Abelmoschus manihot MYB transcription factor and application thereof

Abstract

The invention discloses an Abelmoschus manihot MYB family member HmMYB73 gene and an application of the Abelmoschus manihot MYB family member HmMYB73 gene in Arabidopsis thaliana polysaccharide synthesis and salt stress tolerance. The nucleotide sequence of the HmMYB73 gene is as shown in SEQ ID NO: 1. The recombinant vector pCAMBIA1303-HmMYB73 comprises a nucleotide sequence as shown in SEQ ID NO: 1 (Sequence Identity Number 1). The gene participates in synthesis of polysaccharide in transgenic arabidopsis thaliana and tolerance to salt stress, the transgenic arabidopsis thaliana is created through a genetic engineering means, and a basis is provided for development and utilization of abelmoschus manihot.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a MYB transcription factor of golden sunflower and application thereof in improving polysaccharide content and salt tolerance of Arabidopsis thaliana. Background Art

[0002] Sugar plays an indispensable role in the basic physiological processes of living organisms, especially in energy production and metabolism. Plants use sugar to build their structural components, and sugar can also act as a signaling molecule to participate in the key processes of plant life stages from germination to aging. Active polysaccharides have anti-tumor, immunomodulatory, antioxidant, antiviral and liver protection effects. The wide range of biological activities and unique properties of active polysaccharides make them the focus of research in the fields of biochemistry, agricultural science and medicine.

[0003] Hibiseu manihot L., an annual herbaceous plant of the Malvaceae family, also known as hollyhock and okra, has great 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, and has excellent performance in anti-oxidation, inhibiting free radicals, resisting cancer, regulating the immune system, and lowering blood lipids and blood pressure. However, the current research on Hibiseu manihot in my country is still in its early stages, and its potential value still needs to be deeply explored.

[0004] The MYB transcription factor family of plants is a class of vital DNA-binding proteins. Their structural characteristics are highly conserved peptides consisting of 50 to 52 amino acids, which appear repeatedly. The core DNA-binding domain is the MYB domain. According to the number of its 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 the growth and development of 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 MYBC1 transcription factors can regulate the content of anthocyanins in leaves, flowers and seeds of corn. However, there is no functional research report on the MYB transcription factor family in golden sunflower. Summary of the invention

[0005] In view of the problems in the prior art, the present invention cloned the HmMYB73 gene of Helichrysum chinensis, constructed a plant overexpression vector pCAMBIA1303-HmMYB73, transformed wild-type Arabidopsis to obtain transgenic Arabidopsis, and improved the soluble sugar content and salt stress tolerance in the transgenic Arabidopsis.

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

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

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

[0009] (c) a polynucleotide having at least 90% homology with 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, and the protein encoded by the polynucleotide mutant still has the activity of increasing polysaccharide content and salt tolerance.

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

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

[0013] (b) an amino acid having at least 90% homology with the amino acid shown in SEQ ID No: 2; or

[0014] (c) A protein mutant obtained by deleting, replacing or inserting one or more amino acids on the basis of 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 comprising the HmMYB73 gene.

[0016] An engineered bacterium containing a vector of the HmMYB73 gene.

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

[0018] The HmMYB73 gene is used in cultivating Arabidopsis thaliana with high polysaccharide content.

[0019] The application of the HmMYB73 gene in cultivating salt-tolerant Arabidopsis thaliana.

[0020] A method for increasing the polysaccharide content in Arabidopsis thaliana, and increasing the expression level of HmMYB73 protein in Arabidopsis thaliana.

[0021] A method for improving the salt tolerance of Arabidopsis thaliana, and improving the expression level of HmMYB73 protein in Arabidopsis thaliana.

[0022] Beneficial effects of the present invention: The present invention cloned a gene HmMYB73, a member of the MYB family of golden sunflower, and found that it affects the polysaccharide synthesis of Arabidopsis thaliana and enhances tolerance to salt stress. Transgenic Arabidopsis thaliana was created through genetic engineering means, providing a basis for the development and utilization of golden sunflower. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the relative expression level of HmMYB73 gene in different tissues of Helichrysum chinensis.

[0024] Figure 2 This is the gel electrophoresis diagram of HmMYB73 gene cloning, and M represents DL 2000 Marker.

[0025] Figure 3 It is a phylogenetic evolutionary tree diagram, where Hm is Heliotropium chrysantha, At is Arabidopsis thaliana, Ga is soybean, Gr is cotton, Ht is Cottonseed, Pb is Populus alba, Ca is Hazelnut, Qs is Boxwood Oak, Mi is Mango, Zj is Date, Hb is Rubber Tree, Jc is Jatropha Curcas, 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 To identify the soluble sugar content and polysaccharide yield of wild-type and transgenic Arabidopsis, A represents the soluble sugar content of wild-type and transgenic Arabidopsis; B represents the polysaccharide yield of wild-type and transgenic Arabidopsis; Col and OH-1, OH-2, and OH-3 represent wild-type Arabidopsis and transgenic Arabidopsis with HmMYB73 gene, respectively.

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

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

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0031] Example 1

[0032] 1. Extraction of total RNA from Helichrysum chrysantha and quantitative PCR detection

[0033] RNA was extracted from the roots, stems, leaves and flowers of Helichrysum chrysantha during the flowering period according to the instructions of RNAprep Pure Plant Total RNA Extraction Kit of Beijing TIANGEN Company. The RNA content was detected using Nano100C instrument of Hangzhou Aosheng Company, and the integrity was detected by 1.2% agarose gel electrophoresis.

[0034] Strictly follow TaKaRa's PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit instructions: Reverse transcribe the RNA of Helichrysum truncatum to obtain cDNA. Store the reverse transcribed cDNA in a -20℃ refrigerator for later use.

[0035] By consulting the transcriptome data of Helichrysum chinensis, the fluorescent quantitative primers (HmMYB73-qF and HmMYB73-qR) were designed using Primer 5.0 software, and the relative expression of HmMYB73 was analyzed using Bio-Rad's SsoAdvanced Universal SYBR Green Supermix. The Helichrysum chinensis AeActin sequence was used as the internal reference gene, and quantitative analysis was performed on the CFX96 Real-Time PCRDetection System instrument (Bio-Rad, USA). The real-time fluorescence quantitative PCR (RT-qPCR) reaction was as follows: 95℃30s, 95℃10s, 60℃20s, 40 cycles.

[0036] The primer sequences used for HmMYB73 fluorescence quantitative verification are:

[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:

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

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

[0042] Adoption 2 -△△CT The relative expression of HmMYB73 gene in different tissues was calculated by Figure 1 : HmMYB73 is expressed in stems, leaves, flowers and seeds.

[0043] 2. Cloning of HmMYB73 gene from Heliotropium chrysantha

[0044] By consulting the transcriptome data of Helianthus annuus, cloning primers (HmMYB73-F and HmMYB73-R) were designed for the HmMYB73 gene. HmMYB73 was cloned by PCR using Helianthus annuus cDNA as a template. The polymerase used for cloning the HmMYB73 gene was Phanta Super-Fidelity DNA Polymerase from Nanjing Vazyme Company. The reaction system was as follows: 1.0 μL cDNA (template), 6 μL Premix Taq (containing dNTAs, Mg 2+ ), 1.0 μL forward and reverse primers (10 μM), 16 μL sterile water, total volume 25 μL. PCR reaction was as follows: 95°C for 1 min, 95°C for 10 s, 60°C for 20 s, 72°C for 50 s for 30 cycles, 72°C for 10 min, 4°C for 9 min.

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

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

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

[0048] The PCR product was detected by 1% agarose gel electrophoresis, and then the target fragment was recovered and purified by Shanghai Biotech's SanPrep column DNA gel recovery kit. The purified product was connected to the pMD19-T-PfABI4 and pMD19-T-PfWRI1 vectors and transformed into competent E. coli DH5α. The transformed E. coli was cultured on IXA solid medium for 16 hours, and a single white colony was picked and cultured in LB liquid medium (containing Amp 50mg / mL) on a shaker for 12 hours. After the bacterial liquid was verified by PCR, it was sent to Shanghai Biotech for sequencing. The results are as follows Figure 2 The nucleic acid sequence of HmMYB73 is shown in SEQ ID NO.1, which can encode an amino acid sequence of 290 in length as shown in SEQ ID NO.2.

[0049] The following sequences were downloaded from TAIR and NCBI databases: Arabidopsis thaliana (AT4G37260.1), Glycine max (NP_001237778.1), Gossypium raimondii (XP_012451049.1), Hibiscus trionum (GMI77414.1), Populus alba x Populus xberolinensis (KAJ6939820.1), Corylus avellana (XP_059454602.1), Quercus suber (XP_044476069.1), Mangifera indica (XP_044476069.1), Ziziphus jujuba, XP_015884347.2), rubber tree (Hevea brasiliensis, XP_021678755.2), Jatropha curcas (Jatropha curcas, NP_001295712.1), almond (Prunus dulcis, XP_034200303.1), durian (Durio zibethinus, XP_022772895.1), wheat (Triticum aestivum, XP_044420055.1) protein sequences of MYB subfamily S22. After multiple sequence alignment by BLAST, MEGA4.0 was used to construct the phylogeny of HmMYB73. The results are shown in Figure 3As shown, HmMYB73 is most closely related to MtMYB73 of Caryophyllaceae.

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

[0051] Primers were designed using pMD19-T-HmMYB73 plasmid as a template. By aligning the restriction site of pCAMBIA1303, restriction sites and protective bases were added at both ends. The primer sequences are:

[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 solution sequence was subjected to bacterial solution PCR, and after electrophoresis detection, it was purified and recovered using a gel recovery kit to obtain the HmMYB73 gene sequence containing part of the vector sequence. pCAMBIA1303 was double-digested with 50 μL of the following system: 10X QuickCut Buffer 5 μL, DNA 5 μL, Bgl II 1 μL, Spe I 1 μL, ddH2O 38 μL, and reacted at 37°C for 5 min. Then, the HmMYB73 gene with restriction sites and protective bases was connected to the linear pCAMBIA1303 vector after restriction digestion. The connection system was: 2 μL of linear pCAMBIA1303 plasmid, 3 μL of template DNA, 0.2 μL of 5X In-Fusion HDEnzyme Premix, and 4.8 μL of sterile water. The constructed plant expression vector pCAMBIA1303-HmMYB73 was transformed into Agrobacterium GV3101 by freeze-thaw method, plated on a resistant plate containing kanamycin, and single colonies were picked and sequenced correctly to extract plasmid pCAMBIA1303-HmMYB73. The identification primers selected were 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 Obtaining transgenic Arabidopsis thaliana using Agrobacterium transformation

[0056] 1. Integration of plant expression vectors into the Arabidopsis genome

[0057] After the Arabidopsis thaliana with good growth condition blooms for 7 days, cut off the blooming flowers and fruit pods on the Arabidopsis thaliana plants and use them for the infection experiment the next day. Inoculate the Agrobacterium containing the target gene vector in 10mL LB+Kan liquid culture medium and culture for 12h for activation. The constant temperature shaking condition is set to: 200rpm, 28℃. Prepare a sterilized shaking bottle again, add 20mL LB+Kan liquid culture medium, then absorb 1mL of activated Agrobacterium, shake and culture for 6h, add 40mL LB liquid culture medium to the shaking bottle, and continue to culture until OD 600 =0.8. Collect Agrobacterium cells by centrifugation at 4000rpm for 10min. After centrifugation, discard the supernatant. Add 5g sucrose and 10μL Silwet L-77 to every 100mL ddH2O to prepare a suspension. Mix well before use. Then add the suspension to the Agrobacterium precipitate and use a disposable rubber-tipped dropper to suspend the cells until the solution is turbid and the OD is 600 =0.8. Soak the inflorescence of Arabidopsis in the suspension containing Agrobacterium for 90s, blowing the liquid surface to make it flow, give enough water to the infected Arabidopsis, use a spray bottle to spray water on the inner wall of the plastic cover to ensure the internal environmental humidity, and cover the infected Arabidopsis with a light-proof plastic cover. After 15 hours of light-proof treatment, the Arabidopsis will resume normal growth under long-day conditions. After 7 days, repeat the above operation once. Harvest the mature seeds of Arabidopsis after infection, use a medium containing 1 / 2MS+Hyg to screen the T1 generation of positive plants that can root normally and have green leaves, and transplant them to polycomposite soil for further cultivation. Repeat the 1 / 2MS+Hyg resistance plate screening, PCR molecular identification and GUS staining operations until the T4 generation of homozygotes with no trait separation is harvested.

[0058] 2. Identification of transgenic plants overexpressing HmMYB73

[0059] gDNA was extracted from the leaves of 25-day-old Arabidopsis thaliana transfected with the HmMYB73 gene in the T0 generation, and the gDNA of wild-type Arabidopsis thaliana was used as a negative control for bacterial liquid 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 the transgenic Arabidopsis thaliana could be successfully amplified, so it was initially considered that the Arabidopsis thaliana overexpressing HmMYB73 was successfully transformed. Then, after three 1 / 2MS+Hyg resistance plate screenings and molecular identification, the T4 homozygous line was obtained. We obtained a total of three T4 generation homozygous transgenic positive seedlings, named OH-1, OH-2, and OH-3. Subsequent experiments were all based on T4 homozygous seedlings.

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

[0061] 1. Identification of polysaccharide content in HmMYB73-transfected Arabidopsis

[0062] After the T4 positive seedlings were treated with seedlings, they were transplanted into pots for cultivation. They were grown naturally, and when they reached maturity, the total sugars of the overexpressing HmMYB73 strain and the wild-type Arabidopsis were extracted and compared. Figure 5 The results showed that the yield of transgenic Arabidopsis polysaccharides increased by 15.90-38.63% compared with that of wild-type Arabidopsis; the average polysaccharide content of wild-type Arabidopsis was 17.30%, and the polysaccharide contents of the three transgenic Arabidopsis strains OH-1, OH-2, and OH-3 increased by 26.60%, 27.9%, and 34.68%, respectively, compared with Col.

[0063] 2. Identification of salt stress tolerance of HmMYB73 transgenic Arabidopsis

[0064] The seeds of OH strain were planted on 1 / 2MS medium containing 0 mM, 50 mM and 100 mM NaCl for germination and growth. Figure 6 As shown in Figures AC, when 50mM NaCl is present, the germination of wild-type Arabidopsis seeds is affected and the germination time is prolonged. When 100mM NaCl is present, the germination of wild-type Arabidopsis seeds is more affected. In general, high salt concentrations inhibit the germination of Col and prolong the germination time, while the OH strain is less affected by salt stress, only showing slow germination in the early and middle stages. Figure 6 D and Figure 7 As shown in the figure, when high concentration of salt exists, the cotyledon greening rate of the transgenic plant line overexpressing HmMYB73 is greater than that of the wild type, indicating that the HmMYB73 gene can improve the resistance of plants to salt stress.

[0065] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. HmMYB73 gene, characterized in that The polynucleotide sequence of the HmMYB73 gene is as shown in (a), (b), (c) or (d): (a) a polynucleotide as shown in SEQ ID No: 1 in the sequence listing; or (b) a polynucleotide that can hybridize with the complementary sequence of SEQ ID No: 1 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of increasing polysaccharide content and salt tolerance; (c) a polynucleotide having at least 90% homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, and the protein encoded by the polynucleotide mutant still has the activity of increasing polysaccharide content and salt tolerance.

2. HmMYB73 protein, characterized in that The amino acid sequence of the HmMYB73 protein is as shown in (a), (b) or (c): (a) the amino acid sequence shown in SEQ ID No: 2 in the sequence listing; or (b) an amino acid having at least 90% homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, replacing or inserting one or more amino acids on the basis of the protein shown in SEQ ID No: 2, and the protein still has the activity of increasing polysaccharide content and salt tolerance.

3. A vector comprising the HmMYB73 gene according to claim 1.

4. An engineered bacterium comprising a vector containing the HmMYB73 gene according to claim 3.

5. Primers for detecting any fragment of the HmMYB73 gene according to claim 1.

6. Use of the HmMYB73 gene according to claim 1 in cultivating Arabidopsis thaliana with high polysaccharide content.

7. Use of the HmMYB73 gene according to claim 1 in cultivating salt-tolerant Arabidopsis thaliana.

8. A method for increasing the polysaccharide content of Arabidopsis thaliana, characterized in that: Improve the expression level of HmMYB73 protein in Arabidopsis thaliana.

9. A method for improving salt tolerance of Arabidopsis thaliana, characterized in that: Improve the expression level of HmMYB73 protein in Arabidopsis thaliana.

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