Peppermint non-specific lipid transfer protein, genes and uses thereof

Abstract

The present application provides a peppermint non-specific lipid transporter, gene and application thereof, relates to the technical field of plant molecular biology, and provides a non-specific lipid transporter, gene and biological material of peppermint, which are overexpressed in the peppermint through a transgenic means, can significantly improve the resistance of a transgenic strain to thrips, realize precision breeding, and play a positive role in improving the breeding efficiency of plants of the genus Mentha. The application of the gene of the peppermint itself realizes the resistance of the peppermint to thrips by using the gene engineering technology, is safer, reduces the harm of chemical pesticides to the natural environment and human health, and is of great significance to the sustainable development of the peppermint industry.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to a peppermint nonspecific lipid transporter, its gene, and its applications. Background Technology

[0002] Thrips, a general term for insects belonging to the order Thysanoptera, encompass many species. The main types that damage flowers and vegetables include the flower thrips and onion thrips, as well as rice thrips and western flower thrips. Thrips are agricultural pests that adults and nymphs feed by rasping and sucking sap from the tender tissues of plants (twigs, leaves, flowers, fruits, etc.). Damaged young leaves and shoots harden, curl, and wither, with dense white spots or grayish-white and grayish-brown patches appearing on the leaves. Plant growth slows, and the plants gradually shrivel and dry out. Young fruits (such as eggplants, cucumbers, and watermelons) harden after being damaged, and in severe cases, fruit drop occurs, significantly impacting agricultural yield and value. Thrips are also important vectors for plant viruses. In recent years, thrips infestations have become severe, occurring year-round. Production control measures primarily rely on the use of chemical pesticides, such as imidacloprid, acetamiprid, nitenpyram, flonicamid, spirotetramat, spinosad, abamectin, pyriproxyfen, and lambda-cyhalothrin. Chemical pesticides play an indispensable role in effectively controlling pests; however, the control effect of pesticide spraying is limited, and long-term use can have impacts on human health and the ecological environment. Finding insect-resistant genes, utilizing plants' own resistance to control pests, and reducing the use of chemical pesticides are crucial for agricultural development.

[0003] Peppermint is a perennial herbaceous plant belonging to the Lamiaceae family, used as a spice due to its unique aroma. Peppermint is rich in essential oil components, such as menthol, menthone, isomenthone, menthol, and limonene. Peppermint essential oil is widely used in the food, chemical, and pharmaceutical industries, possessing significant economic value. Peppermint is also a traditional Chinese medicine, with effects such as dispelling wind-heat and clearing the head and eyes. my country is the world's largest consumer of peppermint essential oil and menthol. As the source plant for peppermint essential oil extraction and a widely used medicinal herb, peppermint faces strict requirements regarding its quality, appearance, and pesticide residues. Peppermint thrives in warm and humid environments, making it susceptible to pests and diseases. Therefore, cultivating highly resistant and superior peppermint germplasm resources to produce pollution-free peppermint and reduce economic losses is of great importance.

[0004] Previously, the discovery of resistant germplasm resources in mint mainly relied on traditional hybridization breeding, which was slow, time-consuming, and complex. With the development and application of high-throughput omics technologies, significant progress has been made in the discovery and functional analysis of new genes, as well as in the study of the molecular mechanisms underlying important traits such as yield, quality, and resistance. This has led to the development and continuous improvement of molecular breeding and molecular design breeding techniques. Therefore, identifying functional genes in mint that can enhance resistance traits and utilizing new genetic breeding technologies, such as molecular breeding, genetic engineering, and gene editing, can play a positive role in improving the breeding efficiency of mint plants and achieving precision breeding.

[0005] By identifying insect-resistant genes and using them to breed insect-resistant transgenic plants, it is possible to suppress pest populations, improve plant insect resistance, and achieve this through low cost, high specificity, and minimal environmental impact. Conventional insect-resistant crop breeding mainly focuses on the insecticidal activity of expressed insect-resistant proteins, such as transgenic Bt insect-resistant cotton. Plants themselves also possess numerous genes related to pest resistance, which can regulate plant development or changes in metabolic products to activate their own chemical and physical defenses, thereby enhancing plant resistance to pests. Mint, being a plant with both medicinal and edible uses, is particularly suitable for identifying and utilizing its own genes to activate defense mechanisms for precision breeding. However, current understanding of insect-resistant genes in mint is still very limited, and their application is also very rare.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a peppermint nonspecific lipid transporter and its encoding gene, so as to at least solve one of the technical problems existing in the prior art.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] This invention provides a peppermint nonspecific lipid transporter, comprising a protein consisting of the amino acid sequence described in SEQ ID NO.2.

[0010] The present invention also provides a gene encoding the above-mentioned peppermint nonspecific lipid transporter.

[0011] Furthermore, the gene comprises a nucleotide sequence as shown in SEQ ID NO.1.

[0012] The present invention also provides biological materials containing the above-mentioned genes;

[0013] Preferably, the biological material includes a recombinant expression vector, an expression cassette, a transgenic cell line, or a recombinant bacterium.

[0014] The present invention also provides the application of the above-mentioned non-specific menthol lipid transporter, gene or biological material in improving menthol thrips resistance.

[0015] Furthermore, the present invention provides a method for cultivating mint with thrips resistance, comprising increasing the expression level of the aforementioned gene or biological material and / or the activity of mint nonspecific lipid transport proteins in recipient mint to obtain positive plants; the positive plants exhibit higher resistance to thrips than the recipient mint.

[0016] Furthermore, by introducing the aforementioned genes or biological materials into the recipient peppermint, peppermint with thrips resistance is obtained.

[0017] Furthermore, the biological material is a plant overexpression vector.

[0018] Furthermore, it includes the following steps:

[0019] (a) The plant overexpression vector was transferred into Agrobacterium tumefaciens and the fermentation broth of positive colonies was used as the inoculum.

[0020] (b) Take a 2-3 mm long sterile pepper seedling stem segment and immerse it in the infection solution obtained in (a) for 15-25 min;

[0021] (c) After infection, the samples were transferred to a co-culture medium and cultured in the dark for 48-60 hours. Then they were transferred to a selection medium and cultured under normal light. After the shoots differentiated, they were transferred to a rooting medium.

[0022] (d) Take seedlings that can take root normally to obtain the thrips-resistant mint.

[0023] Further, the co-culture medium comprises: MS, 30 g / L sucrose, 5 mg / L TDZ, 0.2 mg / L IAA, 20% coconut milk, 200 μmol / L acetylsalicylic acid, and 8 g / L agar, pH 5.8; and / or,

[0024] The screening differentiation medium comprises: MS, 30 g / L sucrose, 5 mg / L TDZ, 0.2 mg / L IAA, 20% coconut milk, 150 mg / L cephalosporin, 30 mg / L Kan, and 8 g / L agar, pH 5.8; and / or,

[0025] The rooting medium comprises: MS, 30 g / L sucrose, 100 mg / L cephalosporin, 50 mg / L kanamycin and 8 g / L agar, pH 5.8.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. This invention provides a non-specific lipid transporter protein, gene, and biological material for mint. By overexpressing this protein in mint through transgenic means, the transgenic lines can be significantly improved in terms of resistance to thrips, thus achieving precision breeding and playing a positive role in improving the breeding efficiency of mint plants.

[0028] 2. By utilizing the peppermint's own genes and employing genetic engineering technology, peppermint can be made more resistant to thrips infestation, which is safer and reduces the harm of chemical pesticides to the natural environment and human health. This is of great significance to the sustainable development of the peppermint industry.

[0029] 3. It provides insect-resistant gene resources for other species. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram illustrating the construction of a plant recombinant vector for genetic transformation provided in an embodiment of the present invention;

[0032] Figure 2 The following are images of the effects of transgenic peppermint on thrips resistance provided in the embodiments of the present invention: (A) the state of wild-type and transgenic peppermint after thrips infestation under the same conditions; (B) the detection of target gene expression in transgenic materials by real-time PCR; (C) the difference in chlorophyll content between wild-type and transgenic peppermint after thrips infestation; (D) the number of adult thrips on wild-type and transgenic peppermint. Detailed Implementation

[0033] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0034] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] According to one aspect of the present invention, a peppermint nonspecific lipid transporter is provided, comprising a protein consisting of the amino acid sequence described in SEQ ID NO. 2. The present invention also provides a gene encoding the aforementioned peppermint nonspecific lipid transporter, preferably comprising the nucleotide sequence shown in SEQ ID NO. 1.

[0037] Through extensive experiments, the inventors of this invention discovered a non-specific lipid transporter protein in mint and its encoding gene that is significantly associated with mint thrips resistance. The function of the gene encoding this non-specific lipid transporter protein was verified using transgenic technology, demonstrating that the protein and its encoding gene can effectively regulate mint resistance to thrips, providing a new approach for breeding insect-resistant mint varieties.

[0038] Among them, SEQ ID NO.1 includes:

[0039] ATGGCTGGAGTCGCGAAGCTAACTTGCGCCGTCCTCATCACCGCCGCACTGATGGCCCGCTTTTGCACCGCCTTGCGAGGCGGCGATAGGATGCGGCCAGGTGGTTTCGTACCTCAACCCCTGCCTCCCTTACGTCACCGGAAAGGGTGCTTTGGGGGGCTGCTGCGGC GGCATTAAGGGCCTGTACGCCGCCGCCAAGAGTACGGCGGACAAGCAGAGCGTCTGCGGCTGCCTGAAAAGCCTCGCCGGTTCAGCTTCCGGCGTCAACTACGGCCAATGCGGCGTCAACATTCCGTACAAGATTAGCCCTTCCACCGACTGCTCCAAGGTGAAGTGA;

[0040] SEQ ID NO.2 includes:

[0041] MAGVAKLTCAVLITAALMAAFAPPCEAAIGCGQVVSYLNPCLPYVTGKGALGGCCGGIKGLYAAAKSTADKQSVCGCLKSLAGSASGVNYGQCGVNIPYKISPSTDCSKVK.

[0042] It should be noted that "comprising" means that the nucleotide sequence of the gene encoding the peppermint nonspecific lipid transporter may only have the nucleotide sequence shown in SEQ ID NO.1, or it may be composed of the nucleotide sequence shown in SEQ ID NO.1 and other nucleotide sequences, such as nucleotide sequences encoding functional units for protein purification tags, fluorescent protein markers, and DNA binding sites, or encoding elements that regulate gene transcription and expression, including but not limited to promoters, strong promoters, enhancers, or transcription factor binding sites; "comprising" may also mean that the nucleotide sequence shown in SEQ ID NO.1 is not continuous in the gene encoding the peppermint nonspecific lipid transporter, but can produce cDNA with the nucleotide sequence shown in SEQ ID NO.1.

[0043] It is understandable that, in addition to the gene encoding the non-specific lipid transport protein in peppermint itself, biological materials containing the aforementioned genes can also be used to regulate peppermint resistance to thrips. These biological materials can be, for example, but are not limited to, recombinant expression vectors, expression cassettes, transgenic cell lines, or recombinant bacteria.

[0044] According to a second aspect of the invention, the use of the above-described peppermint nonspecific lipid transporter, gene, or biological material in improving peppermint thrips resistance is provided.

[0045] Furthermore, based on the applications provided by this invention, this invention also provides a method for cultivating thrips-resistant mint, comprising increasing the expression level of the gene or biological material encoding the mint nonspecific lipid transport protein and / or the activity of the mint nonspecific lipid transport protein in the recipient mint to obtain positive plants; the positive plants exhibit higher resistance to thrips than the recipient mint. This method can more precisely regulate the plant phenotype with high regulatory efficiency, providing a new approach to improving mint resistance to thrips, and has broad application prospects and market potential.

[0046] Various molecular biology techniques can be used to increase the expression level of genes encoding non-specific lipid transport proteins in peppermint, such as increasing promoter strength or inducing expression. In some preferred embodiments, peppermint resistant to thrips is obtained by infecting peppermint with genes encoding non-specific lipid transport proteins in peppermint.

[0047] In some preferred embodiments, the following steps are included:

[0048] (a) The plant overexpression vector was transferred into Agrobacterium tumefaciens and the fermentation broth of positive colonies was used as the inoculum.

[0049] (b) Take a 2-3 mm long sterile pepper seedling stem segment and immerse it in the infection solution obtained in (a) for 15-25 min;

[0050] (c) After infection, the samples were transferred to a co-culture medium and cultured in the dark for 48-60 hours. Then they were transferred to a selection medium and cultured under normal light. After the shoots differentiated, they were transferred to a rooting medium.

[0051] (d) Take seedlings that can take root normally to obtain the thrips-resistant mint.

[0052] Further, the co-culture medium comprises: MS, 30 g / L sucrose, 5 mg / L TDZ, 0.2 mg / L IAA, 20% coconut milk, 200 μmol / L acetylsalicylic acid, and 8 g / L agar, pH 5.8; and / or,

[0053] The screening differentiation medium comprises: MS, 30 g / L sucrose, 5 mg / L TDZ, 0.2 mg / L IAA, 20% coconut milk, 150 mg / L cephalosporin, 30 mg / L Kan, and 8 g / L agar, pH 5.8; and / or,

[0054] The rooting medium comprises: MS, 30 g / L sucrose, 100 mg / L cephalosporin, 50 mg / L kanamycin and 8 g / L agar, pH 5.8.

[0055] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market; experimental methods not specified in the examples are generally performed according to conventional methods, such as those described in Molecular Cloning: A Laboratory Manual (3rd Edition, by J. Sambrook et al.), or according to the methods recommended by the reagent kit manufacturer.

[0056] Example 1: Cloning of a nonspecific lipid transporter gene in peppermint

[0057] (1) Application Total RNA was extracted from peppermint leaves using the Promega Promega Total RNA Extraction Kit (Peppermint leaves were collected in May 2022 from the Germplasm Resource Nursery of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province). Using the extracted total RNA as a template, cDNA was synthesized under the action of reverse transcriptase. Cloning primers were designed and synthesized based on the peppermint transcriptome sequencing results.

[0058] LTP-F: 5'-ATGGCTGGAGTCGCGAAG-3' (SEQ ID NO.3);

[0059] LTP-R: 5'-TCACTTCACCTTGGAGCAGTC-3' (SEQ ID NO. 4).

[0060] Using peppermint cDNA as a template, the gene sequence of the peppermint nonspecific lipid transporter was amplified by PCR, cloned into a T vector, and sequenced. The sequencing was successful to obtain the gene coding sequence (SEQ ID NO.1), and its protein coding sequence (SEQ ID NO.2) was translated.

[0061] Example 2: Construction of a plant recombinant expression vector containing the above gene nucleotide sequence

[0062] The gene sequence obtained in Example 1 was constructed into a plant overexpression vector, and homologous recombination was performed using the XhoI single restriction enzyme site. The ligation product was transformed into competent E. coli cells and cultured overnight at 37°C on plates containing 50 mg / L kanamycin. Positive single colonies were picked, plasmids were extracted, and sequencing was performed for verification. A schematic diagram of the recombinant vector is attached. Figure 1 As shown.

[0063] Example 3: Genetic transformation of peppermint using the above-mentioned plant recombinant expression vector

[0064] The plant expression vector described in Example 2 was transferred into Agrobacterium EHA105. Positive colonies were picked and cultured until the OD600 was 0.6. After collection by low-speed centrifugation, the colonies were resuspended in MS solution as the infection solution (containing 200 μmol / L acetylsyl syringone). The stem segments (without nodes) of sterile peppermint seedlings were cut into 2-3 mm segments and infected in Agrobacterium infection solution for 20 min. After the moisture was absorbed by filter paper, the segments were placed in co-culture medium and cultured in the dark for 48-60 hours. Then, the segments were transferred to selection medium and cultured under normal light. After the shoots differentiated, the segments were transferred to rooting medium. Seedlings that could root normally and grew to about 8 cm were transplanted into an artificial climate chamber.

[0065] Culture medium formulations: co-culture medium (MS + 30 g / L sucrose + 5 mg / L TDZ + 0.2 mg / L IAA + 20% coconut milk + 200 μmol / L acetosyringone + 8 g / L agar, pH 5.8); screening and differentiation medium (MS + 30 g / L sucrose + 5 mg / L TDZ + 0.2 mg / L IAA + 20% coconut milk + 150 mg / L cephalosporin + 30 mg / L Kanamycin + 8 g / L agar, pH 5.8); rooting medium (MS + 30 g / L sucrose + 100 mg / L cephalosporin + 50 mg / L kanamycin + 8 g / L agar, pH 5.8).

[0066] Example 4: Molecular identification and antithrips effect of transgenic peppermint

[0067] RNA was extracted from the leaves of transgenic positive plants, and the gene expression level was detected by real-time quantitative PCR in transgenic positive peppermint to confirm its overexpression in peppermint. Figure 2 The conditions for quantitative real-time PCR were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 25 s, for 40 cycles.

[0068] The primer sequences for quantitative fluorescence are as follows:

[0069] qPCR-F: 5'-GTTTCGTACCTCAACCCCTG-3' (SEQ ID NO.5),

[0070] qPCR-R: 5'-TCACTTCACCTTGGAGCAGT-3' (SEQ ID NO. 6).

[0071] From the plants identified as positive and the wild type, healthy stem tips approximately 3cm long were cut and hydroponically rooted. After rooting, 10 plants (pots) of each type were planted in pots containing sterilized soil. They were then allowed to grow normally in an artificial climate chamber for two weeks (this process ensures consistency between the transgenic and wild types). Next, the plants were placed in insect rearing cages, and 10 second-instar thrips nymphs were released onto each mint plant. After 2-3 weeks of growth, the number of thrips on the leaves, the extent of thrips infestation, and the chlorophyll content were compared. (Appendix) Figure 2 The results showed that the number of flower thrips in the wild-type strain was significantly higher than that in the overexpression strain, the leaves were more severely damaged by thrips, and the chlorophyll content was significantly reduced. Therefore, the overexpression strain significantly improved the resistance of mint to flower thrips.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A peppermint-specific lipid transporter, characterized in that, The amino acid sequence of the peppermint nonspecific lipid transporter is shown in SEQ ID NO.

2.

2. The gene encoding the peppermint nonspecific lipid transporter of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. Biological material containing the gene as described in claim 2 or 3; The biological material is a recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacteria.

5. The use of the peppermint nonspecific lipid transporter of claim 1, the gene of claim 2 or 3, or the biomaterial of claim 4 in improving peppermint thrips resistance.

6. A method for cultivating mint with thrips resistance, characterized in that, The method includes increasing the expression level of the gene described in claim 2 or 3 or the biological material described in claim 4 and / or the activity of the non-specific lipid transport protein of peppermint described in claim 1 in the recipient peppermint to obtain positive plants; the positive plants have higher resistance to thrips than the recipient peppermint.

7. The method according to claim 6, characterized in that, By introducing the gene of claim 2 or 3 or the biological material of claim 4 into the recipient peppermint, peppermint with thrips resistance is obtained.

8. The method according to claim 7, characterized in that, The biological material is a plant overexpression vector.

9. The method according to claim 8, characterized in that, Includes the following steps: (a) The plant overexpression vector was transferred into Agrobacterium, and the fermentation broth of positive colonies was used as the inoculum. (b) Take a 2-3 mm long section of sterile peppermint seedling stem and infect it in the infection solution obtained in (a) for 15-25 min; (c) After infection, the samples were transferred to co-culture medium and cultured in the dark for 48-60 hours, then transferred to selection medium and cultured under normal light. After the shoots differentiated, they were transferred to rooting medium. (d) Take seedlings that can take root normally to obtain the thrips-resistant mint.

10. The method according to claim 9, characterized in that, The co-culture medium comprises: MS, 30 g / L sucrose, 5 mg / L TDZ, 0.2 mg / L IAA, 20% coconut milk, 200 μmol / L acetylsalicylic acid, and 8 g / L agar, pH 5.8; and / or, The screening medium comprises: MS, 30 g / L sucrose, 5 mg / L TDZ, 0.2 mg / L IAA, 20% coconut milk, 150 mg / L cephalosporin, 30 mg / L Kan, and 8 g / L agar, pH 5.8; and / or, The rooting medium comprises: MS, 30 g / L sucrose, 100 mg / L cephalosporin, 50 mg / L kanamycin and 8 g / L agar, pH 5.8.

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