Application of pineapple AcGF14f gene in improving cold resistance of plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses application of a pineapple AcGF14f gene in improving the cold resistance of plants. According to the invention, a Shenwan pineapple is used as a material, and a 14-3-3 protein gene is cloned and is named as AcGF14f, the open reading frame of the gene is 783bp, 260 amino acids are encoded, and the gene contains a typical 14-3-3 protein structural domain. Gene expression analysis finds that AcGF14f can be induced by various adversity stresses such as low temperature, ABA and the like and hormone treatment. Compared with a wild control group, the AcGF14f overexpressed tobacco strain has higher survival rate and Fv / Fm value after low-temperature treatment, CAT, SOD and POD enzyme activity is enhanced, O2 <-> removal capacity is higher, it is indicated that the cold resistance of the plant is remarkably enhanced, the AcGF14f can promote expression of ROS related genes in tobacco, it is indicated that the AcGF14f is a gene with a positive cold resistance regulation effect, and the AcGF14f is a gene with a positive cold resistance regulation effect. A foundation is laid for genetic improvement of cold resistance of pineapples, and an important candidate functional gene is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of pineapple AcGF14f gene in improving plant cold tolerance. Background Art

[0002] Pineapple is one of the famous tropical fruits. During the cultivation process of pineapple, it will be damaged by various abiotic stress factors, among which low temperature stress is the most serious. Continuous extreme low temperature will cause varying degrees of damage such as withered and yellow leaves, necrosis of shoot apical meristems, and rot of stems and leaves in pineapples. Low temperature has become one of the important environmental factors restricting the production distribution and industrial development of pineapples. Low temperature will induce plants to initiate defense mechanisms and make adaptive adjustments at multiple levels such as molecular, cellular, physiological, and biochemical levels to alleviate and resist the damage caused by low temperature. Screening high-quality cold-resistant germplasms, exploring the internal molecular mechanisms related to cold resistance, and carrying out transgenic breeding are important ways to improve the cold resistance of pineapples. Exploring the molecular mechanisms and regulatory networks related to cold resistance in pineapples and screening important genes related to cold resistance are of great significance for carrying out the creation work of transgenic cold-resistant germplasms.

[0003] 14-3-3 proteins are a class of highly conserved regulatory proteins widely present in eukaryotes. They usually appear in the form of homo- or heterodimers and can bind to target proteins through phosphorylation patterns and participate in regulating multiple cell signaling pathways, such as transcription factors, protein kinases, and apoptosis factors. In plants, 14-3-3 proteins were first discovered in Arabidopsis thaliana and are also simply referred to as "G-box factor 14-3-3" or "GF14" because they bind to the cis-acting element G-box. 14-3-3 proteins play important roles in plant growth and development. They affect plant growth by regulating processes such as the cell cycle and division, cell expansion, and organ development. In addition, 14-3-3 proteins help plants adapt to adverse environmental conditions such as drought, high salt, and low temperature by regulating the stress responses of plants. The number of members of the 14-3-3 protein family is numerous, covering multiple aspects such as plant growth and development and stress response. The number of unknown genes and unknown functions in the family is still huge, and their functions show obvious species specificity in different plants.

[0004] So far, there has been no report on the function of the 14-3-3 protein gene in pineapples, and the functional research on genes related to cold resistance regulation in pineapples is also extremely limited. Therefore, it is urgent to explore and identify genes related to cold tolerance in pineapples to lay a foundation for regulating the low-temperature resistance of pineapples by molecular means and at the same time create conditions for carrying out various innovative works of transgenic cold-resistant germplasms using cold-resistant genes. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the primary object of the present invention is to provide a pineapple cold tolerance-related gene AcGF14f and its application to solve the problem of plant tolerance to low temperature stress. Further, overexpression of AcGF14f can improve the ability of related plants to respond to adverse environments such as low temperature stress.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] Application of pineapple AcGF14f gene or its expression product in improving plant cold tolerance, the nucleotide sequence of the pineapple AcGF14f gene is shown in SEQ ID NO: 1 and consists of 783 bases; the amino acid sequence of the protein encoded by the AcGF14f gene is shown in SEQ ID NO.2 and consists of 260 amino acid residues; the application is to improve plant cold tolerance based on overexpression of the AcGF14f gene.

[0008] Further, the primer sequences for synthesizing the pineapple AcGF14f gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively.

[0009] Even further, the plant is pineapple or tobacco.

[0010] The present invention also provides an application of a biological material in a product for improving plant cold tolerance by overexpressing the AcGF14f gene, and the biological material is any one of the following:

[0011] 1) A nucleic acid molecule encoding a protein with an amino acid sequence shown in SEQ ID NO.2;

[0012] 2) An expression cassette containing the nucleic acid molecule described in 1);

[0013] 3) A recombinant vector containing the nucleic acid molecule described in 1), or a recombinant vector containing the expression cassette described in 2);

[0014] 4) A transgenic plant cell line containing the nucleic acid molecule described in 1), or a transgenic plant cell line containing the expression cassette described in 2).

[0015] In addition, the present invention also provides a method for upregulating or enhancing or improving the cold tolerance of a target plant, and the method includes overexpressing the AcGF14f gene to upregulate or enhance or improve the cold tolerance of the target plant, and the plant is pineapple or tobacco.

[0016] Further, the application includes cultivating plant varieties with enhanced cold tolerance under low temperature stress conditions.

[0017] Furthermore, by transforming the vector overexpressing the pineapple AcGF14f gene, the transgenic cell line or the transgenic recombinant bacterium into plant cells, tissues or organs, and then cultivating the transformed plant cells, tissues or organs, cold-tolerant transgenic plants are obtained.

[0018] Furthermore, when the plant is tobacco, the overexpression vector is pBI121-AcGF14f.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] In the present invention, a recombinant expression vector of the pineapple cold-tolerant gene AGF14f is constructed, and the above recombinant expression vector is transferred into tobacco, and an AGF14f gene overexpression line is screened and obtained. Further biological function verification reveals that the cold tolerance of the overexpression line is significantly enhanced compared with that of the wild-type plant, indicating that the AGF14f gene cloned in the present invention has a cold-resistant function. The present invention also provides a method for improving the cold tolerance of plants, laying a foundation for the research on regulating the low-temperature stress resistance of plants by molecular means and the breeding of new varieties, and is of great significance for improving the quality and yield of plants and cultivating cold-resistant plants. Description of the Drawings

[0021] Figure 1 It is the PCR amplification electrophoresis pattern of AcGF14f;

[0022] Figure 2 It is the expression of AcGF14f under various stress conditions such as low temperature, ABA and hormones; the expression of AcGF14f under low temperature, high temperature, NaCl, PEG, ABA, GA, SA, MeJA, NAA and IAA treatments; the asterisk indicates a significant difference from the control plants (P<0.05);

[0023] Figure 3 It is the resistance screening and RT-qPCR verification of AcGF14f transgenic tobacco; (a) Screening of the T3 generation lines of overexpressing AcGF14f tobacco (b) Semi-quantitative RT-qPCR identification of the T3 generation lines of overexpressing AcGF14f tobacco (c) Expression of the T3 generation lines of overexpressing AcGF14f tobacco; WT is the tobacco wild type (col-0), and AcGF14f-OE1, AcGF14f-OE2, AcGF14f-OE3 respectively represent 3 T3 generation transgenic lines. The asterisk indicates a significant difference from the control plants (P<0.05);

[0024] Figure 4Phenotypic identification, survival rate, and changes in Fv / Fm of AcGF14f-overexpressing tobacco and wild-type plants; (a) Phenotypic characteristics of wild-type (WT) and AcGF14f-overexpressing tobacco lines under low-temperature treatment; (b) Fv / Fm chlorophyll fluorescence images; (c) Survival of AcGF14f-overexpressing tobacco and wild-type (WT) lines after cold treatment; (d) Changes in Fv / Fm of AcGF14f-overexpressing tobacco and wild-type (WT) lines after cold treatment; WT is the wild type of tobacco (col-0), and AcGF14f-OE1, AcGF14f-OE2, and AcGF14f-OE3 represent three T3-generation tobacco transgenic lines, respectively; Asterisks indicate significant differences from the control plants (P<0.05);

[0025] Figure 5 Changes in physiological indexes and NBT staining of AcGF14f-overexpressing tobacco and wild-type plants before and after cold treatment; Asterisks indicate significant differences from the control plants (P<0.05);

[0026] Figure 6 Expression analysis diagram of cold-responsive genes in AcGF14f-overexpressing tobacco. Asterisks indicate significant differences from the control plants (P<0.05).

[0027] Detailed implementation manners

[0028] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments and comparative examples of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0030] Example 1 Cloning of AcGF14f gene and construction of overexpression vector

[0031] I. Experimental methods

[0032] 1. RNA extraction

[0033] The materials were selected from the pineapple germplasm resource garden of the College of Horticulture, South China Agricultural University, variety 'Shenwan'. Pineapple plants with good growth status were selected, and 0.1 g of leaf samples were randomly weighed and quickly frozen with liquid nitrogen. The total RNA of 'Shenwan' pineapple was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme). The specific method is as follows:

[0034] (1) Sample preparation: Take 0.1 g of fresh or liquid nitrogen-quick-frozen sample, grind it into powder and transfer it to a centrifuge tube, add 600 μL of Buffer PSL, vortex vigorously for 30 sec, and centrifuge at 12,000 rpm and 4 °C for 5 min.

[0035] (2) Take about 500 μL of the supernatant and transfer it to FastPure gDNA-Filter Columns III (which has been placed in the collection tube), centrifuge at 12,000 rpm and 4 °C for 30 sec, discard FastPure gDNA-Filter Columns III, and collect the filtrate.

[0036] (3) Add 250 μL of absolute ethanol to the collection tube and mix well by shaking for 15 sec.

[0037] (4) Transfer the above mixture to FastPure RNA Columns V (which has been placed in the collection tube), centrifuge at 12,000 rpm and 4 °C for 30 sec, and discard the filtrate.

[0038] (5) Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm and 4 °C for 30 sec, and discard the filtrate.

[0039] (6) Add 500 μL of Buffer RWB to FastPure RNA Columns V, centrifuge at 12,000 rpm and 4 °C for 30 sec, and discard the filtrate.

[0040] (7) Repeat step 6.

[0041] (8) Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm and 4 °C for 2 min.

[0042] (9) Transfer the FastPure RNA Columns V to new 1.5 ml RNase-free Collection Tubes. Pipette 30 - 100 μL of RNase-free ddH2O (preheated at 65 °C) onto the center of the adsorption column membrane, and centrifuge at 12,000 rpm and 4 °C for 1 min. The resulting solution is pineapple RNA.

[0043] (10) Store the extracted pineapple RNA in a -80 °C ultra-low temperature freezer for later use.

[0044] 2. Synthesis of cDNA

[0045] Refer to the operation manual of the YEASEN reverse transcription kit. Product name: AdvanceFast One-step RT-gDNADigestion SuperMix for qPCR. The specific method is as follows:

[0046] Prepare the following mixture (Table 1) in an RNase free centrifuge tube and gently pipette to mix well.

[0047] Table 1

[0048]

[0049] The reaction program is: reverse transcription at 37 °C for 5 min, and enzyme inactivation at 85 °C for 30 sec.

[0050] The reaction product obtained is pineapple cDNA, and the resulting first-strand cDNA is used for the amplification of the AcGF14f gene.

[0051] 3. Cloning of the AcGF14f gene

[0052] Use Phanta Max Super-Fidelity DNA Polymerase (Vazyme) to amplify the target fragment. The specific method is as follows (Table 2):

[0053] Reaction system:

[0054] Table 2

[0055]

[0056] Reaction program: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 sec, annealing at 56 °C for 15 sec, extension at 72 °C for 60 sec, 35 cycles; final extension at 72 °C for 5 min.

[0057] 4. Recovery of the target band

[0058] After the PCR amplification was completed, the target band was detected by 1% agarose gel electrophoresis. After the band size was correct, the DNA gel extraction kit (Qingke Biotech) was used to recover the PCR product and the recovered product was detected by electrophoresis. The specific method was as follows:

[0059] (1) Add Buffer GL (not less than 150 μL) to the PCR product according to the ratio of PCR stock solution:Buffer GL = 1:3, and pipette and mix well;

[0060] (2) Transfer the solution into the adsorption column, centrifuge at 12,000×g for 1 min, discard the waste liquid, and put the adsorption column back into the empty collection tube;

[0061] (3) Add 700 μL of Buffer W2 to the adsorption column (please first check whether the specified volume of absolute ethanol has been added), centrifuge at 12,000×g for 1 min, and discard the waste liquid;

[0062] (4) Repeat step 3

[0063] (5) Put the adsorption column back into the empty collection tube and centrifuge at 12,000×g for 2 min;

[0064] (6) Take out the adsorption column, place it in a clean 1.5 mL centrifuge tube, add 35 μL of Eluent to the middle part of the adsorption membrane (preheating Eluent at 60 - 65 °C has a better effect), place it at 20 - 25 °C for 2 min, and centrifuge at 12,000×g for 2 min to obtain the PCR recovered product.

[0065] 5. Transformation of the target band into DH5α competent cells

[0066] After purifying the PCR product, it was ligated to the pBM23 vector, and then the ligation product was transformed into DH5α competent cells. The specific operation steps were as follows:

[0067] (1) Take 50 μL of Escherichia coli competent cells thawed on ice, add 10 μL of the ligation product to the competent cells, flick the tube wall several times to mix well, and let it stand on ice for 25 min.

[0068] (2) Heat shock at 42 °C for 45 s, and quickly transfer it to ice and let it stand for 2 min.

[0069] (3) Add 700 μL of LB liquid medium without antibiotics, incubate at 37 °C and 200 rpm on a shaker for 60 min.

[0070] (4) Centrifuge at 5000 rpm for 1 min, discard 700 μL of the supernatant, resuspend the bacteria and spread them on the LB + Kan culture plate, and incubate overnight at 37 °C.

[0071] After the bacterial plates grew on the culture plates, pick monoclonal colonies for PCR detection. Use Kangwei reagent 2×Flash PCR MaterMix (Dye) to detect whether the clones are positive. The upstream and downstream primers are 35S-F and Det-AcGF14f-R (Table 5). Send the bacterial liquid of the positive clones to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and compare and analyze the obtained sequencing results with the reference gene sequence. After the sequencing results are compared by BLAST, if the size of the target fragment is consistent with the base sequence and AcGF14f, it indicates that the AcGF14f clone is successful( Figure 1 ), expand and shake the bacterial liquid to extract and prepare plasmids for later use.

[0072] 6. Construction of overexpression vector

[0073] Using the previously obtained pBM23-AcGF14f as a template, construct an AcGF14f overexpression vector using the principle of homologous recombination, and place the target fragment downstream of the 35S promoter of the pBI121 plasmid. The specific steps are as follows:

[0074] (1) Primer design

[0075] Design primers pB-AcGF14f-F and pB-AcGF14f-R, add a restriction enzyme cleavage site for XbaⅠ at the 5' end of the AcGF14f gene fragment, and add a restriction enzyme cleavage site for BamHⅠ at the 3' end.

[0076] (2) Amplification of target gene fragment

[0077] Use Phanta Max Super-Fidelity DNA Polymerase (Vazyme) to amplify the target gene fragment, and use a DNA gel recovery kit (Tsingke Biological) to recover the PCR product and perform electrophoresis to detect the recovered product (the method is the same as above).

[0078] (3) Preparation of linearized vector

[0079] Digest the pBI121 vector with XbaⅠ and BamHⅠ.

[0080] (4) Recombination reaction

[0081] Use Uniclone One Step Seamless Cloning Kit (Genesand) to ligate the amplified target gene fragment with the linearized plasmid to complete homologous recombination. The reaction system (Table 3):

[0082] Table 3

[0083]

[0084] The recombinant reaction products were transformed into Escherichia coli competent cells. Single colonies were picked for PCR detection. The bacterial liquid of the positive clones was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequencing results were compared and analyzed with the reference gene sequence. After BLAST comparison of the sequencing results, if the size of the target fragment and the base sequence were consistent with AcGF14f, it indicated that the pBI121-AcGF14f recombinant cloning vector was successfully constructed.

[0085] 7. Transfer of the overexpression vector into Agrobacterium

[0086] (1) Take the Agrobacterium competent cells stored at -80°C and let them sit at room temperature or in the palm of the hand for a moment until they partially melt. When in an ice-water mixture state, insert them into ice.

[0087] (2) Add 0.01 - 1 μg of plasmid DNA to every 100 μL of competent cells. Gently flick the bottom of the tube to mix evenly. Then let it stand on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes in sequence.

[0088] (3) Add 700 μL of antibiotic-free YEP liquid medium and culture it with shaking at 28°C for 2 - 3 hours.

[0089] (4) Centrifuge at 6000 rpm for 1 minute to collect the bacteria. Leave about 100 μL of the supernatant and gently resuspend the bacterial pellet by pipetting. Spread it on a YEP plate containing kanamycin (Kan) and place it upside down in a 28°C incubator for 2 - 3 days.

[0090] After colonies grow on the culture plate, pick single colonies for PCR detection. Take the bacterial liquid of the positive clones and store it in a -80°C refrigerator for later use.

[0091] II. Experimental Results

[0092] Using the cDNA of 'Shenwan' pineapple leaves as a template, the full-length sequence of AcGF14f was amplified by PCR. The band was consistent with the expectation, about 783 bp ( Figure 1 ). The sequence analysis results showed that AcGF14f (gene ID: LOC109711396) contained a complete open reading frame of 783 bp, encoding 260 amino acids. Protein sequence feature analysis showed that the molecular weight of the AcGF14f protein was 28.95 kDa, the theoretical isoelectric point was 4.78, belonging to an acidic protein; the aliphatic index was 87.27, and the instability index was 44.88, belonging to an unstable protein. The AcGF14f protein was composed of 20 kinds of amino acids, among which the contents of Ala (alanine) and Glu (glutamic acid) were the highest, and the contents of Cys (cysteine) and Trp (tryptophan) were the lowest.

[0093] Taking plasmid pBM23-AcGF14f as a template, an overexpression vector of AcGF14f was constructed using the principle of homologous recombination. PCR detection was performed using primers 35S-F and DET-AcGF14f-R, and a band of the expected size, approximately 1000 bp, was obtained, proving that the vector construction was successful. The recombinant plasmid was named pBI121-AcGF14f.

[0094] Example 2 Expression analysis of AcGF14f under different stresses and hormone treatments

[0095] I. Experimental methods

[0096] Using the callus and tissue culture seedlings of 'Shenwan' pineapple as materials, the callus was cultured under dark conditions at 26±2°C, and the tissue culture seedlings were cultured under conditions of 26±2°C, 16 h light / 8 h dark, maintaining good growth status for subsequent experiments on stress treatments such as low temperature and exogenous hormone treatments such as ABA. After sample collection, they were quickly placed into liquid nitrogen for quick freezing, and all the collected samples were stored in an ultra-low temperature refrigerator at -80°C. The expression of AcGF14f was detected by qRT-PCR, using the pineapple β-actin gene as a reference gene.

[0097] Low temperature treatment: The tissue culture seedlings placed on MS solid medium were put into a culture room at 4°C for culture, and samples were collected after pre-cooling treatment for 0, 1, 2, 4, 8, 12, 24, 48, 72 h;

[0098] High temperature treatment: The tissue culture seedlings placed on MS solid medium were put into a culture room at 42°C for culture, and samples were collected after pre-cooling treatment for 0, 1, 2, 4, 8, 12, 24, 48, 72 h;

[0099] Salt treatment: The callus was treated in a solution containing 150 mM sodium chloride (NaCl) for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h;

[0100] PEG treatment: The callus was treated in a solution containing 20% polyethylene glycol (PEG4000) for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h;

[0101] ABA treatment: The callus was treated in MS liquid medium containing abscisic acid (ABA, 100 μM) for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h respectively;

[0102] GA treatment: The callus was treated in MS liquid medium containing gibberellin (GA, 100 μM) for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h respectively;

[0103] SA treatment: The callus was treated in MS liquid medium containing salicylic acid (SA, 100 μM) for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h, respectively;

[0104] MeJA treatment: The callus was treated in MS liquid medium containing methyl jasmonate (MeJA, 100 μM) for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h, respectively;

[0105] IAA treatment: The callus was treated in MS liquid medium containing indole-3-acetic acid (IAA, 100 μM) for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h, respectively;

[0106] NAA treatment: The callus was treated in MS liquid medium containing 1-naphthaleneacetic acid (NAA, 100 μM) for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h.

[0107] The reaction system (Table 4) was operated according to the instructions of qPCR SYBR Green Master Mix (No Rox) (Yeasen). qRT-PCR was performed on a fluorescence quantitative PCR instrument 384 (BIO-RAD). The reaction system was 10 μL, and the reaction program was: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 sec, annealing at 60 °C for 15 sec, extension at 72 °C for 35 sec, and the number of cycles was 40. The quantitative primer sequences are shown in Table 1, and the 2 -△△Ct -method was used to calculate the relative expression level of the gene, and each sample was repeated 3 times.

[0108] Table 4

[0109]

[0110] II. Experimental results

[0111] The qRT-PCR results showed ( Figure 2) With the increase of low-temperature treatment time, the expression of AcGF14f gene gradually increased and reached the maximum at 4 h, indicating that AcGF14f gene responded strongly to low-temperature stress; in high-temperature treatment, the expression of AcGF14f increased briefly at 1 h, but then decreased immediately and dropped to and maintained at the initial level at 4 h; NaCl treatment induced the expression of AcGF14f, and the expression level of the gene reached the maximum after 8 h of treatment, and the expression level decreased at 12 h but was still higher than the initial level; after PEG treatment, the expression of AcGF14f gene increased rapidly and reached the maximum at 1 h, then the expression level decreased, and there was a recovery phenomenon at 12 h, and finally it was lower than the lowest level at 72 h; after ABA treatment, the expression level increased and reached the maximum at 2 h, and then decreased slowly; after GA treatment, the expression of AcGF14f increased rapidly, reached the maximum at 2 h and then gradually decreased, and then the expression level decreased in a gradient manner, but finally it was still higher than the initial level; after MeJA treatment, the expression of AcGF14f gradually increased, and the expression level at 72 h was about twice that of the initial level; after IAA treatment, the expression of AcGF14f at 4, 8 and 72 h was significantly higher than the initial level; after NAA treatment, AcGF14f increased exponentially, and the expression level at 72 h was 8 times that of the initial level; after SA treatment, the expression of AcGF14f was relatively stable and did not increase until 48 h. It can be seen that the expression of AcGF14f gene can respond to various abiotic stresses and hormone treatments, indicating that AcGF14f may participate in multiple stress signaling pathways and play multiple functional roles.

[0112] Example 3 Tobacco Genetic Transformation and Positive Identification

[0113] I. Experimental Methods

[0114] 1. Genetic Transformation of Tobacco

[0115] 1.1 Sowing and Cultivation of Tobacco

[0116] Disinfect the surface of tobacco seeds in a laminar flow hood: First, rinse with sterile water, and then sequentially treat with 75% ethanol for 1 min and sodium hypochlorite solution (effective chlorine content is 2%) for 15 min. After disinfection, rinse with sterile water 3 - 5 times to remove the residual reagents. After drying with sterile filter paper, place them on MS medium (pH 5.8) for cultivation.

[0117] 1.2 Activation of Agrobacterium tumefaciens

[0118] Take out the stored Agrobacterium liquid from the -80°C ultra-low temperature refrigerator, streak it on YEP solid medium (containing 100 mg / L Kan and 30 mg / L Rif), and culture it at 28°C for 48 - 72 h; pick monoclonal colonies and inoculate them into 3 mL of YEP liquid medium (containing 100 mg / L Kan and 30 mg / L Rif), and culture them overnight with shaking (200 rpm) at 28°C until OD 600 is approximately 0.6, centrifuge (10 min, 5000 rpm) to collect the bacteria, and resuspend the collected bacteria in MS liquid medium to OD 600 = 0.4.

[0119] 1.3 Transformation of tobacco

[0120] For tobacco plants that have been aseptically cultured for 4 - 8 weeks, take their young leaf tissues, remove the leaf margins and main veins, and prepare explants of about 0.5 * 0.5 cm. Place them in the resuspended bacterial liquid, gently shake for 5 min, blot the bacterial liquid on the surface of the explants with sterile filter paper, and inoculate them on the co-culture medium (MS + 6-BA 1 mg / L, pH 5.8) and culture them in the dark for 2 - 3 days.

[0121] After co-culture, place the explants on the bud induction and differentiation medium (pH 5.8) containing 1 mg / L 6-BA, 300 mg / L Timentin, and 100 mg / L Kan, and replace the medium every two weeks. When the resistant buds grow to about 2 cm, cut them off and transfer them to the rooting medium (1 / 2MS + NAA 0.1 mg / L + Timentin 300 mg / L + Kan 100 mg / L, pH 5.8). After the regenerated plants are fully developed, cut a part of the leaf tissue, extract the tobacco leaf RNA, and detect the resistant plants with the gene-specific primers DET-AcGF14f-F and DET-AcGF14f-R. Select the tobacco with positive detection results for seed collection to obtain T1 generation seeds.

[0122] 2. Screening and identification of transgenic tobacco

[0123] The harvested T1 generation seeds were treated with 75% alcohol twice, 30 s each time; the seeds were suspended in absolute ethanol and poured onto sterilized filter paper; after the absolute ethanol had evaporated, the seeds were evenly sown on a selection medium containing 50 mg / L kanamycin for screening. The seeds for resistance screening were cultured under a light cycle of 25°C, 16 h light / 8 h darkness. Two weeks later, the resistant plants grew well on the selection medium, while the negative plants did not germinate or turned white and died soon after. The resistant seedlings growing well on the plate were planted in the substrate. After the tobacco grew up, the lowest leaves were taken to extract the RNA of the tobacco plant leaves using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme) kit, reverse transcribed into cDNA, specific primers for the AcGF14f gene were designed, and RT-qPCR was performed to identify the positive seedlings. Tobacco plants with positive test results were selected for seed collection to obtain T2 generation seeds. Similarly, the T2 generation seeds were screened using the selection medium, and three transgenic plant lines with a segregation ratio close to 3:1 in the T2 generation plants were selected for subsequent analysis and identification, and T3 generation seeds were collected and obtained. Figure 3 , a).

[0124] II. Experimental results

[0125] RT-qPCR positive identification results Figure 3 , b) showed that the transgenic tobacco lines (OE-1, OE-2, and OE-3) and the wild type (WT) both successfully amplified the AtActin band, indicating good cDNA quality. In addition, the transgenic tobacco lines (OE-1, OE-2, and OE-3) could amplify the specific band of AcGF14f, while the wild type (WT) failed to amplify the corresponding band, and the expression levels of AcGF14f in the three transgenic tobacco lines were all more than 6000 times that of the wild type. Figure 3 , c). The above results all proved that the recombinant plasmid pBI121-AcGF14f had been successfully integrated into the tobacco genome.

[0126] Example 4 Cold tolerance analysis of AcGF14f and determination of corresponding indicators

[0127] I. Experimental method

[0128] 1. Cold tolerance analysis and chlorophyll fluorescence measurement of AcGF14f transgenic tobacco

[0129] Wild-type and T3 generation transgenic tobacco at 4 weeks old and with consistent growth were selected, treated at -4°C for 4 h without cold acclimation, and the survival rate was counted after 3 days of recovery at room temperature. Photos were taken and recorded before and after the cold treatment and on the 1st and 3rd days of recovery.

[0130] Use the PlantExplorer (Huinuo Ruide) plant chlorophyll fluorescence imaging system to measure parameters such as Fv / Fm, NPQ, ChlIdx, and AriIdx. Before measurement, the plants need to be dark-treated for 10 min. After measurement, the data is imported into DataAnalysis 5.4.1 software for analysis.

[0131] 2. Determination of physiological indexes of AcGF14f transgenic tobacco

[0132] Select wild-type and T3-generation transgenic tobacco plants at 4 weeks old with consistent growth. Treat them at -4°C for 4 h, and collect leaves before and after cold treatment for the determination of cold resistance-related physiological indexes such as CAT (catalase), SOD (superoxide dismutase), POD (peroxidase), O 2- (superoxide anion). The determination of specific indexes uses a CAT activity assay kit (A007-1-1, Suzhou Keming Biotechnology Co., Ltd.), a total superoxide dismutase (T-SOD) test kit (A001-1-1, Nanjing Jiancheng Bioengineering Institute), a peroxidase (POD) assay kit (A084-3-1, Nanjing Jiancheng Bioengineering Institute), a kit for inhibiting the production of superoxide anion free radicals (O 2- ) (A052-1-1, Nanjing Jiancheng Bioengineering Institute), a malondialdehyde (MDA) assay kit (MDA-1-Y, Suzhou Keming Biotechnology Co., Ltd.), and an NBT staining kit (I023, Nanjing Jiancheng Bioengineering Institute), and operate according to the instructions of the kit. Each experiment has at least 3 biological replicates, and SPSS version 19.0 software is used for t-test to analyze the significance of differences. The specific method is as follows:

[0133] (1) Determination of CAT activity

[0134] Add 9 times the volume of normal saline to 0.1 g of the sample to be measured according to the ratio of weight (g): volume (mL) = 1:9, and prepare a 10% tissue homogenate under ice-water bath conditions. Centrifuge at 2500 rpm for 10 min, and take the supernatant and place it on ice for later measurement.

[0135] Add 1 mL of reagent one and 0.1 mL of reagent two to the control tube and the measurement tube. Add 0.05 mL of the supernatant to the measurement tube, mix immediately, and react accurately at 37°C for 1 min; add 1 mL of reagent three and 0.1 mL of reagent four, and add 0.05 mL of the supernatant to the control tube; mix well, take 200 μL from each tube and place it in a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and measure the absorbance value A at 405 nm with an enzyme-linked immunosorbent assay (ELISA) reader. ΔA = A 对照 -A 测定 .

[0136] Calculation formula: CAT activity (U / g fresh weight) in the tissue = ΔA × 271 / sampling volume (mL) / reaction time (s) / (sample fresh weight (g) / sample pretreatment homogenization medium volume (L)).

[0137] (2) O 2- (Superoxide anion) scavenging ability determination

[0138] Add 9 times the volume of normal saline to 0.1 g of the sample to be tested according to the ratio of weight (g): volume (mL) = 1:9, and prepare a 10% tissue homogenate under ice-water bath conditions. Centrifuge at 2500 rpm for 10 min, and take the supernatant and place it on ice for later measurement.

[0139] Add 0.05 mL of distilled water, Vc standard solution, and supernatant to the control tube, standard tube, and measurement tube respectively; add 1 mL of reagent one application solution, 0.1 mL of reagent two, three, and four application solutions, mix well, and place in a 37 °C constant temperature water bath for 40 min; add 2 mL of color reagent, mix well, let stand at room temperature for 10 min, then take 200 μL from each tube and put it into a 96-well microplate reader. Measure the absorbance value A at 520 nm with a microplate reader.

[0140] Calculation formula: Superoxide anion resistance (U / g fresh weight) = (A 对照 - A 测定 ) / (A 对照 - A 标准 ) × V C Standard solution concentration (mg / mL) × 1000 × (sample fresh weight (g) / extraction solution volume (mL)). One activity unit U is the change value of the superoxide anion radical inhibited by 1 mg of vitamin C per gram of tissue in a 40-minute reaction at 37 °C.

[0141] (3) Determination of SOD activity

[0142] Add 9 times the volume of ultrapure water to 0.1 g of the tobacco leaf sample to be tested according to the ratio of weight (g): volume (mL) = 1:9. Add two small steel balls to each centrifuge tube, and mechanically homogenize at low temperature for 60 s in a grinding machine. Centrifuge at 3500 rpm at low temperature for 10 min, and take the supernatant and place it on ice for later measurement. Add 50 μL of ultrapure water and 50 μL of sample supernatant to the control tube and measurement tube respectively. Add 1 mL of reagent one application solution and 100 μL of reagent two, three, and four application solutions to each tube, vortex and mix well, water bath at 37 °C for 40 min, add 2 mL of color reagent, mix well, let stand at room temperature for 10 min, take 200 μL from each tube and add it to a 96-well microplate hole, and measure the OD value of each tube at 550 nm with a microplate reader.

[0143] The calculation formula for correcting the protein concentration of the sample: SOD activity of the sample to be measured (U / g) = (OD of the control tube - OD of the measurement tube) / OD of the control tube / 50% × total volume of the reaction solution (mL) / sampling volume (mL) / homogenate concentration (g / mL).

[0144] (4) Determination of POD activity

[0145] Add 9 times the volume of ultrapure water to 0.1 g of the tobacco leaf sample to be measured according to the ratio of weight (g): volume (mL) = 1:9. Add two small steel beads to each centrifuge tube, mechanically homogenize at low temperature for 60 s in a grinding machine, centrifuge at 3500 rpm at low temperature for 10 min, and take the supernatant and place it on ice for measurement. Add 2.4 mL of Reagent 1, 300 μL of Reagent 2, and 100 μL of the sample to the control tube and the measurement tube. Add 200 μL of ultrapure water to the control tube and 200 μL of Reagent 3 to the measurement tube. Mix well and react accurately in a 37°C water bath for 30 min. Add 1 mL of Reagent 4, mix well, centrifuge at 3500 rpm for 10 min. Take 200 μL of the supernatant from each tube and add it to a 96-well microplate reader. Measure the OD value at 420 nm with a microplate reader.

[0146] The calculation formula for correcting the protein concentration of the sample: POD activity of the sample to be measured (U / g) = (OD of the measurement tube - OD of the control tube) / 12 colorimetric optical path (1 cm) × total reaction volume (mL) / sampling volume (mL) / reaction time (30 min) / homogenate concentration (g / mL) × 1000.

[0147] (5) NBT staining

[0148] Mix the developer A, developer B, and distilled water according to the volume ratio (mL) = 1:1:50 to prepare the NBT working solution. Select a tobacco plant with good growth conditions, take the third leaf from the bottom and immerse it in the NBT working solution, soak it under vacuum for 8 h in the dark, then discard the working solution, add absolute ethanol and boil it in boiling water for 5 min until the chlorophyll completely fades. After naturally cooling to room temperature, lay the leaf flat on the MS medium plate and take a photo for recording.

[0149] II. Experimental results

[0150] As Figure 4 shown in a, before cold treatment, there was no obvious difference in the growth state between the transgenic lines and the wild-type plants. After cold treatment (-4°C, 4 h), after 3 days of recovery, it was found that: compared with the transgenic lines, the wild-type plants showed large areas of withering and yellowing, while the leaves of the transgenic lines remained bright green, and the leaf margins were slightly dry and curled. After statistics, the survival rate of the wild-type was 10.82%, and the survival rates of the three transgenic lines were 37.46%, 47.20%, and 32.22% respectively. The survival rate of the transgenic lines was significantly higher than that of the wild-type ( Figure 4, c). Under normal growth conditions, there was no significant difference in the maximum photochemical efficiency (Fv / Fm) between the wild type and transgenic lines. After cold treatment, the Fv / Fm of both decreased (Figure), but after 3 days of recovery, the transgenic lines (0.737, 0.738, 0.740) were still significantly higher than the wild type (0.657)( Figure 4 , b and Figure 4 , d).

[0151] Low temperature stress significantly induced the antioxidant enzyme activities in tobacco leaves. Among them, the activities of POD, SOD and CAT in transgenic lines were significantly higher than those in the wild type, indicating that the ability of transgenic tobacco to scavenge reactive oxygen species and affect the intracellular reactive oxygen species level was enhanced. Further research found that after low temperature treatment, the ability of transgenic tobacco lines to resist O 2- anions was significantly higher than that of the wild type, and during the NBT histochemical staining process, the indigo spot area of transgenic plants after low temperature treatment was significantly smaller than that of wild type plants, further indicating that the overexpression tobacco lines of AcGF14f could more efficiently scavenge intracellular reactive oxygen species under low temperature stress to adapt to the low temperature environment( Figure 5 ).

[0152] In summary, overexpressing AcGF14f tobacco enhanced the tolerance of the photosynthetic system to low temperature, activated the antioxidant enzyme activities in cells, and more quickly scavenged the accumulated O 2- in vivo to restore the ROS metabolic balance in the body and enhance the cold resistance of the plants.

[0153] Example 5 Expression Analysis of Cold-Responsive Genes in Transgenic Tobacco

[0154] I. Experimental Methods

[0155] Select 4-week-old transgenic tobacco seedlings with consistent growth, treat them at -6°C for 4 h without cold acclimation, extract the RNA from leaf tissues and reverse transcribe it into cDNA. Using cDNA as a template for qRT-PCR, wild type tobacco as a control and NtActin as a reference gene, detect the expression levels of reactive oxygen species (ROS)-related genes NtRD26, NtSOD and NtCAT in tobacco. The details of the quantitative primers are shown in Table 5.

[0156] The reaction system was operated according to the instructions of HieffqPCR SYBR Green Master Mix (No Rox) (Yeasen), and qRT-PCR was performed on a fluorescence quantitative PCR instrument 384 (BIO-RAD). The reaction system was 10 μL, and the reaction program was: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 35 s, and the number of cycles was 40. The 2 -△△Ct method was used to calculate the relative expression levels of genes, and each sample was repeated 3 times.

[0157] II. Experimental Results

[0158] The qRT-PCR results showed that ( Figure 6 ), after cold treatment, the transcriptional levels of NtRD26, NtSOD, and NtCAT genes in overexpressing plants were significantly up-regulated compared to the wild type (WT), indicating that AcGF14f might be a key regulatory factor upstream of some ROS-related genes, suggesting that overexpression of AcGF14f increased the mRNA levels of reactive oxygen species scavenging-related genes under cold stress treatment.

[0159] Table 5 Primers and Sequences Used in the Experiment

[0160]

[0161]

[0162] The physicochemical indicators in the above embodiments are only for some embodiments, but it cannot be determined that the specific embodiments of the present invention are limited to these examples and these application detections. Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, rather than limitations on the specific embodiments of the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Use of pineapple AcGF14f gene or its expression product in improving cold tolerance of plants, characterized in that, The nucleotide sequence of the pineapple AcGF14f gene is shown as SEQ ID NO: 1; the amino acid sequence of the protein encoded by the AcGF14f gene is shown as SEQ ID NO.2; the application is to improve the cold tolerance of plants based on overexpressing the AcGF14f gene.

2. The application according to claim 1, characterized in that The primer sequences for synthesizing the pineapple AcGF14f gene are shown as SEQ ID NO: 3 and SEQ ID NO: 4 respectively.

3. The application according to claim 1 or 2, characterized in that, The plant is pineapple or tobacco.

4. Use of a biomaterial in a product for improving cold tolerance of plants by overexpressing the AcGF14f gene, characterized in that, The biological material is any one of the following: 1) A nucleic acid molecule encoding a protein with an amino acid sequence shown as SEQ ID NO.2; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A recombinant vector containing the nucleic acid molecule described in 1) or a recombinant vector containing the expression cassette described in 2); 4) A transgenic plant cell line containing the nucleic acid molecule described in 1) or a transgenic plant cell line containing the expression cassette described in 2).

5. A method for up-regulating, enhancing or improving the cold tolerance of a target plant, characterized in that, The method includes overexpressing the AcGF14f gene to up-regulate or enhance or improve the cold tolerance of the target plant, and the plant is pineapple or tobacco.

6. The application according to claim 5, characterized in that, The application includes cultivating plant varieties with enhanced cold tolerance under low temperature stress conditions.

7. The application according to claim 6, wherein By transforming a vector, transgenic cell line or transgenic recombinant bacterium overexpressing the pineapple AcGF14f gene into plant cells, tissues or organs, and then cultivating the transformed plant cells, tissues or organs, cold-tolerant transgenic plants are obtained.

8. The application according to claim 7, wherein When the plant is tobacco, the overexpression vector is pBI121-AcGF14f.

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