Cyrtomium fortunei pp2c19 gene and application

Abstract

This invention discloses a type of tiger nut. CePP2C19 Genes, the ones mentioned CePP2C19 The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing; tiger nuts CePP2C19 The gene was obtained by reverse transcription of RNA from tiger nut leaves subjected to 12 h of drought stress into cDNA, followed by PCR amplification using primers and the cDNA as a template. Tiger nut CePP2C19 Application of genes in improving plant drought resistance: Tiger nuts CePP2C19 After gene silencing, under drought stress, the degree of leaf wilting increases and drought sensitivity is enhanced; CePP2C19 Under drought stress, the overexpressing Arabidopsis thaliana lines showed less leaf wilting and yellowing compared to the control lines, indicating drought tolerance. CePP2C19 Gene expression can improve plant drought resistance, laying the foundation for breeding new drought-resistant tiger nut varieties.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the CePP2C19 gene of tiger nuts and its applications. Background Technology

[0002] Tiger nuts (Cyperus esculentus), also known as oilseed rape, are a herbaceous oilseed crop native to Africa with a long history of cultivation. Tiger nut tubers are rich in oil, which can be extracted for edible oil. Tiger nuts are prone to yield reduction under drought conditions, making it particularly important to improve their drought resistance. Protein phosphatases play a crucial role in plant cell stress signal transduction. Plant phosphatases are divided into two superfamilies of PPs: serine / threonine (Ser / Thr) phosphatases and tyrosine (Tyr) phosphatases. Serine / threonine phosphatases can be further divided into PP1s, PP2As, PP2Bs, and PP2Cs. In most plants, PP2Cs (Protein phosphatase 2C) is the largest classification of PPs. Phylogenetic analysis divides plant PP2Cs into 13 branches, namely AM. In plants such as rice, tomato, and maize, expression analysis has revealed that subfamily A PP2C genes play an important role under abiotic stresses such as drought and ABA and salinity. Studies have shown that subfamily A PP2Cs play an important role in drought stress in Arabidopsis and rice, and can negatively regulate abscisic acid signaling. However, their role in tiger nuts is rarely reported. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems by providing a CePP2C19 gene for tiger nuts and its application.

[0004] The CePP2C19 gene of tiger nuts has its base sequence shown in SEQ ID NO.1 of the sequence listing;

[0005] The CePP2C19 gene of tiger nut was reverse transcribed from RNA in tiger nut leaves after 12 h of drought stress into cDNA. Using the cDNA as a template, primers were then used...

[0006] CePP2C19F: ATGGCGGAGGTTTGTTGCG

[0007] CePP2C19R: TTACAATCTTCTAGCTC

[0008] Obtained by PCR amplification.

[0009] A recombinant plant expression vector into which the gene shown in SEQ ID NO.1 of the sequence listing is inserted.

[0010] The plant expression vector is pCAMBIA3301.

[0011] Application of the CePP2C19 gene in tiger nuts to improve plant drought resistance;

[0012] The plant in question is tiger nut or Arabidopsis thaliana.

[0013] This invention provides the CePP2C19 gene of tiger nut and its applications. The nucleotide sequence of the CePP2C19 gene is shown below. The CePP2C19 gene of this invention is obtained by reverse transcription of RNA from tiger nut leaves subjected to 12 h of drought stress into cDNA, followed by PCR amplification using primers with the cDNA as a template. The invention also includes a TRV virus-mediated gene silencing vector pTRV2 and a plant expression vector pCAMBIA3301. The application of the tiger nut CePP2C19 gene in improving plant drought resistance is also discussed. Results show that under drought stress, tiger nut CePP2C19 gene-silenced lines are more wilted and more drought-sensitive than the control group. In contrast, Arabidopsis thaliana lines overexpressing the CePP2C19 gene show less leaf yellowing and wilting compared to the control group. The tiger nut CePP2C19 gene provided by this invention can enhance plant drought resistance and provides a new candidate gene resource for tiger nut drought resistance research. Attached Figure Description

[0014] Figure 1 RNA extraction from tiger nut leaves;

[0015] Figure 2 PCR identification of CePP2C19 gene-ligated cloning vector;

[0016] Figure 3 PCR identification of the pTRV2-CePP2C19 recombinant vector in bacterial culture;

[0017] Figure 4 Phenotypic diagrams of tiger nuts in the control and gene-silenced groups under drought stress;

[0018] Figure 5 Dry weight, fresh weight, and moisture content of Arabidopsis thaliana control group and CePP2C19 gene-silenced group under drought stress.

[0019] Figure 6 PCR identification of the pCAMBIA3301-CePP2C19 recombinant vector in bacterial culture;

[0020] Figure 7 Phenotypic diagrams of Arabidopsis thaliana control group and CePP2C19 overexpression group under drought stress;

[0021] Figure 8MDA content was measured in the control group and CePP2C19 overexpression group of Arabidopsis thaliana under drought stress.

[0022] Figure 9 Dry weight, fresh weight, and water content of Arabidopsis thaliana control group and CePP2C19 overexpression group under drought stress. Detailed Implementation

[0023] Example 1: RNA extraction and reverse transcription from tiger nuts

[0024] Experimental materials: Tiger nuts HJ2 (Cyperus esculentus) cultivated by Jilin Agricultural University. Leaves were harvested after 12 hours of drought stress. The experimental procedure is as follows:

[0025] 1. RNA extraction from tiger nut leaves under drought stress

[0026] RNA was extracted from tiger nut leaves under drought stress using an RNA extraction kit from Nanjing Novizan Pharmaceutical Co., Ltd. The specific steps are as follows:

[0027] (1) Use liquid nitrogen to grind tiger nut leaf samples thoroughly. Take an appropriate amount of the ground sample and add 500 μL of preheated Buffer PRL at 65℃ (add 5% β-mercaptoethanol before use). Immediately vortex vigorously for 30-60 seconds to fully lyse the sample.

[0028] (2) Incubate the lysate in a water bath at 65°C for 5 min, inverting it 1-2 times during the process to aid in lysis, and centrifuge at 12000 rpm for 10 min.

[0029] (3) Transfer the supernatant to a new 1.5 mL RNase-free centrifuge tube, add 0.5 times the volume of anhydrous ethanol to the supernatant, and immediately mix by pipetting.

[0030] (4) Transfer the above mixture to FastPure gDNA-Filter Column II, centrifuge at 12000 rmp for 2 min, and discard the filtrate.

[0031] (5) Place FastPure gDNA-Filter Column II into a new Collection Tubes 2 mL, add 500 μL Buffer PRLPlus, centrifuge at 12000 rmp for 30 sec, and collect the filtrate.

[0032] (6) Add 0.5 times the volume of anhydrous ethanol to the filtrate and immediately mix by blowing.

[0033] (7) Transfer the above mixture to FastPure RNA Column IV, centrifuge at 12000 rmp for 2 min, and discard the filtrate.

[0034] (8) Add 700 μL of Buffer PRW1 to FastPure RNA Column IV, incubate at room temperature for 1 min, centrifuge at 12000 rmp for 30 sec, and discard the filtrate.

[0035] (9) Add 500 μL of Buffer PRW2 to FastPure RNA Column IV, centrifuge at 12000 rmp for 30 sec, and discard the filtrate.

[0036] (10) Repeat step 9.

[0037] (11) Place the FastPure RNA Column IV adsorption column back into the collection tube, centrifuge at 12000 rmp for 2 min to remove the residual Buffer PRW2 in FastPure RNA Column IV.

[0038] (12) Transfer FastPure RNA Column IV to a new RNase-free Collection Tube 1.5 mL centrifuge tube, add 30-100 μL of RNase-free ddH2O to the center of the adsorption column membrane, incubate at room temperature for 2 min, centrifuge at 12000 rmp for 1 min to obtain RNA.

[0039] Purity was determined using agarose gel electrophoresis, such as... Figure 1 As shown, 28S rRNA and 18S rRNA are clearly visible and bright, with 28S rRNA being approximately twice as bright as 18S rRNA, indicating that RNA extraction was complete.

[0040] 2. Synthesis of the first strand of cDNA

[0041] RNA stored at -80℃ was reverse transcribed according to the instructions of the reverse transcription kit. The reaction system is shown in Table 1. The cDNA after reverse transcription was stored at -20℃ for later use.

[0042] surface reverse transcription reaction system

[0043] reagents Usage template RNA 1 μg <![CDATA[MonScript TM 5Xrtiii All-in-One Mix]]> 4 μL <![CDATA[MonScript TM dsDNase]]> 1 μL Nuclease-Free Water To 20 μL

[0044] Reverse transcription reaction conditions

[0045] 37℃ for 2 min

[0046] 55℃ for 15 min

[0047] 85℃ for 5 min

[0048] The sample was cooled on ice to obtain cDNA for subsequent reactions and then frozen at -20°C for later use.

[0049] Example 2: Cloning of the coding region sequence of the CePP2C19 gene in tiger nuts

[0050] Using cDNA from tiger nut leaves under drought stress as a template, specific primers were designed for RT-PCR amplification. Primers were cloned as follows:

[0051] CePP2C19F: ATGGCGGAGGTTTGTTGCG

[0052] CePP2C19R: TTACAATCTTCTAGCTC

[0053] cDNA from tiger nut leaves under drought stress was used as a template for amplification. The RT-PCR amplification reaction system is shown in Table 2. The amplified product was ligated into the pMD19-T cloning vector and transformed into DH5α competent Escherichia coli. Single colonies were picked and subjected to bacterial PCR amplification. A clear and bright band was obtained at 1146 bp by agarose gel electrophoresis, indicating that the cloning vector was successfully ligated. Figure 2 )

[0054] Table 2 RT-PCR reaction system

[0055] reagents Usage template cDNA 1 μL Forward Primer 1 μL Reverse Primer 1 μL 10x TransTaq-T Buffer 5 μL 2.5 mM dNTPs 4 μL

[0056] Continued table

[0057] reagents Dosage Trans Taq-T DNA Polymerase 0.5 μL Nuclease-free Water 37.5 μL

[0058] Example 3 Construction of pTRV2-CePP2C19 recombinant expression vector and Agrobacterium transformation

[0059] pTRV2 is a gene silencing vector mediated by TRV virus. Homologous recombination primers were designed using the upstream and downstream sequences of the EcoRI and BamHI restriction sites in the pTRV2 vector and the CePP2C19 gene cloning primers.

[0060] Homologous recombination primers:

[0061] pTRV2-CePP2C19F: gtgagtaaggttaccgaattcTATCTGAAGCCATACGTGATCC

[0062] pTRV2-CePP2C19R:

[0063] cgtgagctcggtaccggatccGTTATCGGCGCTCTGCTTTC

[0064] After double digestion of the pTRV2 vector with the two enzymes mentioned above, the product was purified into DNA. The target fragment was ligated to the linearized vector using a single-fragment seamless cloning kit (Mona Biotechnology Co., Ltd.). The ligation product was transformed into *E. coli*, and positive selection was performed using antibiotic plates containing kanamycin (50 mg / L). Single clones were picked and cultured for identification by PCR. The target band size was 1146 bp. Figure 3 This demonstrates the successful construction of the expression vector, named pTRV2-CePP2C19, which was then transformed into Agrobacterium. The specific transformation steps are as follows:

[0065] (1) Take competent Agrobacterium cells stored at -80°C, add 1 μg of plasmid DNA when they are about to thaw, mix well, and then place them on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0066] (2) Add 700 μL of antibiotic-free YEP liquid medium and incubate at 28°C with shaking for 2-3 h.

[0067] (3) Centrifuge at 6000 rpm for 1 min to collect the bacteria, discard 600 μL of supernatant, keep about 100 μL of supernatant, gently resuspend the bacterial block by blowing and spreading it onto YEP solid medium containing kanamycin and rifampicin resistance.

[0068] (4) Incubate upside down in a 28 ℃ incubator for 2-3 days.

[0069] (5) Pick a single colony from the culture medium and inoculate it into resistant YEP liquid medium. Incubate overnight at 28°C and 180 rpm.

[0070] (6) Perform PCR amplification and detection on the bacterial culture. After PCR, analyze the culture by 1% agarose gel electrophoresis, select the bacterial culture with the correct target fragment size, store it in glycerol, label it and store it in a -80℃ refrigerator.

[0071] Example 4: Drought tolerance analysis of CePP2C19 gene-silenced lines under drought stress

[0072] (1) Place tiger nut seeds in a 37℃ incubator to germinate, select tiger nut seedlings with uniform growth and place them in hydroponic solution for cultivation, and inject Agrobacterium tumefaciens solution after 10 days.

[0073] (2) Place the Agrobacterium bacterial suspensions of pTRV1, pTRV2, and pTRV2-CePP2C19 in a shaker at 28°C and shake until OD600 Values ​​range from 0.8 to 1.0.

[0074] (3) Centrifuge at 5000 rpm for 10 min, collect the bacterial cells, resuspend the bacterial cells in a buffer containing 10 mM MgCl2, 10 mM MES, and 200 μM AS, and adjust to OD. 600 Up to 0.8-1.0.

[0075] (4) Mix pTRV1 resuspension with pTRV2 and pTRV2-CePP2C19 resuspension at a ratio of 1:1, let stand at room temperature for 3 h, and then inject into tiger pea leaves.

[0076] (5) After 10 days, drought stress was applied using 30% PEG 6000. The phenotype of the strains showed obvious changes compared with the control strains, and the results were photographed.

[0077] like Figure 4 As shown, compared with the control line, the leaves of the CePP2C19 gene-silenced line under drought stress were more wilted and curled, as... Figure 5 As shown, under drought stress, the control line had a fresh weight of about 0.4 g higher, a dry weight of about 0.02 g higher, and a water content of about 0.04% higher than the CePP2C19 gene-silenced line, indicating that the expression of the CePP2C19 gene can affect the drought resistance of plants.

[0078] Example 5: Construction of the pCAMBIA3301-CePP2C19 recombinant vector and Agrobacterium transformation

[0079] Homologous recombination primers were designed using the upstream and downstream sequences of the Bgl II and BstE II restriction sites in the pCAMBIA3301 vector and the CePP2C19 gene cloning primers.

[0080] Homologous recombination primers:

[0081] pCAMBIA3301-CePP2C19:

[0082] gtcttgaccatggtagatcttATGGGGAGAGGAAGGGTGC

[0083] pCAMBIA3301-CePP2C19:

[0084] cgggaaattcgagctggtcaccTTATTGGTTAATGTGACG

[0085] The pCAMBIA3301 vector was double-digested with the two enzymes mentioned above, and the DNA was purified. The target fragment was ligated to the linearized vector using a single-fragment seamless cloning kit (Mona Biotechnology Co., Ltd.). The ligation product was transformed into E. coli, and positive selection was performed using antibiotic plates containing kanamycin (50 mg / L). Single clones were picked and cultured for identification by PCR. Figure 6 The target band was the correct size and bright, proving that the expression vector was successfully constructed. It was named pCAMBIA3301-CePP2C19 and transformed into Agrobacterium according to the Agrobacterium transformation steps (same as in Example 4).

[0086] Example 6: Transformation of tiger nuts (CePP2C19 gene) into Arabidopsis thaliana and phenotypic analysis and physiological index determination of Arabidopsis thaliana under drought stress

[0087] The constructed pCAMBIA3301-CePP2C19 recombinant expression vector was transformed into Arabidopsis thaliana via inflorescence infection. Homozygous T3 individuals were obtained through Basta screening. The T3 generation Arabidopsis were then subjected to drought stress, and phenotypic changes were observed. Figure 7 As shown, after drought stress, compared with the leaves of the overexpressing Arabidopsis thaliana lines (OE1, OE2), the leaves of the control line (WT) were wilted and yellowed, with a water content reduced to approximately 0.84%. However, compared with the mutant Arabidopsis thaliana line (pp2c19), the control line showed less yellowing and wilting, indicating that overexpression of the CePP2C19 gene makes Arabidopsis thaliana more drought-tolerant. Malondialdehyde (MDA) content can reflect the degree of damage to plant cell membranes. Detecting MDA content in Arabidopsis thaliana leaves, such as... Figure 8 As shown, under drought stress, the malondialdehyde (MDA) content of the OE1 and OE2 lines was lower than that of the root mean maximal (WT), while the MDA content of the pp2c19 mutant line was 1.3 times that of the WT, indicating that the OE1 and OE2 lines suffered less damage from drought stress. Key indicators reflecting the degree of drought stress on plants, such as dry weight, fresh weight, and water content, were measured. Figure 9 As shown, under drought stress, the dry weight, fresh weight, and water content of the OE1 and OE2 lines were all higher than WT, with the dry weight being about 0.03g higher than WT, the fresh weight being about 0.5g higher than WT, and the water content being about 0.02% higher than WT. In contrast, the dry weight, fresh weight, and water content of the pp2c19 mutant line were all lower than WT, with the dry weight being about 0.03g lower than WT, the fresh weight being about 0.4g lower than WT, and the water content being about 0.02% lower than WT. These results combined indicate that the CePP2C19 gene can enhance the drought resistance of plants.

Claims

1. The CePP2C19 gene of tiger nuts, its base sequence is shown in SEQ ID NO.1 of the sequence listing.

2. The method for preparing the CePP2C19 gene from tiger nuts according to claim 1, comprising: RNA from tiger nut leaves subjected to drought stress for 12 h was reverse transcribed into cDNA. The cDNA was then used as a template for PCR amplification with primers: CePP2C19F:ATGGCGGAGGTTTGTTGCG CePP2C19R: TTACAATCTTCTAGCTC.

3. A recombinant plant expression vector, wherein the gene shown in SEQ ID NO.1 is inserted into the plant expression vector.

4. A recombinant plant expression vector according to claim 3, characterized in that: The plant expression vector is pCAMBIA3301.

5. The application of the CePP2C19 gene of tiger nuts as described in claim 1 in improving plant drought resistance; The improvement in plant drought resistance is achieved by overexpressing the CePP2C19 gene in tiger nuts. The plant in question is tiger nut or Arabidopsis thaliana.

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