Golden sunflower MYB transcription factor and its application

Abstract

This invention discloses a gene of HmMYB73, a member of the MYB family of Arabidopsis thaliana, and its application in polysaccharide synthesis and salt stress tolerance in Arabidopsis thaliana. The nucleotide sequence of the HmMYB73 gene is shown in SEQ ID NO: 1. The recombinant vector pCAMBIA1303-HmMYB73 contains the nucleotide sequence shown in SEQ ID NO: 1. This gene participates in polysaccharide synthesis and salt stress tolerance in transgenic Arabidopsis thaliana, providing a foundation for the development and utilization of Arabidopsis thaliana through genetic engineering.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a golden hibiscus MYB transcription factor and its application in increasing the polysaccharide content and salt tolerance of Arabidopsis thaliana. Background Technology

[0002] Sugars play an indispensable role in the basic physiological processes of living organisms, especially in energy production and metabolism. Plants use sugars to build their structural components, and sugars also act as signaling molecules, participating in the regulation of key processes throughout the plant's life cycle, from germination to senescence. Bioactive polysaccharides possess anti-tumor, immunomodulatory, antioxidant, antiviral, and hepatoprotective effects. The broad bioactivity and unique properties of bioactive polysaccharides have made them a focus of research in biochemistry, agricultural science, and medicine.

[0003] Golden okra (Hibiseu manihot L.), an annual herbaceous plant belonging to the Malvaceae family and the Hibiscus genus, also known as yellow okra or simply okra, has significant development and utilization value in my country's food, medicine, agriculture, and industry. It is rich in flavonoids, unsaturated fatty acids, polysaccharides, and various trace elements, exhibiting remarkable performance in anti-oxidation, free radical inhibition, anti-cancer activity, immune system regulation, and lowering blood lipids and blood pressure. However, current research on golden okra in my country is still in its early stages, and further in-depth exploration of its potential value is needed.

[0004] The MYB transcription factor family in plants is a crucial class of DNA-binding proteins characterized by highly conserved peptides of 50 to 52 amino acids, which appear repeatedly. The core DNA-binding domain is the MYB domain. Based on the number of MYB domains, the MYB transcription factor family can be divided into four subfamilies: 1R-MYB (including MYB-related and R3 MYB), R2R3-MYB, 3R-MYB (R1R2R3-MYB), and the extremely rare 4R-MYB subfamily. The functional diversity of the MYB transcription factor family involves important areas such as growth and development in horticultural plants, primary and secondary metabolic processes, strategies for coping with biotic and abiotic stresses, and hormone synthesis and signal transduction. Studies have shown that the MYBC1 transcription factor can regulate the anthocyanin content in maize leaves, flowers, and seeds. However, there are currently no reports on the function of the MYB transcription factor family in *Hippophae rhamnoides*. Summary of the Invention

[0005] To address the problems in the existing technology, this invention cloned the HmMYB73 gene of *Hymenochloa chinensis* and constructed a plant overexpression vector pCAMBIA1303-HmMYB73. After transforming wild-type *Arabidopsis thaliana*, transgenic *Arabidopsis thaliana* was obtained, and the soluble sugar content and salt stress tolerance in the transgenic *Arabidopsis thaliana* were improved.

[0006] The HmMYB73 gene, the polynucleotide sequence of which is shown in (a), (b), (c) or (d):

[0007] (a) a polynucleotide as shown in SEQ ID No: 1; or

[0008] (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of increasing polysaccharide content and salt tolerance;

[0009] (c) A polynucleotide that is at least 90% homologous to the polynucleotide shown in SEQ ID No: 1; or

[0010] (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the activity of increasing polysaccharide content and salt tolerance.

[0011] The HmMYB73 protein, the amino acid sequence of which is shown in (a), (b), or (c):

[0012] (a) The amino acid sequence as shown in SEQ ID No: 2; or

[0013] (b) Amino acids that are at least 90% homologous to the amino acid shown in SEQ ID No: 2; or

[0014] (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the activity of increasing polysaccharide content and salt tolerance.

[0015] A vector containing the HmMYB73 gene.

[0016] Engineered bacteria containing the HmMYB73 gene.

[0017] Primers for detecting any fragment of the HmMYB73 gene.

[0018] Application of the HmMYB73 gene in the cultivation of Arabidopsis thaliana with high polysaccharide content.

[0019] Application of the HmMYB73 gene in the cultivation of salt-tolerant Arabidopsis thaliana.

[0020] A method to increase the polysaccharide content in Arabidopsis thaliana and improve the expression level of HmMYB73 protein in Arabidopsis thaliana.

[0021] A method to improve salt tolerance in Arabidopsis thaliana by increasing the expression level of HmMYB73 protein in Arabidopsis thaliana.

[0022] The beneficial effects of this invention are as follows: This invention cloned the HmMYB73 gene, a member of the MYB family of Arabidopsis thaliana, and found that it affects the synthesis of Arabidopsis thaliana polysaccharides and enhances tolerance to salt stress. By using genetic engineering, transgenic Arabidopsis thaliana was created, providing a basis for the development and utilization of Arabidopsis thaliana. Attached Figure Description

[0023] Figure 1 The relative expression levels of the HmMYB73 gene in different tissues of *Hippophae rhamnoides*.

[0024] Figure 2 This is a gel electrophoresis image of the HmMYB73 gene clone, where M represents the DL 2000 Marker.

[0025] Figure 3 This is a phylogenetic tree diagram, where Hm is golden hibiscus, At is Arabidopsis thaliana, Ga is soybean, Gr is American cotton, Ht is sage, Pb is silver poplar, Ca is hazelnut, Qs is boxwood, Mi is mango, Zj is jujube, Hb is rubber tree, Jc is jatropha, Pd is almond, Dz is durian, and Ta is wheat.

[0026] Figure 4 To analyze the expression level of HmMYB73 in transgenic Arabidopsis thaliana using PCR.

[0027] Figure 5 The soluble sugar content and polysaccharide yield of wild-type and transgenic Arabidopsis thaliana were identified. In this study, A represents the soluble sugar content of wild-type and transgenic Arabidopsis thaliana; B represents the polysaccharide yield of wild-type and transgenic Arabidopsis thaliana; and Col and OH-1, OH-2, and OH-3 represent wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana, respectively.

[0028] Figure 6 The effect of NaCl on Arabidopsis seed germination is shown. A represents the seed germination rate of wild-type and transgenic Arabidopsis on 1 / 2 MS medium containing 0 mM NaCl; B represents the seed germination rate of wild-type and transgenic Arabidopsis on 1 / 2 MS medium containing 50 mM NaCl; C represents the seed germination rate of wild-type and transgenic Arabidopsis on 1 / 2 MS medium containing 100 mM NaCl; and D represents the cotyledon greening rate of wild-type and transgenic Arabidopsis on day 8 under different salt concentrations in culture media.

[0029] Figure 7Phenotypic observation of the effect of NaCl on Arabidopsis seed germination; where A represents the growth of wild-type Arabidopsis and transgenic Arabidopsis on 1 / 2 MS medium containing 0 mM NaCl; B represents the growth of wild-type Arabidopsis and transgenic Arabidopsis on 1 / 2 MS medium containing 50 mM NaCl; and C represents the growth of wild-type Arabidopsis and transgenic Arabidopsis on 1 / 2 MS medium containing 100 mM NaCl. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0031] Example 1

[0032] 1. Extraction and quantitative real-time PCR detection of total RNA from *Hippophae rhamnoides*

[0033] RNA was extracted from the roots, stems, leaves, and flowers of *Hibiscus rosa-sinensis* during its flowering period, following the instructions of the RNAprep Pure plant total RNA extraction kit from Beijing TIANGEN Company. The RNA content was detected using a Nano100C instrument from Hangzhou Aosheng Company, and the integrity was assessed using 1.2% agarose gel electrophoresis.

[0034] Strictly in accordance with TaKaRa's PrimeScript TM The instructions for the RT reagent kit with gDNA Eraser (Perfect Real Time) show how to reverse transcribe *Hymenochloa crus-galli* RNA to obtain cDNA. The resulting cDNA should be stored at -20°C for later use.

[0035] By reviewing the transcriptome data of *Hymenochloa crus-galli*, quantitative real-time PCR primers (HmMYB73-qF and HmMYB73-qR) were designed using Primer 5.0 software. The relative expression level of HmMYB73 was analyzed using Bio-Rad's SsoAdvanced Universal SYBR Green Supermix. The *Hymenochloa crus-galli* AeActin sequence was used as an internal reference gene, and quantitative analysis was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). The real-time quantitative PCR (RT-qPCR) reaction was as follows: 95℃ for 30 s, 95℃ for 10 s, 60℃ for 20 s, for 40 cycles.

[0036] The primer sequence used for HmMYB73 fluorescence quantitative verification is as follows:

[0037] HmMYB73-qF: 5'-AAGTCGTGTAGGCTTCGGTG-3' (SEQ ID NO.3)

[0038] HmMYB73-qR:5'-CGTGGGGCTCGGATAATCGTT-3'(SEQ ID NO.4)

[0039] The primer sequences used for the internal reference gene AeActin are as follows:

[0040] AeActin-F:5'-TCTTTCATCGGGATGGAAGC-3'(SEQ ID NO.5)

[0041] AeActin-R:5'-ACTGAGCACAATGTTACCGTAGAG-3'(SEQ ID NO.6)

[0042] Use 2 -△△CT The relative expression levels of the HmMYB73 gene in different tissues were calculated using the method described above. The results are shown in [Figure 1]. Figure 1 HmMYB73 is expressed in stems, leaves, flowers, and seeds.

[0043] 2. Cloning of the HmMYB73 gene in *Hymenochloa chinensis*

[0044] By reviewing the transcriptome data of *Hibiscus syriacus*, cloning primers (HmMYB73-F and HmMYB73-R) targeting the HmMYB73 gene were designed. Using *Hibiscus syriacus* flower cDNA as a template, HmMYB73 was cloned via PCR. The polymerase used for HmMYB73 gene cloning was Phanta Super-Fidelity DNA Polymerase from Vazyme (Nanjing). The reaction system was as follows: 1.0 μL cDNA (template), 6 μL Premix Taq (containing dNTAs, Mg... 2+ The reagents included 1.0 μL of forward and reverse primers (10 μM), 16 μL of sterile water, and a total volume of 25 μL. The PCR reaction was performed as follows: 30 cycles of 95℃ for 1 min, 95℃ for 10 s, 60℃ for 20 s, and 72℃ for 50 s, followed by 72℃ for 10 min and 4℃ for 9 min.

[0045] The primers used for PCR amplification of the full-length HmMYB73 gene are:

[0046] HmMYB73-F:5'-CTTGAGCAGTTGAGAATTAGGTTC-3'(SEQ ID NO.7)

[0047] HmMYB73-R:5'-CAACCACTCACCATGAGCG-3'(SEQ ID NO.8)

[0048] PCR products were detected by 1% agarose gel electrophoresis, and the target fragment was then recovered and purified using the SanPrep column-based DNA gel extraction kit from Shanghai Sangon Biotech. The purified product was ligated into the pMD19-T-PfABI4 and pMD19-T-PfWRI1 vectors and transformed into competent *E. coli* DH5α. The transformed *E. coli* were cultured on IXA solid medium for 16 h, and white single colonies were picked and cultured on LB liquid medium (containing 50 mg / mL of ampicillin) in a shaker for 12 h. After verification by colony PCR, the culture was sent to Shanghai Sangon Biotech for sequencing. The results are as follows: Figure 2 As shown in SEQ ID NO.1, the HmMYB73 nucleic acid sequence encodes a 290-amino acid sequence as shown in SEQ ID NO.2.

[0049] Downloaded the following species from the TAIR and NCBI databases: Arabidopsis thaliana (AT4G37260.1), soybean (Glycine max, NP_001237778.1), American cotton (Gossypium raimondii, XP_012451049.1), sweet privet (Hibiscus trionum, GMI77414.1), silver poplar (Populus alba x Populus xberolinensis, KAJ6939820.1), hazelnut (Corylus avellana, XP_059454602.1), boxwood oak (Quercus suber, XP_044476069.1), mango (Mangifera indica, XP_044476069.1), and jujube (Ziziphus jujuba). Protein sequences of the MYB subfamily S22 from the following trees were collected: *Jujuba jujuba* (XP_015884347.2), *Hevea brasiliensis* (XP_021678755.2), *Jatropha curcas* (NP_001295712.1), *Prunus dulcis* (XP_034200303.1), *Durio zibethinus* (XP_022772895.1), and *Triticum aestivum* (XP_044420055.1). Multiple sequence alignment was performed using BLAST, and a phylogenetic model of HmMYB73 was constructed using MEGA 4.0. The results are as follows: Figure 3As shown, HmMYB73 is most closely related to MtMYB73 of the Chinese bellflower.

[0050] Example 2: Construction of the pCAMBIA1303 recombinant vector and transformation of Agrobacterium GV3101

[0051] Primers were designed using pMD19-T-HmMYB73 plasmid as a template. Enzyme restriction sites and protective bases were added to both ends of the pCAMBIA1303 plasmid by alignment with its restriction sites. The primer sequences are as follows:

[0052] HmMYB73-IF-F:5'-TTGACCATGGTAGATCTGACTAGTGCTTCTACACGGAAAGATGTGGAT-3'(SEQ ID NO.9)

[0053] HmMYB73-IF-R:5'-GGTTTCTACAGGACGTAAACTAGTCTCGATTTTGCTAATGCCAATACG-3'(SEQ ID NO.10)

[0054] The pMD19-T-HmMYB73 bacterial culture sequence was subjected to bacterial PCR. After electrophoresis, the sequence was purified and recovered using a gel extraction kit to obtain the HmMYB73 gene sequence containing a portion of the vector sequence. pCAMBIA1303 was double-digested with the following 50 μL solutions: 5 μL 10X QuickCut Buffer, 5 μL DNA, 1 μL Bgl II, 1 μL Spe I, and 38 μL ddH2O. The reaction was carried out at 37°C for 5 min. The HmMYB73 gene with restriction sites and protective bases was then ligated to the digested linear pCAMBIA1303 vector. The ligation system consisted of: 2 μL linear pCAMBIA1303 plasmid, 3 μL template DNA, 0.2 μL 5X In-Fusion HDEnzyme Premix, and 4.8 μL sterile water. The constructed plant expression vector pCAMBIA1303-HmMYB73 was transformed into Agrobacterium GV3101 using the freeze-thaw method. The transformed cells were plated on kanamycin-containing resistant plates, and after successful sequencing of single colonies, the plasmid pCAMBIA1303-HmMYB73 was extracted. Identification primers included HmMYB73-IF-F and HmMYB73-IF-R, MI-F (5'-TTTCATTTGGAGAGAACACGGGGGA-3'SEQ ID NO.11) and MI-R (5'-CGCTGATCAATTCCACAGTTTTCGC-3'SEQ ID NO.12).

[0055] Example 3: Transgenic Arabidopsis thaliana was obtained using Agrobacterium-mediated transformation.

[0056] 1. Plant expression vector integrated into the Arabidopsis genome

[0057] Seven days after flowering, the fully opened flowers and pods of the Arabidopsis thaliana plants in good condition were removed, and the plants were used for the inoculation experiment the following day. Agrobacterium containing the target gene vector was inoculated into 10 mL of LB+Kan liquid medium and cultured for 12 h for activation. The constant temperature shaking conditions were set to 200 rpm and 28℃. A new sterile shaking flask was prepared, and 20 mL of LB+Kan liquid medium was added. Then, 1 mL of the activated Agrobacterium was aspirated and cultured on a shaking flask for 6 h. 40 mL of LB liquid medium was added to the shaking flask, and the culture was continued until OD (Organic Discharge) was reached. 600 =0.8. Collect Agrobacterium cells by centrifugation at 4000 rpm for 10 min. Discard the supernatant after centrifugation. Add 5 g sucrose and 10 μL Silwet L-77 to each 100 mL ddH2O to prepare a suspension, mix well and use. Then add the suspension to the Agrobacterium precipitate and suspend the cells using a disposable dropper until the solution is turbid and OD is 0.8. 600 =0.8. The inflorescences of Arabidopsis thaliana were immersed in a suspension containing Agrobacterium for 90 seconds, with continuous agitation to maintain flow. Sufficient water was applied to the infected Arabidopsis, and water was sprayed onto the inside of a plastic lid to ensure humidity. The infected Arabidopsis was then covered with a light-proof plastic lid and kept in the dark for 15 hours before resuming normal growth under long-day conditions. This process was repeated once after 7 days. Mature seeds of the infected Arabidopsis were harvested. Positive plants (T1 generation) with normal rooting and green leaves were selected using 1 / 2 MS + Hyg medium and transplanted into multi-layered soil for further cultivation. The 1 / 2 MS + Hyg resistance plate selection, PCR molecular identification, and GUS staining were repeated until homozygous T4 generation plants that did not exhibit phenotypic segregation were harvested.

[0058] 2. Identification of transgenic plants overexpressing HmMYB73

[0059] gDNA was extracted from leaves of 25-day-old Arabidopsis thaliana plants transfected with the HmMYB73 gene in the T0 generation. gDNA from wild-type Arabidopsis thaliana was used as a negative control for colony PCR verification. PCR amplification was performed using HmMYB73-IF-F and HmMYB73-IF-R, MI-F and MI-R as primers, respectively. Figure 4 The results showed that all transgenic Arabidopsis thaliana strains were successfully amplified, indicating that the overexpression of HmMYB73 Arabidopsis thaliana was successfully transformed. Then, after three rounds of 1 / 2 MS+Hyg resistance plate screening and molecular identification, T4 homozygous lines were obtained. We obtained three T4 generation homozygous transgenic positive seedlings, named OH-1, OH-2, and OH-3, respectively. Subsequent experiments all used T4 homozygous seedlings as material.

[0060] Example 4: Identification of polysaccharide content and salt stress tolerance in T4 generation Arabidopsis thaliana transgenic HmMYB73.

[0061] 1. Identification of polysaccharide content in Arabidopsis thaliana strain HmMYB73

[0062] T4 generation positive seedlings were treated with seedling cultivation and then transplanted into flower pots for culture. They were allowed to grow naturally until maturity. Total sugars were extracted from and compared between the HmMYB73 overexpressing line and wild-type Arabidopsis. Results are as follows... Figure 5 The results showed that the yield of transgenic Arabidopsis polysaccharides was 15.90–38.63% higher than that of wild-type Arabidopsis. The average polysaccharide content of wild-type Arabidopsis was 17.30%, while the polysaccharide content of the three transgenic Arabidopsis lines OH-1, OH-2, and OH-3 was 26.60%, 27.9%, and 34.68% higher than that of Col, respectively.

[0063] 2. Tolerance assessment of HmMYB73 Arabidopsis thaliana under salt stress

[0064] Seeds of the OH strain were planted on 1 / 2 MS medium containing 0 mM, 50 mM, and 100 mM NaCl, respectively, for germination and growth. Figure 6 As shown in Figure AC, the germination of wild-type Arabidopsis thaliana seeds was affected and the germination time was prolonged in the presence of 50 mM NaCl. The impact was even greater in the presence of 100 mM NaCl. In general, high salt concentrations inhibited Col germination and prolonged the germination time, while the OH strain was less affected by salt stress, only exhibiting slow germination in the early and middle stages. Figure 6 D and Figure 7 As shown, when high salt concentrations are present, the cotyledon chlorophyll rate of transgenic plants overexpressing HmMYB73 is greater than that of wild-type plants. This indicates that the HmMYB73 gene can enhance the plant's resistance to salt stress.

[0065] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. HmMYB73 Genes, characterized by, The HmMYB73 The polynucleotide sequence of the gene is shown in SEQ ID No:

1.

2. HmMYB73 protein, characterized in that, The amino acid sequence of the HmMYB73 protein is shown in SEQ ID No:

2.

3. Containing the contents of claim 1 HmMYB73 The carrier of genes.

4. Containing the contents of claim 3 HmMYB73 Engineered bacteria that act as vectors for genes.

5. The claim 1 HmMYB73 Application of genes in the cultivation of Arabidopsis thaliana with high polysaccharide content.

6. The claim 1 HmMYB73 Application of genes in the breeding of salt-tolerant Arabidopsis thaliana.

7. A method for increasing the polysaccharide content of Arabidopsis thaliana, characterized in that, To increase the expression level of the HmMYB73 protein described in claim 2 in Arabidopsis thaliana.

8. A method for improving the salt tolerance of Arabidopsis thaliana, characterized in that, To increase the expression level of the HmMYB73 protein described in claim 2 in Arabidopsis thaliana.

Images