Rapeseed transcription factor BnFUS3 related to plant fatty acid and fat metabolism and coding gene and application thereof

Abstract

The invention discloses a rapeseed transcription factor BnFUS3 related to the plant fatty acid and fat metabolism and a coding gene and application thereof. The amino acid sequence of the rapeseed transcription factor BnFUS3 is represented as the SEQ ID NO.1, and the coding gene is represented as the SEQ ID NO. 2 or 3. The plant oil content can be increased after the protected gene BnFUS3 induced expression. According to transgenic inducing expression tests, by the aid of the gene, the plant oil content can be increased significantly, and a valid path for increasing the plant fatty acid content by the transgenic method is provided; important theoretical and practical significance is provided for increasing the plant (especially oil crops) fatty acid content and optimizing the related traits, and the application and market prospect is promised in the field of agriculture.

Description

technical field

[0001] The invention belongs to the field of genetic engineering, and relates to rapeseed transcription factor BnFUS3 related to plant fatty acid and oil metabolism, its encoding gene and application. Background technique

[0002] Fatty acids in plants are also known as vegetable oils, which are widely found in the seeds of plants. Vegetable oil is widely used, and the use of biotechnology to improve the composition and content of fatty acids in plants has important economic value. Among them, the operating gene selected by genetic engineering technology must be the key gene in the process of fatty acid biosynthesis in plants, but people still don't know much about the molecular regulation mechanism of plant fatty acid biosynthesis. Quantitative work is currently progressing slowly. Taking the dicot model plant Arabidopsis thaliana as an example, the embryogenesis of plants is mainly divided into two stages: early morphogenesis and late maturation. The ent...

Examples

Embodiment 1

[0055] Embodiment 1, the acquisition of BnFUS3 gene

[0056] Collect rapeseed seeds that have grown to 30 days, and extract 30-day-old rapeseed Zhongyou 821 seeds (Xie Zhucun. Characteristics and cultivation of Zhongyou 821 [J]. Guangxi Agricultural Science, 1991, (05): 213) total mRNA. The obtained mRNA was synthesized into cDNA by a reverse transcription kit (Takara Company, the specific operation was performed according to the kit instructions). The obtained cDNA was amplified with the following primer sequences to amplify the BnFUS3 gene cDNA sequence: (the underline is the restriction site, and the first three bases are the protection bases)

[0057] BnFUS3 F(XhoI):5'-CGA CTCGAG CACACACTGTTTCCACACTTCCT-3' (SEQ ID NO. 4);

[0058] BnFUS3 R(SpeI):5'-TTA ACTAGT TCACTGCATTTTTATATATCTTG-3' (SEQ ID NO. 5).

[0059]Under the guidance of PCR amplification, after the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and the target fragm...

Embodiment 2

[0060] Embodiment 2, the construction of BnFUS3 gene induction expression vector

[0061] Select the positive clone with the correct sequence of pGEM-BnFUS3 in step 1, prepare a large amount of plasmid DNA, use XhoI / SpeI double enzymes purchased from NEB Company to obtain the BnFUS gene fragment, and recover BnFUS3 with a gel recovery kit (purchased from Dingguo Company) Gene fragments, fragments after purification were used T4 ligase (Roche Company) and the XVE-induced expression vector (abbreviated as XVE) (Gateway Company, http: / / www.gateway .com / worldwide / ) to form an inducible expression vector for XVE::BnFUS3. Transform Escherichia coli (E.coli) DH5α strain by XVE::BnFUS3 heat shock method, select positive colonies and add them to 5ml LB liquid medium containing 50mg / L kanamycin, culture at 37℃, 200rpm for 12-16 hours, extract Plasmids were identified by PCR and enzyme digestion. For the construction diagram of the expression vector, see figure 2 .

Embodiment 3

[0062] Embodiment 3, the acquisition of transgenic Arabidopsis

[0063] 1) Transformation of Agrobacterium

[0064] The inducible expression vector XVE::BnFUS3 obtained in step 2 was transformed into Agrobacterium by electroporation. A single colony of the transgenic Agrobacterium was picked and inoculated in 20 ml of LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin), and cultured for 2 days at 28° C. with shaking at 150 rpm. Then, the obtained bacterial solution was inoculated into 300 ml LB medium containing rifampicin and kanamycin with 2% inoculum amount, and cultured by shaking according to the above conditions for 16-18 hours. The obtained bacterial solution was centrifuged at 5,000 rpm for 20 minutes to collect the bacterial cells, and the bacterial cells were resuspended in 250ml of 5% sucrose and Silwet L-77 transformation solution, and shaken slowly to obtain the transformation solution of Agrobacterium containing the XVE::BnFUS3 plasmid .

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technical field

[0001] The invention belongs to the field of genetic engineering, and relates to rapeseed transcription factor BnFUS3 related to plant fatty acid and oil metabolism, its encoding gene and application. Background technique

[0002] Fatty acids in plants are also known as vegetable oils, which are widely found in the seeds of plants. Vegetable oil is widely used, and the use of biotechnology to improve the composition and content of fatty acids in plants has important economic value. Among them, the operating gene selected by genetic engineering technology must be the key gene in the process of fatty acid biosynthesis in plants, but people still don't know much about the molecular regulation mechanism of plant fatty acid biosynthesis. Quantitative work is currently progressing slowly. Taking the dicot model plant Arabidopsis thaliana as an example, the embryogenesis of plants is mainly divided into two stages: early morphogenesis and late maturation. The ent...

Examples

Embodiment 1

[0055] Embodiment 1, the acquisition of BnFUS3 gene

[0056] Collect rapeseed seeds that have grown to 30 days, and extract 30-day-old rapeseed Zhongyou 821 seeds (Xie Zhucun. Characteristics and cultivation of Zhongyou 821 [J]. Guangxi Agricultural Science, 1991, (05): 213) total mRNA. The obtained mRNA was synthesized into cDNA by a reverse transcription kit (Takara Company, the specific operation was performed according to the kit instructions). The obtained cDNA was amplified with the following primer sequences to amplify the BnFUS3 gene cDNA sequence: (the underline is the restriction site, and the first three bases are the protection bases)

[0057] BnFUS3 F(XhoI):5'-CGA CTCGAG CACACACTGTTTCCACACTTCCT-3' (SEQ ID NO. 4);

[0058] BnFUS3 R(SpeI):5'-TTA ACTAGT TCACTGCATTTTTATATATCTTG-3' (SEQ ID NO. 5).

[0059]Under the guidance of PCR amplification, after the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and the target fragm...

Embodiment 2

[0060] Embodiment 2, the construction of BnFUS3 gene induction expression vector

[0061] Select the positive clone with the correct sequence of pGEM-BnFUS3 in step 1, prepare a large amount of plasmid DNA, use XhoI / SpeI double enzymes purchased from NEB Company to obtain the BnFUS gene fragment, and recover BnFUS3 with a gel recovery kit (purchased from Dingguo Company) Gene fragments, fragments after purification were used T4 ligase (Roche Company) and the XVE-induced expression vector (abbreviated as XVE) (Gateway Company, http: / / www.gateway .com / worldwide / ) to form an inducible expression vector for XVE::BnFUS3. Transform Escherichia coli (E.coli) DH5α strain by XVE::BnFUS3 heat shock method, select positive colonies and add them to 5ml LB liquid medium containing 50mg / L kanamycin, culture at 37℃, 200rpm for 12-16 hours, extract Plasmids were identified by PCR and enzyme digestion. For the construction diagram of the expression vector, see figure 2 .

Embodiment 3

[0062] Embodiment 3, the acquisition of transgenic Arabidopsis

[0063] 1) Transformation of Agrobacterium

[0064] The inducible expression vector XVE::BnFUS3 obtained in step 2 was transformed into Agrobacterium by electroporation. A single colony of the transgenic Agrobacterium was picked and inoculated in 20 ml of LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin), and cultured for 2 days at 28° C. with shaking at 150 rpm. Then, the obtained bacterial solution was inoculated into 300 ml LB medium containing rifampicin and kanamycin with 2% inoculum amount, and cultured by shaking according to the above conditions for 16-18 hours. The obtained bacterial solution was centrifuged at 5,000 rpm for 20 minutes to collect the bacterial cells, and the bacterial cells were resuspended in 250ml of 5% sucrose and Silwet L-77 transformation solution, and shaken slowly to obtain the transformation solution of Agrobacterium containing the XVE::BnFUS3 plasmid .

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