Chili heat-resistant gene and application thereof

Abstract

The invention provides a hot pepper heat-resistant gene and application thereof, and relates to the technical field of plant genetic engineering, the hot pepper heat-resistant gene is a PP2C51 gene, and the nucleotide sequence of the hot pepper heat-resistant gene is shown as SEQ ID NO.1. The invention further provides a recombinant vector containing the PP2C51 gene, and the PP2C51 gene and the recombinant vector are applied to breeding for improving heat resistance of plants. According to the invention, functional identification and analysis are carried out on the PP2C51 gene to find that the PP2C51 gene has the function of regulating and controlling the heat resistance of plants, a recombinant vector is prepared, the recombinant vector is applied to the cultivation of new plant heat-resistant varieties, a PP2C51 overexpression plant is successfully screened and cultivated, the plant can resist high-temperature stress, and the growth condition of the plant is obviously superior to that of a common plant; the invention not only deepens a plant high-temperature response mechanism in theory, but also provides a theoretical basis for early identification of heat-resistant cultivation and new heat-resistant varieties of pepper in production practice.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a pepper heat-resistant gene and application thereof. Background Art

[0002] Pepper (Capsicum annuum L.) is one of the important economic crops in the Solanaceae family. It is widely planted around the world and is also one of the most important vegetable crops in China, with the largest planting area and yield among vegetable crops. Pepper has a wide range of application values ​​and can be used as medicine, natural dye and cosmetics, ornamental plants, or as an active ingredient in insect repellents; pepper can provide a variety of vitamins, minerals and nutrients necessary for human health.

[0003] Peppers like warmth but are afraid of heat. The suitable temperature for the seedling stage is 25-30℃. High temperature stress will occur when the temperature exceeds 32℃, and high temperature exceeding 35℃ often causes irreversible physiological damage to their growth. Facility autumn-extended cultivation is a major method of pepper production in my country. Autumn-extended pepper seedlings are usually raised in the hot summer, and the seedling stage often encounters high temperature adversity, which makes pepper seedling raising difficult and the seedling quality low. Breeding pepper varieties with strong heat resistance is one of the effective means to resist high temperature adversity. Therefore, it is necessary to screen and identify heat-resistant germplasm resources and heat-resistant genes of pepper seedlings, and explore the heat-resistant mechanism of seedlings, so as to provide a reference for heat-resistant cultivation and biological breeding of peppers. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies in the prior art, the present invention provides a heat-resistant gene for pepper and its application, which solves the technical problem that the existing pepper varieties cannot withstand high temperature adversity, resulting in difficulty in raising pepper seedlings and low seedling quality.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] The invention provides a heat-resistant gene for pepper, which is a PP2C51 gene, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] The present invention also provides a recombinant vector containing the PP2C51 gene.

[0010] Preferably, the vector is obtained by inserting the PP2C51 gene into the BamHI and XbaI restriction sites of the pCambia1305-35S-nFLAG-cMYC expression vector.

[0011] Preferably, the construction method of the recombinant vector is as follows:

[0012] (1) Using a single cloned fragment, Bam HI and Xba I as double restriction sites, using double restriction linearization to design recombinant primers PP2C51-1305-F and PP2C51-1305-R, using pepper cDNA as a template, PCR amplification was performed to obtain a PCR amplification product, and the obtained PCR amplification product was purified;

[0013] (2) extracting the plasmid of the overexpression vector pCambia1305-35S-nFLAG-cMYC, double-digesting the extracted plasmid with BamHI and XbaI enzymes, and purifying it using a gel recovery kit;

[0014] (3) The purified pCambia1305-35S-nFLAG-cMYC overexpression vector plasmid and the target gene PP2C51 fragment were connected to form a recombinant vector.

[0015] Preferably, in step (1), the nucleotide sequence of primer PP2C51-1305-F is shown as SEQ ID NO.2, and the nucleotide sequence of primer PP2C51-1305-R is shown as SEQ ID NO.3.

[0016] Preferably, the PCR amplification system in step (1) is: 12.5 μL of 2×Phanta Max Master Mix (Dye Plus), 9.5 μL of ddH2O, 1 μL of cDNA, and 1 μL each of PP2C51-1305-F and PP2C51-1305-R;

[0017] The PCR amplification program was 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 1 min, 35 cycles, and 72°C full extension for 5 min.

[0018] Preferably, the double enzyme digestion system in step (2) is: 10×Quiekcut Buffer 5 μL, BamHI quick digester 2.5 μL, XbaI quick digester 2.5 μL, plasmid 15 μL, ddH2O 25 μL.

[0019] The present invention also provides the use of the PP2C51 gene or recombinant vector in breeding for improving plant heat tolerance.

[0020] Preferably, the specific standard of heat resistance is 42±1°C, and the plant is pepper or Arabidopsis thaliana.

[0021] Preferably, the method for cultivating heat-resistant plants comprises the following steps:

[0022] (1) Transform the pCambia1305-35S-PP2C51-nFLAG-cMYC overexpression vector plasmid into GV3101 Agrobacterium;

[0023] (2) Using two pairs of primers, PP2C51-1305-F and PP2C51-1305-R, and PP2C51-1305-F and 1305R, the Agrobacterium culture obtained in the above steps was used as a template for PCR amplification verification;

[0024] (3) resuscitating the Agrobacterium bacterial solution and preparing a resuspension with a buffer solution, and infecting the Arabidopsis thaliana inflorescence using an inflorescence infection method to obtain infected T0 generation Arabidopsis thaliana seeds;

[0025] (4) T0 generation Arabidopsis seeds were sown on 1 / 2MS agar plates containing 50 ug / mL hygromycin B to select overexpressing Arabidopsis plants, and the selected transgenic plants were sown again on 1 / 2MS agar plates containing 50 ug / mL hygromycin B until T3 generation transgenic plants were selected.

[0026] (III) Beneficial effects

[0027] The present invention provides a heat-resistant gene for pepper and its application. Compared with the prior art, it has the following beneficial effects:

[0028] 1. The present invention identified the function of the PP2C51 gene and found that it has the function of regulating plant heat resistance. A recombinant vector was prepared and applied to the cultivation of new heat-resistant plant varieties. PP2C51 overexpressing plants were successfully screened and cultivated. The plants can withstand high temperature adversity and have significantly better growth conditions than ordinary plants.

[0029] 2. The present invention not only deepens the high temperature response mechanism of plants in theory, but also provides a theoretical basis for the heat-resistant cultivation of peppers and the early identification of new heat-resistant varieties in production practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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 or the description of the prior art 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.

[0031] Figure 1 The electrophoresis diagram of the gene PP2C51 and the overexpression plasmid in Example 1 of the present invention;

[0032] Figure 2 This is a diagram showing the culture status of wild-type Arabidopsis thaliana and transgenic plants in a culture dish in Example 2 of the present invention;

[0033] Figure 3 The electrophoresis diagram of the expression level of PP2C51 in wild-type Arabidopsis and T3 transgenic Arabidopsis in Example 3 of the present invention;

[0034] Figure 4 This is a comparison chart of the growth conditions of wild-type Arabidopsis and T3 transgenic Arabidopsis after high temperature treatment in Example 3 of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The embodiments of the present application provide a heat-resistant pepper gene and its application, thereby solving the technical problem that existing pepper varieties cannot withstand high temperature adversity, resulting in difficulty in raising pepper seedlings and low seedling quality.

[0037] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0038] Example 1 - Construction of recombinant vector:

[0039] (1) In order to obtain the gene sequence of PP2C51, CE Design V1.04 software was used to design the recombinant primers PP2C51-1305-F and PP2C51-1305-R by using a single cloning fragment and Bam HI and Xba I as double restriction sites. The cDNA of HDL from red lantern pepper germplasm was used as a template for PCR amplification to obtain the PCR amplification product.

[0040] The PCR amplification system was: 2× Phanta Max Master Mix (Dye Plus) 12.5 μL, ddH2O 9.5 μL, cDNA 1 μL, PP2C51-1305-F and PP2C51-1305-R 1 μL each;

[0041] The PCR amplification program was 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 1 min, 35 cycles, and 72°C full extension for 5 min.

[0042] The nucleotide sequence of primer PP2C51-1305-F is shown in SEQ ID NO.2;

[0043] The nucleotide sequence of primer PP2C51-1305-R is shown in SEQ ID NO.3.

[0044] Table 1 Nucleotide sequences of primers for PP2C51 gene cloning and vector plasmid identification

[0045] Primers Sequence (5'-3') PP2C51-1305-F gacgatgacgataagggatccATGATTGATAACGTTAATAGTGTGCCG PP2C51-1305-R tgcctgcaggtcgactctagaTTATGCTCCGTCAGTGGATGAA

[0046] The obtained PCR amplification product was purified (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and the concentration of the recovered product was measured using a nucleic acid quantifier after purification.

[0047] (2) A plasmid extraction kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) was used to extract the plasmid of the overexpression vector pCambia1305-35S-nFLAG-cMYC according to the instructions, and the extracted plasmid was double-digested with Bam HI and Xba I enzymes.

[0048] The double enzyme digestion system is: 10×Quiekcut Buffer 5μL, BamHI quick digester 2.5μL, XbaI quick digester 2.5μL, plasmid 15μL, ddH2O 25μL.

[0049] Then, the gel recovery kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) was used for purification, and the concentration of the recovered product was measured using a nucleic acid quantifier.

[0050] (3) Using a gene recombination kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), the purified pCambia1305-35S-nFLAG-cMYC vector plasmid and the target gene PP2C51 fragment were connected to form a recombinant vector.

[0051] Example 2—Recombinant vector construction verification

[0052] Verification 1:

[0053] (4) The recombinant vector was transformed into DH5α Escherichia coli (the transformation process was carried out according to the instructions of DH5α Escherichia coli competent cells), and the Escherichia coli culture solution was sequenced. If the sequencing result was the same as the target gene, it indicated that the PP2C51 gene was successfully inserted into the pCambia1305-35S-nFLAG-cMYC vector.

[0054] Verification 2:

[0055] (5) The pCambia1305-35S-PP2C51-nFLAG-cMYC overexpression vector plasmid was transformed into GV3101 Agrobacterium (purchased from Weidi Biotechnology Co., Ltd.);

[0056] (6) Using two pairs of primers, PP2C51-1305-F and PP2C51-1305-R, and PP2C51-1305-F and 1305R, the Agrobacterium culture obtained in step (5) was used as a template for PCR amplification verification;

[0057] The PCR amplification system is: 2×Phanta Max Master Mix (Dye Plus) 12.5 μL, ddH2O 9.5 μL, cDNA (Agrobacterium liquid obtained in step (5)) 1 μL, PP2C51-1305-F and PP2C51-1305-R 1 μL each;

[0058] 2×Phanta Max Master Mix (Dye Plus) 12.5 μL, ddH2O 9.5 μL, cDNA (Agrobacterium culture obtained in step (5)) 1 μL, PP2C51-1305-F and 1305R 1 μL each;

[0059] The PCR amplification program was 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 1 min, 32 cycles, and 72°C full extension for 5 min.

[0060] PCR amplification verification results:

[0061] It was found that the PCR product containing the vector fragment was longer than the PP2C51 fragment ( Figure 1 ), which indicates that the gene PP2C51 has been successfully inserted into the overexpression vector pCambia1305-35S-nFLAG-cMYC.

[0062] Example 3 - Cultivation of overexpressing Arabidopsis plants

[0063] (7) resuscitating the Agrobacterium bacterial solution and preparing it into a resuspension with an OD560 of 0.8-1 using a buffer solution, and infecting the Col-0 Arabidopsis inflorescence using an inflorescence infection method to obtain infected T0 generation Arabidopsis seeds;

[0064] The buffer system is: 1 / 2MS 200mL, sucrose 10g, 50μL Silwet-77, and the pH is adjusted to 5.8.

[0065] (8) T0 generation Arabidopsis thaliana were sown on 1 / 2MS agar plates containing 50 ug / mL hygromycin B (Invitrogen, Carlsbad, California) to select overexpressing Arabidopsis thaliana plants. The selected transgenic plants were sown again on 1 / 2MS agar plates containing 50 ug / mL hygromycin B (Invitrogen, Carlsbad, California) until T3 generation transgenic plants were selected.

[0066] like Figure 2 As shown, the upper part is the wild-type Arabidopsis thaliana, and the lower part is the T3 transgenic plant. It can be seen that only the T3 transgenic plant can grow normally on the 1 / 2MS agar plate containing hygromycin B.

[0067] Example 4—Verification of genetic transformation of transgenic Arabidopsis plants

[0068] (9) PCR was performed on T3 transgenic plants and wild-type Arabidopsis using specific primers (PP2C51-1305-F and PP2C51-1305-R) to detect the expression level of the T3 transgenic plants.

[0069] like Figure 3 As shown, the electrophoresis bands of the PP2C51 gene overexpressing Arabidopsis plants are brighter than those of the wild type and empty vector (water), indicating that pCambia1305-35S-PP2C51-nFLAG-cMYC has been successfully introduced into the Arabidopsis genome and successfully expressed. These results all indicate that the genetic transformation of transgenic Arabidopsis plants is successful, and transgenic plants can be used for subsequent experiments.

[0070] Example 5 - Comparison of heat tolerance between transgenic Arabidopsis plants and wild-type Arabidopsis plants

[0071] (10) WT and OE seedlings were grown on 1 / 2MS plates at 25°C / 18°C, 16h / 8h (day and night), relative humidity of 75%, and light intensity of 15000LUX, and then transplanted into nutrient pots for three weeks. The plants were then moved to an environment with the same conditions at 42±1°C for 13h.

[0072] This experiment used wild-type Arabidopsis and T3 transgenic plants, and the experimental data were repeated three times. After three weeks of normal cultivation and 13 hours of high temperature treatment, the growth status of the plants was as follows: Figure 4 As shown, the leaves of the transgenic plants tended to grow normally, while the leaves of the wild-type plants wilt due to water loss. This shows that the transgenic plants have the ability to resist high temperature adversity at 42±1℃, and their growth is significantly better than that of ordinary plants.

[0073] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0074] 1. The present invention identified the function of the PP2C51 gene and found that it has the function of regulating plant heat resistance. A recombinant vector was prepared and applied to the cultivation of new heat-resistant plant varieties. PP2C51 overexpressing plants were successfully screened and cultivated. The plants can withstand high temperature adversity and have significantly better growth conditions than ordinary plants.

[0075] 2. The present invention not only deepens the high temperature response mechanism of plants in theory, but also provides a theoretical basis for the heat-resistant cultivation of peppers and the early identification of new heat-resistant varieties in production practice.

[0076] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A heat-resistant gene for pepper, characterized in that: It is the PP2C51 gene, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The heat-resistant gene of pepper according to claim 1, characterized in that A recombinant vector containing the PP2C51 gene according to claim 1.

3. The heat-resistant gene of pepper according to claim 2, characterized in that The recombinant vector is obtained by inserting the PP2C51 gene into the BamHI and XbaI restriction sites of the pCambia1305-35S-nFLAG-cMYC overexpression vector.

4. The heat-resistant gene of pepper according to claim 3, characterized in that The construction method of the recombinant vector is as follows: (1) Using a single cloned fragment, Bam HI and Xba I as double restriction sites, using double restriction linearization to design recombinant primers PP2C51-1305-F and PP2C51-1305-R, using pepper cDNA as a template, PCR amplification was performed to obtain a PCR amplification product, and the obtained PCR amplification product was purified; (2) extracting the plasmid of the overexpression vector pCambia1305-35S-nFLAG-cMYC, double-digesting the extracted plasmid with Bam HI and Xba I enzymes, and purifying it using a gel recovery kit; (3) The purified pCambia1305-35S-nFLAG-cMYC vector plasmid and the target gene PP2C51 fragment were connected to form a recombinant vector.

5. The heat-resistant gene of pepper according to claim 4, characterized in that In step (1), the nucleotide sequence of primer PP2C51-1305-F is shown in SEQ ID NO.2, and the nucleotide sequence of primer PP2C51-1305-R is shown in SEQ ID NO.

3.

6. The heat-resistant gene of pepper according to claim 4, characterized in that The PCR amplification system in step (1) is: 12.5 μL of 2×Phanta Max Master Mix (Dye Plus), 9.5 μL of ddH2O, 1 μL of cDNA, and 1 μL each of PP2C51-1305-F and PP2C51-1305-R; The PCR amplification program was 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 1 min, 35 cycles, and 72°C full extension for 5 min.

7. The heat-resistant gene of pepper according to claim 4, characterized in that The double enzyme digestion system in step (2) is: 10×Quiekcut Buffer 5 μL, BamHI quick digester 2.5 μL, XbaI quick digester 2.5 μL, plasmid 15 μL, ddH2O 25 μL.

8. Use of the PP2C51 gene according to claim 1 and the recombinant vector according to any one of claims 2 to 8 in breeding for improving plant heat tolerance.

9. The use according to claim 8, characterized in that The specific standard of heat resistance is 42±1°C, and the plant is pepper or Arabidopsis thaliana.

10. The use according to claim 9, characterized in that A method for cultivating heat-resistant plants comprises the following steps: (1) Transform the pCambia1305-35S-PP2C51-nFLAG-cMYC overexpression vector plasmid into GV3101 Agrobacterium; (2) Using two pairs of primers, PP2C51-1305-F and PP2C51-1305-R, and PP2C51-1305-F and 1305R, the Agrobacterium culture obtained in the above steps was used as a template for PCR amplification verification; (3) resuscitating the Agrobacterium bacterial solution and preparing a resuspension with a buffer solution, and infecting the Arabidopsis thaliana inflorescence using an inflorescence infection method to obtain infected T0 generation Arabidopsis thaliana seeds; (4) T0 generation Arabidopsis seeds were sown on 1 / 2MS agar plates containing 50 ug / mL hygromycin B to select overexpressing Arabidopsis plants, and the selected transgenic plants were sown again on 1 / 2MS agar plates containing 50 ug / mL hygromycin B until T3 generation transgenic plants were selected.

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