A sweet potato salt tolerance-related gene IbCHR10 and its application
By identifying and overexpressing the IbCHR10 gene in sweet potatoes, the problem of hindering development of sweet potatoes under salt stress was solved, and the salt tolerance of sweet potatoes was significantly improved, providing a way to apply salt tolerance genes.
Patent Information
- Application Number
- CN202510825676.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-08-22
- Estimated Expiration
- 2045-06-19
AI Technical Summary
There are no related reports on the IbCHR10 gene in sweet potatoes in the prior art, resulting in the hindered development of sweet potato plants under salt stress, reduced yield and quality, and lack of effective salt-tolerant gene improvement methods.
By identifying the sweet potato DC1 protein family, the salt-tolerant gene IbCHR10 was obtained, and the gene overexpression vector was constructed, and the Agrobacterium mediated method was used to transform it into sweet potatoes, which increased the tolerance of crops to salt stress.
Overexpression of IbCHR10 significantly enhances the salt tolerance of sweet potatoes, improves the tolerance of sweet potatoes to salt stress, and provides application prospects for salt-tolerant-related genes.
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Figure CN120350030B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of nucleic acid technology, and particularly relates to a sweet potato salt tolerance-related gene IbCHR10 and an application thereof. Background Art
[0002] The information disclosed in this background technology section is only intended to enhance some understanding of the overall background of the invention and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.
[0003] Salt stress can cause ion imbalance, nutrient deficiency, osmotic stress, ion toxicity, and oxidative stress in sweet potato plants, hindering their development and reducing root yield and quality. Therefore, cultivating sweet potato varieties with high yield, high nutritional value, and greater salt tolerance is an effective guarantee for efficient production.
[0004] In recent years, a class of zinc finger proteins containing a C1 domain has been discovered in plants. The C1 domain is a zinc finger domain rich in cysteine and histidine, consisting of approximately 50 amino acids. Plant C1 domain proteins often contain multiple C1 domains and are therefore also known as plant DC1 domain proteins. They play an important role in plant growth, development, and stress resistance. C1 domains were originally discovered in PKCs by binding to phosphatidylcholine (DAG) and phenolic esters. They were later found to bind proteins by transporting them to membranes, where they interact with signaling complexes, substrates and regulators, substrate-enzyme activation, and membrane trafficking, thereby regulating the expression of genes involved in stress and, consequently, the plant's ability to cope with stress (Bhaskar RV, Mohanty B., Verma V., Wijaya E., Kumar PP. A hormone-responsive C1-domain-containing protein At5g17960 mediates stress response in Arabidopsis thaliana [J]. PLoS ONE, 2015, 10, e0115418). Several DC1 domain-containing proteins have been identified in plants. Although named differently in different plant species, they all play a role in plant disease resistance, stress tolerance, and growth and development.The Arabidopsis DC1 domain genes ULI3, VLG, and BNP are involved in photomorphogenesis, seed development, and pollen development, respectively. At5g17960 is induced by certain hormones and stresses and may be involved in plant hormone-induced stress responses (Suesslin C., Frohnmeyer H. An Arabidopsis mutant defective in UV-B light-mediated responses[J]. Plant Journal. 2003, 33, 591–601; D'Ippolito S., Arias LA, Casalongué CA, Pagnussat GC, Fiol DF The DC1-domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis[J]. Plant Journal. 2017, 90, 261-275; Brownfield L. Pollen helps reveal a role for DC1 domain proteins[J]. Plant Cell Physiology. 2022, 63, The DC1 domain gene TaCHP in wheat is differentially expressed between salt-tolerant and salt-sensitive varieties. Overexpression of TaCHP significantly improves wheat production performance under salt stress (Li CL, Lv J., Zhao X., Ai XH, Zhu XL, Wang MC, Zhao SY, Xia GM TaCHP: A wheat zinc finger protein gene down-regulated by abscisic acid and salinitystress plays a positive role in stress tolerance[J]. Plant Physiology. 2010, 154, 211-221).
[0005] In the prior art, there are no reports related to the IbCHR10 gene in sweet potato. Summary of the Invention
[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a salt tolerance related gene IbCHR10 and its application to fill the gaps in the existing technology.
[0007] The technical solution adopted in the present invention is as follows:
[0008] In a first aspect of the present invention, a salt-tolerance-related gene IbCHR10 is provided. The gene is obtained by identifying the sweet potato DC1 protein family and combining salt-tolerance transcriptome analysis. The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0009] In a second aspect of the present invention, the use of the IbCHR10 gene in improving the tolerance of crops to salt stress is provided.
[0010] Specifically, the tolerance of crops to salt stress is improved by overexpressing IbCHR10; the method for overexpressing IbCHR10 comprises the following steps:
[0011] The cloned IbCHR10 was constructed into a gene overexpression vector to obtain a recombinant vector;
[0012] The obtained recombinant vector is transformed into Agrobacterium tumefaciens;
[0013] Agrobacterium tumefaciens is inoculated into crops to achieve gene overexpression.
[0014] Preferably, the crop is sweet potato.
[0015] Preferably, the gene overexpression vector is a pCAMBIA1301 vector.
[0016] In a third aspect of the present invention, there is provided a use of the polypeptide encoded by the IbCHR10 gene in improving the tolerance of crops to salt stress.
[0017] In the fourth aspect of the present invention, a specific primer for detecting the salt tolerance-related gene IbCHR10 is provided, wherein the sequence from the 5' end to the 3' end of the upstream primer is shown as SEQ ID NO.2, and the sequence from the 5' end to the 3' end of the downstream primer is shown as SEQ ID NO.3.
[0018] In a fifth aspect of the present invention, there is provided the use of the amplification primers for detecting the salt-tolerance-related gene IbCHR10 in crop trait-related research for use in salt-tolerance-assisted crop breeding.
[0019] In a sixth aspect of the present invention, a method for cultivating highly salt-tolerant crops is provided, the method comprising the following steps: transforming the crops using a vector-mediated method to obtain transgenic crops overexpressing IbCHR10.
[0020] Preferably, the vector-mediated method is Agrobacterium-mediated method.
[0021] Preferably, the method specifically comprises the following steps:
[0022] IbCHR10 was cloned into a gene overexpression vector to obtain a recombinant vector;
[0023] The obtained recombinant vector is transformed into Agrobacterium tumefaciens;
[0024] Agrobacterium tumefaciens is inoculated into crops to achieve gene overexpression.
[0025] Further preferably, the gene overexpression vector is a pCAMBIA1301 vector.
[0026] Compared with the related art known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:
[0027] The IbCHR10 gene provided by the present invention lacks a conserved DST element region downstream of its 3'UTR region, and its mRNA is stable after translation, confirming that the gene is expressed at higher levels in sweet potato fibrous roots than in other tissues. The present invention successfully cloned IbCHR10 from sweet potato and transformed the gene into sweet potato via Agrobacterium-mediated transfection, demonstrating for the first time that overexpression of IbCHR10 enhances salt stress tolerance in sweet potato. This further demonstrates that the IbCHR10 gene is associated with salt tolerance in sweet potato. This suggests that the gene IbCHR10 of the present invention has broad application prospects in research related to salt stress tolerance and trait selection in sweet potato. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The accompanying drawings, which constitute a part of the specification of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.
[0029] Figure 1 for IbCHR expression patterns of genes in sweet potato varieties with different salt tolerance;
[0030] Figure 2 for IbCHR10 Amplification results of the full-length cDNA sequence of the gene;
[0031] Figure 3 for IbCHR10 gene expression patterns in different tissues;
[0032] Figure 4 for IbCHR10 expression patterns of genes under different stress and hormone treatments;
[0033] Figure 5 Sweet potato IbCHR10Identification of transgenic plants;
[0034] Figure 6 Sweet potato IbCHR10 Phenotypic identification of salt tolerance in transgenic plants. DETAILED DESCRIPTION
[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.
[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof.
[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0038] Example 1 Gene identification and amplification
[0039] Twelve DC1 domain family members were identified from the sweet potato genome data and named IbCHR1-IbCHP12 according to their location on the chromosome. IbCHR The expression of genes in different salt-tolerant sweet potato varieties under salt treatment was different. Figure 1 shown.
[0040] Depend on Figure 1 It can be seen that IbCHR10 The expression level was higher in the salt-tolerant sweet potato variety Jishu 26.
[0041]
[0042] IbCHR10-F: 5'-ATGGAGTATAAGCATTTC-3' (as shown in SEQ ID NO. 2)
[0043] IbCHR10-R: 5'-TCACTCATGAAATCGAGT-3' (as shown in SEQ ID NO. 3)
[0044] Extraction of total RNA from sweet potato: Total RNA was extracted from the fibrous root samples of the sweet potato variety 'Jishu 25' using the RNA rapid extraction kit (product number: AC0307) from Sikoje. The RNA quality was tested by agarose gel electrophoresis.
[0045] Reverse transcription to obtain cDNA: TaKaRa reverse transcription kit (product number: RR047Q) was used to reverse transcribe sweet potato RNA to obtain total cDNA of sweet potato fiber roots.
[0046] The full-length sequence of IbCHR10 was amplified using the high-fidelity enzyme ApexHF HS DNA polymerase-FS from Acryl Corporation and the cDNA of sweet potato hairy roots as a template.
[0047] PCR amplification system: 50 μL total system (DNA Polymerase-FS 1 μL; 5× Buffer 10 μL; dNTPMix 1 μL; cDNA 1 μL; Primer-F 1 μL; Primer-R 1 μL; ddH2O 35 μL).
[0048] PCR amplification program: pre-denaturation: 94°C, 30 s; [denaturation: 98°C, 10 s; annealing: 55°C, 10 s; extension: 72°C, 5 s] × 35 cycles; cooling to 4°C.
[0049] The obtained PCR products were detected by 1.2% agarose gel electrophoresis ( Figure 2 The target fragments were recovered using the Novozymes FastPure® GelDNA Extraction Mini Kit (Cat. No. DC301) and ligated into the cloning vector VS007 (Qingke Biotechnology) at a 4:1 ratio of target fragment to cloning vector. After ligation at room temperature for 5 minutes, the fragments were transformed into competent E. coli DH5α cells and cultured overnight at 37°C. Single clones were identified and sequenced.
[0050] Example 2 IbCHR10 gene expression and separation in different tissues
[0051] The sweet potato variety 'Jishu 25' was used as the material. The fourth expanded leaf, stem tip, young stem, old stem, fibrous root and tuber of the sweet potato cultivar 'Jishu 25' were selected after 125 days of field growth. They were washed twice with distilled water, dried with absorbent paper, ground in liquid nitrogen and stored in cryovials at -80℃.
[0052] Primer Premier 5.0 software was used to design qRT-PCR primers for the non-conserved region of the IbCHR10 gene ORF sequence:
[0053] IbCHR10-qRT-F: 5'-ACATGTGCCAAAAATGCCCC-3' (as shown in SEQ ID NO. 4)
[0054] IbCHR10-qRT-R: 5'-TCTGGGACGAGAGGTCTAGC-3' (as shown in SEQ ID NO. 5)
[0055] The steps of total RNA extraction and reverse transcription were the same as in Example 1.
[0056] qRT-PCR experiments were performed using ChamQSYBR qPCR Master Mix (Product No.: Q311) in the CFX Connect™ Fluorescence Quantitative PCR Detection System (BIO-RAD, USA), and the internal reference gene used was IbActin. -ΔΔCT The relative expression levels of genes were calculated using the method. The average Ct value of each gene was obtained from three biological replicates. The qRT-PCR reaction conditions were as follows: pre-denaturation: 95°C, 3 min; cycling reaction: [95°C, 10 s; 60°C, 30 s] × 40 cycles. The results are shown in Figure 2. Figure 3 shown.
[0057] Depend on Figure 3 It can be seen that the expression level of IbCHR10 gene was highest in the fibrous roots of 'Jishu 25', followed by the stem and tuberous roots, and the lowest in the stem tip.
[0058] Example 3 Expression pattern of IbCHR10 gene under different stress and hormone treatments
[0059] Stem segments of 'Jishu 25' with uniform growth and 5-6 functional leaves were selected and treated with 1 / 2 Hoagland solution containing 20% PEG6000, 150 mM NaCl, 100 mM ABA and 150 μM GA3. Leaves were collected at 0 h, 3 h, 6 h, 12 h, 24 h and 48 h after treatment, quick-frozen in liquid nitrogen, and stored at -80℃ for later use.
[0060] The fluorescence quantitative experimental steps are the same as in Example 2, and the results are as follows Figure 4 shown.
[0061] Depend on Figure 4 It can be seen that the expression level of IbCHR10 gene first increased and then decreased after salt, drought and ABA treatment, and the expression level was the highest after 6 or 12 hours of treatment; after GA3 treatment, the expression level of IbCHR10 gene gradually increased.
[0062] Example 4 Construction of plant expression vector
[0063] The pCAMBIA1301-IbCHR10 recombinant vector was constructed using the ClonExpress® II One Step Cloning Kit (Cat. No. C112) from Novozymes. The primer sequences are as follows:
[0064] IbCHR10-1301-F: 5'-GGGGTACCCCGCCACCATGGAGTATAAG-3' (as shown in SEQ ID NO. 6)
[0065] IbCHR10-1301-R: 5'-GCTCTAGATCACTCATGAAATCGAGT-3' (as shown in SEQ ID NO. 7)
[0066] The vector plasmid was double-digested with restriction endonucleases Kpn I and Xba I, and the IbCHR10 ORF sequence was amplified using the high-fidelity enzyme FS. The target fragment was detected and recovered by agarose gel electrophoresis. The following reaction system was set up on ice: 0.03 pmol of linearized vector; 0.06 pmol of insert; 4 μL of 5× CE II Buffer; 2 μL of Exnase II; and 20 μL of ddH2O. The reaction was incubated at 37°C for 30 min and then cooled on ice. The recombinant plasmid was transformed into DH5α medium, and sequencing yielded the positive plasmid pCAMBIA1301-IbCHR10.
[0067] Example 5 Transformation of competent Agrobacterium
[0068] Take competent Agrobacterium tumefaciens EHA105 cells stored at -80°C and let them stand at room temperature for a short while. Once the competent cells thaw, immediately add 10 μL of the recombinant plasmid pCAMBIA1301-IbCHR10 and gently pipette to mix thoroughly. Incubate on ice for 5 minutes, in liquid nitrogen for 5 minutes, heat shock at 37°C for 5 minutes, and let stand on ice for 5 minutes. Add an appropriate amount of resistance-free LB liquid medium and allow the cells to recover at 28°C for 2-3 hours. Harvest the cells by centrifugation at 6000 rpm, discard some of the medium, resuspend the cells, and spread them onto YEP solid medium supplemented with 50 mg / L Kan and 50 mg / L Rif. Incubate inverted at 28°C for 2-3 days. Identify positive colonies by colony PCR and shake the cells for later use.
[0069] Example 6 Transgenic Function Verification - Sweet Potato Transformation Screening
[0070] Sweet potato embryonic callus culture: Use tubers from 'Jishu 25' tubers to germinate healthy seedlings. Cut stem segments approximately 7 cm in diameter and excise the 2 cm stem tip. Clean and disinfect the stem tip with 70% ethanol and then 2% sodium hypochlorite solution, then rinse three times with sterile water. Excise the white meristem encapsulated within the stem tip and place on a MS solid plate supplemented with 2,4-D at 26°C in the dark. After culturing the embryonic callus in MS liquid medium for 8 weeks, filter out smaller cell clusters using a sieve. Gently break up the larger cell clusters (>1 mm) remaining on the sieve with forceps and continue culturing for 3-5 days before Agrobacterium infection and transformation.
[0071] Agrobacterium infection of sweet potato callus: Pick positive colonies from the plate and inoculate them into YEP liquid medium for overnight culture. Inoculate the overnight culture solution into 100 mL YEP liquid medium supplemented with Kan (100 mg / L) at a ratio of 1:100 (v / v) and incubate at 28°C, 200 rpm for 6-8 h until the OD value of the solution reaches 0. 600 Between 0.8 and 1.2. Collect the cells by centrifugation at 5000 rpm for 5 minutes, resuspend in YEP liquid medium, wash twice, and then centrifuge and resuspend in 50 mL of infection solution (MS medium supplemented with 2 mg / L 2, 4-D and 200 μM acetosyringone (AS). Incubate at 28°C for 2 hours. Resuspend the sweet potato embryonic callus in the infection solution and shake slowly at 40 rpm for 20 minutes. Remove the excess solution with a pipette, and spread the infected callus onto solid co-culture medium (containing 2 mg / L 2, 4-D and 200 μM AS). Co-cultivate at 26°C in the dark for 3-5 days (depending on the growth status of the callus).
[0072] Screening and regeneration of transgenic plants: Calli after co-cultivation were transferred to sterile water supplemented with 300 mg / L Cef and gently shaken for 20 minutes to remove Agrobacterium from the callus surface. Repeat three times. Calli were then transferred to MS solid medium (containing 2 mg / L 2,4-D) and incubated at 26°C in the dark for 1 week. Calli after delayed culture were transferred to MS selective solid medium (containing 10 mg / L HYG and 300 mg / L Cef) and incubated under low light for 8 weeks, with the medium changed every 2 weeks. Calli that grew well on the selective medium were transferred to MS solid medium and cultured until complete plants emerged from the calli. The newly emerged transgenic plants were transferred to tissue culture flasks containing MS solid medium and numbered for multiplication.
[0073] Example 7 Transgenic Function Verification - Phenotypic Analysis
[0074] Identification of positive transgenic plants: DNA and total RNA were extracted from leaves of the transgenic sweet potato plants, and positive lines were further identified by PCR and qRT-PCR. Figure 5 The PCR detection primers are as follows:
[0075] HYG-F: 5'-TTCTACACAGCCATCGGTCC-3' (as shown in SEQ ID NO. 8)
[0076] HYG-R: 5'-CCCATGTGTATCACTGGCAA-3' (as shown in SEQ ID NO. 9)
[0077] Salt tolerance of transgenic plants: Positive overexpressing plants and 'Jishu 25' test tube seedlings were acclimated in the greenhouse for 4 weeks and then transplanted to the isolated field. Two months later, stem segments approximately 10 cm long were cut from the transgenic and WT lines and cultured in nutrient soil (26°C, photoperiod of 16 h light, 8 h dark). They were treated with Hoagland's medium containing 200 mM NaCl for 2 weeks. Plant growth was observed and recorded, and plant fresh weight and root length after salt treatment were measured. The results are shown in Table 1 (data are expressed as means ± SE (n = 3), *P ≤ 0.05) and Figure 6 (WT is the 'Jishu 25' control, and IbCHR10-OE is the transgenic line) as shown.
[0078] Table 1 Phenotypic assay results
[0079] strain WT IbCHR10-OE Fresh weight of plants treated with 200 mM NaCl (g) 5.13±0.26 8.33±1.20* Root length of plants treated with 200 mM NaCl (cm) 8.27±0.39 12.96±0.33*
[0080] Depend on Figure 6As shown in Table 1, after 2 weeks of treatment with 200 mM NaCl, the growth and rooting conditions of the IbCHR10-OE line were significantly better than those of the WT.
[0081] As shown in the above examples, the IbCHR10 gene provided by the present invention lacks a conserved DST element region downstream of its 3'UTR region, and its mRNA is stable after translation, confirming that the gene is expressed at higher levels in sweet potato fibrous roots than in other tissues. The present invention successfully cloned IbCHR10 from sweet potato and transformed it into sweet potato using Agrobacterium-mediated transfection. This study demonstrated for the first time that overexpression of IbCHR10 enhances salt tolerance in sweet potato. This further demonstrates that the IbCHR10 gene is associated with salt tolerance in sweet potato.
[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A gene related to salt tolerance IbCHR10 , which is characterized by, The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. The method according to claim 1 IbCHR10 The application of genes in improving crop tolerance to salt stress is characterized by: Through overexpression IbCHR10 To improve the tolerance of crops to salt stress; the crop is sweet potato.
3. The use according to claim 2, characterized in that: The method for overexpressing IbCHR10 comprises the following steps: The cloned IbCHR10 was constructed into a gene overexpression vector to obtain a recombinant vector; The obtained recombinant vector is transformed into Agrobacterium tumefaciens; Agrobacterium tumefaciens is inoculated into crops to achieve gene overexpression.
4. The method according to claim 1 IbCHR10 The invention relates to an application of a polypeptide encoded by a gene in improving the tolerance of crops to salt stress, wherein the crop is sweet potato.
5. A method for cultivating high salt-tolerant crops, characterized in that: The method comprises the following steps: using a vector-mediated method to transform crops, obtaining IbCHR10 Overexpressed transgenic crop; the crop is sweet potato.
6. The method for cultivating high salt-tolerant crops according to claim 5, wherein: The vector-mediated method is an Agrobacterium-mediated method.
7. The method for cultivating high salt-tolerant crops according to claim 5, wherein: The method specifically comprises the following steps: Will IbCHR10 Cloning into a gene overexpression vector to obtain a recombinant vector; The obtained recombinant vector is transformed into Agrobacterium tumefaciens; Agrobacterium tumefaciens is inoculated into crops to achieve gene overexpression.