Application of t5h gene from hippophae rhamnoides and its encoded protein in improving cold tolerance of plants

Abstract

The application discloses a T5H gene from Hippophae rhamnoides and application of a protein coded by the T5H gene in improving cold resistance of plants, and belongs to the technical field of biotechnology. The T5H gene from Hippophae rhamnoides and the protein coded by the T5H gene can enhance survival rate and cold resistance of plants (such as crops) under low-temperature stress, and have wide application prospect and popularization value in agricultural production practice.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the T5H gene and its encoded protein derived from sea buckthorn in improving plant cold resistance. Background Technology

[0002] Serotonin (5-hydroxytryptamine, or 5-HT) is a typical indoleamine bioactive substance widely found in animals and plants. In plants, the synthesis pathway of 5-HT is relatively well-defined: first, tryptophan decarboxylase (TDC) catalyzes the formation of tryptophan to tryptamine, which is then hydroxylated by tryptamine 5-hydroxylase (T5H) to form 5-HT. Studies have shown that endogenous 5-HT in plants plays an important role in multiple biological processes, including root growth and development, fruit ripening, leaf senescence, seed formation, and defense against biotic and abiotic stresses.

[0003] In existing technologies, exogenous application of 5-HT or heterologous regulation of related metabolic pathway genes can alleviate stress damage and improve antioxidant levels in plants to some extent. However, there are currently no published studies reporting the applications of the T5H gene from sea buckthorn (Hippophaerhamnoides L.). As a naturally abundant plant resource rich in 5-HT, sea buckthorn's T5H gene possesses unique sequence characteristics and high catalytic activity. Therefore, developing novel molecular tools based on the sea buckthorn T5H gene has significant application value. Summary of the Invention

[0004] In view of one or more problems existing in the prior art, the first aspect of the present invention provides the application of the T5H gene derived from sea buckthorn in improving the cold resistance of plants, wherein the nucleotide sequence of the T5H gene derived from sea buckthorn is shown in SEQ ID NO:1.

[0005] In some embodiments, the cold hardiness of the plant is improved by expressing the T5H gene derived from sea buckthorn in the plant.

[0006] A second aspect of the present invention provides the application of a protein encoded by the T5H gene from sea buckthorn in improving plant cold resistance, wherein the amino acid sequence of the protein encoded by the T5H gene from sea buckthorn is shown in SEQ ID NO:2.

[0007] A third aspect of the present invention provides the application of an expression cassette, expression vector, or recombinant bacteria containing a T5H gene derived from sea buckthorn in improving plant cold resistance, wherein the nucleotide sequence of the T5H gene derived from sea buckthorn is shown in SEQ ID NO:1.

[0008] The fourth aspect of the present invention provides the application of the T5H gene derived from sea buckthorn, an expression cassette containing the T5H gene derived from sea buckthorn, an expression vector, or a recombinant bacterium in the cultivation of cold-resistant plants, wherein the nucleotide sequence of the T5H gene derived from sea buckthorn is shown in SEQ ID NO:1.

[0009] The fifth aspect of the present invention provides a method for improving the cold resistance of plants, comprising transferring the T5H gene derived from sea buckthorn into the plant and causing it to be expressed or overexpressed;

[0010] The nucleotide sequence of the T5H gene derived from sea buckthorn is shown in SEQ ID NO:1.

[0011] In some embodiments, in the above-described methods for improving plant cold resistance, the plants are made to express or overexpress the T5H gene derived from sea buckthorn by means of transgenic, hybrid, backcross, self-pollination or asexual reproduction.

[0012] In some embodiments, in the above-described methods for improving plant cold resistance, the transgenic method includes introducing a recombinant expression vector containing the T5H gene from sea buckthorn into the plant using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electrical conductivity, or Agrobacterium-mediated transformation. Attached Figure Description

[0013] Figure 1 The results of qRT-PCR detection of the expression levels of HrT5H1 (image a) and HrT5H2 (image b) in sea buckthorn after low-temperature treatment are shown.

[0014] Figure 2 The plasmid map of expression vector 1302-HrT5H1 is shown.

[0015] Figure 3 The image shows a gel electrophoresis image of HrT5H1 detected by PCR in transgenic tobacco.

[0016] Figure 4 The results of qRT-PCR detection of HrT5H1 expression level in transgenic tobacco are shown.

[0017] Figure 5 The results of the detection and comparison of survival rate and physiological indicators of transgenic tobacco plants and wild-type control plants after low temperature stress are shown.

[0018] Figure 6The results show the comparative effects of HrT5H1 on improving cold resistance in the hairy roots of Hippophae rhamnoides after low-temperature stress. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0020] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0021] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., 3rd edition, 2001, NY, Cold SpringHarbor).

[0022] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0023] Example 1: Cloning and sequence analysis of the sea buckthorn T5H gene

[0024] According to the analysis of the sea buckthorn genome, there are two T5H genes (named HrT5H1 and HrT5H2, respectively) in the sea buckthorn genome. This example aims to clone and analyze these two T5H genes in the sea buckthorn genome, specifically including the following operations.

[0025] (1) Total RNA extraction and cDNA synthesis. Healthy sea buckthorn leaves were selected as experimental materials. Total RNA was extracted using the Tiangen Plant RNA Extraction Kit (Tiangen Biotech Co., Ltd., China) according to the instructions. After detecting RNA integrity by agarose gel electrophoresis, first-strand cDNA was synthesized by reverse transcription using the Takara PrimeScript™ RT Reagent Kit (with gDNA Eraser) (Takara, Japan) for subsequent amplification.

[0026] (2) Gene amplification and sequencing verification. Based on the reference genome sequence of Hippophae rhamnoides, specific primers were designed. The PCR product of the HrT5H1 gene was obtained by amplification using specific primers HrT5H1-F: 5′-ATGGACCATTTTACTCTTCAGTCTATCT-3′ (SEQ ID NO:6); HrT5H1-R: 5′-CTAAAGTATCTGAATACCTTGAGGCTCA-3′ (SEQ ID NO:7). The PCR product of the HrT5H2 gene was obtained by amplification using specific primers HrT5H2-F: 5′-ATGGAGTATTTTACACCCGAGTCTCTCT-3′ (SEQ ID NO:8); HrT5H2-R: 5′-TCAGGAACTAGTACTTGTTGTCTCCTTG-3′ (SEQ ID NO:9). Specifically, PCR amplification was performed using sea buckthorn cDNA as a template. The reaction system was prepared according to standard procedures (25 μL of 2×Taq Master Mix, 1 μL each of forward and reverse primers, 2 μL of template cDNA, and ddH2O to a final volume of 50 μL). The amplification program was as follows: 95°C pre-denaturation for 3 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min; and extension at 72°C for 5 min. The PCR products were detected by 1% agarose gel electrophoresis, purified using a Takara DNA purification kit, and then sequenced for verification. Sequencing results showed that the nucleotide sequence of the obtained HrT5H1 gene is shown in SEQ ID NO:1, encoding 534 amino acids (amino acid sequence shown in SEQ ID NO:2); the nucleotide sequence of the obtained HrT5H2 gene is shown in SEQ ID NO:3, encoding 536 amino acids (amino acid sequence shown in SEQ ID NO:4).

[0027] SEQ ID NO:1

[0028]

[0029] SEQ ID NO:2

[0030] *。

[0031] SEQ ID NO:3

[0032]

[0033] SEQ ID NO:4

[0034] *

[0035] (3) Amino acid sequence analysis. The amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4 were analyzed using the NCBI Conserved Domain Database (CDD) and SMART database. The results showed that both HrT5H1 and HrT5H2 proteins contain typical cytochrome P450 conserved domains, with the core conserved motif being PFGGGRRGCPG (SEQ ID NO:5), belonging to the P450 monooxygenase family. The conserved domains of cytochrome P450 proteins are typically involved in heme binding and mediate a wide range of redox reactions, and are extensively involved in hormone regulation, membrane lipid metabolism, and secondary metabolite synthesis in plants.

[0036] (4) Gene expression analysis. To analyze the expression response of Hippophae rhamnoides T5H family members under low temperature stress, 2-month-old Hippophae rhamnoides seedlings were subjected to low temperature treatment, and the expression levels of HrT5H1 and HrT5H2 were detected. Specifically, 2-month-old Hippophae rhamnoides seedlings with uniform growth and free from pests and diseases were selected and cultivated in a greenhouse. The seedlings were transferred from room temperature (25°C) to a low temperature incubator at 4°C for treatment at 0, 6, 12, 24, 48, and 72 h. At each time point, the second to third unfolded leaves were taken, cut, and immediately flash-frozen in liquid nitrogen and stored at −80°C for subsequent RNA extraction. Three biological replicates were set up for each time point. RNA was extracted using a plant total RNA extraction kit (Tiangen Biotech Co., Ltd., China) according to the instructions. Genomic DNA was removed by DNase I treatment, and then cDNA was synthesized from the RNA using a reverse transcription kit (Takara, Japan) for RT-PCR. Specific primers were designed for HrT5H1 and HrT5H2: RT-HrT5H1-F: 5′-GAATGGGCAATGACAGAG-3′ (SEQ ID NO:10); RT-HrT5H1-R: 5′-TGAAGGTGTTGAAGATGGT-3′ (SEQ ID NO:11); RT-HrT5H2-F: 5′-AGTAGCGTGTTGCGTGTG-3′ (SEQ ID NO:12); RT-HrT5H2-R: 5′-TCCTCCTCTTTCTCTTCCAT-3′ (SEQ ID NO:13). Using the sea buckthorn internal reference gene (18S) as a control, the primer sequence was RT-18s-F: 5′-gccaaggaagtttgaggcaa-3′ (SEQ ID NO:14); RT-18s-R: 5′-ttcaaagattacccgggcct-3′ (SEQ ID NO:15) was amplified by RT-PCR using the SYBR Green assay. Three technical replicates were performed for each sample, and both the amplification and melting curves met the single-peak requirement. (Based on 2...) ^−ΔΔ The relative expression levels of HrT5H1 and HrT5H2 at each time point were calculated using the Ct method. The obtained data were statistically analyzed using one-way ANOVA, with a significance level set at P < 0.05. The results are as follows: Figure 1 As shown, the letters a, b, and c represent significant differences between different time points: the same letter indicates no significant difference, and different letters indicate a significant difference.

[0037] Depend on Figure 1The results show that although the sea buckthorn genome contains two T5H genes (HrT5H1 and HrT5H2), and the proteins encoded by these two T5H genes both contain typical cytochrome P450 conserved domains, only HrT5H1 was significantly upregulated after 48 hours of low-temperature treatment, while HrT5H2 expression remained unchanged during the detection period. These results indicate that HrT5H1 plays an important role in sea buckthorn's response to low-temperature stress and serotonin biosynthesis, while HrT5H2 lacks this function. Therefore, the following examples only focus on studying and verifying the role of HrT5H1 in improving or enhancing cold resistance.

[0038] Example 2: Expression vector construction and plant transformation

[0039] like Figure 2 The plasmid map shown illustrates the process of cloning the HrT5H1 gene (SEQ ID NO:1) downstream of the CaMV 35S promoter in the plant binary expression vector pCAMBIA1302 (from CAMBIA, http: / / www.cambia.org) to construct the recombinant vector, named 1302-HrT5H1. Sequencing confirmed the correct insertion direction and sequence. The recombinant vector was introduced into tobacco (Nicotiana tabacum) using the Agrobacterium tumefaciens GV3101-mediated leaf disc method. This method followed the standard operating procedure reported by Horsch et al. (Plant Molecular Biology, 1985, 5:357–371), with slight modifications: leaves from aseptically grown tobacco seedlings for 4 weeks were cut into leaf discs of approximately 1 cm²; these discs were then placed in a suspension containing recombinant Agrobacterium tumefaciens (OD200). 600 Soak in an infection solution of ≈0.6 mg / L for 10 min; after removing excess bacterial solution, transfer to antibiotic-free co-culture medium (MS + 2 mg / L 6-BA + 0.1 mg / L NAA) and culture in the dark for 2 days; then transfer to a selective medium containing 50 mg / L kanamycin and 250 mg / L cephalosporin and culture under light at 28°C; when the regenerated shoots grow to about 2–3 cm, cut them off and transfer them to a rooting medium containing antibiotics (1 / 2 MS + 0.1 mg / L NAA) for further culture to obtain transgenic plants with healthy roots.

[0040] Example 3: Expression analysis of HrT5H1 in transgenic plants

[0041] Screening and verification of positive plants: Genomic DNA was extracted from the obtained transgenic tobacco leaves using the Tiangen Plant DNA Extraction Kit (Tiangen Biotech Co., Ltd., China). PCR detection was performed using specific primers (hyg-F: 5′-CTGCCCGCTGTTCTACAACCGG-3′ (SEQ ID NO:16); hyg-R: 5′-GGAGCATATACGCCCGGAGTC-3′ (SEQ ID NO:17)). Samples showing the target band were identified as positive plants, indicating that the target gene had been integrated into the genome. Figure 3 As shown, the PCR test results for 17 positive transgenic tobacco plants (numbered OE1–OE17) are exemplarily presented, and the target bands can be seen in all of them.

[0042] Based on the PCR electrophoresis results, positive plants with strong bands (OE10, OE11, and OE12) were selected for further analysis. Subsequently, qRT-PCR (RT-F: 5′-GAATGGGCAATGACAGAG-3′ (SEQ ID NO:18); RT-R: 5′-TGAAGGTGTTGAAGATGGT-3′ (SEQ ID NO:19)) was used to detect the transcriptional level of HrT5H1 in these transgenic plants to verify the stable expression of the exogenous gene. The results are as follows: Figure 4 As shown, HrT5H1 was expressed at a high level in all three transgenic plants, indicating that the exogenous gene was stably expressed in the plants.

[0043] The above results indicate that this embodiment successfully obtained multiple tobacco plants overexpressing HrT5H1, which can provide a material basis for subsequent functional verification experiments.

[0044] Example 4: Identification of cold resistance in transgenic plants

[0045] (1) Preparation of experimental materials. Positive transgenic tobacco plants (OE10, OE11 and OE12) verified by PCR were inoculated on MS solid medium containing kanamycin (50 mg / L) and screened until robust and phenotypic stable transgenic plants were obtained. The stable transgenic plants were transplanted into flowerpots containing nutrient soil and vermiculite (volume ratio 3:1) and cultured in an artificial climate chamber under the following conditions: temperature 25 ± 1 ℃, light intensity 100 μmol·m⁻²·s⁻¹, light duration 16 h / day, darkness 8 h / day, and relative humidity 60%. When the plants were about 4 weeks old, 6–8 cm tall, and had 6–8 true leaves, stable transgenic plants with consistent growth and wild-type control plants were selected for low-temperature stress treatment.

[0046] (2) Low temperature stress and recovery culture. Four-week-old transgenic plants and wild-type control plants were placed together in a constant temperature incubator and exposed to a low temperature of 4 ℃ for 6 days (light conditions were the same as in culture), and then recovered to 25 ℃ for 1 day. After 1 day of recovery, the survival rate of each group of plants was counted, and serotonin content and physiological indicators were measured.

[0047] (3) Determination of serotonin (5-HT) content. Samples were taken from the same leaf position of transgenic plants (OE10, OE11, and OE12) and wild-type controls, weighing approximately 1 g of fresh leaf (at least three biological replicates per sample). The samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C. All samples were processed within the same batch to reduce batch variation. The samples were thoroughly mixed, ground in liquid nitrogen, and approximately 0.4 g (accurate to 0.0001 g) of the sample was accurately weighed into a 15 mL transparent centrifuge tube. 2 mL of pre-cooled methanol was added, and the tube was thoroughly shaken for 1 min. The mixture was then sonicated at low temperature for 30 min. After the sonication, the tube was removed and centrifuged at 4000 r / min for 10 min. The supernatant was transferred to a new centrifuge tube. This process was repeated twice, and the supernatants from the three centrifuge tubes were combined. The supernatant was dried to 0.5 mL under nitrogen at room temperature. The supernatant was filtered through a 0.22 μm filter membrane for liquid chromatography analysis. Test conditions: Detector: Variable wavelength detector (VWD); Column: C18 reversed-phase column (250 mm × 4.6 mm, particle size 5 μm); Column temperature: 38 ℃; Detection wavelength: 360 nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Mobile phase: Acetonitrile and 0.05% formic acid aqueous solution at a volume ratio of 18:82 (v / v).

[0048] (4) Determination of malondialdehyde (MDA) content. The MDA content was determined by the thiobarbituric acid (TBA) method. The specific steps were as follows: 0.3 g of fresh leaves were taken, 5 mL of 10% trichloroacetic acid (TCA) solution was added, and the mixture was ground into a homogenate. The mixture was centrifuged at 12000 rpm for 10 min. 2 mL of the supernatant was taken and mixed with 2 mL of 0.6% TBA solution. The mixture was heated in a water bath at 100 ℃ for 15 min, and the reaction was immediately terminated by ice bath. After cooling, the mixture was centrifuged at 12000 rpm for 10 min, and the absorbance (A) of the supernatant was measured. 450 A 532 A 600 The MDA content is calculated using the following formula: MDA (μmol·g⁻¹ FW) = 6.45 × (A 532 – A 600 ) – 0.56 × A 450 .

[0049] (5) Relative conductivity determination. Take 10 perforated leaf sections (1 cm in diameter) from fresh leaves, place them in 10 mL of deionized water, shake at room temperature for 1 h, and measure the initial conductivity C1; then heat the samples in a 100 ℃ water bath for 10 min, cool them, and measure the total conductivity C2; relative conductivity (%) = (C1 / C2) × 100%. Relative conductivity reflects the degree of cell membrane damage, and the lower the value, the higher the membrane stability.

[0050] (6) Determination of chlorophyll fluorescence parameter Fv / Fm. The maximum photochemical efficiency (Fv / Fm) of photosystem II was determined using a portable chlorophyll fluorometer (Walz MINI-PAM, Germany). Before the test, the leaves were allowed to acclimatize in the dark for 30 min; then the minimum fluorescence value Fo and the maximum fluorescence value Fm were recorded. The calculation formula was: Fv / Fm = (Fm – Fo) / Fm. A higher Fv / Fm value indicates higher photosystem II activity and lower photoinhibition.

[0051] The results are as follows Figure 5 As shown, image a presents photographs of transgenic plants before and after low-temperature treatment, and wild-type control plants; image b shows the results of 5-HT content measurement; image c shows the results of chlorophyll fluorescence parameter Fv / Fm measurement; image d shows the statistical results of plant survival rates in each group; image e shows the results of relative conductivity measurement; and image f shows the results of malondialdehyde (MDA) content measurement. Figure 5 The results show that after 6 days of low-temperature stress at 4 °C, the survival rate of transgenic tobacco was significantly higher than that of the wild-type control. Simultaneously, the 5-HT content in the leaves of transgenic plants was significantly increased. The MDA content and relative conductivity of transgenic plants were significantly lower than those of the control, while the Fv / Fm value was significantly higher, indicating that overexpression of HrT5H1 can effectively alleviate low-temperature-induced membrane lipid peroxidation damage, maintain photosynthetic system stability, and thus enhance the plant's cold resistance.

[0052] Example 5: Cold resistance assessment of transgenic hairy roots of Hippophae rhamnoides

[0053] (1) Preparation of experimental materials. Mature sea buckthorn (Hippophae rhamnoides) seeds were selected, soaked in tap water for 24 h, and then placed on moist filter paper to germinate. After germination, the seeds were transplanted into nutrient pots containing sterilized soil and cultured at room temperature (25±1 ℃) for about 2 months to obtain robust seedlings, which were used as hairy root induction materials.

[0054] (2) Obtaining and culturing hairy roots. The recombinant plasmid 1302-HrT5H1, confirmed by sequencing, was transformed into Agrobacterium rhizogenes K599. Positive single colonies were picked and inoculated into TY liquid medium containing kanamycin (50 mg / L) and streptomycin (25 mg / L), and cultured on a shaker at 28 ℃ and 200 rpm until OD. 600 It reaches 0.6 to 0.8.

[0055] Two-month-old sea buckthorn seedlings were used, and the bacterial solution was injected into the base of the seedling stem using a syringe. After infection, the seedlings were continuously cultured under greenhouse conditions, with the substrate kept moist. After about 2-3 months, white, branching hairy roots grew from the injection site. Using a portable fluorescent excitation light source, positive transgenic hairy roots carrying the target gene were screened for subsequent experiments. Hairy roots obtained from transformation with the 1302 empty vector were used as controls.

[0056] (3) Low temperature stress treatment. Transgenic hairy root plants (1302 empty vector and 1302-HrT5H1) were placed in a constant temperature incubator and treated at 4 ℃ for 1 day (light conditions were the same as those for conventional culture), and then the conductivity was measured.

[0057] The results are as follows Figure 6 As shown: a) Phenotypic images of Hippophae rhamnoides hairy roots under natural light and fluorescent excitation light; b) Detection results of HrT5H1 expression levels in transgenic and control hairy roots; c) Changes in electrical conductivity of hairy roots before and after treatment. Compared with the control, the electrical conductivity of HrT5H1 transgenic hairy roots was significantly reduced under low temperature stress, indicating that its cells were less damaged and it showed stronger cold resistance.

[0058] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of the T5H gene derived from sea buckthorn in improving plant cold resistance, wherein the nucleotide sequence of the T5H gene derived from sea buckthorn is shown in SEQ ID NO:1; The plant in question is either tobacco or sea buckthorn.

2. The application according to claim 1, wherein the cold resistance of the plant is improved by expressing the T5H gene derived from sea buckthorn in the plant.

3. Application of the protein encoded by the T5H gene from sea buckthorn in improving plant cold resistance, wherein the amino acid sequence of the protein encoded by the T5H gene from sea buckthorn is shown in SEQ ID NO:2; The plant in question is either tobacco or sea buckthorn.

4. The application of expression cassettes, expression vectors, or recombinant bacteria containing the T5H gene derived from sea buckthorn in improving plant cold resistance, wherein the nucleotide sequence of the T5H gene derived from sea buckthorn is shown in SEQ ID NO:1; The plant in question is either tobacco or sea buckthorn.

5. The application of the T5H gene derived from sea buckthorn, an expression cassette containing the T5H gene derived from sea buckthorn, an expression vector or recombinant bacteria in the cultivation of cold-resistant plants, wherein the nucleotide sequence of the T5H gene derived from sea buckthorn is shown in SEQ ID NO:1; The plant in question is either tobacco or sea buckthorn.

6. A method for improving the cold resistance of plants, comprising transferring a T5H gene derived from sea buckthorn into the plant and expressing or overexpressing it; the nucleotide sequence of the T5H gene derived from sea buckthorn is shown in SEQ ID NO:1; The plant in question is either tobacco or sea buckthorn.

7. The method according to claim 6, wherein the plant is expressed or overexpressed with a T5H gene derived from sea buckthorn by means of transgenic methods.

8. The method according to claim 7, wherein the transgenic method comprises introducing a recombinant expression vector containing the T5H gene from sea buckthorn into a plant using a Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated method.

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