Sugarcane transcription factor ScbZIP72 and application of coding gene thereof

Abstract

The invention discloses a sugarcane transcription factor ScbZIP72 and application of a coding gene of the sugarcane transcription factor ScbZIP72. The amino acid sequence of the sugarcane transcription factor ScbZIP72 is as shown in SEQ ID NO: 2, the nucleotide sequence of the coding gene of the sugarcane transcription factor ScbZIP72 is as shown in SEQ ID NO: 1, and the sugarcane transcription factor ScbZIP72 can significantly improve the drought resistance of plants. The sugarcane ScbZIP72 transcription factor and the coding gene thereof are ideal choices for transgenic molecular breeding engineering, are used for improving the drought resistance of the existing cultivated sugarcane variety and increasing the yield of sugarcane, can also be used for drought-resistant adversity stress transgenic breeding of monocotyledonous and dicotyledonous plants, and have important significance on transgenic crops for cultivating drought-resistant varieties. The method provides theoretical support for improving stress resistance of crops, and has important research value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to application of a sugarcane transcription factor ScbZIP72 and a coding gene thereof in improving plant drought resistance. Background Art

[0002] bZIP transcription factors are widely present in a variety of eukaryotic organisms such as plants, animals and insects, and are one of the largest transcription factor families in plants. The conserved domain of bZIP protein consists of a DNA binding domain and a leucine zipper dimer, which regulates plant growth and development and stress responses to various stresses by participating in the plant hormone signal transduction pathway. Previous studies have shown that the functions of the A subfamily members in the plant bZIP gene family are different from those of other subfamily members. Under the activation of ABA, the A subfamily members can bind to the ABRE cis-acting element and regulate the corresponding downstream genes to resist various abiotic stresses. Overexpression of bZIP transcription factor A subfamily members can enhance the tolerance of transgenic plants to abiotic stresses. Although the biological functions of many bZIP transcription factors have been clarified, there are still some new genes with functions to be developed, or new functions of known genes to be verified.

[0003] As a large grass C4 plant, sugarcane has a long growth cycle and a large demand for water. In my country's main sugarcane producing areas such as Guangxi Zhuang Autonomous Region, Yunnan and Guangdong, more than 85% of sugarcane is planted on dry slopes and hillsides, which are easily affected by drought. Drought can cause a reduction in sugarcane production of more than 60%, and drought has become an important limiting factor that seriously restricts the healthy development of my country's sugarcane industry.

[0004] At present, there is almost no research on the role of bZIP transcription factors in drought resistance in sugarcane, and there is no report on the research of sugarcane drought resistance-related genes. Therefore, studying the regulatory pathways and action mechanisms of related genes under drought stress is of great significance for breeding new drought-resistant varieties. Summary of the invention

[0005] Based on this, the object of the present invention is to provide a sugarcane transcription factor ScbZIP72 and an application of its encoding gene, wherein the transcription factor and the encoded protein can be used to improve the drought resistance of plants.

[0006] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] In a first aspect of the present invention, a use of a sugarcane transcription factor ScbZIP72 in improving plant drought resistance or plant drought resistance genetic breeding is provided. The amino acid sequence of the sugarcane transcription factor ScbZIP72 is shown in SEQ ID NO:2.

[0008] In a second aspect, the present invention provides an application of a gene encoding a sugarcane transcription factor ScbZIP72 in improving plant drought resistance or genetic breeding of plant drought resistance, wherein the nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO:1.

[0009] In a third aspect of the present invention, there is provided a primer for amplifying a gene encoding a sugarcane transcription factor ScbZIP72 for use in improving plant drought resistance or plant drought resistance genetic breeding, wherein the primer comprises an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO: 4.

[0010] In a fourth aspect, the present invention provides the use of a recombinant expression vector containing a gene encoding the sugarcane transcription factor ScbZIP72 in improving plant drought resistance or genetic breeding of plant drought resistance, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1.

[0011] The fifth aspect of the present invention provides the use of an engineered bacterium transformed with the above-mentioned recombinant expression vector in improving plant drought resistance or in genetic breeding of plant drought resistance.

[0012] The sixth aspect of the present invention provides a preparation for improving drought resistance of plants, wherein the active ingredient of the preparation includes the above-mentioned recombinant expression vector.

[0013] In a seventh aspect, the present invention provides a method for improving plant drought resistance, comprising the following steps: increasing the expression level of a gene encoding a sugarcane transcription factor ScbZIP72 in a plant, wherein the nucleotide sequence of the gene encoding the sugarcane transcription factor ScbZIP72 is shown in SEQ ID NO:1.

[0014] The present invention has the following beneficial effects:

[0015] The inventors of the present invention discovered a bZIP transcription factor ScbZIP72 involved in the regulation of sugarcane drought tolerance when studying the sugarcane genome. The drought resistance of plants overexpressing the sugarcane ScbZIP72 gene was significantly improved, and the action mechanism of the sugarcane ScbZIP72 gene involved in the regulation of sugarcane drought tolerance was revealed. Therefore, the sugarcane ScbZIP72 transcription factor and its encoding gene are ideal choices for transgenic molecular breeding engineering, which are used to improve the drought resistance of existing cultivated sugarcane varieties and increase the yield of sugarcane, provide genetic resources and molecular basis for cultivating new drought-resistant sugarcane varieties, and open up a new way to improve the drought resistance of sugarcane through genetic engineering. At the same time, it can also be used in the transgenic breeding of monocotyledonous and dicotyledonous plants resistant to drought stress, and provide theoretical support for cultivating drought-resistant transgenic crops and improving the stress resistance of crops, which has important research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1This is the gel electrophoresis diagram of the amplification of the sugarcane ScbZIP72 gene in Example 1 of the present invention, wherein M represents Trans2K Plus DNA Marker, and lanes 1 and 2 are PCR amplification samples.

[0017] Figure 2 It is an amino acid sequence alignment diagram of the sugarcane ScbZIP72 transcription factor in Example 2 of the present invention and the bZIP transcription factors of other species; wherein the bZIP conserved domain is represented by a red box, and the underline represents the nuclear localization signal.

[0018] Figure 3 This is a phylogenetic tree diagram of the sugarcane ScbZIP72 transcription factor in Example 2 of the present invention.

[0019] Figure 4 The expression levels of the sugarcane ScbZIP72 gene in various tissues of sugarcane plants under drought stress (A) and ABA (B) treatments in Example 3 of the present invention; the values ​​in the figure are mean values ​​± standard errors (n = 3), and different lowercase letters indicate significant differences compared with the control (P < 0.05).

[0020] Figure 5 The phenotypic analysis and ROS detection of transgenic Arabidopsis plants overexpressing sugarcane ScbZIP72 under drought stress in Example 4 of the present invention are shown; wherein A is the phenotypic observation result; B is the survival rate comparison result; C is the nitro blue tetrazolium (NBT) staining result, and D is the diaminobenzidine (DAB) staining result; the scale bar in the figure is 2 mm, and the data are expressed as mean ± SE (n = 3), and * and ** indicate that the difference reaches a significant level (P < 0.05) and an extremely significant level (P < 0.01), respectively.

[0021] Figure 6 The statistical results of the survival rate of transgenic sugarcane overexpressing sugarcane ScbZIP72 under drought stress in Example 5 of the present invention are shown, wherein ** indicates that the difference is at an extremely significant level (P<0.01). DETAILED DESCRIPTION

[0022] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. 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.

[0023] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0024] Unless otherwise specified, the examples are based on conventional experimental conditions, such as Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or the conditions recommended by the manufacturer's instructions. The raw materials and reagents used in the present invention are all commercially available, and any biological germplasm material can be provided to the outside for scientific research.

[0025] The present invention cloned the ScbZIP72 gene (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:2) from the sugarcane variety ROC22. After quantitative analysis, it was found that the gene was significantly induced by drought stress and ABA in the root and leaf tissues of sugarcane, indicating that ScbZIP72 is a drought stress-induced transcription factor. By overexpressing the sugarcane ScbZIP72 gene in Arabidopsis and sugarcane, it was verified that the gene has the function of responding to drought stress and can significantly improve the survival rate of transgenic Arabidopsis and transgenic sugarcane under drought stress. The ScbZIP72 transcription factor and its encoded protein discovered by the present invention have practical guiding significance in breeding new drought-resistant sugarcane varieties, provide a molecular basis for breeding new drought-resistant sugarcane varieties, open up a new way to improve the drought resistance of sugarcane by genetic engineering means, and also provide gene resources for drought-resistant molecular breeding and variety improvement of other crops.

[0026] In some embodiments of the present invention, a use of a sugarcane transcription factor ScbZIP72 in improving plant drought resistance is disclosed. The amino acid sequence of the sugarcane transcription factor ScbZIP72 is shown in SEQ ID NO:2.

[0027] In other embodiments of the present invention, an application of sugarcane transcription factor ScbZIP72 in plant drought resistance genetic breeding is disclosed.

[0028] In other embodiments of the present invention, disclosed is a use of a gene encoding a sugarcane transcription factor ScbZIP72 in improving plant drought resistance, wherein the nucleotide sequence of the gene encoding the sugarcane transcription factor ScbZIP72 is shown in SEQ ID NO:1.

[0029] In other embodiments of the present invention, disclosed is the use of a protein encoded by the sugarcane transcription factor ScbZIP72 in plant drought resistance genetic breeding.

[0030] In other embodiments of the present invention, disclosed is a use of primers for amplifying a gene encoding a sugarcane transcription factor ScbZIP72 in improving plant drought resistance, wherein the primers include an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO: 4.

[0031] In other embodiments of the present invention, disclosed is the use of a recombinant expression vector containing the gene encoding the sugarcane transcription factor ScbZIP72 in improving plant drought resistance.

[0032] In other embodiments of the present invention, disclosed is the use of an engineered bacterium transformed with the above-mentioned recombinant expression vector in improving the drought resistance of plants.

[0033] In other embodiments of the present invention, a preparation for improving drought resistance of plants is disclosed, wherein the active ingredient of the preparation includes the above-mentioned recombinant expression vector.

[0034] In other embodiments of the present invention, a method for improving plant drought resistance is disclosed, comprising the following steps: increasing the expression level of a gene encoding a sugarcane transcription factor ScbZIP72 in a plant.

[0035] In some embodiments, the plant is Arabidopsis thaliana or Sugarcane.

[0036] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Example 1 Cloning of Sugarcane ScbZIP72 Gene

[0038] The following steps are involved:

[0039] 1. Total RNA extraction from sugarcane leaves

[0040] Leaf samples of the sugarcane variety ROC22 were collected, cleaned and disinfected with 75% alcohol, placed in self-sealing bags, and stored in an ultra-low temperature refrigerator at -80°C.

[0041] The total RNA from sugarcane leaves was extracted using Trizol reagent. The absorbance and concentration of total RNA were determined using the American NanoVue ultra-micro spectrophotometer (GE Healthcare) protein and nucleic acid analyzer, and the integrity of total RNA was detected by electrophoresis.

[0042] 2. Sugarcane cDNA Synthesis

[0043] The DNase-treated sugarcane total RNA was purified by PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa, China) was used for reverse transcription into cDNA. The reaction system was 10 μL, including 1.0 μL Oligo dT Primer, 1.0 μL Random 6mers, 1.0 μL dNTP Mixture (10 mM each), and 2 μg total RNA. The reaction conditions were 65°C, 5 min. After cooling on ice, 4.0 μL 5× PrimeScript II Buffer, 0.5 μL RNase Inhibitor, 1.0 μL PrimeScript II RTase, and 4.5 μL RNase Free H2O were added to the above reaction solution. The reaction conditions were 30°C, 10 min; 42°C, 60 min; 70°C, 10 min. After the reaction, the next step of the experiment was carried out directly or stored at -80°C for future use.

[0044] 3. Cloning of ScbZIP72 gene

[0045] A pair of specific primers ScbZIP72-F (SEQ ID NO: 3): 5'-ATGGACGAGCTGCTCCGGAGCATC-3' and ScbZIP72-R (SEQ ID NO: 4): 5'-TCAAGATAATATACAGGGGTC-3' were designed by Primer Premier 5 software for cloning the ScbZIP72 gene sequence. 200 ng of total DNA was used as a template and PCR amplification was performed using the LATaq kit (TaKaRa, China). The PCR reaction system was as follows: 10×LA PCR Buffer (MgSO4) 2+ Plus) 5.0μL, dNTP Mixture (each 2.5mM) 8.0μL, ScbZIP72-F (10μM) 2.0μL, ScbZIP72-R (10μM) 2.0μL, LA Taq (5U / μL) 0.5μL, Pure H2O 31.5μL, DNA 1.0μL. The PCR reaction procedure is as follows: 94℃ pre-denaturation for 6min; 94℃ denaturation for 1min, 55℃ annealing for 1min, 72℃ extension for 1min, a total of 35 amplification cycles; final extension at 72℃ for 10min. After the PCR amplification reaction, 5μL of the PCR reaction product was taken for 1% agarose gel electrophoresis detection. The results are shown in Figure 1 As shown, the fragment size is between 800 bp and 900 bp, which is consistent with the expected target fragment size.

[0046] PCR reaction products were After purification with the Gel Extraction Kit (Omega, USA), the plasmid was connected to the pMD19-T cloning vector and transformed into the competent cells of the Escherichia coli host bacteria DH5α. 100 μL of the transformed bacterial solution was spread on a LB solid culture medium plate containing ampicillin (50 μg / mL). After culturing at 37°C in the dark overnight, several white single colonies were picked and inoculated into LB liquid culture medium containing ampicillin (50 μg / mL). The culture was shaken at 37.4°C in the dark for 6 to 8 hours, and 0.5 μL of the bacterial solution was taken for bacterial solution PCR detection. The reaction system was as follows: 10×LA PCRBuffer (Mg 2+ Plus) 2.5μL, dNTP Mixture (2.5mM each) 4.0μL, ScbZIP72-F (10μM) 1.0μL, ScbZIP72-R (10μM) 1.0μL, LA Taq (5U / μL) 0.25μL, Pure H2O 15.75μL, bacterial solution 0.5μL. The reaction procedure is as follows: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, a total of 30 amplification cycles; final extension at 72℃ for 10min. After bacterial solution PCR detection, 3 positive clones were selected for sequencing verification. After sequencing, the ligation product has the nucleotides shown in SEQ ID NO:1, which is named ScbZIP72, and the amino acid sequence of the transcription factor ScbZIP72 encoded by it is shown in SEQ ID NO:2.

[0047] SEQ ID NO:1

[0048] ATGGACGAGCTGCTCCGGAGCATCTGGACCGCGGAGGAGACCCAGGCCATGGCCTCCGCCTCTGCCGCAGGAGCGGGCGCGGGGATGCCGCCCACGCCGCTGCAGCGGCAGGGATCGTCCCTCACACTGCCCCGGACGCTCAGCGCCAAGACGGTCGACGAGGTGTGGCGCAATCTCGTGAGCGACGAGCCGCCGCAGGCGCAGGGGGCGGACGGCGGCGGACACCAGCAGCACCACCGCCAGTCGACGCTCGGGGAGATGACTCTGGAGGAGTTCTTGGTCAGAGCTGGCGTGGTCAGGGAGAACCCCGCTCCAGCTCCTCCAGCGCCACCACCCATGATGCCGCCGCGGCCGGTACCTGTTGCTCCTAAAAGCTCCGCCTTTTTCGGGAATTTACCGGGTGCCGACGATGCCGCAGCTGCGGCAGCGCTGGGGTTTGCACCTGTCGGCATGGGGGATCTGGCCATGATACCGCCCAGGGCGGCAGGCATGGGAGGCAGCGCCATGGCTGTGCAAACAGCGGTGAATCAGCAGCTTGATTCTGGCGGGAAGGGGTACAGCGACCTGTCGTCGCCGACGGAGTCGCTGCCTTTTTCGTTTGAAGGGATGATTCGGGGGAGAAGGCATGATGGAGGCGGAGTGGAGAAAGTGGTGGAGAGGCGGCAGAGGAGGATGATCAAGAACAGGGAGTCCGCCGCGAGGTCCCGAGCTCGCAAACAGGCTTATACAATGGAGTTAGAAGCTGAAGTTCAGAAACTCAAGGAGCAGAATCAGGAACTGGAGAGGAAACAGGCAGAGATTATGGAAATGCAGAAGAACGAGGTAACTGTTAATTCCAAACTTCCTGTGTTTGACCCCTGTATATTATCTTGA

[0049] SEQ ID NO:2

[0050] MDELLRSIWTAEETQAMASASAAGAGAGMPPTPLQRQGSSLTLPRTLSAKTVDEVWRNLVSDEPPQAQGADGGGHQQHHRQSTLGEMTLEEFLVRAGVVRENPAPAPPAPPPMMPPRPVPVAPKSSAFFGNLPGADDAAAAAALG FAPVGMGDLAMIPRAAGMGGSAMAVQTAVNQQLDSGGKGYSDLSSPTESLPFSFEGMIRGRRHDGGGVEKVVERRQRRMIKNRESAARSRARKQAYTMELEAEVQKLKEQNQELERKQAEIMEMQKNEVTVNSKLPVFDPCILS

[0051] Example 2 Bioinformatics analysis of sugarcane ScbZIP72 gene sequence

[0052] The sequence was aligned and spliced ​​using DNAMAN8.0 software, the open reading frame (ORF) of the ScbZIP72 transcription factor was confirmed using ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ), and the conserved domain of the transcription factor was predicted using SMART online software (http: / / smart.embl-heidelberg.de / ). Figure 2 As shown, Figure 2 The results showed that the ScbZIP72 protein has a typical bZIP conserved domain and belongs to a bZIP transcription factor. The amino acid sequence identity with maize ZmbZIP72 (HQ328839) and rice OsbZIP72 (NP_001063362) was 81.06% and 56.96%, respectively. The size and isoelectric point (pI) of the protein were predicted using the ProtParam tool online website (https: / / web.expasy.org / protparam / ). The predicted relative molecular mass of the protein was 31.03 kDa and the isoelectric point (pI) was 6.16.

[0053] The protein sequences of bZIP transcription factor A subfamily members in rice, corn and Arabidopsis were downloaded from the NCBI website, and aligned with the ScbZIP72 protein sequence using the Muscle operation method in MEGA6.0 software. The phylogenetic tree was constructed using the neighbor-joining method, and the bootstrap analysis was repeated 1000 times to evaluate its stability. The nodes with support less than 60% were omitted. The phylogenetic tree is shown in the figure below. Figure 3As shown, the analysis revealed that ScbZIP72 belongs to the A subfamily group of bZIP transcription factors and is most closely related to ZmbZIP72 and OsbZIP72 in evolution.

[0054] Example 3 Expression analysis of sugarcane ScbZIP72 gene under drought stress and ABA treatment

[0055] The following steps are involved:

[0056] 1. Sugarcane drought stress treatment and ABA treatment

[0057] The robust sugarcane variety New Taiwan Sugar No. 22 was selected for single bud warm water detoxification treatment, 52°C water bath for 30 minutes, and 1g / L carbendazim was added. After that, it was placed in an incubator for germination. The culture conditions were 16h light / 8h dark, 65% humidity and 30°C. The next stress treatment was carried out when the plant grew 3 to 5 leaves.

[0058] Sugarcane seedlings with similar growth were moved to clean water for cultivation. After new roots grew out in about 5 to 7 days, they were cultured in Hongland nutrient solution for 2 weeks. Seedlings with the same growth were selected and cultured in Hongland nutrient solution containing 25% PEG 6000 (simulating drought stress), and the sampling time points were 0h, 3h, 6h and 12h; in addition, 50μM ABA (containing 0.01% Tween-20, v / v) was sprayed on the leaves, and the sampling time points were 0h, 3h, 6h and 12h.

[0059] 2. Real-time fluorescence quantitative PCR detection

[0060] (1) RNA was extracted from sugarcane seedlings treated with drought stress and ABA using the TRIzol method. For specific steps, please refer to the instruction manual of TRIzol reagent (Invitrogen, USA).

[0061] (2) Remove residual DNA from total RNA. The reaction system is 10 μL, including 2.0 μL 5×gDNA Eraser Buffer, 1.0 μL gDNAEraser and 1 μg total RNA. The reaction conditions are 42°C and 2 min.

[0062] (3) Using the extracted total RNA as a template, PrimeScript TMRT reagent Kit with gDNAEraser kit (TaKaRa) was used for reverse transcription to obtain cDNA. 10 μL of reverse transcription solution was added to the above reaction solution for removing the residual DNA in the total RNA, including 1.0 μL PrimeScript RT Enzyme Mix I, 1.0 μL RT Primer Mix, 2.0 μL 5× PrimeScript Buffer 2, and 4.0 μL RNase Free dH2O. The reaction conditions were 37°C, 15 min; 85°C, 5 s. After the reaction was completed, the product was diluted 5 times and directly proceeded to the next step or stored at -80°C for later use.

[0063] (4) Real-time fluorescence quantitative PCR experiments were performed using QuantStudio 3 quantitative PCR instrument (Applied Biosystems, USA). Premix Ex Taq TM Fluorescence kit (TaKaRa) was used for qRT-PCR detection, and sugarcane GAPDH was selected as the internal reference gene to quantify the expression of ScbZIP72 gene. ScbZIP72 quantitative primers qScbZIP72-F (SEQ ID NO: 5): 5'-CAG GCTTATACAATGGAGTTAG-3' and qScbZIP72-R (SEQ ID NO: 6): 5'-AACA GTTACCTCGTTCTTCT-3', GAPDH quantitative primers qGAPDH-F (SEQ ID NO: 7): 5'-CACGGCCACTGGAAGCA-3' and qGAPDH-R (SEQ ID NO: 8): 5'-TCCTC AGGGTTCCTGATGCC-3'. The qRT-PCR reaction system contained 10μLTB Green Premix Ex Taq (2×), 0.4μL upstream and downstream primers (10μM, final concentration 0.2μM), 0.4μL ROX Reference Dye II and 1.0μL cDNA template. After mixing, centrifuge slightly and place in QuantStudio 3 quantitative PCR instrument for real-time fluorescence PCR reaction. The reaction procedure was: 95℃ pre-denaturation for 30s; 95℃ reaction for 15s, 60℃ reaction for 34s, 40 cycles. The experiment was set up with 3 biological replicates and 3 technical replicates.

[0064] The quantitative results of ScbZIP72 are shown in Figure 4 As shown in A, from Figure 4A in the figure shows that in the 25% PEG 6000 simulated drought stress experiment, after 3-6h of sugarcane plant treatment, the expression level of ScbZIP72 in the root tissue reached a high level, which was 2.6-2.7 times that of the 0h treatment group, and then the expression level decreased to the expression level of the 0h treatment group; in the stem tissue, the expression level of ScbZIP72 was significantly downregulated in the 3h treatment group, but increased sharply in the 6h treatment group, which was 4 times that of the 0h treatment group, and then the expression level in the 12h treatment group was downregulated to the same level as the 0h treatment group; in the leaf tissue, the expression level of ScbZIP72 was significantly higher than that of the 0h treatment group after 3-12h of PEG osmotic stress, especially in the 3h treatment group, reaching the highest level, which was 2.6 times that of the 0h treatment group.

[0065] ABA treatment results are as follows Figure 4 As shown in B, Figure 4 B in the figure shows that after 3 to 12 hours of ABA treatment, the expression of ScbZIP72 in root and leaf tissues was significantly upregulated, especially in root tissue, where the expression level of ScbZIP72 was 44 to 56 times that of the 0 hour treatment group; in leaf tissue, the expression level of ScbZIP72 was 3 to 17 times that of the 0 hour treatment group; there was no significant change in the expression level of ScbZIP72 in stem tissue.

[0066] The above results showed that the expression of ScbZIP72 in sugarcane root and leaf tissues was significantly induced by drought stress and ABA, indicating that sugarcane ScbZIP72 is a drought stress-induced transcription factor.

[0067] Example 4 Sugarcane ScbZIP72 gene enhances tolerance of transgenic Arabidopsis plants to drought stress

[0068] 1. Construction of ScbZIP72 recombinant expression vector

[0069] Primers 35S-ScbZIP72-F (SEQ ID NO: 9): 5′-GAGAGAACACGGATCCCCGGGTGGTCAGTCCCTTATGGACGAGCTGCTCCGGAGCATC-3′ and 35S-ScbZIP72-R (SEQ ID NO: 10): 5′-GATCGGGGAAATTCGAGCTCTCAAGATAATATACAGGGGTC-3′ designed by the seamless cloning primer design tool (http: / / 123.56.75.195 / ) were used to amplify the PCR fragment containing the linker. The vector pSK-35S-GUS-NOS / pCAMBIA was linearized with rapid restriction endonucleases BamHI and SacI (Fermentas) and ligated using the In-Fusion kit (TaKaRa, Dalian, China) to obtain the pSK-35S-ScbZIP72-NOS recombinant plasmid.

[0070] 2. Plasmid transformation into Agrobacterium competent cells

[0071] The plasmid pSK-35S-ScbZIP72-NOS extracted and preserved above was used to transform Agrobacterium competent cells GV3101. The transformation steps are as follows: (1) Take 1 μg of plasmid DNA and add it to 200 μL of prepared GV3101 competent cells, and gently flick to mix; (2) Place the mixed solution in liquid nitrogen for 10 minutes and place it on ice for 5 minutes; (3) Take 200 μL of antibiotic-free LB liquid culture medium and add it to the mixed solution, and activate it in a shaker at 28°C and 200 rpm / min for 2 hours; (4) Place the activated transformation solution in a sterile clean bench, take 100 μL and spread it on an LB solid plate culture medium containing 50 μg / mL kanamycin and rifampicin, and invert and culture at 28°C for 2 days; (5) Pick a single colony, shake the bacteria, perform bacterial solution PCR, run a gel to detect whether there is a target band, select the positive Agrobacterium bacterial solution with the target plasmid for expansion culture, and store it in a -80°C refrigerator for later use.

[0072] 3. Arabidopsis transformation and transgenic seedling screening

[0073] The specific steps are as follows: (1) Take 10 μL of the above positive Agrobacterium bacterial solution into 10 mL LB liquid medium (50 μg / mL kanamycin and rifampicin), and activate it at 28°C, 200 rpm / min overnight; (2) Inoculate 10 mL of Agrobacterium bacterial solution into 200 mL LB liquid medium (50 μg / mL kanamycin and rifampicin), and activate it at 28°C, 200 rpm / min until OD 600=0.8~1.0, centrifuge at 5000rpm / min at room temperature for 5min, and collect the bacteria; (3) discard the supernatant, add 10mL infection solution (1 / 2MS, 2.215mg / mL; sucrose, 5% (W / V); Silwet 77, 0.02% (W / V)) to resuspend the bacteria, centrifuge at 5000rpm / min at room temperature for 5min, and collect the bacteria; (4) discard the supernatant, add 200mL infection solution to resuspend the bacteria; (5) select healthy Arabidopsis in the early fruiting stage, remove the fruit pods, and only keep the unopened inflorescences, lay the plants flat so that the inflorescences are completely immersed in the infection solution, soak for 1min, then lay the plants flat in a new tray, cover with plastic wrap and culture in a dark room for 24h; (6) After 1 day of dark treatment, The plants were removed and placed in an artificial climate culture room for normal culture. After about one month, the plants matured and T0 generation seeds were harvested for transgenic plant screening. (7) Resistance plates were prepared and the sterilized T0 generation seeds were spread on the plates. After 2-4 days of vernalization in the dark at 4°C, the seeds were removed and cultured in a constant temperature incubator at 23°C for 2-3 weeks. The positive seedlings were transplanted into nutrient soil and the positive seedlings (OE1, OE3 and OE4) were identified again by PCR. The culture was continued until T3 homozygous seeds were obtained for subsequent experiments.

[0074] 4. Drought stress treatment of transgenic Arabidopsis

[0075] Thirty transgenic and wild-type Arabidopsis seedlings were selected, and the ratio of nutrient soil was peat soil: vermiculite = 1:1. After normal watering for 21 days, they were drought-stricken for 14 days, then re-watered and continued to be cultivated under normal conditions for 7 days. Three independent experiments were conducted, and three replicates were set up for each experiment.

[0076] 5. Drought tolerance analysis

[0077] After 14 days of drought, the leaves and shape of the plants were observed. After 7 days of cultivation under normal conditions, the survival rate of the plants was calculated.

[0078] Compared with wild-type Arabidopsis, the leaves of transgenic Arabidopsis plants overexpressing ScbZIP72 basically maintained their original leaf color and shape under 14-day soil water shortage treatment, while the leaves of wild-type Arabidopsis showed severe wilt and yellowing ( Figure 5 A in Figure 1). After 7 days of rehydration, the transgenic plants showed a significantly stronger ability to recover growth than wild-type Arabidopsis, with a survival rate of over 83.3%, 15.7% higher than that of the wild type ( Figure 5 B). The results showed that overexpression of ScbZIP72 could significantly increase the drought tolerance of transgenic Arabidopsis.

[0079] 6. Active oxygen detection

[0080] To investigate whether ScbZIP72 gene is involved in the scavenging of ROS, DAB and NBT staining were used to detect H2O2 and O2 in transgenic and wild Arabidopsis plants, respectively.ˉ level.

[0081] (1) DAB staining: Place the leaves in a 6-well plate, add an appropriate amount of diaminobenzidine (DAB) staining solution to immerse the leaves, and stain overnight at room temperature in the dark; discard the DAB staining solution and decolorize with 75% anhydrous ethanol; observe and photograph under a stereo microscope. The results showed that under soil drought stress, the leaves of wild Arabidopsis thaliana showed a darker dark blue color, while the leaves of transgenic Arabidopsis thaliana plants overexpressing ScbZIP72 showed a lighter blue color (e.g. Figure 5 C).

[0082] (2) NBT staining: Place the leaves in a 6-well ELISA plate, add an appropriate amount of nitro blue tetrazolium (NBT) staining solution to immerse the leaves, and stain at room temperature in the dark for 8 hours; discard the NBT staining solution and add 75% anhydrous ethanol to decolorize; observe and photograph under a stereo microscope. The results showed that under soil drought stress, the leaves of wild Arabidopsis thaliana showed a darker brown color, while the leaves of transgenic Arabidopsis thaliana plants overexpressing ScbZIP72 showed a lighter brown color (e.g. Figure 5 D).

[0083] The experimental results preliminarily confirmed that under drought stress, the H2O2 and O2 ˉ The content was low, suggesting that ScbZIP72 might be involved in activating the ROS scavenging system.

[0084] Example 5 Sugarcane ScbZIP72 gene enhances tolerance of transgenic sugarcane to drought stress

[0085] 1. Construction of ScbZIP72 recombinant expression vector

[0086] Primers Ubi-ScbZIP72-F (SEQ ID NO: 11): 5′-CGACTCTAGAGGATCCATGGACGAGCTGC TCCGGAG-3′ and Ubi-ScbZIP72-R (SEQ ID NO: 12): 5′-GGGAAATTCGAGCTCT CAAGATAATATACAGGGGT-3′ designed by the seamless cloning primer design tool (http: / / 123.56.75.195 / ) were used to amplify the PCR fragment containing the linker. The vector pSK-Ubi-GUS-NOS / pCAMBIA was linearized with rapid restriction endonucleases BamHI and SacΙ (Fermentas) and ligated using the In-Fusion kit (TaKaRa, Dalian, China) to obtain the pSK-Ubi-ScbZIP72-NOS recombinant plasmid.

[0087] 2. Genetic transformation of sugarcane and screening and identification of transgenic plants

[0088] (1) The tops of the sugarcane variety New Taiwan Sugar No. 22, which is growing normally and free of pests and diseases, were taken as materials. The old leaves and the upper leaves that grew out were removed, and the leaves were disinfected with 5% sodium hypochlorite for 10 min and washed twice with sterile water. The young heart leaf tissue of the sugarcane was selected and sliced ​​into 1 mm thick discs, which were spread flat in an induction medium (4.4 g / L MS medium base salt (containing vitamins) (Cooliebo, China), 2 mg / L 2,4-D, 20 g / L sucrose, 6 g / L agar powder, pH 5.8-6.2), and cultured at 28°C in the dark for 10-14 days.

[0089] (2) Select clean, loose, and light yellow callus tissue and spread it on an infiltration medium (4.4 g / L MS medium base salt (containing vitamins), 36.4 g / L sorbitol, 36.4 g / L mannitol, 0.6 mg / L 2,4-D, 25 g / L sucrose, 6 g / L agar powder, pH 5.8-6.2) and pretreat for 4 h at 28°C in the dark.

[0090] (3) Plasmid DNA coating: Take the prepared tungsten powder suspension (60 μg / uL), vortex for 1 min, then add plasmid DNA (1 μg / gun), 2.5 M CaCl2 and 0.1 M spermidine in sequence, continue vortexing for 2 to 3 min to mix thoroughly, and let it stand on ice for 5 min until the tungsten powder sinks to the bottom of the centrifuge tube; add 800 μl of anhydrous ethanol, remove the supernatant, repeat this process, and then add an appropriate amount of anhydrous ethanol to make the concentration of tungsten powder 60 μg / uL.

[0091] (4) Pipette 10 μL of the tungsten powder-ethanol mixture coated with plasmid DNA onto a microparticle slide and dry it. Place the cleavable membrane (1100 psi) soaked in 80% isopropanol into a metal ring. After checking the air tightness, evacuate the pressure to above 28 inHg and use high-pressure helium to introduce the plasmid DNA into the young leaf cells of sugarcane. The bombardment parameters are helium pressure of 1300 psi and bombardment distance of 6 cm.

[0092] (5) After the completion of the gene gun bombardment, the callus tissue was cultured on the infiltration medium for 24 hours and then transferred to the recovery medium (4.4 g / L MS medium base salt (containing vitamins), 0.6 g / L 2,4-D, 20 g / L sucrose, 6 g / L agar powder, pH 5.8-6.2) and cultured in the dark at 28°C for 5-7 days.

[0093] (6) The callus tissue was crushed and transferred to a differentiation medium containing 4 mg / L Bialaphos (4.4 g / L MS medium base salt (containing vitamins), 0.5 mg / L NAA, 0.5 mg / L 6-BA, 1 mg / L KT, 25 g / L sucrose, 6 g / L agar powder, pH 5.8-6.2), 28°C, light culture (16 h light / 8 h dark), and the medium was changed every two weeks until 3-5 cm long and relatively strong seedlings grew. Bialaphos can be added only during the first and last transfer of the differentiation medium.

[0094] (7) The sugarcane seedlings were transferred to rooting medium (4.4 g / L MS medium basal salt (containing vitamins), 3 mg / L NAA, 0.2 mg / L 6-BA, 0.2 mg / L KT, 5 g / L sucrose, 0.25 g / L activated carbon, pH 5.8-6.2) and cultured at 28°C, 16 h light / 8 h dark conditions. The medium was replaced every two weeks until roots were formed and the seedlings were strong.

[0095] (8) Transplant the robust sugarcane seedlings into nutrient soil and culture them at 28°C, 60% relative humidity and 16 h light / 8 h dark conditions.

[0096] (9) Herbicide screening of transgenic plants: After the sugarcane seedlings grow in the nutrient soil for 7 to 10 days and turn green, spray them with 6.6 mL / L of glufosinate-ammonium herbicide (Baoshida), and spray them again 7 days later. After 7 to 10 days, the surviving sugarcane seedlings with green leaves are transplanted into pots for cultivation; PCR molecular identification: When the sugarcane seedlings grow stronger, cut the leaves, extract the total DNA, and use the PCR detection method to screen positive plants.

[0097] 3. Drought stress treatment of transgenic sugarcane

[0098] 20 transgenic sugarcane plants and wild-type plants were selected, and the ratio of nutrient soil was peat soil: vermiculite = 1:1. After normal watering for 21 days, the plants were left dry for 10 days, then re-watered and continued to be cultivated under normal conditions for 7 days. Three replicates were set for each experiment.

[0099] 4. Evaluation of drought tolerance of transgenic sugarcane

[0100] After 7 days of rehydration, the survival rate of the plants was counted. The results are as follows: Figure 6 As shown, under 10 days of soil water shortage treatment, after 7 days of rehydration, compared with wild-type sugarcane, transgenic sugarcane plants overexpressing ScbZIP72 showed a significantly stronger ability to recover growth, with a survival rate of more than 66.6%, 28.3% higher than the wild type. The results showed that overexpression of ScbZIP72 can significantly increase the drought tolerance of transgenic sugarcane.

[0101] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0102] 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. An application of a sugarcane transcription factor ScbZIP72 in improving plant drought resistance or plant drought resistance genetic breeding, wherein the amino acid sequence of the sugarcane transcription factor ScbZIP72 is shown in SEQ ID NO:

2.

2. An application of a gene encoding a sugarcane transcription factor ScbZIP72 in improving plant drought resistance or plant drought resistance genetic breeding, wherein the nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO:

1.

3. Use of primers for amplifying a gene encoding a sugarcane transcription factor ScbZIP72 in improving plant drought resistance or plant drought resistance genetic breeding, the primers comprising an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO:

4.

4. Use of a recombinant expression vector containing a gene encoding a sugarcane transcription factor ScbZIP72 in improving plant drought resistance or genetic breeding of plant drought resistance, wherein the nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO:

1.

5. Use of an engineered bacterium transformed with the recombinant expression vector according to claim 4 in improving plant drought resistance or in genetic breeding of plant drought resistance.

6. The use according to any one of claims 1 to 5, characterized in that: The plant is Arabidopsis thaliana or sugarcane.

7. A preparation for improving drought resistance of plants, characterized in that The active ingredient of the preparation includes the recombinant expression vector according to claim 4.

8. The preparation according to claim 7, characterized in that The plant is Arabidopsis thaliana or sugarcane.

9. A method for improving drought resistance of plants, characterized in that: The following steps are involved: The expression level of the gene encoding the sugarcane transcription factor ScbZIP72 in the plant is increased, and the nucleotide sequence of the gene encoding the sugarcane transcription factor ScbZIP72 is shown in SEQ ID NO:

1.

10. The method according to claim 9, characterized in that The plant is Arabidopsis thaliana or sugarcane.

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