CN116254291B - Method for inhibiting potato solanine and application - Google Patents

Abstract

The invention discloses a method for inhibiting potato solanine and application thereof. In particular, the overexpression of the ethylene response factor can effectively inhibit the expression of the potato tuber solanine without affecting the solanine content of other parts of the potato, and the gene plays an important role in potato breeding.

Description

A method for inhibiting potato solanine and its application

Technical Field

The invention belongs to the field of plant genetic engineering, and in particular relates to a method for inhibiting potato solanine and its application.

Background technique

Potato (Solanum tuberosum) is an annual crop of the Solanaceae family. In addition to providing a large amount of high-quality starch, potatoes are also rich in vitamin C, amino acids, minerals and dietary fiber, which can meet the Chinese people's pursuit of diversified nutrition and healthy diet. It is the world's third largest food crop after rice and wheat. However, during the storage process, potatoes often cause tuber germination and rot due to environmental discomfort, resulting in the production of toxic substances mainly solanine, which produces an unpleasant bitter taste (10-20mg/100g·FW). Excessive intake can also cause poisoning in humans and animals (2-5mg/kg body weight), which is a food safety issue that needs to be paid great attention to.

Solanine, also known as solanine, is a general term for plant steroidal glycoalkaloids (SGAs), which are composed of hydrophobic aglycones (glycosides) and hydrophilic oligosaccharide chains. The aglycone part is composed of a cyclopentane polyhydrophenanthrene (non-polar steroidal unit) and a nitrogen-containing heterocycle (nitrogen nucleus). There are more than 100 known solanines, most of which are found in plants of the Solanaceae and Liliaceae families. In addition to α-solanine (C ₄₅ H ₇₃ O ₁₅ N) and α-chaconine (C ₄₅ H ₇₃ O ₁₄ N), the main components of solanine in potatoes also include small amounts of tricholamine, camosanine, lepasoline and lepasoline. However, since α-solanine and α-chaconine account for more than 95% of the total solanine in potato tubers, the total solanine content of potatoes is usually represented by the sum of the contents of α-solanine and α-chaconine.

Although solanine in potatoes has caused serious food safety problems to a large extent, the accumulation of solanine in stems and leaves can effectively prevent damage from diseases and insects. Therefore, it is necessary to effectively reduce the solanine content in tubers without affecting the growth of potato plants. This is also one of the important goals of potato breeding.

Summary of the invention

In view of this, in order to solve the drawbacks of the above-mentioned prior art, the present invention cloned the ethylene response factor gene and found that it can be used as the StTSI (tuber SGAs inhibitor) gene, which can effectively reduce the solanine content in tubers. Overexpression of the StTSI gene can significantly reduce the solanine content in tubers, but does not affect the solanine content in the leaves of the plant, thereby effectively avoiding food safety problems caused by solanine. The ethylene response factor can be used as an effective inhibitor of the solanine content in potato tubers.

A first aspect of the present invention provides a method for constructing a transgenic plant, wherein the transgenic plant overexpresses an ethylene response factor.

Preferably, the coding sequence of the ethylene response factor comprises a nucleotide sequence having more than 80% homology with SEQ ID NO:1 or comprises the nucleotide sequence shown in SEQ ID NO:1.

Preferably, the amino acid sequence of the ethylene response factor has more than 80% homology with SEQ ID NO: 2 or comprises the amino acid sequence shown in SEQ ID NO: 2.

Preferably, the construction method comprises introducing a nucleotide sequence encoding an amino acid sequence having more than 80% homology with SEQ ID NO: 2 or comprising SEQ ID NO: 2 into the genome of the plant.

Preferably, the construction method comprises introducing a nucleotide sequence having more than 80% homology with SEQ ID NO: 1 or comprising SEQ ID NO: 1 into the genome of the plant.

Preferably, the construction method comprises constructing a transgenic plant using a vector comprising a sequence encoding an ethylene response factor.

Preferably, the vector is a plant binary vector.

Preferably, the vector comprises a 35S promoter.

Preferably, the ethylene response factor is expressed on a chromosome or on a vector.

Preferably, the plant is a potato plant, and further preferably, the potato plant variety is CIP65.

Preferably, the solanine content of the roots, stems, leaves, flowers, fruits and/or seeds of the potato plants is reduced, further preferably, the solanine content of the potato stems is reduced, and more preferably, the solanine content of the potato tubers is reduced.

Preferably, the construction method comprises the following steps:

1) introducing the coding sequence of the ethylene response factor into the vector by homologous recombination technology;

2) Plant pre-cultivation;

3) Infection and co-cultivation: The vector in 1) is introduced into the plant by Agrobacterium transformation;

4) After callus screening, differentiation, rooting, transplanting and identification, plants overexpressing ethylene response factors are obtained.

Preferably, the identification step comprises:

(1) DNA extraction;

(2) Vector transfer identification: PCR amplification identification, followed by agarose gel electrophoresis;

(3) RNA extraction and reverse transcription;

(4) qPCR was used to measure the expression level changes.

The second aspect of the present invention provides a plant obtained by the above-mentioned construction method, wherein the plant is a potato plant, and preferably, the potato plant variety is CIP65.

Preferably, the solanine content of the roots, stems, leaves, flowers, fruits and/or seeds of the potato plants is reduced, further preferably, the solanine content of the potato stems is reduced, and more preferably, the solanine content of the potato tubers is reduced.

Preferably, the solanine includes α-solanine and α-chaconine.

The third aspect of the present invention is a method for inhibiting solanine in potatoes, wherein the method comprises cultivating the transgenic plants obtained by the above-mentioned construction method, and then obtaining potatoes.

Preferably, the solanine content of the roots, stems, leaves, flowers, fruits and/or seeds of the potato plants is reduced, further preferably, the solanine content of the potato stems is reduced, and more preferably, the solanine content of the potato tubers is reduced.

Preferably, the solanine includes α-solanine and α-chaconine.

A fourth aspect of the present invention provides a cell, wherein the cell overexpresses an ethylene response factor.

Preferably, the coding sequence of the ethylene response factor comprises a nucleotide sequence having more than 80% homology with SEQ ID NO:1 or comprises the nucleotide sequence shown in SEQ ID NO:1.

Preferably, the amino acid sequence of the ethylene response factor has more than 80% homology with SEQ ID NO: 2 or comprises the amino acid sequence shown in SEQ ID NO: 2.

Preferably, the cell comprises a vector comprising a sequence encoding an ethylene response factor.

Preferably, the vector is a plant binary vector.

Preferably, the vector comprises a 35S promoter.

Preferably, the cells are cells of potato plants, and further preferably, the potato plant variety is CIP65.

The fifth aspect of the present invention provides the use of an ethylene response factor in inhibiting solanine in plants, wherein the plants overexpress the ethylene response factor.

Preferably, the coding sequence of the ethylene response factor comprises a nucleotide sequence having more than 80% homology with SEQ ID NO:1 or comprises the nucleotide sequence shown in SEQ ID NO:1.

Preferably, the amino acid sequence of the ethylene response factor has more than 80% homology with SEQ ID NO: 2 or comprises the amino acid sequence shown in SEQ ID NO: 2.

Preferably, the plant comprises a vector comprising a sequence encoding an ethylene response factor.

Preferably, the vector is a plant binary vector.

Preferably, the vector comprises a 35S promoter.

Preferably, the plant is a potato plant, and further preferably, the potato plant variety is CIP65.

Preferably, the solanine content of the roots, stems, leaves, flowers, fruits and/or seeds of the potato plants is reduced, further preferably, the solanine content of the potato stems is reduced, and more preferably, the solanine content of the potato tubers is reduced.

Preferably, the solanine includes α-solanine and α-chaconine.

In a sixth aspect of the present invention, a solanine inhibitor is provided, wherein the coding sequence of the solanine inhibitor comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 1 or comprises the nucleotide sequence shown in SEQ ID NO: 1.

Preferably, the amino acid sequence of the solanine inhibitor has more than 80% homology with SEQ ID NO: 2 or comprises the amino acid sequence shown in SEQ ID NO: 2.

Preferably, the solanine inhibitor is a potato plant solanine inhibitor.

Preferably, the solanine inhibitor inhibits solanine in roots, stems, leaves, flowers, fruits and/or seeds of potato plants. Further preferably, the solanine inhibitor inhibits solanine in potato stems. More preferably, the solanine inhibitor inhibits solanine in potato tubers.

The seventh aspect of the present invention provides a pharmaceutical composition, which includes the solanine inhibitor mentioned above and pharmaceutically acceptable excipients.

Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

The "overexpression" and "reduced content" mentioned in the present invention are all based on the normal growth potatoes that have not been modified as the control, among which, the content of solanine inhibitor is higher than that of normal growth potatoes, which is "overexpression".

When the term “comprising” as used in the present invention is used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

The "homology" mentioned in the present invention refers to that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39 %, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology.

The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

All patents and publications mentioned in this specification are incorporated herein by reference as a whole. Those skilled in the art will recognize that certain changes may be made to the present invention without departing from the concept or scope of the present invention.

Beneficial effects of the present invention:

The invention discloses a method for inhibiting solanine in potato. After the ethylene response factor is over-expressed, only the solanine content in potato tubers is reduced, and no obvious effect is exerted on above-ground organs such as leaves, thereby preventing potato plants from being easily harmed by diseases and insect pests due to the reduction of solanine content in above-ground parts, and solving the food safety problem caused by excessive solanine content in edible parts, namely potato tubers.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

Figure 1: Growth status of each plant, where a is a CIP65 plant (control group), with a plant height of 97 cm and a plant width of 1.15 cm; b is an OE-1 plant, with a plant height of 90 cm and a plant width of 1.18 cm; c is an OE-2 plant, with a plant height of 75 cm and a plant width of 1.11 cm; d is an OE-3 plant, with a plant height of 85 cm and a plant width of 1.02 cm;

Figure 2: Candidate gene expression and solanine content in transgenic plants, where a is the relative expression of StTSI in leaves of CIP65 (control group) and three overexpression plants (experimental group), and b is the solanine content in leaves of the corresponding plants; c is the relative content of StTSI in tubers of CIP65 (control group) and three overexpression plants (experimental group), and d is the solanine content in tubers of the corresponding plants;

Figure 3: pCAMBIA 1305.4 vector map.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Example 1 Preparation of potatoes overexpressing solanine inhibitor

After genome-wide association analysis of tuber solanine content in different diploid potato materials, it was found that the genetic locus on chromosome 1 was highly correlated with tuber solanine content. After analyzing the functional genes in this locus, it was found that a gene encoding an ethylene response factor may be the tuber solanine inhibitor (tuber SGAsinhibitor, StTSI) we are looking for. Among them, the coding sequence of the ethylene response factor is shown in SEQ ID NO: 1, and the protein sequence is shown in SEQ ID NO: 2. In order to verify whether the ethylene response factor gene has the function of inhibiting tuber solanine content, the gene was overexpressed.

First, in order to obtain the coding sequence of the StTSI gene, the applicant planted the sequenced potato variety CIP65 for one month, and then used the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN Company) to extract the total RNA of CIP65 leaves (the extraction method was based on the instructions of the above kit), and used the reverse transcription kit PrimeScriptTM RT reagent Kit with gDNA Eraser (purchased from TaKaRa Company) to reverse transcribe the RNA into cDNA. The reaction conditions were divided into two steps, the first step was: 42°C 2min; the second step was: 37°C 15min, 85°C 5s. Using this cDNA as a template, PCR amplification was performed with primers StTSI-F: 5’-GGACTCTAGAGGATCCATGGATTACAAGGACGACGATGACAAGGAAGATCA TAATGAATTCTC-3’ (SEQ ID NO: 3) and StTSI-R: 5’-GTCACCAATTCACACGTGCTAATAGCACTCCCACAAAA-3’ (SEQ ID NO: 4) to obtain the full-length coding sequence (CDS sequence) of the StTSI gene with a FLAG tag added (807 bp in total).

PCR reaction conditions: 98°C for 10 sec, 60°C for 15 sec, 68°C for 50 sec, for 34 cycles.

The PCR product obtained by amplification was connected into the plant binary vector pCAMBIA 1305.4 through homologous recombination technology as shown in Figure 3, which contains a strong 35S promoter. The positive clones were screened and sequenced to confirm, and the recombinant clone plasmid OE-StTSI was obtained and used for transformation. That is, it was introduced into the potato variety CIP65 through the Agrobacterium-mediated potato genetic transformation system, and transgenic plants were obtained after pre-culture, infection, co-cultivation, screening of callus with hygromycin resistance, differentiation, rooting, transplanting, and identification. A total of 3 independent transgenic potato plants were obtained in the transformation vector.

The specific steps of genetic modification include:

(1) Cultivating sterile seedlings: The stem tips of the donor potato material CIP65 preserved aseptically were cultured on MS30 basal medium (an average of 6-7 stem tips per bottle for propagation), with a culture temperature of 2322°C, a humidity of 40%-50%, a light intensity of 2000-2500 lx, and alternating cultivation with 16 h of light and 8 h of darkness.

(2) Pre-culture of seedlings: When the sterile seedlings mature (about one month), select internodes of plants with emerald green color and compact tissue as explants, spread them on MS20 plate culture medium for pre-culture for 2 days, with 100 explants per culture dish;

(3) Agrobacterium culture: Agrobacterium GV3101 (from Shanghai Weidi Biotechnology, commercial strain) was pre-cultured on LB solid medium with corresponding resistance selection for two days at a culture temperature of 28° C. The Agrobacterium was transferred to LB liquid medium and cultured on a shaker at 28° C. for 12 hours;

(4) Agrobacterium infection and co-cultivation: The Agrobacterium bacterial liquid was centrifuged (4°C, 4000 rpm, 10 min), the bacterial precipitate was collected, and resuspended in MS20 liquid medium (1% AS was added, 10 μL AS was added to every 10 ml liquid MS20 medium), and the Agrobacterium suspension was adjusted to OD600 0.5-0.6. The explants pre-cultured for 2 days were gently shaken and cultured in the Agrobacterium suspension for 10 min, then transferred to sterilized filter paper to be blotted dry, and placed on the co-cultivation medium for dark culture for 48 h;

(5) Recovery culture: transfer the stem segments on the co-culture medium to the recovery medium and culture for one week;

(6) Screening culture: After one week, transfer the stem segments on the recovery medium to the screening medium. Change the medium every two weeks until adventitious buds grow from the callus tissue.

(7) Rooting: Cut off the grown adventitious buds, remove all surrounding callus tissue, keep them as intact as possible, and transfer them to rooting medium (insert medium) for rooting culture;

(8) Transplantation: Select healthy plants with roots and transplant them into nutrient soil. Avoid excessive water loss for 5 days after transplanting. Identify the plants after they grow normally.

The specific steps of identification are as follows:

(1) DNA extraction: Take an appropriate amount of fresh leaves and extract DNA using the CTAB method. First, use 2% CTAB extraction solution to heat and extract, then add chloroform and isoamyl alcohol to separate the organic phase, and then use 75% ethanol to wash and precipitate the DNA to obtain a DNA solution;

(2) Vector transformation identification: In order to verify whether the pCAMBIA 1305.4 overexpression vector was transformed into the plant, PCR amplification was performed using vector-specific primers 1305.4test-F: 5'-GGTCCCAAAGATGGACCCCC-3' (SEQ ID NO: 5) and StTSI-R: 5'-GTCACCAATTCACACGTGCTAATAGCACTCCCACAAAA-3' (SEQ ID NO: 4).

PCR reaction conditions: pre-denaturation at 94°C for 3 min; 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min, 34 cycles; extension at 72°C for 5 min.

Then agarose gel electrophoresis was performed, and bands indicated vector transfer;

(3) RNA extraction and reverse transcription: Total RNA was extracted from leaves of plants into which the vector was transferred and leaves of wild type plants using the RNA extraction and reverse transcription methods described above, and cDNA was obtained;

(4) qPCR determination of expression level changes: qPCR detection was performed using the SYBR qPCR quantitative PCR detection kit (purchased from TAKARA) (the method of use was in accordance with the instructions of the kit), using real-time fluorescence quantitative PCR software with potato gene actin as the internal reference, and the detection primer sequences were St-actin-qPCR-F: 5'-GGATCTTGCTGGTCGTGATTTAAC-3' (SEQ ID NO: 6), St-actin-qPCR-R: 5'-CATAGGCAAGCTTTTCCTTCATGT-3' (SEQ ID NO: 7), and the ΔΔCt method was used to calculate the relative expression level of StTSI in the transgenic plants. An increase in expression level indicates that an overexpression plant is obtained, and the identification is completed, and finally 3 diploid positive plants (OE-1, OE-2 and OE-3) are obtained.

Example 2 Growth of potatoes overexpressing solanine inhibitor

The three StTSI gene overexpressing plants obtained in Example 1 were planted in nutrient soil, and mature leaves and tubers were obtained from the experimental group and the control group at the tuber maturity stage. Solanine was extracted by methanol method and the solanine content was determined by LC-QTof-MS.

Solanine extraction method: ① Grind the sample (mixed sampling) stored at -80℃ into powder with liquid nitrogen, weigh about 0.1g and put it into a 1.5mL centrifuge tube, and repeat 3 times; ② Add 1mL of extract [80% (v/v) methanol + 0.1% (v/v) formic acid] to the centrifuge tube, vortex mix for 30s, ultrasonic extract for 60min at room temperature, and then vortex for 30s; ③ Centrifuge at 12,000rpm for 10min, take 25μL of supernatant (the remaining supernatant is saved for later use) and add it to 975μL of 100% methanol solution for dilution (placed in a 1.5ml centrifuge tube); ④ Centrifuge at 12000rpm for 10min, carefully aspirate 800ul of supernatant and put it into the sample bottle for testing (this step is similar to the function of membrane purification, and be careful not to aspirate to the bottom).

Solanine determination conditions: The instrument used was Agilent Technologies 6530 Accurate-Mass Q-TOF L-C-MS, and the separation column was ACQUITY UPLC BEH Amide column ( 1.7μm 2.1mm×100mm1/pk), column temperature 35℃. Mobile phase A is ultrapure water containing 0.1% (v/v) formic acid, mobile phase B is pure acetonitrile. Mobile phase flow rate is 0.200mL/min, and the injection volume per needle is 10μL. The mobile phase gradient changes of the liquid phase are shown in Table 1 below.

Table 1 Mobile phase gradient changes

The final test results are shown in Figure 1. Overexpression of the StTSI gene does not affect the growth of potato plants, and the solanine content in the leaves does not change significantly (Figure 2a and b), but the solanine content in the tubers is significantly reduced (Figure 2c and d), which shows that the gene does have the effect of inhibiting the solanine content in the tubers. At the same time, it shows that overexpression of the gene not only avoids the potato plants from being easily harmed by pests and diseases due to the reduction of solanine content in the aboveground part, but also solves the food safety problem caused by the excessive solanine content in the edible part, that is, the potato flesh, which can be said to kill two birds with one stone. Therefore, the gene will play an important role in potato breeding.

The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims (5)

1. A method for inhibiting solanine in potato tubers, characterized in that the method comprises introducing an ethylene response factor having a nucleotide sequence as shown in SEQ ID NO: 1 and an amino acid sequence as shown in SEQ ID NO: 2 into the genome of a potato plant, and cultivating transgenic potatoes. 2. The method according to claim 1, characterized in that the method comprises constructing transgenic potatoes using a vector comprising a sequence encoding an ethylene response factor. 3. The method according to claim 1 or 2, characterized in that the ethylene response factor is expressed on a chromosome or on a vector. 4. The method according to claim 1, characterized in that the method comprises the following steps: 1) introducing the coding sequence of the ethylene response factor into the vector by homologous recombination technology; 2) Potato pre-cultivation; 3) Infection and co-cultivation: The vector in 1) is introduced into potatoes by Agrobacterium transformation; 4) The transgenic potatoes are obtained after callus screening, differentiation, rooting, transplanting and identification. 5. Use of an ethylene response factor in inhibiting solanine in potato tubers, characterized in that the potato overexpresses an ethylene response factor having a nucleotide sequence as shown in SEQ ID NO: 1 and an amino acid sequence as shown in SEQ ID NO: 2.