Gene gbTG1 for improving salt tolerance of ginkgo biloba, expressed protein and application thereof

Abstract

The application discloses a gene GbTG1 for improving salt tolerance of ginkgo, an expression protein thereof and application, and belongs to the field of plant molecular biology. The applicant screens and clones the ginkgo GbTG1 gene based on previous ginkgo transcriptome data, a nucleotide sequence of the gene is shown as SEQ ID NO. 1, and an amino acid sequence of the expression protein of the gene is shown as SEQ ID NO. 2. The transformation of the GbTG1 into tobacco shows that, compared with tobacco strains into which EV is transformed, the leaves of transgenic tobacco strains have no obvious dryness phenomenon observed by naked eyes. Measurement shows that the relative conductivity in the leaves of the transgenic tobacco strains is significantly reduced, and the average reduction is 9.4% compared with the leaves of the tobacco into which EV is transformed. Meanwhile, the content of malondialdehyde in the leaves and the relative conductivity show the same trend, and the content of the transgenic strains is averagely reduced by 18%. It is shown that the GbTG1 is a salt stress resistance gene of ginkgo, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, specifically relating to a gene GbTG1 that enhances the salt tolerance of Ginkgo biloba, its expressed protein, and its applications. Background Technology

[0002] Ginkgo (Ginkgo biloba L.) is a relict plant belonging to the single genus of the Ginkgoaceae family, originating from the Jurassic period 170 million years ago. It is the only extant species in the class Ginkgoales and is hailed as a "living fossil" of the plant kingdom. This species possesses ornamental, edible, and medicinal value. Its leaves are rich in flavonoids and terpene lactones, exhibiting pharmacological properties that improve microcirculation and inhibit thrombus formation. It has now become one of the world's most valuable functional plant resources for development. Paleontological studies show that Ginkgo exhibits significant evolutionary conservation during its morphological evolution, confirming its unique adaptive mechanisms to environmental stresses.

[0003] As a typical stress-resistant gymnosperm, Ginkgo exhibits multiple stress resistance characteristics, including drought resistance, cold resistance, and heavy metal accumulation. However, its molecular response mechanism to combined saline-alkali stress (high salinity-high pH) remains unclear. Given the urgent need for biological improvement of saline-alkali land, systematically elucidating its genetic mechanisms of salt-alkali stress resistance is of significant value for the targeted breeding of new salt-alkali tolerant varieties. As an important medicinal resource, developing endogenous stress-resistant genes from Ginkgo has significant advantages over exogenous gene introduction strategies: First, endogenous genes are naturally compatible with the genetic background of Ginkgo, avoiding metabolic disorders and expression instability caused by exogenous genes; second, the regulatory network of endogenous genes conforms to the inherent physiological mechanisms of Ginkgo, effectively reducing the risk of unintended phenotypes; and third, molecular breeding techniques based on endogenous genes are more in line with biosafety standards, which is beneficial for shortening the approval cycle for Ginkgo varieties.

[0004] Currently, there are few reports on genes related to salt stress resistance in Ginkgo biloba. Discovering key genes in Ginkgo biloba's response to salt and alkali stress will not only deepen our understanding of its stress resistance mechanisms but also provide target gene resources for establishing a precise molecular breeding system. This is of great significance for expanding suitable growing areas for Ginkgo biloba, improving the benefits of economic forest construction, and ecological restoration of saline-alkali soils. Summary of the Invention

[0005] One of the technical problems to be solved by this invention is to provide the Ginkgo gene GbTG1 and its expressed protein. Another technical problem to be solved by this invention is to provide specific applications of the aforementioned GbTG1 gene, providing an effective molecular tool for improving the ornamental traits of plants through genetic engineering.

[0006] To address the aforementioned problems in the existing technology, the technical solution adopted by the present invention is as follows:

[0007] A gene, GbTG1, that enhances the salt tolerance of Ginkgo biloba has the nucleotide sequence shown in SEQ ID NO.1.

[0008] The amino acid sequence of the GbTG1 gene, which enhances the salt tolerance of Ginkgo biloba, is shown in SEQ ID NO.2.

[0009] A biomaterial comprising an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the gene GbTG1 that enhances the salt tolerance of Ginkgo biloba.

[0010] The application of the GbTG1 gene for improving salt tolerance in Ginkgo biloba, or the expression protein of the GbTG1 gene for improving salt tolerance in Ginkgo biloba, or the biological material described herein, in plant breeding.

[0011] The application of the GbTG1 gene for improving salt tolerance in Ginkgo biloba, or the expression protein of the GbTG1 gene for improving salt tolerance in Ginkgo biloba, or the biological material described herein, in improving plant salt tolerance.

[0012] One method to enhance salt tolerance in plants is to overexpress the GbTG1 gene, which is said to enhance salt tolerance in Ginkgo biloba, in plants.

[0013] In some embodiments, the method includes the following steps:

[0014] (1) Construct an overexpression vector for the gene GbTG1 that enhances salt tolerance in Ginkgo biloba;

[0015] (2) Transform the constructed expression vector of GbTG1, which enhances the salt tolerance of Ginkgo biloba, into plants or plant tissues;

[0016] (3) Cultivate and screen transgenic plants or plant tissues with improved salt tolerance.

[0017] In some embodiments, the plant is tobacco or ginkgo.

[0018] In some embodiments, the overexpression vector for the gene GbTG1 that enhances salt tolerance in Ginkgo biloba is Super1300-GbTG1.

[0019] In some embodiments, the transformation is mediated by Agrobacterium.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] Based on previous Ginkgo transcriptome data, the applicant screened and cloned the Ginkgo GbTG1 gene. On this basis, an overexpression vector, Super1300-GbTG1, was constructed and transferred into Nicotiana macrophylla using Agrobacterium tumefaciens to obtain transgenic plants. Compared with tobacco plants transferred with the EV (empty vector), the leaves of the transgenic tobacco lines showed no obvious signs of wilting. Measurements showed a significant decrease in the relative electrical conductivity of the leaves of the transgenic tobacco lines, averaging a 9.4% reduction compared to the leaves of tobacco plants transferred with the EV. Simultaneously, the malondialdehyde (MDA) content in the leaves showed the same trend as the relative electrical conductivity, with the MDA content in the transgenic lines decreasing by an average of 18%. The experiments demonstrate that GbTG1 is a salt stress resistance gene in Ginkgo and has good application value. This invention provides an effective molecular tool for understanding the salt tolerance mechanism and molecular breeding of Ginkgo, contributing to the acquisition of new Ginkgo varieties with stronger stress resistance, and therefore has promising application prospects. Attached Figure Description

[0022] Figure 1 Diagram showing GbTG1 gene amplification;

[0023] Figure 2 The image shows the electrophoresis results of E. coli transfected with the GbTG1 vector.

[0024] Figure 3 Image showing the bacterial test results of GbTG1 vector transfected with GV3101;

[0025] Figure 4 This is a graph showing the salt tolerance of GbTG1 transgenic tobacco. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. Molecular biology experimental methods not specifically described in the following embodiments can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] The materials used in this application were leaves from 10-year-old Ginkgo biloba trees grown in the Ginkgo germplasm resource bank. In April 2024, the leaves were placed in sterilized centrifuge tubes, immediately flash-frozen in liquid nitrogen, and then stored at -80°C. The large-flowered tobacco seedlings used were provided by the research group of Cao Fuliang at Nanjing Forestry University.

[0029] In this embodiment, total RNA was extracted from plants using the TIANGEN Plant RNA Extraction Kit (DP432). TaKaRa PrimeScript was used. TMThe RT Master Mix (Perfect Real Time) reverse transcription kit reverse transcribes the extracted RNA into cDNA. The resulting cDNA is then diluted 10 times with water and stored at -20°C.

[0030] Example 1: Construction of an overexpression vector for the GbTG1 gene

[0031] (1) Obtaining the target gene

[0032] Based on the previously published Ginkgo whole genome database, one gene sequence was screened and named GbTG1.

[0033] (2) Design primers

[0034] The full-length nucleotide sequence of the gene was analyzed using BioXM software to determine restriction enzyme sites. KpnI and SmaI enzymes were selected as the two restriction endonucleases. Primers were designed using CE design software. The required information was filled in, including the sequence near the restriction sites on the vector, the full length of the target gene, and the two restriction sites (5' and 3' ends) in sequence. The designed primers were then sent to Jereh Biotech for synthesis.

[0035] F 5'-aagcttctgcaggggcccgggATGATGTTGGGTGTTTCAGGGA-3',

[0036] R 5'-gcccttgctcaccatggtaccCTCAATTGTGTTTGTGTTTGTGTTTG-3'.

[0037] (3) Vector double enzyme digestion

[0038] The Super1300 vector was activated and cultured after being removed from the -80℃ ultra-low temperature freezer. The Super1300 vector plasmid was extracted according to the kit, followed by double enzyme digestion. The 20μL system is as follows:

[0039] Restriction endonuclease 1 1 μL, restriction endonuclease 2 μL, buffer 2 μL, vector plasmid X μL, ddH2O 6 μL.

[0040] Where X(μL) = 1000 ng / vector plasmid concentration (ng / μL). Gently shake the centrifuge tube to mix, centrifuge briefly for 6 seconds, and incubate in a water bath at 37°C for 1 hour. Perform agarose gel electrophoresis on the obtained double-digested vector, and then use a kit for gel extraction and recovery.

[0041] (4) Target gene amplification

[0042] Using cDNA diluted 10-fold as a template, PCR amplification was performed in a 20 μL volume as follows:

[0043] Forward Primer 1μL, Forward Primer 1μL, cDNA 1μL, Prime STAR 10μL, ddH2O 7μL.

[0044] Three 20 μL systems were prepared for each gene. The reaction conditions were: 98℃ denaturation for 10 s; 58℃ annealing for 15 s; 72℃ extension for 1 min, 35 cycles; total extension at 72℃ for 10 min; reaction terminated at 16℃. The obtained amplification products were subjected to agarose gel electrophoresis. Figure 1 Then, the gel was cut and recovered using a kit.

[0045] (5) Connection conversion

[0046] The connection system is as follows:

[0047] 200 ng of target gene recovery product, 100 ng of plasmid double enzyme digestion recovery product, 2 μL of ligase, 4 μL of buffer, X μL, and ddH2O added to 20 μL.

[0048] Gently shake the centrifuge tube to mix it, centrifuge briefly for 6 seconds, incubate in a 37°C water bath for 30 minutes, and then on ice for 2 minutes.

[0049] Transformation: In a clean bench, use a pipette to take 5 μL of the ligation product and add it to 50 μL of Trelief™ 5α competent cells. Gently mix, incubate on ice for 5 min, incubate in water at 42°C for 60 s, then incubate on ice for 2 min. Add 250 μL of liquid LB (without Kana) and incubate at 37°C and 200 rpm in a shaker for 30 min.

[0050] Spreading: Take 200 μL of the incubated bacterial solution, spread it evenly on LB solid medium (containing 50 mg / L Kana) with a sterile glass rod, and let it dry. After sealing with film, invert the plate and incubate in a 37°C constant temperature incubator for 12-14 h.

[0051] (6) Detection and sequencing of positive single colonies

[0052] After bacteria have grown on the culture medium, single colony detection is performed in a clean bench. Eight plump single colonies are picked from each gene and sequentially backed up on LB solid medium containing Kana resistance. The corresponding single colonies are then transferred to the following 20 μL system for bacterial testing using a sterile toothpick:

[0053] 35sF 1μL, Gene R 1μL, Green Mix 10μL, ddH2O 8μL.

[0054] The PCR reaction conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ total extension for 10 min; and termination of the reaction at 16℃. The obtained amplification products were subjected to agarose gel electrophoresis. Figure 2 Three correct positive colonies were selected and sent for testing. The nucleotide sequence of the GbTG1 gene was determined as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein was shown in SEQ ID NO.2.

[0055] (7) Double enzyme digestion verification

[0056] The plasmid with the correct sequence obtained from sequencing was verified by double enzyme digestion. The 20 μL system is as follows:

[0057] Restriction endonuclease 1 1 μL, restriction endonuclease 2 μL, buffer 2 μL, vector plasmid X μL, ddH2O 6 μL.

[0058] Where X(μL) = 1000 ng / vector plasmid concentration (ng / μL). Gently shake the centrifuge tube to mix, centrifuge briefly for 6 seconds, and incubate in a water bath at 37℃ for 1 hour. Perform agarose gel electrophoresis on the obtained double-digested vector to detect the double digestion status.

[0059] Example 2: Transformation of Agrobacterium GV3101

[0060] (1) Take out the GV3101 competent cells stored in the -80℃ ultra-low temperature freezer and thaw them on ice. Add 1μL of plasmid to every 33μL of competent cells, mix well by pipetting, and then sequentially in an ice bath for 20min, quick freeze with liquid ammonia for 5min, in a 37℃ water bath for 5min, and in an ice bath for 5min.

[0061] (2) Add 500 μL of antibiotic-free LB liquid medium and incubate at 28°C and 200 rpm on a shaker for 1 h;

[0062] (3) After the culture is completed, centrifuge the bacterial solution at 6000r for 1min, discard part of the supernatant, and leave 100μL to spread evenly on LB solid medium (containing 50mg / LKana), seal with sealing film, and invert in an incubator at 28℃ for 40-48h.

[0063] (4) Microbial testing and backup: The target band in the microbial test is correct and the brightness is consistent. Figure 3 If the bacteria are in the backup plate, pick the corresponding colonies from the backup plate into LB liquid medium (containing 50 mg / L Kana) and shake to inoculate. Then, mix the bacterial solution with 50% glycerol at a volume ratio of 3:7 to preserve the bacteria. After quick-freezing in liquid nitrogen, store in an ultra-low temperature freezer at -80°C.

[0064] Example 3: Transgenic plants were obtained by infecting large-flowered tobacco and screening them.

[0065] (1) Explant disinfection: After harvesting the tender leaves of Nicotiana macrophylla, the surface of the leaves was cleaned with detergent to remove the dust layer. After rinsing with running water for 30 minutes, the leaves were transferred to a clean bench for disinfection. First, 75% ethanol was poured into a beaker and shaken to ensure that the ethanol fully contacts the surface of the tender leaves for 30 seconds. The leaves were then rinsed three times with sterile water. Next, the leaves were soaked in 5% NaCl for 10 minutes and rinsed four times with sterile water. The moisture on the surface of the leaves was then absorbed with sterile filter paper. After disinfection, the leaf edges and veins were removed with a sterile scalpel. The remaining leaves were then cut into small pieces of 0.5 × 0.5 cm for infection.

[0066] (2) Shaking culture: Take out the Super1300 empty vector and the vector containing the target gene and thaw it on ice. Use a pipette to add the bacterial culture to 20 mL of LB liquid medium (containing 50 mg / L Kana) and incubate in the dark on a shaker at 28°C and 200 rpm until the bacterial culture reaches OD. 600 = Between 0.5 and 0.6;

[0067] (3) Infection: Use sterile tweezers to transfer the cut tobacco leaves into the infection solution and infect for 10 minutes, shaking the conical flask once every 2 minutes;

[0068] (4) Co-culture: After the leaves have been infected, they are taken out and laid flat on sterile filter paper. After the bacterial solution has dried slightly, the leaves are laid flat on the symbiotic culture medium and incubated in the dark at 25°C for 3 days.

[0069] (5) Screening culture: After co-culturing for 3 days, the leaves are transferred to the screening medium for culture. The medium is replaced every 15 days until resistant callus and resistant buds grow.

[0070] (6) Rooting culture: When the length of the sprouted adventitious buds reaches more than 5cm, cut them off from the tissue and remove the callus tissue connected to the stem tissue. Then transfer them to the rooting culture medium to induce rooting.

[0071] (7) Hardening off and transplanting: When the taproot of the transgenic seedlings has grown to about 5 cm and 7-8 leaves have emerged, remove the tobacco seedlings from the culture medium. Without damaging the roots, rinse away any remaining agar gel between the root tissues and place them in tissue culture bottles containing deionized water for acclimatization culture for 2 days. Change the water regularly during acclimatization to prevent contamination and damage to the tobacco seedlings. After acclimatization, label the tobacco seedlings with numbers and transplant them into sterilized substrate soil for further cultivation.

[0072] (8) Screening of transgenic plants: When the transgenic tobacco seedlings were 30 days old, RNA was extracted from the leaves using a kit and reverse transcribed into cDNA. The cDNA was then diluted 10 times and semi-quantitative PCR was performed. After confirming that the cDNA quality was up to standard, qRT-PCR was performed to detect the gene expression level. Transgenic seedlings with high expression levels and consistent growth status were selected for subsequent functional verification.

[0073] Example 4: Observation of salt tolerance in GbTG1 transgenic plants

[0074] Three identical plantlets, each transformed with the Super1300 empty vector (EV), were selected as transgenic controls. Each plantlet was irrigated with 200 mL of 500 mmol / L saline solution. Repeat irrigation was performed every 7 days until the phenotype appeared. Figure 4 As shown, after 15 days of salt stress, the GbTG1 gene-transgenic plants showed better condition compared to the EV-transgenic tobacco plants. Many leaves of the EV-transgenic tobacco plants withered, while the transgenic plants did not show obvious leaf withering.

[0075] Example 5: Determination of physiological indicators of transgenic plants

[0076] Leaf samples were taken 24 hours after stress, flash-frozen in liquid nitrogen for 5 minutes, and then stored at -80°C. The physiological parameters measured included relative conductivity and malondialdehyde (MDA) content.

[0077] (1) Relative conductivity: Leaves from the same location in the empty control and transgenic tobacco were washed with water, and 0.1g of each leaf was added to 20mL of deionized water and soaked at room temperature for 24 hours, shaking 3-5 times during this period. The conductivity EC0 of the deionized water was measured using a conductivity meter. Then, the conductivity EC1 of each experimental group was measured. After heating in a 100℃ water bath for 30 minutes and cooling to room temperature, the water was shaken well, and the conductivity EC2 was measured again. Relative conductivity = (EC1-EC0 / EC2-EC0)×100%.

[0078] (2) Malondialdehyde content: The thiobarbituric acid colorimetric method was used. 0.2 g of sample was weighed, mixed with 5% trichloroacetic acid (TCA) solution, and centrifuged at 4℃ and 6000 r / min for 10 min. 2 mL of 0.67% TBA solution was added to a test tube, and 2 mL of the centrifuged supernatant was mixed. 2 mL of 5% TCA was added to the control group. The reaction solution was boiled in a water bath for 30 min and then cooled in water. After cooling, the reaction solution was centrifuged at 4℃ and 6000 r / min for 10 min. The absorbance of the reaction solution supernatant at wavelengths of 450 nm, 532 nm, and 600 nm was measured using a UV spectrophotometer. The absorbance was calculated using the following formula: C(μmol / g)=[6.452×(A 532 -A 600 )×-0.56×A450] ×Vt / (V0×W)Vt: Total volume of extract (mL); V0: Volume of test solution (mL); W: Weight of plant tissue (g).

[0079] Table 1. Relative electrical conductivity of leaves and malondialdehyde content

[0080]

[0081] Table 1 shows that after salt treatment, the relative electrical conductivity of the leaves of the three transgenic tobacco lines decreased significantly, averaging a 9.4% reduction compared to EV leaves, indicating a very low degree of salt stress. Simultaneously, the malondialdehyde (MDA) content in the leaves showed the same trend as the relative electrical conductivity, with the MDA content in the transgenic lines decreasing by an average of 18%. These results indicate that overexpression of GbTG1 can significantly improve the salt tolerance of tobacco, suggesting that GbTG1 is a salt stress resistance gene in Ginkgo biloba with significant application value.

Claims

1. A gene GbTG1 derived from Ginkgo biloba, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expressed protein encoded by the gene GbTG1 of claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that, The biological material is an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the gene GbTG1 described in claim 1.

4. The application of the gene GbTG1 of claim 1, the expressed protein of claim 2, or the biomaterial of claim 3 in improving the salt tolerance of tobacco.

5. A method for improving the salt tolerance of tobacco, characterized in that, Overexpression of the gene GbTG1 as described in claim 1 in tobacco.

6. The method according to claim 5, characterized in that, Includes the following steps: (1) Construct an overexpression vector for the gene GbTG1 as described in claim 1; (2) Transform the constructed expression vector into tobacco or tobacco tissue; (3) Cultivate and screen transgenic tobacco or tobacco tissue with improved salt tolerance.

7. The method according to claim 6, characterized in that, The overexpression vector is Super1300-GbTG1.

8. The method according to claim 6, characterized in that, The transformation was mediated by Agrobacterium.

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