Application of mutation transcription factor AHL14 gene in regulation and control of oil content of plant seeds and method for improving oil content of plant seeds
Through the overexpression and transformation technology of the mutant transcription factor AHL14 gene, the problem of failure to effectively increase the oil content of plant seeds in the prior art is solved, and the effect of significantly increasing the total oil yield of seeds is achieved, while maintaining the advantages of seed size and 1,000 grain weight.
Patent Information
- Application Number
- CN202510342121.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-21
- Publication Date
- 2025-06-10
AI Technical Summary
The influence of the AHL14 gene on the oil content in plant seeds has not been disclosed in the prior art, making it difficult to effectively increase the oil content of plant seeds.
Through the application of the mutant transcription factor AHL14 gene, the mutant transcription factor AHL14 was overexpressed, and homozygous mutants ahl14-1 and ahl14-2 were identified by PCR and RT-qPCR. The recombinant plasmid was further transformed into the host plant through Agrobacterium-mediated flower leaching method, increasing the oil content of plant seeds.
The total oil production of plant seeds has been significantly improved, while the seed size and 100-grain weight have not been reduced or decreased compared with wild type, providing new and excellent genetic resources and providing good reference value and application prospects for high-oil breeding of oil crops.
Smart Images
Figure CN120118947A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant genetic engineering, and specifically relates to the application of the mutant transcription factor AHL14 gene in regulating the oil content of plant seeds and a method for increasing the oil content of plant seeds. Background Art
[0002] Vegetable oil is the main source of human edible oil and an important chemical raw material, which can be used to manufacture pharmaceuticals, cosmetics, etc. The main chemical component of vegetable oil is triacylglycerol, which is formed by the esterification of 1 molecule of glycerol and 3 molecules of fatty acid chains. As an important source of human dietary lipids and chemical raw materials, vegetable oil contains 5 common fatty acid components, namely saturated fatty acids: palmitic acid (palmitic acid, C16:0) and stearic acid (C18:0), and unsaturated fatty acids: oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Among them, appropriate intake of palmitic acid can help reduce cholesterol levels in the blood and reduce the risk of cardiovascular diseases. Palmitic acid also has certain antioxidant effects, can scavenge free radicals, reduce oxidative damage, and thus protect the skin and cells. Stearic acid is stable at high temperatures and is commonly used in the production of soaps, cosmetics, industrial lubricants, etc. Polyunsaturated fatty acids (linoleic acid and linolenic acid) are important sources of essential fatty acids in humans and some animals, and have the effects of reducing cholesterol, softening blood vessels to prevent arteriosclerosis, and inhibiting body aging. Linolenic acid can be further converted into eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the human body. DHA is also known as "brain gold" and plays an important role in protecting eyesight, preventing cancer and cardiovascular diseases. Therefore, comprehensively increasing the total fat content in plant seeds has important health value and economic value.
[0003] Rapeseed (Brassica napus) is one of the most important oil crops in the world and the largest source of domestic edible vegetable oil in China. The high-quality edible oil provided annually accounts for more than 50% of the oil production of domestic oil crops; at the same time, rapeseed provides about 8 million tons of high-protein rapeseed meal for the livestock and poultry breeding industry in China every year. The research on the regulation mechanism of rapeseed seed oil accumulation has always been an important research topic in the rapeseed field. Arabidopsis thaliana and rapeseed belong to the same family of Brassicaceae plants and are ideal model materials for studying the molecular mechanism of rapeseed seed oil accumulation and its regulatory network. AHL14 belongs to the AT-HOOK MOTIF NUCLEAR LOCALIZED (AHL) transcription factor family, and this family has been reported to be involved in regulating multiple growth and development processes of plants.
[0004] The influence of the AHL14 gene on the oil content in plant seeds is not disclosed in the prior art. Summary of the Invention
[0005] In view of the above technical problems, the present invention for the first time discovers the application of the mutated transcription factor AHL14 gene in regulating the oil content of plant seeds, and provides a method for increasing the oil content of plant seeds.
[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] The first technical solution protected by the present invention is: the application of the mutated transcription factor AHL14 gene in regulating the oil content of plant seeds, and the nucleotide sequence of the mutated transcription factor AHL14 is as shown in SEQ ID NO.1.
[0008] The second technical solution protected by the present invention is: the application of the mutated transcription factor AHL14 gene in cultivating transgenic plants with high seed oil content, and the nucleotide sequence of the mutated transcription factor AHL14 is as shown in SEQ ID NO.1.
[0009] The third technical solution protected by the present invention is: the application of a preparation for promoting the expression of the mutated transcription factor AHL14 gene in regulating the oil content of plant seeds or in cultivating transgenic plants with high seed oil content, and the preparation is a plasmid containing the mutated transcription factor AHL14 gene as shown in the sequence SEQ ID NO.1.
[0010] Another object of the present invention is to provide a method for increasing the oil content of plant seeds.
[0011] A method for increasing the oil content of plant seeds, which includes overexpressing the mutated transcription factor AHL14 in a plant, and the nucleotide sequence of the mutated transcription factor AHL14 is as shown in SEQ ID NO.1.
[0012] Furthermore, it includes amplifying a target fragment from a host plant DNA template using the primer sequences shown in SEQ ID NO.2 and SEQ ID NO.3, then digesting and ligating the target fragment into the pHY105 vector, and thereafter transforming the recombinant plasmid into the host plant by the agrobacterium flower dipping method.
[0013] Furthermore, the host plant is Arabidopsis thaliana or Brassica napus.
[0014] Specifically, the nucleotide sequence of the mutated transcription factor AHL14 is as shown in SEQ ID NO.1:
[0015]
[0016] The primer sequence shown in SEQ ID NO.2: GTCGACGGTATCGATaagcttGGAGTCAGAATATTTTATTTTTGTCAATTTTG.
[0017] The primer sequence shown in SEQ ID NO.3: TCCCCCGGGCTGCAGgaattcATCCGGTATTTGCTGCTCGTAAT.
[0018] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:
[0019] The present invention provides the application of the mutant transcription factor AHL14 gene in regulating the oil content of plant seeds and a method for increasing the oil content of plant seeds. Through the identification of two T-DNA insertion mutants of the AHL14 transcription factor at the DNA and RNA levels, the homozygous mutants ahl14-1 and ahl14-2 were obtained. Phenotypic analysis showed that compared with the wild type Col-0, the total oil yield of the seeds of the ahl14-1 and ahl14-2 mutant plants was significantly increased, and at the same time, the seed size and 1000-grain weight of the mutant plants were not less than those of the wild type Col-0. The present invention discloses that the AHL14 gene provides a new excellent gene resource for the molecular design breeding of oil crops, and has good reference value and application prospects in the high-oil breeding of oil crops. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 It is a data graph of the expression pattern of the AHL14 gene in Arabidopsis thaliana.
[0021] Figure 2 It is a data graph of the identification of the AHL14 mutant and the complementation line.
[0022] Figure 3 It is a data graph of the phenotypic analysis of the seeds of the AHL14 mutant and the complementation line. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0023] The present invention will be described in detail below with reference to the drawings.
[0024] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0025] Embodiment
[0026] The solution provided in this example is a method and application for increasing the oil content of seeds based on the Arabidopsis transcription factor AHL14. In this invention, two homozygous T-DNA insertion mutants, ahl14-1 and ahl14-2, were identified based on PCR and RT-qPCR; the full-length gDNA sequence of AHL14 was constructed into the complementation vector pHY105 and transferred into the homozygous mutant ahl14-2 by the Agrobacterium-mediated floral dip method; through glufosinate resistance screening, T1 generation positive transgenic plants were obtained; further, positive plants were identified at the DNA and RNA levels using resistance screening and specific primers, and finally, the T3 generation homozygous transgenic complementation line ahl14-2 ProAHL14:AHL14–3HA (#1-#3) was obtained.
[0027] The total fatty acid content, 1000-seed weight, and size of the seeds of wild-type Col-0, homozygous mutants ahl14-1 and ahl14-2, and the complementation line ahl14-2 ProAHL14:AHL14–3HA (#1-#3) were measured. The results showed that compared with Col-0, the oil content of the mutant seeds increased significantly, while the 1000-seed weight and size of the seeds did not decrease or become smaller. The total fatty acid content of the complementation line could be restored to the wild-type level. Therefore, we found that the mutation of AHL14 could significantly increase the oil content of seeds.
[0028] Specifically as follows:
[0029] 1. Sequence acquisition and mutant identification of the Arabidopsis AHL14 gene
[0030] 1.1 Sequence acquisition and expression analysis of the Arabidopsis AHL14 gene:
[0031] The nucleic acid sequence of AHL14 (AT3G04590) was searched and obtained using the TAIR database (https: / / www.arabidopsis.org);
[0032] Specific primers (AHL14-qPCR-F / AHL14-qPCR-R) for RT-qPCR were designed using the coding sequence (CDS) as a template, and the CDS sequence was constructed into the pGreen-35S–GFP vector. 35S-F / AHL14-GFP-R was used as the identification primer for transient expression in Nicotiana benthamiana leaves for subcellular localization analysis. The full-length promoter sequence of 1350 base pairs upstream of its start codon was cloned using specific primers (AHL14-GUS-F / AHL14-GUS-R) and constructed into the pGreen–HY107 vector. HY107-F / AHL14-GUS-R was used as the identification primer, and then it was stably transformed into Arabidopsis wild-type Col-0 plants by the Agrobacterium-mediated floral dip method. GUS staining analysis was performed on the obtained positive plants to determine its expression status.
[0033] 1.2 Identification of T-DNA insertion mutants of Arabidopsis AHL14 gene
[0034] Seeds of two T-DNA insertion mutants ahl14-1 (SALK_093612C) and ahl14-2 (SALK_081411C) were obtained from the Arabidopsis mutant library Arashare (https: / / www.arashare.cn / index). Arabidopsis seeds were placed in a 1.5 mL sterile centrifuge tube and sterilized with 75% (v / v) alcohol three times for 1 min each time in a laminar flow hood; then washed three times with sterilized ddH 2 O. The sterilized seeds were spotted on MS solid medium and placed in the dark at 4 °C for 3 days for synchronization. Then they were placed in an artificial climate chamber for normal light cultivation. After about 10 days, the seedlings with consistent growth were transplanted into pots containing nutrient soil (nutrient soil: vermiculite = 3:1) and placed in an artificial climate chamber for normal growth. Genomic DNA (gDNA) was extracted from the leaves of Col-0 and mutant plants at the seedling stage for subsequent identification.
[0035] Use the online tool T-DNA Primer Design (http: / / signal.salk.edu / tdnaprimers.2.html) to obtain the specific left primer (Left Primer, LP), right primer (Right Primer, RP), and vector primer LBb1.3 (BP) for mutant identification. Use Col-0, ahl14-1, and ahl14-2 gDNA as templates for PCR. Each mutant identification is divided into two independent PCR combinations: LP+RP and BP+RP. The length of the PCR amplification product of the LP+RP combination is approximately 900 bp, and the length of the amplification product of the BP+RP combination is 400 - 700 bp. Wild-type, heterozygous mutants, and homozygous mutants can be distinguished according to the length of the PCR amplification product. In each PCR combination during material identification, if the LP+RP combination can amplify a band (approximately 900 bp), while the BP+RP combination cannot amplify any band, it is a wild-type; if both the LP+RP combination (approximately 900 bp) and the BP+RP combination (400 - 700 bp) can amplify bands, it is a heterozygous mutant; when only the BP+RP combination (approximately 400 - 700 bp) amplifies a band, it indicates that the T-DNA insertion mutant is a homozygous mutant.
[0036] The primers used are shown in Table 1:
[0037] Table 1 Primer information for AHL14 RT-qPCR, mutant identification, and vector construction
[0038]
[0039]
[0040] Among them, the expression pattern of the AHL14 gene in Arabidopsis thaliana is as Figure 1 shown, where Figure 1 A in Figure 1 is the expression level of AHL14 in roots, stems, rosette leaves, cauline leaves, flower buds, and open flowers of Arabidopsis thaliana analyzed by RT-qPCR. EF1αA4 is used as an internal reference gene, and the measured values of each index are the mean ± standard deviation (n = 3); Figure 1 B in Figure 1In D, GUS staining was used to analyze the expression of AHL14 in developing seeds at 12 and 14 days after pollination. Scale bar = 200 μm; Figure 1 In E, in situ hybridization was used to analyze the transcriptional level of AHL14 in Arabidopsis thaliana embryos at 12 days after pollination. Scale bar = 50 μm.
[0041] 2. Construction of the complementation vector of Arabidopsis thaliana AHL14 gene and identification of complementation lines
[0042] 2.1 Construction of the complementation vector of Arabidopsis thaliana AHL14 gene
[0043] Specific primers (AHL14-res-F / AHL14-res-R) were designed according to the gDNA reference sequence of the AHL14 gene to amplify its full-length sequence (3680 bp), and the cloned gDNA sequence was constructed into the pHY105 vector. HY105-F / AHL14-res-R was used as the identification primer.
[0044] The nucleotide sequence of the mutant transcription factor AHL14 is as shown in SEQ ID NO.1:
[0045]
[0046] 2.2 Transformation, Screening and Identification of the Complemented Lines of Arabidopsis thaliana AHL14 Gene
[0047] The complementation vector was introduced into the homozygous ahl14-2 mutant by the Agrobacterium-mediated floral dip method. The seeds of the transformed plants harvested were treated at 4°C for 3 days and evenly sown on nutrient soil. After 1 week of germination, basta (the main component is glufosinate ammonium) was sprayed on the leaves for resistance screening to obtain the T1 generation of positive transgenic plants. PCR was used to identify the presence of the target gene in the positive plants, and RT-qPCR was used to detect the transcriptional level of the target gene;
[0048] Subsequently, 10 T 1 -generation positive seedlings with relatively high transcriptional levels were continuously screened using MS solid medium containing 10 μg / mL glufosinate ammonium until it was identified that all the seedlings of a single plant in the T 2 -generation offspring had glufosinate ammonium resistance. At this time, the seeds harvested from the offspring of this single plant were the T 3 -generation homozygous seeds, which could be used for subsequent experiments.
[0049] The primers used are shown in Table 2:
[0050] Table 2 Primer Information for the Construction of AHL14 Complementation Vector
[0051]
[0052] All Arabidopsis thaliana materials in this study were planted as follows: Arabidopsis thaliana seeds were placed in a 1.5 mL sterile centrifuge tube and sterilized 3 times with 75% (v / v) alcohol for 1 min each time in a laminar flow hood; then washed 3 times with sterilized ddH 2 O. The sterilized seeds were spread on MS solid medium and placed in the dark at 4°C for 3 days for synchronization. Then they were placed in a programmable climate chamber for normal light cultivation. After about 10 days, the seedlings with consistent growth were transplanted into pots containing nutrient soil (nutrient soil: vermiculite = 3:1) and placed in the programmable climate chamber for normal growth. The plants were transferred back to the previous programmable climate chamber and continued to be cultivated until the seeds matured. The photoperiod of the programmable climate chamber was set to 16 h light / 8 h dark, the light intensity was set to 160 μmol·m -2 ·s -1 , and the day / night temperature was set to 22°C.
[0053] Specifically, the identification data of the AHL14 mutant and the complemented lines are as Figure 2 shown, where Figure 2 A is a schematic diagram of the AHL14 gene structure and the T-DNA insertion site. The gray squares represent the UTR regions, the black squares represent the CDS, and the black lines represent the introns; Figure 2In B, PCR combined with agarose gel electrophoresis was used to identify the ahl14-1 and ahl14-2 homozygous mutants; Figure 2 In C, PCR combined with agarose gel electrophoresis was used to identify the complemented positive lines after introducing the ProAHL14:AHL14–3HA vector in the ahl14-2 background; Figure 2 In D, RT-qPCR was used to analyze the expression levels of AHL14 in wild-type, mutant, and complemented lines.
[0054] 3. Statistics of seed fatty acid content and seed-related traits of each material
[0055] The mature seeds of wild-type Col-0, homozygous mutants ahl14-1 and ahl14-2, and complemented line ahl14-2ProAHL14:AHL14–3HA (#1-#3) were naturally dried until the weight remained unchanged. The seed fatty acid content was analyzed by gas chromatography (Shimadzu GC-2010plus) using the internal standard method (the internal standard was methyl heptadecanoate C17:0). The results showed that compared with wild-type Col-0, the total fatty acid content in the seeds of mutants ahl14-1 and ahl14-2 increased significantly, indicating that AHL14 negatively regulates seed fatty acid accumulation.
[0056] Furthermore, the related traits of mature seeds of wild-type Col-0, homozygous mutants ahl14-1 and ahl14-2, and complemented line ahl14-2ProAHL14:AHL14–3HA (#1-#3) were statistically analyzed. The results showed that compared with wild-type Col-0, the 1000-seed weight and size of the seeds of AHL14 homozygous mutants ahl14-1 and ahl14-2 were both reduced or decreased. It was shown that the AHL14 homozygous mutants ahl14-1 and ahl14-2 were two excellent materials with high oil content.
[0057] Specifically, as Figure 3 shown, among them, Figure 3 in A, the total fatty acid content in mature seeds of wild-type, mutant, and complemented lines, DW represents dry weight, and the measured values of each index are the mean ± SD (n = 5). The asterisk * indicates a significant difference in phenotype between the mutant and wild-type Col-0, and *** represents P < 0.001; Figure 3 in B, the content of each fatty acid component in mature seeds of wild-type, mutant, and complemented lines; Figure 3 in C, microscopic observation of the phenotypes of mature seeds of wild-type, mutant, and complemented lines, scale bar = 1 mm; Figure 3 in D, the 1000-seed weight of mature seeds of wild-type, mutant, and complemented lines; Figure 3In this study, E represents the mature seed size of wild type, mutants and complementation lines, expressed as length and width.
[0058] The present invention provides the application of the mutant transcription factor AHL14 gene in regulating the oil content of plant seeds and a method for increasing the oil content of plant seeds. Through the identification of two T-DNA insertion mutants of the AHL14 transcription factor at the DNA and RNA levels, the homozygous mutants ahl14-1 and ahl14-2 were obtained. Phenotypic analysis showed that compared with the wild type Col-0, the total oil yield of the seeds of the ahl14-1 and ahl14-2 mutant plants was significantly increased, and at the same time, the seed size and 1000-grain weight of the mutant plants were not less than those of the wild type Col-0. The present invention discloses that the AHL14 gene provides a new excellent gene resource for the molecular design breeding of oil crops, and has good reference value and application prospects in the high-oil breeding of oil crops.
[0059] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. Application of mutant transcription factor AHL14 gene in regulating oil content of plant seeds, characterized in that: The nucleotide sequence of the mutant transcription factor AHL14 is shown in SEQ ID NO.
1.
2. Application of mutant transcription factor AHL14 gene in cultivating transgenic plants with high seed oil content, characterized in that: The nucleotide sequence of the mutant transcription factor AHL14 is shown in SEQ ID NO.
1.
3. Use of a preparation for promoting the expression of mutant transcription factor AHL14 gene in regulating plant seed oil content or in cultivating transgenic plants with high seed oil content, characterized in that: The preparation is a plasmid containing the mutant transcription factor AHL14 gene as shown in the sequence SEQ ID NO.
1.
4. A method for increasing the oil content of plant seeds, characterized in that: The method comprises overexpressing a mutant transcription factor AHL14 in a plant, wherein the nucleotide sequence of the mutant transcription factor AHL14 is shown in SEQ ID NO.
1.
5. The method for increasing the oil content of plant seeds according to claim 4, characterized in that: The method comprises amplifying a target fragment in a host plant DNA template using primer sequences shown in SEQ ID NO.2 and SEQ ID NO.3, then enzymatically cutting the target fragment and connecting it to a pHY105 vector, and then transforming the recombinant plasmid into the host plant using the Agrobacterium floral dipping method.
6. The method for increasing the oil content of plant seeds according to claim 5, characterized in that: The host plant is Arabidopsis thaliana or rapeseed.