Application of Osmanthus fragrans OfProTs gene in improvement of plant stress resistance

Abstract

The invention discloses application of Osmanthus fragrans OfProTs genes in improvement of plant stress resistance, and relates to the technical field of plant genetic engineering. The Osmanthus fragrans OfProTs gene disclosed by the invention comprises an OfProT1 gene, or / and an OfProT2 gene, and the OfProTs gene comprises an OfProT1 gene and / or an And the nucleotide sequences of the primers are respectively as shown in SEQ ID NO.1 and SEQ ID NO.3. The method comprises the following steps: constructing an overexpression vector of OfProT1 or OfProT2 genes of osmanthus fragrans; transforming the constructed overexpression vector into the nicotiana benthamiana; and cultivating and screening to obtain a transgenic Bensi tobacco plant with improved salt stress resistance. Results show that after high salt stress, the relative conductivity, the proline content and the malondialdehyde content in a plant with overexpressed OfProT1 and OfProT2 genes are remarkably reduced, and the salt stress coping capacity of the plant is improved through overexpressed OfProT1 and OfProT2 genes.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to the application of a class of osmanthus OfProTs genes in improving plant stress resistance. Background Art

[0002] ProTs genes are a family of proteins involved in proline transport in plants. Proline transport plays an important role in plant adaptation to stress. 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 enhance stress resistance. Taking potato as an example, the StProT3 gene is expressed in the roots, stems, leaves, runners and tubers of potato, and is regulated by hormones, heavy metals, salt, drought and low temperature, indicating that it is involved in the hormone signal transduction and abiotic stress response of potato. At present, in Arabidopsis, three member genes of the ProTs subfamily have been found, namely AtProT1, AtProT2 and AtProT3. The genes of this subfamily mainly mediate the transport of compounds such as proline, GABA (γ-aminobutyric acid) and betaine. AtProT1 is mainly expressed in the thin-walled cells of the phloem or phloem of Arabidopsis, and may be involved in the long-distance transport of amino acids. When plants are subjected to stress, the expression of the AtProT2 gene increases and the main expression site is in the root. AtProT3 is mainly expressed in the epidermal cells of leaves in the aboveground part, and may be involved in regulating the transport of proline in leaves.

[0003] Sweet osmanthus (Osmanthus fragrans) is common in subtropical areas. It is not salt-tolerant and difficult to grow in saline-alkali land, which seriously restricts its application in coastal and high-salt areas. ProTs genes play a key role in plant adaptation to stress. They help plants cope with various adverse conditions by regulating the transport and accumulation of proline, but the specific research on them in sweet osmanthus is still unclear. Research on ProTs genes helps to gain a deeper understanding of the molecular mechanism of plant adaptation to stress and provide new ideas and methods for crop stress resistance breeding. ProTs, a key transporter gene in the proline transport process, plays an important role in plant response to salt stress. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a class of Osmanthus fragrans OfProTs genes. Another technical problem to be solved by the present invention is to provide an expression protein of Osmanthus fragrans OfProTs genes. Another technical problem to be solved by the present invention is to provide an application of Osmanthus fragrans OfProT1 gene or / and OfProT2 gene in regulating plant stress resistance, for regulating Osmanthus fragrans stress resistance.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

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

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

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

[0009] The expressed protein of Osmanthus fragrans OfProTs gene includes OfProT1 protein and / or OfProT2 protein;

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

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

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

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

[0014] The method of regulating plant stress resistance is to improve the plant's ability to resist salt stress, and the specific steps include:

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

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

[0017] 3) Cultivate, screen and obtain transgenic Nicotiana benthamiana plants with improved resistance to salt stress.

[0018] The overexpression vector is pSuper1300-OfProT1 or / and pSuper1300-OfProT2 plant overexpression vector.

[0019] Application of the Osmanthus fragrans OfProT1 gene and / or OfProT2 gene in regulating relative electrical conductivity in plants.

[0020] Application of the osmanthus fragrans OfProT1 gene and / or the OfProT2 gene in regulating the proline content in plants.

[0021] Application of the osmanthus fragrans OfProT1 gene and / or the OfProT2 gene in regulating the malondialdehyde content in plants.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) A class of Osmanthus fragrans OfProTs genes disclosed in the present invention includes OfProT1 gene and / or OfProT2 gene; the nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.3 respectively. The present invention constructs an overexpression vector of Osmanthus fragrans OfProT1 or OfProT2 gene; transforms the constructed overexpression vector into Nicotiana benthamiana; cultivates, screens and obtains transgenic Nicotiana benthamiana plants with improved salt stress resistance.

[0024] 2) The results of the examples of the present invention show that after being subjected to high salt stress, the relative conductivity, proline content and malondialdehyde content of the plants overexpressing the OfProT1 and OfProT2 genes are significantly reduced, and the results show that overexpressing the OfProT1 and OfProT2 genes improves the ability of plants to cope with salt stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result diagram of OfProT1 and OfProT2 gene amplification verification;

[0026] Figure 2 This is the result of double enzyme digestion verification;

[0027] Figure 3 This is the result of bacterial examination;

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

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be carried out with reference to the methods listed in the book "Molecular Cloning Experiment Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0030] The materials used in this application are leaves of Osmanthus fragrans growing in the Osmanthus fragrans National Germplasm Resource Bank. In November 2022, the flowers of Osmanthus fragrans were placed in a sterilized centrifuge tube, immediately frozen in liquid nitrogen, and then stored in a -80°C refrigerator. The Nicotiana benthamiana seedlings used were provided by Wang Lianggui's research group at Nanjing Forestry University.

[0031] Example 1

[0032] 1. Total RNA extraction and cDNA acquisition

[0033] use The total RNA of the leaves of Cinnamomum cassia was extracted using the Super Total RNA Extraction Kit (Promega (Beijing) Biotechnology Co., Ltd.). The extracted total RNA was used as a template and the reverse transcription reagent HiScriptⅢ1st Strand cDNA Synthesis Kit (Nanjing Novogene Biotechnology Co., Ltd.) was used for reverse transcription to obtain cDNA.

[0034] 2. Primer design

[0035] According to the Osmanthus fragrans genome database published by the previous research group, two gene sequences were screened and named OfProT1 and OfProT2. The full-length nucleotide sequences of the two genes were analyzed for restriction sites using BioXM software, and SmaⅠ and SpeⅠ were selected as two restriction endonucleases. Primers were designed using CE design software. Fill in the relevant information as required, including the sequence near the restriction site on the vector, the full length of the target gene, and fill in the two restriction sites (5' and 3' ends) in order to obtain the amplification primers. The designed sequence was sent to Jierui Biotechnology Company for synthesis. The primer sequences are as follows:

[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] PCR amplification of the target gene was performed using 10-fold diluted cDNA as template.

[0046] The PCR reaction system is: Forward Primer 1μL, Forward Primer 1μL, cDNA 1μL, PrimeSTAR 10μL, ddH2O 7μL. Three 20μL systems are made 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 of the reaction at 16°C.

[0048] The obtained amplified products were subjected to agarose electrophoresis and then recovered by gel excision using a kit. The nucleotide sequences of OfProT1 and OfProT2 genes obtained by sequencing were shown in SEQ ID NO.1, and the amino acid sequences of the expressed proteins were shown in SEQ ID NO.2.

[0049] Example 2

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

[0051] 1) Double restriction digestion of vector

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

[0053] The 20μL restriction enzyme digestion reaction system is: SmaⅠ1μL, SpeⅠ1μL, Buffer 2μL, vector plasmid XμL, ddH2O 6μL.

[0054] Where X (μL) = 1000ng / vector plasmid concentration (ng / μL). Slightly shake the centrifuge tube to mix it, centrifuge it for 6 seconds, and incubate it in a 37°C water bath for 1 hour. Perform agarose electrophoresis on the double-enzyme-digested vector, and then use the kit to cut the gel for recovery.

[0055] 2) Connection conversion

[0056] The target gene fragment is recombined and connected with the linearized vector fragment.

[0057] The ligation reaction system is: 200ng of target gene recovery product, 100ng of plasmid double enzyme digestion recovery product, 2μL of ligase, 4μL of Buffer, XμL of linearized vector, and ddH2O added to 20μL.

[0058] The centrifuge tube was shaken slightly to mix, centrifuged for 6 seconds, and incubated in a 37°C water bath for 30 minutes, and placed on ice for 2 minutes.

[0059] Transformation: In a clean bench, use a pipette to take 5 μL of the ligation product and add it to 50 μL of Trelief TM 5α competent cells, flick to mix, place on ice for 5 min, place in a 42°C water bath for 60 s, place on ice for another 2 min, add 250 μL of liquid LB (without Kana), and incubate in a shaker at 37°C, 200 rppm for 30 min.

[0060] Plate coating: Take 200 μL of the incubated bacterial solution and spread it evenly on LB solid culture medium (containing 50 mg / L Kana) with a sterilized glass rod and let it dry. Cover with sealing film and invert it in a 37°C constant temperature incubator for 12-14 hours.

[0061] 3) Positive single colony detection and sequencing

[0062] When bacteria grow on the culture medium, single colony detection is performed in the clean bench. Pick 8 full single colonies for each gene, and backup them in sequence on LB solid culture medium containing Kana resistance, and use a sterile toothpick to dip the corresponding single colony into the following system for bacterial detection:

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

[0064] The PCR reaction conditions were as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, 35 cycles; 72°C total extension for 10 min; 16°C termination. The obtained amplified products were subjected to agarose electrophoresis, and 3 correct positive colonies were selected to extract the plasmid.

[0065] 4) Double enzyme digestion verification

[0066] The plasmid with the correct sequence obtained by sequencing was verified by double enzyme digestion.

[0067] The double enzyme digestion system was: SmaⅠ1μL, SpeⅠ1μL, Buffer 2μL, vector plasmid XμL, ddH2O 6μL.

[0068] Where X (μL) = 1000ng / vector plasmid concentration (ng / μL). Slightly shake the centrifuge tube to mix it, centrifuge it for 6 seconds, and incubate it in a 37°C water bath for 1 hour. Perform agarose electrophoresis on the double-enzyme-digested vector to detect the double-enzyme-digestion status ( Figure 2 ).

[0069] 2. Transformation of Agrobacterium GV3101

[0070] Take out the competent cells of GV3101 stored in the -80℃ ultra-low temperature refrigerator and put them on ice to melt. Add 1μL plasmid to every 33μL competent cells, mix well by pipetting, and then place in ice bath for 20min, quick freeze with liquid ammonia for 5min, water bath at 37℃ for 5min, and ice bath for 5min; add 500μL of LB liquid medium without resistance, and culture on a shaker at 28℃ and 200rppm for 1h; after the culture is completed, centrifuge the bacterial liquid at 6000r for 1min, discard part of the supernatant, and evenly spread 100μL on LB solid culture medium (containing 50mg / L Kana), seal with sealing film, and invert in a 28℃ incubator for 40-48h.

[0071] Bacteria inspection and backup: The target bands in the bacteria inspection are correct and have consistent brightness ( Figure 3 ), then pick the corresponding colony in the backup plate into LB liquid culture medium (containing 50 mg / L Kana), shake the bacteria, and then preserve the bacteria with 50% glycerol at a volume ratio of 3:7, quickly freeze in liquid nitrogen and store in a -80℃ ultra-low temperature refrigerator.

[0072] 3. Infection with Nicotiana benthamiana

[0073] Shake the bacteria: Take out the Super1300 empty vector and the vector culture solution with the target gene and thaw it on ice. Use a pipette to add the culture solution to 20 mL of LB liquid medium (containing 50 mg / L Kana) and culture it in the dark on a shaker at 28°C and 200 rppm until the culture solution OD reaches 0. 600 = between 0.5 and 0.6;

[0074] Infection: After shaking, transfer the bacterial solution to a centrifuge tube and centrifuge for 10 minutes. Discard the bacterial solution and add an equal volume of buffer to resuspend. The ratio of bacterial solution to buffer is 1:1. After standing in the dark for 2-3 hours, use a syringe to inject the bacterial solution into Nicotiana benthamiana seedlings of about 30 days old.

[0075] Screening of transgenic plants: RNA from leaves was extracted using a kit and reverse transcribed into cDNA. The cDNA was then diluted 10 times and tested by PCR ( Figure 4 ), and transgenic seedlings with high expression levels and consistent growth status were selected for subsequent functional verification.

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

[0077] Three transgenic Nicotiana benthamiana plants of the same status were selected and transferred into the strain (EV) of Super1300 empty vector as transgenic control. After watering thoroughly, they were cultured in the dark for 10 hours, and then cultured in normal light and dark alternation. After 2 days, they were irrigated with salt water with a concentration of 500mmol / L, 200mL per pot.

[0078] 1) Relative conductivity

[0079] Take the leaves of the empty control and transgenic Nicotiana benthamiana at the same position, wash with water, weigh 0.1g and add 20mL deionized water, soak at room temperature for 24h, shake 3-5 times during the period. Use a conductivity meter to measure the conductivity EC0 of deionized water, then measure the conductivity EC1 of each experimental group, heat in a 100℃ water bath for 30min, cool to room temperature, shake well, and measure the conductivity EC2 again. Relative conductivity = (EC1-EC0 / EC2-EC0)×100%.

[0080] 2) Proline

[0081] Weigh 0.2g of frozen tobacco leaf sample and place it in a 10mL centrifuge tube, add 5mL 3% sulfosalicylic acid, and boil in boiling water for 10min. After cooling, draw 2mL of filtrate into a new test tube, add 2mL glacial acetic acid and 2mL 2.5% acidic ninhydrin respectively, and color the mixture in boiling water for 30min. After cooling to room temperature, add 4mL toluene, shake thoroughly, and after extraction, draw the upper red toluene solution into a colorimetric cup, use toluene as a control, and measure the absorbance at a wavelength of 520nm.

[0082] 3) Malondialdehyde content

[0083] Use thiobarbituric acid colorimetry. Weigh 0.2g of sample, add 5% trichloroacetic acid (TCA) solution and mix, centrifuge at 4℃, 6000r / min for 10min. Add 2mL of 0.67% TBA solution to the test tube, take 2mL of the supernatant after centrifugation and mix, and add 2mL of 5% TCA to the control group. Boil the reaction solution in a water bath for 30min and then cool it in water. After cooling, centrifuge the reaction solution at 4℃, 6000r / min for 10min. Use a UV spectrophotometer to measure the absorbance of the supernatant of the reaction solution at wavelengths of 450nm, 532nm, and 600nm. The following formula was used for calculation: C (μmol / g) = [6.452×(A532-A600)×-0.56×A450]×Vt / (V0×W) Vt is the total volume of the extract (mL); V0 is the volume of the assay solution (mL); W is the weight of the plant tissue (g).

[0084] The results are shown in Table 1. After salt stress, the relative conductivity of the leaves of OfProT1 and OfProT2 transgenic lines was significantly reduced, which were 0.85 and 0.92 times of the empty load, respectively; the proline content was significantly lower than that of the control group, which were 0.70 and 0.49 times of the empty load, respectively; the malondialdehyde content was significantly lower than that of the control group, which were 0.90 and 0.84 times of the empty load, respectively. In summary, after high salt stress, the relative conductivity, proline content, and malondialdehyde content of 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 parameters 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 (μg / g) 3.0699±0.2878a 2.1467±0.2212b 1.4955±0.1016c Malondialdehyde (μmol / g) 0.4956±0.0151a 0.4447±0.0336b 0.4168±0.0175c

[0087] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

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

3.

2. The expression protein of the Osmanthus fragrans OfProTs gene of claim 1, comprising OfProT1 protein and / or OfProT2 protein; The amino acid sequence of the OfProT1 protein is shown in SEQ ID NO.2; The amino acid sequence of the OfProT2 protein is shown in SEQ ID NO.

4.

3. A vector or recombinant bacterium containing the Osmanthus fragrans OfProT1 gene or OfProT2 gene according to claim 1.

4. Use of the sweet osmanthus OfProT1 gene and / or the OfProT2 gene according to claim 1 in regulating plant stress resistance.

5. The use according to claim 4, characterized in that: The regulating plant stress resistance is to improve the plant's ability to resist salt stress.

6. The use according to claim 4, characterized in that: The specific steps include: 1) constructing an overexpression vector of the Osmanthus fragrans OfProT1 or OfProT2 gene; 2) Transforming the constructed overexpression vector into Nicotiana benthamiana; 3) Cultivate, screen and obtain transgenic Nicotiana benthamiana plants with improved resistance to salt stress.

7. The use according to claim 6, characterized in that: The overexpression vector is pSuper1300-OfProT1 or / and pSuper1300-OfProT2 plant overexpression vector.

8. Use of the Osmanthus fragrans OfProT1 gene and / or the OfProT2 gene according to claim 1 in regulating relative conductivity in a plant.

9. Use of the Osmanthus fragrans OfProT1 gene and / or OfProT2 gene according to claim 1 in regulating the proline content in plants.

10. Use of the Osmanthus fragrans OfProT1 gene and / or the OfProT2 gene according to claim 1 in regulating the content of malondialdehyde in plants.

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