Application of Biella CIPK12 gene in improving salt tolerance of hybrid Taxodium distichum

Abstract

The application discloses application of a Nitraria billingsii CIPK12 gene in improving salt tolerance of a hybrid Liriodendron chinense, and belongs to the technical field of gene breeding.The Nitraria billingsii CIPK12 gene positively responding to stress is cloned from the Nitraria billingsii, the Nitraria billingsii CIPK12 gene is overexpressed in hybrid Liriodendron chinense embryogenic callus and hybrid Liriodendron chinense plants, and the results show that the Nitraria billingsii CIPK12 gene can positively regulate the salt tolerance of the hybrid Liriodendron chinense, and can be used as a candidate gene for molecular improvement breeding of forest salt tolerance, thereby providing important gene resources for cultivating new forest varieties suitable for planting in saline-alkali land, and having good practicability.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to the application of the Bilatia nigra NbCIPK12 gene in improving the salt tolerance of hybrid tulip trees. Background Technology

[0002] Plants of the genus *Nitraria* are typical halophytic shrubs, widely distributed in arid deserts and saline-alkali areas of Northwest my country, exhibiting strong tolerance to extreme environments such as drought and high salinity. Among them, *Nitraria billardieri*, as an important germplasm resource of the genus, has developed a unique salt tolerance mechanism during its long-term adaptation to saline habitats. Studies have shown that *Nitraria* can transfer sodium... + Transported to the leaves and translocated to the vacuoles for compartmentalization to reduce the toxicity of Na⁺ in the cytoplasm, while simultaneously regulating K. + Ca 2+ Mg 2+ The absorption, transport, and distribution of mineral elements are essential for maintaining normal metabolic processes. Furthermore, under salt stress, *Nitraria tangutorum* can enhance its adaptability to saline environments by increasing leaf succulence and accumulating osmotic regulators. Given its outstanding salt tolerance, *Nitraria tangutorum* contains valuable salt-tolerant gene resources in its genome. In recent years, researchers have cloned several genes related to salt stress responses from *Nitraria tangutorum*, such as plasma membrane Na+. + / H + Antiporter gene SOS1, vacuolar membrane Na + / H + The reverse transporter gene NHX1 and the L-galactose-1-phosphate phosphatase gene GPP are among the key components. However, there is limited research on the salt tolerance function of CBL-CIPK signaling pathway members, especially CIPK family genes, in *Nitraria tangutorum*, and the role of CIPK12 in the salt stress response of *Nitraria tangutorum* has not been reported.

[0003] Hybrid tulip trees (Liriodendron tulipifera) are excellent artificial hybrids belonging to the genus Liriodendron in the family Magnoliaceae. They are characterized by rapid growth, straight trunks, beautiful tree shapes, and strong adaptability, making them an important timber and landscaping tree species in southern my country. With the increasing demand for greening and afforestation of saline-alkali land in my country, cultivating new hybrid tulip tree varieties with higher salt tolerance is of great significance for expanding their planting range and improving the effectiveness of ecological management of saline-alkali land. Currently, genetic improvement of salt tolerance in hybrid tulip trees still relies on conventional breeding, which suffers from problems such as long breeding cycles and low efficiency in improving salt tolerance traits. Discovering superior exogenous salt-tolerant genes for molecular breeding of hybrid tulip trees is an effective way to improve their salt tolerance. This application provides new gene resources and application schemes to address this need. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the problem to be solved by this application is to provide the application of the Bilatia nigra NbCIPK12 gene in improving the salt tolerance of hybrid tulip trees.

[0005] To solve the above-mentioned technical problems, the technical solution adopted in this application is as follows:

[0006] Application of the Biladdi white thorn NbCIPK12 gene in improving the salt tolerance of hybrid tulip tree, the nucleotide sequence of the Biladdi white thorn NbCIPK12 gene is shown in SEQ ID NO.1.

[0007] The expressed protein of the NbCIPK12 gene of *Nelumbo nucifera* is shown in SEQ ID NO.2.

[0008] The application described follows these steps:

[0009] 1) Construct a recombinant plasmid of the NbCIPK12 gene of *Bilacos chinensis*;

[0010] 2) Transfection of hybrid tulip tree callus;

[0011] 3) Induce callus tissue of hybrid tulip tree to form somatic embryos, and obtain regenerated plants of hybrid tulip tree with improved salt tolerance.

[0012] The recombinant plasmid used in the aforementioned application is pRI 101-NbCIPK12.

[0013] The application described uses hybrid tulip tree embryogenic callus that has been subcultured for 18 days as material. It is infected with Agrobacterium tumefaciens transformed with NbCIPK12, and after two days of co-culture, the callus is destered. One week after destering, the callus is placed on a subculture medium supplemented with G418 and cephalosporin (M13 medium supplemented with 2 mg / L 2,4-D) for screening. After the emergence of transgenic positive callus, samples are taken for DNA extraction and PCR verification to confirm whether the selected callus is a positive transgenic callus.

[0014] The application described above uses NbCIPK12 transgenic hybrid tulip tree callus as material to induce somatic embryogenesis. After the somatic embryos mature, they are inoculated onto MS medium. After the somatic embryos germinate and grow into seedlings, DNA is extracted from the leaves. The DNA is then verified by PCR product gel electrophoresis and sequencing to determine whether the regenerated plants are NbCIPK12 transgenic regenerated plants.

[0015] This application uses the halophyte *Nitraria pedunculata* as material to clone the NbCIPK12 gene, which was significantly upregulated after salt treatment. An NbCIPK12 overexpression vector was constructed and transformed into hybrid *Liriodendron chinense*. Salt-tolerance function of the NbCIPK12 gene was verified through salt stress treatment at both the callus and regenerated plant levels. This application confirms the role of the NbCIPK12 gene in the regulation of salt tolerance in *Nitraria pedunculata*, providing gene resources and theoretical basis for molecular breeding of salt-tolerant woody plants, and also laying the foundation for a deeper understanding of the salt tolerance regulatory mechanism of the CBL-CIPK signaling network in halophytes.

[0016] Beneficial effects: Compared with the prior art, the technical advantages of this application are as follows:

[0017] 1) This application cloned the NbCIPK12 gene, which actively responds to stress, from *Nitraria pekinensis*. Gene expression detection results confirmed that after treating *Nitraria pekinensis* seedlings with 400 mM NaCl for 2 hours, the expression level of NbCIPK12 in the root, stem, and leaf organs was significantly upregulated compared with untreated plants.

[0018] 2) The results of the overexpression experiment of NbCIPK12 gene in embryogenic callus of hybrid tulip tree confirmed that NbCIPK12 gene can promote the growth of transgenic callus under salt stress treatment.

[0019] 3) The results of the overexpression experiment of NbCIPK12 gene in hybrid tulip tree plants confirmed that NbCIPK12 gene can reduce the wilting rate of leaves of transgenic seedlings under salt treatment and improve the salt tolerance of transgenic seedlings.

[0020] 4) The results of this application show that the NbCIPK12 gene of *Liriodendron bilatum* can positively regulate the salt tolerance of hybrid tulip trees. It can be used as a candidate gene for molecular improvement breeding of salt-tolerant forest trees, providing important gene resources for breeding new forest tree varieties adapted to saline-alkali land, and also providing a research basis for further analysis of the molecular mechanism of CIPK gene regulation of plant salt tolerance. Attached Figure Description

[0021] Figure 1 This is an image of the pRI 101-NbCIPK12 vector;

[0022] Figure 2 Figure 1 shows the results of the analysis of the relative expression of NbCIPK12 in response to salt in *Nitraria bilaudiana*. In the figure, (A) is the phenotype of two-month-old *Nitraria bilaudiana* seedlings, and (B) is the change in the relative expression of NbCIPK12 in the root, stem, and leaf tissues and organs of two-month-old *Nitraria bilaudiana* seedlings after treatment with 400 mM NaCl for 2 hours using real-time quantitative PCR (qRT-PCR).

[0023] Figure 3 The figure shows the growth analysis results of wild-type and NbCIPK12 overexpression hybrid callus treated with salt. In the figure, (A) shows the growth phenotype of the callus after 5 days of subculture using culture media supplemented with 0 mM NaCl and 150 mM NaCl, respectively; (B) shows the statistical analysis results of the area of ​​the callus after 5 days of treatment with 0 mM NaCl and 150 mM NaCl.

[0024] Figure 4 The figures show the phenotypic results of wild-type hybrid tulip trees and NbCIPK12 overexpression hybrid tulip trees treated with salt. In the figures, (A) shows the phenotypic results of wild-type hybrid tulip trees and NbCIPK12 overexpression hybrid tulip trees treated with 0 mM NaCl, 150 mM NaCl and 200 mM NaCl for 16 days; (B) shows the statistical results of the proportion of wilted leaves of wild-type hybrid tulip trees and NbCIPK12 overexpression hybrid tulip trees treated with different concentrations of NaCl for 16 days. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0026] The materials used in the following examples are as follows: Seeds of *Nitraria pekinensis* were vernalized in sand at 4°C for two months, and then germinated in a germination box. After the seeds germinated and sprouted into seedlings, the seedlings were transplanted into nutrient soil and placed in a light incubator at 23°C for 16 hours of light and 8 hours of darkness for two months, after which they were used for salt stress treatment.

[0027] The data analysis method in the following examples is as follows: initial data is organized and statistically analyzed using Excel, and charts are drawn using Graphpad Prism 11.0.0 software.

[0028] Example 1: Gene Cloning and Overexpression Vector Construction

[0029] Based on the genomic data of *Nitraria bildis*, the NbCIPK12 gene was identified. RNA was extracted from *Nitraria bildis* leaves, and cDNA was synthesized using a reverse transcription kit (catalog number: R312-01) purchased from Nanjing Novizan Biotechnology Co., Ltd. This cDNA served as the template for cloning the NbCIPK12 gene. The gene cloning primers are as follows:

[0030] NbCIPK12 F: 5'-ATGGCAAGCTCCGCCGCC-3',

[0031] NbCIPK12 R: 5'-TGTATCTGATGGCAGATGCAAATC-3';

[0032] After cloning the NbCIPK12 gene fragment, this fragment was used as a template for PCR. PCR was performed using primers carrying the homologous arms of the target vector to add homologous arms to the NbCIPK12 gene fragment. Primers carrying the homologous arms of the target vector:

[0033] pRI 101-NbCIPK12 F: 5'-ttgatacatatgcccgtcgacATGGCAAGCTCCGCCGCC-3',

[0034] pRI 101-NbCIPK12 R:

[0035] 5'-gatgaattcggatccggtaccTGTATCTGATGGCAGATGCAAATC-3'.

[0036] The complete coding region of NbCIPK12 was ligated into the overexpression vector pRI101-AN using restriction enzyme digestion and homologous recombination ligation. Figure 1 The vector carries the CaMV 35S promoter and a kanamycin resistance selection tag. The vector was double-digested with SalI and KpnI, and the restriction endonuclease reaction system was prepared according to the instructions. After digestion, the linearized vector and the NbCIPK12 gene fragment with added homologous arms were ligated using recombinase. The recombinase ligation kit, ClonExpress Ultra One Step Cloning Kit V3, was purchased from Novizan (catalog number: C117-01 / 02). The ligation product was then transformed into *E. coli* DH5α competent cells, plated on LB agar medium supplemented with kanamycin, and incubated upside down at 37°C for 12–16 h. Single clones were picked for PCR identification. Positive clones were sent to a biotechnology company for sequencing verification. After confirming the sequence was correct, the recombinant plasmid was extracted and transformed into *Agrobacterium* for subsequent genetic transformation experiments of hybrid tulip trees. The NbCIPK12 gene obtained by sequencing has a nucleotide sequence as shown in SEQ ID NO.1, and its expressed protein has an amino acid sequence as shown in SEQ ID NO.2.

[0037] Example 2 Real-time quantitative PCR (qRT-PCR) analysis

[0038] To analyze the expression changes of NbCIPK12 in *Nitraria bildis* during salt stress response, two-month-old seedlings were treated with 400 mM NaCl solution. qRT-PCR was used to detect the expression changes of NbCIPK12 in the root, stem, and leaf tissues of *Nitraria bildis*. Using the expression level of NbCIPK12 in the root of *Nitraria bildis* under normal growth conditions as a reference, the relative expression levels of NbCIPK12 in each tissue after salt treatment were calculated.

[0039] The expression of the NbCIPK12 gene in the response of *Bilacera bistorta* salt was detected by qRT-PCR. The experimental procedures were performed according to the instructions of the AceQ qPCR SYBR Green Master Mix (Without ROX) kit from Novizan Pharmaceuticals (Nanjing). A LightCycler 480 II (Roche, Switzerland) instrument was used for quantitative PCR. The reaction program was: 1. 95℃ for 30 s, 95℃ for 10 s; 3. 60℃ for 34 s, with steps 2 and 3 repeated 40 times. After the reaction, 2... -ΔΔCt The relative expression levels of genes were calculated using the qRT-PCR method. The qRT-PCR primers are shown in Table 2. The qRT-PCR primer sequences are as follows:

[0040] NbCIPK12 qPCR F: 5'-AAGTTACTTCTTTGCCCAGACCG-3',

[0041] NbCIPK12 qPCR R: 5'-CCTCCTTCTTCAAATAGTCCCGAT-3';

[0042] NbActin qPCR F: 5'-TACAGTGTCATCACAATCGAGAGGC-3',

[0043] NbActin qPCR R: 5'-AACTCCAGATGACACAAGGCAAG-3'.

[0044] Figure 2 This study analyzed the relative expression changes of NbCIPK12 in salt-responsive *Nitraria bildis* seedlings. (A) Phenotype of two-month-old *Nitraria bildis* seedlings. (B) Detection of the relative expression changes of NbCIPK12 in root, stem, and leaf tissues and organs of two-month-old *Nitraria bildis* seedlings treated with 400 mM NaCl for 2 hours using quantitative real-time PCR (qRT-PCR). Actin2 gene was used as an internal reference gene. -ΔΔCt The fold change in relative expression of NbCIPK12 before and after salt treatment was calculated. One-way ANOVA was used to analyze the significance of the differences in the data. This means P < 0.01.

[0045] Figure 2 The results showed that NbCIPK12 was upregulated in the root, stem, and leaf tissues after salt treatment, with the largest upregulation in the leaf tissue. The expression level after salt treatment was about 2.7 times that in the control root tissue, while the expression levels in the stem and leaves were 1.7 times and 1.6 times that in the control root tissue, respectively. This indicates that NbCIPK12 can respond to salt treatment in different tissues of Nitraria bildis, and the response is the highest in the leaves under salt stress.

[0046] Example 3: Treatment of callus and regenerated plants from NbCIPK12 overexpression hybrid tulip tree

[0047] 1. To analyze the effect of the NbCIPK12 gene on the salt tolerance of hybrid tulip tree, a 35S:NbCIPK12 overexpression vector was constructed, and genetic transformation was performed using embryogenic callus tissue of hybrid tulip tree. NbCIPK12 transgenic hybrid tulip tree callus tissue was obtained through resistance screening and identification of positive transgenic callus tissue. Wild-type and NbCIPK12 transgenic callus tissues were treated with subculture media supplemented with 0 mM NaCl and 150 mM NaCl, respectively.

[0048] Using hybrid tulip tree embryogenic callus subcultured for 18 days as material, *Agrobacterium tumefaciens* EHA105 transformed with NbCIPK12 was used for infection. After two days of co-culturing, the callus was destered. One week after destering, the callus was transferred to M13 medium supplemented with G418 and cephalosporin for selection. Once transgenic positive callus had grown, samples were taken for DNA extraction and PCR verification to confirm whether the selected callus was a positive transgenic callus. Subculture was used to expand the callus tissue. The subculture cycle was 20-25 days. When the transgenic callus tissue proliferated to a sufficient size for subsequent experiments, wild-type callus tissue and NbCIPK12 transgenic callus tissue were subcultured for 20 days and placed on subculture medium supplemented with 0 mM NaCl and 150 mM NaCl. After treatment for 5 days, the callus tissue was observed and photographed using a stereomicroscope. The size of the callus tissue was analyzed using ImageJ to clarify the effect of NbCIPK12 overexpression on the salt tolerance of hybrid tulip tree callus tissue.

[0049] Figure 3This study analyzes the growth of callus from wild-type and NbCIPK12-overexpressing hybrid tulip tree treated with salt. (A) The growth phenotype of the callus was observed after 5 days of subculture using culture media supplemented with 0 mM NaCl and 150 mM NaCl, respectively. (B) The area of ​​the callus was statistically analyzed after 5 days of treatment with 0 mM NaCl and 150 mM NaCl. One-way ANOVA was used to analyze the significance of data differences; ns represents no significant difference. This means P < 0.01.

[0050] Figure 3 The results showed that on culture medium without NaCl, the growth of wild-type and transgenic callus was consistent, and the size difference of callus was not significant. On subculture medium supplemented with 150 mM NaCl, the growth of NbCIPK12 transgenic callus was significantly better than that of wild-type callus, and its callus size was significantly larger than that of wild-type.

[0051] 2. To further verify the effect of the NbCIPK12 gene on the salt tolerance of hybrid tulip trees, somatic embryogenesis technology was used to induce somatic embryos and regenerated plants from wild-type and NbCIPK12 transgenic callus tissues. Wild-type and NbCIPK12 overexpression regenerated plants with consistent growth status were selected and transplanted into culture media supplemented with different concentrations of NaCl. The treatment was continued for 15 days, and the plant phenotype was observed and the proportion of wilted leaves was counted.

[0052] Somatic embryogenesis was induced using wild-type and NbCIPK12 transgenic hybrid callus tissue. After the somatic embryos matured, they were inoculated onto MS medium. After the somatic embryos germinated and formed seedlings, DNA was extracted from the leaves. The DNA was then extracted by PCR product gel electrophoresis and sequencing to verify whether the regenerated plants were NbCIPK12 transgenic regenerated plants.

[0053] Two-month-old wild-type and NbCIPK12 transgenic hybrid tulip tree seedlings with uniform growth were selected and transferred to MS medium supplemented with 0, 150, and 200 mM NaCl. Phenotypic changes in the plants were dynamically monitored. After 16 days of salt treatment, the wilting rate of the plant leaves was statistically analyzed to determine the effect of NbCIPK12 overexpression on the salt tolerance of the hybrid tulip tree plants.

[0054] Figure 4The figures show the phenotypes of wild-type hybrid tulip trees and NbCIPK12 overexpression hybrid tulip trees treated with salt. In the figure, (A) shows the phenotypes of wild-type hybrid tulip trees and NbCIPK12 overexpression hybrid tulip trees treated with 0 mM NaCl, 150 mM NaCl, and 200 mM NaCl for 16 days; (B) shows the statistical results of the proportion of wilted leaves in wild-type hybrid tulip trees and NbCIPK12 overexpression hybrid tulip trees treated with different concentrations of NaCl for 16 days. One-way ANOVA was used to analyze the significance of data differences. This means P < 0.01.

[0055] Figure 4 The results showed that after 16 days of treatment with 150 mM NaCl, 67% of wild-type plants exhibited obvious leaf wilting and yellowing, with some plants dying completely. In contrast, only 19% of NbCIPK12-overexpressing plants showed leaf wilting, and their overall growth was significantly better than that of wild-type plants. After 16 days of treatment with 200 mM NaCl, more than 95% of wild-type plants showed obvious leaf wilting, with most plants dying completely. In contrast, the wilting rate of NbCIPK12-overexpressing plants was 56%, significantly lower than that of wild-type plants.

[0056] The above results indicate that overexpression of NbCIPK12 can significantly improve the salt tolerance of hybrid tulip trees.

Claims

1. Application of the Biladdi white thorn NbCIPK12 gene in improving the salt tolerance of hybrid tulip tree, wherein the nucleotide sequence of the Biladdi white thorn NbCIPK12 gene is shown in SEQ ID NO.

1.

2. The expressed protein of the NbCIPK12 gene of *Bryonymus bilatum* as described in claim 1, with the nucleotide sequence shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that: The steps are as follows: 1) Construct a recombinant plasmid of the NbCIPK12 gene of *Bilacos chinensis*; 2) Transfection of hybrid tulip tree callus; 3) Induce transgenic callus tissue of hybrid tulip tree to form somatic embryos, and obtain regenerated plants of hybrid tulip tree with improved salt tolerance.

4. The application according to claim 3, characterized in that: The recombinant plasmid was pRI 101-NbCIPK12.

5. The application according to claim 3, characterized in that: Using hybrid tulip tree embryogenic callus that had been subcultured for 18 days as material, EHA105 Agrobacterium transformed with NbCIPK12 was used for infection. After two days of co-culture, the callus was destered. One week after destering, the callus was placed on a subculture medium supplemented with G418 and cephalosporin for screening. The subculture medium was M13 medium supplemented with 2 mg / L 2,4-D. After the transgenic positive callus grew, samples were taken for DNA extraction and PCR verification to confirm whether the selected callus was a positive transgenic callus.

6. The application according to claim 3, characterized in that: Somatic embryogenesis was induced using NbCIPK12 transgenic hybrid tulip tree callus. After the somatic embryos matured, they were inoculated onto MS medium. After the somatic embryos germinated and formed seedlings, DNA was extracted from the leaves. The DNA was then verified by PCR product gel electrophoresis and sequencing to confirm whether the regenerated plants were NbCIPK12 transgenic regenerated plants.

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