A FaTRAB1 gene for regulating the growth, development and resistance of strawberry plants and its application

Abstract

The invention discloses a FaTRAB1 gene for regulating the growth, development and resistance of strawberry plants and its application, and belongs to the field of genetic engineering. The invention provides a gene FaTRAB1 related to regulating the growth, development and resistance of strawberry plants, and by constructing a plant expression vector for overexpressing the FaTRAB1 gene, transforming the leaf discs of 'Rugen' strawberry by Agrobacterium-mediated method, the plant stably overexpressing the FaTRAB1 gene is obtained to be shorter, smaller in crown width, shorter in petiole, thicker in leaf, darker in leaf color, higher in chlorophyll content, and multiple non-glandular trichomes on the back of the leaf, and the plant overexpressing the FaTRAB1 gene is more resistant to two-spotted spider mites. It can be seen that the invention provides a gene capable of regulating the growth, development and resistance of strawberry plants, provides a theoretical basis and technical means for the ability of FaTRAB1 to regulate the growth, development and resistance of strawberry plants, and has great application value.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a FaTRAB1 gene for regulating the growth, development and resistance of strawberry plants and an application thereof. Background Art

[0002] Strawberry (Fragaria×ananassa Duch.) is a perennial herbaceous plant of the genus Fragaria in the family Rosaccae. my country ranks first in the world in terms of strawberry cultivation area and yield. Strawberry fruit is known as the "Queen of Fruits" because of its bright color, sweet and sour flavor, rich aroma, delicious taste, and rich minerals, vitamins, and a variety of amino acids. However, during the growth and development of strawberries, it is easily invaded by pests and diseases such as powdery mildew, gray mold, and two-spotted spider mites, which affect the normal growth and development of strawberries, resulting in serious consequences such as poor quality of strawberry fruits and reduced yields. Among them, the two-spotted spider mite is the main pest that affects the growth of strawberry plants. It parasitizes on the back of strawberry leaves or other young and tender tissues to suck juice, causing white spots or red rust-like spots on strawberry leaves, which has an adverse effect on the photosynthesis of the leaves, and can even cause the leaves to fall off and die and the fruits to be deformed. Therefore, cultivating strawberry plants with high resistance to pests and diseases is of great significance for the management and pest control of cultivated strawberries.

[0003] Basic region leucine zipper (bZIP), TRAB1 is an abscisic acid (ABA)-related transcription factor that can bind to ABA response elements, mediate ABA signal activation transcription, regulate plant maturation and dormancy, and respond to osmotic stress in rice. In strawberry research, FaTRAB1 can bind to the FaMADS1 promoter and downregulate the expression of FaMADS1, thereby inhibiting the ripening of ABA-dependent strawberry fruits. However, the function of FaTRAB1 in participating in the growth and development of strawberry plants and responding to stress remains to be explored. Summary of the invention

[0004] The purpose of the present invention is to provide a FaTRAB1 gene for regulating the growth and development of strawberry plants and their application, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a theoretical basis and technical means for the ability of FaTRAB1 gene to regulate plant growth and development and improve plant resistance, and has great application value.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application for overexpressing the FaTRAB1 gene, including any of the following applications:

[0007] (1) Changing the agronomic traits of strawberry plants;

[0008] (2) Promote the occurrence of non-glandular trichomes in strawberry leaves;

[0009] (3) Improve strawberry's resistance to two-spotted spider mites;

[0010] (4) Cultivating transgenic strawberries with altered agronomic traits;

[0011] (5) Cultivating transgenic strawberries that promote the formation of non-glandular trichomes on strawberry leaves;

[0012] (6) Cultivate transgenic strawberries with strong resistance to two-spotted spider mites;

[0013] Wherein, the nucleotide sequence of the FaTRAB1 gene is shown as SEQ ID NO.1;

[0014] The agronomic traits of the strawberry plant are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of non-glandular trichomes;

[0015] The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of non-glandular trichomes is increased.

[0016] The present invention also provides applications of the protein encoded by the FaTRAB1 gene, including any of the following applications:

[0017] (1) Changing the agronomic traits of strawberry plants;

[0018] (2) Promote the occurrence of non-glandular trichomes in strawberry leaves;

[0019] (3) Improve strawberry's resistance to two-spotted spider mites;

[0020] (4) Cultivating transgenic strawberries with altered agronomic traits;

[0021] (5) Cultivating transgenic strawberries that promote the formation of non-glandular trichomes on strawberry leaves;

[0022] (6) Cultivate transgenic strawberries with strong resistance to two-spotted spider mites;

[0023] Wherein, the nucleotide sequence of the FaTRAB1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO.2;

[0024] The agronomic traits of the strawberry plant are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of non-glandular trichomes;

[0025] The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of non-glandular trichomes is increased.

[0026] The present invention also provides an application of a recombinant vector for overexpressing the FaTRAB1 gene, including any of the following applications:

[0027] (1) Changing the agronomic traits of strawberry plants;

[0028] (2) Promote the occurrence of non-glandular trichomes in strawberry leaves;

[0029] (3) Improve strawberry's resistance to two-spotted spider mites;

[0030] (4) Cultivating transgenic strawberries with altered agronomic traits;

[0031] (5) Cultivating transgenic strawberries that promote the formation of non-glandular trichomes on strawberry leaves;

[0032] (6) Cultivate transgenic strawberries with strong resistance to two-spotted spider mites;

[0033] Wherein, the nucleotide sequence of the FaTRAB1 gene is shown as SEQ ID NO.1;

[0034] The agronomic traits of the strawberry plant are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of non-glandular trichomes;

[0035] The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of non-glandular trichomes is increased.

[0036] The present invention also provides applications of the recombinant bacteria overexpressing the FaTRAB1 gene, including any of the following applications:

[0037] (1) Changing the agronomic traits of strawberry plants;

[0038] (2) Promote the occurrence of non-glandular trichomes in strawberry leaves;

[0039] (3) Improve strawberry's resistance to two-spotted spider mites;

[0040] (4) Cultivating transgenic strawberries with altered agronomic traits;

[0041] (5) Cultivating transgenic strawberries that promote the formation of non-glandular trichomes on strawberry leaves;

[0042] (6) Cultivate transgenic strawberries with strong resistance to two-spotted spider mites;

[0043] Wherein, the nucleotide sequence of the FaTRAB1 gene is shown as SEQ ID NO.1;

[0044] The agronomic traits of the strawberry plant are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of non-glandular trichomes;

[0045] The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of non-glandular trichomes is increased.

[0046] The present invention also provides a method for changing agronomic traits of strawberry, comprising using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to regulate the growth and development of the strawberry plant; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1;

[0047] The agronomic traits of the strawberry plants are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of non-glandular trichomes.

[0048] The present invention also provides a method for promoting the occurrence of non-glandular trichomes on strawberry leaves, comprising using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to promote the occurrence of non-glandular trichomes on the back of the leaves of the strawberry plant; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1.

[0049] The present invention also provides a method for improving the resistance of strawberry to two-spotted spider mites, comprising using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to improve the resistance of the strawberry plant to two-spotted spider mites; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1.

[0050] The present invention also provides a method for cultivating transgenic strawberries with altered agronomic traits, comprising using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to obtain transgenic strawberries with altered agronomic traits; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1;

[0051] The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of non-glandular trichomes is increased.

[0052] The present invention also provides a method for cultivating transgenic strawberries that promote the formation of non-glandular trichomes on strawberry leaves, comprising using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to obtain transgenic strawberries that promote the formation of non-glandular trichomes on strawberry leaves; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1.

[0053] The present invention also provides a method for cultivating transgenic strawberries with strong resistance to two-spotted spider mites, comprising using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to obtain transgenic strawberries with strong resistance to two-spotted spider mites; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1.

[0054] The present invention discloses the following technical effects:

[0055] The present invention constructs a plant expression vector for overexpressing the FaTRAB1 gene through a stable genetic transformation technology, and the constructed plant expression vector is transformed into a leaf disc of 'Ruegen' strawberry by an Agrobacterium-mediated method to obtain a plant overexpressing the FaTRAB1 gene, wherein the plant as a whole exhibits dwarfing, shorter petioles, thicker leaves, darker leaf color, higher chlorophyll content, multiple non-glandular trichomes on the back of the leaves, and better resistance to two-spotted spider mites. The present invention obtains new germplasm resources with different leaf morphological characteristics, and improves plant resistance while regulating plant growth. Therefore, the present invention uses genetic engineering technology to provide a theoretical basis and technical means for the ability of the FaTRAB1 gene to regulate plant growth and development and improve plant resistance, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0057] Figure 1 Figure 1 is the construction process of the overexpression vector, wherein A is a schematic diagram of the vector for constructing the 35S overexpression of the FaTRAB1 gene; B is a spectrum of the P1300-35s-SBXA-2XFlag-YFP-KP-NOS overexpression vector; C is an electrophoresis diagram of the double-enzyme-cut vector (the first lane is Maker, and the second lane is the sample); D is an electrophoresis diagram of the amplification of the FaTRAB1 gene (the first lane is Maker, and the second lane is the sample);

[0058] Figure 2 Diagram of the process of obtaining transgenic materials overexpressing FaTRAB1, where A is the pre-culture of strawberry leaf discs; B is the fully resistant callus and young shoots; C is the resistant seedling; D is the transplanted seedling;

[0059] Figure 3 is the FaTRAB1 expression level of the wild type and five overexpression transgenic lines;

[0060] Figure 4Subcellular localization of FaTRAB1 in tobacco leaf epidermal cells, scale bar is 40 μm;

[0061] Figure 5 Comparison of agronomic traits between wild type and overexpressed FaTRAB1 in cultivated strawberry, where A is plant phenotype; B is leaf phenotype; C is plant height, D is crown width, E is petiole length, F is petiole thickness, G is compound leaf petiole length, H is leaf thickness, I is chlorophyll a content, J is chlorophyll b content, and K is carotenoid content; the scale bars in Figures A and B represent 1 cm;

[0062] Figure 6 The anatomical structure of leaves of wild type and overexpressing FaTRAB1 (A) and the statistical results of leaf thickness (B), upper epidermal cell thickness (C), lower epidermal cell thickness (D) and palisade tissue thickness (E) were observed under a microscope at 200× magnification; in Figure A, a is the upper epidermis, b is the palisade tissue, c is the spongy tissue, d is the lower epidermis, and the scale bar represents 100 μm;

[0063] Figure 7 The lower epidermal trichomes of leaves overexpressing FaTRAB1 and wild type (A), the number of non-glandular trichomes under 50× SEM (B), the number of glandular trichomes under 50× SEM (C), and the length of glandular trichomes under 500× SEM (D); in Figure A, a is glandular trichomes, b is non-glandular trichomes, and c is stomata; the scale bar in 50× SEM represents 1 mm, in 100× SEM represents 500 μm, and in 500× SEM represents 100 μm;

[0064] Figure 8 Overexpression of FaTRAB1 can enhance resistance to two-spotted spider mites, including the phenotypes on days 0 and 7 after inoculation with two-spotted spider mites (A), maximum photochemical efficiency (Fv / Fm) (B), net photosynthetic rate (C), intercellular CO 2 concentration (D), stomatal conductance (E), transpiration rate (F), relative conductivity (G), Malondialdehyde (MDA) content (H) and H 2 O 2 Content (I); scale bars in Figure A represent 1 cm and 1 mm. DETAILED DESCRIPTION

[0065] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0066] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0067] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0068] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0069] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0070] Example

[0071] 1. Overexpression vector construction

[0072] The schematic diagram of constructing the 35S overexpression vector of FaTRAB1 gene is shown in Figure 1 As shown in A, wherein the sequence of the FaTRAB1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0073] SEQ ID NO.1:

[0074]

[0075] SEQ ID NO.2:

[0076] MGSNINFKNYGDVQNGDVNGGKLGGNFSLARQTSVYSLTFDEFQNTMGGLGKDFGSMNMDELLKNIWSAEETQGITTSTSGAGEGNAPVGNLQRQGSLTLPRTLSQKTV DDVWKELNRESGDVKFGNVGVGSDLPQEQRQQTLGEMTLEEFLARAGVVREDAQEMARPSNSGFFGELFRHSNNNTGLAPGFQQASRNNGLLGSRVESNNNSVPHQSP NLALNVGGVRTSQQQTHQLPPQQQPIFPKPATLSFAPSMHLVSNAQLTSPRSRGPMSAVVEPSRNTAFSQGGGFPGAGIGMGGLGAGGVTVASRSPVNKISPDVIVKS SADTSSLSPIPYMFTRGRKANGALEKVVERRQRRMIKNRESAARSRARKQAYTLELEAEVQKLKEMNEELQKKQAEIMEMQKDQILETMNREWGGKRQCLRRTLTGPW.

[0077] The specific construction is as follows:

[0078] 1.1 FaTRAB1 gene cloning

[0079] Using SnapGene 2.3.3 software, a pair of specific primers were designed at both ends according to the ORF sequence of the FaTRAB1 gene, and the overexpression vector P1300-35s-SBXA-2XFlag-YFP-KP-NOS (vector map see Figure 1 The specific Xba I and Bgl II restriction sites and their protective base sequences on B) were used to synthesize the following primers containing restriction sites: 35S FaTRAB1-F and 35S FaTRAB1-R.

[0080] 35S FaTRAB1-F (SEQ ID NO. 3):

[0081] 5'-ACGAGCTCGGATCCTCTAGAGTAAATGGTGGGAAGCTAGGTGG-3';

[0082] 35S FaTRAB1-R (SEQ ID NO.4):

[0083] 5'-TCGTCCTTGTAGTCAGATCT CAATGTTCGTCTCAAGCACTGC-3';

[0084] The FaTRAB1 gene was amplified using the above primers. The electrophoresis of the amplified product is shown in Figure 1 As shown in D, the amplified product is recovered for later use.

[0085] 1.2 Enzyme digestion of overexpression vector

[0086] The overexpression vector P1300-35s-SBXA-2XFlag-YFP-KP-NOS was double-digested with Xba I and Bgl II restriction endonucleases to recover the linearized vector large fragment ( Figure 1 C). Enzyme digestion system: 5 μL 10× 1 μL each of Buffe, BamHⅠ and Xba, 2 μg of P1300-35s-SBXA-2XFlag-YFP-KP-NOS plasmid, add water to 50 μL and mix well, and digest at 37℃ for 0.5-1h; when terminating the reaction, add 10× DNA Loading Buffer to a final concentration of 1× and recover.

[0087] 1.3 Connection of target gene and vector

[0088] The recovered and tested complete target fragment with homology arms and linearized vector large fragment were mixed according to the following formula: 5 μL 2×Basic Assembly Mix, 2 μL DNA fragment (target fragment: linearized vector large fragment = 3:1, V / V), ddH 2 O to a total volume of 10 μL and mixed according to the Transgen homologous recombination kit ( -Basic Seamless Cloning and Assembly Kit) instructions, gently mix, react at 50℃ for 20min. After the reaction, place the centrifuge tube on ice to cool. Transform the ligation product into E. coli Trans T1 competent state, spread on LB solid medium containing 50mg / L kanamycin, culture overnight at 37℃, select single colonies, and perform colony PCR. The reaction program is 95℃ pre-denaturation for 3min, 95℃ denaturation for 25s, 58℃ annealing for 25s, 72℃ extension for 30s, cycle 35 times, 72℃ final extension for 5min, and store at 12℃. Amplification system: 1μL bacterial liquid template, 1μL primers 35S FaTRAB1-F and 35S FaTRAB1-R, 10μL PCR enzyme MIX, and make up the total system to 20μL with sterile water. 200 μL of the recombinant bacteria verified as positive clones by electrophoresis was sent to a biological company for sequencing. Successful sequencing indicated that the vector containing the FaTRAB1 sequence was successfully constructed. The recombinant bacteria were then expanded and cultured, and the plasmids were extracted and stored at -20°C for future use.

[0089] 2. Genetic transformation

[0090] 2.1 Pre-cultivation of strawberry leaves

[0091] Distilled water was placed in a sterile culture dish; tender and flat leaves of 10-30d old 'Ruegen' strawberry tissue culture seedlings were taken, the leaf tips, leaf margins and main veins were removed, and the leaves were cut into 5mm×3mm strips (or with a 4mm punch) in a direction perpendicular to the main veins, and the obtained leaves were promptly transferred to a culture dish containing 25mL of liquid culture medium (MS culture medium containing vitamin B5 + TDZ 2.0mg / L + IBA 0.5mg / L + 3% sucrose, pH 5.8) (sterile water), and 100-150 leaves were cut each time; thereafter, the leaves were placed on filter paper to absorb the surface water, and placed in a culture dish containing pre-culture medium (MS culture medium containing vitamin B5 + TDZ 2.0mg / L + IBA 0.5mg / L + 3% sucrose + 0.7% agar, pH 5.8), with the back of the leaves in contact with the culture medium (such as Figure 2 A), put 20-30 in each culture dish, seal with parafilm, and culture in the dark at 25℃ for 7-10 days. (Note: The regeneration rate of adventitious buds on leaves with a leaf age of 10-30 days is higher, and it is basically difficult to regenerate adventitious buds on old leaves.)

[0092] 2.2 Plasmid transformation into GV3101 Agrobacterium

[0093] Take the GV3101 Agrobacterium competent cells stored at -80℃ and place them at room temperature or in the palm of your hand for a while until they partially melt. When they are in an ice-water mixing state, insert them into ice. Add 1μg of plasmid DNA to every 100μL competent cells, stir the bottom of the tube by hand to mix, and then place them on ice for 30min, liquid nitrogen for 5min, 37℃ water bath for 5min, and ice bath for 5min. Add 700μL of YEB liquid medium without antibiotics and culture at 28℃ with shaking for 2-3h. Centrifuge at 6000rpm for 1min to collect the bacteria, keep 100μL of supernatant, gently blow and resuspend the bacteria, and apply it on the YEB plate containing the corresponding antibiotics (50μg / mL kanamycin + 20μg / mL rifampicin + 40μg / mL gentamicin), and place it upside down in a 28℃ incubator for 2-3 days.

[0094] 2.3 Cultivation of Agrobacterium tumefaciens GV3101

[0095] Single clones were picked and cultured in 800 μL YEB liquid medium containing antibiotics (50 μg / mL kanamycin + 20 μg / mL rifampicin + 40 μg / mL gentamicin) and shake-cultured at 28°C and 150 rpm overnight in the dark. The next day, all were inoculated in 25 mL YEB liquid medium containing antibiotics (50 μg / mL kanamycin + 20 μg / mL rifampicin + 40 μg / mL gentamicin) and shake-cultured at 28°C and 150 rpm in the dark until OD 600 = 0.5-1.0, centrifuged at 4000 × g for 5 min to collect the bacteria, washed the bacterial pellet three times with the culture medium described below, and resuspended the bacterial pellet in a culture medium (MS medium containing vitamin B5 + 2% sucrose + 10 mM 2-morpholineethanesulfonic acid (MES) + 200 μM acetosyringone, pH 5.5) to an OD of 0. 600 =0.4-0.5, 28℃50rpm shaking culture for 2-3h, this is the bacterial solution used for infection (MES and acetosyringone are both filtered and sterilized, and stored after subpackaging).

[0096] 2.4 Infection and co-culture

[0097] After 7-10 days of pre-culture, the leaves were placed in the prepared Agrobacterium suspension (Silwetl-77 was added to the suspension at a final concentration of 0.01% before infection), and the leaves were infected for 20 minutes, and gently shaken several times every 5 minutes; after the infection, the leaves were placed on filter paper to absorb (blow dry) the bacterial liquid on the surface, and placed in a culture dish containing 25 mL of co-culture medium (MS medium containing vitamin B5, vitamin B5 + TDZ 2.0 mg / L + IBA 0.5 mg / L + 3% sucrose + 0.7% agar + 100 μM AS, pH 5.5), 20-30 leaves were placed in each culture dish, sealed, and cultured in the dark at 25°C for 2-4 days.

[0098] 2.5 Delayed Screening

[0099] Wash the leaves after co-cultivation for 3 times (washing solution: MS medium containing vitamin B5 + 3% sucrose + 250mg / L carbenicillin (Carb) + 250mg / L cefotaxime (Cefotaxime), pH 5.2), 20min each time (wash on a dedicated horizontal shaker) and then place on filter paper to absorb (blow dry) the surface liquid. If there is little bacterial liquid on the leaves, you can also directly inoculate the leaves after co-cultivation on a culture dish containing 25mL delayed screening medium (MS medium containing vitamin B5 + 3% sucrose + 7% agar + 250mg / L carbenicillin (Carb) + 250mg / L cefotaxime (Cefotaxime, pH 5.2), put 20-30 in each culture dish, seal it, and culture it in the dark at 22℃ for 14d (after this step, you need to carefully observe whether Agrobacterium is completely inhibited. If Agrobacterium has been inhibited, the corresponding antibiotic should be reduced in the culture medium in the subsequent steps; otherwise, it should be added).

[0100] 2.6 Screening

[0101] After the delayed screening, the leaves were transferred to a culture dish containing 25 mL of screening medium (MS medium containing vitamin B5 + TDZ 2.0 mg / L + IBA 0.5 mg / L + 3% sucrose + 0.7% agar + 200 mg / L timentin + 5 mg / L hygromycin, pH 5.8), with 20-30 leaves placed in each culture dish, and cultured in the dark at 22°C for 20-30 days. The surviving young white and yellow callus were transferred to a new screening medium and cultured continuously until adventitious buds (such as Figure 2 B).

[0102] 2.7 Rooting

[0103] The adventitious buds of 2-3 cm in height were cut off and transferred to rooting medium (1 / 2MS + 5 mg / L hygromycin + 3% sucrose + 0.7% agar, pH 5.8) under the same culture conditions as above to obtain complete plants (such as Figure 2 C).

[0104] 2.8 Hardening and transplanting

[0105] When the tissue culture seedlings grow to 3-4cm and the root system grows to 2-3cm, harden the seedlings. First, open the seal of the tissue culture bottle containing the rooted seedlings and place it in a room with a temperature of 25℃ and a humidity of 75% for 2 days. Then take out the seedlings and wash the culture medium attached to the roots. Plant them in clusters in a sterilized (autoclaved) substrate (mass ratio of nutrient soil: perlite: vermiculite = 1:1:1) ( Figure 2D). After planting the seedlings, water them thoroughly immediately and cover them with polyethylene film to keep them warm and moist. Spray them once a day to maintain a relative humidity of 80% to 85% and a temperature of (25±2)℃. Remove the film after 7 days.

[0106] 3. Identification of overexpression plants

[0107] Five wild-type (WT) and five overexpressing (OE1-FaTRAB1 to OE5-FaTRAB1) transgenic strawberry plant lines were transplanted and identified.

[0108] 3.1 Identification of overexpression positive plants

[0109] The mRNA extracted from the leaves of the overexpressing plants by CTAB method was reverse transcribed, and the synthesis of the first-chain cDNA was based on the reverse transcription kit of Beijing Transgen Biotechnology Co., Ltd. All-in-One First-Strand cDNA Synthesis SuperMix for PCR User Manual, reverse transcription system: 1000ng TotalRNA, 4μL 5× All-in-one SuperMix for PCR, 1μL gDNA remover, RNase-free Water to a total volume of 20μL. The PCR program is: 42℃, 30min; 85℃, 5s, 12℃∞, and quantitative primers are designed for quantification. The quantitative primers are as follows:

[0110] qFaTRAB1-F (SEQ ID NO.5): GAATTGAATAGGGAGAGTGGTG;

[0111] qFaTRAB1-R (SEQ ID NO. 6): GTTGCAGGCTTGGGAAAG.

[0112] The results showed that the expression levels of FaTRAB1 in the five overexpression transgenic lines were significantly increased, with relative expression levels of 15.24, 1.21, 13.71, 13.79 and 1.55 ( Figure 3 ), indicating that it is a positive plant. The present invention selected strains OE1, OE3 and OE4 with higher relative expression levels for subsequent experiments.

[0113] 4. Subcellular localization analysis of FaTRAB1

[0114] The full-length cds sequence of FaTRAB1 (SEQ ID NO. 1) was constructed into the PBI221-eGFP vector, and eGFP fused to FaTRAB1, cytoplasmic marker (RPP3AmChery) and nuclear marker (red fluorescent protein, RFP) were injected into the epidermis of tobacco plants in equal proportions using Agrobacterium strain GV3101 and standard transient transformation protocols. The infiltration buffer was 10 mM MgCl 2 , 10 mM MES, 200 μM acetosyringone (OD600 = 0.8). Three days later, the fluorescence signals of eGFP, RPP3A-mCherry and RFP were captured using a FV3000 confocal laser scanning microscope (Olympus, Japan). RPP3A-mCherry and RFP ( Figure 4 ). The fluorescence of GFP-fused FaTRAB1 completely overlaps with the cytoplasm and nucleus ( Figure 4 ), indicating that FaTR4B1 is expressed in the cytoplasm and nucleus.

[0115] 5. Determination of morphological indicators

[0116] To clarify the role of FaTRAB1 in the growth and development of strawberry plants, we planted three WT and three overexpression lines (OE1-FaTRAB1, OE3-FaTRAB1 and OE4-FaTRAB1, three lines in each line) in the substrate for three months, and then observed the growth status of the plants and leaves, and performed paraffin section and scanning electron microscopy observations. The leaf discs of wild-type and FaTRAB1 overexpression plants were immersed in paraffin section fixative (pure ethanol: water: glacial acetic acid: formaldehyde = 63:30:5:2, V / V / V) and scanning electron microscopy fixative (2.5% glutaraldehyde) and sent to a biological company for subsequent experiments. The cross-sectional characteristics of the leaves and the occurrence of trichomes were observed under an optical microscope (Nikon Eclipse E100, Nikon, Japan) and a scanning electron microscope (JSMIT800, Japan), and the images were collected and analyzed using an imaging system (Nikon DS-U3, Nikon, Japan).

[0117] The results showed that stable overexpression of FaTRAB1 resulted in altered phenotypes of diploid strawberry plants compared with WT ( Figure 5 Stable overexpression of FaTRAB1 significantly reduced plant height, crown width, and petiole length in strawberry plants, while there was no difference in petiole thickness ( Figure 5 In addition, overexpression of FaTRAB1 also led to a reduction in the petiole length of each compound leaf in strawberry plants ( Figure 5 G) and the increase in blade thickness ( Figure 5Since the color of strawberry leaves deepened after overexpression of FaTRAB1, this study measured the pigments of strawberry leaves. Overexpression of FaTRAB1 significantly increased the content of chlorophyll a, chlorophyll b and carotenoids in strawberry leaves ( Figure 5 The above results indicate that stable overexpression of FaTRAB1 in cultivated strawberry will seriously change the agronomic morphology of strawberry plants, thereby affecting the growth and development of plants.

[0118] 6. Analysis of leaf anatomical structure and dorsal scanning electron microscopy of FaTRAB1-overexpressing leaves

[0119] The basic cell structure of the leaf is mainly composed of the upper and lower epidermis, palisade and spongy tissue. The palisade tissue is a columnar parenchyma tissue containing many chloroplasts, while the spongy cell tissue is loose. Microscopic observation showed that the thickness of the leaf overexpressing FaTRAB1 was significantly higher than that of the wild type ( Figure 6 A and B), there was no significant difference in the thickness of the upper and lower epidermis ( Figure 6 C and D), while the length of palisade tissue in FaTRAB1-overexpressing cells was significantly higher than that in wild-type cells ( Figure 6 E). These results indicate that stable overexpression of FaTRAB1 in cultivated strawberry affects the anatomical structure of leaves.

[0120] To understand the differences in trichomes between overexpressed FaTRAB1 and wild-type, scanning electron microscopy (SEM) analysis was performed on the lower epidermis of strawberry leaves. The results showed that the number of non-glandular hairs in the lower epidermis of wild-type and stably overexpressed FaTRAB1 leaves was 47 and 118, respectively ( Figure 7 A in Ⅰ-Ⅱ and Figure 7 However, the number of glandular hairs in the FaTRAB1-overexpressing cells was not significantly different from that in the WT cells, which was approximately 170 ( Figure 7 C). Further observations under 100x and 500x SEM revealed no difference in the structure of the non-glandular hairs and glandular hair trichomes of the two materials, which were linear and rod-shaped, respectively ( Figure 7 In addition, we also observed under 500 times SEM that the stalk of the glandular hair trichomes overexpressing FaTRAB1 was longer, about 81.3μm, while the WT was only 42.3μm. At the same time, the cells in the leaves overexpressing FaTRAB1 were significantly smaller than those of the wild type ( Figure 7 A in Ⅴ-Ⅵ and Figure 7 D). Therefore, the above results indicate that stable overexpression of FaTRAB1 increases leaf thickness, promotes the occurrence of non-glandular trichomes, and lengthens the stalk of glandular trichomes, which may help respond to stress environments.

[0121] 7. Overexpression of FaTRAB1 makes it resistant to two-spotted spider mite

[0122] Given that overexpression of FaTRAB1 leads to thicker leaves and more trichomes, in order to explore whether it has a defensive effect against red spider mites, we inoculated red spider mites on the back of strawberry leaves. Seven days after inoculation, yellow spots appeared on the front of wild-type strawberry leaves, and there were no obvious symptoms on the back. However, leaves overexpressing FaTRAB1 had only sporadic yellow spots or no spots on the front, and no obvious symptoms on the back of the leaves ( Figure 8 In addition, we found that the maximum photochemical efficiency (Fv / Fm), net photosynthetic rate, stomatal conductance, and intercellular CO 2 concentration, transpiration rate, relative conductivity, H 2 O 2 There were no significant differences in the content of MDA and PH value between the wild type and the overexpressed FaTRAB1. However, after 7 days of inoculation with red spider mites, the maximum photochemical efficiency (Fv / Fm) of the overexpressed FaTRAB1 was not significantly different from that of the wild type, but the net photosynthetic rate, stomatal conductance, and intercellular CO 2 The concentration and transpiration rate were increased, while the relative conductivity, H 2 O 2 and MDA contents decreased ( Figure 8 These results indicate that inoculation with spider mites affects the photosynthesis and growth of strawberry plants, whereas overexpression of FaTRAB1 can alleviate the stress of spider mites on strawberry plants.

[0123] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of overexpressing FaTRAB1 gene, characterized in that: This includes any of the following applications: (1) Changing the agronomic traits of strawberry plants; (2) Promote the occurrence of trichomes on strawberry leaves; (3) Improve strawberry's resistance to red spider mites; (4) Cultivating transgenic strawberries with altered agronomic traits; (5) Cultivating transgenic strawberries that promote the formation of trichomes on strawberry leaves; (6) Cultivate transgenic strawberries with strong resistance to spider mites; Wherein, the nucleotide sequence of the FaTRAB1 gene is shown as SEQ ID NO.1; The agronomic traits of the strawberry plants are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of trichomes; The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of trichomes is increased; The trichomes are non-glandular trichomes.

2. Application of increasing the protein content encoded by the FaTRAB1 gene, characterized in that: This includes any of the following applications: (1) Changing the agronomic traits of strawberry plants; (2) Promote the occurrence of trichomes on strawberry leaves; (3) Improve strawberry's resistance to red spider mites; (4) Cultivating transgenic strawberries with altered agronomic traits; (5) Cultivating transgenic strawberries that promote the formation of trichomes on strawberry leaves; (6) Cultivate transgenic strawberries with strong resistance to spider mites; Wherein, the nucleotide sequence of the FaTRAB1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO.2; The agronomic traits of the strawberry plants are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of trichomes; The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of trichomes is increased; The trichomes are non-glandular trichomes.

3. Application of a recombinant vector for overexpressing the FaTRAB1 gene, characterized in that: This includes any of the following applications: (1) Changing the agronomic traits of strawberry plants; (2) Promote the occurrence of trichomes on strawberry leaves; (3) Improve strawberry's resistance to red spider mites; (4) Cultivating transgenic strawberries with altered agronomic traits; (5) Cultivating transgenic strawberries that promote the formation of trichomes on strawberry leaves; (6) Cultivate transgenic strawberries with strong resistance to spider mites; Wherein, the nucleotide sequence of the FaTRAB1 gene is shown as SEQ ID NO.1; The agronomic traits of the strawberry plants are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of trichomes; The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of trichomes is increased; The trichomes are non-glandular trichomes.

4. Application of recombinant bacteria overexpressing the FaTRAB1 gene, characterized in that: This includes any of the following applications: (1) Changing the agronomic traits of strawberry plants; (2) Promote the occurrence of trichomes on strawberry leaves; (3) Improve strawberry's resistance to red spider mites; (4) Cultivating transgenic strawberries with altered agronomic traits; (5) Cultivating transgenic strawberries that promote the formation of trichomes on strawberry leaves; (6) Cultivate transgenic strawberries with strong resistance to spider mites; Wherein, the nucleotide sequence of the FaTRAB1 gene is shown as SEQ ID NO.1; The agronomic traits of the strawberry plant are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of non-glandular trichomes; The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of non-glandular trichomes is increased; The trichomes are non-glandular trichomes.

5. A method for changing agronomic traits of strawberry, characterized in that: The method comprises using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to regulate the growth and development of the strawberry plant; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1; The agronomic traits of the strawberry plants are changed by reducing plant height, crown width and petiole length, increasing leaf thickness and chlorophyll content, and promoting the occurrence of non-glandular trichomes.

6. A method for promoting the occurrence of non-glandular trichomes in strawberry leaves, characterized in that: The method comprises using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to promote the occurrence of non-glandular trichomes on the back of leaves of the strawberry plant; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.

1.

7. A method for improving strawberry's resistance to red spider mites, characterized in that: The method comprises using genetic transformation technology to stably over-express the FaTRAB1 gene in the strawberry plant to improve the resistance of the strawberry plant to red spider mites; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.

1.

8. A method for cultivating transgenic strawberries with altered agronomic traits, characterized in that: The method comprises using genetic transformation technology to stably overexpress the FaTRAB1 gene in the strawberry plant to obtain transgenic strawberries with altered agronomic traits; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1; The agronomic traits are changed as follows: plant height, crown width and petiole length are reduced, leaf thickness and chlorophyll content are increased, and the occurrence of non-glandular trichomes is increased.

9. A method for cultivating transgenic strawberries that promotes the formation of non-glandular trichomes in strawberry leaves, characterized in that: The method comprises using genetic transformation technology to stably over-express the FaTRAB1 gene in the strawberry plant to obtain transgenic strawberries that promote the formation of non-glandular trichomes in strawberry leaves; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.

1.

10. A method for cultivating transgenic strawberries with strong resistance to red spider mites, characterized in that: The method comprises using genetic transformation technology to stably over-express the FaTRAB1 gene in the strawberry plant to obtain transgenic strawberries with strong resistance to red spider mites; the nucleotide sequence of the FaTRAB1 gene is shown in SEQ ID NO.1.

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