Application of NtCrRLK04 gene in relieving tobacco rhizosphere microenvironment imbalance
By knocking out the tobacco NtCrRLK04 gene through gene editing and constructing a highly adaptable mutant, the problem of imbalance in the tobacco rhizosphere microenvironment was solved, and the growth indicators and yield of tobacco were improved.
Patent Information
- Application Number
- CN202510883968.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-10-10
AI Technical Summary
The rhizosphere microenvironment of tobacco is prone to imbalance during long-term cultivation, leading to hindered root growth, compacted soil structure, and reduced microbial diversity, affecting tobacco leaf quality and yield. Existing regulation methods are unstable.
The tobacco NtCrRLK04 gene was knocked out through gene editing technology, and mutants with low or no expression of the NtCrRLK04 gene were constructed. The CRISPR/Cas9 system was used for site-directed editing to cultivate tobacco mutants with strong adaptability and improve the rhizosphere microenvironment.
It can effectively alleviate the imbalance of tobacco rhizosphere microenvironment, improve tobacco growth indicators such as fresh weight, stem length, leaf area, and enhance the planting adaptability and yield of tobacco in specific plots.
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Abstract
Description
Technical Field
[0001] The present invention relates to the fields of biotechnology and plant breeding technology, and particularly to application of the NtCrRLK04 gene in alleviating imbalance in the rhizosphere microenvironment of tobacco. Background Art
[0002] Tobacco is an important cash crop in my country, contributing positively to increasing farmers' income. Data from the National Bureau of Statistics shows that between 2013 and 2023, my country's tobacco planting area decreased by 43.1%. To address the trend of limited planting space, extending the tobacco planting cycle on a single plot has become a practical option for the industry. However, tobacco is sensitive to changes in the rhizosphere microenvironment. With increasing years of continuous planting on the same plot, harmful heavy metals may accumulate in the soil, beneficial microbial abundance may decrease, and root growth and development may be hindered, seriously hindering the high quality and sustainable production of tobacco leaves. Therefore, scientifically regulating the tobacco rhizosphere microenvironment, optimizing root growth and development, and balancing soil trace elements has become a key issue for ensuring the sustainable development of the industry. Soil physical and chemical parameters and fertility have a significant impact on tobacco growth, development, and quality. However, with extended planting years, the stability of the tobacco rhizosphere microenvironment may be challenged, manifested by soil topsoil degradation, imbalanced nutrient supply, increased structural compaction, and reduced rhizosphere microbial diversity. These changes not only affect the normal growth and function of the root system, but may also lead to the weakening of the aroma and flavor of the tobacco leaves, which is not conducive to the sustainable production of high-quality tobacco.
[0003] Plant cells possess a unique subfamily of membrane receptors—CrRLK1Ls. These receptors serve as key nodes for signal perception, recognizing multiple signals from both inside and outside the cell and integrating changes in metabolic activity, physiological state, and molecular processes to finely regulate plant growth and development. Once activated, CrRLK1Ls trigger a complex series of downstream responses, encompassing adjustments in physiological state, dynamic assembly and dissociation of protein complexes, reprogramming of metabolic pathways, alterations in gene expression patterns, and modulation of phytohormone activity. In tobacco cultivation, extending the cropping cycle of specific plots has become a practical strategy for coping with shrinking cropping space. To ensure the sustainability of this strategy, in-depth understanding of tobacco's adaptive mechanisms is crucial. Studies in model plants such as Arabidopsis thaliana and rice have revealed the key role of CrRLK receptor family members in plant adaptation to long-term cultivation environments, particularly their ability to regulate root system development and the microecological balance of the rhizosphere. Therefore, systematically identifying and elucidating the functions of CrRLK family genes in major tobacco varieties is crucial for understanding and improving tobacco root health and its rhizosphere microenvironment. There are multiple strategies to improve the adaptability of tobacco to long-term cultivation in specific plots. Among the many strategies, cultivating cultivars that are more adaptable to changes in the soil environment is given great significance. Compared with other methods (such as biological regulation or chemical intervention), breeding adaptable varieties shows unique advantages: stable and reliable effects, high environmental compatibility, reduced dependence on chemical fertilizers, help improve soil physical and chemical properties, and ultimately improve production efficiency. Despite its broad prospects, this approach still faces challenges such as insufficient basic knowledge (such as unclear key mechanisms), high heterogeneity of field soils, and a lack of key adaptive gene resources.
[0004] The means of regulating the imbalance of the tobacco rhizosphere microenvironment are still facing many challenges. Due to the significant differences in soil properties and the high complexity of the rhizosphere microecosystem, traditional regulatory methods are extremely susceptible to environmental fluctuations. Although studies have shown that some microorganisms can improve the rhizosphere microenvironment to a certain extent, their effects show obvious instability under different soil types and environmental conditions. Therefore, it is a key technical problem that needs to be overcome to explore new genetic resources that can effectively regulate the homeostasis of the rhizosphere microenvironment and cultivate cultivars with the ability to optimize the rhizosphere microenvironment so that they can maintain the health of the rhizosphere while ensuring the stability of tobacco leaf yield and quality. Summary of the Invention
[0005] The purpose of the present invention is to provide an application of the NtCrRLK04 gene in regulating the tobacco rhizosphere microenvironment, and to provide a method for effectively alleviating the imbalance of the tobacco rhizosphere microenvironment.
[0006] To achieve the above objectives, the present invention provides a method for alleviating imbalances in the tobacco rhizosphere microenvironment. The invention provides a method for alleviating imbalances in the tobacco rhizosphere microenvironment. Specifically, the method involves sowing tobacco in soil previously grown with a tobacco mutant in which the NtCrRLK04 gene has been knocked out. This method alleviates imbalances in the tobacco rhizosphere microenvironment. The tobacco NtCrRLK04 gene can be used in tobacco cultivation to increase tobacco fresh weight, stem length, and leaf area, among other applications, demonstrating significant practical value.
[0007] The specific technical solutions are as follows:
[0008] The present invention discloses the application of the NtCrRLK04 gene or an NtRLK04 mutant in alleviating the imbalance of the rhizosphere microenvironment of tobacco and eliminating continuous cropping obstacles. The NtCrRLK04 gene in tobacco is subjected to site-directed mutagenesis by gene editing to construct an NtRLK04 mutant with low or no expression of the NtCrRLK04 gene. The nucleotide sequence of the NtCrRLK04 gene is shown in SEQ ID NO.1.
[0009] SEQ ID NO.1:
[0010]
[0011] Preferably, the specific method of gene editing is: taking the tobacco NtCrRLK04 gene as the target, designing an sgRNA sequence based on CRISPR / Cas9, and connecting a DNA fragment encoding the sgRNA into a CRISPR / Cas9 vector to transform tobacco, thereby achieving site-specific editing of the tobacco NtCrRLK04 gene and obtaining a mutant with low or no expression of the NtCrRLK04 gene.
[0012] Preferably, the nucleotide sequence of the sgRNA sequence is at least one of the sequences shown in SEQ ID NO. 8-10.
[0013] The present invention discloses a breeding method for an NtRLK04 mutant for alleviating the imbalance of the tobacco rhizosphere microenvironment and eliminating the continuous cropping obstacle. The method comprises the following steps:
[0014] (1) Gene editing was performed on the tobacco NtCrRLK04 gene to construct a knockout vector, and the knockout vector was transformed into Agrobacterium; the nucleotide sequence of the NtCrRLK04 gene is shown in SEQ ID NO.1;
[0015] (2) The Agrobacterium in step (1) is genetically transformed into tobacco to obtain an NtRLK04 mutant with the NtCrRLK04 gene knocked out.
[0016] Preferably, the specific method of gene editing is: taking the tobacco NtabCrRLK04 gene as the target, designing an sgRNA sequence based on CRISPR / Cas9, and connecting the DNA fragment containing the encoding above sgRNA into the CRISPR / Cas9 vector to transform tobacco, thereby achieving site-specific editing of the tobacco NtabCrRLK12 gene and obtaining a mutant with low or no expression of the NtabCrRLK04 gene.
[0017] Preferably, the sgRNA nucleotide sequence is at least one of the sequences shown in SEQ ID NO. 8-10.
[0018] Preferably, the tobacco is cultivated tobacco Yunyan 87.
[0019] The present invention has the following technical effects:
[0020] The present invention discloses for the first time a new use of the tobacco NtCrRLK04 gene. By knocking out the tobacco NtCrRLK04 gene, the imbalance of the tobacco rhizosphere microenvironment can be effectively alleviated. The present invention also discloses a method for breeding NtRLK04 mutants to alleviate the imbalance of the tobacco rhizosphere microenvironment and resolve continuous cropping obstacles. By knocking out or interfering with the expression of the gene in tobacco, or constructing a tobacco mutant strain with the NtCrRLK04 gene knocked out, and then planting tobacco in soil after the mutant material is planted, the method can effectively alleviate the imbalance of the tobacco rhizosphere microenvironment, resolve continuous cropping obstacles, and increase tobacco yield, showing application prospects in the field of tobacco cultivation. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is the CRISPR editing vector for tobacco gene mutants in the present invention.
[0022] Figure 2 The tobacco CrRLK mutant gene sequence mutation type of the present invention is
[0023] Figure 3 This is the phenotypic comparison result of the tobacco CrRLK gene mutant material in the present invention.
[0024] Figure 4 These are the test results of various indicators of the tobacco CrRLK gene mutant material in the present invention. DETAILED DESCRIPTION
[0025] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.
[0026] Experimental Example 1 Identification of Tobacco CrRLK Family Genes
[0027] Based on the Yunyan 87 genome sequence from the Chinese Tobacco Genome Database, we systematically identified members of the CrRLK1L gene family. The specific process was as follows: First, using previously reported CrRLK1L protein sequences from Arabidopsis, tomato, and potato as queries, we performed a Blastp homology alignment of the Yunyan 87 genome to identify potential homologous genes. Subsequently, Pfam domain analysis was performed on the proteins encoded by the initially identified candidate genes. Genes containing both malectin domains (PF12819.7 and PF11721) and protein kinase (Pkinase) domains (PF00069 and PF07714) were rigorously selected and ultimately confirmed as the genes encoding CrRLK proteins in Yunyan 87.
[0028] A total of 58 genes encoding CrRLK proteins with conserved domains were identified in tobacco cultivar Yunyan 87. These genes were named NtCrRLK01 to NtCrRLK58 according to their chromosomal locations, and their specific database numbers are shown in Table 1.
[0029] Table 1 Tobacco names and database numbers
[0030]
[0031]
[0032]
[0033] Experimental Example 2 Creation and Phenotypic Identification of Tobacco CrRLK Gene Mutant Materials
[0034] (1) Creation of tobacco CrRLK gene mutant materials
[0035] Based on the complete CDS sequences of 58 tobacco NtCrRLK genes identified, gRNA design and analysis were performed. This analysis revealed that the coding sequences (CDS) of several phylogenetically adjacent NtCrRLK genes exhibited high sequence homology. Leveraging this property, a collaborative knockout strategy was developed: targeting highly homologous gene pairs, a single shared CRISPR target was designed to simultaneously edit both genes. Based on this strategy, 17 NtCrRLK genes with high sequence homology (constituting eight potential homologous gene pairs) were identified from the 58 genes, and 27 specific gRNA targets were designed based on these targets. Subsequently, CRISPR editing vectors were constructed using these gRNAs. The selection and identification of gRNA targets were primarily based on the online tools CRISPR-DB (https: / / crispr.dbcls.jp / ) and CCTop (https: / / cctop.cos.uni-heidelberg.de / ). The final target selection was based on a combination of analysis and prediction results from these two platforms. The specific design principle is to design three gRNA target sites for each target NtCrRLK gene, with two target sites approximately 100 bp apart for each gene. The selected genes and corresponding gRNA information are shown in Table 2, and the primer designs for CRISPR vector construction are shown in Table 3.
[0036] (1) CRISPR vector construction: 5 μL of each synthesized single-stranded adapter primer was pipetted into a PCR tube, and ddH2O was added to prepare a 50 μL reaction system. The reaction was denatured at 95°C for 10 min, annealed at 55°C for 10 min, and then cooled to room temperature for 5 min. The single-stranded sgRNA sequence primer was denatured and annealed to obtain the corresponding target sequence. The annealed product was ligated with the pHSb-dcas9 linearized fragment digested with BsaⅠ and added to the Golden Gate reaction system to construct the intermediate vector. T4 DNA ligase was used for ligation at 16°C overnight.
[0037] (2) Transformation of ligation product: Add 10 μL of ligation fragment to 100 μL of E. coli DH5α competent cells, ice bath for 30 minutes, heat shock for 45 seconds, and then place the competent cells on ice for 5 minutes. Then add 900 μL of LB medium and incubate for 1 hour. Then spread the bacterial solution on LB plate containing kanamycin resistance. After overnight culture, colonies were selected for colony PCR identification, and positive colonies were sequenced to obtain CRISPR vector construction ( Figure 1 ).
[0038] (3) Agrobacterium transformation: Add 1 μg of plasmid DNA to every 100 μL of competent Agrobacterium, stir the bottom of the tube by hand to mix, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes. Add 700 μL of LB liquid medium without antibiotics and shake and culture at 28°C for 3 hours. Collect the bacteria by centrifugation at 6000 rpm, take 100 μL of supernatant, gently blow and resuspend the bacteria, spread it on LB solid medium containing kanamycin + rifampicin antibiotics, and invert it in a 28°C incubator for 23 days. After a single colony grows, pick a single colony for PCR identification, and store the positive colony by shaking.
[0039] (4) CrRLK editing vector transformation
[0040] Activated Agrobacterium was selected and placed in 50 mL of Kan+Rif-resistant liquid LB medium. Cultured with shaking at 180 rpm at 28°C until the OD600 was close to 0.6-0.8. The culture was then collected and centrifuged twice at 4000 rpm for 10 minutes at 4°C. The cells were resuspended in pre-chilled MS liquid medium to an OD600 of 0.6, and then AS was added to a final concentration of 20 mg / L to prepare for infection. Using a clean bench, the edges of sterile leaves were trimmed with scissors. The leaves were cut along the main vein into 1.0×1.0 cm leaf discs and placed in the Agrobacterium infection solution for infection for 5 minutes. The infected leaves were removed, the Agrobacterium solution was blotted dry on sterile filter paper, and the leaves were spread flat, leaf side down, onto co-cultivation medium (G). The leaves were placed in a climate chamber (temperature 26°C, humidity 40%) and incubated in the dark for 3 days.
[0041] (5) Cultivation of genetically transformed materials: After the leaves are cultured in the co-culture medium for 2 days, they are transferred to the differentiation medium. After 20 to 30 days, small buds can be seen growing from the callus tissue. When the leaves grow to about 2 cm, they are transferred to the rooting medium. The well-growing seedlings are transplanted into the soil.
[0042] (6) Positive detection of genetically transformed materials: PCR detection was performed on the vector-transformed T0 generation transgenic materials, and the target band size was compared. The PCR products were sent for testing and compared with the wild-type gene sequence to analyze the gene mutation. The results showed that all 17 target CrRLK genes were mutants, and the types of gene mutations included base mutations, fragment deletions, and base insertions ( Figure 2 ), of which 9 homozygous mutants were obtained.
[0043] Table 2 NtCrRLK knockout vector design
[0044]
[0045]
[0046] Table 3 Primer design for CRISPR vector construction
[0047]
[0048]
[0049] CRISPR / Cas9 gene knockout technology was used to create mutants, and the function of the gene was analyzed by analyzing the mutant phenotype. Given the complexity of the plant rhizosphere microenvironment response process and the possible functional redundancy among CrRLK family members, multiple CRISPR targets were designed for the evolutionarily adjacent and highly similar NtCrRLK genes, and editing vectors capable of simultaneously knocking out single and double genes were constructed ( Figure 1 ), thereby maximizing knockout efficiency. NtCrRLK01 / 52, NtRLK06 / 16, NtCrRLK14 / 20, NtCrRLK07 / 37, NtCrRLK03 / 08, NtCrRLK05 / 50, NtCrRLK15 / 46, and NtCrRLK10 / 11, which have high homology, were used to construct double mutants, while single mutants were constructed for NtCrRLK04. Multi-gene knockout of tobacco CrRLKs can significantly reduce the physiological functions shared by similar CrRLK protein members, thereby helping the knockout mutants exhibit more pronounced growth phenotypes.
[0050] (2) Phenotypic identification of tobacco CrRLK gene mutants
[0051] Under controlled indoor conditions (long daylight, 24°C), the obtained tobacco CrRLK gene homozygous mutants were phenotypically identified. The core goal was to evaluate the effects of their rhizosphere microenvironmental characteristics on the subsequent growth and development of tobacco plants. The experiment used 9-cm-diameter nutrient pots, each filled with 110 g of nutrient soil and moistened, and sowed seeds of wild-type tobacco Yunyan 87 (control) and 9 different CrRLK mutants (treatment group), respectively, with 3 technical replicates per group. The nutrient pots were placed in a growth chamber for culture and watered regularly. One week after sowing, thinning and transplanting were carried out to ensure that one seedling was retained in each pot. After 4 weeks of growth, the above-ground parts of the plants were removed, and the roots were thoroughly mixed with the nutrient soil in the pot to form a rhizosphere soil mixture representing the characteristics of each genotype. Subsequently, the one-week-old Yunyan 87 seedlings (as unified recipients) were transplanted into the rhizosphere soil mixture of the above-mentioned genotypes and continued to be cultured in the growth chamber for 4 weeks. After incubation, the growth indicators of the recipient Yunyan 87 plants, including plant fresh weight, leaf area, and root length, were systematically measured and recorded. By comparing the growth differences between recipient plants grown in rhizosphere soils derived from wild-type plants and those derived from different CrRLK mutants, the authors systematically evaluated the effects of CrRLK genotype variation on the characteristics of the tobacco rhizosphere microenvironment and its ability to shape subsequent plant growth. Based on this, the changes in the rhizosphere microenvironment of each mutant were summarized and analyzed.
[0052] After obtaining the treatment results of each group, the growth phenotype of transplanted Yunyan 87 was Figure 3 As shown in A, the leaf phenotypes of Yunyan 87 in each group are compared. Figure 3 As shown in B. The growth phenotype analysis showed that planting in the soil after the mutant material Ntcrrlk was planted had the most significant effect in alleviating the imbalance of the rhizosphere microenvironment.
[0053] Among them, after each treatment, the fresh weight, stem length, root length and leaf area of transplanted Yunyan 87 were measured respectively. Figure 4 As shown in Figures AD, compared to the control group, Yunyan 87 grown in soil inoculated with the mutant material NTRLK04 had a 47.5% higher fresh weight, 25.4% higher stem length, 26.3% higher root length, and 29.5% higher leaf area. These results demonstrate that the NtCrRLK04 mutation effectively promotes tobacco root development, enabling it to adapt to changes in the rhizosphere microenvironment. Three replicates of the experiment demonstrated consistent results.
[0054] In summary, the present invention provides an application of the tobacco NtCrRLK04 gene in alleviating the imbalance of the tobacco rhizosphere microenvironment. By knocking out or interfering with the expression of the gene in tobacco, or constructing a tobacco mutant strain with the tobacco NtCrRLK04 gene knocked out, tobacco is planted in the soil after the mutant material is planted. This can effectively alleviate the imbalance of the tobacco rhizosphere microenvironment and increase tobacco yield, and has application prospects in the field of tobacco cultivation.
[0055] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.
Claims
1. Application of the NtCrRLK04 gene or NtRLK04 mutant in alleviating the imbalance of the tobacco rhizosphere microenvironment and eliminating the continuous cropping obstacle, characterized in that: The NtCrRLK04 gene in tobacco was subjected to site-directed mutagenesis using gene editing to construct an NtRLK04 mutant with low or no expression of the NtCrRLK04 gene. The nucleotide sequence of the NtCrRLK04 gene is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The specific gene editing method is as follows: taking the tobacco NtCrRLK04 gene as the target, designing an sgRNA sequence based on CRISPR / Cas9, and connecting a DNA fragment encoding the sgRNA into a CRISPR / Cas9 vector to transform tobacco, thereby achieving site-specific editing of the tobacco NtCrRLK04 gene and obtaining a mutant with low or no expression of the NtCrRLK04 gene.
3. The use according to claim 2, characterized in that The nucleotide sequence of the sgRNA sequence is at least one of the sequences shown in SEQ ID NO.8-10.
4. A method for breeding NtRLK04 mutants to alleviate the imbalance of tobacco rhizosphere microenvironment and eliminate continuous cropping obstacles, characterized in that: The method comprises the following steps: (1) Gene editing was performed on the tobacco NtCrRLK04 gene to construct a knockout vector, and the knockout vector was transformed into Agrobacterium; the nucleotide sequence of the NtCrRLK04 gene is shown in SEQ ID NO.1; (2) The Agrobacterium in step (1) is genetically transformed into tobacco to obtain an NTRLK04 mutant with the NtCrRLK04 gene knocked out.
5. The method according to claim 4, characterized in that The specific gene editing method is as follows: taking the tobacco NtabCrRLK12 gene as the target, designing an sgRNA sequence based on CRISPR / Cas9, and connecting a DNA fragment containing the encoding of the above sgRNA into a CRISPR / Cas9 vector to transform tobacco, thereby achieving site-specific editing of the tobacco NtabCrRLK12 gene and obtaining a mutant with low or no expression of the NtabCrRLK12 gene.
6. The method according to claim 5, characterized in that The sgRNA nucleotide sequence is at least one of the sequences shown in SEQ ID NO.8-10.
7. The method according to any one of claims 4 to 6, characterized in that: The tobacco is Yunyan 87.