Barley hvwox8 gene and use thereof

Abstract

The present application relates to the field of biological gene engineering, and particularly relates to application of a barley WOX family gene HvWOX8 in promoting plant salt tolerance. The CDS region nucleotide sequence of the barley HvWOX8 gene is shown as SEQ ID No. 1. The present application performs cloning and analysis on the barley HvWOX8 gene, and performs genetic transformation on Arabidopsis thaliana to verify the function of the gene. It is found that overexpression of the exogenous barley HvWOX8 gene can significantly improve the salt tolerance of Arabidopsis thaliana. Therefore, HvWOX8 can be used for genetic engineering breeding, and provides a theoretical basis and related genes for barley salt tolerance breeding and production.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to the barley HvWOX8 gene and its application in promoting plant salt tolerance. Background Technology

[0002] Soil salinization has become one of the major abiotic stresses facing agriculture worldwide. High salt content in salinized soils hinders plant water absorption, impeding root development and affecting nutrient uptake and growth. Secondly, the alkaline substances in salinized soils damage soil structure, affecting aeration and permeability, which in turn affects root respiration and water absorption. Furthermore, salinized soil conditions also affect plant physiological metabolism, inhibiting growth and development, leading to reduced yield and quality. Therefore, improving crop salt tolerance not only increases the utilization rate of salinized soils but is also crucial for protecting global food security. Plant salt tolerance is a complex regulatory mechanism. Although significant progress has been made in the research of salt tolerance-related genes, the related signal transduction and regulatory mechanisms remain unclear. Discovering superior salt-tolerant germplasm and genes is of great significance for improving crop salt tolerance, and breeding new salt-tolerant crop varieties is an effective way to address soil salinization.

[0003] The WUSCHEL-related homebox (WOX) gene family consists of plant-specific transcription factors containing highly conserved homologous domains. They primarily branch into three clades: the ancient clade, the intermediate clade, and the WUS clade. Members of the WOX transcription factor family are expressed in almost all plant organs and play crucial roles in key plant developmental processes. WOX family members have been identified and studied in numerous species, including Arabidopsis thaliana, rice, maize, soybean, sorghum, cotton, poplar, and cucumber. Studies have shown that WOX family genes participate in the growth and development of different stages and organs. Many WOX genes interact with hormones to jointly regulate plant growth and development. Furthermore, WOX genes not only participate in plant growth and development but also play important roles in abiotic stress responses. Research indicates that after 12 hours of drought treatment, the expression levels of almost all rice WOX genes were significantly upregulated; NaCl treatment induced the expression of OsWOX3 and OsWOX5; and low-temperature treatment increased the expression of OsWOX9B and OsWOX12B.

[0004] Cereals are a vital food crop worldwide, and their functional genomics research is of paramount importance to global food security. Barley (Hordeum vulgare L.), belonging to the genus Hordeum in the family Poaceae, is the world's fourth largest cereal crop and an important model plant for genetic and physiological research. Barley exhibits stronger salt tolerance than other Poaceae crops, and currently, there are no reports on barley WOX genes. Therefore, identifying superior salt tolerance-related genes in barley and exploring their specific molecular mechanisms of action will lay the foundation for research on the regulatory mechanisms of barley salt tolerance and provide important references for breeding new crop varieties with strong stress resistance. Summary of the Invention

[0005] The purpose of this invention is to provide the barley HvWOX8 gene, a recombinant expression vector containing the barley HvWOX8 gene, and the application of the gene in promoting plant salt tolerance.

[0006] In a first aspect, the present invention provides the application of the barley HvWOX8 gene in plant breeding, wherein the nucleotide sequence of the CDS region of the barley HvWOX8 gene is shown in SEQ ID No. 1.

[0007] Cloning and analysis of the HvWOX8 gene: The present invention identifies and clones the gene HvWOX8 from cultivated barley “Hua 30”. The full length of the CDS region of the gene is 624 bp, which encodes a 208aa protein sequence with a molecular weight of 23.42 kDa and an isoelectric point pI = 7.11.

[0008] Furthermore, the plant breeding described herein is for promoting salt tolerance in plants.

[0009] Furthermore, the plant in question is barley or Arabidopsis thaliana.

[0010] In a second aspect, the invention provides the application of primer pairs for amplifying the barley HvWOX8 gene in plant breeding, the nucleotide sequences of which are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

[0011] In a third aspect, the present invention provides the application of a recombinant expression vector containing the barley HvWOX8 gene in plant breeding, wherein the nucleotide sequence of the CDS region of the barley HvWOX8 gene is shown in SEQ ID No. 1.

[0012] Furthermore, the recombinant expression vector comprises an original vector and a target gene inserted into the original vector, the base sequence of which is shown in SEQ ID No. 1.

[0013] Furthermore, the promoter that initiates the expression of the target gene is the 35S strong promoter.

[0014] Furthermore, the original carrier is pH2GW7.

[0015] Furthermore, the recombinant expression vector was constructed as follows: the barley HvWOX8 gene was ligated into the plant expression vector pH2GW7 using the Gateway system (Invitrogen). The promoter for initiating the expression of the barley HvWOX8 gene was a strong promoter. A strong promoter can initiate the overexpression of the barley HvWOX8 gene. Preferably, the strong promoter is 35S.

[0016] In a fourth aspect, the present invention provides the application of a transformant containing a recombinant expression vector of the barley HvWOX8 gene in plant breeding.

[0017] Furthermore, the host bacteria of the transformant are Agrobacterium GV3101, LBA4404, AGL1, or EHA105.

[0018] This invention provides the application of the barley HvWOX8 gene in enhancing plant salt tolerance. The invention involves cloning the barley HvWOX8 gene and genetically transforming it in Arabidopsis thaliana. Overexpression of the exogenous barley HvWOX8 gene, followed by treatment with 50mM and 100mM NaCl, resulted in significantly longer root lengths compared to the wild type, demonstrating a significant improvement in the salt tolerance of Arabidopsis thaliana. Therefore, HvWOX8 holds promise for use in genetic engineering breeding, potentially enhancing the salt tolerance of barley. This invention provides a theoretical basis and related genes for salt-tolerant barley breeding and production.

[0019] The advantages of this invention are:

[0020] This invention, through the cloning and analysis of the barley HvWOX8 gene, combined with phenotypic analysis in Arabidopsis thaliana, demonstrates that the barley HvWOX8 gene can significantly increase the salt tolerance of Arabidopsis thaliana. This invention provides a theoretical basis and related genes for barley salt-tolerant breeding and production. Attached Figure Description

[0021] Figure 1 The expression level of the HvWOX8 gene in the Arabidopsis transgenic material that overexpressed the HvWOX8 gene in Example 2 is shown.

[0022] Figure 2 The root length phenotype of Arabidopsis transgenic material overexpressing the HvWOX8 gene in Example 3 after treatment with different concentrations of NaCl for 7 days (bar = 1 cm).

[0023] Figure 3 The changes in root length of Arabidopsis thaliana transgenic material overexpressing the HvWOX8 gene in Example 3 after treatment with different concentrations of NaCl. Detailed Implementation

[0024] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0025] Example 1: Construction of barley HvWOX8 gene overexpression vector and genetic transformation in Arabidopsis thaliana

[0026] (1) Take barley “Hua 30” immature embryos, extract RNA, and use the Trizol method (Invitrogen) according to the instructions. Take 1 μg of RNA for reverse transcription to obtain cDNA.

[0027] (2) Design specific primers based on the HvWOX8 gene sequence, and add restriction enzyme sites to the 5' end of the primers, as shown in the following sequences:

[0028] HvWOX8-CDS-F:5'-CGGAATTCTACGCCCTCAATCACCTTTA-3' (SEQ ID No. 2);

[0029] HvWOX8-CDS-R:5'-CGGGATCCGCCTTCACCAAAGAAGTCAT-3' (SEQ ID No. 3);

[0030] (3) PCR amplification was performed using full-length cDNA as a template.

[0031] The KOD high-fidelity enzyme (TOYOBO) system was used.

[0032]

[0033] The PCR cycling conditions were as follows: 94℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 68℃ extension for 90 s, 35 cycles of amplification, and a final extension at 68℃ for 5 min to terminate the reaction.

[0034] (4) After the PCR product was recovered by gel electrophoresis, it was ligated into the Gateway cloning system entry vector NT007 by enzyme digestion, transformed into Escherichia coli DH5α, and the positive clones were sequenced and analyzed. The HvWOX8 gene sequence is shown in SEQ ID No.1.

[0035] (5) The NT007 plasmid containing the full-length CDS of HvWOX8 that was correctly sequenced was named HvWOX8-NT007. The plasmid was then subjected to LR exchange reaction with the overexpression vector pH2GW7.

[0036] Using Gateway LR Clonase™ Enzyme Mix (Invitrogen, Cat. No. 11791-019), the system is as follows:

[0037]

[0038] Mix briefly by vortexing twice, centrifuge gently, incubate at 25°C for 2 hours, then add 1 μL of Proteinase K solution and mix well. Incubate at 37°C for 10 minutes to end the reaction.

[0039] 5 μL of the reaction product was transformed into E. coli DH5α. Positive clones were selected for PCR verification and sequenced. After the sequencing was confirmed to be correct, the plasmid was extracted and named 35S:HvWOX8.

[0040] (6) Take out LBA4404 competent cells from the -80℃ freezer and transform Agrobacterium LBA4404 using the heat shock method.

[0041] Thaw competent cells on ice, add 5 μL of the target plasmid to the competent cells, mix well, and place on ice for 30 min. Freeze centrifuge tubes in liquid nitrogen for 1 min, then place them in a 37°C water bath until thawed. Add 1 mL of LB liquid nutrient medium and shake to culture for 2-3 h. After centrifugation, spread the culture onto YEP resistant plates containing 50 mg / L Rifampicin and 50 mg / L Spectinomycin Hydrochloride, and incubate upside down at 28°C for 2 nights until single colonies grow.

[0042] (7) Pick a single Agrobacterium positive clone verified by PCR and add it to 5 ml of YEP liquid medium containing the corresponding antibiotic. Incubate at 28°C with shaking for 48 h. Add the shaken bacteria to YEP liquid medium (containing the corresponding antibiotic) at a volume ratio of 1:50 and incubate at 28°C until OD reaches 0.50. 600 =1.0-1.2.

[0043] (8) Arabidopsis thaliana was transformed by the Arabidopsis thaliana flower soaking method.

[0044] Remove the pods, leaving only the inflorescence, and place the whole Arabidopsis plant, along with the seedling tray, upside down in the bacterial solution that has been transformed with Agrobacterium tumefaciens. Soak the seedlings for 5 minutes, shaking the bacterial solution continuously during this time.

[0045] After infection, remove the plant, place it on its side in a tray, cover it with a black plastic sheet to block light, and place it in an incubator. Uncover the plastic sheet after 24 hours.

[0046] Arabidopsis plants were cultivated under natural light and watered 1-2 times a week. Once the seeds matured, the T0 generation seeds were harvested.

[0047] Example 2: Screening of transgenic barley lines overexpressing HvWOX8

[0048] (1) After sterilizing the harvested T0 generation seeds, they were planted in MS medium supplemented with 35 mg / L hygromycin. After vernalization at 4°C in the dark for 3 days, the culture dishes were placed in an artificial climate chamber for cultivation, and the growth of Arabidopsis plants was observed. Transgenic seeds with hygromycin resistance will grow in the selection medium, while non-transgenic seeds will not grow after germination.

[0049] (2) Transplant the selected transgenic plants into nutrient soil. When the plants bolt, cut about 100 mg of Arabidopsis thaliana leaves and place them in a 1.5 ml centrifuge tube to extract DNA. Perform PCR verification to check whether the selected plants are positive.

[0050] (3) After harvesting T1 generation seeds from individual plants that tested positive, sterilize and disinfect them, plant them in MS + 35 mg / L hygromycin solid medium, vernalize them in the dark at 4℃ for 3 days, and then place the culture dishes in an artificial climate chamber for cultivation.

[0051] (4) Transgenic plants resistant to hygromycin were transplanted into nutrient soil. When the plants bolted, about 100 mg of Arabidopsis thaliana leaves were cut from each line to extract RNA, which was then reverse transcribed to obtain cDNA. The expression of the HvSERK1 gene in different Arabidopsis thaliana transgenic lines was analyzed by real-time quantitative PCR using SYBR Green and a PCR instrument, and the expression values ​​were corrected using the internal reference gene TUB2.

[0052] The real-time PCR system is as follows:

[0053]

[0054] The PCR program was: 95℃ for 1 min, 95℃ for 5 s, 60℃ for 20 s, for 40 cycles. Each experiment was repeated three times. The primer sequences for quantitative real-time PCR are as follows:

[0055] HvWOX8-RT-PCR-F: 5'-GGAGGCCACAAGATCACAGC-3' (SEQ ID

[0056] No. 4);

[0057] HvWOX8-RT-PCR-R: 5'-GGTCTCCGAGATCTGTCCGT-3' (SEQ IDNo. ​​5);

[0058] AtTUB2-F: 5'-GTTCTCGATGTTGTTCGTAAG-3' (SEQ ID No. 6);

[0059] AtTUB2-R: 5'-TGTAAGGCTCAACCACAGTAT-3' (SEQ ID No. 7).

[0060] (5) The relative expression levels of HvWOX8 in different Arabidopsis transgenic lines are shown in the following results. Figure 1 As shown in the figure. The two lines with the highest expression levels, 35S:HvWOX8-2 and 35S:HvWOX8-18, were selected, and T2 generation seeds were harvested.

[0061] Example 3: Observation of salt tolerance phenotype in Arabidopsis thaliana

[0062] (1) Wild-type Arabidopsis thaliana and T2 generation HvWOX8 transgenic seeds (35S:HvWOX8-2, 5S:HvWOX8-18) were sterilized and vernalized in the dark at 4℃ for 3 days. They were then sown on MS solid medium containing different concentrations (0, 50, 100, 150 mM) of NaCl. The culture dishes were placed vertically and cultured in an artificial climate chamber (temperature: 20±2℃; light intensity: 90-120 μmol / m²). -2 sec -1 Photoperiod: 16 hours of light / 8 hours of darkness.

[0063] (2) After culturing in an artificial climate chamber for 3 days, the root length of Arabidopsis thaliana was measured until it reached 7 days, and the changes in root length were observed daily.

[0064] (3) Image Pro Plus software was used to perform statistical analysis on root length, with at least 20 roots measured for each line and treatment. For example... Figure 2 and Figure 3 As shown in the figure. The results showed that in the 0 mM NaCl treatment, there was no difference in root length between wild-type and overexpression lines; in the 50 mM NaCl treatment, the root length of the overexpression lines was significantly longer than that of the wild-type; in the 100 mM NaCl treatment, starting from day 5, the root length of the overexpression lines was significantly longer than that of the wild-type; in the 150 mM NaCl treatment, there was no difference in root length between the wild-type and overexpression lines. This indicates that overexpression of HvWOX8 can enhance the salt tolerance of Arabidopsis thaliana.

[0065] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Barley HvWOX8 The application of genes in plant breeding is characterized by, The barley HvWOX8 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID No. 1; the plant is Arabidopsis thaliana; the barley is... HvWOX8 The gene is overexpressed in plant breeding; the plant breeding mentioned is for promoting salt tolerance in plants.

2. The application according to claim 1, characterized in that, Used to expand barley HvWOX8 The nucleotide sequences of the primer pairs for the gene are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

3. A substance containing barley HvWOX8 The application of recombinant gene expression vectors in plant breeding is characterized by, The plant in question is Arabidopsis thaliana, and the barley is... HvWOX8 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID No. 1; the barley mentioned HvWOX8 The gene is overexpressed in plant breeding; the plant breeding mentioned is for promoting salt tolerance in plants.

4. The application according to claim 3, characterized in that, The recombinant expression vector includes an original vector and a target gene inserted into the original vector, the base sequence of which is shown in SEQ ID No.

1.

5. The application according to claim 4, characterized in that, The promoter for initiating the expression of the target gene is a strong 35S promoter; the original vector is pH2GW7.

6. A substance containing barley HvWOX8 The application of transformants from recombinant gene expression vectors in plant breeding is characterized by, The plant in question is Arabidopsis thaliana, and the barley is... HvWOX8 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID No. 1; the barley mentioned HvWOX8 The gene is overexpressed in plant breeding; the plant breeding mentioned is for promoting salt tolerance in plants.

7. The application according to claim 6, characterized in that, The host bacteria of the transformants are Agrobacterium GV3101, LBA4404, AGL1 or EHA105.

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