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Perilla is a 1a-year-old herb of the Lamiaceae family, which is a traditional medicinal and edible homologous plant, which is widely cultivated in eastern Asian countries, especially China, South Korea and Japan.
Its seed oil yield is as high as 46%~58%, and the content of unsaturated fatty acids is abundant, especially the content of α-linolenic acid is much higher than that of soybean, rape, corn, sunflower and other oil crops.
The extremely low omega-6/ω-3FAs ratio makes perilla oil an ideal source of vegetable oil in the human diet, and can also be used to make dry paints, varnishes, inks, etc.
Therefore, as a multi-purpose cash crop, perilla has potential application prospects in pharmaceutical, food and industrial fields.
Materials and methods of the experiment
If perilla and tobacco seeds are taken at a suitable time, the roots, stems, leaves, flowers and seeds at different development stages should be stored in a -80 °C refrigerator for later use, and the tobacco should be cultured in a constant temperature light incubator.
Using the Arabidopsis thaliana AtPAH1 protein sequence as the retrieval sequence, BLAST homology alignment was performed in the perilla genome and transcriptome database, and the PAH1 gene sequence of perilla was screened by ORF and CDD functional domain analysis and alignment.
The CDS sequence of PfPAH1-1 gene was used as a template, and the PCR amplification primer PfPAH1-1-ORF-F/R was designed.
PCR amplification was performed using KOD enzyme, and the reaction system was 50 μL: 2 μL of template cDNA, 1.5 μL of upstream and downstream primers, 5 μL of 10× PfuBuffer, 5 μL of dNTPs, 42 μL of MgSO, 1 μL of KOD-Plus-Neo, and 32 μL of ddH2O.
The reaction conditions were: pre-denaturation at 94°C for 2min; Denaturation at 94°C for 15s, annealing at 50°C for 30s, extension at 68°C for 2min30s, and 35 cycles; Final extension at 68°C for 10min.
After the reaction, the amplified product was detected by agarose gel electrophoresis, the correct product was purified, recovered, and ligated to the pMD18-T vector, transformed into E. coli DH5α competent state, and monoclonal was selected for PCR identification and screening of positive clones.
Analysis of experimental results
Using the Arabidopsis thaliana AtPAH1 protein sequence as the retrieval sequence, two Perilla PfPAH1 gene sequences were screened and obtained through ORF and CDD databases, which were named PfPAH1-1 and PfPAH1-2, respectively.
Protein functional domain prediction showed that both PfPAH1-1 and PfPAH1-2 contained three conserved domains, namely Lipin_N domain, Lipin_mid domain, and LNS2 domain belonging to the HAD_like superfamily.
Gene structure analysis showed that the PfPAH1-1 gene contained 8 introns and 9 exons, and the PfPAH1-2 gene contained 9 introns and 10 exons, which were similar to those of Arabidopsis thaliana and sesame PAH1 genes.
The number of amino acids encoded by PfPAH1-1 and PfPAH1-2 genes was 904, and the molecular formulas of the encoded proteins were C4375H6819N1191O1419S32, respectively.
The molecular weights were 10.08 ku and 9.98 ku, and the theoretical isoelectric pI were 4.73 and 4.96, respectively, and the hydrophilicity and hydrophobic coefficients of both were less than 0, indicating that they were hydrophilic aqueous proteins.
The results showed that the PfPAH1 protein had no transmembrane region and no signal peptide, and it was speculated that it was a non-secretory protein.
The secondary structure of PfPAH1 protein was predicted by SOPMA software, and the results showed that the secondary structure of PfPAH1-1 and PfPAH1-2 protein was mainly composed of four elements.
Pf PAH1-1 contained 56.08% random coil, 23.01% α-helix, 16.37% straight chain elongation, and 4.54% β-turn.
However, Pf PAH1-2 contained 56.75% random coil, 22.9% α-helix, 15.93% linear chain elongation, and 4.42% β-turn angle, suggesting that random coil and α-helix were the main structural elements of PfPAH1 protein.
The homology modeling method was used to predict the tertiary structure of Perilla PfPAH1 protein through the SWISS-MODEL database, and 6tzy.2 protein was selected as the template protein for homology modeling.
The results showed that the sequence coverage of PfPAH1-1 and PfPAH1-2 protein and template protein 6tzy.2 was 36.75% and 35.05%, respectively, and the conserved sequence range was 589~876aa and 578~878aa, respectively.
Both the PfPAH1 protein and the template protein are composed of a polypeptide chain, and the template protein contains two CA ligands, while the PfPAH1-1 and PfPAH1-2 proteins contain only one CA ligand.
The amino acid sequences of PAH1 protein in perilla, Arabidopsis, sesame, olea europaea and a bunch of red were compared.
The analysis showed that the DXDX catalytic motif in the published plant PAH1 protein sequence was highly conserved in the PfPAH1 protein sequence, and it was inferred that the DXDX domain played an important role in the PAH1 protein.
MEGA7.0 software was used to construct a phylogenetic tree for Perilla PfPAH1 protein and Arabidopsis thaliana, sesame, tobacco and olive proteins.
Perilla PfPAH1-1 protein is clustered with a bunch of red clusters, which belongs to the Lamiaceae family, and is far from other species such as grapes and mustards, and its evolution is basically in line with plant evolutionary classification, and PfPAH1-2 protein is the most closely related to sesame SiPAH1.
The second is Olive, which is distantly related to Bergamot spp. and Pepper, and it is speculated that the function of Perilla PfPAH1-2 gene may be similar to that of Sesame SiPAH1 gene.
After 2 days of transient infection by Agrobacterium, total RNA was extracted from tobacco leaves, and RT-PCR was used to detect whether the PfPAH1-1 gene was effectively expressed.
The results showed that the target gene PfPAH1-1 was normally transcribed into mRNA in tobacco leaves after transient transformation, and the effect of transient expression of Perilla Pf PAH1-1 gene on the lipid content of tobacco leaves was analyzed by taking tobacco leaves after transient infection for 5 days.
The total fatty acids in wild tobacco leaves accounted for 15.29% of the dry weight of the leaves, and the total fatty acids in the leaves of tobacco that were transferred to pCAM-BIA1303 accounted for 15.30% of the dry weight DW of the leaves.
However, the content of total fatty acids in the leaves of tobacco transgenic with PfPAH1-1 gene was 17.35%, and the content of total fatty acids increased significantly.
The analysis of lipid composition showed that the glycolipid content of PfPAH1-1 gene transiently expressed in tobacco leaves did not change significantly, accounting for about 3.55%~3.56% of the dry weight of the leaves, and the phospholipid content decreased by 0.3% compared with WT, while the neutral lipid content increased by 1.86%.
Discussion of experimental results
As a multi-purpose cash crop, perilla is a good substitute for deep-sea fish oil because of its rich nutrients and high seed oil content and unsaturated fatty acid content.
Triacylglycerol is the main storage form of seed oil in oil crops, and PAH is a key enzyme in the TAG synthesis pathway.
The substrate PA that mediates the reaction is the direct precursor of the synthesis of the libonucleotide CDP-DAG, and the product DAG is the direct precursor of the synthesis of TAG.
It has been shown that PAH activity is dependent on the DXDX catalytic motif of the halogenated acid dehalogenase-like domain.
On the catalytic motif of Saccharomyces cerevisiae PAH1DXDX (T/V), the activity of aspartic acid D398 and D400 was mutated at the same time, and its activity would be reduced by more than 99.9%.
Two PfPAH1-1 and PfPAH1-2 genes encoding 904 amino acid residues were identified in perilla, both of which contained HAD_like domains and had the unique active site DXDX of PAH.
The sequence was the same as that of published plants such as Arabidopsis thaliana and sesame, indicating that PfPAH1-1 and PfPAH1-2 could catalyze their protein functions, and sequence characteristic analysis showed that Perilla PfPAH1 protein had no transport peptide or transmembrane region.
Analysis of the expression profile of Arabidopsis PAH1 gene showed that the gene was expressed in all tissues and organs of Arabidopsis thaliana and seeds at different developmental stages, and the expression level reached the highest in mature seeds.
qRT-PCR analysis of Arabidopsis thaliana PAH1 showed that the gene was expressed in seedlings, leaves, roots, stems and flowers, and the expression of PAH1 in seeds at different developmental stages showed a trend of first decreasing and then increasing, and the expression level in mature seeds was significantly higher than that in other tissues.
The results showed that PAH gene was expressed in flowers of Arabidopsis thaliana at different developmental stages.
The spatiotemporal expression pattern of Pf PAH1 gene was analyzed, and the expression of PfPAH1-1 was expressed in all tissues of Perilla perilla, and the expression level of Pf PAH1-1 decreased first and then increased with the maturity of seeds.
The expression level of PfPAH1-2 gene was lower in different tissues of perilla in different tissues.
It was speculated that Pf PAH1-1 played a leading role in the regulation of perilla lipid synthesis and metabolism, and may be more involved in the synthesis of PI and PG with PA as substrate in mid-development seeds, and then played a role in the synthesis of TAG and phospholipids at the same time.
PAH converts PA into DAG to provide substrates for phospholipid and TAG biosynthesis and is an important step in glyceride assembly.
In the study of Saccharomyces cerevisiae, the expression of phospholipid biosynthesis-related genes in pah1Δ mutant was up-regulated, and the phospholipid levels increased, but the levels of DAG and TAG decreased significantly.
On the contrary, the overexpression of PAH1 leads to the inhibition of phospholipid biosynthesis gene expression and impairs phospholipid biosynthesis, but it can effectively enhance the lipid synthesis in Saccharomyces cerevisiae cells.
In plants, the Arabidopsis thaliana Δpah1pah2 double mutant contained twice as many phospholipids as the wild type, several key enzyme genes encoding phospholipid synthesis were up-regulated, and the total fatty acid content was reduced by 15%.
In order to further analyze the function of Perilla perilla PfPAH1-1 gene, the target gene was transiently expressed in tobacco leaves by Agrobacterium infection, and the content of each lipid component in tobacco leaves after transient expression was determined.
The results showed that the total fatty acid content in the leaves of tobacco transgenic PfPAH1-1 gene was significantly increased, and its lipid composition was also changed, among which the neutral lipid level increased, the phospholipid content decreased, and the glycolipid content remained basically unchanged.
It was inferred that the transient expression of Perilla PfPAH1-1 gene effectively strengthened the transition from phospholipid PA to DAG in the TAG synthesis pathway, and the level of neutral lipids increased.
However, the overexpression of this gene can consume the phospholipid synthesis substrate PA, which hinders the phospholipid synthesis process and reduces the phospholipid content, which may also be related to the decrease in the expression of phospholipid synthase gene based on the results of previous studies.
In the long-term evolution of plants, changing membrane lipid composition is an important way for plants to adapt to adversity.
Among them, glycerides are the main components of membrane lipids, which are composed of two hydrophobic fatty acids attached to sn-1 and sn-2, and phosphorus or sugar molecules at sn-3 of the glycerol backbone.
The transient expression of Pf PAH1-1 gene in tobacco leaves can cause changes in membrane lipid composition in tobacco leaves, so it is speculated that it may play a regulatory role in response to plant stress.
Using the Arabidopsis thaliana AtPAH1 protein sequence as the reference sequence, two Perilla PfPAH1 genes were identified, named Pf PAH1-1 and PfPAH1-2, respectively, and their sequence characteristics and expression characteristics were analyzed.
The cDNA clone of Perilla PfPAH1-1 gene with high expression in tissues was successfully obtained by RT-PCR, and the effect of heterologous expression of this gene on the lipid composition of tobacco in tobacco was preliminarily discussed.
In order to lay a theoretical foundation for the in-depth study of the molecular mechanism of PfPAH1-1 gene in the synthesis and accumulation of vegetable oils, and to provide beneficial genetic elements for changing the lipid composition of perilla and other oil crops by genetic engineering.