Cold-resistant gene BcACL5.2 of black cabbage and application of cold-resistant gene BcACL5.2

Abstract

The invention relates to the technical field of plant genetic engineering, and provides a cold-resistant gene BcACL5.2 of black cabbage, and the nucleotide sequence of the cold-resistant gene BcACL5.2 is as shown in SEQ ID NO.1. The invention also provides an overexpression vector containing the gene and a host cell. Meanwhile, the invention also provides an application of the gene BcACL5.2 in breeding for improving the cold tolerance of plants and a method for improving the cold tolerance of the plants. According to the invention, a key gene BcACL5.2 related to the cold resistance is screened from black cabbage, and the gene is over-expressed in an arabidopsis thaliana plant, so that the positive regulation of the gene on the cold resistance of the plant is verified, and the growth ability of the plant under low-temperature stress can be improved; the invention lays a foundation for cultivating a new plant variety with strong cold resistance, especially a new black cabbage variety, and has important value for expanding a black cabbage planting area and promoting industrial development.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a cold-resistant gene for *Brassica oleracea*. BcACL5.2 And its applications. Background Technology

[0002] black cabbage ( Brassica campestris L. ssp. chinensis var. rosularis Tsen, also known as Chinese cabbage, is a variety of Brassica oleracea subspecies in the Brassicaceae family. It is an important autumn and winter vegetable native to my country and mainly cultivated in the Yangtze-Huaihe River basin. Tsen has soft, juicy leaves that can be stir-fried or used in soups, offering a delicious flavor and rich nutritional value. It is also known as a "vitamin" vegetable because every 100 grams of fresh leaves contains 70 mg of vitamin C, 180 mg of calcium, and abundant minerals such as iron, phosphorus, and magnesium, making it very popular with consumers.

[0003] Although spinach thrives in cool climates with an optimal growth temperature of 15-20℃, lower temperatures are still detrimental to its normal growth and development, especially in the seedling stage, leading to reduced yields. In recent years, the demand for spinach has been increasing, and the cultivation area has been gradually expanding. However, with the intensification of climate change and the frequent occurrence of extreme high and low temperatures, low temperatures have severely restricted the northward expansion of spinach cultivation areas and the development of the industry.

[0004] Breeding cold-resistant varieties of *Brassica juncea* is one of the key means to resist low-temperature stress. Therefore, it is necessary to screen and identify cold-resistant germplasm resources in the seedling stage of *Brassica juncea*, explore cold-resistant genes, and investigate the mechanism of cold resistance in the seedling stage, so as to provide a reference for cold-resistant cultivation and bio-breeding of *Brassica juncea*. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a cold-resistant gene for black spinach. BcACL5.2 And its applications.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A cold-resistant gene in black cabbage BcACL5.2 The BcACL5.2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] As one of the preferred embodiments of the present invention, the BcACL5.2 Genes enhance a plant's ability to grow under low-temperature stress by positively regulating its cold tolerance.

[0008] An overexpression vector containing the aforementioned cold-resistant gene from *Brucea javanica*. BcACL5.2 .

[0009] As one of the preferred embodiments of the present invention, the overexpression vector is pCAMBIA1305-35S- BcACL5.2 -nFLAG-cMYC, by BcACL5.2 The gene fragment was obtained by inserting it into the linearized vector pCAMBIA1305-35S-nFLAG-cMYC, which was double-digested with SacI and KpnI.

[0010] As one of the preferred embodiments of the present invention, the overexpression vector pCAMBIA1305-35S- BcACL5.2 The specific construction method for -nFLAG-cMYC is as follows: (1) with BcACL5.2 Using the fragment as a template, PCR amplification was performed using homologous arm primers, and the PCR product was purified. (2) The plasmid of the overexpression vector pCAMBIA1305-35S-nFLAG-cMYC was extracted, and the digested product was purified after double digestion with SacI and KpnI enzymes. (3) The purified pCAMBIA1305-35S-nFLAG-cMYC vector plasmid and the target gene were combined. BcACL5.2 The fragments are ligated through homologous recombination to form a recombinant plasmid.

[0011] As one of the preferred embodiments of the present invention, the homologous arm primers are 1305-BcACL5.2-F and 1305-BcACL5.2-R, with sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0012] A genetically engineered host cell containing the aforementioned cold-resistant gene from *Brucea javanica*. BcACL5.2 The constructed overexpression vector.

[0013] A cold-resistant gene of the above-mentioned black spinach BcACL5.2 Application in breeding to improve plant cold tolerance.

[0014] As one of the preferred embodiments of the present invention, the cold resistance standard is 4℃ / 0℃ (day / night), and the plant is Arabidopsis thaliana or Brassica oleracea var. thaliana in the seedling stage.

[0015] One method to improve the cold tolerance of plants involves using genetic engineering to modify the plant's... BcACL5.2 Gene overexpression; the aforementioned BcACL5.2 The nucleotide sequence of the gene is shown in SEQ ID NO.1. It enhances the plant's growth ability under low temperature stress by positively regulating the plant's cold tolerance.

[0016] The advantages of this invention compared to the prior art are: This invention screened key genes related to cold resistance in *Brucea javanica*. BcACL5.2 Furthermore, by overexpressing the gene in Arabidopsis thaliana plants, the positive regulation of plant cold tolerance by the gene was verified, which can improve the plant's growth ability under low temperature stress. This invention lays the foundation for breeding new plant varieties with strong cold tolerance, especially new varieties of black cabbage, and is of great value for expanding the planting area of ​​black cabbage and promoting industrial development. Attached Figure Description

[0017] Figure 1 This is a plasmid structure diagram of pCAMBIA1305-35S-nFLAG-cMYC in Example 3 of the present invention; Figure 2 This is a verification electrophoresis image of the overexpression recombinant plasmid in Example 4 of this invention (in the image, from right to left, the marker, the plasmid with homologous arms, etc.) BcACL5.2 (fragments and overexpression recombinant plasmids); Figure 3 This is a growth diagram of T0 generation Arabidopsis thaliana seeds in a culture dish containing hygromycin in Example 5 of the present invention (in the diagram, plants with green leaves and vigorous growth are positive plants). Figure 4 This is in embodiment 6 of the present invention. BcACL5.2 Relative expression levels in wild-type Arabidopsis thaliana (WT) and different overexpressing Arabidopsis thaliana lines (OE9, OE6, OE2); Figure 5 This is a comparison of the growth status of wild-type Arabidopsis thaliana (WT) and T3 generation transgenic Arabidopsis thaliana (OE9, OE6, OE2) plants under different treatments in Example 7 of the present invention (in the figure, CK is the normal temperature treatment group: 25 / 18℃, 14 / 10h, day / night; LT is the low temperature treatment group: 4 / 0℃, 14 / 10h, day / night). Detailed Implementation

[0018] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents, kits, and other materials used below are all commercially available products conventional in the art; the experimental methods used are all conventional methods in the art and will not be described in detail further.

[0019] Example 1 BcACL5.2 Gene: This invention screened key genes related to cold resistance in *Brucea javanica*. BcACL5.2 Its nucleotide sequence is shown in SEQ ID NO.1.

[0020] Example 2 BcACL5.2 Gene cloning and purification: 1. Using CE Design V1.04 software, single-fragment cloning was employed, with SacI and KpnI as double restriction sites. Homologous arm primers 1305-BcACL5.2-F (SEQ ID NO.2) and 1305-BcACL5.2-R (SEQ ID NO.3) were designed using the gene sequence and the vector sequence near the restriction sites.

[0021] 2. Based on synthesis BcACL5.2 Using the fragment as a template, PCR amplification was performed using the aforementioned designed homologous arm primers (1305-BcACL5.2-F, 1305-BcACL5.2-R) to obtain the PCR product (containing homologous arms). BcACL5.2 (Gene fragments).

[0022] The PCR amplification system (50 μL) consisted of: 1 μL DNA fragment, 25 μL 2×PrimeSTAR Max DNA Polymerase, 2 μL primer 1305-BcACL5.2-F (10 μmol / L), 2 μL primer 1305-BcACL5.2-R (10 μmol / L), and 20 μL ddH2O.

[0023] The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 56℃ annealing for 10 s, 72℃ extension for 2 min, 35 cycles, and 72℃ final extension for 5 min.

[0024] 3. The obtained PCR products were purified using a DNA product purification kit (Beijing Tiangen Biotech Co., Ltd.), and the concentration of the recovered products was determined using a nucleic acid quantification instrument after purification.

[0025] Example 3: Overexpression vector pCAMBIA1305-35S- BcACL5.2 Build with -nFLAG-cMYC: 1. Use a plasmid extraction kit (Beijing Qingke Biotechnology Co., Ltd.) to extract the plasmid of vector pCAMBIA1305-35S-nFLAG-cMYC (structure as shown in the image). Figure 1 As shown in the figure, it was double digested with SacI and KpnI enzymes.

[0026] The double digestion system (50 μL) consisted of: 5 μL plasmid (plasmid concentration 200 ng / μL), 1 μL SacI rapid digestion enzyme, 1 μL KpnI rapid digestion enzyme, 5 μL 10×Quiekcut Buffer, and 38 μL ddH2O.

[0027] 2. The product was then purified using a product purification kit (Beijing Tiangen Biotech Co., Ltd.), and the concentration of the recovered product was determined using a nucleic acid quantification instrument.

[0028] 3. Using a homologous recombination kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.), the purified pCAMBIA-35S-nFLAG-cMYC vector plasmid and the target gene were combined. BcACL5.2 Fragment concatenation forms pCAMBIA1305-35S- BcACL5.2 -nFLAG-cMYC recombinant plasmid.

[0029] Example 4: Overexpression vector pCAMBIA1305-35S- BcACL5.2 Build verification with -nFLAG-cMYC: Verification 1: The recombinant plasmid was transformed into DH5α Escherichia coli (competent cells purchased from Beijing Qingke Biotechnology Co., Ltd.). Colonies were picked, cultured, and the bacterial culture was sequenced. If the sequencing result is the same as the target gene sequence, it indicates that... BcACL5.2 The gene was successfully inserted into the pCAMBIA1305-35S-nFLAG-cMYC vector.

[0030] Verification 2: (1) The pCAMBIA1305-35S-BcACL5.2-nFLAG-cMYC overexpression recombinant plasmid was transformed into GV3101 Agrobacterium (competent cells purchased from Weidi Biotechnology Co., Ltd.), and colonies were picked and shaken.

[0031] (2) Using the primer pair 1305-BcACL5.2-F (SEQ ID NO.2) and 1305-R (SEQ ID NO.4) or 1305-F (SEQ ID NO.5) and 1305-BcACL5.2-R (SEQ ID NO.3), the Agrobacterium tumefaciens culture obtained in step (1) was used as a template for PCR amplification verification.

[0032] The bacterial culture PCR amplification system was as follows: 1 μL of Agrobacterium tumefaciens bacterial culture obtained in the above steps, 10 μL of 2×Rapid Taq PlusMaster Mix (Dye Plus), 1 μL each of 1305-BcACL5.2-F (10 μmol / L) and 1305-R (10 μmol / L), and 7 μL of ddH2O.

[0033] The PCR amplification program was as follows: 95℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 1 min, 35 cycles, and 68℃ final extension for 5 min.

[0034] PCR amplification verification results: Agarose gel electrophoresis revealed that the positions of the bands in the PCR products were similar to those in the standard PCR product. BcACL5.2 The corresponding position of the segment ( Figure 2 This is consistent, which indicates BcACL5.2 The gene has been successfully inserted into the overexpression vector pCAMBIA1305-35S-nFLAG-cMYC.

[0035] Example 5: Cultivation of Arabidopsis thaliana plants overexpressing Arabidopsis thaliana: (1) After the Agrobacterium tumefaciens culture was revived, it was prepared into an OD by using a suspension. 600 The inflorescence of Col-0 Arabidopsis thaliana was infected with a 0.6-0.8 pH solution to obtain infected T0 generation Arabidopsis thaliana seeds. The buffer solution was prepared as follows: 500 mL of 1 / 2 MS, 25 g of sucrose, and 100 μL of Silwet-77, with the pH adjusted to 5.8.

[0036] (2) T0 generation Arabidopsis seeds were sown on 1 / 2 MS medium containing 50 mg / L hygromycin to screen for positive plants (T1 generation, Figure 3 Seeds were collected from individual plants. Seeds from each plant were sown on 1 / 2 MS medium containing 50 mg / L hygromycin. Lines with phenotypic segregation of 1:2:1 / 3:1 were selected, and relatively robust green plants (T2 generation) were transplanted into the substrate for culture. Seeds from each line were collected individually. T2 generation seeds were sown again on 1 / 2 MS plates containing 50 mg / L hygromycin to screen out T3 generation transgenic lines with relatively uniform growth.

[0037] like Figure 3 As shown, healthy plants growing on the culture medium are positive plants, while wild-type plants have yellow leaves, grow slowly, and may even fail to germinate seeds.

[0038] Example 6: Validation of genetic transformation of transgenic Arabidopsis plants: The overexpression level of T3 transgenic plants was detected by qRT-PCR using specific primers 1305-MYC-F (SEQ ID NO. 6) and 1305-MYC-R (SEQ ID NO. 7) in T3 transgenic plants and wild-type Arabidopsis thaliana.

[0039] The results are as follows Figure 4 As shown, BcACL5.2 The expression levels of pCAMBIA1305-35S- in the overexpression lines OE9, OE6, and OE2 were significantly higher than those in the wild type, and the expression levels gradually increased in these three lines, indicating that pCAMBIA1305-35S- BcACL5.2 The -nFLAG-cMYC overexpression recombinant plasmid has been successfully introduced into the Arabidopsis genome and expressed successfully, with varying overexpression levels among different lines. These results indicate that the genetic transformation of transgenic Arabidopsis plants has been successful, and subsequent experiments can be conducted using these transgenic plants.

[0040] Example 7: Cold tolerance control between transgenic Arabidopsis thaliana lines and wild-type Arabidopsis thaliana: After sterilization, seeds of wild-type and overexpression lines were sown on 1 / 2 MS medium and grown under the following conditions: 23℃ / 18℃, 10h / 14h (day / night), 75% relative humidity, and 3000 Lux light intensity. Two weeks later, plants with uniform growth were selected and transplanted into nutrient pots and cultured under the same conditions for three weeks. Subsequently, the plants were divided into two groups and treated with low temperature (4℃ day / 0℃ night, 14h light / 10h dark) and normal temperature (23℃ day / 18℃ night, 14h light / 10h dark) for 7 days.

[0041] The growth status of Arabidopsis plants in the normal temperature treatment group and the low temperature treatment group is as follows: Figure 5 As shown in Figure 5, the results indicate that after low-temperature treatment, the leaves of wild-type plants wilted and their growth was significantly inhibited, while the transgenic lines were less affected, with the OE2 line, which had the highest overexpression level, showing the best growth. This suggests that the transgenic plants have the ability to resist low-temperature stress at low temperatures (4℃ / 0℃) and their growth is significantly better than that of the wild type.

[0042] In summary, this invention has screened key genes related to cold tolerance in *Brucea javanica*. BcACL5.2 Furthermore, by overexpressing the gene in Arabidopsis thaliana plants, we verified that the gene positively regulates plant cold tolerance and can improve the plant's growth ability under low temperature stress, laying the foundation for breeding new plant varieties with strong cold tolerance, especially new varieties of black cabbage.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cold-resistant gene in black spinach BcACL5.2 Its characteristics are, The BcACL5.2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The cold-resistant gene of *Brucea javanica* according to claim 1 BcACL5.2 Its characteristics are, The BcACL5.2 Genes enhance a plant's ability to grow under low-temperature stress by positively regulating its cold tolerance.

3. An overexpression vector, characterized in that, Contains the cold-resistant gene of black spinach as described in claim 1 or 2 BcACL5.2 .

4. The overexpression vector according to claim 3, characterized in that, The overexpression vector is pCAMBIA1305-35S- BcACL5.2 -nFLAG-cMYC, by BcACL5.2 The gene fragment was obtained by inserting it into the linearized vector pCAMBIA1305-35S-nFLAG-cMYC, which was double-digested with SacI and KpnI.

5. The overexpression vector according to claim 4, characterized in that, The overexpression vector pCAMBIA1305-35S- BcACL5.2 The specific construction method for -nFLAG-cMYC is as follows: (1) with BcACL5.2 Using the fragment as a template, PCR amplification was performed using homologous arm primers, and the PCR product was purified. (2) The plasmid of the overexpression vector pCAMBIA1305-35S-nFLAG-cMYC was extracted, and the digested product was purified after double digestion with SacI and KpnI enzymes. (3) The purified pCAMBIA1305-35S-nFLAG-cMYC vector plasmid and the target gene were combined. BcACL5.2 The fragments are ligated through homologous recombination to form a recombinant plasmid.

6. The overexpression vector according to claim 5, characterized in that, The homologous arm primers are 1305-BcACL5.2-F and 1305-BcACL5.2-R, with sequences shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

7. A genetically engineered host cell, characterized in that, Contains the cold-resistant gene of black spinach as described in claim 1 or 2 BcACL5.2 The constructed overexpression vector.

8. A cold-resistant gene for *Brucea javanica* as described in any one of claims 1 to 2 BcACL5.2 Application in breeding to improve plant cold tolerance.

9. The application according to claim 8, characterized in that, The cold tolerance standard is 4℃ daytime / 0℃ nighttime, and the plant is Arabidopsis thaliana or Brassica oleracea var. spinosa seedlings.

10. A method for improving the cold tolerance of plants, characterized in that, Using genetic engineering methods to make plants... BcACL5.2 Gene overexpression; the aforementioned BcACL5.2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. It enhances the plant's growth ability under low temperature stress by positively regulating the plant's cold tolerance.

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