Perilla PfLACS1 gene for regulating and controlling synthesis of vegetable fat, recombinant expression vector, recombinant bacterium and application

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to a perilla frutescens PfLACS1 gene for regulating and controlling vegetable fat synthesis, a recombinant expression vector, recombinant bacteria and application, and the nucleotide sequence of the perilla frutescens PfLACS1 gene is shown as SEQ ID NO.1; the perilla key enzyme differential expression gene PfLACS1 is screened and identified for the first time, a molecular biology means is used for constructing a recombinant overexpression vector of the perilla PfLACS1 gene, the recombinant overexpression vector is converted into a yeast strain, the total oil content of overexpression yeast is analyzed, and the result shows that the perilla PfLACS1 gene can remarkably improve the total oil content of tobacco plant leaves. Theoretical reference is provided for further understanding of a perilla fatty acid synthesis accumulation mechanism and improvement of perilla oil yield and quality, and meanwhile, an excellent gene element is provided for specific enrichment of target fatty acid for other oil crops through a gene engineering technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology, and particularly relates to a Perilla frutescens PfLACS1 gene for regulating plant oil synthesis, a recombinant expression vector, a recombinant bacterium and applications thereof, which are used to encode a key enzyme involved in plant oil synthesis. Background Art

[0002] Perilla frutescens (L.) Britt., belonging to the genus Perilla of the Lamiaceae family, is an annual self-pollinating herbaceous plant with a specific aroma and has important development and utilization value in the fields of medicine and food. Its seed oil content is as high as 46% - 58%, especially the health-beneficial fatty acid α-linolenic acid is more than 65%. Long-term consumption of Perilla oil can prevent cardiovascular and cerebrovascular diseases, improve immunity, delay aging, etc. With the continuous growth of the market demand for oil use, more and more research work focuses on using molecular biology and genetic engineering technologies to improve the oil yield and quality of crops. Therefore, in-depth exploration of the detailed process of plant oil metabolism and its underlying molecular regulation mechanism has become the research cornerstone for improving the oil yield and quality of crops. Plant oils are mainly stored in the form of TAG, and long-chain acyl-Coenzyme A synthetase (LACS) is a key enzyme in the process of TAG synthesis. LACS can bind fatty acids to coenzyme A to form acyl-CoA, and then participate in various physiological processes such as β-oxidation of fatty acids, phospholipid synthesis, and signal transduction. Therefore, it is necessary to discover a coding gene from Perilla frutescens to encode a key enzyme that can participate in plant oil synthesis. Summary of the Invention

[0003] To solve the above problems, the present invention provides a Perilla frutescens PfLACS1 gene for regulating plant oil synthesis, a recombinant expression vector, a recombinant bacterium and applications thereof, which are used to encode a key enzyme that can participate in plant oil synthesis.

[0004] The present invention is realized by the following technical solutions:

[0005] A Perilla frutescens PfLACS1 gene for regulating plant oil synthesis, wherein the nucleotide sequence of the Perilla frutescens PfLACS1 gene is as shown in SEQ ID NO.1.

[0006] A recombinant expression vector, which comprises the above-mentioned coding gene and a backbone plasmid connecting the coding gene.

[0007] Preferably, the backbone plasmid is pBI121 or pYES2.0.

[0008] Preferably, the method for constructing the recombinant expression vector is to amplify the perilla PfLACS1 gene using a specific primer pair, recover the PCR product and ligate it with the backbone plasmid pBI121 to obtain the recombinant expression vector of plants; recover the PCR product and ligate it with the backbone plasmid pYES2.0 to obtain the recombinant expression vector of yeast.

[0009] Preferably, the nucleotide sequences of the specific primer pair are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0010] A recombinant bacterium, which is obtained by transforming the recombinant expression vector into an engineering bacterium.

[0011] Preferably, the engineering bacterium is Agrobacterium tumefaciens GV3101.

[0012] Application of the perilla PfLACS1 gene for regulating plant oil synthesis, the recombinant expression vector or the recombinant bacterium in increasing the total oil content of plant leaves.

[0013] Preferably, the plant is tobacco.

[0014] Preferably, the heterologous expression of the perilla PfLACS1 gene can complement the LACS enzyme deficiency phenotype of YB525 yeast and has acyl-CoA enzyme activity.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a perilla PfLACS1 gene for regulating plant oil synthesis, and the nucleotide sequence of the perilla PfLACS1 gene is shown in SEQ ID NO.1. The present invention firstly screened and identified the differentially expressed gene PfLACS1 of the key enzyme of perilla, constructed a recombinant overexpression vector of the perilla PfLACS1 gene by molecular biology means, transformed the recombinant overexpression vector into a yeast strain, and analyzed the total oil content of the overexpressed yeast; and carried out the complementary function analysis of the PfLACS gene transformed into the defective yeast YB525. The results show that the perilla PfLACS1 gene can significantly increase the total oil content of yeast, and the heterologous expression of the PfLACS gene can complement the LACS enzyme deficiency phenotype of YB525 yeast and has acyl-CoA enzyme activity. Further, the constructed recombinant overexpression vector was transformed into tobacco by Agrobacterium-mediated transformation, and the total oil content of the overexpressed tobacco was analyzed; the results show that the perilla PfLACS1 gene can significantly increase the total oil content of the leaves of tobacco plants.

[0017] The present invention deeply explores the functional characteristics of the key enzyme gene PfLACS1 in perilla oil metabolism, laying a theoretical foundation for deeply understanding the biological mechanism of perilla fatty acid synthesis and accumulation, improving perilla oil yield and optimizing its quality. In addition, the research results also provide excellent gene resources for other oil crops to directionally increase the content of target fatty acids through genetic engineering technology. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 Relative expression levels of the PfLACS1 gene of the present invention in different tissues of "Jin Zisu 1".

[0020] Figure 2 PCR amplification of the PfLACS1 gene of the present invention. In the figure, M: D2000 II DNA Marker.

[0021] Figure 3 PCR detection of the bacterial solution of the present invention. In the figure, M: D2000 II DNA Marker; 1-5: Monoclonal bacteria detection of PfLACS1.

[0022] Figure 4 Map of the yeast expression vector pYES2.0 of the present invention.

[0023] Figure 5 Positive identification of transgenic yeast of the present invention. In the figure, M: D2000 II DNA Marker; 1-3: Yeast transformed with the empty pYES2.0 vector; 4-6: Yeast transformed with PfLACS1.

[0024] Figure 6 Total lipid content of transgenic yeast of the present invention. Different lowercase letters indicate significant differences at the p<0.05 level.

[0025] Figure 7 Complementation test of defective yeast of the present invention. In the figure, A: Yeast transformed with the empty pYES2.0 vector; B: Yeast transformed with pYES2.0-PfLACS1.

[0026] Figure 8 Map of the plant expression vector pBI121 of the present invention.

[0027] Figure 9This is the positive detection map of transgenic Agrobacterium tumefaciens of the present invention. In the figure, M: 8000bp Marker; 1-3: Agrobacterium tumefaciens transformed with pBI121-PfLACS1.

[0028] Figure 10 This is the tobacco genetic transformation process of the present invention. In the figure, A: Pre-culture of tobacco leaves; B: Dedifferentiation to induce callus; C: Callus forming buds; D: Rooting culture; E: Growth culture; F: Acclimatization and transplantation.

[0029] Figure 11 This is the genomic level detection map of transgenic tobacco of the present invention. In the figure, 1: WT tobacco, 2-7: Tobacco transformed with PfLACS1.

[0030] Figure 12 This is the transcriptional level detection map of transgenic tobacco of the present invention. In the figure, 1: WT tobacco, 2-7: Tobacco transformed with PfLACS1.

[0031] Figure 13 This is the total lipid content of transgenic tobacco leaves of the present invention. Different lowercase letters indicate significant differences at the p<0.05 level. Detailed implementation manners

[0032] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0033] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0034] The beneficial effects of the present invention are illustrated by the following specific embodiments:

[0035] Example 1: Cloning of Perilla frutescens PfLACS1 gene

[0036] 1. Total RNA extraction and cDNA synthesis: Use the EASYspin plant RNA rapid extraction kit to extract the total RNA of different tissues of Perilla frutescens. Using the total RNA of each sample of Perilla frutescens obtained as a template, use Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR reverse transcription kit to reverse transcribe to obtain cDNA.

[0037] 2. Analysis of the expression characteristics of the Perilla frutescens PfLACS1 gene

[0038] Specific primers for real-time fluorescence quantitative PCR were designed according to the Perilla frutescens PfLACSs family genes. The nucleotide sequence of the Perilla frutescens PfLACS1 gene is shown in SEQ ID NO.1. Using Perfect Green qPCR SuperMix kit for real-time fluorescence quantitative PCR analysis, and a real-time fluorescence PCR instrument (BIO-RAD CFX Maestro) was used to detect the expression patterns of PfLACSs genes in different periods of Perilla frutescens tissues. The 2 -ΔΔCT method was used to calculate and analyze the relative expression levels of the genes, and three biological replicates were set. To ensure the amplification efficiency, the cDNA was diluted about 10-fold before the operation. The reaction system is shown in Table 1.

[0039] SEQ ID NO.1:

[0040]

[0041] Table 1 qRT-PCR system:

[0042]

[0043] The qRT-PCR program is: 94°C for 30 sec; 94°C for 5 sec, 50°C for 15 sec, 72°C for 10 sec, 40 cycles.

[0044] 3. Design primers: The primer information is shown in Table 2.

[0045] Table 2 Cloning primer information of PfLACS1 gene

[0046]

[0047] Note: The black underline indicates the restriction enzyme site.

[0048] 4. PCR reaction: The amplification system is shown in Table 3.

[0049] Table 3 PCR amplification system:

[0050]

[0051]

[0052] The PCR program is: 98°C for 1 min; 98°C for 10 s, 60°C for 5 s, 72°C for 12 s, 30 cycles; 72°C for 5 min.

[0053] After the PCR reaction, the target band is detected by agarose gel electrophoresis.

[0054] 5. Recovery and purification of the target fragment

[0055] Cut the target band obtained by amplification in step 4 above from the agarose gel, and operate according to the DNA Gel Extraction Kit general DNA purification and recovery kit to recover and purify the target fragment, and store the product in a -20°C refrigerator.

[0056] 6. Preparation of linearized vector

[0057] Inoculate DH5α Escherichia coli carrying the pBI121 empty vector plasmid into LB liquid medium containing 50 μg / mL kanamycin (Kan), and culture overnight at 37°C. Extract the pBI121 empty plasmid using a plasmid DNA extraction kit. Double digest the pBI121 plasmid with the corresponding restriction endonucleases. The 50 μL double digestion system is shown in Table 4.

[0058] Table 4 50 μL double digestion system

[0059] pBI121 empty plasmid 23 μL Restriction enzyme 1 1 μL Restriction enzyme 2 1 μL Endonuclease Buffer 5 μL Nuclease-free Water Make up to 50 μL

[0060] The PCR program is as follows: 37°C for 30 min, 4 cycles; 65°C for 20 min; 16°C for 59 min 59 s.

[0061] After the restriction digestion reaction, use a PCR product purification kit to recover the linearized vector, and store the obtained vector fragment at -20°C.

[0062] 7. Cloning and Transformation

[0063] 7.1 Cloning of the target gene

[0064] Use the ClonExpress Ultra One Step Cloning kit to complete the cloning of the target gene. Mix 2 μL of the PCR purified product in step 5, 3 μL of the linearized pBI121 vector in step 6, and 5 μL of 2×ClonExpress Mix, and perform a recombination reaction at 50°C for 5 min. Immediately perform the transformation operation after the reaction ends.

[0065] 7.2 Preparation and transformation of E. coli competent cells

[0066] Inoculate the activated E. coli DH5α into 50 mL of LB liquid medium and culture it with shaking at 37°C until the OD 600 = 0.4. Place the bacterial solution on ice for 20 min, centrifuge at 4°C and 5000 r / min to collect the bacterial cells. Add 30 mL of pre-cooled 0.1 M CaCl2 solution to the collected cells, mix well repeatedly, centrifuge at 4°C and 5000 r / min to remove the supernatant. Then add 4 mL of 0.1 M CaCl2 solution containing 80% (v / v) glycerol to the precipitate to resuspend and mix the bacterial solution to obtain competent cells. Aliquot 200 μL of the competent cells into a sterile centrifuge tube for one transformation.

[0067] Add 10 μL of the cloning product in step 7.1 to the freshly thawed E. coli DH5α competent cells, mix well and incubate on ice for 30 min. Then place the centrifuge tube in a 42°C metal bath for 90 s, and immediately incubate on ice for 5 min after completion. Add 800 μL of LB medium to the obtained product, culture it with shaking at 37°C and 200 rpm for more than 45 min. Pipette 25 μL of the bacterial solution and spread it on an LB solid screening plate containing 50 μg / mL kanamycin, and incubate it in the dark at 37°C for 12 h until colonies appear.

[0068] 7.3 Detection of positive clones

[0069] Pick a single colony from the screening plate and inoculate it into 1 mL of LB medium containing 50 μg / mL kanamycin. Shake and culture until the bacterial solution is obtained. Pipette the bacterial solution for PCR detection. The primers used are those in Table 1, and the PCR program is 94°C for 3 min; 94°C for 30 s, annealing temperature for 30 s, 72°C for 2 min 15 s, 30 cycles; 72°C for 5 min.

[0070] Send a part of the bacterial solution detected as positive to Beijing Tsingke Biotechnology Co., Ltd. (Xi'an) for sequencing, and store a part of the bacteria for subsequent experiments.

[0071] 8. Results and Analysis

[0072] Using the mixed cDNA of different tissues of Perilla frutescens as a template, the fluorescence quantitative qRT-PCR technique was used to analyze the expression of the PfLACS1 gene of Perilla frutescens in different tissues of "Jin Zisu 1", as Figure 1 shown. The results showed that the PfLACS gene was expressed in different tissues of Perilla frutescens, and the relative expression levels were diverse. The expression level of PfLACS1 showed a trend of first increasing and then decreasing, with the highest expression level at 20 days after flowering and the lowest expression level in the stem. Through analysis, it was found that the PfLACS1 gene was highly expressed in the developing seeds of Perilla frutescens. Therefore, the PfLACS1 gene was selected for research. A band with a length of about 2000 bp was amplified with the primers in Table 1, as Figure 2 shown, which was consistent with the expected length, and it was preliminarily judged to be the target band. After recovering and purifying the target fragment, it was ligated to the pBI121 vector and transferred into Escherichia coli. Monoclonal colonies were obtained through kanamycin screening, and positive clones were identified by PCR detection, as Figure 3 shown. The bacterial solution with successful detection was sent to the company for sequencing. After comparison, it was found to be consistent with the ORF sequence of the PfLACS1 gene of Perilla frutescens, confirming successful cloning.

[0073] Example 2. Construction and Transformation of Yeast Expression Vector

[0074] 1. Construction of Recombinant Expression Vector

[0075] 1.1 Plasmid Extraction

[0076] The plasmid used for constructing the vector was extracted from the propagated bacterial solution using a rapid plasmid DNA mini kit. Extract the pYES2.0 empty plasmid, as Figure 4 shown, and the successfully sequenced pBI121-PfLACS1 recombinant plasmid, and store them in the -20°C refrigerator for later use. The specific operation was carried out according to the instructions.

[0077] 1.2 Amplification and Purification of Target Fragment

[0078] Design PCR primers containing corresponding restriction sites based on the target sequence, as shown in Table 5. Use the successfully cloned pBI121-PfLACS1 plasmid as a template for PCR amplification, detect the amplification products by agarose gel electrophoresis, and recover and purify the target bands.

[0079] Table 5 Primer Information

[0080]

[0081] Note: The black underlines indicate restriction sites.

[0082] 1.3 Construction of Recombinant Vectors

[0083] 1.3.1 Double Digestion of Vector

[0084] Digest the empty pYES2.0 plasmid with two restriction endonucleases at 37 °C for 1 h. The enzyme digestion reaction system is shown in Table 6.

[0085] Table 6 Enzyme Digestion Reaction System

[0086]

[0087]

[0088] Detect the enzyme digestion products by agarose gel electrophoresis and purify and recover them.

[0089] 1.3.2 Construction of Recombinant Vectors

[0090] Prepare the recombinant reaction system according to the reaction system shown in Table 7 below.

[0091] Table 7 Reaction System

[0092] 2×ClonExpress Mix 5 μL Digested vector fragment 2 μL Target gene fragment 2 μL <![CDATA[ddH2O]]> Make up to 10 μL

[0093] The recombinant reaction program is 50 °C for 5 min.

[0094] 1.3.3 Detection of Recombinant Plasmids

[0095] Transfer the above ligation products into competent Escherichia coli cells, coat them on LB solid medium containing 50 μg / mL ampicillin for screening, and pick monoclonal colonies for PCR detection after the colonies grow.

[0096] 2. Preparation and Transformation of Yeast Competent Cells

[0097] 2.1 Cultivation of Yeast Strains

[0098] INVSc1 Saccharomyces cerevisiae is an ideal protein expression strain with ampicillin and kanamycin resistance, and grows best in YPD medium at 30 °C. INVSc1 Saccharomyces cerevisiae is a His, Leu, Trp, and Ura auxotrophic strain, so it cannot grow in a defective medium lacking histidine, leucine, tryptophan, and uracil. The pYES2.0 vector has ampicillin resistance and contains the URA3 locus, and can grow on a medium lacking uracil. Therefore, positive transformants can be screened using a medium lacking uracil supplemented with kanamycin (SC-URA).

[0099] 2.2 Preparation and transformation of yeast competent cells

[0100] Inoculate the activated INVSc1 yeast cells into 30 mL of YPD liquid medium, and place them in a shaker at 30 °C and shake culture until the OD of the bacterial solution 600 = 0.4, centrifuge at 3000 rpm for 5 min to collect the cells; resuspend the precipitate with 10 mL of Y1 solution and then centrifuge again to collect the cells. Add 1 mL of Y2 solution to the precipitate to resuspend the cells, and the preparation of competent cells is completed. Aliquot 50 μL into a sterile centrifuge tube for transforming one plasmid. To ensure the transformation efficiency, the competent cells are preferably prepared and used immediately.

[0101] The transformation operation is carried out with reference to the yeast transformation kit. First, prepare the premix according to the following system, and then add the competent cells to the prepared premix respectively, and pipette repeatedly to completely suspend the competent cells. Incubate in a metal bath at 30 °C for 60 min, and mix once every 10 min during this period. Centrifuge the reaction solution to remove the supernatant, add 200 μL of sterile deionized water to the precipitate, mix well and then pipette 10 μL and spread it on a solid SC-URA medium containing 50 μg / mL of kanamycin and 2% (v / v) glucose, and culture it inverted at 30 °C for 2 days.

[0102] The premix system is shown in Table 8.

[0103] Table 8 Premix system

[0104] Y3 solution 350 μL Recombinant vector plasmid linked with target gene 5 μL <![CDATA[ddH2O]]> Make up to 360 μL

[0105] 2.3 Identification of positive recombinant yeast

[0106] Pick single colonies from the SC-URA screening plate cultured for 2 days, inoculate them into 5 mL of SC-URA liquid medium containing 2% (v / v) glucose, and culture overnight at 30 °C until the bacterial solution becomes turbid. Perform PCR detection using the primers with restriction sites in Table 5 (SEQ ID NO.4 and SEQ ID NO.5) to identify positive recombinants.

[0107] Because the yeast cell wall is relatively thick, cell wall breaking treatment is required before detection. Take 1 mL of the bacterial liquid and place it in a 1.5 mL centrifuge tube, centrifuge at 13000 rpm for 1 min to remove the supernatant. Then add 500 μL of ddH2O and centrifuge again to collect the cells. Add 200 μL of TE buffer to the precipitate, place it in a 100 °C metal bath for 10 min, then ice-bath at -20 °C for 10 min, and centrifuge to collect the supernatant for PCR detection.

[0108] 2.4 Induced expression of transgenic yeast

[0109] The pYES2.0 vector drives the expression of the target gene with the GAL1 promoter, and galactose is needed to induce the expression of the recombinant protein. Take 1 mL of the bacterial liquid with positive PCR detection and inoculate it into 100 mL of SC-URA liquid medium containing 2% (v / v) glucose, and culture it overnight with shaking at 30 °C. Centrifuge to collect the bacterial cells, wash them 3 times with sterile water, and then transfer the bacterial cells to 300 mL of SC-URA liquid medium supplemented with 2% (w / v) galactose for inducing the expression of recombinant yeast. Culture with shaking at 30 °C for 2 days. At this time, the OD of the bacterial liquid 600 is about 1.0 or so, and the activity is good. Then centrifuge to remove the supernatant, wash the precipitate 3 times with sterile water, and collect the bacterial cells. The collected yeast cells are treated with a freeze dryer and then ground into powder, placed in a centrifuge tube and stored at room temperature for subsequent experiments.

[0110] 2.5 Analysis of lipid synthesis in transgenic yeast

[0111] Extract the total fatty acids of transgenic yeast, and the specific operation for measuring the total lipids is as follows:

[0112] Weigh 150 mg of freeze-dried yeast cells and place them in a 50 mL centrifuge tube. Add 7.5 mL of a methanol:chloroform mixture to it, and the volume ratio of methanol to chloroform is 2:1. After extraction at 37 °C and 200 rpm for 24 h, centrifuge to collect the upper organic phase into a new centrifuge tube. Add another 7.5 mL of the methanol:chloroform mixture to the precipitate, with the volume ratio of methanol to chloroform being 2:1. Repeat the extraction for 12 h and collect the upper organic phase. Mix the two collected upper organic phases, then add 5 mL of chloroform solution and 9 mL of 1% (v / v) NaCl solution, so that Vchloroform:Vmethanol:Vwater = 2:2:1.8. Mix well, and centrifuge to collect the lower organic phase into a clean, pre-weighed empty glass finger tube (m0). Place it in an oven to dry the organic reagent, and the lipid remains on the wall of the finger tube. Weigh it again (m1). The total fatty acid content of yeast = (m1 - m0) / 0.15. Each sample is set with 3 replicates.

[0113] 3. Complementary function analysis of the PfLACS gene transformed into the defective yeast YB525

[0114] 3.1 Cultivation of yeast strains

[0115] The yeast strain YB525 (faa1Δfaa4Δ) lacks LACSs necessary for activating exogenous fatty acids and cannot grow normally in minimal medium. It is often used for the analysis of exogenous LACSs activity. Saccharomyces cerevisiae YB525 is an ideal protein expression strain with kanamycin resistance and grows best in YPDA medium at 30 °C.

[0116] 3.2 Preparation and transformation of YB525 yeast competent cells

[0117] The preparation and transformation of Saccharomyces cerevisiae YB525 competent cells are the same as those in Example 2.2 of this embodiment. Inoculate the activated YB525 yeast cells into 30 mL of YPD liquid medium and place them in a shaker at 30 °C for shaking culture until the OD 600 of the bacterial solution = 0.4. Centrifuge at 3000 rpm for 5 min to collect the cells; resuspend the precipitate with 10 mL of Y1 solution and then centrifuge again to collect the cells. Add 1 mL of Y2 solution to the precipitate to resuspend the cells, and the preparation of competent cells is completed. Aliquot 50 μL into a sterile centrifuge tube for transforming one plasmid. To ensure the transformation efficiency, it is best to prepare and use the competent cells immediately. The transformation operation is carried out with reference to the yeast transformation kit.

[0118] 3.3 Identification of recombinant yeast positive

[0119] Pick single colonies from the SC-URA screening plate cultured for 2 days and inoculate them into 5 mL of SC-URA liquid medium containing 2% (v / v) glucose. Culture overnight at 30 °C until the bacterial solution becomes turbid. Take 1 mL of the bacterial solution for cell wall breaking treatment and perform PCR detection with the primers (SEQ ID NO.4 - SEQ ID NO.5) with restriction enzyme sites in Table 5 to identify positive recombinants.

[0120] 3.4 Complementation experiment of perilla LACS1-deficient yeast

[0121] Select positive clones and culture yeast complementation in liquid SC-URA medium lacking uracil. Yeast complementation test is to add galactose and cerulenin to the SC-URA medium to induce the overexpression of exogenous genes, and then transfer the bacterial solution into SC-URA medium with C18:1 fatty acid (oleic acid) as the sole carbon source for culture.

[0122] 4. Results and analysis

[0123] 4.1 The cloned target fragment was ligated with the pYES2.0 vector to obtain a recombinant plasmid containing the target gene, which was then transferred into Escherichia coli and screened with ampicillin, followed by PCR detection. Monoclonal colonies identified as positive by PCR were amplified and the plasmids were extracted for double digestion verification. The plasmids successfully verified by double digestion were sent to the company for sequencing and comparison. The results showed that the sequence was correct and there were no base mutations, proving that the Perilla frutescens PfLACS1 gene had been ligated to the pYES2.0 vector and the recombinant vector was successfully constructed.

[0124] 4.2 The empty pYES2.0 plasmid and the constructed pYES2.0-PfLACS1 recombinant plasmid were transferred into INVSc1 Saccharomyces cerevisiae. The transformed bacterial solution was evenly spread on an SC-URA solid medium containing 2% (v / v) glucose and kanamycin for screening. Single colonies were picked for PCR detection to identify positive clones. The primers with restriction enzyme sites in Table 2 were used as the detection primers for transgenic yeast. The PCR detection results were as Figure 5 shown. The band of the yeast transformed with the pYES2.0-PfLACS1 recombinant vector was around 2000 bp, and the bands obtained by electrophoresis were all consistent with the expected length, indicating that the recombinant plasmid had been successfully transferred into INVSc1 yeast.

[0125] 4.3 The recombinant vector was transferred into INVSc1 Saccharomyces cerevisiae to heterologously express PfLACS1 in yeast. The total fatty acids of the transgenic yeast were extracted for total lipid content analysis. The results of the total lipid content analysis were as Figure 6 shown. Compared with the yeast transformed with the empty vector, the total lipid content of the yeast transformed with PfLACS1 increased significantly by 5.04%. This further indicated that the overexpression of the PfLACS1 gene increased the total oil synthesis and accumulation level of the yeast strain, demonstrating that the expression of the Perilla frutescens PfLACS1 gene could significantly improve the oil synthesis ability of yeast.

[0126] 4.4 To determine whether the encoded protein of the cloned Perilla frutescens PfLACS gene has LACS enzyme activity, it was analyzed using the YB525 yeast complementary expression system. Yeast cells need to absorb the carbon source in the medium to de novo synthesize fatty acids. Cerulenin inhibits this de novo fatty acid synthesis process, and yeast has to absorb exogenous fatty acids from the medium to maintain growth. Yeast strain YB525 is a strain lacking LACS activity. This defective yeast cannot grow in a medium with fatty acids as the sole carbon source unless a gene with LACS enzyme activity is expressed. The yeast complementary test measured the growth status of yeast cells transfected with different vectors after being induced for 84 h in an SC-URA medium with C18:1 fatty acid as the sole carbon source, as Figure 7As shown in the figure. The test results showed that the YB525 yeast cells transformed with the pYES2-PfLACS vector could grow normally, indicating that they could grow normally in the SC-URA medium with only fatty acid C18:1 as the sole carbon source, while the yeast cells transformed with the pYES2 empty vector could not grow normally, indicating that they could not grow in the SC-URA with fatty acid C18:1 as the sole carbon source. It shows that the heterologous expression of the PfLACS gene can complement the LACS enzyme deficiency phenotype of YB525 yeast and has acyl-CoA enzyme activity.

[0127] Example 3. Construction of a constitutive plant expression vector of Perilla frutescens PfLACS1 gene and genetic transformation of tobacco

[0128] 1. Construction of plant recombinant expression vector

[0129] The Agrobacterium tumefaciens GV3101 strain and the pBI121 empty plasmid used in this experiment are as Figure 8 shown and stored in the Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University. The construction steps of the pBI121-PfLACS1 recombinant vector were carried out according to Example 1. The primers used are shown in Table 2 (SEQ ID NO.2~SEQ ID NO.3).

[0130] 2. Agrobacterium transformation and screening of positive strains

[0131] 2.1 Cultivation of Agrobacterium tumefaciens GV3101 strain

[0132] The Agrobacterium tumefaciens GV3101 strain grows best in LB medium at 28°C. Since the GV3101 strain contains the rifampicin resistance gene, it can grow normally on the medium containing rifampicin.

[0133] 2.2 Preparation of Agrobacterium competent cells

[0134] Inoculate the activated Agrobacterium tumefaciens into 50 mL of LB liquid medium containing 50 μg / mL rifampicin, place it in a shaker at 28°C and shake it until the OD of the bacterial solution 600 = 0.4, centrifuge to collect the bacterial cells, resuspend the precipitate with 10 mL of pre-cooled 0.15 M NaCl and centrifuge again. After removing the supernatant, add 10 mL of 20 mM CaCl2 containing 15% (v / v) glycerol to resuspend the cells to make competent cells. Aliquot the competent cells into 200 μL per tube and store them in a -80°C refrigerator for later use.

[0135] 2.3 Transformation of Agrobacterium by freeze-thaw method

[0136] Absorb 5 μL of the recombinant plasmid and add it to 200 μL of competent cells. After ice-bathing for 5 min, place it in liquid nitrogen for 5 min, and then transfer it to a 37°C metal bath for heat shock for 5 min. After cooling, add 800 μL of YEB liquid medium, and culture it with slow shaking at 28°C for 4 h. Then centrifuge to enrich the bacteria, absorb 25 μL and spread it on a solid YEB screening plate containing 50 μg / mL rifampicin and 50 μg / mL kanamycin, and incubate it upside down at 28°C for 48 h.

[0137] 2.4 Screening of positive transformants

[0138] Pick single colonies from the screening plate and inoculate them into 800 μL of YEB medium containing rifampicin and kanamycin, culture them overnight at 28°C, perform bacterial liquid PCR detection, and store the bacterial liquid with positive results after agarose gel electrophoresis amplification for subsequent experiments. The detection primers are the primers with restriction enzyme sites in Table 1, and the PCR detection operation refers to Example 1.

[0139] 3. Tobacco genetic transformation

[0140] 3.1 Agrobacterium-mediated tobacco genetic transformation

[0141] In a laminar flow hood, take common tobacco leaves with a leaf age of 30 days and good growth, cut off the leaf margins and main veins with sterile scissors, and cut the leaves into small pieces of 0.5 cm 2 in size, place them face up on the pre-culture medium, and pre-culture them in an incubator for 48 h; culture the Agrobacterium with the successfully transformed pBI121-PfLACS1 recombinant plasmid until OD 600 = 0.5, and use it to infect the cultured tobacco leaves; soak the pre-cultured leaves in the bacterial liquid for 8 min, stir them several times during this period to improve the infection efficiency of the bacterial liquid, rinse them with sterile water for 1 min, and then place the leaves face up on a new co-culture medium and culture them in the dark for 48 h; transfer the leaves to the screening medium for differentiation culture, replace the fresh medium every 10 days until buds differentiate. Transfer each individual bud to the rooting medium, transfer it to the growth medium after roots grow, and acclimatize and transplant the seedlings when they grow to an appropriate size.

[0142] 3.2 Identification of transgenic tobacco

[0143] Take transgenic tobacco leaves, extract DNA by the CTAB method, use the DNA as a template, add primers with restriction enzyme sites, and perform PCR amplification as shown in Table 1. Detect the target band by agarose gel electrophoresis to determine whether the target gene is integrated into the tobacco genome. Plants with positive DNA detection are further extracted with RNA, reverse transcribed and then PCR amplified to detect whether the target gene is effectively expressed in tobacco. The PCR system and procedure refer to Example 1.

[0144] 3.3 Determination of oil content in transgenic tobacco

[0145] Take the leaves of positive transgenic tobacco plants, freeze-dry them and grind them into powder, extract the total lipids and determine their content. The specific operation steps are as follows:

[0146] Weigh 150 mg of freeze-dried tobacco leaf powder and place it in a 50 mL centrifuge tube. Add 7.5 mL of a mixed solution of methanol and chloroform to it, with the volume ratio of methanol to chloroform being 2:1. After extraction at 37 °C and 200 rpm for 24 h, centrifuge and collect the upper organic phase into a new centrifuge tube. Add another 7.5 mL of the mixed solution of methanol and chloroform to the precipitate, with the volume ratio of methanol to chloroform being 2:1. Repeat the extraction for 12 h and then collect the upper organic phase. Mix the two collected upper organic phases, add 5 mL of chloroform solution and 9 mL of 1% (v / v) NaCl solution, so that V chloroform:V methanol:V water = 2:2:1.8. After thorough mixing, centrifuge and collect the lower organic phase into a clean, pre-weighed empty glass finger tube (m0). Place it in an oven to dry the organic reagent, and the oil remains on the wall of the finger tube. Weigh it again (m1). The total fatty acid content of yeast = (m1 - m0) / 0.05. Set 3 replicates for each sample.

[0147] 4. Results and Analysis

[0148] 4.1 The result analysis sequence is correct and there are no base mutations, proving that the Perilla frutescens PfLACS1 gene has been successfully ligated to the pBI121 vector and the recombinant vector construction is successful.

[0149] 4.2 Use the freeze-thaw method to transfer the constructed pBI121-PfLACS1 recombinant expression vector into Agrobacterium tumefaciens. Pick single colonies on the screening plate for PCR verification. The results are as Figure 9 shown. The position of the electrophoresis band is consistent with the expectation, and the Agrobacterium transformation is successful.

[0150] 4.3 Use the Agrobacterium-mediated leaf disc method to transform tobacco, with kanamycin as the screening marker, and conduct genetic transformation of tobacco as Figure 10 shown. Transfer PfLACS1 into tobacco. Take the leaves of transgenic plants, extract DNA, and the results are as Figure 11 shown. The electrophoresis band is consistent with the length of the target gene. For the plants with positive PCR detection, extract the leaf RNA again, perform PCR amplification after reverse transcription, and detect the expression of the target gene by electrophoresis. The results are as Figure 12 shown. The electrophoresis band is consistent with the length of the target gene, indicating that the target gene has been successfully integrated into tobacco and is effectively expressed.

[0151] 4.4 Take the leaves of transgenic tobacco, vacuum freeze-dry them and grind them into powder, extract the total lipids, and analyze the change in the total lipid content of tobacco leaves. The results are as Figure 13, compared with the tobacco leaves under no-load rotation, the total lipid content of the tobacco leaves with the PfLACS1 gene transferred increased significantly by 3.85%, indicating that the expression of the Perilla frutescens PfLACS1 gene can significantly improve the lipid synthesis ability of tobacco.

[0152] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0153] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A Perilla frutescens PfLACS1 gene for regulating plant oil synthesis, characterized in that, The nucleotide sequence of the perilla PfLACS1 gene is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that, The recombinant expression vector contains the coding gene described in claim 1 and the backbone plasmid ligating the coding gene.

3. The recombinant expression vector according to claim 2, characterized in that, The backbone plasmid is pBI121 or pYES2.

0.

4. The recombinant expression vector according to claim 2, wherein The method for constructing the recombinant expression vector is to amplify the perilla PfLACS1 gene using a specific primer pair, recover the PCR product and ligate it with the backbone plasmid pBI121 to obtain the recombinant expression vector for plants; recover the PCR product and ligate it with the backbone plasmid pYES2.0 to obtain the recombinant expression vector for yeast.

5. The recombinant expression vector according to claim 4, wherein The nucleotide sequences of the specific primer pair are shown in SEQ ID NO.2 and SEQ ID NO.

3.

6. A recombinant bacterium, characterized in that, The recombinant bacterium is obtained by transforming the recombinant expression vector described in claim 2 into an engineering bacterium.

7. The recombinant bacterium according to claim 6, wherein The engineering bacterium is Agrobacterium tumefaciens GV 3101.

8. The application of the perilla PfLACS1 gene for regulating plant oil synthesis described in claim 1, the recombinant expression vector described in claim 2 or the recombinant bacterium described in claim 6 in increasing the total oil content in plant leaves.

9. The application according to claim 8, characterized in that, The plant is tobacco.

10. The application according to claim 8, wherein The heterologous expression of the perilla PfLACS1 gene can complement the LACS enzyme deletion phenotype of YB525 yeast and has acyl-CoA enzyme activity.

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