Poney CwPHL7 gene, cloning method, expression vector and application

Abstract

The invention relates to the technical field of gene engineering, in particular to a trifoliate orange CwPHL7 gene, a cloning method, an expression vector and application. According to the invention, a CwPHL7 gene high-expression arabidopsis thaliana transgenic plant is successfully obtained through cloning of the trifoliate CwPHL7 gene and construction and transformation of an over-expression vector, and drought stress treatment experiments find that the chlorophyll content, malondialdehyde (MDA) content, active oxygen accumulation and other key physiological indexes of the transgenic plant under drought conditions are all significantly superior to those of a non-transgenic plant, so that the CwPHL7 gene has a good application prospect. The Poncirus trifoliate CwPHL7 gene can significantly enhance the drought tolerance of plants, and is a potential drought-resistant breeding gene resource. The invention not only provides new experimental data for plant stress resistance gene function research, but also provides efficient technical means and gene resources for crop drought resistance genetic improvement, and has important theoretical significance and application value.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a Citrus wilsonii Tanaka CwPHL7 gene, a cloning method, an expression vector and an application thereof. Background Art

[0002] Citrus wilsonii Tanaka, also known as Xiangyuan, is a plant of the genus Citrus in the Rutaceae family and is a unique local hybrid variety in southern Shaanxi. Citrus wilsonii Tanaka shows significant resistance to iron deficiency chlorosis in production, which is superior to the widely used Poncirus trifoliata rootstock at present, and is an important excellent rootstock resource in southern Shaanxi. At the same time, Citrus wilsonii Tanaka is also an important citrus medicinal plant. Its green fruits are sliced and dried for treating cough, inflammation, excessive phlegm and other symptoms, and it is one of the sources of Citrus medica L. in the Chinese Pharmacopoeia, with extremely high economic and medicinal values. In addition, Citrus wilsonii Tanaka also shows significant stress resistance traits such as drought resistance and salt tolerance, providing valuable resources for exploring stress resistance genes.

[0003] With the intensification of global climate change, abiotic stresses such as drought and salinity have become the main factors restricting crop yields. Using genetic engineering technology to explore plant drought resistance-related genes and improve crop stress resistance is an important way to ensure food security. At present, multiple stress resistance-related genes have been cloned from model plants such as Arabidopsis thaliana and Oryza sativa, and it has been confirmed that their overexpression can significantly improve the drought resistance and salt tolerance of plants. Therefore, studying the drought resistance genes of Citrus wilsonii Tanaka not only has important value for improving the quality and yield of citrus under seasonal stress, but also provides a reference for drought resistance breeding and drought resistance mechanism analysis of Citrus wilsonii Tanaka. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Citrus wilsonii Tanaka CwPHL7 gene, a cloning method, an expression vector and an application thereof in view of the above-mentioned deficiencies of the prior art. The CwPHL7 gene is cloned from Citrus wilsonii Tanaka, and an efficient plant expression vector pK7WG2D-CwPHL7 is constructed by combining two rounds of PCR amplification with Gateway technology, and transgenic plants are obtained by Agrobacterium-mediated transformation of Arabidopsis thaliana.

[0005] The present invention provides a Citrus wilsonii Tanaka CwPHL7 gene, and the nucleotide sequence of the Citrus wilsonii Tanaka CwPHL7 gene is as shown in SEQ ID NO:1.

[0006] The present invention provides a cloning method for the above-mentioned Citrus wilsonii Tanaka CwPHL7 gene, comprising the following steps:

[0007] S1. Using trifoliate orange - sparrowhawk leaf as the material, extract total RNA with an RNA extraction kit. Using the extracted total RNA as a template, synthesize cDNA with the Evo M - MLV reverse transcription kit;

[0008] S2. Design primers for amplifying the full - length CDS of the CwPHL7 gene. The upstream and downstream primers are CwPHL7 - F and CwPHL7 - R respectively. Using cDNA as a template, perform PCR amplification with the primers to obtain the trifoliate orange - sparrowhawk CwPHL7 gene.

[0009] According to the cloning method of the trifoliate orange - sparrowhawk CwPHL7 gene provided by the present invention, the nucleotide sequence of CwPHL7 - F in S2 is shown as SEQ ID NO:2, and the nucleotide sequence of CwPHL7 - R is shown as SEQ ID NO:3.

[0010] According to the cloning method of the trifoliate orange - sparrowhawk CwPHL7 gene provided by the present invention, the reaction system of the PCR amplification in S2 is: 2×SYBR Green Pro Taq HS Premix 10μL, cDNA 2μL, CwPHL7 - F 0.5μL, CwPHL7 - R 0.5μL, ddH2O 7μL;

[0011] The reaction program of the PCR amplification is: 95°C for 5 min; 95°C for 30 s, 54°C for 30 s, 72°C for 80 s (35 cycles); 72°C for 10 min.

[0012] The present invention provides a recombinant plant expression vector of the above - mentioned trifoliate orange - sparrowhawk CwPHL7 gene. The trifoliate orange - sparrowhawk CwPHL7 gene is inserted into the plant expression vector pK7WG2D by Gateway cloning technology to construct the expression vector pK - CwPHL7.

[0013] The present invention also provides an application of the above - mentioned trifoliate orange - sparrowhawk CwPHL7 gene. Over - expression of the trifoliate orange - sparrowhawk CwPHL7 gene can significantly improve the drought resistance of plants.

[0014] The present invention has the following advantages compared with the prior art:

[0015] Through the cloning, overexpression vector construction and transformation of the CwPHL7 gene of Poncirus trifoliata×Citrus sinensis, Arabidopsis transgenic plants with high expression of the CwPHL7 gene were successfully obtained. Through drought stress treatment experiments, it was found that the key physiological indicators of transgenic plants, such as chlorophyll content, malondialdehyde (MDA) content, and reactive oxygen species accumulation, were significantly better than those of non-transgenic plants under drought conditions. The CwPHL7 gene of Poncirus trifoliata×Citrus sinensis can significantly enhance the drought tolerance of plants and is a potential gene resource for drought-resistant breeding. This invention not only provides new experimental data for the study of plant stress-resistant gene functions but also provides efficient technical means and gene resources for crop drought-resistant genetic improvement, with important theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the cloning verification result of the CwPHL7 gene;

[0017] Figure 2 is the construction and PCR detection of the pK-CwPHL7 overexpression vector;

[0018] Figure 3 is the PCR identification of CwPHL7 overexpressing Arabidopsis thaliana;

[0019] Figure 4 is the comparison of morphological characteristics of CwPHL7 overexpressing Arabidopsis thaliana;

[0020] Figure 5 is the morphological characteristic analysis of CwPHL7 overexpressing Arabidopsis thaliana after drought stress;

[0021] Figure 6 is the determination of leaf physiological indexes of WT and CwPHL7 overexpressing Arabidopsis thaliana after drought stress;

[0022] Figure 7 is the histological chemical staining of leaves of WT and CwPHL7 overexpressing Arabidopsis thaliana under drought stress. DETAILED DESCRIPTION OF THE INVENTION

[0023] Example 1

[0024] This example provides a CwPHL7 gene of Poncirus trifoliata×Citrus sinensis, and the nucleotide sequence of this gene is shown in SEQ ID NO:1;

[0025] This example also provides a cloning method for the above-mentioned CwPHL7 gene of Poncirus trifoliata×Citrus sinensis:

[0026] S1. Total RNA extraction and cDNA synthesis

[0027] The total RNA of Zhique leaves was extracted using the RNA-prep pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit, and the purity of the RNA was detected. Using the extracted RNA as a template, the first strand of cDNA was synthesized according to the instructions of the Evo M-MLV Reverse Transcription Kit and stored at -20°C for later use;

[0028] PCR Amplification of S2 and CwPHL7 Genes

[0029] Primers for amplifying the full-length CDS of the CwPHL7 gene were designed. The nucleotide sequence of the upstream primer CwPHL7-F is shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer CwPHL7-R is shown in SEQ ID NO:3;

[0030] The CwPHL7 gene was obtained by PCR amplification, and its nucleotide sequence is shown in SEQ ID NO:1;

[0031] The reaction system for the above PCR amplification was: 2×SYBR Green Pro Taq HS Premix

[0032] 10 μL, cDNA 2 μL, CwPHL7-F 0.5 μL, CwPHL7-R 0.5 μL, ddH2O

[0033] 7 μL, for a total of 20 μL;

[0034] The reaction program for the above PCR amplification was: 95°C for 5 min; 95°C for 30 s, 54°C for 30 s, 72°C for 80 s (35 cycles); 72°C for 10 min;

[0035] S3, Detection and Purification of PCR Products

[0036] The obtained PCR products were subjected to 1% agarose gel electrophoresis (120 V, 30 min). The results observed by the gel imaging system showed that the target band was approximately 776 bp, as Figure 1 shown in A below. It is the agarose gel electrophoresis diagram of the PCR amplification products. The target band was cut out, and the PCR products were purified using the Gel DNA Recovery Kit;

[0037] S4, Ligation of pTOPO Vector and Transformation of Escherichia coli

[0038] The purified PCR products were ligated with the pTOPO vector to obtain ligation products. The reaction system for ligation was: recovered PCR products 0.5 μL, pTOPO-TA / Blunt Simple Vector 1 μL, 10×Enhancer 1 μL, ddH2O 7.5 μL;

[0039] After connection at room temperature for 5 min, take 2 μL of the ligation product and add it to 100 μL of Escherichia coli DH5α competent cells. Incubate on ice for 30 min, perform heat shock at 42 °C for 45 sec, incubate on ice for 5 min, add 700 μL of antibiotic-free LB liquid medium, and culture with shaking at 37 °C and 200 rpm for 1 h. Take 100 μL of the bacterial solution and spread it on an LB plate containing 100 μg / mL ampicillin, and incubate it upside down at 37 °C for 16 h;

[0040] S5. Screening of positive clones and sequencing verification

[0041] Pick 5 single colonies and suspend them separately in 20 μL of sterile water. Take 2 μL as a template for colony PCR verification;

[0042] The colony PCR primers are the universal vector primers M13-F / R. The nucleotide sequence of M13-F is shown in SEQ ID NO:4, and the nucleotide sequence of M13-F is shown in SEQ ID NO:5;

[0043] The reaction program for the above PCR verification is: 95 °C for 5 min; 95 °C for 30 s, 50 °C for 30 s, 72 °C for 70 s (35 cycles); 72 °C for 10 min;

[0044] The results of electrophoretic detection of positive clone colonies are as shown in Figure 1 shown in B. Inoculate the positive colonies into 3 mL of LB liquid medium containing 100 μg / mL ampicillin, and culture with shaking at a temperature of 37 °C and a rotation speed of 200 rpm for 12 h. Take 500 μL of the bacterial solution and send it to the company for sequencing. Add 60% glycerol to the remaining bacterial solution at a ratio of 2:1 and store it at -80 °C;

[0045] Example 2

[0046] This example provides a method for constructing an overexpression vector of the CwPHL7 gene of trifoliate orange, which specifically includes the following steps:

[0047] S1. Design of PCR primers and two rounds of amplification

[0048] Based on the Gateway cloning technology, specific primers containing attB adapters were designed, including attB1-CwPHL7-F, attB1-CwPHL7-R, Adapter-attB1-F, and Adapter-attB1-R. The nucleotide sequence of attB1-CwPHL7-F is shown in SEQ ID NO:6, the nucleotide sequence of attB1-CwPHL7-R is shown in SEQ ID NO:7, the nucleotide sequence of Adapter-attB1-F is shown in SEQ ID NO:8, and the nucleotide sequence of Adapter-attB1-R is shown in SEQ ID NO:9. The CwPHL7 gene was amplified by two rounds of PCR;

[0049] For the first-round PCR amplification system, the pTOPO-CwPHL7 recombinant plasmid was used as a template, and primers attB1-CwPHL7-F and attB1-CwPHL7-R were used for amplification;

[0050] The reaction system for the first-round PCR amplification was: P525Mix 10 μL, bacterial solution 2 μL, attB1-CwPHL7-F 0.5 μL, attB1-CwPHL7-R 0.5 μL, and ddH2O 7 μL;

[0051] The reaction program for the first-round PCR amplification was: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min (35 cycles); 72°C for 10 min;

[0052] For the second-round PCR, the product of the first-round PCR was used as a template, and primers Adapter-attB1-F and Adapter-attB1-R were used for amplification to introduce a complete attB adapter;

[0053] The reaction system for the second-round PCR amplification was: P525Mix 10 μL, bacterial solution 2 μL, Adapter-attB1-F 0.5 μL, Adapter-attB1-R 0.5 μL, and ddH2O 7 μL;

[0054] The reaction program for the second-round PCR amplification was the same as that of the first-round PCR;

[0055] S2. BP reaction to construct an entry vector

[0056] The product of the second-round PCR was mixed with the pDONR221 vector. The mixing system included: 1 μL of the product of the second-round PCR, 0.4 μL of pDONR221, 0.6 μL of BP clonase, and 1 μL of ddH2O. The agarose gel electrophoresis pattern of the product of the second-round PCR is shown as Figure 2 shown in C;

[0057] Perform the BP reaction: React in a metal bath at 25 °C for 4 hours to construct the entry vector pD-CwPHL7. The construction results are as Figure 2 shown in A below;

[0058] S3. Transformation and verification of the entry vector

[0059] Use the freeze-thaw method to transform the constructed entry vector pD-CwPHL7 into Escherichia coli DH5α competent cells. Incubate on ice for 30 min, heat shock at 42 °C for 45 sec, incubate on ice for 5 min, add 700 μL of antibiotic-free LB liquid medium, shake culture at 37 °C and 200 rpm for 1 h. Take 100 μL of the bacterial solution and spread it on an LB plate containing 50 μg / mL kanamycin. Incubate inverted at 37 °C for 16 h. The PCR verification primers are attB1-CwPHL7-F and attB1-CwPHL7-R;

[0060] S4. Construct the expression vector by LR reaction

[0061] Extract the entry vector pD-CwPHL7 and mix it with the plant expression vector pK7WG2D. The mixing system includes: 1 μL of pD-CwPHL7, 0.4 μL of pK7WG2D, 0.6 μL of LR clonase, and 1 μL of ddH2O;

[0062] Perform the LR reaction: React in a metal bath at 25 °C for 4 hours to construct the expression vector pK7WG2D-CwPHL7 (denoted as pK-CwPHL7). The construction results of the expression vector are as Figure 2 shown in B below;

[0063] S5. Transformation and verification of the expression vector

[0064] Transform the LR reaction product into Escherichia coli DH5α, pick a single colony from the plate for colony PCR verification. The colony PCR detection results of DH5α-pD-CwPHL7 are as Figure 2 shown in D below, and the colony PCR detection results of DH5α-pK-CwPHL7 are as Figure 2 shown in E below. The bacterial solution with positive verification is sent to the company for sequencing. The sequencing primers are Adapter-attB1-F and Adapter-attB1-R;

[0065] S6. Transform Agrobacterium tumefaciens GV3101

[0066] Extract the recombinant plasmid pK-CwPHL7 and transform it into Agrobacterium tumefaciens GV3101. The specific steps are as follows:

[0067] S61. Take 1 μg of the plasmid and add it to 100 μL of competent cells, incubate on ice for 30 min;

[0068] S62. Quick-freeze in liquid nitrogen for 5 min, water bath at 37 °C for 5 min, and ice bath for 2 min;

[0069] S63. Add 700 μL of LB liquid medium and incubate with shaking at 28 °C for 3 h;

[0070] S64. Spread on an LB plate containing 50 μg / mL spectinomycin and culture at 28 °C for 48 h to obtain GV3101-pK-CwPHL7 colonies;

[0071] S65. Pick a single colony for colony PCR verification. The colony PCR detection results of GV3101-pK-CwPHL7 are as Figure 2 shown in F below. Add 60% glycerol to the positively verified bacterial liquid and store it at -80 °C.

[0072] Example 3

[0073] This example provides the identification of the drought resistance function of transgenic Arabidopsis thaliana.

[0074] Genetic transformation of Arabidopsis thaliana

[0075] S1. Activation of bacterial strain

[0076] Pick single colonies of Agrobacterium tumefaciens GV3101 containing the empty vector pK7WG2D and the recombinant expression vector pK-CwPHL7, and inoculate them into 3 mL of LB liquid medium containing 50 μg / mL spectinomycin respectively. Incubate with shaking at 28 °C and 200 rpm for 16 h for the first activation. Take 1 mL of the bacterial liquid and transfer it to 50 mL of LB liquid medium containing 50 μg / mL spectinomycin, and continue to culture at 28 °C and 200 rpm until OD 600 = 0.8 - 1.0 for the second activation;

[0077] S2. Treatment of bacterial cells

[0078] Centrifuge the bacterial liquid at 5000×g for 10 min at room temperature to collect the bacterial cells, and resuspend them with an infection solution containing 25 mL of 10×MS macroelements, 2.5 mL of VB, 25 g of sucrose, 1 g of MgCl2, 200 μL of Silwet-77, and make up the volume to 500 mL with ddH2O, and the pH value is 5.8 until OD 600 = 0.6 - 0.8, and let it stand at room temperature for 2 h;

[0079] S3. Inflorescence infiltration

[0080] Select wild-type Arabidopsis thaliana Columbia-0 that has grown to the bolting stage, remove the already opened flowers and pods, immerse the inflorescence in the infiltration solution for 30 - 60 s, gently shake off the excess liquid, cover with plastic wrap to keep moist, culture in the dark for 24 h, then resume normal lighting of 16 h light / 8 h dark and a constant temperature of 22 °C. Repeat the infection once after 7 d, and collect the T1 generation seeds after the seeds mature.

[0081] Screening and Identification of Transgenic Arabidopsis thaliana

[0082] S1. Resistance Screening

[0083] Surface sterilize the T1 generation seeds with 75% ethanol for 1 min, 10% NaClO for 5 min, rinse with sterile water 5 times, sow on MS solid medium containing 50 μg / mL kanamycin, vernalize at 4 °C for 3 d, then transfer to a light culture room at 22 °C. After 14 d, observe the green fluorescent protein (GFP) signal through a fluorescence microscope, and screen positive seedlings and transplant them into nutrient soil (peat soil: vermiculite = 3:1).

[0084] S2. PCR Verification

[0085] Extract the genomic DNA of T2 generation Arabidopsis thaliana leaves, and perform PCR verification using vector-specific primers. The vector-specific primers include pKPH-F and pKPH-R. The nucleotide sequence of pKPH-F is as shown in SEQ ID NO:10, and the nucleotide sequence of pKPH-R is as shown in SEQ ID NO:11.

[0086] The reaction system for the above PCR verification is: 2×Phanta Max Master Mix (Dye Plus) 25 μL, leaf DNA 1 μL, pKPH-F 2 μL, pKPH-R 2 μL, and ddH2O 20 μL;

[0087] The reaction program for the above PCR verification is: 95 °C for 3 min; 95 °C for 15 s, 60 °C for 15 s, 72 °C for 1 min (35 cycles); 72 °C for 5 min;

[0088] Electrophoresis detection shows that the target band is as Figure 3 shown, and the T2 generation transgenic plants are obtained. Figure 3 In it, 1 is WT, 2 - 3 are Arabidopsis thaliana transformed with pK, and 4 - 10 are Arabidopsis thaliana transformed with pK-CwPHL7.

[0089] Analysis of Drought Resistance Function of Transgenic Arabidopsis thaliana

[0090] S1. Morphological Observation

[0091] Wild-type (WT), empty vector control (pK), and overexpression line (pK-CwPHL7) Arabidopsis thaliana were transplanted into nutrient soil. After 30 days, the rosette leaf morphology, rosette diameter, number of rosette leaf layers, leaf length, and leaf width of the largest leaf were observed. Three biological replicates were set for each group, and data analysis was performed using SPSS 22.0 software.

[0092] S2. Drought stress experiment

[0093] S21. Osmotic stress treatment: Seven-day-old seedlings were transferred to MS medium containing 0, 50, or 100 mM mannitol and cultured for an additional 15 days. Root length, fresh weight, and leaf wilting degree were observed.

[0094] S22. Natural drought treatment: Four-week-old plants were divided into a control group watered every 7 days and a treatment group with watering stopped. After 20 days, relative electrical conductivity, chlorophyll content, malondialdehyde (MDA) content, and reactive oxygen species accumulation were measured: NBT (O2 -. ) and DAB (H2O2) histochemical staining.

[0095] Through morphological comparison and physiological index determination of wild-type (WT), empty vector control (pK), and Arabidopsis thaliana overexpressing CwPHL7 (pK-CwPHL7) grown for 30 days, the results showed that:

[0096] Compared with WT, the rosette diameter of pK-CwPHL7 Arabidopsis thaliana increased by 47%, the number of rosette leaf layers increased by 92%, the leaf width increased by 30%, and the leaf length of rosette leaves increased by 54%. As Figure 4 shown, Figure 4 in which A is the comparison of rosette diameter, B is the comparison of the number of rosette leaf layers, C is the comparison of leaf width, and D is the comparison of leaf length. Different letters in the figure indicate significant differences, P < 0.05;

[0097] Under 50 mM and 100 mM mannitol treatments, the primary root length of pK-CwPHL7 plants increased by 74% and 35% compared with WT, respectively. As Figure 5 shown in A, it is the analysis of the primary root length of Arabidopsis thaliana;

[0098] At 100 mM treatment, the lateral root length of pK-CwPHL7 increased by 62% compared with WT. As Figure 5 shown in B, it is the analysis of the average lateral root length of Arabidopsis thaliana;

[0099] After 20 days of drought stress, the relative water content and chlorophyll content of the leaves of pK-CwPHL7 Arabidopsis thaliana increased by 41% and 57% compared with WT, respectively. As Figure 6 shown in A and B, A is the relative water content of the leaves; B is the chlorophyll content of the leaves; the MDA content and relative electrical conductivity decreased by 35% and 42% compared with WT, respectively. As Figure 6As shown in C and D, C is the MDA content of leaves; D is the relative electrical conductivity of leaves;

[0100] NBT and DAB staining showed that the accumulation level of reactive oxygen species (ROS) in WT leaves was significantly higher than that in pK-CwPHL7 plants, as Figure 7 shown;

[0101] Overexpression of the CwPHL7 gene significantly promoted the growth of Arabidopsis, including rosette expansion and leaf enlargement, and enhanced its tolerance to drought stress, including main root elongation, reduced ROS accumulation, and reduced membrane damage, indicating that this gene has important application value in the genetic improvement of plant drought resistance.

[0102] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent variations made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A CwPHL7 gene of Poncirus trifoliata, characterized in that: The nucleotide sequence of the CwPHL7 gene of the Pteris trifoliata is shown in SEQ ID NO:

1.

2. A method for cloning the CwPHL7 gene of P. trifoliata according to claim 1, characterized in that: The following steps are involved: S1. Using the leaves of Prunus trifoliata as the material, the total RNA was extracted using an RNA extraction kit, and the extracted total RNA was used as a template to synthesize cDNA using the Evo M-MLV reverse transcription kit; S2. Design primers for amplifying the full-length CDS of the CwPHL7 gene. The upstream and downstream primers are CwPHL7-F and CwPHL7-R, respectively. Using cDNA as a template, PCR amplification is performed using the primers to obtain the CwPHL7 gene of the trifoliate orange sparrow.

3. The method for cloning the CwPHL7 gene of P. trifoliata according to claim 2, characterized in that: The nucleotide sequence of CwPHL7-F in S2 is shown in SEQ ID NO:2, and the nucleotide sequence of CwPHL7-R is shown in SEQ ID NO:

3.

4. The method for cloning the CwPHL7 gene of P. trifoliata according to claim 2, characterized in that: The reaction system for PCR amplification described in S2 is: 2×SYBR Green Pro TaqHS Premix 10 μL, cDNA 2 μL, CwPHL7-F 0.5 μL, CwPHL7-R 0.5 μL, ddH2O 7 μL; The reaction procedure of the PCR amplification is: 95°C for 5 min; 95°C for 30 s, 54°C for 30 s, 72°C for 80 s (35 cycles); 72°C for 10 min.

5. A recombinant plant expression vector of the CwPHL7 gene of P. trifoliata according to claim 1, characterized in that: The CwPHL7 gene of the trifoliate orange was inserted into the plant expression vector pK7WG2D by Gateway cloning technology to construct the expression vector pK-CwPHL7.

6. A use of the CwPHL7 gene of Poncirus trifoliata as claimed in claim 1, characterized in that: Overexpression of the CwPHL7 gene of P. trifoliata can significantly improve the drought resistance of plants.

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