CN115807028B - Application of transcription factor SlDOG1 in improving plant steroid alkaloid content - Google Patents

Abstract

The invention discloses an application of a transcription factor SlDOG1 in improving the content of plant steroid alkaloids, belonging to the technical field of biology. The amino acid sequence of the transcription factor SlDOG1 is shown in SEQ ID NO:4 is shown in the figure; the nucleotide sequence of the gene for encoding the transcription factor SlDOG1 is shown in SEQ ID NO: 3. According to the invention, the natural variation of the steroid alkaloid content of 362 parts of tomatoes is analyzed, and a transcription factor SlDOG1 on chromosome 10 is positioned by combining with GWAS data, and experiments prove that the transcription factor is over-expressed in plants, so that the steroid alkaloid content can be obviously improved. Therefore, the invention provides theoretical basis for the synthesis and molecular assisted breeding of tomato steroid alkaloid.

Description

Application of transcription factor SlDOG1 in increasing phytosteroidal alkaloid content

technical field

The invention relates to the field of biotechnology, in particular to the application of transcription factor SlDOG1 in increasing the content of phytosteroid alkaloids.

Background technique

Plants produce and accumulate many biologically active natural products during evolution, including alkaloids, terpenes, flavonoids and other metabolites. Steroidal alkaloids (Steroidals: SAs) and their glycosylated forms Steroidal glycoalkaloids (Steroidals glycoalkaloids: SGAs) are nitrogen-containing toxic compounds, mainly found in Solanaceae and Liliaceae plants, about 1350 species. α-solanine and α-chaconine are the main steroidal alkaloids in Solanaceae vegetable crops (Solanum tuberosum L.), accounting for about 90% of the total steroidal alkaloids (SAs and SGAs). Steroidal alkaloids help humans and plants defend against external pathogens and predators, however, due to their toxic effects, some steroidal alkaloids are considered anti-nutritional compounds in humans. Therefore, it is particularly important to study the synthesis and regulation of steroidal alkaloids in plants.

Studies have shown that tomato has become one of the most popular fruits and vegetables in the world because of its unique flavor and high nutritional value, and it is also an important source of micronutrients in human diet. The content of steroidal alkaloids in tomato is directly related to the taste, flavor and nutritional quality of the fruit. Changing the content of tomato steroidal alkaloids not only improves the quality of tomato fruit, but also contributes to human health. Therefore, it is of great significance to study the transcription factors involved in regulating the synthesis of tomato steroidal alkaloids for improving the content of tomato steroidal alkaloids.

Contents of the invention

The purpose of the present invention is to provide the application of transcription factor SlDOG1 in improving the content of phytosteroidal alkaloids, to solve the problems in the prior art mentioned above, overexpressing the SlDOG1 transcription factor gene in Solanaceae plants to obtain plants with improved steroidal alkaloids content resistance, providing a theoretical basis for the synthesis of tomato steroidal alkaloids and molecular assisted breeding.

To achieve the above object, the present invention provides the following scheme:

The invention provides the application of transcription factor SlDOG1 in increasing the content of phytosteroidal alkaloids, the amino acid sequence of the transcription factor SlDOG1 is shown in SEQ ID NO: 4: MTGGSCSSHDDNFFEVFLVGWFIRQEQFQNELVVAQDTFDDQENDMRGLIHRVLAHYQQYYEEKSRMTHRNVFRVFSPTWFSPLERSFLWITGFNPGLVFNLVTNSINDLSEHQVERLNRL KQETKAQERSLTKELAQIQESVASPPLVDLARRLGTQLLYTDNINTDIEEVDGDIDQLKTALENVVTDADRLRRTTAERVVGLLSPLQSLKFLSAVGQLQLRARRMMGMEREVERQQQRNEDTNGW.

The present invention also provides the application of the gene encoding the transcription factor SlDOG1 in increasing the content of plant steroidal alkaloids, the nucleotide sequence of the gene is shown in SEQ ID NO: 3: ATGACAGGAGGATCATGTAGTAGTCATGATGATAATTTTTTTGAGGTGTTTCTAGTAGGTTGGTTCATTAGACAAGAACAATTTCAAAATGAACTTGTTGTAGCACAAGATACTTTTGATGATCAAGAAAATGATATGAGAGGCC TTATTCATAGGGTTTTTAGCTCATTATCAACAATATTATGAAGAAAAATCAAGAATGACTCATAGGAATGTTTTTAGAGTCTTTTTCACCTACTTGGTTTCTCCACTTGAAAGAAGCTTCTTATGGATCACAGGGTTCAATCCGGCTTAGTTTTTAACCTGGTAACTAATTCCATAAACGATTTGAGTGAACATCAAGTTGAAAGGCTCAACAGGTTGAAACAAGAGACAA AGGCTCAAAGAAAGGTCCTTAACGAAGGAGTTGGCACAAATCCAAGAGAGTGTGGCCTCACCACCGTTGGTGGATCTAGCACGTCGATTAGGAACGCAACTTTTTACACTGACAATATAAACACAGATATCGAGGAGGTTGATGGAGACATAGACCAATTGAAGACGGCCTTGGAGAATGTGGTGACGGATGCTGATAGGTTAAGGACAAGAACAGCTGA AAGAGTTGTAGGGTTACTTAGTCCCTTGCAAAGTTTTGAAGTTTTTTGAGTGCTGTAGGTCAGTCCAATTGAGGGCAAGGATGATGGGAATGGAAAGAGAAGTTGAGAGGCAACAACAAAGAAATGAAGATACAAATGGATGGTGA.

The invention also provides the application of the recombinant vector containing the gene in increasing the content of phytosteroid alkaloids.

The invention also provides the application of the host bacteria containing the recombinant vector in increasing the content of phytosteroid alkaloids.

Preferably, the steroidal alkaloids include tomatine, α-tomatine, hydroxylated tomatine, Lycoperoside B and (Lycoperoside H)FA.

Preferably, the plant is a Solanaceae plant.

Preferably, the plant of the family Solanaceae is tomato.

The present invention also provides a method for increasing the content of phytosteroidal alkaloids, by overexpressing the transcription factor SlDOG1 in plants to obtain transgenic plants with increased phytosteroidal alkaloids content; wherein, the amino acid sequence of the transcription factor SlDOG1 is as shown in SEQ ID NO: 4.

Preferably, the plant is tomato.

Preferably, the steroidal alkaloids include tomatine, α-tomatine, hydroxylated tomatine, Lycoperoside B and (Lycoperoside H)FA.

The invention discloses the following technical effects:

The invention provides that the SlDOG1 transcription factor has a positive regulatory effect on the synthesis of tomato steroidal alkaloids, further regulates the accumulation of steroidal alkaloids by regulating the expression of tomato steroidal alkaloid synthesis genes, and reveals the metabolic pathway of tomato steroidal alkaloids Therefore, overexpression of the SlDOG1 transcription factor gene in plants, especially in Solanaceae plants, can obtain plant resistance to increased steroidal alkaloid content, providing a theoretical basis for the synthesis of tomato steroidal alkaloids and molecular assisted breeding .

Description of drawings

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 accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

Figure 1 shows the expression of SlDOG1 in tomato stable transgenic leaves detected by real-time fluorescent quantitative PCR; OE#3, OE#27 and OE#29 are three over-expression plants; WT: wild type;

Figure 2 is the detection of steroidal alkaloid content in tomato stable transgenic leaves by LC-MS; SIDOG1 OE#3, SIDOG1 OE#27 and SIDOG1 OE#29 are three transgenic leaves; CK-1 and CK-2 are controls.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. The 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.

It should be understood that the terminology described in the present invention is only used to describe specific embodiments, and is not used to limit the present invention. In addition, regarding the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents are described. In case of conflict with any incorporated document, the contents of this specification control.

It will be apparent to those skilled in the art that various modifications and changes can be made in the specific embodiments of the present invention described herein without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the present invention. The specification and examples in this application are exemplary only.

As used herein, "comprising", "comprising", "having", "comprising" and so on are all open terms, meaning including but not limited to.

Cloning of embodiment 1 tomato SlDOG1 gene

(1) Extraction of total RNA from tomato leaves

Take an appropriate amount of tomato leaves, quick-frozen in liquid nitrogen, and use a rapid grinder to fully pulverize three times under the conditions of 45Hz and 30s, add lysate, and extract total RNA according to the instructions of Plant RNA Extraction Kit V1.5 (BIOFIT). The RNA quality was detected by agarose gel electrophoresis, and the RNA concentration was detected by a NanoDrop 2000 spectrophotometer.

(2) Cloning of tomato SlDOG1 gene

Using the extracted total RNA as a template, cDNA was synthesized according to the instructions of the Takara reverse transcription kit; gene-specific primers were designed according to the sequence of the SlDOG1 gene, and the specific sequence was as follows:

SlDOG1 upstream primer: 5'-AAAAAGCAGGCTTAATGACAGGAGGATCATGTAGTAGTCATG-3' (SEQ ID NO: 1);

SlDOG1 downstream primer: 5'-AGAAAGCTGGGTATCACCATCCATTTGTATCTTCATTTC-3' (SEQ ID NO: 2).

The SlDOG1 gene was amplified by PCR with Takara high-fidelity enzyme Primerstar Max, then connected to the pDONR207 vector, transformed into Escherichia coli DH5α, and positive clones were picked and sequenced.

The results showed that the full-length coding sequence of tomato SlDOG1 was amplified, and the nucleotide sequence was shown in SEQ ID NO: 3; the protein-coding amino acid sequence was shown in SEQ ID NO: 4, and the start codon was ATG, and the stop codon was ATG The sub is TGA.

Embodiment 2 Construction of the plant overexpression vector containing SlDOG1

In order to study the effect of SlDOG1 gene on the content of steroidal alkaloids in tomato, the overexpression vector 35S-SlDOG1-PBI121 vector was constructed. The non-specific arms of the respective vectors were added to the forward and reverse primers, and the primers are shown in Table 1.

Table 1 SlDOG1-PBI121 vector construction primer sequence

Using the correctly sequenced T4 vector containing the SlDOG1 gene as a template, PCR amplification was performed using the Primerstar Max high-fidelity enzyme from Takara Company with SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The reaction conditions of PCR were pre-denaturation at 98°C for 5min, denaturation at 98°C for 30s, annealing at 60°C for 20s, extension at 72°C for 1min30s, 30 cycles, and extension at 72°C for 5min. The expression vector 35S-SlDOG1-PBI121 was obtained by connecting the gateway and goldenbraid into the SlDOG1-PBI121 vector for sequencing verification.

In this example, the tomato 35S-SlDOG1-PBI121 gene is operably linked to the expression control sequence, and the vector can be used to regulate the content of tomato steroidal alkaloids through a transcriptional regulation strategy.

Example 3 Agrobacterium tumefaciens mediated genetic transformation of 35S-SlDOG1-PBI121 overexpression vector to obtain transgenic tomato plants

1. Acquisition of Agrobacterium tumefaciens Engineering Bacteria Containing SlDOG1-PBI121 Expression Vector

The 35S-SlDOG1-PBI121 expression vector obtained in Example 2 was transformed into Agrobacterium tumefaciens (taking EHA105 as an example) by electroporation, and then verified by PCR. The method was as described above in the PCR procedure.

The results showed that the 35S-SlDOG1-PBI121 expression vector had been successfully constructed into the Agrobacterium tumefaciens strain.

2. Transformation of tomato with 35S-SlDOG1-PBI121 gene mediated by Agrobacterium tumefaciens

The specific operation steps are as follows:

(1) Tomato sterile cotyledon preparation and pre-cultivation

The seeds were sterilized with 20% sodium hypochlorite solution for 15 minutes, about 30 seeds per bottle, spread on 1/2MS (Murashige and Skoog, 1962) solid medium, and cultivated at 25°C, 16h/8h (light/dark) for 7-10 days, tomato sterile cotyledons can be obtained. Cut off both ends of the cotyledons in the seedling cutting solution (MS+KH ₂ PO ₄ 200mg/L+2,4-D 0.2mg/L+KT 0.1mg/L), and place them in the pre-medium (1/2MS+IAA 1mg /L+ZR 2mg/L) for subsequent transformation.

(2) Co-cultivation of Agrobacterium and explants

Add the explants obtained in step (1) into the activated Agrobacterium tumefaciens suspension (MS+KH ₂ PO ₄ 200 mg/L+acetosyringone (AS) 200 μmol/L) containing the SlDOG1-PBI121 expression vector, The bacterial solution was in full contact with the explants for 18 minutes, transferred to the co-cultivation medium (MS+AS 200 μmol/L), wrapped in toilet paper, and placed in a 25°C incubator for 2 days under low light.

(3) rebirth culture and rooting culture of explant

The explants in step (2) are placed in the regeneration medium (MS+IAA 1mg/L+ZR 2mg/L+Kan100mg/L+Tim 320mg/L), and the medium is changed every two weeks until sprouting; Afterwards, the callus is stripped off, transferred to the rooting medium (MS+Kan 50mg/L+Tim 320mg/L), and the medium is replaced every 4 weeks until roots grow out to form a complete plant.

(4) transplanting transgenic plants

The sterile and stable transgenic plants obtained in step (3) were trained overnight in an incubator at 25°C, and then moved into soil pots until strong roots, stems, leaves and other tissues grew.

3. Identification of positive transgenic plants

According to the 35S-SlDOG1-PBI121 expression vector, forward and reverse detection primers were designed to detect the transgenic plants. The specific primer sequences are shown in Table 2.

Table 2 Primer sequences for identification of 35S-SlDOG1-PBI121 positive transgenic tomato plants

Primer name Primer sequence (5'-3') Kan-F (SEQ ID NO: 7) CCGGATCTGGATCGTTTCGCATG Kan-R (SEQ ID NO: 8) GGTCATTTCGAACCCCAGAGTCCC

Transgenic plants containing the SlDOG1-PBI121 expression vector were detected with SEQ ID NO: 9 and SEQ ID NO: 10.

The test results showed that with the designed primer sequences, the positive transgenic plants could amplify specific DNA fragments, while the negative and wild-type tomatoes did not amplify any fragments.

4. Quantitative detection of SlDOG1-PBI121 gene expression in transgenic tomato plants

The transgenic tomato leaves of each transgenic tomato line containing the 35S-SlDOG1-PBI121 expression vector were harvested, quick-frozen in liquid nitrogen, and RNA was extracted according to the method in Example 1 using a rapid grinder to obtain cDNA. The cDNA was diluted 20 times with RNA-free water, and the SYBR Supermix of Bio-Rad Company was used to perform fluorescent quantitative PCR, and SlUBI was used as a control gene to detect the expression level of transgenic tomato fruit, wherein the primer sequences of the target gene were as SEQ ID NO: 9 and SEQ ID NO: 10, and the sequence of the control UBI primer is shown in Table 3.

Table 3 Primer sequences for detection of gene expression in 35S-SlDOG1-PB121 transgenic tomato hairy roots

PCR program: PCR reaction conditions: 94°C pre-denaturation for 5min, 94°C denaturation for 30s, 60°C annealing for 20s, 68°C extension for 1min30s, 30 cycles, 68°C extension for 5min.

The result is shown in Figure 1. The results showed that the expression level of SlDOG1-PBI121 leaves of the transgenic tomato with 35S promoter was higher than that of the wild type, indicating that the SlDOG1 gene was successfully transferred into tomato, and the tomato plants OE#3, OE#27 and OE# overexpressing the SlDOG1 gene were successfully obtained. 29.

In this example, the plant expression vector was transformed into Agrobacterium tumefaciens to obtain the engineering strain of Agrobacterium tumefaciens used to transform the 35S-SlDOG1-PBI121 expression vector of tomato, and the constructed Agrobacterium tumefaciens was used to transform tomato to obtain the Transgenic tomato plants with positive PCR results. The acquisition of transgenic tomato plants provides direct material and theoretical basis for screening high-content steroidal alkaloids.

Example 4 LC-MS Determination of Steroidal Alkaloid Content in Transgenic Tomato

(1) Sample processing

The 35S-SlDOG1-PBI121 transgenic leaves were harvested, quick-frozen in liquid nitrogen, freeze-dried at a low temperature in a freeze dryer to constant weight, and ground into a powder using a mixing mill (MM400, Retsch) and zirconia beads at 30 Hz for 1 minute.

Weigh 100 mg of the powder, and extract metabolites with 1.0 mL of 70% aqueous methanol (methanol:H ₂ O, 70:30, v/v) at 4°C overnight. After centrifugation at 12,000 rpm for 10 minutes, all supernatants were pooled and filtered (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China). In addition, to ensure the stability and applicability consistency of mass spectrometry analysis, all filtered supernatants were mixed in equal volumes (10 μL) to prepare quality control (QC) samples before LC-MS analysis. In addition, relative signal intensities of metabolites were also divided and normalized using an internal standard (0.1 mg/L lidocaine).

(2) UHPLC-TQ-MS analysis

For each sample, three biological replicates were analyzed independently. UHPLC-MS/MS analysis was performed using a 7500 Triple Quadrupole Linear Ion Trap Mass Spectrometer (AB Sciex, Framingham, MA, USA). The analysis conditions are as follows:

The HPLC column is Waters ACQUITY UPLC HSS T3 C18 (1.8μm, 2.1mm×100mm);

Solvent system: water (0.04% acetic acid): acetonitrile (0.04% acetic acid);

Gradient program: 95:5v/v at 0 minutes, 5:95v/v at 11.0 minutes, 5:95v/v at 12.0 minutes, 95:5v/v at 12.1 minutes, 95:5v/v at 14.0 minutes;

Flow rate: 0.35mL/min; temperature, 40°C;

Injection volume: 2 μL.

The effluent was alternately connected to the esi-triple quadrupole linear ion trap (Q trap)-MS. Linear ion trap (LIT) and triple quadrupole (QQQ) scans were performed on an ESI 7500QTRAP LC/MS/MS system equipped with an ESI turbo ion spray interface, working in positive ion mode, by Sciex OS 2.1.6 software (AB Sciex) controls. The working parameters of the ESI source are: ion source, turbo spray; source temperature, 500°C; ion spray (IS) voltage, 2500V; ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) are set to 45, 75, and 45.0 psi; collision gas (CAD) set at 11 psi. Instrument tuning and mass calibration were performed in QQQ and LIT modes with 10 μmol/L and 100 μmol/L polypropylene glycol solutions, respectively. The QQQ scans were acquired in multiple reaction monitoring (MRM) mode, and the collision energy (CE) of each MRM transition was determined and further optimized. A specific set of MRM transitions is monitored based on the elution of metabolites at each stage. After obtaining the metabolite spectral analysis data of different samples, MultiQuant 3.0.3 was used to integrate the peak area of the mass spectrum peaks of all metabolites to obtain the metabolite content in these samples. The mass spectrum peaks were analyzed according to the metabolite retention time and peak shape information to ensure the accuracy of quantification.

As a result, as shown in Figure 2, the leaves of the 35S-SlDOG1-PBI121 overexpression vector significantly increased the content of some steroid alkaloids, for example, tomatidine (tomatidine glycoside), α-tomatine (α-tomatine), Hydroxytomatidine (hydroxytomatidine Lycoperoside B and (Lycoperoside H)FA. Among them, Hydroxytomatidine (hydroxylated tomatidine), Lycoperoside B and (Lycoperoside H) FA were all increased by about 2-3 times in the 35S-SlDOG1-PBI121 strain.

In this example, the UHPLC-MS/MS method was used to measure the steroid alkaloid content in the transgenic leaves of the 35S-SlDOG1-PBI121 overexpression vector, and the metabolic engineering strategy of the successful transformation of the SlDOG1 overexpression vector was used to find that the expression level of the SlDOG1 gene was correlated with the steroid The content of tomato steroidal alkaloids has a significant positive correlation, which provides a strong experimental basis for the use of this gene for overexpression research to improve the content of tomato steroidal alkaloids.

The above-mentioned embodiments are only to describe the preferred mode of the present invention, and are not intended to limit the scope of the present invention. Variations and improvements should fall within the scope of protection defined by the claims of the present invention.

Claims (5)

1. The application of the overexpression transcription factor SlDOG1 in improving the content of phytosteroid alkaloids, characterized in that the amino acid sequence of the transcription factor SlDOG1 is as shown in SEQ ID NO: 4, the plant is tomato, and the steroid The alkaloids are alpha-tomatine, hydroxylated tomatine, Lycoperoside B and (Lycoperoside H)FA. 2. the application of the gene of the transcription factor SlDOG1 described in coding claim 1 in improving phytosteroid alkaloid content, it is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO: 3, described The plant is tomato and the steroidal alkaloids are alpha-tomatine, hydroxylated tomatine, Lycoperoside B and (Lycoperoside H)FA. 3. the application of the recombinant vector comprising the gene described in claim 2 in improving the content of phytosteroidal alkaloids is characterized in that, the plant is tomato, and the steroidal alkaloids are α-tomatine, hydroxylated Tomatine, Lycoperoside B, and (Lycoperoside H)FA. 4. The application of the host bacterium comprising the recombinant vector described in claim 3 in improving the content of phytosteroidal alkaloids, the plant is tomato, and the steroidal alkaloids are α-tomatine, hydroxylated tomatine, Lycoperoside B and (Lycoperoside H)FA. 5. A method for improving phytosteroid alkaloid content, characterized in that, the transcription factor SlDOG1 is overexpressed in plants to obtain transgenic plants that the phytosteroid alkaloid content improves; wherein the amino acid sequence of the transcription factor SlDOG1 As shown in SEQ ID NO: 4, the plant is tomato, and the steroidal alkaloids are α-tomatine, hydroxylated tomatine, Lycoperoside B and (Lycoperoside H)FA.

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