Application of NtbHLH128 transcription factor in improving heavy metal cadmium resistance of plants

Abstract

The invention discloses application of an NtbHLH128 transcription factor in improving heavy metal cadmium resistance of plants, and belongs to the technical field of gene engineering. According to the invention, an NtbHLH128 gene knockout mutant plant is constructed through a gene editing technology, and the knockout mutant plant can maintain normal growth in a heavy metal cadmium polluted environment and shows remarkable cadmium stress resistance; meanwhile, the knockout mutant plant also has high enrichment capacity, cadmium ions in the environment can be enriched, and the heavy metal concentration in the environment is reduced. The regulation effect of the NtbHLH128 gene in heavy metal stress is defined, an important target gene is provided for cultivating new varieties of crops which are highly resistant to heavy metal stress and used for contaminated soil remediation, a theoretical basis is laid for ecological remediation work, and the NtbHLH128 gene has important application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the NtbHLH128 transcription factor in improving plant resistance to the heavy metal cadmium. Background Technology

[0002] Heavy metal pollutants in soil mainly originate from two sources: natural emissions and human activities. Rock weathering is the primary natural source of heavy metal pollution in the environment. However, human activities, including mining, smelting, chemical production, and agriculture, all generate large amounts of toxic and harmful heavy metals. Many heavy metal compounds may enter agricultural systems through the excessive use of fertilizers and pesticides. Once in farmland soil, heavy metals directly reduce crop yields and indirectly decrease land use efficiency.

[0003] Phytoremediation technology has attracted much attention due to its economic and eco-friendly nature, but existing plants generally suffer from low tolerance to heavy metals and insufficient accumulation efficiency. Current applications of heavy metal resistance genes are mostly concentrated in hyperaccumulating plants (such as Arabidopsis thaliana and Indian mustard), while tobacco, as a model crop with high biomass and strong adaptability, has insufficient development of its gene resources. Discovering tobacco's own resistance genes and constructing efficient expression systems is of great significance for improving phytoremediation efficiency. The bHLH (basicHelix-Loop-Helix) family is a class of transcription factors widely involved in plant development and abiotic stress responses. However, research on bHLH genes in tobacco responding to heavy metal stress is limited, especially the role of the bHLH128 gene in the response to cadmium stress, which has not been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the NtbHLH128 transcription factor in enhancing plant resistance to the heavy metal cadmium, thereby addressing the problems existing in the prior art. This invention utilizes gene editing technology to construct NtbHLH128 gene knockout mutant plants. These knockout mutant plants can maintain normal growth under cadmium pollution, exhibiting significant resistance to cadmium stress. Simultaneously, the knockout mutant plants also possess high accumulation capacity, capable of accumulating cadmium ions in the environment and reducing the concentration of heavy metals. This invention clarifies the regulatory role of the NtbHLH128 gene in heavy metal stress, providing an important target gene for breeding new crop varieties with high resistance to heavy metal stress and for soil remediation, laying a theoretical foundation for ecological restoration work, and possessing significant application value.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of NtbHLH128 transcription factor or related biological materials in improving plant resistance to the heavy metal cadmium. The improvement method is to reduce the expression level of the NtbHLH128 transcription factor in plants or inhibit the expression of the gene encoding the NtbHLH128 transcription factor to improve plant resistance to the heavy metal cadmium.

[0007] The amino acid sequence of the NtbHLH128 transcription factor is shown in SEQ ID NO.2.

[0008] The present invention also provides the application of NtbHLH128 transcription factor or related biological materials in improving the enrichment capacity of plants for heavy metal cadmium. The method of improvement is to reduce the expression level of NtbHLH128 transcription factor in plants or inhibit the expression of the gene encoding NtbHLH128 transcription factor to improve the enrichment capacity of plants for heavy metal cadmium.

[0009] The amino acid sequence of the NtbHLH128 transcription factor is shown in SEQ ID NO.2.

[0010] Optionally, the relevant biomaterial is any one of the following:

[0011] 1) The gene encoding the NtbHLH128 transcription factor;

[0012] 2) Targeted knockout of the gRNA encoding the gene described in 1);

[0013] 3) Recombinant vectors or recombinant microorganisms containing the gRNA described in 2);

[0014] 4) Targeted knockout of recombinant vectors or recombinant microorganisms encoding the genes described in 1).

[0015] Optionally, the plant includes tobacco.

[0016] The present invention also provides a method for improving the resistance of plants to the heavy metal cadmium, including the step of using gene editing technology to inhibit the expression of the NtbHLH128 gene in plants to improve the resistance of plants to the heavy metal cadmium;

[0017] The nucleotide sequence of the NtbHLH128 gene is shown in SEQ ID NO.1.

[0018] The present invention also provides a method for improving the ability of plants to accumulate heavy metal cadmium, including the step of using gene editing technology to inhibit the expression of the NtbHLH128 gene in plants and improve the ability of plants to accumulate heavy metal cadmium.

[0019] The nucleotide sequence of the NtbHLH128 gene is shown in SEQ ID NO.1.

[0020] The present invention also provides a method for cultivating plant lines with high resistance to heavy metal cadmium, including the step of using gene editing technology to knock out or silence the NtbHLH128 gene in plants to cultivate plant lines with high resistance to heavy metal cadmium.

[0021] The nucleotide sequence of the NtbHLH128 gene is shown in SEQ ID NO.1.

[0022] The present invention also provides a method for cultivating plant lines that can efficiently accumulate heavy metal cadmium, including the step of using gene editing technology to knock out or silence the NtbHLH128 gene in plants to cultivate plant lines that can efficiently accumulate heavy metal cadmium.

[0023] The nucleotide sequence of the NtbHLH128 gene is shown in SEQ ID NO.1.

[0024] Optionally, the plant includes tobacco.

[0025] The present invention also provides an application of plant strains cultivated by the above method in the remediation of heavy metal polluted environments.

[0026] The present invention discloses the following technical effects:

[0027] This invention clarifies for the first time the regulatory role of the tobacco NtbHLH128 gene in heavy metal stress. Using gene editing technology, this invention constructed an NtbHLH128 gene knockout mutant plant. This knockout mutant plant maintained normal growth under cadmium pollution, demonstrating significant resistance to cadmium stress. Simultaneously, the knockout mutant plant also exhibited high accumulation capacity, with its roots possessing stronger heavy metal absorption or retention capabilities, enabling it to accumulate cadmium ions from the environment and thus reduce heavy metal concentrations. Utilizing this NtbHLH128 gene, plant strains for heavy metal pollution remediation can be constructed, achieving ecological restoration and remediation of heavily polluted environments.

[0028] This invention provides important target genes for breeding new crop varieties with high resistance to heavy metal stress and for remediation of polluted soil, laying a theoretical foundation for ecological restoration work and having significant application value. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1Results of NtbHLH128 gene expression level detection in wild-type tobacco plants and gene-edited tobacco plants;

[0031] Figure 2 Phenotypic images of wild-type tobacco plants and gene-edited tobacco plants after treatment with the heavy metal cadmium;

[0032] Figure 3 The figures show the cadmium content in the roots and leaves of wild-type tobacco plants and gene-edited tobacco plants after cadmium treatment; in the legend, 128-Cas represents gene-edited tobacco. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0039] Example 1: Cloning of the tobacco NtbHLH128 gene

[0040] Using cDNA from common tobacco safflower as a template, the nucleotide coding sequence of the NtbHLH128 gene was amplified. The full-length coding sequence of the NtbHLH128 gene is 1113 bp, and its nucleotide sequence is shown in SEQ ID NO.1; the gene encodes 370 amino acids, and its amino acid sequence is shown in SEQ ID NO.2.

[0041] SEQ ID NO.1:

[0042]

[0043] SEQ ID NO.2:

[0044] MYPSSTSSSSQNSMSHSTTAGGGSNGGGGLTRYGSAPGAFLTTAVESVIGANNHDFNLHGSHHQHLGPSRYFSPNMTSSNSLNSESTSKAKEQSSLQRSIGFNDLTIGSGGGGGGGGGCGVMPAASSSTTPLVRHSSSPARFLNQLAAAAGDTGFSVSMGRGSYNSKGVADSSRGITRLNSQLSFT RQEALSQIAEENEDVEGTSTDNGHRKSTHSYATASSFAMGSWEDNNSIMFSVTPSKRAKHISNDIVNGLDDGETQFQFGLSQTALEMASMDRLLHIPEDSVPCKIRAKRGCATHPRSIAERERRTRISGKLKKLQDLVPNMDKQTSYADMLDLAVQHIRTLQDQVQNLNTELENCKCGCKKSSQ*.

[0045] Example 2: Obtaining NtbHLH128 gene-edited tobacco

[0046] Referring to the description in patent ZL202410553856.5 (publication date: April 11, 2025), NtbHLH128 gene-edited tobacco was constructed. The specific process is as follows:

[0047] CRISPR multi-targeter software was used to design gRNAs for target sites. The designed target sites + PAM regions were compared with the tobacco genome database to select target sites without intron crossings and with high specificity (no off-target effects). The gRNA sequences were annealed from single-stranded Oligo DNA to form double-stranded DNA via PCR. Using the pORE-Cas9 vector as the backbone, the annealed double-stranded DNA was ligated into the backbone vector after enzyme digestion to construct the knockout vector, pORE-Cas9-128. After constructing the knockout vector, the expression vector containing the target gene was introduced into tobacco leaf explants via Agrobacterium-mediated transformation. Following tissue culture, antibiotic resistance screening, and target site PCR detection, gene-editing positive materials were obtained.

[0048] The designed gRNA sequence is shown below:

[0049] NtbHLH128-gRNA: GAAGGGACTAGTACAGACAA, SEQ ID NO. 3.

[0050] The primer sequences for target site PCR detection are as follows:

[0051] CACGACCTCTTACGTGCTCTGAT, SEQ ID NO.4;

[0052] AAGATTACCTGAGTTTCCCCGTC, SEQ ID NO.5.

[0053] After constructing the NtbHLH128 knockout vector using CRISPR-Cas9 technology, it was transformed into tobacco leaf explants, successfully obtaining the homozygous gene-edited material bHLH128-Cas. Sequencing analysis revealed that the knockout mutation in bHLH128-Cas involved the insertion of one T base at position 620. Figure 1 This leads to the mutation of the 208th amino acid from asparagine to glutamine, causing premature termination of translation.

[0054] Example 3: Verification of resistance to heavy metal cadmium stress

[0055] 1. Comparison of heavy metal resistance between wild-type and gene-edited tobacco

[0056] Wild-type *Tobacco Monotyphimurium* 'Honghua Dajinyuan' (HD) and gene-edited tobacco (bHLH128-Cas) were subjected to heavy metal cadmium stress via hydroponics. The growth phenotypes of tobacco under different cadmium ion concentrations (0 μM and 200 μM CdCl2) were observed. After 15 days of continuous cadmium ion treatment, the phenotypes of wild-type and gene-edited tobacco were observed. During hydroponic treatment, the hydroponic system and methods were optimized based on previous research experience: the hydroponic system was enlarged (from a one-liter hydroponic box to a two-liter hydroponic tray) to increase the oxygen content in the hydroponic solution and reduce the risk of root rot. In the initial stage of hydroponics, the seedlings were hardened off with clean water for 3-5 days. After the seedlings adapted, a low-concentration nutrient solution was used to improve their condition before heavy metal treatment.

[0057] The results showed that under 0 μM cadmium ion treatment, there was no significant difference in phenotype between the two types of tobacco plants; however, under 200 μM cadmium ion treatment, the two types of tobacco plants exhibited significant differences in stress phenotypes, with the wild-type plants showing significantly weaker growth than the transgenic plants. Older leaves of the wild-type plants showed obvious yellowing first, with severe chlorophyll degradation. With prolonged treatment, leaf edges or tips became scorched. Leaves generally wilted and drooped, losing their normal luster and uprightness, exhibiting obvious dehydration. The overall vitality of the plants was low, showing obvious weakness and near-death. In contrast, the gene-edited tobacco plants showed better overall vitality, were able to maintain basic physiological functions, and demonstrated stronger survival ability. Figure 2This indicates that knocking out the NtbHLH128 gene can improve the survival status of plants under heavy metal cadmium stress and enhance their resistance to heavy metal cadmium stress.

[0058] 2. Comparison of heavy metal content between wild-type and gene-edited tobacco

[0059] Using hydroponics, wild-type Red Flower Dajinyuan (HD) and gene-edited tobacco (bHLH128-Cas) at the 3-4 leaf stage were treated with a specific concentration (200 μM CdCl2) of heavy metals for 15 days. The heavy metal content in the roots and leaf tissues of the tobacco plants was then measured to observe the heavy metal content in the leaves and roots of tobacco under cadmium treatment.

[0060] The results showed that under 200 μM cadmium treatment, the cadmium concentration in wild-type tobacco leaves was 0.206 μg / mg, and in roots it was 0.29 μg / mg, while in gene-edited tobacco leaves it was 0.255 μg / mg, and in roots it was 0.68 μg / mg. The heavy metal content per unit mass (μg / g DW) of root tissue in wild-type tobacco was significantly lower. In contrast, although the roots of gene-edited tobacco showed relatively better tolerance, the concentration of heavy metals in their root tissue was significantly higher than that of wild-type. This indicates that the roots of gene-edited plants have a stronger capacity for heavy metal absorption or retention. A similar trend was observed in leaf tissue: the heavy metal content in wild-type tobacco leaves was significantly lower than that in gene-edited tobacco leaves. Figure 3 ).

[0061] The above results indicate that the NtbHLH128 gene plays an important regulatory role in tobacco's resistance to heavy metal cadmium stress. By knocking out the NtbHLH128 gene and causing its function to be lost, the survival ability of tobacco in cadmium stress environment can be improved, its normal growth can be enhanced, and the absorption of cadmium ions by tobacco roots and the translocation capacity of cadmium ions by the plant can be improved, thereby enriching cadmium ions in the environment into the plant and realizing the ecological restoration of heavy metal polluted environment.

[0062] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of NtbHLH128 transcription factor or related biomaterials in improving plant resistance to the heavy metal cadmium, characterized in that, The method of improvement is to increase the plant's resistance to the heavy metal cadmium by reducing the expression level of the NtbHLH128 transcription factor in the plant or inhibiting the expression of the gene encoding the NtbHLH128 transcription factor. The amino acid sequence of the NtbHLH128 transcription factor is shown in SEQ ID NO.

2.

2. The application of NtbHLH128 transcription factor or related biomaterials in enhancing the accumulation capacity of plants for the heavy metal cadmium, characterized in that... The method of improvement is to increase the plant's ability to accumulate heavy metal cadmium by reducing the expression level of the NtbHLH128 transcription factor in the plant or by inhibiting the expression of the gene encoding the NtbHLH128 transcription factor. The amino acid sequence of the NtbHLH128 transcription factor is shown in SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that, The relevant biomaterial is any one of the following: 1) The gene encoding the NtbHLH128 transcription factor; 2) Targeted knockout of the gRNA encoding the gene described in 1); 3) Recombinant vectors or recombinant microorganisms containing the gRNA described in 2); 4) Targeted knockout of recombinant vectors or recombinant microorganisms encoding the genes described in 1).

4. The application according to claim 1 or 2, characterized in that, The plant mentioned includes tobacco.

5. A method for improving plant resistance to the heavy metal cadmium, characterized in that, This includes steps such as using gene editing technology to suppress the expression of the NtbHLH128 gene in plants and improve the resistance of plants to the heavy metal cadmium; The nucleotide sequence of the NtbHLH128 gene is shown in SEQ ID NO.

1.

6. A method for improving the ability of plants to accumulate the heavy metal cadmium, characterized in that, This includes steps such as using gene editing technology to suppress the expression of the NtbHLH128 gene in plants and improve the plants' ability to accumulate the heavy metal cadmium. The nucleotide sequence of the NtbHLH128 gene is shown in SEQ ID NO.

1.

7. A method for cultivating plant lines highly resistant to the heavy metal cadmium, characterized in that, This includes the steps of using gene editing technology to knock out or silence the NtbHLH128 gene in plants to cultivate plant lines with high resistance to the heavy metal cadmium; The nucleotide sequence of the NtbHLH128 gene is shown in SEQ ID NO.

1.

8. A method for cultivating plant lines capable of efficiently accumulating the heavy metal cadmium, characterized in that, This includes the steps of using gene editing technology to knock out or silence the NtbHLH128 gene in plants to cultivate plant lines that can efficiently accumulate the heavy metal cadmium. The nucleotide sequence of the NtbHLH128 gene is shown in SEQ ID NO.

1.

9. The method according to any one of claims 5-8, characterized in that, The plant mentioned includes tobacco.

10. The application of a plant strain cultivated by the method described in claim 8 in the remediation of heavy metal contaminated environments.

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