CN102978172A - Brassica carinata A. braun fatty acid elongase and coding gene thereof - Google Patents

Abstract

The invention provides a Brassica carinata A. braun fatty acid elongase and a coding gene thereof (BcFAE1). The gene is introduced in to yeast; and the erucic acid content in the recombinant yeast reaches 0.27+/-0.24%. The protein and the coding gene thereof have important theoretical and practical significance for the research of a genetic control mechanism of plant erucic acids, the increasement of the erucic acid content and the improvement of related traits, can play an important role in the genetic engineering improvement of the plant erucic acid genes, and have wide application prospects.

Description

Fatty acid elongase and its coding gene from Ethiopian mustard

technical field

The present invention relates to the field of molecular biology and genetic engineering, more specifically, relates to Ethiopian mustard (Brassica carinata A. Braun) fatty acid elongase (FAE1) and its coding gene.

Background technique

Erucic acid (C22:1) is a very long-chain unsaturated fatty acid mainly found in the seeds of Brassicaceae and Tropaeolaceae. As a metabolite of plants, erucic acid also affects the growth and development of plants to a certain extent, and participates in the synthesis of biofilm cutin or wax in the interaction between plants and the environment. In terms of nutritional value, erucic acid is an anti-nutritional component of edible oil, which is not easy to be digested and absorbed by the human body, and it is easy to cause diseases such as coronary heart disease and fatty liver. However, in industry, erucic acid and its derivatives, such as erucic acid amide, have good plasticity and hydrophobicity, so they have a wide range of uses and are important raw materials for the production of lubricants, preservatives, chemical fiber raw materials and cosmetics and other chemical products .

The FAE1 (Fatty Acid Elongase 1) gene is a key gene that regulates erucic acid synthesis. Although the FAE1 gene was first cloned from Arabidopsis by James et al., complete FAE1 genes have been isolated from various cruciferous plants. However, at present, the cloning of FAE1 gene is limited to a few plants of Arabidopsis and Brassica, and Brassicaceae is the main germplasm resource of erucic acid distribution. There are many types of plants and complex erucic acid content. Suitable material for gene cloning.

The invention obtains active protein through the eukaryotic expression of BcFAE1 gene, and at the same time, detection and analysis of fatty acid content reveals that the protein encoded by the gene has catalytic activity for erucic acid synthesis.

Contents of the invention

The object of the present invention is to provide a fatty acid elongase (Fatty Acid Elongase 1, FAE1) and its coding gene from Ethiopian mustard with higher erucic acid content.

The FAE1 gene of the Ethiopian thaliana of the present invention consists of 1251 bases, contains a complete open reading frame, and is the No. 1 sequence in the sequence list, and the gene is named BcFAE1.

The Ethiopian thaliana FAE1 protein of the present invention consists of 506 amino acids, the start codon of the open reading frame is ATG, and the stop codon is TAA. The theoretical molecular weight of BcFAE1 protein is 56.37kDa, and the isoelectric point is 9.54.

The vector containing the polynucleotide of the above sequence 1 and its open reading frame, as well as the biological cells transformed with the polynucleotide of the above sequence 1 and its open reading frame, are the contents to be protected in the present invention.

The discovery of the gene of the present invention makes it possible to regulate the synthesis of erucic acid through transgene, which is of great significance for the cultivation of transgenic cruciferous crops with high erucic acid.

The present invention will be further described below in conjunction with specific examples.

Description of drawings

Figure 1 is the Ethiopian thaliana genome DNA

Figure 2 is the full-length PCR amplification product of BcFAE1 gene

Fig. 3 is the construction flowchart of eukaryotic expression vector pYES-BcFAE1

Figure 4 is the restriction map of vector pYES-BcFAE1

Figure 5 is the Western Blot analysis of BcFAE1 expression products in yeast

Detailed ways

Embodiment 1, the cloning of gene BcFAE1

To clone BcFAE1, the specific steps are as follows:

1. Acquisition of BcFAE1 gene

Ethiopian mustard is cultivated under normal conditions. After the true leaves grow, the leaves are collected, DNA is extracted from the leaves, polymerase chain reaction is carried out, and the BcFAE1 gene is finally obtained.

1. Extraction of Ethiopian thaliana genomic DNA

Take about 0.2g of fresh young living plant leaves, fully grind them under liquid nitrogen freezing conditions, transfer them to a 1.5mL centrifuge tube, add 500μL CTAB extract, bathe in water at 65°C for 45min, mix by inverting 3 times during the period; add 500μL volume Chloroform-isoamyl alcohol with a ratio of 24:1, mixed evenly, and centrifuged at 12000r·min ^-1 for 15min; taking the supernatant, adding 2 times the volume of absolute ethanol, and putting it in a refrigerator at -20°C overnight; centrifuging at 4000r·min ^-1 for 15min DNA precipitation: wash twice with 700 μL 70% ethanol, centrifuge ^at 4000 r min for 10 min, wash once with 700 μL absolute ethanol, centrifuge at 4000 r ^min for 10 min, and dry the DNA precipitate; finally add 100 μL deionized water to make the precipitate dissolve. The results of electrophoresis detection are shown in Figure 1.

2. Polymerase chain reaction PCR

Using the Ethiopian thaliana genomic DNA obtained above as a template, using TFK (5′CGGGGTACCGCAATGACGTCCGTTAAC 3′) and TRB (5′CGCGGATCCGGACCGACCGTTTTGGAC 3′) with KpnI and BamHI restriction sites as primers, amplify BcFAE1 according to the following reaction Gene. The reaction system is as follows:

The amplification reaction was carried out according to the following procedure: first, 94°C pre-denaturation for 3 min; then 94°C denaturation for 30 sec, 53°C annealing for 30 sec, 72°C extension for 1 min, 35 cycles; finally, 72°C extension for 5 min. The PCR product was detected by electrophoresis, as shown in Figure 2, there was a band with a size of about 1500bp, which was consistent with the theoretical value.

The 1500bp band was recovered, and the DNA fragment was connected to the pMD18-T vector to generate the pMD-BcFAE1 recombinant vector, which was transformed into Escherichia coli DH5. After sequencing, the Blastn comparison was carried out in GenBank. The results showed that the DNA was compatible with a variety of plant FAE1 The genes have high homology, so it is speculated that the amplified DNA is a FAE1 gene. After sequencing, BLAST search was performed, and the results showed that the gene sequence of Ethiopian thaliana FAE1 had been isolated.

2. Homology analysis and secondary structure analysis of BcFAE1 gene

In order to analyze the homology between the protein sequence of the FAE1 gene of Ethiopian thaliana and the protein encoded by the FAE1 gene of other higher plants, Arabidopsis thaliana, Brassica juncea, Brassica napus), cauliflower (Brassica oleracea), turnip (Brassica rapa), Camelina hispida, Cardamine graeca, crabgrass (Crambeabyssinica), woad (Isatis tinctoria), green unicorn (Lepidium campestre), silver fan grass (Lunaria annua) , White mustard (Sinapis alba), Central European mustard (Teesdalia nudicaulis) and other plant homologous gene protein sequences. The result of analysis by Megalign software shows that the amino acid sequence of the FAE1 protein of Ethiopian thaliana is 97.2% homologous to the FAE1 protein in Crabapple, about 80%-90% homologous to other plants, and FAE1 to the FAE1 of Cardamine graeca. The protein homology is the lowest, only 81.5%.

The results of Pfam showed that the protein encoded by the BcFAE1 gene had the typical FAE1 protein-specific FAE1_CUT1_RppA and ACP_syn_III_C domains.

Example 2, Functional Verification of BcFAE1 Gene

1. High expression of BcFAE1 gene in yeast

In order to demonstrate the coding function of the BcFAE1 gene, the present invention clones the BcFAE1 gene between the BamHI and KpnI sites of pYES2/NT C from Invitrogen, and transforms the yeast strain InvSc1 to obtain high-level expression.

The present invention utilizes the multiple cloning sites of the pYES2/NT C high-efficiency expression vector of Invitrogen Company to realize the high-efficiency expression of BcFAE1. The enzyme cleavage site used in this experiment makes the 5' end of the product expressed by the pYES2/NT C expression vector add 6 continuous His fusion proteins, and these 6 continuous His constitute the specific binding site of i-NTA gel. Therefore, the expression product can be purified by affinity chromatography.

The pMD-BcFAE1 recombinant vector carrying the BcFAE1 gene and the pYES2/NT C expression vector were digested with the same enzymes (BamHI and KpnI), then ligated, and the ligated product was transformed into yeast InvSc1 strain, and added with 2% (w/v ) glucose but without uracil medium (SC-ura) culture and selection. The construction process of the vector is shown in Figure 3. After plasmid digestion and identification (as shown in Figure 4), 1 and 2 are plasmids pYES-BcFAE1 digested with BamHI and KpnI, and then identified by PCR. The reading frame of the DNA sequence is correct. The expression vector constructed with BcFAE1 gene was named pYES-BcFAE1 and used for induced expression analysis.

The recombinants screened and identified were inoculated into SC-ura liquid medium supplemented with 2% (w/v) glucose, and cultured overnight at 28° C. with shaking. The overnight culture was diluted with SC-ura liquid medium supplemented with 2% (w/v) galactose to an OD600 value of 0.02, and continued shaking until the OD600 value was 1.4. Collect the bacteria, extract the total protein of the cells according to the method provided by KGI, then use HisBind resin to purify and perform 10% SDS-PAGE electrophoresis detection, the results are shown in Figure 5, it can be seen from the figure that the BcFAE1 gene is in InvSc was expressed efficiently.

2. Function identification

In order to detect the function of the BcFAE1 gene, the present invention measures the erucic acid content of the yeast cells transformed with the pYES-BcFAE1 plasmid, the BcFAE1 gene promotes the erucic acid synthesis, and the erucic acid is accumulated in the yeast cells.

The fatty acid elongase activity of the yeast strain InvSc used in the present invention is very low, and only a small amount of ultra-long chain fatty acid is synthesized, so it is a common system for studying the activity of the protein encoded by FAE1. The recombinants screened and identified in Step 1 were inoculated into SC-ura liquid medium supplemented with 2% (w/v) glucose, and cultured with shaking at 28° C. overnight. The overnight culture was diluted with SC-ura liquid medium supplemented with 2% (w/v) galactose to an OD600 value of 0.02, and continued shaking until the OD600 value was 1.4. The cells were collected and washed with ultrapure water. Yeast cells were saponified in potassium hydroxide-methanol solution (10% (w/v) KOH, 5% (v/v) H2O in methanol) at 80°C for 2h. After saponification, the samples were frozen on ice and washed with n-hexane to remove unsaponifiable matter. The remaining aqueous phase was acidified with 6mol/L HCl. Free fatty acids were extracted in n-hexane, and the solvent was removed by vacuum. Free fatty acids were methylated in 2ml of methanol solution containing 1% H2SO4 at 60°C for 1h. Yeast fatty acid methyl esters (FAMEs) were extracted in n-hexane, the solvent was removed by vacuum, and the residue was dissolved in n-hexane for gas chromatography analysis. The results showed that the content of erucic acid in the yeast transformed with pYES-BcFAE1 plasmid was 0.27±0.24%, and the empty plasmid pYES2/NT C as a control had no accumulation of erucic acid. The results confirmed the in vitro activity of the protein encoded by the BcFAE1 gene.

Claims (7)

1. A fatty acid elongase polypeptide, which is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of Sequence 2; (b) A polypeptide having the function of fatty acid elongase formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence of Sequence 2. 2. according to the coding gene of fatty acid elongase described in claim 1, it is characterized in that the gene cDNA of described fatty acid elongase is one of following nucleotide sequence: (a) the nucleotide sequence of sequence 1 in the sequence listing; (b) a nucleotide sequence complementary to polynucleotide (a); (c) Nucleotide sequence encoding the amino acid sequence of the protein of Sequence 2 in the Sequence Listing. 3. A recombinant expression vector containing the gene of any one of claim 2. 4. A transgenic cell line containing the gene of any one of claim 2. 5. the engineering bacterium containing the arbitrary described gene of claim 2. 6. Use of the protein according to any one of claims 1 in cultivating fatty acid-improved plants. 7. The use of the gene according to any one of claims 2 in cultivating fatty acid-improved plants.

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