NO.9) and CeABI3BR3 (SEQ ID NO.8) as primers, respectively. After PCR amplification, the product was excised from the gel and cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI3BTAD. Using the PCR product diluted 100-fold in Example 1 as a template, the C-terminus (i.e., the 1366-1707 bp of the nucleotide sequence shown in SEQ ID NO.1, downstream of the B3 domain) of CeABI3BF3 (SEQ ID NO.7) and CeABI3BR2 (SEQ ID NO.6) were amplified respectively. After PCR amplification, the gel was excised and the product was cloned into pNC-GBKT7 using the NC kit to construct the yeast two-hybrid bait vector pNC-GBKT7-CeABI3BC. The PCR amplification reaction system, reaction procedure, and NC ligation system described above are the same as those in Example 2. (3) The empty vector (pNC-GBKT7) and 5 recombinant plasmids were transferred into Y2HGold yeast cells. The transformed yeast cells were then selected on tryptophan-deficient medium (purchased from Beijing Coolabor Biotechnology Co., Ltd.). Positive bacteria were selected and screened on tryptophan and histidine-deficient medium (purchased from Beijing Coolabor Biotechnology Co., Ltd.). Subsequently, the positive bacteria were subjected to X-α-gal colorimetric reaction. (4) The results showed that the engineered bacteria transformed with pNC-GBKT7-CeABI3BF, pNC-GBKT7-CeABI3BN1, pNC-GBKT7-CeABI3BN2, and pNC-GBKT7-CeABI3BTAD could survive on tryptophan- and histidine-deficient media and showed a blue color, while yeast cells transformed with empty vector and pNC-GBKT7-CeABI3BC failed to show β-galactosidase activity. This indicates that the CeABI3B protein has transcriptional activation function, and its activation domain is located upstream of the B3 domain, i.e., positions 15-142 of the protein, corresponding to positions 45-426 of the nucleotide sequence (…). Figure 5 ).

[0032] Example 4 CeABI3B Identification of gene regulation of lipids in tobacco (1) For identification CeABI3B The function of the gene in the regulation of plant oil was investigated by using the PCR product diluted 100 times in Example 1 as a template and CeABI3BF5 (SEQ ID NO.11) and CeABI3BR5 (SEQ ID NO.12) as primers for PCR amplification. After the target fragment was excised and recovered from the gel, it was cloned into the plant overexpression vector pCAMBIA1301 using homologous recombination to construct the recombinant vector pCAMBIA1301-CeABI3B. (2) The recombinant vector pCAMBIA1301-CeABI3B and the empty vector pCAMBIA1301 were transformed into Agrobacterium strain GV3101 (containing pSoup-P19), and the above-mentioned Agrobacterium engineered strain containing the empty vector and the plant overexpression vector pCAMBIA1301-CeABI3B was transformed into 4-week-old tobacco leaves by micro-injection. Samples were collected 1 day, 3 days and 5 days after transformation for oil content determination. (3) Oil content determination results based on Agilent 7890A (HP-FFAP, 30m×0.25mm ID, 0.25μm, Santa Clara, CA, USA) showed that, compared with the control group transformed with empty vector, the TAG content in the experimental group increased by 2.0 times, 2.5 times, and 3.5 times after 1 day, 3 days, and 5 days of transformation, respectively. Figure 6 ).

[0033] Example 5 CeABI3B Application of genes in the regulation of lipids in Arabidopsis seeds and vegetative tissues (1) For evaluation CeABI3B To explore the potential of genes in regulating plant lipids, the empty vector and the plant overexpression vector pCAMBIA1301-CeABI3B constructed in Example 4 were transformed into Agrobacterium strain GV3101 (containing pSoup-P19), and wild-type Arabidopsis thaliana was transformed using the inflorescence immersion method. The specific steps are as follows: (1-1) Positive bacteria were cultured overnight in LB liquid containing 50 mg / L kanamycin and 100 mg / L rifampin until OD reached. 600 The bacterial cells were collected by centrifugation at 12,000 rpm for 5 minutes with a concentration of 0.6. (1-2) Resuspend in staining buffer (1 / 2 MS solution containing 5% sucrose and 0.02% Silwet-77) and adjust to OD. 600 It is 0.8; (1-3) Place the Arabidopsis thaliana upside down in the infusion solution and soak for half a minute. Try to avoid getting the leaves in the infusion solution. You can use a pipette to draw the solution and then drip it onto the inflorescence. (1-4) After the inoculation is completed, spray a small amount of water and cover the inflorescence with a black plastic bag for dark culture. After 1 day, remove the plastic bag and culture normally. (1-5) To improve the efficiency of the infiltration, the infiltration can be repeated once or twice (once a week), and the seeds can be harvested under the same conditions.

[0034] (1-6) T1 seeds were screened for resistance on MS solid plate medium containing 30 µg / mL hygromycin to obtain T2 generation positive transgenic plants. After they matured, they were screened for resistance again to obtain stable T3 generation positive transgenic plants.

[0035] (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, compared with the control group (WT) containing the empty vector, CeABI3B The accumulation of TAG in overexpressed seeds was significantly increased, by approximately 20.8%. Figure 7 (A in the middle) (3) For evaluation CeABI3B To investigate the application value of oil regulation in plant vegetative tissues, leaves of the aforementioned T3 generation transgenic Arabidopsis thaliana were collected for oil content determination. The results showed that, compared with the control group (WT) containing empty vector, oil content was significantly higher. CeABI3B The TAG content in the overexpressing leaves increased by 2.47 times. Figure 7 (B in the middle).

[0036] (4) The above results indicate that, CeABI3B It has the function of promoting oil accumulation and shows its application prospect in increasing the oil content of seeds and nutrient tissues.

[0037] 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. A method for regulating oil content in Arabidopsis thaliana or tobacco. CeABI3B Genes, characterized by, The CeABI3B The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The claim 1 CeABI3B The CeABI3B protein encoded by the gene is characterized by, The amino acid sequence of the CeABI3B protein is shown in SEQ ID NO.

2.

3. Containing the contents of claim 1 CeABI3B Gene expression vectors, characterized in that, The expression vector includes one or more of the following: cell localization vector pNC-Cam1304-CeABI3B, yeast hybridization vector, and plant overexpression vector pCAMBIA1301-CeABI3B; The yeast hybridization vector is one or more of the following: pNC-GBKT7-CeABI3BF, pNC-GBKT7-CeABI3BN1, pNC-GBKT7-CeABI3BN2, pNC-GBKT7-CeABI3BC, and pNC-GBKT7-CeABI3BTAD.

4. The expression vector according to claim 3, characterized in that, pNC-Cam1304-CeABI3B was constructed using primers CeABI3BF2 and CeABI3BR2; Alternatively, pCAMBIA1301-CeABI3B can be constructed using primers CeABI3BF5 and CeABI3BR5; pNC-GBKT7-CeABI3BF was constructed using primers CeABI3BF2 and CeABI3BR2; pNC-GBKT7-CeABI3BN1 was constructed using primers CeABI3BF2 and CeABI3BR3; pNC-GBKT7-CeABI3BN2 was constructed using primers CeABI3BF2 and CeABI3BR4; pNC-GBKT7-CeABI3BC was constructed using primers CeABI3BF3 and CeABI3BR2; pNC-GBKT7-CeABI3BTAD was constructed using primers CeABI3BF4 and CeABI3BR3; The nucleotide sequence of CeABI3BF2 is shown in SEQ ID NO.5; The nucleotide sequence of CeABI3BR2 is shown in SEQ ID NO.6; The nucleotide sequence of CeABI3BF3 is shown in SEQ ID NO.7; The nucleotide sequence of CeABI3BR3 is shown in SEQ ID NO.8; The nucleotide sequence of CeABI3BF4 is shown in SEQ ID NO.9; The nucleotide sequence of CeABI3BR4 is shown in SEQ ID NO.10; The nucleotide sequence of CeABI3BF5 is shown in SEQ ID NO.11; The nucleotide sequence of CeABI3BR5 is shown in SEQ ID NO.

12.

5. For amplifying the device described in claim 1 CeABI3B A primer pair for a gene, characterized in that, The primer pair includes CeABI3BF1 and CeABI3BR1; The nucleotide sequence of CeABI3BF1 is shown in SEQ ID NO.3; The nucleotide sequence of CeABI3BR1 is shown in SEQ ID NO.

4.

6. The claim 1 CeABI3B The application of the gene, the CeABI3B protein of claim 2, the expression vector of claim 3 or 4, or the primer pair of claim 5 in the regulation of Arabidopsis thaliana or tobacco oil; The oil regulation includes: By increasing the content of Arabidopsis thaliana or tobacco CeABI3B The level of gene expression promotes the accumulation of oil in seeds or plant tissues of Arabidopsis or tobacco, thereby increasing the oil content.

7. The application according to claim 6, characterized in that, The plant tissues include one or more of the following: leaves, stems, tubers, and rhizomes.

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 CeABI3B Genes are introduced into target plants to increase the oil content or promote oil accumulation in the target plants. The target plant is one or more of Arabidopsis thaliana and tobacco.

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