A key enzyme gene for photosynthetic carbon assimilation in celery AgRbcS3 and its application under drought stress

Abstract

The invention discloses a celery photosynthetic carbon assimilation key enzyme gene AgRbcS3 and its application under drought stress, and relates to the field of biotechnology. The nucleotide sequence of the celery photosynthetic carbon assimilation key enzyme gene AgRbcS3 is shown in SEQ ID NO.1. The invention clones a new RbcS gene, AgRbcS3, from the celery variety 'Jinyun Baoqin', and describes its function in plant drought resistance. The invention first verifies that AgRbcS3 transgenic yeast has stronger drought resistance through yeast expression, and then transfers the AgRbcS3 gene into Arabidopsis thaliana and 'Liuhe Huangxinqin' celery variety callus tissue by Agrobacterium tumefaciens-mediated method. As a result, it is found that the drought resistance of the obtained transgenic plants and transgenic callus is significantly improved, which proves that the AgRbcS3 gene provided by the invention can improve the tolerance of plants to drought stress and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a key enzyme gene for celery photosynthetic carbon assimilation AgRbcS3 and its application under drought stress. Background Art

[0002] celery( Apium graveolens L.) is an annual or perennial herb of the Umbelliferae family. It is native to the swamps along the Mediterranean coast and is widely cultivated in countries around the world. It is an important vegetable crop. Celery is rich in carotene, vitamins and volatile aromatic components. In addition to being a vegetable, it also has excellent medicinal functions. Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) is a key enzyme for photosynthetic carbon assimilation. Rubisco is conservatively present in algae, plants and some photosynthetic bacteria. Rubisco is the most abundant protein in plant leaves and may also be the most abundant protein on earth. Rubisco has a very low efficiency in fixing carbon and is the rate-limiting enzyme for photosynthesis. RbcS The gene is a gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase. Some studies have shown that plants increase the activity of ribulose-1,5-bisphosphate carboxylase / oxygenase under abiotic stresses such as drought, high salt and low temperature. The present invention intends to clone a new RbcS Genes to solve the problem of how to improve the plant's resistance to drought stress. Summary of the invention

[0003] The purpose of the present invention is to provide a celery photosynthetic carbon assimilation key enzyme gene AgRbcS3 The invention relates to a method for preparing a celery plant for use in a drought stress environment to solve the problems existing in the prior art. AgRbcS3 It can improve the tolerance of plants to drought stress and has broad application prospects

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The invention provides a key enzyme gene for celery photosynthetic carbon assimilation AgRbcS3 , the nucleotide sequence is shown in SEQ ID NO.1.

[0006] The present invention also provides a recombinant expression vector, comprising the above-mentioned celery photosynthetic carbon assimilation key enzyme gene AgRbcS3 .

[0007] The present invention also provides a recombinant host cell, comprising the above-mentioned recombinant expression vector.

[0008] Furthermore, the recombinant host cell is a recombinant Agrobacterium.

[0009] The present invention also provides the above-mentioned celery photosynthetic carbon assimilation key enzyme gene AgRbcS3 , the use of recombinant expression vectors or recombinant host cells in improving the tolerance of plants to drought stress.

[0010] Furthermore, the plant is celery or Arabidopsis thaliana.

[0011] The present invention also provides the above-mentioned celery photosynthetic carbon assimilation key enzyme gene AgRbcS3 , the use of recombinant expression vectors or recombinant host cells in improving the tolerance of yeast to drought stress.

[0012] The present invention also provides a method for improving the tolerance of plants to drought stress, comprising: AgRbcS3 Transformed into plants to construct overexpression of the key enzyme gene for photosynthetic carbon assimilation of celery AgRbcS3 The steps for producing transgenic plants.

[0013] Furthermore, the plant is celery or Arabidopsis thaliana.

[0014] The present invention also provides a method for improving the tolerance of yeast to drought stress, comprising: AgRbcS3 Transformed into yeast, constructed to overexpress the key enzyme gene of celery photosynthetic carbon assimilation AgRbcS3 Steps for genetically modifying yeast.

[0015] The present invention discloses the following technical effects:

[0016] The present invention cloned a new RbcS Gene-- AgRbcS3 , and expounded its function in plant drought tolerance. The present invention firstly verifies through yeast expression AgRbcS3 The transgenic yeast has stronger drought tolerance, and then the Agrobacterium tumefaciens-mediated method is used to AgRbcS3 The gene was transferred into Arabidopsis thaliana and callus of celery variety 'Liuhe Huangxinqin', and the drought tolerance of the obtained transgenic plants and callus was significantly improved, confirming that the AgRbcS3 The gene can improve the plant's tolerance to drought stress and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a comparison of the growth of the control strain (pYES2) and the recombinant strain (pYES2-AgRbcS3) under PEG 3350 stress;

[0019] Figure 2 The PCR detection diagram of transgenic Arabidopsis T2 generation and callus tissue; NNJ-1~3 are Arabidopsis T2 generation; YS-1~3 are callus tissue;

[0020] Figure 3 GUS staining images of wild-type Arabidopsis plants (WT) and transgenic Arabidopsis T2 plants (OE-AgRbcS3);

[0021] Figure 4 The results of the root system of wild-type Arabidopsis plants (WT) and transgenic Arabidopsis T2 plants (OE-AgRbcS3) under 20% PEG6000 treatment;

[0022] Figure 5 This is a comparison of the growth status of wild-type Arabidopsis plants (WT) and transgenic Arabidopsis T2 plants (OE-1 and OE-2) under drought stress;

[0023] Figure 6 The figure is a statistical diagram of the physiological data of wild-type Arabidopsis plants (WT) and transgenic Arabidopsis T2 plants (OE-AgRbcS3) under drought stress; AF stands for proline, SOD, soluble protein, POD, malondialdehyde and CAT;

[0024] Figure 7 Statistical graph of photosynthesis-related data of wild-type Arabidopsis plants (WT) and transgenic Arabidopsis T2 plants (OE-AgRbcS3) under drought stress; AD represents chlorophyll, F v / F m , ribulose-1,5-bisphosphate carboxylase (Rubisco), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH);

[0025] Figure 8 This is a comparison of the growth status of wild-type callus (WT) and transgenic callus (OE-1, OE-2 and OE-3) under drought stress;

[0026] Figure 9 Statistical graphs of the growth and physiological data of wild-type callus (WT) and transgenic callus (AgRbcS3) under drought stress; AD represents the amount of substance, chlorophyll, Rubisco and GAPDH, respectively. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1 Materials and methods

[0034] 1.1 Test materials

[0035] The 'Liuhe Huangxinqin' variety was selected as the material for celery callus preparation. The competent E. coli DH5α used in the experiment was provided by Beijing Qingke Biotechnology Co., Ltd., the competent Agrobacterium GV3101 was provided by Shanghai Shenggong Biogene Technology Co., Ltd., and the competent yeast INVSc1 was provided by Beijing Coolbo Technology Co., Ltd. The plant overexpression vector used pCAMBIA1301 (purchased from Shanghai Shenggong Biogene Technology Co., Ltd.) and the yeast expression vector used pYES2 (purchased from Beijing Coolbo Technology Co., Ltd.).

[0036] 1.2 Test methods

[0037] 1.2.1 AgRbcS3 Screening and cloning

[0038] 1.2.1.1 AgRbcS3 Gene amplification

[0039] AgRbcS3 Nucleotide sequence of the gene (SEQ ID NO.1):

[0040] .

[0041] according to AgRbcS3The nucleotide sequence of the gene was determined, and the primers were designed using Primer Premier 6.0 software. The annealing temperature was set to 55 ± 3 °C, and the GC content was set to 40% to 60%. The following specific primers were designed based on the screening conditions:

[0042] Upstream primer F- AgRbcS3 :5'-ATGGCTTCCTCGATGATCTC-3' (SEQ ID NO.2),

[0043] Downstream primer R- AgRbcS3 :5'-TTATTCTGGCTGGTGGGCA-3' (SEQ ID NO. 3).

[0044] Specific primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0045] Using the cDNA of 'Jinyun Baoqin' as a template, the fragment was amplified using the premix of ExTaq PCR polymerase (SangonBiotech, Shanghai, China). The reaction system was prepared in 50 μL: Ex Taq PCR Master Mix 25µL, cDNA template 2 μL (concentration of 100 ng / μL), upstream primer 2 μL (concentration of 10 μmol / μL), downstream primer 2 μL (concentration of 10 μmol / μL), ddH2O 19 μL. After mixing the prepared system, it was placed in a PCR instrument for amplification. The reaction program was: pre-denaturation at 94℃ for 3min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for a total of 35 cycles; finally, extension at 72℃ for 10 min, and retention at 4℃. After the program is completed, 50 μL of the amplified product obtained by PCR is mixed with 5 μL of 10× Loading Buffer. When loading, 3 μL of 10000 DNA Marker is added as a ruler. Use 1.0% agarose gel and set the electrophoresis instrument to 120 V for 20 min. After the end, use ultraviolet light on the gel imager to observe the size of the cloned target band. If it is correct, cut the gel and recover it. AgRbcS3 Fragment.

[0046] 1.2.1.2 Rubber Recycling

[0047] DNA was recovered using a column DNA gel recovery kit (Sangon Biotech, Shanghai, China). The correct band was cut off and placed in a centrifuge tube. Buffer B2 solution of equal weight to the gel block was added and heated in a 55°C metal bath for 10 min to ensure that the gel was completely melted. After the gel solution cooled to room temperature, it was pipetted into the adsorption column with a pipette and allowed to stand for 1 min. Centrifuged at 9000×g for 1 min, the solution in the collection tube was repeatedly loaded onto the column, and the liquid in the collection tube was discarded after centrifugation. 650 μL of Wash Solution was added to the adsorption column, and the column was centrifuged at 9000×g for 1 min. The waste liquid in the collection tube was discarded. The empty adsorption column and collection tube were placed back into the centrifuge and centrifuged at 9000×g for 2 min. Finally, the adsorption column was replaced in a new centrifuge tube and allowed to stand and dry at room temperature for 5 min to completely evaporate the anhydrous ethanol. Add 40 μL of elution solution to the center of the adsorption column, place at room temperature for 1 min, centrifuge at 8000×g for 1 min, and measure the obtained DNA with an ultra-micro nucleic acid protein analyzer and adjust the concentration to 200 ng / μL. Store the recovered DNA in a -20℃ refrigerator.

[0048] 1.2.1.3 Conversion sequencing

[0049] Will obtain AgRbcS3 The fragment was connected to the pUCm-T vector (purchased from Beijing Qingke Biotechnology Co., Ltd.) and the reaction system was prepared to 10 μL: AgRbcS3 1.5 μL of fragment, 1 μL of pUCm-T vector, 1 μL of 10×Ligation Buffer, 1 μL of 50% PEG 4000, 1 μL of T4 DNA Ligase, 4.5 μL of ddH2O. Mix the prepared reaction system and place it in a PCR instrument, set it at 16℃ for 4 h. Then add the ligation product to 100 μL of E. coli competent cells, place it on ice for 30 min, heat shock it in a 42℃ water bath for 90 s, and place it on ice for 5 min. Add 700 μL of LB liquid culture medium without antibiotics to the mixture, culture it in a 37℃ air bath shaking incubator at 180 r / min for 1 h, centrifuge it at 12000 rpm for 4 min to collect the bacteria, discard 600 μL of supernatant, keep 100 μL of supernatant, gently blow and resuspend the precipitate, and then apply it to the LB (Amp resistance) plate with a spreader, and culture it upside down at 37℃ for 14 h. Use a sterile pipette tip to pick a single clone from the plate, place it in 400 μL of LB liquid medium containing Amp antibiotics, and culture it in a 37°C air bath shaking incubator at 180 r / min for 6 h to make the bacterial solution concentration OD 600The value was between 0.6 and 0.8. The bacterial solution was used as a template for PCR detection. The PCR system configuration and reaction procedure were the same as above. After the reaction was completed, the bacterial solution was sent to Qingke Biotechnology Co., Ltd. for sequencing after being identified by 1.0% agarose gel electrophoresis.

[0050] 1.2.1.4 Plasmid DNA extraction

[0051] After the sequencing is returned, it is compared with the original sequence information. When the comparison result is correct, 200 μL of the test bacterial solution is inoculated into 50 mL of LB liquid culture medium with Amp resistance, and cultured in a 250 mL Erlenmeyer flask at 37°C in an air bath shaking incubator at 180 r / min for 16 h. 600 When the value was between 0.6 and 0.8, 6 mL of bacterial solution was taken for plasmid DNA extraction. Plasmid extraction was performed using the SanPrep column plasmid DNA mini-extraction kit (Sangon Biotech, Shanghai, China). The obtained plasmid DNA was measured using an ultra-micro nucleic acid protein analyzer and the concentration was adjusted to 200 ng / μL. The recovered plasmid DNA was stored in a -20°C refrigerator. The remaining bacterial solution was stored using the glycerol method: 300 μL of bacterial solution was mixed with 200 μL of 50% glycerol, mixed well, and stored in a -80°C refrigerator.

[0052] 1.2.2 AgRbcS3 Gene vector construction

[0053] 1.2.2.1 Enzyme digestion of vector

[0054] The plant expression vector pCAMBIA1301 and the yeast expression vector pYES2 were linearized separately: each expression vector was double-digested with Sac I and BamH I restriction enzymes, and the 20 μL reaction system was configured as follows: plasmid DNA (200 ng / μL) 5 μL, enzyme Sac I (15 U / μL) 1 μL, enzyme BamH I (15 U / μL) 1 μL, 10×FastDigest Buffer 2 μL, ddH2O 11 μL. The mixed reaction solution was placed in a PCR instrument for enzyme digestion and inactivation, and the enzyme digestion program was set to: incubate at 37℃ for 30 min and inactivate at 80℃ for 10 min. After the reaction was completed, the reaction solution was quickly cooled on ice, and then the bands were detected in 1.0% agarose gel electrophoresis. The reaction solution obtained by detection was a linearized vector mixture with a concentration of about 50 ng / μL.

[0055] 1.2.2.2 Homologous recombination

[0056] Homologous recombination primers were designed according to the expression vector sequence, restriction sites and gene fragment sequence. Sac I and BamH I restriction sites were reconstructed in the primers respectively, and the two ends of the primers were the same terminal sequences of the vector and the fragment respectively.

[0057] Yeast expression vector pYES2 homologous recombination primers:

[0058] Upstream primer: 5'-ATTAAGCTTGGTACCGAGCTCATGGCTTCCTCGATGATCTC-3' (SEQ ID NO. 4),

[0059] Downstream primer: 5′-GCGGCCGTTACTAGTGGATCCTTATTCTGGCTGGTGGGCA-3′ (SEQ ID NO. 5).

[0060] Plant expression vector pCAMBIA1301 homologous recombination primers:

[0061] Upstream primer: 5'-TTTACAATTACCATGGGATCCATGGCTTCCTCGATGATCTC-3' (SEQ ID NO.6),

[0062] Downstream primer: 5′-ACCGATGATACGAACGAGCTCTTATTCTGGCTGGTGGGCA-3′ (SEQ ID NO. 7).

[0063] Use homologous recombination primers to amplify the gene fragment using the plasmid extracted in 1.2.1.4 as a template to obtain AgRbcS3 Homologous recombination fragments, amplification reaction system and cycle program are the same as 1.2.1.1. The amplified product was purified and recovered using the SanPrep column PCR product purification kit (Sangon Biotech, Shanghai, China). The recovered purified product was measured using an ultra-micro nucleic acid protein analyzer and the concentration was adjusted to 200 ng / μL; the recombinase SeamLesscloning MasterMix (Sangon Biotech, Shanghai, China) was used to recombinant the fragment into the pCAMBIA1301 vector or pYES2 vector. According to the instructions, a 10 μL reaction system was prepared: AgRbcS3Homologous recombination fragment (200 ng / μL) 1.5 μL, pCAMBIA1301 or pYES2 linearized vector (50 ng / μL) 1 μL, 2× SeamLess cloning Master Mix 5μL, ddH2O 2.5 μL. Mix the reaction system and react on a PCR instrument at 50℃ for 60 min. After the reaction is completed, immediately place the centrifuge tube on ice for 5 min for transformation.

[0064] 1.2.2.3 Transformation and sequencing

[0065] 10 μL of the recombinant ligation reaction solution was added to 100 μL of E. coli competent cells. After the transformant colonies grew out, a single clone was picked up on the plate with a sterile pipette tip the next day and placed in 400 μL of LB liquid medium containing Kan antibiotics. The culture was incubated in a 37°C air bath shaking incubator at 180 r / min for 6 h until the bacterial solution concentration reached OD 600 The value was between 0.6 and 0.8. The bacterial solution was used as a template for PCR detection. The PCR system configuration and reaction procedure were the same as above. After the reaction was completed and detected by 1.0% agarose gel electrophoresis, the bacterial solution was sent to Sangon Biotech Co., Ltd. for sequencing. After successful identification, the recombinant plasmids were recorded as pCAMBIA1301-AgRbcS3 and pYES2-AgRbcS3.

[0066] 1.2.3 Yeast expression verification

[0067] 1.2.3.1 Yeast transformation

[0068] Take 100 µL of INVSc1 competent cells melted on ice, add 5 µg of pre-cooled recombinant plasmid pYES2-AgRbcS3, 10 µL of Carrier DNA (95-100℃, 5 min, quick ice bath, repeat once), and 500 µL of PEG / LiAc, pipette and mix several times, and place in a 30℃ water bath for 30 min (invert 6 times at 15 min to mix). Place the tube in a 42℃ water bath for 15 min (invert 6 times at 7.5 min to mix). Centrifuge at 10,000 rpm for 30 s, discard the supernatant, resuspend in 400 µL of ddH2O, centrifuge for 30 s, discard the supernatant. Resuspend in 50 µL of ddH2O, apply to the corresponding SD-Ura defective screening plate, and culture at 29℃ for 3 h to obtain the recombinant strain pYES2-AgRbcS3.

[0069] The control strain pYES2 was constructed by transforming the empty pYES2 vector using the same method as above.

[0070] 1.2.3.2 Yeast drought verification

[0071] The control strain (pYES2) and the recombinant strain (pYES2-AgRbcS3) were resuspended with 2 mL of sterile water, spotted on SG-Ura plates and plates containing 120 mM PEG 3350 SG-Ura, respectively, and cultured in an incubator at 29°C. The plates were observed and photographed after 3 days.

[0072] 1.2.4 Plant expression vector transformation into Agrobacterium

[0073] Add 1 μg of recombinant plasmid pCAMBIA1301- AgRbcS3 to 100 μL GV3101 competent cells, let stand on ice for 30 min, put the mixture in liquid nitrogen for quick freezing for 3 min, and immediately put it in a 37℃ water bath for 5 min, taking it out and mixing it. Add 700 μL YEB liquid medium (containing 25 μg / mL Rif) and shake and culture it at 28℃, 180 r / min in a constant temperature shaking box for 6 h. Take 100 μL of bacterial solution and spread it on a YEB plate (containing 50 μg / mL Kan and 25 μg / mL Rif), invert it and culture it in a 28℃ incubator for 48 h. Pick out the single colony grown for PCR identification. The positive one is the recombinant GV3101 Agrobacterium carrying the recombinant plasmid pCAMBIA1301-AgRbcS3, which is stored in an ultra-low temperature refrigerator at -70℃ for use.

[0074] 1.2.5 Expression vector transformation into Arabidopsis

[0075] 1.2.5.1 Planting of Arabidopsis seedlings

[0076] In the clean bench, soak the dried Arabidopsis seeds in 75% ethanol for 1 min, rinse the seeds with sterile water 3 times to wash away the residual ethanol, and then soak them in 0.1% mercuric chloride for 10 min and shake them repeatedly during the process. After disinfection, rinse them with sterile water 5 times. Evenly sow the disinfected seeds on the MS solid culture medium and seal them. After the culture medium is placed in a 4℃ environment for vernalization for 4 days, continue to culture in a light incubator. After the seeds germinate and grow for about 10 days, transplant the seedlings into small pots filled with vermiculite: nutrient soil = 2:3, plant 4 seedlings in each small box, and cover them with transparent plastic wrap to keep the seedlings in a relatively humid environment.

[0077] 1.2.5.2 Activation of Agrobacterium culture and preparation of infection solution

[0078] Take out the recombinant GV3101 Agrobacterium constructed in 1.2.4 from the -70℃ ultra-low temperature refrigerator and activate the strain in the clean bench. After the clean bench is sterilized for 30 minutes, use a sterile inoculation loop to dip the recombinant GV3101 Agrobacterium culture liquid, draw a "Z" shape in the prepared YEB solid culture medium, seal the culture medium and invert it in a constant temperature incubator, culture at 28℃, and after a single colony grows, pick up a single colony with a 10 μL pipette tip in the clean bench, place it in a triangular flask containing 10 mL YEB activation culture medium, seal it, place it on a shaker, and shake it overnight at 28℃ and 180 rpm. Take the activated bacterial liquid at a ratio of 1:100 for expansion culture, shake it on a shaker at 28℃ and 180 rpm, and measure the OD 600 When the concentration is about 1.5, take out the bacterial solution and set aside.

[0079] 1.2.5.3 Activation of Agrobacterium culture and preparation of infection solution

[0080] After Arabidopsis thaliana bolts, blooms, and enters the bud stage, the top inflorescence is cut off to promote the growth of more lateral inflorescences. Continue to culture for 5 days. When there are more flower buds, Agrobacterium tumefaciens transformation is performed. Water Arabidopsis thaliana fully one day before transformation. In the clean bench, add the Agrobacterium cultured in 1.2.5.2 to the sterilized 50 mL centrifuge tube, centrifuge at 28°C and 6000 rpm for 10 min in a refrigerated high-speed centrifuge, discard the supernatant in the clean bench, repeatedly shake the precipitated bacteria, evenly suspend them in the buffer, and adjust the buffer OD 600 Adjust to about 0.8. Invert the wild-type Arabidopsis inflorescence and immerse it in the buffer solution containing the strain. During this process, shake the Arabidopsis inflorescence slightly to make it fully immersed. After 1 minute, cover the infected Arabidopsis with plastic wrap to keep moisture. After 1 day of cultivation in the dark, remove the plastic wrap and place it in the light culture room for further cultivation. A week later, new flower buds grow on Arabidopsis. In order to increase the transformation rate, the transformation of Arabidopsis is repeated once. The transformed Arabidopsis continues to be cultivated. When the Arabidopsis siliques turn brown, the seeds are mature and collected. Rub the seeds of the Arabidopsis inflorescence onto paper by hand, pick out the impurities, and store them in a dry environment in a sealed container.

[0081] 1.2.5.4 Screening of Arabidopsis transformed plants

[0082] The T1 generation seeds of the transformed Arabidopsis obtained in the above steps were sterilized. In the clean bench, the seeds were soaked in 70% ethanol for 1 min, the residual ethanol was washed off with sterile water, and then soaked in 0.1% mercuric chloride for 10 min (to ensure that the seeds were thoroughly disinfected), and then rinsed with sterile water for 5 times to end the sterilization process. The seeds were sown on MS solid medium with a concentration of 30 mg / L Kan for screening. After about 15 days, the growth of Arabidopsis in the medium was observed. The non-transgenic seeds had poor radicle development due to lack of kanamycin resistance, and the leaves turned yellow and died. The seeds that were successfully transformed germinated normally and the cotyledons were tender green. They were transplanted to the soil with a ratio of nutrient soil: vermiculite = 1:1 for continued cultivation. After the Arabidopsis seeds matured, the T2 generation seeds were collected.

[0083] 1.2.5.5 PCR detection of T2 transgenic Arabidopsis plants

[0084] When the T2 positive plants were grown on MS medium for 10 days, they were taken out with tweezers and stained with GUS staining kit (Beijing Coolbo Technology Co., Ltd.). At the same time, the positive plants were transplanted and the leaf genomic DNA was extracted for PCR molecular identification to determine the T2 positive plants.

[0085] 1.2.6 AgRbcS3 Obtaining transgenic callus

[0086] 1.2.6.1 Aseptic culture of celery callus

[0087] The hypocotyls of 21-day-old sterile celery seedlings obtained from the culture medium were used as explant sources for callus induction. The hypocotyls were cut into 0.3-0.5 cm segments with sterilized surgical scissors and placed in an induction medium containing 2.0 mg / L IBA and 2.0 mg / L 6BA for 30 days to form callus tissue.

[0088] 1.2.6.2 Celery callus genetic transformation process

[0089] Dilute the Agrobacterium culture solution cultured in 1.2.5.2 to OD 600 = 0.3, select well-growing celery callus and place it at the above OD 600= 0.3 of Agrobacterium liquid, shake slowly, and infect for 15 min. After infection, filter out the callus with a filter, place it on sterile filter paper, absorb the excess bacterial liquid, and use tweezers to spread the callus on the culture medium for co-culture for 2 days. Wash the dark-cultured callus with cephalosporin water 3 times, 10 min each time, and then wash it with ddH2O 2 times, 5 min each time. Filter with a filter and spread it on the screening medium, and put it in the incubator for culture. About a month later, new callus tissue begins to grow on the screening medium, and it is transferred to the new screening medium for continued growth. When the callus grows to the size of a broad bean, GUS staining is performed and DNA is extracted for PCR detection. After the detection is correct, the transgenic callus obtained is transferred to a tissue culture bottle with a screening medium for large-scale propagation.

[0090] 2 Results and analysis

[0091] 2.1 AgRbcS3 Yeast expression verification

[0092] The yeast was cultured in SG-Ura medium for three days. Figure 1 As shown. The control strain (pYES2) and the recombinant strain (pYES2-AgRbcS3) can grow normally in SG-Ura medium. However, the control strain no longer grows under the action of 120 mM PEG 3350, while the transgenic strain still grows in 120 mM PEG 3350. This shows that AgRbcS3 Genes respond to drought stress in yeast.

[0093] 2.2 PCR detection of transgenic Arabidopsis plants and callus

[0094] The genomic DNA was extracted from the obtained Arabidopsis positive plants and callus tissues, and PCR molecular identification was performed to determine the T2 generation positive plants and callus. The PCR identification results were as follows: Figure 2 As shown, it proves that the genetic modification was successful.

[0095] 2.3 AgRbcS3 Responses of Arabidopsis mutants to drought stress

[0096] First of all AgRbcS3 Arabidopsis mutants and wild type (WT) were subjected to GUS staining, and the results are shown in Figure 3 .Will AgRbcS3 Arabidopsis mutant (OE-AgRbcS3) and WT seeds were grown on MS solid medium for 7 days and then transferred to MS medium containing 20% ​​PEG6000 and soil, respectively. The results after culturing in 20% PEG 6000 medium for 10 days are as follows: Figure 4 The Arabidopsis transplanted into the soil continued to grow for 30 days under normal light and sufficient water conditions. Figure 5As shown in the figure, after drought treatment, WT plants almost withered and died due to severe water loss, while AgRbcS3 The mutant plants grew well. This shows that under drought stress conditions AgRbcS3 The mutant plants have stronger drought tolerance. Afterwards, the present invention measured the physiological and photosynthetic indices after drought stress, and the results are shown in Figures 6 - 7 . AgRbcS3 Compared with WT, the mutant has significantly higher ribulose-1,5-bisphosphate carboxylase / oxygenase activity than WT, and also has higher POD, SOD, CAT and other enzyme activities and higher photosynthetic efficiency.

[0097] 2.4 Effects of drought stress on AgRbcS3 Effects of transgenic celery wound healing

[0098] WT callus and AgRbcS3 mutant callus with the same growth conditions were transferred to MS medium containing 4% PEG 6000 and cultured for 7 days. Figure 8 As shown, after drought treatment, WT callus grew slowly, while AgRbcS3 The mutant callus grew well. The present invention then measured the growth and physiological indicators after drought stress, and the results were shown in Figure 9 The results showed that compared with WT callus, AgRbcS3 After 7 days of drought stress, the mutant callus had a larger mass and higher Rubisco and GAPDH activities, indicating that under drought stress conditions AgRbcS3 The mutant plants were more drought tolerant.

[0099] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene encoding a key enzyme for photosynthetic carbon assimilation in celery AaS , characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: Comprising the key enzyme gene of celery photosynthetic carbon assimilation according to claim 1 AaS .

3. A recombinant host cell, characterized in that Comprising the recombinant expression vector according to claim 2; the recombinant host cell is a recombinant Agrobacterium.

4. A celery photosynthetic carbon assimilation key enzyme gene as claimed in claim 1 AaS , Use of the recombinant expression vector according to claim 2 or the recombinant host cell according to claim 3 in improving the tolerance of plants to drought stress, characterized in that: The plant is celery or Arabidopsis thaliana; By expressing the key enzyme gene of celery photosynthetic carbon assimilation AaS Overexpression in the plant can improve the plant's tolerance to drought stress.

5. A celery photosynthetic carbon assimilation key enzyme gene as claimed in claim 1 AaS , Use of the recombinant expression vector according to claim 2 or the recombinant host cell according to claim 3 in improving the tolerance of yeast to drought stress, characterized in that: By expressing the key enzyme gene of celery photosynthetic carbon assimilation AaS Overexpression in the yeast can improve the tolerance of the yeast to drought stress.

6. A method for improving the tolerance of plants to drought stress, characterized in that: The invention comprises the following steps: AaS Transformed into plants to construct overexpression of the key enzyme gene for photosynthetic carbon assimilation of celery AaS The steps of producing transgenic plants; The plant is celery or Arabidopsis thaliana.

7. A method for improving the tolerance of yeast to drought stress, characterized in that: The invention comprises the following steps: AaS Transformed into yeast, constructed to overexpress the key enzyme gene of celery photosynthetic carbon assimilation AaS Steps for genetically modifying yeast.

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