Gene GbTG1 for improving salt tolerance of ginkgo biloba as well as expression protein and application of gene GbTG1

Abstract

The invention discloses a gene GbTG1 for improving salt tolerance of gingko and an expression protein and application thereof, and belongs to the field of plant molecular biology. The gingko GbTG1 gene is screened and cloned based on early-stage gingko transcriptome data, the nucleotide sequence of the gingko GbTG1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein of the gingko GbTG1 gene is as shown in SEQ ID NO.2. Experimental results of transforming the GbTG1 into tobacco show that compared with a tobacco strain into which EV is transferred, leaves of a transgenic tobacco strain have no obvious withering phenomenon when observed by naked eyes. Measurement shows that the relative conductivity in the leaf of the transgenic tobacco line is remarkably reduced and is averagely reduced by 9.4% compared with that of the tobacco leaf with the EV transferred, meanwhile, the content of malondialdehyde in the leaf has the same trend as the relative conductivity, and the content of the transgenic line is averagely reduced by 18%. Therefore, the GbTG1 is a salt stress resistant gene of the gingko and has an important application value.

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular biology, and particularly relates to a gene GbTG1 for improving the salt tolerance of Ginkgo biloba, its expressed protein, and applications thereof. Background Art

[0002] Ginkgo biloba L. is a relic plant of a single genus in the Ginkgoaceae family. It originated in the Jurassic period 170 million years ago and is the only extant species of Ginkgoopsida, known as the "living fossil" in the plant kingdom. This species has ornamental, edible, and medicinal values. Its leaves are rich in active ingredients such as flavonoids and terpenoids, with pharmacological properties of improving microcirculation and inhibiting thrombus formation, and has now become one of the most valuable functional plant resources for development globally. Paleontological studies have shown that Ginkgo biloba exhibits significant evolutionary conservatism during morphological evolution, confirming its unique adaptation mechanism to environmental stresses.

[0003] As a typical stress-resistant gymnosperm, Ginkgo biloba exhibits multiple stress tolerance characteristics including drought tolerance, cold tolerance, and heavy metal enrichment, but the molecular response mechanism to combined salt-alkali stress (high salinity - high pH) remains unclear. Currently, there is an urgent need for biological improvement of saline-alkali land. Systematically analyzing its genetic mechanism of salt-alkali stress tolerance is of great value for the directional cultivation of new salt-tolerant varieties. As an important medicinal resource, compared with the strategy of introducing foreign genes, developing its endogenous stress-resistant genes has significant advantages: First, endogenous genes are naturally compatible with the genetic background of Ginkgo biloba, avoiding metabolic disorders and expression instability caused by foreign genes; Second, the endogenous gene regulatory network conforms to the inherent physiological mechanism of Ginkgo biloba, effectively reducing the risk of unexpected phenotypes; Third, the molecular breeding technology route based on endogenous genes is more in line with biosafety regulations, facilitating the shortening of the Ginkgo biloba variety approval cycle.

[0004] Currently, there are few reports on Ginkgo biloba genes resistant to salt stress. Exploring the key genes in Ginkgo biloba responding to salt-alkali stress can not only deepen the understanding of the stress resistance mechanism of Ginkgo biloba, but also provide target gene resources for establishing a precise molecular breeding system, which is of great significance for expanding the suitable growing area of Ginkgo biloba, improving the economic benefits of forestry construction, and ecological restoration of saline soil. Summary of the Invention

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

[0006] In view of the above problems existing in the prior art, the technical solutions adopted by the present invention are as follows:

[0007] A gene GbTG1 for improving the salt tolerance of Ginkgo biloba, the nucleotide sequence of which is shown in SEQ ID NO.1.

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

[0009] A biological material, which is an expression cassette, recombinant vector, recombinant bacterium or recombinant cell containing the gene GbTG1 for improving the salt tolerance of Ginkgo biloba.

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

[0011] The application of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba or the expressed protein of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba or the biological material in improving the salt tolerance of plants.

[0012] A method for promoting the improvement of plant salt tolerance is to overexpress the gene GbTG1 for improving the salt tolerance of Ginkgo biloba in plants.

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

[0014] (1) Construct an overexpression vector of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba;

[0015] (2) Transform the constructed expression vector of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba into plants or plant tissues;

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

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

[0018] In some embodiments, the overexpression vector of the gene GbTG1 for improving the salt tolerance of 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:

[0021] Based on the previous Ginkgo biloba transcriptome data, the applicant screened and cloned the GbTG1 gene of Ginkgo biloba. On this basis, its overexpression vector Super1300-GbTG1 was constructed and transferred into Nicotiana tabacum by Agrobacterium tumefaciens to obtain transgenic plants. Compared with the tobacco lines transfected with EV (empty vector), there was no obvious withering phenomenon in the leaves of the transgenic tobacco lines observed by the naked eye. After measurement, the relative electrical conductivity in the leaves of the transgenic tobacco lines decreased significantly, with an average reduction of 9.4% compared with the tobacco leaves transfected with EV. At the same time, the content of malondialdehyde in the leaves showed the same trend as the relative electrical conductivity, and the content of the transgenic lines decreased by 18% on average. Experiments showed that GbTG1 is a salt stress resistance gene of Ginkgo biloba and has good application value. The present invention provides an effective molecular tool for the salt tolerance mechanism and molecular breeding of Ginkgo biloba, which helps to obtain new Ginkgo biloba varieties with stronger stress resistance, so it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the amplification diagram of GbTG1 gene;

[0023] Figure 2 It is the electrophoresis result diagram of the detection of GbTG1 vector transfected into Escherichia coli;

[0024] Figure 3 It is the detection result diagram of GbTG1 vector transfected into GV3101;

[0025] Figure 4 It is the observation diagram of the salt tolerance of GbTG1 transgenic tobacco. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be further described below in conjunction with specific embodiments. For the molecular biology experimental methods not specifically described in the following embodiments, they can be referred to the methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product manuals.

[0027] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0028] The materials used in this application are the leaves of 10-year-old Ginkgo biloba growing in the Ginkgo biloba germplasm resource bank. In April 2024, the leaves were put into sterilized centrifuge tubes, immediately frozen in liquid nitrogen, and then stored in a -80°C refrigerator. The Nicotiana tabacum seedlings used were provided by the research group of Cao Fuliang from Nanjing Forestry University.

[0029] In this embodiment, TIANGEN plant RNA extraction kit (DP432) was used to extract total plant RNA. TaKaRa PrimeScript TMThe RT Master Mix (Perfect Real Time) reverse transcription kit reverse transcribes the extracted RNA into cDNA, and the finally obtained cDNA is diluted 10 times with water and stored in a -20°C refrigerator.

[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 of the research group, 1 gene sequence was screened and named GbTG1.

[0033] (2) Designing primers

[0034] The BioXM software was used to analyze the restriction enzyme sites of the full-length nucleotide sequence of this gene, and KpnⅠ and SmaⅠ enzymes were selected as the two restriction endonucleases. The CE design software was used to design primers. Fill in the relevant information as required, including the sequence near the restriction enzyme site on the vector, the full length of the target gene, and fill in 2 restriction enzyme sites (5' end and 3' end) in order, and the amplification primers can be obtained. The designed sequence was sent to the email for synthesis by GenScript Biotech Corporation.

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

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

[0037] (3) Double digestion of the vector

[0038] The Super1300 vector was taken out from the -80°C ultra-low temperature refrigerator in advance for activation and shaking culture. The Super1300 vector plasmid was extracted according to the kit, and then a double digestion experiment was carried out. The 20 μL 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] Among them, X (μL) = 1000 ng / vector plasmid concentration (ng / μL). The centrifuge tube was gently shaken to mix it, centrifuged instantaneously for 6 s, and placed in a 37°C water bath for 1 h. The double-digested vector obtained was subjected to agarose electrophoresis, and then gel extraction was carried out using the kit.

[0041] (4) Amplification of the target gene

[0042] Using the cDNA diluted 10 times as a template, PCR amplification was carried out. The 20 μL system was as follows:

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

[0044] Three 20 μL systems were prepared for each gene. The reaction conditions were: denaturation at 98 °C for 10 s; annealing at 58 °C for 15 s; extension at 72 °C for 1 min, for 35 cycles; final extension at 72 °C for 10 min; termination at 16 °C. The obtained amplification products were subjected to agarose gel electrophoresis ( Figure 1 ), and then gel extraction was carried out using a kit.

[0045] (5) Ligation and transformation

[0046] The ligation system was as follows:

[0047] 200 ng of the recovered product of the target gene, 100 ng of the recovered product of the plasmid digested with two enzymes, 2 μL of ligase, 4 μL of Buffer, X μL, ddH2O added to 20 μL.

[0048] The centrifuge tube was gently shaken to mix it evenly, centrifuged instantaneously for 6 s, placed in a water bath at 37 °C for 30 min, and then on ice for 2 min.

[0049] Transformation: In a laminar flow hood, 5 μL of the ligation product was taken with a pipette and added to 50 μL of Trelief TM 5α competent cells, gently flicked to mix, ice-bathed for 5 min, water-bathed at 42 °C for 60 s, then ice-bathed for 2 min, and 250 μL of liquid LB (without Kana) was added, and incubated in a shaker at 37 °C and 200 rppm for 30 min.

[0050] Plating: 200 μL of the incubated bacterial solution was taken, evenly spread on an LB solid medium (containing 50 mg / L of Kana) with a sterilized glass rod and air-dried, sealed with a sealing film and inverted in a constant temperature incubator at 37 °C for 12 - 14 h.

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

[0052] After colonies grew on the medium, single colony detection was carried out in a laminar flow hood. Eight plump single colonies were picked for each gene, successively backed up on an LB solid medium containing Kana resistance, and the corresponding single colonies were picked up with a sterile toothpick and transferred to the following 20 μL system for bacterial detection:

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

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

[0055] (7) Double digestion verification

[0056] The plasmid with the correct sequenced sequence was verified by double digestion. The 20 μL system was as follows:

[0057] Restriction enzyme 1 1 μL, restriction enzyme 2 μL, Buffer 2 μL, vector plasmid X μL, ddH2O 6 μL.

[0058] Among them, X (μL) = 1000 ng / vector plasmid concentration (ng / μL). The centrifuge tube was gently shaken to mix it evenly, centrifuged instantaneously for 6 s, and placed in a water bath at 37°C for 1 h. The obtained vector after double digestion was subjected to agarose gel electrophoresis to detect the double digestion situation.

[0059] Example 2: Transformation of Agrobacterium tumefaciens GV3101

[0060] (1) The GV3101 competent cells stored in an ultra-low temperature freezer at -80°C were taken out and melted on ice. 1 μL of plasmid was added to every 33 μL of competent cells. After pipetting and mixing evenly, it was ice-bathed for 20 min, quick-frozen in liquid ammonia for 5 min, water-bathed at 37°C for 5 min, and ice-bathed for 5 min in sequence;

[0061] (2) 500 μL of LB liquid medium without resistance was added, and it was cultured on a shaker at 28°C and 200 rppm for 1 h;

[0062] (3) After the culture was completed, the bacterial solution was centrifuged at 6000 r for 1 min, and part of the supernatant was discarded. 100 μL was left and evenly spread on the LB solid medium (containing 50 mg / L Kana), sealed with a sealing film, and inverted and cultured in an incubator at 28°C for 40 - 48 h;

[0063] (4) Bacterial inspection and backup: If the target bands in the bacterial inspection are correct and have the same brightness ( Figure 3 ), then the corresponding colonies on the backup plate were picked into the LB liquid medium (containing 50 mg / L Kana) for shaking culture, and then the bacterial solution and 50% glycerol were used for bacteria preservation according to a volume ratio of 3:7. After quick-freezing in liquid nitrogen, it was stored in an ultra-low temperature freezer at -80°C.

[0064] Example 3: Infecting Nicotiana alata and screening to obtain transgenic plants

[0065] (1) Explant disinfection: After picking the young leaves of Nicotiana alata, wash the ash layer on the leaf surface with dishwashing liquid, transfer it to a laminar flow hood for disinfection treatment after rinsing with running water for 30 min. First, pour 75% ethanol into a beaker, shake it to make the ethanol fully contact with the surface of the young leaves for 30 s, and wash it 3 times with sterile water. Then soak it in 5% Naclo for 10 min, rinse it 4 times with sterile water, and dry the water on the leaf surface with sterile filter paper. After disinfection treatment, cut off the leaf edges and veins with a sterile scalpel, and then cut the remaining leaves into small pieces of 0.5×0.5 cm for infection.

[0066] (2) Bacterial shaking: Take out the Super1300 empty vector and the vector bacterial liquid ligated with the target gene and melt it on ice. Use a pipette to add the bacterial liquid into 20 mL of LB liquid medium (containing 50 mg / L Kana), and place it in a shaker at 28 °C and 200 rppm for dark culture until the OD 600 of the bacterial liquid is between 0.5 and 0.6;

[0067] (3) Infection: Use sterile forceps to transfer the cut tobacco leaves to the infection solution for 10 min, and shake the conical flask every 2 min;

[0068] (4) Co-culture: Take out the infected leaves and spread them on sterile filter paper. After the bacterial liquid dries slightly, spread the leaves on the symbiotic medium and culture them in the dark at 25 °C for 3 d;

[0069] (5) Screening culture: After co-culturing for 3 d, transfer the leaves to the screening medium for culture, and change it every about 15 d until resistant callus and resistant buds grow.

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

[0071] (7) Hardening off and transplanting: When the main root of the transgenic seedling elongates to about 5 cm and 7-8 leaves grow, take out the tobacco seedling from the medium. Under the condition of not damaging the root system, wash off the agar gel remaining between the root tissues and put it into a tissue culture bottle containing deionized water for acclimation culture for 2 d. During the acclimation period, change the water regularly to prevent contamination from damaging the tobacco seedlings. After acclimation, mark the tobacco seedlings with numbers and transplant them into the sterilized substrate soil for continuous culture.

[0072] (8) Screening of transgenic plants: When the transgenic tobacco seedlings grew for 30 days, a kit was used to extract RNA from the leaves and reverse-transcribe it into cDNA. Then, the cDNA was diluted 10-fold and subjected to semi-quantitative PCR detection. After determining that the quality of the cDNA was qualified, qRT-PCR was performed to detect the gene expression level, and transgenic seedlings with high expression levels and consistent growth states were selected for subsequent functional verification.

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

[0074] Three lines (EV) with consistent states and transfected with the Super1300 empty vector were selected as transgenic controls. Salt water with a concentration of 500 mmol / L was used for irrigation, 200 mL per pot. Supplementary irrigation was carried out every 7 days until phenotypes appeared. As Figure 4 shown, at 15 days of salt stress, compared with the EV-transfected tobacco plants, the GbTG1 gene-transfected plants showed a better state. Multiple leaves of the EV-transfected tobacco plants withered and dried, while there was no obvious phenomenon of leaf withering and drying in the transgenic plants.

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

[0076] After 24 hours of stress, leaf samples were taken, quickly frozen in liquid nitrogen for 5 minutes, and then transferred to a -80 °C refrigerator for storage. The physiological indexes to be measured included relative electrical conductivity and malondialdehyde content.

[0077] (1) Relative electrical conductivity: Take leaves at the same position of the empty vector control and transgenic tobacco, wash them with water, weigh 0.1 g, add 20 mL of deionized water, and soak at room temperature for 24 hours, shaking 3 - 5 times during this period. Use a conductivity meter to measure the conductivity EC0 of the deionized water, then measure the conductivity EC1 of each experimental group. After heating in a water bath at 100 °C for 30 minutes and cooling to room temperature, shake well and measure the conductivity EC2 again. Relative electrical conductivity = (EC1 - EC0 / EC2 - EC0) × 100%.

[0078] (2) Malondialdehyde content: Use the thiobarbituric acid colorimetric method. Weigh 0.2 g of the sample, add 5% trichloroacetic acid (TCA) solution and mix well. Centrifuge at 4 °C and 6000 r / min for 10 minutes in a centrifuge. Add 2 mL of 0.67% TBA solution to the test tube, take 2 mL of the centrifuged supernatant and mix well. Add 2 mL of 5% TCA to the control group. The reaction solution was boiled in a water bath for 30 minutes and then taken out and cooled in water. After cooling, centrifuge the reaction solution at 4 °C and 6000 r / min for 10 minutes. Use a UV spectrophotometer to measure the absorbance values of the supernatant of the reaction solution at wavelengths of 450 nm, 532 nm, and 600 nm. Calculate using the following formula: C (μmol / g) = [6.452 × (A 532 - A 600 ) × -0.56 × A450] ×Vt / (V0×W) where Vt is the total volume of the extraction solution (mL); V0 is the volume of the determination solution (mL); W is the weight of the plant tissue (g).

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

[0080]

[0081] As can be seen from Table 1, after salt treatment of the three transgenic tobacco lines, the relative electrical conductivity in the leaves decreased significantly, with an average reduction of 9.4% compared to the EV leaves, indicating that the degree of salt stress on the plants was very low. At the same time, the content of malondialdehyde in the leaves showed the same trend as the relative electrical conductivity, with an average reduction of 18% in the transgenic lines. The research results show that overexpression of GbTG1 can significantly improve the salt tolerance of tobacco, indicating that GbTG1 is a salt stress resistance gene in ginkgo and has important application value.

Claims

1. A gene GbTG1 for improving the salt tolerance of Ginkgo biloba, whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The expressed protein of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A biological material, characterized in that, The biological material is an expression cassette, recombinant vector, recombinant bacterium or recombinant cell containing the gene GbTG1 for improving the salt tolerance of Ginkgo biloba according to claim 1.

4. The application of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba according to claim 1 or the expressed protein of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba according to claim 2 or the biological material according to claim 3 in plant breeding.

5. The application of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba according to claim 1 or the expressed protein of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba according to claim 2 or the biological material according to claim 3 in improving the salt tolerance of plants.

6. A method for promoting the improvement of plant salt tolerance, characterized in that, Overexpress the gene GbTG1 for improving the salt tolerance of Ginkgo biloba according to claim 1 in plants.

7. The method according to claim 6, wherein It includes the following steps: (1) Construct an overexpression vector of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba according to claim 1; (2) Transform the constructed expression vector of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba into plants or plant tissues; (3) Cultivate and screen to obtain transgenic plants or plant tissues with improved salt tolerance.

8. The method according to claim 7, wherein The plant is tobacco or Ginkgo biloba.

9. The method according to claim 7, wherein The overexpression vector of the gene GbTG1 for improving the salt tolerance of Ginkgo biloba is Super1300-GbTG1.

10. The method according to claim 7, characterized in that The transformation is mediated by Agrobacterium.

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