Application of a Class of Osmanthus fragrans OfProTs Genes in Improving Plant Stress Resistance

Abstract

The application discloses application of a kind of Osmanthus fragrans OfProTs gene in improving plant stress resistance, and relates to the technical field of plant genetic engineering.The application discloses a kind of Osmanthus fragrans OfProTs gene, including OfProT1 gene, or / and OfProT2 gene;Nucleotide sequence is shown as SEQ ID NO.1 and SEQ ID NO.3 respectively.The application constructs the overexpression vector of Osmanthus fragrans OfProT1 or OfProT2 gene;The constructed overexpression vector is transformed into Nicotiana benthamiana;Transgenic Nicotiana benthamiana plants with improved salt stress resistance are obtained by cultivation and screening.The results show that after being subjected to high salt stress, the relative conductivity, proline and malondialdehyde content in the plants overexpressing OfProT1 and OfProT2 genes are significantly reduced, and the results show that the plants overexpressing OfProT1 and OfProT2 genes improve the ability to cope with salt stress.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and more particularly relates to application of Osmanthus fragrans OfProTs genes in improving plant stress resistance. BACKGROUND

[0002] ProTs genes are a family of proteins involved in proline transport in plants. Proline transport plays an important role in plant stress adaptation. The transport proteins encoded by ProTs genes are responsible for the transport of proline between various organs, which helps plants accumulate proline under stress and thus enhances stress resistance. For example, in potato, StProT3 gene is expressed in potato roots, stems, leaves, stolons and tubers, and is regulated by hormones, heavy metals, salt, drought and low temperature, indicating that it is involved in hormone signal transduction and non-biological stress response in potato. Currently, in Arabidopsis, three members of the ProTs subfamily, AtProT1, AtProT2 and AtProT3, have been found. The genes in this subfamily mainly mediate the transport of compounds such as proline, GABA (gamma-aminobutyric acid) and betaine. AtProT1 is mainly expressed in the phloem or parenchymal cells of the phloem, and may be involved in long-distance transport of amino acids. When plants are subjected to stress, the expression of AtProT2 gene increases and the main expression site is in the roots. AtProT3 is mainly expressed in the epidermal cells of the leaves of the aboveground part, and may be involved in regulating the transport of proline in the leaves.

[0003] Osmanthus fragrans is commonly found in subtropical regions and is not salt-tolerant, making it difficult to grow in saline-alkali soils, which severely restricts its application in coastal and high-salt areas. ProTs genes play a key role in plant stress adaptation by regulating the transport and accumulation of proline to help plants cope with various stress conditions, but the specific research on ProTs genes in Osmanthus fragrans is not clear. Research on ProTs genes helps to better understand the molecular mechanisms of plant stress adaptation and provides new ideas and methods for crop stress resistance breeding. Key transport protein genes ProTs play an important role in plant response to salt stress. SUMMARY

[0004] To solve the above problems in the prior art, the technical problems to be solved by the present application are to provide a class of Osmanthus fragrans OfProTs genes. Another technical problem to be solved by the present application is to provide an expression protein of the Osmanthus fragrans OfProTs gene. The present application also solves the technical problem of providing application of the Osmanthus fragrans OfProT1 gene or / and OfProT2 gene in regulating plant stress resistance for regulating stress resistance of Osmanthus fragrans.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0006] an OfProTs gene of Osmanthus fragrans, including an OfProT1 gene, or / and an OfProT2 gene;

[0007] the nucleotide sequence of the OfProT1 gene is shown as SEQ ID NO. 1;

[0008] the nucleotide sequence of the OfProT2 gene is shown as SEQ ID NO. 3.

[0009] an expression protein of the OfProTs gene of Osmanthus fragrans, including an OfProT1 protein, or / and an OfProT2 protein;

[0010] the amino acid sequence of the OfProT1 protein is shown as SEQ ID NO. 2;

[0011] the amino acid sequence of the OfProT2 protein is shown as SEQ ID NO. 4.

[0012] a vector containing the OfProT1 gene or the OfProT2 gene of Osmanthus fragrans, or a recombinant bacterium.

[0013] application of the OfProT1 gene, or / and the OfProT2 gene of Osmanthus fragrans in regulating plant stress resistance.

[0014] the regulating plant stress resistance is to improve the salt stress resistance of the plant, and the specific steps include:

[0015] 1) constructing an overexpression vector of the OfProT1 or OfProT2 gene of Osmanthus fragrans;

[0016] 2) transforming the constructed overexpression vector into Nicotiana benthamiana;

[0017] 3) cultivating, screening and obtaining a transgenic Nicotiana benthamiana plant with improved salt stress resistance.

[0018] the overexpression vector is a pSuper1300-OfProT1, or / and a pSuper1300-OfProT2 plant overexpression vector.

[0019] application of the OfProT1 gene, or / and the OfProT2 gene of Osmanthus fragrans in regulating the relative electrical conductivity in a plant.

[0020] application of the OfProT1 gene, or / and the OfProT2 gene of Osmanthus fragrans in regulating the proline content in a plant.

[0021] application of the OfProT1 gene, or / and the OfProT2 gene of Osmanthus fragrans in regulating the malondialdehyde content in a plant.

[0022] Compared with the prior art, the application has the beneficial effects of:

[0023] 1) The application discloses a type of Osmanthus fragrans OfProTs genes, including OfProT1 genes or / and OfProT2 genes; the nucleotide sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 3 respectively. The application constructs an overexpression vector of the OfProT1 or OfProT2 gene of Osmanthus fragrans; the constructed overexpression vector is transformed into Nicotiana benthamiana; and transgenic Nicotiana benthamiana plants with improved salt stress resistance are obtained through cultivation and screening.

[0024] 2) The embodiment results of the application show that, after high-salt stress, the relative conductivity, proline content and malondialdehyde content in the plants overexpressing the OfProT1 and OfProT2 genes are significantly reduced, and the results show that the OfProT1 and OfProT2 genes improve the ability of plants to cope with salt stress. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Fig. 1 is a diagram of OfProT1 and OfProT2 gene amplification verification results;

[0026] Figure 2 Fig. 2 is a diagram of double enzyme digestion verification results;

[0027] Figure 3 Fig. 3 is a diagram of bacterial detection results;

[0028] Figure 4 Fig. 4 is a diagram of positive OfProT1 transgenic plant detection (Nbaction is an internal reference gene of Nicotiana benthamiana, L1-3 represents three tobacco plants injected with pSuper1300 empty vector; L4-6 represents three tobacco plants injected with pSuper1300-OfProT2 overexpression vector). DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be further described below in combination with specific embodiments. If no detailed description is given in the following embodiments, the technical means used are all conventional means familiar to those skilled in the art. If no specific description is given, the molecular biology experimental methods can be performed according to the methods listed in the book of J. Sambrook, Molecular Cloning Experiment Guide (third edition) or conventional methods in the art, or according to the instructions of the kit and product.

[0030] The material used in the application is the leaf of Osmanthus fragrans grown in the national germplasm resource library of Osmanthus fragrans. In November 2022, the flowers of Osmanthus fragrans were placed in a sterilized centrifuge tube and immediately frozen in liquid nitrogen, and then stored in a-80℃ refrigerator. The Nicotiana benthamiana seedlings used were provided by the Wang Lianggui research group of Nanjing Forestry University.

[0031] Example 1

[0032] 1. Total RNA extraction and cDNA acquisition

[0033] use Total RNA was extracted from Osmanthus fragrans leaves using the Super Total RNA Extraction Kit (Promega (Beijing) Biotechnology Co., Ltd.). Using the extracted total RNA as a template, reverse transcription experiments were performed using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain cDNA.

[0034] 2. Design primers

[0035] Based on the previously published Osmanthus fragrans genome database, two gene sequences were screened and named OfProT1 and OfProT2. Restriction enzyme sites of the full-length nucleotide sequences of the two genes were analyzed using BioXM software, and SmaⅠ and SpeⅠ 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, to obtain the amplification primers. The designed sequences were sent to Jereh Biotech for synthesis. The primer sequences are shown below:

[0036] OfProT1-F:

[0037] 5'-aagcttctgcaggggcccgggATGGACGACGGCCAAGGA-3',

[0038] OfProT1-R:

[0039] 5'-catggtaccggatccactagtTAAATCAGCAAAAACATCATAATTTTTAG-3'.

[0040] OfProT2-F:

[0041] 5'-aagcttctgcaggggcccgggATGAACCGCCAAGGAACTAAAG-3',

[0042] OfProT2-R:

[0043] 5'-catggtaccggatccactagtTAAATCAGCAAAAACATGATAAGTTTTAG-3'.

[0044] 3. Target gene amplification

[0045] The cDNA was diluted 10 times as a template for PCR amplification of the target gene.

[0046] The PCR reaction system was as follows: Forward Primer 1 μL, Forward Primer 1 μL, cDNA 1 μL, PrimeSTAR 10 μL, ddH2O 7 μL. Three 20 μL systems were prepared for each gene.

[0047] The PCR reaction conditions were as follows: denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, 35 cycles, total extension at 72°C for 10 min, and termination at 16°C.

[0048] The obtained amplification product was subjected to agarose electrophoresis, and then gel recovery was performed using a kit. The nucleotide sequence of the OfProT1 and OfProT2 genes obtained by final sequencing is shown in SEQ ID NO. 1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO. 2.

[0049] Example 2

[0050] 1. Construction of the superexpression vector of the OfProT1 and OfProT2 genes

[0051] 1) Vector double enzyme digestion

[0052] The Super1300 vector was taken out from the -80°C ultra-low temperature freezer in advance for activation and shaking, the Super1300 vector plasmid was extracted according to the kit, and then the double enzyme digestion experiment was performed.

[0053] The 20 μL enzyme digestion reaction system was as follows: Sma I 1 μL, Spe I 1 μL, Buffer 2 μL, vector plasmid X μL, ddH2O 6 μL.

[0054] X (μL) = 1000 ng / vector plasmid concentration (ng / μL). The centrifuge tube was slightly shaken to mix, centrifuged for 6 s, and incubated in a 37°C water bath for 1 h. The obtained double enzyme-digested vector was subjected to agarose electrophoresis, and then gel recovery was performed using a kit.

[0055] 2) Ligation transformation

[0056] The target gene fragment was recombined with the linearized vector fragment.

[0057] The ligation reaction system was as follows: target gene recovery product 200 ng, plasmid double enzyme digestion recovery product 100 ng, ligase 2 μL, Buffer 4 μL, linearized vector X μL, ddH2O Add to 20 μL.

[0058] Vortex the centrifuge tube briefly to mix, spin down briefly, and incubate in a 37°C water bath for 30 min, and on ice for 2 min.

[0059] Transformation: Take 5 μL of ligation product and add to 50 μL of Trelief™ 5α competent cells. Vortex briefly, and incubate on ice for 5 min. Heat at 42°C for 60 s, and then incubate on ice for 2 min. Add 250 μL of LB liquid medium (without Kana), and incubate at 37°C for 30 min at 200 rpm.

[0060] Plating: Take 200 μL of the incubated bacteria, and spread evenly on LB solid medium (with 50 mg / L of Kana). Allow to dry, and incubate at 37°C for 12-14 h.

[0061] 3) Positive single colony detection and sequencing

[0062] When the bacteria grow on the medium, perform single colony detection in a clean bench. Pick 8 full single colonies for each gene, and sequentially perform backup on LB solid medium with Kana resistance. Use a sterile toothpick to dip the corresponding single colony into the following system for bacterial detection:

[0063] The PCR reaction system is: 35 sF 1 μL, Gene R 1 μL, Green Mix 10 μL, and ddH2O 8 μL.

[0064] The PCR reaction conditions are: 94°C for 3 min for pre-denaturation; 94°C for 30 s for denaturation, 58°C for 30 s for annealing, and 72°C for 1 min for extension, for 35 cycles; 72°C for 10 min for total extension; and 16°C for termination. Perform agarose electrophoresis on the obtained amplification product, and pick 3 correct positive colonies for plasmid extraction.

[0065] 4) Double enzyme digestion verification

[0066] Perform double enzyme digestion verification on the correct plasmid obtained by sequencing.

[0067] The double enzyme digestion system is: Smal 1 μL, Spel 1 μL, Buffer 2 μL, vector plasmid X μL, and ddH2O 6 μL.

[0068] X (μL) = 1000 ng / vector plasmid concentration (ng / μL). Vortex the centrifuge tube briefly to mix, spin down briefly, and incubate in a 37°C water bath for 1 h. Perform agarose electrophoresis on the obtained double enzyme-digested vector to detect the double enzyme digestion situation. Figure 2

[0069] ​2. Transformation of Agrobacterium GV3101

[0070] The GV3101 competent cells stored in a -80°C ultra-low temperature refrigerator were taken out and thawed on ice. 1 μL of plasmid was added to each 33 μL of competent cells, which were mixed by pipetting and then sequentially placed in an ice bath for 20 min, frozen in liquid nitrogen for 5 min, placed in a 37°C water bath for 5 min, and placed in an ice bath for 5 min. 500 μL of LB liquid medium without antibiotics was added, and the mixture was incubated at 28°C for 1 h on a 200 rpm shaker. After incubation, the bacterial solution was centrifuged at 6000 r for 1 min, and part of the supernatant was discarded. 100 μL of the solution was evenly spread on LB solid medium (containing 50 mg / L Kana), sealed with parafilm, and incubated in a 28°C incubator for 40-48 h.

[0071] Bacterial detection and backup: the target band in the bacterial detection was correct and had consistent brightness ( Figure 3 ), and the corresponding colonies in the backup plate were picked into LB liquid medium (containing 50 mg / L Kana) and shaken. The bacterial solution and 50% glycerol were mixed at a volume ratio of 3:7, frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature refrigerator.

[0072] 3. Infection of Nicotiana benthamiana

[0073] Shaking: the bacterial solution of Super1300 empty vector and the vector with the inserted target gene were taken out and thawed on ice. The bacterial solution was added to 20 mL of LB liquid medium (containing 50 mg / L Kana) using a pipette, and the mixture was incubated in the dark at 28°C and 200 rpm until the OD 600 of the bacterial solution was between 0.5 and 0.6.

[0074] Infection: after shaking, the bacterial solution was transferred to a centrifuge tube and centrifuged for 10 min. The bacterial solution was discarded and an equal volume of buffer was added for resuspension. The ratio of bacterial solution to buffer was 1:1. After standing in the dark for 2-3 h, the bacterial solution was injected into Nicotiana benthamiana seedlings of about 30 d old using a syringe.

[0075] Selection of transgenic plants: the RNA in the leaves was extracted using a kit and reverse-transcribed into cDNA. The cDNA was then diluted 10-fold and subjected to PCR detection ( Figure 4 ). Transgenic seedlings with high expression and consistent growth were selected for subsequent functional verification.

[0076] 4. Physiological indicators of OfProT1 and OfProT2 transgenic plants

[0077] Three transgenic N. benthamiana with same state were selected, and the strain (EV) of the transgenic control with Super1300 empty vector was selected. After watering, it was dark cultured for 10 h, and then it was normally light and dark alternately cultured. After 2 d, it was irrigated with 500 mmol / L saline, 200 mL per pot.

[0078] 1) Relative conductivity

[0079] The same position leaves of empty control and transgenic N. benthamiana were washed with water, 0.1 g was weighed and added into 20 mL deionized water, and soaked at room temperature for 24 h, and shaken 3-5 times during the period. The conductivity EC0 of deionized water was measured by using a conductivity meter, then the conductivity EC1 of each experimental group was measured, and the conductivity EC2 was measured again after 30 min of heating in a 100 ℃ water bath and cooling to room temperature. Relative conductivity = (EC1-EC0 / EC2-EC0) x 100%.

[0080] 2) Proline

[0081] 0.2 g of tobacco leaf frozen sample was weighed and placed in a 10 mL centrifuge tube, 5 mL of 3% sulfosalicylic acid was added, and boiled in boiling water for 10 min. After cooling, 2 mL of filtrate was taken into a new test tube, 2 mL of glacial acetic acid and 2 mL of 2.5% acid ninhydrin were added, and the mixed solution was colored in boiling water for 30 min. After cooling to room temperature, 4 mL of toluene was added, shaken well, and after extraction, the upper red toluene solution was taken into a colorimetric cup, toluene was used as a control, and the absorbance at 520 nm was measured.

[0082] 3) Malondialdehyde content

[0083] The thiobarbituric acid colorimetric method was used. 0.2 g of sample was weighed and 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 the test tube, and 2 mL of 5% TCA was added to the supernatant. The reaction solution was boiled in a water bath for 30 min, then taken out and cooled in water. After cooling, the reaction solution was centrifuged at 4 ℃ and 6000 r / min for 10 min. The absorbance values of the supernatant at 450 nm, 532 nm and 600 nm were measured by using a UV spectrophotometer. The following formula was used for calculation: C (μmol / g) = [6.452 x (A532-A600) x-0.56 x A450] x Vt / (V0 x W) Vt total volume of extract (mL); V0 measured volume (mL); W plant tissue weight (g).

[0084] The results are shown in Table 1. After salt stress, the relative conductivity of OfProT1 and OfProT2 transgenic lines was significantly reduced, which was 0.85 and 0.92 times of the empty control, respectively; the proline content was extremely significantly lower than that of the control, which was 0.70 and 0.49 times of the empty control, respectively; and the malondialdehyde content was extremely significantly lower than that of the control, which was 0.90 and 0.84 times of the empty control, respectively. In summary, after high salt stress, the relative conductivity, proline content and malondialdehyde content of the plants overexpressing OfProT1 and OfProT2 genes were significantly reduced, indicating that overexpression of OfProT1 and OfProT2 genes improved the ability of plants to cope with salt stress.

[0085] Table 1 Physiological indicators of OfProT1 and OfProT2 transgenic plants

[0086] Physiological indicators EV OfProT1 OfProT2 Relative conductivity (%) 63.6335±2.4034a 54.2695±0.4023c 58.5096±1.7298b Proline (pg / g) 3.0699±0.2878a 2.1467±0.2212b 1.4955±0.1016c Malondialdehyde (pmol / g) 0.4956±0.0151a 0.4447±0.0336b 0.4168±0.0175c

[0087] The above description is only illustrative and is not restrictive, and those skilled in the art understand that many modifications, changes or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present application.

Claims

1. A class of Osmanthus fragrans OfProTs genes, as OfProT1 genes or OfProT2 genes; The OfProT1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1 ; The OfProT2 The nucleotide sequence of the gene is shown as SEQ ID NO.

3.

2. The expression protein of the Osmanthus fragrans OfProTs gene according to claim 1 is OfProT1 protein or OfProT2 protein. The amino acid sequence of the OfProT1 protein is shown as SEQ ID NO.

2. The amino acid sequence of the OfProT2 protein is shown as SEQ ID NO.

4.

3. A composition comprising the osmanthus fragrans of claim 1 OfProT1 gene or OfProT2 vector, recombinant bacteria of the gene.

4. The tea of claim 1 OfProT1 gene or OfProT2 application of the gene in regulating plant stress resistance, characterized in that, The regulation of plant stress resistance is to improve the salt stress resistance of Nicotiana benthamiana.

5. Use according to claim 4, characterized in that, The specific steps include: 1) Construction of overexpression vectors for the genes of Osmanthus fragrans OfProT1 or OfProT2 Osmanthus fragrans 2) transforming the constructed overexpression vector into Nicotiana benthamiana; 3) cultivating, screening and obtaining transgenic Nicotiana benthamiana plants with improved salt stress resistance.

6. Use according to claim 5, characterized in that, The overexpression vector is pSuper1300- OfProT1 or pSuper1300- OfProT2 Plant overexpression vectors.

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