Use of BnFAE1new gene in improving production of erucic acid in plants

Abstract

This invention discloses BnFAE1new The application of genes in increasing erucic acid yield in plants belongs to the fields of plant genetic engineering and oilseed crop genetic breeding technology. This invention, while maintaining the complete integrity of the BnFAE1 protein amino acid sequence, modifies the nucleotide sequence of its encoding gene through synonymous codon substitution, resulting in the sequence shown in SEQ ID NO. 1. BnFAE1new Genes. Experimental results show that this invention successfully avoids natural [problems] through nucleotide synonym substitution. FAE1 Heterologous overexpression of this gene can easily lead to gene co-suppression. Overexpression of this gene in Arabidopsis thaliana increased erucic acid accumulation in transgenic Arabidopsis seeds from 2% to over 20%, an increase of more than 10 times. The breeding method provided by this invention is simple to operate, requires no gene editing or mutation, has a short breeding cycle, and can be directly applied to the breeding of high erucic acid varieties, demonstrating significant economic and application value.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and oilseed crop genetic breeding technology, and particularly to... BnFAE1new Application of genes in increasing erucic acid production in plants. Background Technology

[0002] Erucic acid is an important long-chain fatty acid found in the seeds of cruciferous oil crops. It is widely used in high-end lubricants, chemical industry, pharmaceutical intermediates and other fields, and has extremely high industrial application value. FAE1 The gene is a key rate-limiting gene that controls the biosynthesis of erucic acid in plants, and overexpression of this gene is the core pathway to increase the erucic acid content in plant seeds.

[0003] Existing conventional techniques can directly clone natural high-erucic acid rapeseed. FAE1 The gene was heterologously overexpressed in plants such as Arabidopsis thaliana and rapeseed. However, studies have confirmed that the natural gene... FAE1 When the gene is overexpressed in wild-type Arabidopsis thaliana Col-0, it strongly induces gene co-repression, causing simultaneous silencing of the exogenous gene and endogenous homologous genes, resulting in a significant decrease in gene expression levels and an inability to increase erucic acid content in seeds. Current technologies have the following drawbacks: 1. Natural... FAE1 1. Heterologous gene overexpression is prone to co-inhibition, which cannot effectively increase erucic acid content; 2. Existing methods to solve gene silencing mostly involve mutating gene sequences, replacing strong promoters, and RNA interference, which can easily change the protein amino acid sequence and reduce enzyme activity, resulting in poor modification effects; 3. There is no mature technical solution in this field that can break co-inhibition and significantly increase erucic acid accumulation by synonymous codon base substitution without changing the amino acid sequence of FAE1 protein.

[0004] Therefore, there is an urgent need to provide a method to overcome gene co-suppression and achieve... FAE1 Methods for efficient and stable gene expression in plants. Summary of the Invention

[0005] The purpose of this invention is to provide BnFAE1new The application of genes in increasing erucic acid yield in plants addresses the problems existing in the above-mentioned technologies. The breeding method provided by this invention is simple to operate, requires no gene editing or mutation, has a short breeding cycle, and can be directly applied to the breeding of high erucic acid varieties, with outstanding economic and application value.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides BnFAE1new The application of genes in any of the following: (1) Application in regulating plant erucic acid production; (2) Application in the cultivation of transgenic plants with increased erucic acid yield; (3) Application in the preparation of products that increase the yield of erucic acid in plants; The BnFAE1new The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, the aforementioned [condition] was upregulated in plants. BnFAE1new The expression level of the gene increases the erucic acid production of the plant.

[0008] Furthermore, the plant in question is Arabidopsis thaliana.

[0009] The present invention also provides an application of a recombinant vector, the recombinant vector comprising: BnFAE1new Gene; The BnFAE1new The nucleotide sequence of the gene is shown in SEQ ID NO.1; The application is any one of the following: (1) Application in regulating plant erucic acid production; (2) Application in the cultivation of transgenic plants with increased erucic acid yield; (3) Application in the preparation of products that increase the yield of erucic acid in plants.

[0010] The present invention also provides the use of engineered bacteria comprising the above-described recombinant vector in any of the following: (1) Application in regulating plant erucic acid production; (2) Application in the cultivation of transgenic plants with increased erucic acid yield; (3) Application in the preparation of products that increase the yield of erucic acid in plants.

[0011] The present invention also provides a method for increasing erucic acid production in plants, comprising upregulating erucic acid in plants. BnFAE1new The steps to increase the erucic acid yield of the plant by adjusting the gene expression level; The BnFAE1new The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0012] Furthermore, the aforementioned upward adjustment BnFAE1new Methods for assessing gene expression levels, including overexpression of the gene in plants. BnFAE1new Gene.

[0013] Furthermore, the plant in question is Arabidopsis thaliana.

[0014] This invention also provides a breeding method for plants with increased erucic acid yield, comprising the following steps: Overexpression in plant cells BnFAE1new Genes are then used to cultivate plant cells, and the plant cells are used to regenerate plants, resulting in plants with increased erucic acid production. The BnFAE1new The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] Furthermore, the plant in question is Arabidopsis thaliana.

[0016] The present invention discloses the following technical effects: In response to the existing technology of natural FAE1 Heterologous overexpression of genes is prone to co-inhibition and poor erucic acid production enhancement. Furthermore, traditional gene modification methods can easily alter protein structure and affect enzyme activity. This invention addresses these issues by modifying the nucleotide sequence of the encoding gene of the BnFAE1 protein through codon synonym substitution while maintaining the amino acid sequence completely unchanged, resulting in the protein shown in SEQ ID NO. 1. BnFAE1new Genes. Experimental results show that this invention successfully avoids natural genes through nucleotide synonym substitution. FAE1 Heterologous overexpression of this gene is prone to gene co-suppression; however, overexpression of this gene in Arabidopsis thaliana increased erucic acid accumulation in transgenic Arabidopsis seeds from 2% to over 20%, an increase of more than 10 times. The breeding method provided by this invention is simple to operate, requires no gene editing or mutation, has a short breeding cycle, and can be directly applied to the breeding of high erucic acid varieties, demonstrating significant economic and application value. The gene modification approach of this invention can be extended to the heterologous expression of other ultra-long chain fatty acid synthesis genes, exhibiting strong versatility. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Natural BnFAE1 Genes and artificially synthesized BnFAE1new Gene sequence alignment diagram; Figure 2 For the transfer BnFAE1new A statistical diagram of fatty acid composition in Arabidopsis thaliana and wild-type Arabidopsis thaliana; where 16:0 is palmitic acid; 18:0 is stearic acid; 18:1 is oleic acid; 18:2 is linoleic acid; 18:3 is linolenic acid; 20:0 is arachidic acid; 20:1 is 11-eicosadecanoic acid; 20:2 is eicosadecanodienoic acid; and 22:1 is erucic acid. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The technical solution of this invention is summarized as follows: 1. Core Principles Utilizing the degeneracy of biological codons, the same amino acid can be encoded by multiple different codons. This invention does not alter the amino acid sequence of the FAE1 protein, but only optimizes the codons and substitutes bases in the gene coding region, changing the gene mRNA sequence characteristics, eliminating post-transcriptional gene silencing caused by homologous sequences, thereby breaking co-repression, ensuring efficient gene expression, and continuously catalyzing erucic acid synthesis.

[0025] 2. Specific operating steps 1) First, clone natural rapeseed from high-erucic acid rapeseed. BnFAE1 Complete gene coding sequence; 2) Based on the host plant's codon usage preferences, [the following is done]: BnFAE1Synonymous codon substitutions were performed on the entire coding region of the gene, changing only the DNA bases while preserving the complete amino acid sequence, resulting in the nucleotide sequence shown in SEQ ID NO.1. BnFAE1new Gene; SEQ ID NO.1:

[0026] 3) The artificially modified BnFAE1new Gene construction of plant overexpression vectors; 4) Introduce the modified gene into the target plant through Agrobacterium-mediated transformation, gene gun method or pollen tube pathway method; 5) Stable genetically derived homozygous transgenic lines were obtained through resistance screening and self-pollination. 6) Gas chromatography was used to detect the erucic acid content in seeds, and superior strains with an erucic acid content of more than 20% were screened.

[0027] 3. Alternative technical solutions 1) Targeted optimization can be performed based on the codon preferences of different crops such as rapeseed, soybeans, and sea cabbage. BnFAE1 Gene sequences adapted to various oilseed crops; 2) Genetic transformation methods can be interchangeably used, including Agrobacterium infection, gene gun, and pollen tube pathway methods, to suit different recipient varieties.

[0028] Example 1 BnFAE1 Cloning of genes 1. Preparation of plant materials Select seeds from high-erucic acid rapeseed that have formed 22-26 days after flowering (25 days being optimal). After removing the embryos, flash-freeze them in liquid nitrogen and store them in an ultra-low temperature freezer (-80℃) for later use.

[0029] The Arabidopsis thaliana used in this invention is the wild-type Col-0. After being synchronized by placing the Arabidopsis seeds at 4℃ for 2-4 days, they were cultured in an artificially controlled culture chamber under the following conditions: temperature 22℃, photoperiod of 16 hours light / 8 hours dark, and light intensity of 100-130 μE·m. -2 ·S -1 .

[0030] 2. Extraction of total RNA from rapeseed embryos Total RNA was extracted from rapeseed embryos using the LiCl-PVP method to remove polysaccharides and polyphenols.

[0031] 3. RNA reverse transcription synthesizes first-strand cDNA Reverse transcription was performed using the Takara kit (Code NO. RR047A).

[0032] 4. Primer design and gene cloning Based on the transcriptome results, BnFAE1 primers were designed, and the upstream and downstream primer sequences are as follows: BnFAE1-F: 5'-ATGACGTCCGTTAACGTAAGCTCCTT-3', SEQ ID NO.2; BnFAE1-R: 5'-TTAGGACCGACCGTTTTGGACA-3', SEQ ID NO. 3.

[0033] Using the cDNA obtained from reverse transcription as a template, and BnFAE1-F and BnFAE1-R as primers, the natural cDNA was obtained by PCR. BnFAE1 Full-length gene. Obtained through sequencing. BnFAE1 The full-length sequence was analyzed using bioinformatics software based on the host plant's codon usage preferences. BnFAE1 Synonymous codon substitutions were performed on the entire coding region of the gene, meaning only the DNA bases were changed while the complete amino acid sequence remained unchanged, resulting in the nucleotide sequence shown in SEQ ID NO.1. BnFAE1new Genes, sequence pairs, for example Figure 1 As shown.

[0034] 5. Construction of expression vectors The cloned above BnFAE1new The gene was ligated into the cloning vector pMD19-T (Takara), transformed, and positive clones were screened and sequenced. The gene was then ligated into the Gateway entry vector pGW-MCS using double digestion with restriction endonucleases BamHI and PstI, resulting in pGW-MCS+. BnFAE1new .

[0035] The GFP expression cassette on pK7WG2D was excised using the restriction endonuclease HindIII, treated with dephosphorylase, and ligated with the DsRED expression cassette using the T4 ligase reaction. The GFP expression cassette and 35S promoter on pK7WG2D were then excised using HindIII and SpeI, and ligated with the Phaseolin promoter using the T4 ligase reaction, resulting in pK7WG2D-Pha. pK7WG2D-Pha was then digested with HindIII, treated with dephosphorylase, and ligated with the DsRED expression cassette using the T4 ligase reaction. The constructed vector was named pK7WG2D-Pha-DsRED.

[0036] Using LR enzyme, pGW-MCS+ BnFAE1new After undergoing an LR reaction with the aforementioned pK7WG2D-Pha-DsRED and being screened for Spec resistance, the resulting plant expression vector was named pK7WG2D-Pha-DsRED+. BnFAE1new .

[0037] Example 2 Agrobacterium-mediated rapeseed BnFAE1new Transformation of genes in Arabidopsis thaliana and screening of transgenic lines 1. Construction of recombinant bacteria The plant expression vector pK7WG2D-Pha-DsRED+ prepared in Example 1 was processed using a freeze-thaw method. BnFAE1new Transformed into Agrobacterium GV3101 strain.

[0038] 2. Genetic transformation in Arabidopsis thaliana Genetic transformation of Arabidopsis thaliana was carried out using the inflorescence dipping method.

[0039] 3. Obtaining and homozygous transgenic Arabidopsis plants The transformed Arabidopsis T0 generation seeds were collected in 1.5 mL centrifuge tubes and emitted red fluorescence under green light (excitation wavelength 543 nm). Through a red filter, the red fluorescence emitted by the positively transformed seeds was visible to the naked eye, compared to the untransformed wild-type plants (Col-0). Based on this, T1 generation transgenic plants were selected and transferred to pots containing vermiculite, perlite, and peat moss as a substrate, allowing them to continue growing in a culture chamber until the seeds were harvested.

[0040] T1 generation seeds were germinated to become T1 generation transgenic plants. These plants were then transferred to pots containing vermiculite, perlite, and peat moss as a substrate and allowed to continue growing in a culture room until harvest, becoming T2 generation seeds. T2 generation seeds obtained from different transgenic lines were numbered and named line1, line2, etc., until all lines were labeled. All lines were randomly obtained independent transgenic lines.

[0041] Example 3: Fatty acid composition analysis of seeds from transgenic Arabidopsis thaliana lines Mature Arabidopsis seeds (including wild-type Col-0, T2 generation seeds obtained in Example 2, and homozygous seeds harvested from T2 generation single plants) were air-dried under natural light until their weight remained unchanged. 10 mg of seeds were weighed and added to 4 mL of 1 M concentrated sulfuric acid methanol solution for extraction. The mixture was then placed in a water bath for fatty acid extraction and esterification. After cooling to room temperature, 2 mL of 0.9 g / mL NaCl was added to terminate the reaction. Then, 2 mL of n-hexane was added, and the mixture was shaken and centrifuged at 2300 rpm for 3 min to extract fatty acid methyl esters. The upper organic phase was pipetted into a GC vial rinsed with n-hexane for gas chromatography analysis of the fatty acid components. The analytical method was based on (Ma...). et al ., Plant Direct, 2020, 4(8):e00253.).

[0042] like Figure 2 The fatty acid composition analysis results showed that: BnFAE1new The erucic acid content in seeds of genetically homozygous lines remained stable between 20.2% and 23.5%; while that of naturally occurring lines... FAE1In the control group of the gene, due to the co-inhibition effect, the erucic acid content was maintained at only 2.1%-2.8%, with no significant yield increase.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A kind BnFAE1new The application of genes in any of the following: (1) Application in regulating plant erucic acid production; (2) Application in the cultivation of transgenic plants with increased erucic acid yield; (3) Application in the preparation of products that increase the yield of erucic acid in plants; The BnFAE1new The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, Upregulation in plants BnFAE1new The expression level of the gene increases the erucic acid production of the plant.

3. The application as described in claim 1 or 2, characterized in that, The plant in question is Arabidopsis thaliana.

4. An application of a recombinant vector, characterized in that, The recombinant vector includes BnFAE1new Gene; The BnFAE1new The nucleotide sequence of the gene is shown in SEQ ID NO.1; The application is any one of the following: (1) Application in regulating plant erucic acid production; (2) Application in the cultivation of transgenic plants with increased erucic acid yield; (3) Application in the preparation of products that increase the yield of erucic acid in plants.

5. The use of an engineered bacterium comprising the recombinant vector of claim 4 in any of the following: (1) Application in regulating plant erucic acid production; (2) Application in the cultivation of transgenic plants with increased erucic acid yield; (3) Application in the preparation of products that increase the yield of erucic acid in plants.

6. A method for increasing erucic acid yield in plants, characterized in that, Including upregulation in plants BnFAE1new The steps to increase the erucic acid yield of the plant by adjusting the gene expression level; The BnFAE1new The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The method as described in claim 6, characterized in that, The upward adjustment BnFAE1new Methods for assessing gene expression levels, including overexpression of the gene in plants. BnFAE1new Gene.

8. The method as described in claim 6 or 7, characterized in that, The plant in question is Arabidopsis thaliana.

9. A breeding method for a plant with increased erucic acid yield, characterized in that, Includes the following steps: Overexpression in plant cells BnFAE1new Genes are then used to cultivate plant cells, and the plant cells are used to regenerate plants, resulting in plants with increased erucic acid production. The BnFAE1new The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

10. The breeding method as described in claim 9, characterized in that, The plant in question is Arabidopsis thaliana.

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