Cotton GhMYB36b promoter for regulating and controlling formation of Kjeldahl band and application of cotton GhMYB36b promoter in cadmium stress tolerance

Abstract

The invention discloses a cotton GhMYB36b promoter for regulating and controlling formation of a Kjeldahl band and application of the cotton GhMYB36b promoter in cadmium stress tolerance. The GhMYB36b promoter disclosed by the invention is high in specificity, high in regulation and control efficiency, safe in source and good in adaptability. The GhMYB36b promoter obviously activates downstream gene expression only under a cadmium stress condition, almost has no basic expression under a non-stress condition, can realize cadmium stress specific regulation and control of a target gene, and avoids burden on cotton growth caused by continuous expression of the gene. Under cadmium stress, the expression quantity and enzyme activity of the GUS gene driven by the promoter are obviously higher than those of no-load control, and the regulation efficiency of the promoter is obviously superior to that of a conventional constitutive promoter. The promoter is a cotton endogenous promoter, when the promoter is applied to plants such as cotton, the risks of gene silencing and the like caused by an exogenous promoter can be reduced, the adaptability is higher, and a high-quality element is provided for cadmium-tolerant gene engineering improvement of the cotton and other plants.

Description

Technical Field

[0001] This invention relates to a cotton method for regulating the formation of Casparian strips. GhMYB36b Promoters and their application in cadmium stress tolerance belong to the field of regulation of plant cadmium stress resistance. Background Technology

[0002] With the increasing prominence of heavy metal pollution in soil, cadmium (Cd), as a typical heavy metal pollutant, not only damages soil ecosystems but also enters the food chain through plant accumulation, threatening human health. Utilizing genetic engineering to cultivate cadmium-tolerant and low-cadmium-accumulation plant varieties is an effective way to solve this problem, and specific promoters are key components for achieving precise expression of target genes. Currently, some plant promoters have been found to have heavy metal-responsive characteristics, but most promoters suffer from insufficient response specificity and limited regulatory efficiency. Cotton, as an important economic crop, has relatively scarce resources of specific promoters related to cadmium stress response. Existing technologies lack endogenous cotton promoters that can efficiently and specifically respond to cadmium stress, making it difficult to meet the needs of cadmium-tolerant genetic engineering improvement in cotton. For example, some reported heavy metal-responsive promoters also exhibit basal expression under non-stress conditions, failing to achieve precise spatiotemporal expression of target genes and increasing the growth burden on genetically engineered plants. Summary of the Invention

[0003] Purpose of the invention: The purpose of this invention is to provide a cotton method for regulating the formation of the Casparian strip. GhMYB36b The promoter and its application in cadmium stress tolerance will solve the problems of insufficient specificity and low regulatory efficiency of plant cadmium response promoters in existing technologies, and provide key components for breeding new plant varieties that are cadmium tolerant and have low cadmium accumulation.

[0004] Technical solution: This invention provides the nucleotide sequence shown in SEQ ID NO.1. GhMYB36b Application of gene promoters in regulating plant resistance to cadmium stress.

[0005] This includes improving plants' resistance to cadmium stress.

[0006] The present invention also provides the following GhMYB36b The application of gene promoters in response to cadmium stress in plants, the GhMYB36b The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] The present invention also provides the following GhMYB36b The application of gene overexpression vectors in regulating plant resistance to cadmium stress. GhMYB36b The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Wherein, the overexpression vector includes the... GhMYB36bThe gene was constructed into the pBI101 vector.

[0009] The present invention also provides a product containing the aforementioned GhMYB36b Application of gene overexpression strains in regulating plant resistance to cadmium stress. GhMYB36b The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] The overexpression strain is obtained by introducing the overexpression vector of claim 4 into Agrobacterium.

[0011] The plant mentioned includes tobacco.

[0012] The present invention also provides a method for improving the resistance of plants to cadmium stress, the method comprising promoting or enhancing the expression of the GhMYB36b promoter in plants.

[0013] The enhancement of plant GhMYB36b promoter expression includes: overexpression in the plant genome. GhMYB36b Genes; or upregulation GhMYB36b Gene transcription, transfer into plants, or by using GhMYB36b Plant varieties with overexpressed genes are hybridized with ordinary plant varieties.

[0014] The nucleotide sequence of the GhMYB36b promoter is shown in SEQ ID NO.1, and it can be obtained as follows: Primer design: Specific amplification primers 2203P-1979F (sequence gaccatgattacgccaagcttTTTAAAATCTGCATGCCC) and 2203P-1518F (sequence gaccatgattacgccaagcttGCCTACTTTTCTAGATTAC) were designed and PCR amplification was performed using cotton genomic DNA as a template. ① Vector construction: The amplified GhMYB36b promoter fragment was ligated into the plant expression vector pBI101 through a restriction endonuclease site (such as Hind III), replacing the original promoter, to construct the pBI101:GhMYB36bP recombinant expression vector. This vector contains NPTII selection marker genes, GUS reporter genes, and NOS terminators. ② Transient expression verification: The pBI101:GhMYB36bP recombinant vector was transformed into Nicotiana benthamiana using the Agrobacterium injection method, while the empty vector pBI101:EV was set as a control. The vectors were treated with Mock (water) and CdCl2, respectively, and the activity of the cadmium-specific inducible promoter was detected.

[0015]

[0016] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. High Specificity: The GhMYB36b promoter significantly activates downstream gene expression only under cadmium stress conditions, with almost no basal expression under non-stress conditions. This enables cadmium stress-specific regulation of the target gene, avoiding the burden of continuous gene expression on cotton growth. 2. High Regulatory Efficiency: GUS enzyme activity assays and relative gene expression level detection show that under cadmium stress, the GUS gene expression level and enzyme activity driven by this promoter are significantly higher than the empty vector control, demonstrating a clear advantage in regulatory efficiency compared to conventional constitutive promoters. 3. Safe Source and Good Adaptability: This promoter is an endogenous promoter for cotton. When used in cotton and other plants, it can reduce the risks of gene silencing caused by exogenous promoters, exhibiting stronger adaptability and providing a high-quality component for cadmium-tolerant genetic engineering improvement in cotton and other plants. Attached Figure Description

[0017] Figure 1 For pBI101: GhMYB36bP Schematic diagram of the structure of a recombinant plant expression vector; Figure 2 Electrophoresis diagram of GhMYB36b promoter amplification; Figure 3 Phenotypic diagram of the GhMYB36b promoter cadmium-responsive activity of transient expression of *Nymphaea benthamiana*. Figure 4 The graph shows the relative expression levels of the reporter gene driven by the GhMYB36b promoter under cadmium stress. Figure 5 The image shows the results of GUS enzyme activity assay after CdCl2 treatment under the GhMYB36b promoter. Figure 6 Fold-up of gene expression after treatment with Cd, Cr, and Pb: A: GhMYB36b Gene expression at different time points induced by cadmium; B: GhMYB36b Gene induction by Cr and Pb. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0019] 1. Amplification of the GhMYB36b promoter ① Template Preparation: Genomic DNA was extracted from cotton leaves and purified using the CTAB method. The DNA concentration and purity were then tested before use. ② PCR Amplification System: A 25 μL amplification system was prepared, containing 2.5 μL of 10×PCR buffer, 2 μL of dNTP mixture (2.5 mM), 1 μL each of forward and reverse primers (10 μM), 0.2 μL of Taq DNA polymerase, and 1 μL of template DNA. Water was added to a final volume of 25 μL. The specific amplification primers were 2203P-1979F (sequence: gaccatgattacgccaagcttTTTAAAATCTGCATGCCC) and 2203P-1518F (sequence: gaccatgattacgccaagcttGCCTACTTTTCTAGATTAC). ③ PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 35 cycles; 72℃ final extension for 10 min, and storage at 4℃. ④ Product verification: Agarose gel electrophoresis was performed on the amplified products. Figure 2 The target band (approximately 2000 bp) was recovered and purified.

[0020] 2. Construction of recombinant expression vectors ① Double enzyme digestion treatment: The purified [product / material] was subjected to double enzyme digestion treatment. GhMYB36b The promoter fragment (nucleotide sequence shown in SEQ ID NO.1) and the pBI101 vector were digested with Hind III and BamHI restriction endonucleases. After digestion at 37°C for 2 hours, the digestion products were recovered. ② Ligation reaction: Using T4 DNA ligase, the digested promoter fragment was ligated to the pBI101 vector. Ligation was performed overnight at 16°C to construct pBI101. GhMYB36b Recombinant vector ( Figure 1 ③ Transformation and identification: The ligation product was transformed into Escherichia coli DH5α competent cells, plated on LB medium containing 50 μg / mL kanamycin, positive clones were screened, and the correctness of the vector construction was verified by enzyme digestion and sequencing.

[0021] 3. Agrobacterium-mediated transient transformation of Nicotiana benthamiana ① Agrobacterium transformation: The verified recombinant vector was transformed into Agrobacterium GV3101 competent cells, and positive Agrobacterium strains were screened. ② Infection solution preparation: The infection solution was prepared according to the specified ratio, containing 10 mM 2-morpholinoethanesulfonic acid (MES), 0.1 mM acetylsuccinone (As), and 10 mM MgCl2, and the pH was adjusted to 5.6. The activated Agrobacterium was resuspended in the infection solution, and the OD was adjusted. 600Up to 0.8. ③ Leaf injection: Select healthy leaves of *Tobacco Benzovia* and inject the infection solution into the underside of the leaves using a needleless syringe. At the same time, set up an empty vector pBI101:EV (the pBI101 empty vector plasmid was transformed into competent *E. coli* DH5α cells, and positive strains were obtained by screening on LB medium containing 50 μg / mL kanamycin. The pBI101 empty vector plasmid was then extracted and purified, and transformed into *Agrobacterium* GV3101 competent cells. Positive *Agrobacterium* strains were screened by double resistance of 20 μg / mL rifampicin + 50 μg / mL kanamycin) as a control. After 24 h of dark culture, normal light was restored.

[0022] 4. Promoter activity detection ① Stress treatment: Three days after transformation, leaves were treated with Mock (water) and CdCl2 (100 μM) via leaf injection. Three biological replicates were set up for each group, with three leaves selected from each replicate. The treatment time was 24 hours. ② GUS staining and enzyme activity assay: Treated leaves were subjected to GUS histochemical staining (…). Figure 3 ), and the activity of GUS enzyme was detected by fluorescence method ( Figure 5 The results showed that pBI101 in the CdCl2-treated group: GhMYB36b The leaves showed deeper staining and significantly higher enzyme activity than the control group. ③ Gene expression level detection: Total RNA was extracted from the leaves and reverse transcribed into cDNA using the TaKaRa PrimeScript™ RT reagent Kit (Perfect RealTime) (Takara Bio Inc.). The relative expression level of the GUS gene (GI:529334, GenBank: U12668.1) was detected by qRT-PCR. The qRT-PCR system (20 μL) consisted of: 10 μL of 2×SYBR Premix ExTaq, 0.8 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, and 6.4 μL of ddH2O. The GUS gene primer sequences were: forward primer 5′-GGTGGGAAAGCGCGTTACAAG-3′, and reverse primer 5′-GTTTACGCGTTGCTTCCGCCA-3′. The reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for a total of 40 cycles. The results showed that ( Figure 4 Under cadmium stress, the gene expression level in the pBI101:GhMYB36b group was significantly higher than that in the empty vector group, proving that the promoter has cadmium-responsive activity.

[0023] 5. Multi-stress handling settings ① Leafy samples of *Nicotiana benthamiana* var. *benziosodium* 3 days after transient transformation were selected. In addition to the Mock (water) and CdCl2 (100 μM) treatment groups, CrCl3 (50 μM) and Pb(NO3)2 (50 μM) treatment groups were added. Each group had three biological replicates, and the treatment duration was 24 h, with consistent treatment conditions. ② RNA extraction and cDNA synthesis: Leaf samples from each treatment group were collected, and total RNA was extracted using the Trizol method. After testing the RNA integrity and purity, the first strand of cDNA was synthesized according to the reverse transcription kit instructions. ③ qRT-PCR detection: Using cDNA as a template, qRT-PCR was performed on selected samples... GhMYB36b Genes were used as detection targets, with the Tobacco Benzoviae internal reference gene (… NtActin The amplification was performed using [a specific reagent] as a reference. The qRT-PCR reaction system consisted of 20 μL: 10 μL of 2×SYBR Premix Ex Taq, 0.8 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, and 5.6 μL of ddH2O; the forward and reverse primers were [specific reagents]. GhMYB36b Upstream and downstream primers for NtActin, GhMYB36 Primer sequences: forward primer 5'-ATGTGCCACTCCAAAGACATCAGG-3', reverse primer 5'-TGGCTTCCGCCTTGACCCGAAT-3'; NtActin Primer sequences: forward primer 5′-CAAGGAAATCACGGCTTTGG-3′, reverse primer 5′-AAGGGATGCGAGGATGGA-3′; reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s; 60℃ annealing for 30 s, for a total of 40 cycles; melting curve analysis was also performed to verify amplification specificity. ④ Data statistics and analysis: Calculations were made for each treatment group. GhMYB36b The relative expression levels of genes were compared with the fold upregulation of genes under Cd, Cr, and Pb treatments. The results showed ( Figure 6 CdCl2 treatment group GhMYB36b Gene upregulation can reach more than 60 folds. Figure 6 A), while the gene upregulation folds in the CrCl3 and Pb(NO3)2 treatment groups were both below 0.08, far lower than those in the cadmium treatment group ( Figure 6 (B) further verified the specificity of the promoter's response to cadmium stress.

Claims

1. Application of the GhMYB36b gene promoter, whose nucleotide sequence is shown in SEQ ID NO.1, in regulating the plant's resistance to cadmium stress.

2. The application according to claim 1, characterized in that, This includes improving plants' resistance to cadmium stress.

3. The application according to claim 1, characterized in that, This includes responding to plant stress.

4. The application of the overexpression vector containing the GhMYB36b gene as described in claim 1 in regulating the plant's resistance to cadmium stress, characterized in that, The nucleotide sequence of the GhMYB36b gene is shown in SEQ ID NO.

1.

5. The application according to claim 4, characterized in that, The overexpression vector is obtained by constructing the GhMYB36b gene into the pBI101 vector.

6. The application of the strain containing the GhMYB36b gene overexpression according to claim 1 in regulating the plant's resistance to cadmium stress, characterized in that, The nucleotide sequence of the GhMYB36b gene is shown in SEQ ID NO.

1.

7. The application according to claim 6, characterized in that, The overexpression strain is obtained by introducing the overexpression vector of claim 4 into Agrobacterium.

8. The application according to any one of claims 1 to 7, characterized in that, The plant mentioned includes tobacco.

9. A method for improving the resistance of plants to cadmium stress, characterized in that, The method includes promoting or enhancing the expression of the plant GhMYB36b promoter.

10. The method according to claim 9, characterized in that, The method of enhancing the expression of the plant GhMYB36b promoter involves: overexpressing the GhMYB36b gene in the plant genome; or upregulating the transcription of the GhMYB36b gene; or hybridizing a plant variety that overexpresses the GhMYB36b gene with a common plant variety.

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