BcKH14 gene for regulating and controlling development of leaf epidermal hair of black cabbage and application of BcKH14 gene

Abstract

The invention relates to the technical field of genetic breeding, and provides a BcKH14 gene for regulating and controlling growth of leaf epidermal hair of black cabbage, and the nucleotide sequence of the BcKH14 gene is as shown in SEQ ID NO.1. The invention also provides an overexpression vector containing the gene and a genetically engineered host cell. In addition, the invention also provides application of the gene in cultivation of a new variety of multi-epidermal black cabbage, and a method for promoting development of epidermal hair of black cabbage leaves. According to the invention, the key gene BcKH14 related to epidermal hair development is screened from the black cabbage, and the gene is over-expressed in an arabidopsis thaliana plant, so that the positive regulation of the gene on the epidermal hair of the leaf of the black cabbage is verified, the growth of the epidermal hair of the leaf of the plant can be promoted, and a foundation is laid for cultivating a new variety of the black cabbage with multiple epidermal hair; the method has important value for improving the stress resistance and disease and pest resistance of the black cabbage.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic breeding, and in particular to a BcKH14 gene for regulating the development of epidermal hairs in Wucai leaves and an application thereof. Background Art

[0002] Wucai (Brassica campestris L.ssp.chinensis (L.) Makino var.rosularis Tsenet Lee), a subspecies of Chinese cabbage (B.campestris L.), is native to China and primarily distributed in the Yangtze and Huaihe River basins. As an important cultivated variety of the cabbage family, epidermal hair structures are commonly found on the surfaces of its leaves, stems, and flowers. Epidermal hairs are common specialized epidermal cell derivatives in terrestrial plants and play a key role in protecting against biotic stresses (such as insect pests and pathogens) and abiotic stresses (such as drought, ultraviolet light, and extreme temperatures).

[0003] Therefore, studying the key genes that regulate the development of epidermal hairs and cultivating new varieties of black cabbage with multiple epidermal hairs are of great value in improving the stress resistance and disease and pest resistance of black cabbage. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a BcKH14 gene for regulating the development of epidermal hairs in Wucai leaves and an application thereof.

[0005] The present invention adopts the following technical solutions to solve the above technical problems:

[0006] A BcKH14 gene regulates the development of epidermal hairs in Wucai leaves. The nucleotide sequence of the BcKH14 gene is shown in SEQ ID NO.1.

[0007] As one of the preferred embodiments of the present invention, the BcKH14 gene promotes the growth of epidermal hairs on Wucai leaves by positively regulating the development of epidermal hairs on Wucai leaves.

[0008] An overexpression vector contains the BcKH14 gene for regulating the development of epidermal hairs in Wucai leaves.

[0009] As one of the preferred embodiments of the present invention, the overexpression vector is specifically pCAMBIA1305-35S-BcKH14, and the construction method is as follows:

[0010] Using the cDNA of Wucai as a template, the BcKH14 gene was amplified by PCR using specific primers, and its full-length coding sequence was successfully cloned. Then, the nucleotide sequence of the BcKH14 gene was inserted into the vector pCambia1305-35S through homologous recombination to obtain pCAMBIA1305-35S-BcKH14.

[0011] As one of the preferred embodiments of the present invention, the specific primers are BcKH14-F and BcKH14-R, and their sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0012] A genetically engineered host cell contains an overexpression vector constructed with the BcKH14 gene.

[0013] The invention relates to an application of the above-mentioned BcKH14 gene for regulating the development of epidermal hairs on wucai leaves in breeding new varieties of wucai with multiple epidermal hairs.

[0014] A method for promoting the development of epidermal hairs in Wucai leaves, comprising overexpressing the BcKH14 gene in Wucai by genetic engineering methods; the nucleotide sequence of the BcKH14 gene is shown in SEQ ID NO.1, and promoting the growth of epidermal hairs in Wucai leaves by positively regulating the development of epidermal hairs in Wucai leaves.

[0015] The advantages of the present invention over the prior art are:

[0016] The present invention screened out the key gene BcKH14 related to the development of epidermal hairs in Wucai, and by overexpressing the gene in Arabidopsis plants, verified that the gene positively regulates the epidermal hairs of Wucai leaves, which can promote the growth of epidermal hairs on the leaves of the plant; the present invention lays the foundation for the cultivation of new varieties of Wucai with multiple epidermal hairs, and is of great value in improving the stress resistance and disease and pest resistance of Wucai. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the PCR verification result of the recombinant plasmid transformed with Agrobacterium in Example 4 (in the figure, M is a marker and BcKH14-1 is a qualified PCR product);

[0018] Figure 2 1 is a graph showing the expression of BcKH14 in Arabidopsis thaliana BcKH14-overexpressing plants (BcKH14-OE) and wild-type Arabidopsis thaliana (WT) in Example 5;

[0019] Figure 3 These are the observation results of leaf epidermal hairs of Arabidopsis thaliana BcKH14-overexpressing plants (BcKH14-OE) and wild-type Arabidopsis thaliana (WT) in Example 5 (Figure A shows the observation results of wild-type Arabidopsis thaliana, and Figure B shows the observation results of Arabidopsis thaliana BcKH14-overexpressing plants). DETAILED DESCRIPTION

[0020] The following examples of the present invention are described in detail. These examples are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operating procedures are given. However, the scope of protection of the present invention is not limited to the following examples. At the same time, the reagents, kits and other materials used in the following examples, unless otherwise specified, are all conventional commercial products in the field; the experimental methods used, unless otherwise specified, are all conventional methods in the field and will not be repeated here.

[0021] Example 1, BcKH14 gene:

[0022] The present invention screened out a key gene BcKH14 related to epidermal hair development in Wucai, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0023] Example 2: Cloning and purification of BcKH14 gene:

[0024] 1. Select Hei Xin Wu (one of the cultivated varieties of Wucai) as the material. Hei Xin Wu seedlings were grown in a greenhouse at 26±2℃ (daytime) and 20±2℃ (nighttime) with a light intensity of 300μmol m -2 s -1 , relative humidity is 70-80%.

[0025] 2. Take the leaves of Rhizoma Citri Reticulatae and perform total RNA extraction and cDNA synthesis:

[0026] The RNA of the black-hearted blackbird sample was extracted using the TakaRa RNA kit. After quantification, the RNA was reverse transcribed using the TRAN reverse transcription kit. The system is shown in Table 1.

[0027] Table 1. Reverse transcription system

[0028]

[0029] 3. Using CE-Design software, homology arm primers BcKH14-F (SEQ ID NO. 2) and BcKH14-R (SEQ ID NO. 3) were designed based on the sequence of the BcKH14 gene (SEQ ID NO. 1) and the pCambia1305-35S vector sequence, and the primer sequences were sent to Universal Biotechnology Co., Ltd. for synthesis.

[0030] 4. Add samples according to the system in Table 2 and perform PCR amplification. The PCR program is 95°C for 5 minutes, 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 2 minutes, and 72°C for 5 minutes. After PCR amplification, identify the PCR product by 1% agarose gel electrophoresis. Recover the target DNA fragment using the TIANGEN Agarose Gel DNA Recovery Kit.

[0031] Table 2. PCR amplification system

[0032]

[0033] Example 3: Construction of overexpression vector pCAMBIA1305-35S-BcKH14 and transformation of E. coli:

[0034] 1. Add samples according to the system in Table 3, pipette and mix, and ligate at 37°C for 30 min. Immediately place on ice to obtain the ligation product, i.e., the overexpression vector pCAMBIA1305-35S-BcKH14.

[0035] Table 3. Connection system

[0036] Components volume Vector pCAMBIA1305-35S 4 μL target gene 1 μL 5×CE Buffer 2μL Exnase II 1 μL <![CDATA[ddH2O]]> Up to 10μL

[0037] 2. Add 10 μL of the ligation product to 100 μL of E. coli, gently pipette to mix, let stand on ice for 5 minutes, heat shock at 42°C for 45-60 seconds, quickly transfer to ice, and let stand for 2 minutes. Then, add 700 μL of antibiotic-free LB to the centrifuge tube, mix thoroughly, and resuscitate the cells at 37°C at 200 rpm for 20 minutes. Spread 100 μL of the resuscitation solution evenly on a culture medium containing 100 mg / mL Kan antibiotic. Then, place the culture medium in an incubator and incubate it upside down at 37°C overnight. The next day, pick 8 single colonies and place them in a centrifuge tube containing 500 μL of LB containing 100 mg / mL Kan. Shake at 37°C for 5 hours until the culture solution becomes turbid. This will obtain the bacterial suspension.

[0038] 3. Take the bacterial suspension obtained in the previous step and use the gene upstream primer BcKH14-F (SEQ ID NO. 2) and the vector downstream primer 1305-R (SEQ ID NO. 4) according to the system in Table 4. The PCR program is: 95°C for 5 minutes, 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 2 minutes, and 72°C for 5 minutes. After the reaction, the PCR product is confirmed by 1% agarose gel electrophoresis and sent to Universal for sequencing. If the alignment is successful, the gene is successfully linked to the vector. Return the bacterial suspension and plasmid, mix the bacterial suspension with glycerol in a 1:1 ratio, snap-freeze with liquid nitrogen, and store at -80°C.

[0039] Table 4. Bacterial liquid PCR system

[0040]

[0041]

[0042] Example 4, Agrobacterium transformation:

[0043] Take 100 μL of competent Agrobacterium GV3101 and add 5 μL of the recombinant plasmid pCAMBIA1305-35S-BcKH14 obtained in the previous example. Mix thoroughly by pipetting. Place on ice for 5 minutes, in liquid nitrogen for 5 minutes, and in a 37°C water bath for 5 minutes. Then, add 700 μL of antibiotic-free LB and shake at 28°C for 3 hours. Harvest the bacteria by centrifugation at 6000 rpm for 1 minute. Collect 100 μL of the supernatant and gently pipette the resuspended bacteria onto an LB plate containing 100 mg / mL Kan (kanamycin) and 25 mg / mL rifampicin (rifampicin). Incubate the plate upside down at 28°C for 2 days. Pick 8 individual colonies and place them in a centrifuge tube containing 500 μL of LB supplemented with 100 mg / mL Kan and 25 mg / mL rif. Shake at 28°C for 24 hours until the suspension becomes turbid. The sample was added according to the system in Table 4. The PCR program was 95℃ for 5 min, 95℃ for 30 s, 60℃ for 30 s, 72℃ for 2 min, and 72℃ for 5 min. After the reaction, the PCR products were identified by 1% agarose gel electrophoresis ( Figure 1 ), mix the qualified bacterial solution with glycerol in a 1:1 ratio, freeze it quickly with liquid nitrogen, and store it at -80°C to obtain Agrobacterium transformants.

[0044] Example 5: Screening of BcKH14-OE overexpressing plants and verification of BcKH14 gene function:

[0045] 1. Arabidopsis thaliana cultivation

[0046] Wild-type Arabidopsis thaliana was cultured in an artificial climate incubator under the following conditions: temperature 24°C / 16°C, photoperiod 16 h / 8 h, and light intensity 300 μmol·m -2 ·s -1 , relative humidity 70%. Cultivate for about 4 weeks until the plants are bolted for subsequent Agrobacterium infection and transformation.

[0047] 2. Infection of Arabidopsis thaliana

[0048] First, resuscitate the preserved Agrobacterium transformant (prepared in Example 4) and add 40 μL of bacterial solution to a 10 mL sterile centrifuge tube containing 4 mL of liquid LB medium, 4 μL of Kan, and 8 μL of Rif. Culture with shaking at 28°C and 220 rpm for 2 days. After turbidity, expand the culture according to the same system and use a UV spectrophotometer to measure the OD value at 560 nm. If it is 1-2, the infection conditions are met. In addition, prepare the infection buffer, the system is shown in Table 5. Centrifuge the bacterial solution that meets the conditions: 4°C, 8000 rpm, 5 minutes, then discard the supernatant, elute with infection buffer and dilute to OD 560 It is 0.8~1 and set aside.

[0049] Table 5. Infection buffer preparation system

[0050] 1 / 2MS(mL) 5% sucrose (g) Silwet L-77 (μL) 500 25 125 400 20 100 200 10 50

[0051] Infect Arabidopsis inflorescences with the diluted bacterial solution for 2 minutes, then place in the dark for 24 hours. After the seedlings have established, incubate under normal conditions. Repeat the infection after 10-14 days. Harvest the seeds when the pods are ripe.

[0052] 3. Screening of transgenic Arabidopsis

[0053] Seeds harvested after infection with Arabidopsis thaliana were disinfected by washing with 2% available chlorine in NaClO for 10 minutes and then 7-9 times with ddH2O. Using an autoclaved pipette tip, the disinfected seeds were spotted onto 1 / 2 MS medium containing 50 μg / mL (50% effective concentration) hygromycin B and placed in an artificial climate incubator for normal growth. After 7 days of incubation, growth was observed to determine whether the plants were transgenic. After 10 days, seedlings that met the requirements and showed good growth were transferred to nutrient pots containing substrate and cultured normally in an incubator.

[0054] When the seedlings reached one month old, 0.1 g of fresh leaves were collected from each plant for total RNA extraction. RNA was then reverse-transcribed into cDNA, and expression levels were determined by qRT-PCR using primers BcKH14-qRT-F (SEQ ID NO. 5) and BcKH14-qRT-R (SEQ ID NO. 6) as templates, with At-actin-F (SEQ ID NO. 7) and At-actin-R (SEQ ID NO. 8) as internal reference primers. Seeds from three high-expressing Arabidopsis thaliana (T1) plants were selected for subsequent experiments.

[0055] After disinfecting and cleaning the seeds of the selected high-expressing Arabidopsis thaliana (T1) plants, they were further screened on 1 / 2 MS medium containing 30 μg / mL (30% effective concentration) hygromycin B. According to Mendel's law of segregation, the T2 generation plants will have a segregation ratio of 3:1. Plants with good growth were selected and their expression levels were detected by qRT-PCR (the method is the same as above). The expression results are shown in Figure 2. Figure 2 As shown, it was demonstrated that the desired target was formally obtained in Arabidopsis BcKH14 overexpressing plants (BcKH14-OE).

[0056] 4. Phenotypic observation

[0057] Using wild-type Arabidopsis plants (WT) as a control, it was found that Arabidopsis BcKH14 overexpressing plants (BcKH14-OE) had significantly more leaf burrs than wild-type plants (WT) ( Figure 3 ).

[0058] In summary, the present invention screened out the key gene BcKH14 related to the development of epidermal hairs in Wucai, and by overexpressing this gene in Arabidopsis plants, verified that this gene positively regulates the epidermal hairs of Wucai leaves, which can promote the growth of epidermal hairs on plant leaves, laying the foundation for breeding new varieties of Wucai with multiple epidermal hairs, and is of great value for improving Wucai's stress resistance and disease and pest resistance.

[0059] 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 in the scope of protection of the present invention.

Claims

1. A BcKH14 gene that regulates the development of epidermal hairs in Wucai leaves, characterized in that: The nucleotide sequence of the BcKH14 gene is shown in SEQ ID NO.

1.

2. The BcKH14 gene for regulating the development of epidermal hairs in Wucai leaves according to claim 1, characterized in that: The BcKH14 gene promotes the growth of epidermal hairs on the leaves of Wucai by positively regulating the development of epidermal hairs on the leaves of Wucai.

3. An overexpression vector, characterized in that Contains the BcKH14 gene for regulating the development of epidermal hairs in Wucai leaves as claimed in claim 1.

4. The overexpression vector according to claim 3, characterized in that The overexpression vector is specifically pCAMBIA1305-35S-BcKH14, and the construction method is as follows: Using the cDNA of Wucai as a template, the BcKH14 gene was amplified by PCR using specific primers, and its full-length coding sequence was successfully cloned. Then, the nucleotide sequence of the BcKH14 gene was inserted into the vector pCambia1305-35S through homologous recombination to obtain pCAMBIA1305-35S-BcKH14.

5. The overexpression vector according to claim 4, characterized in that The specific primers are BcKH14-F and BcKH14-R, and their sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

6. A genetically engineered host cell, characterized in that An overexpression vector containing the BcKH14 gene constructed according to claim 1.

7. Use of the BcKH14 gene for regulating the development of epidermal hairs in Wucai leaves as claimed in claim 1 or 2 in breeding new varieties of Wucai with multiple epidermal hairs.

8. A method for promoting the development of epidermal hairs in Wucai leaves, characterized in that: The BcKH14 gene is overexpressed in Wucai by genetic engineering methods; the nucleotide sequence of the BcKH14 gene is shown in SEQ ID NO.1, which positively regulates the development of epidermal hairs on Wucai leaves and promotes the growth of epidermal hairs on Wucai leaves.

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