CN114107333B - Application of barley receptor kinase HvSERK1 in root hair growth - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to barley HvSERK1 gene, barley receptor kinase HvSERK1, a recombinant expression vector containing the gene, and an application thereof in promoting the growth of plant root hairs. The invention clones the barley HvSERK1 gene, carries out genetic transformation in Arabidopsis thaliana, and overexpresses the exogenous barley HvSERK1 gene, which can significantly increase the root hair length of the Arabidopsis thaliana. Therefore, HvSERK1 is expected to be used in genetic engineering breeding, and its introduction into short root hair barley varieties is expected to improve the ability of barley to absorb water and nutrients. The invention provides a theoretical basis and related genes for drought-resistant breeding and production of barley.

Description

Application of a Barley Receptor Kinase HvSERK1 in Root Hair Growth

technical field

The invention relates to the technical field of genetic engineering, specifically barley HvSERK1 gene and its expression vector, barley receptor kinase HvSERK1 and application thereof.

Background technique

Root hairs are highly specialized cells in the root epidermis of plants, which can increase the surface area of the root and improve the ability of the plant to obtain water and nutrients; it can also improve the stress resistance of the plant through its own adaptive response and root exudates. At present, in order to increase the yield of crops, a large amount of chemical fertilizers and pesticides are used in agricultural production, which has caused serious environmental pollution and energy waste, and also increased the cost of farmers. Therefore, it is necessary to study how to obtain crops with more and longer root hairs New varieties, improved crop yields and efficient use of nutrients and water are the basis and key to improving crop yields, stress resistance and sustainable agricultural development. Studying the genes related to the regulation of root hair growth and development has important theoretical significance and practical application value for understanding the genetic regulation mechanism of plant growth and development.

Receptor-like kinases are a class of transmembrane proteins that are ubiquitous in plants and play an important role in transducing extracellular signals to regulate plant development, self-incompatibility and defense responses. SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) belong to LRR-RLKs subfamily II, the first class of receptor kinases discovered during somatic embryogenesis in carrot. The involvement of SERKs in Arabidopsis includes somatic embryogenesis, seedling development, brassinosteroids (BRs) signal transduction, plant immunity and stomatal development. The Arabidopsis SERKs family includes five homologous genes, namely SERK1, SERK2, SERK3/BKA1, SERK4/BKK1 and SERK5. The amino acid sequences of SERK1 and SERK2 are very similar, and the double mutant serk1serk2 leads to abortion because it cannot form tapetum cells, indicating that SERK1 and SERK2 are very important for the formation of Arabidopsis anther tapetum. BRs can interact with auxin and other plant hormones to affect Arabidopsis root development. The triple mutant serk1bak1bkk1 can not only block the BR signaling pathway, but also affect auxin polar transport, cell cycle, endothelial development, and root development. Gene expression related to meristem differentiation was down-regulated, thereby affecting Arabidopsis root development. SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1 are all involved in regulating stomatal development in Arabidopsis thaliana independently of BR signaling.

Wheat crops are important food crops in the world, and its functional genome research is of great significance to global food security. Barley (Hordeum vulgare L.) belongs to the Gramineae family and is the fourth largest cereal crop in the world. It is mainly used as raw material for beer industry, animal husbandry feed and food processing raw material. It is also an important model plant for genetics and physiology research. However, the drought has seriously affected the cultivation of barley and has become one of the most important abiotic factors restricting the increase of barley production. Therefore, it is urgent to excavate the genes related to root hairs to breed barley varieties with multiple root hairs and long root hairs.

Contents of the invention

The object of the present invention is to provide barley HvSERK1 gene, barley receptor kinase HvSERK1, a recombinant expression vector containing the barley HvSERK1 gene, and the application of the gene in promoting the growth of Arabidopsis root hairs.

The first aspect of the present invention provides the barley HvSERK1 gene, the nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1.

The second aspect of the present invention provides the protein encoded by the above barley HvSERK1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

Cloning and analysis of the HvSERK1 gene: the present invention identified and cloned the gene HvSERK1 from the cultivated barley "Hua 30". The CDS region of the gene is 1884bp in full length, encoding a protein sequence of 627aa, the molecular weight of the protein is 69.0769KDa, and the isoelectric point pI =5.70.

The third aspect of the present invention provides a pair of primers for amplifying the aforementioned barley HvSERK1 gene, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

The fourth aspect of the present invention provides a recombinant expression vector containing the above-mentioned barley HvSERK1 gene.

Further, the recombinant expression vector includes an original vector and a target gene inserted into the original vector, and the base sequence of the target gene is shown in SEQ ID NO.1.

Further, the promoter for promoting the expression of the target gene is a strong 35S promoter.

Further, the original vector is pH2GW7.

Further, the construction of the recombinant expression vector: use the Gateway system (Invitrogen Company) to connect the barley HvSERK1 gene into the plant expression vector pH2GW7. The promoter that drives the expression of the barley HvSERK1 gene is a strong promoter. The strong promoter can promote the overexpression of barley HvSERK1 gene. Preferably, the strong promoter is 35S.

The fifth aspect of the present invention provides a transformant containing the above-mentioned recombinant expression vector.

Further, the host bacteria of the transformants are Agrobacterium GV3101, LBA4404, AGL1 or EHA105.

The sixth aspect of the present invention provides the use of the above-mentioned barley HvSERK1 gene, the protein encoded by the above-mentioned barley HvSERK1 gene, the above-mentioned primer pair, the above-mentioned recombinant expression vector, or the above-mentioned transformant in plant breeding.

Further, the plant breeding is to regulate the growth of plant root hairs.

Further, the plant breeding is to promote the growth of plant root hairs. In one embodiment of the present invention, the plant is Arabidopsis.

The invention provides the use of the barley HvSERK1 gene in regulating root hair growth. The invention clones the barley HvSERK1 gene and performs genetic transformation in Arabidopsis thaliana to overexpress the exogenous barley HvSERK1 gene, which can significantly increase the root hair length of the Arabidopsis thaliana. Therefore, HvSERK1 is expected to be used in genetic engineering breeding, and its introduction into short root hair barley varieties is expected to improve the ability of barley to absorb water and nutrients. The invention provides a theoretical basis and related genes for drought-resistant breeding and production of barley.

The present invention has the advantage that:

The invention proves that the barley HvSERK1 gene can significantly increase the root hair length of the Arabidopsis through the cloning and analysis of the barley HvSERK1 gene and the analysis of the Arabidopsis phenotype. The invention provides a theoretical basis and related genes for barley long root hair breeding and production.

Description of drawings

Fig. 1 shows the expression level of HvSERK1 gene in the transgenic material of Arabidopsis thaliana overexpressing HvSERK1 gene according to the present invention.

Fig. 2 shows that the root hair length of Arabidopsis thaliana transgenic material overexpressing HvSERK1 gene according to the present invention is significantly increased.

Detailed ways

The specific implementation modes provided by the present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings.

Example 1: Construction of overexpression vector of barley HvSERK1 gene and genetic transformation of Arabidopsis thaliana

(1) The immature embryos of barley "Hua 30" were taken, and RNA was extracted by Trizol method (Invitrogen), and the operation steps were carried out according to the instructions. 1 μg of RNA was reverse-transcribed to obtain cDNA.

(2) Design specific primers according to the HvSERK1 gene sequence, add a restriction enzyme site at the 5' end of the primers, the sequence is as follows:

HvSERK1-CDS-F:5'-CCATCGATCTGCGGCTTTGAGACGG-3' (SEQ ID NO.3);

HvSERK1-CDS-R: 5'-CCCCCGGGAGAAATTCTGCAACGAGGGG-3' (SEQ ID NO.4);

(3) Perform PCR amplification using the full-length cDNA as a template.

Using KOD high-fidelity enzyme (TOYOBO) system:

Add sterile distilled water to a total volume of 50 μL.

The PCR cycling conditions were: pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 68°C for 90 s, amplification for 35 cycles, and finally extension at 68°C for 5 min to end the reaction.

(4) After the PCR product was recovered by gel running, it was ligated to Gateway cloning system entry vector NT007 by enzyme digestion, transformed into Escherichia coli DH5α, and the positive clone was sequenced and analyzed. The HvSERK1 gene sequence is shown in SEQ ID NO.1.

(5) The correctly sequenced NT007 plasmid containing the full-length CDS of HvSERK1 was named HvSERK1-NT007, and the plasmid was subjected to LR exchange reaction with the overexpression vector pH2GW7.

Using Gateway LR Clonase™ Enzyme Mix (Invitrogen Company, Cat.No.11791-019), the system is as follows:

Briefly vortex twice to mix well, centrifuge slightly, place in 25°C water bath for 2h, then add 1 μL Proteinase Ksolution (proteinase K solution) to mix well, and place at 37°C for 10 minutes to end the reaction.

Take 5 μL of the reaction product to transform Escherichia coli DH5α, pick positive clones for PCR verification, and sequence the positive clones for verification. After the sequencing is correct, extract the plasmid and name it 35S:HvSERK1.

(6) The LBA4404 competent cells were taken out from the -80°C refrigerator, and Agrobacterium LBA4404 was transformed by the heat shock method.

Thaw the competent cells on ice, add 5 μL of the target plasmid to the competent cells, mix well and place on ice for 30 minutes; place the centrifuge tube in liquid nitrogen to freeze for 1 minute, then put it in a water bath at 37°C until it melts . Add 1mL of LB liquid nutrient medium to shake and culture for 2-3h, after centrifugation, spread on the resistant YEP plate containing Rif (rifampicin) 50mg/L, Spec (spectinomycin hydrochloride) 50mg/L, and invert at 28°C Incubate for 2 nights to grow single colonies.

(7) Pick the Agrobacterium-positive single clones verified by PCR, put them into 5ml YEP liquid medium containing corresponding antibiotics, and culture them with shaking at 28°C for 48h. Add the shaken bacteria into YEP liquid medium (containing corresponding antibiotics) at a volume ratio of 1:50, and cultivate at 28°C until OD ₆₀₀ =1.0-1.2.

(8) Transform Arabidopsis thaliana by Arabidopsis flower dipping method.

Remove the fruit pods and place the whole Arabidopsis thaliana plant with only the inflorescence upside down together with the plug in the transformed Agrobacterium bacterium solution, soak the seedlings for 5 minutes, and shake the bacterium solution continuously during the period;

After infestation, take out the plants, place them sideways in a tray, cover them with black plastic cloth to avoid light, place them in an incubator, and uncover them after 24 hours;

The Arabidopsis plants were cultivated under natural light conditions, watered 1-2 times a week, and the T ₀ generation seeds were harvested after the seeds matured.

Example 2: Screening of barley HvSERK1 overexpression transgenic lines

(1) After sterilizing the harvested T ₀ generation seeds, they were planted in the solid medium of MS+35mg/L hygromycin, and after vernalization in the dark at 4°C for 3 days, the petri dishes were placed in an artificial climate box for cultivation, and observed Growth status of Arabidopsis plants. Transgenic seeds with hygromycin resistance will grow on the selection medium, while non-transgenic seeds will not grow after germinating.

(2) Transplant the screened transgenic plants into nutrient soil. When the plants are bolting, cut about 100 mg of Arabidopsis leaves and place them in a 1.5 ml centrifuge tube, extract DNA, and perform PCR verification to detect whether the screened plants are is positive.

(3) Harvest the _T1 generation seeds of the positive plants, sterilize them, plant them in the solid medium of MS+35mg/L hygromycin, and after vernalization in the dark at 4°C for 3 days, put the petri dishes in artificial Cultivated in a climate chamber.

(4) Transgenic plants with hygromycin resistance were transplanted into nutrient soil. When the plants were bolting, about 100 mg of Arabidopsis leaves were cut from each line to extract RNA, and cDNA was obtained by reverse transcription. The expression of HvSERK1 gene in different transgenic lines of Arabidopsis thaliana was analyzed by fluorescent quantitative PCR using SYBR green and PCR instrument, and the expression value was corrected by the internal reference gene TUB2.

The fluorescence quantitative PCR system is as follows:

The PCR program is: 95°C for 1min, 95°C for 5s, 60°C for 20s, 40 cycles. Each group of experiments was repeated three times, and the sequences of fluorescent quantitative PCR primers were as follows:

HvSERK1-RT-PCR-F: 5'-CTTGGCAGACGGAACTCTTG-3' (SEQ ID NO.5);

HvSERK1-RT-PCR-R: 5'-TCTCGGTCGGTGTCATACAG-3' (SEQ ID NO.6);

AtTUB2-F: 5'-GTTCTCGATGTTGTTCGTAAG-3' (SEQ ID NO.7);

AtTUB2-R: 5'-TGTAAGGCTCAACCACACAGTAT-3' (SEQ ID NO. 8).

(5) The relative expression levels of HvSERK1 in different Arabidopsis transgenic lines, the results are shown in FIG. 1 . The three lines with the highest expression levels, 35S:HvSERK1-4, 35S:HvSERK1-7, and 35S:HvSERK1-9, were selected to harvest _T2 generation seeds.

Example 3: Observation of Arabidopsis root hair phenotype

(1) Sterilize Arabidopsis wild-type and _T2 generation HvSERK1 transgenic seeds (35S:HvSERK1-4, 35S:HvSERK1-7, 35S:HvSERK1-9), sow on MS solid medium at 4°C After 3 days of vernalization in the dark, the petri dishes were cultured vertically and placed in an artificial climate chamber (temperature: 20±2°C; light intensity 90-120 μmolm ^-2 sec ^-1 ; photoperiod: 16h light/8h dark).

(2) After being cultured in an artificial climate box for 7 days, the root hair phenotypes about 4 mm above the root tip were photographed with a stereo microscope.

(3) Statistical analysis was carried out on the length of root hairs using Image pro plus software, the longest 20 root hairs were measured for each root, and at least 20 roots were measured for each strain. as shown in picture 2. It was found that overexpression of HvSERK1 can promote root hair growth.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. These equivalent modifications or replacements are all included within the scope defined by the claims of the present application.

SEQUENCE LISTING

<110> Shanghai Academy of Agricultural Sciences

<120> Application of a Barley Receptor Kinase HvSERK1 in Root Hair Growth

<130> /

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<170> PatentIn version 3.3

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Claims (4)

1. The application of a recombinant expression vector containing a barley HvSERK1 gene in promoting the growth of Arabidopsis root hair is characterized in that the recombinant expression vector comprises an original vector and a target gene inserted into the original vector, and the base sequence of the target gene is shown as SEQ ID NO. 1; the promoter for promoting the expression of the target gene is a 35S strong promoter.

2. The use according to claim 1, wherein the original carrier is pH2GW7.

3. Use of a transformant containing a recombinant expression vector of barley HvSERK1 gene for promoting growth of root hairs of arabidopsis; the recombinant expression vector comprises an original vector and a target gene inserted into the original vector, and the base sequence of the target gene is shown as SEQ ID NO. 1; the promoter for promoting the expression of the target gene is a 35S strong promoter.

4. The use according to claim 3, wherein the host bacterium of the transformant is Agrobacterium GV3101, LBA4404, AGL1 or EHA105.

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