Sweet potato salt tolerance related gene IbCHR10 and application thereof

Abstract

The invention provides a sweet potato salt tolerance related gene IbCHR10 and application thereof, and belongs to the technical field of molecular biology, the sweet potato salt tolerance related gene IbCHR10 provided by the invention has no conservative DST element region fragment at the downstream of the 3 'UTR region, the mRNA is stable after translation, and the vacancy of the prior art is filled. IbCHR10 is successfully cloned from sweet potatoes, it is proved that the expression quantity of the gene in fibrous roots of the sweet potatoes is higher than that of other tissues, it is found for the first time that overexpression of IbCHR10 enables the salt tolerance of the sweet potatoes to be enhanced when the gene is transformed into the sweet potatoes through an agrobacterium-mediated method, and it is further proved that the IbCHR10 gene is related to the salt tolerance of the sweet potatoes. Therefore, the gene IbCHR10 disclosed by the invention has a wide application prospect in the related research of the stress resistance of the sweet potatoes.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acids, and particularly relates to a sweet potato salt tolerance-related gene IbCHR10 and its application. Background Art

[0002] Disclosing the information of this background art section is only intended to increase some understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Salt stress can cause problems such as ion imbalance, nutrient deficiency, osmotic stress, ion toxicity, and oxidative stress in sweet potato plants, thereby hindering the development of sweet potato plants and causing a decrease in the yield and quality of tuberous roots. Therefore, cultivating high-yield, high-nutritional-value, and more salt-tolerant sweet potato varieties is an effective guarantee for its efficient production.

[0004] In recent years, a class of zinc finger proteins containing the 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. C1 domain proteins in plants usually contain multiple C1 domains and are thus also known as DC1 domain proteins in plants, playing important roles in plant growth, development, and stress resistance. The C1 domain was initially discovered in PKC through its binding to phosphatidyl DAG and phenolic esters. Subsequently, it was found that it binds proteins by transporting them to the membrane, where they can interact with signal complexes, substrates, and regulators, activate substrate enzymes, and mediate intermembrane transport, thereby regulating the expression of stress-related genes and further modulating the plant's resistance to stress (Bhaskar R. V., Mohanty B., Verma V., Wijaya E., Kumar P. P. A hormone-responsive C1-domain-containing protein At5g17960 mediates stress response in Arabidopsis thaliana[J]. PLoS ONE, 2015, 10, e0115418). Some DC1 domain proteins have been discovered in plants. Although they are named differently in different plants, they all participate in plant disease resistance, stress resistance, growth, and development.The Arabidopsis DC1 domain genes ULI3, VLG, and BNP are involved in Arabidopsis photomorphogenesis, seed development, and pollen development respectively. At5g17960 can be 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 L. A., Casalongué C. A., Pagnussat G. C., Fiol D. F. 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, 1761-1763). The expression of the wheat DC1 domain gene TaCHP differs between salt-tolerant and salt-sensitive varieties. Overexpression of TaCHP can significantly improve the production performance of wheat under salt stress (Li C. L., Lv J., Zhao X., Ai X. H., Zhu X. L., Wang M. C., Zhao S. Y., Xia G. M. TaCHP: A wheat zinc finger protein gene down-regulated by abscisic acid and salinity stress 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] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a salt tolerance-related gene IbCHR10 and its application, so as to fill the gaps in the prior art.

[0007] The technical solutions adopted in the present invention are as follows: In the first aspect of the present invention, a salt tolerance-related gene IbCHR10 is provided. The gene is obtained through the identification of the sweet potato DC1 protein family combined with salt tolerance transcriptome analysis. The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] In the second aspect of the present invention, the application of the IbCHR10 gene in improving the salt stress tolerance of crops is provided.

[0009] Specifically, the salt stress tolerance of crops is improved by overexpressing IbCHR10. The method for overexpressing IbCHR10 includes the following steps: Clone the IbCHR10 and construct it into a gene overexpression vector to obtain a recombinant vector; Transform the obtained recombinant vector into Agrobacterium tumefaciens; Inoculate the Agrobacterium tumefaciens into the crops to achieve overexpression of the gene.

[0010] Preferably, the crop is sweet potato.

[0011] Preferably, the gene overexpression vector is the pCAMBIA1301 vector.

[0012] In the third aspect of the present invention, the application of the polypeptide encoded by the IbCHR10 gene in improving the salt stress tolerance of crops is provided.

[0013] In the fourth aspect of the present invention, a pair of specific primers for detecting the above salt tolerance-related gene IbCHR10 is provided. The sequence of the 5'-end to 3'-end of the upstream primer is shown in SEQ ID NO.2, and the sequence of the 5'-end to 3'-end of the downstream primer is shown in SEQ ID NO.3.

[0014] In the fifth aspect of the present invention, the application of the amplification primers for detecting the salt tolerance-related gene IbCHR10 in crop trait-related research is provided, which is used for salt tolerance-assisted breeding of crops.

[0015] In the sixth aspect of the present invention, a method for cultivating highly salt-tolerant crops is provided. The method includes the following steps: transforming the crops by vector-mediated method to obtain transgenic crops with overexpression of IbCHR10.

[0016] Preferably, the vector-mediated method is the Agrobacterium-mediated method.

[0017] Preferably, the method specifically includes the following steps: Clone IbCHR10 into an overexpression vector to obtain a recombinant vector; Transform the obtained recombinant vector into Agrobacterium tumefaciens; Inoculate Agrobacterium tumefaciens into crops to achieve overexpression of the gene.

[0018] Further preferably, the gene overexpression vector is the pCAMBIA1301 vector.

[0019] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: There is no conserved DST element region fragment downstream of the 3'UTR region of the IbCHR10 gene provided by the present invention. After translation, the mRNA is stable, which proves that the expression level of the gene in sweet potato fibrous roots is higher than that in other tissues. The present invention successfully cloned IbCHR10 from sweet potato and transformed this gene into sweet potato by the Agrobacterium-mediated method. It was first discovered that overexpression of IbCHR10 enhances the salt stress tolerance of sweet potato. Further evidence shows that the IbCHR10 gene is related to the salt tolerance of sweet potato. It shows that the gene IbCHR10 of the present invention has broad application prospects in the research related to salt stress tolerance of sweet potato and trait breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0021] Figure 1 For IbCHR The expression pattern of the gene in different sweet potato varieties with different salt tolerances; Figure 2 For IbCHR10 The amplification result of the full-length cDNA sequence of the gene; Figure 3 For IbCHR10 The expression pattern of the gene in different tissues; Figure 4 For IbCHR10 The expression pattern of the gene under different stresses and hormone treatments; Figure 5 For sweet potato IbCHR10 Identification of transgenic plants; Figure 6 For sweet potato IbCHR10 Phenotypic identification of the salt tolerance of transgenic plants. DETAILED DESCRIPTION OF THE INVENTION

[0022] It should be noted that the following detailed description is exemplary and is 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 of ordinary skill in the technical field to which the present invention belongs.

[0023] 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 also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0024] 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 in conjunction with specific embodiments.

[0025] Example 1 Gene Identification and Amplification Twelve members of the DC1 domain family were identified from the sweet potato genomic data and named IbCHR1 - IbCHP12 according to their positions on the chromosome. Combining the transcriptome data of the salt - tolerant sweet potato variety Jishu 26 (J26) and the salt - sensitive variety Nonglin 54 (NL54) under salt stress, analyze IbCHR the expression differences of genes in different salt - tolerant sweet potato varieties under salt treatment. The results are as Figure 1 shown.

[0026] As Figure 1 can be seen, IbCHR10 it has a higher expression level in the salt - tolerant sweet potato variety Jishu 26.

[0027] IbCHR10 - F: 5’-ATGGAGTATAAGCATTTC-3’ (as shown in SEQ ID NO.2) IbCHR10 - R: 5’-TCACTCATGAAATCGAGT-3’ (as shown in SEQ ID NO.3) Total RNA extraction from sweet potato: Use the RNA Rapid Extraction Kit from Cisco Jie Co., Ltd. (product number: AC0307) to extract total RNA from the fibrous root samples of sweet potato variety 'Jishu 25', and detect the RNA quality by agarose gel electrophoresis.

[0028] Reverse transcription to obtain cDNA: Use the reverse transcription kit from TaKaRa Co., Ltd. (product number: RR047Q) to perform reverse transcription of sweet potato RNA to obtain the total cDNA of sweet potato fibrous roots.

[0029] Use the high - fidelity enzyme ApexHF HS DNA Polymerase - FS from Aikery Co., Ltd. to amplify the full - length sequence of IbCHR10 with the cDNA of sweet potato fibrous roots as the template.

[0030] 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).

[0031] PCR amplification program: Pre - denaturation: 94℃, 30 s; [Denaturation: 98℃, 10 s; Annealing: 55℃, 10 s; Extension: 72℃, 5 s]×35 cycles; Cool down to 4℃.

[0032] Use 1.2% agarose gel electrophoresis to detect the obtained PCR products ( Figure 2 , where the Maker is DL2000, and the bands are 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp respectively). Use the FastPure® GelDNA Extraction Mini Kit from Novoprotein Co., Ltd. (product number: DC301) to recover the target fragment. ligate the recovered target fragment to the cloning vector VS007 (Qingke Biotechnology), and the ligation system is target fragment: cloning vector = 4:1. After ligating at room temperature for 5 minutes, transform Escherichia coli competent DH5α, culture overnight at 37℃, and pick monoclonal colonies for sequencing identification.

[0033] Example 2 Expression isolation of IbCHR10 gene in different tissues Using the sweet potato variety 'Jishu 25' as the material, the fourth expanded leaf, shoot tip, young stem, old stem, fibrous root and tuberous root at 125 days of field growth were selected, washed twice with distilled water, dried with absorbent paper, ground in liquid nitrogen, placed in a cryotube and stored in a -80 °C refrigerator.

[0034] The qRT-PCR primers were designed for the non-conserved region of the IbCHR10 gene ORF sequence using Primer Premier 5.0 software: IbCHR10-qRT-F: 5’-ACATGTGCCAAAAATGCCCC-3’ (as shown in SEQ ID NO.4) IbCHR10-qRT-R: 5’-TCTGGGACGAGAGGTCTAGC-3’ (as shown in SEQ ID NO.5) The steps for total RNA extraction and reverse transcription were the same as in Example 1.

[0035] In the CFX ConnectTM real-time fluorescence quantitative PCR detection system (BIO-RAD, USA), the qRT-PCR experiment was carried out using Novizan ChamQ SYBR qPCR Master Mix (product number: Q311), and the internal reference gene used was IbActin. The 2 -ΔΔCT method was used to calculate the relative expression level of the gene. The average Ct value of each gene was obtained from three biological replicate experiments. The qRT-PCR reaction conditions were as follows: pre-denaturation: 95 °C, 3 min; cycle reaction: [95 °C, 10 s; 60 °C, 30 s] × 40 cycles, and the results were as Figure 3 shown.

[0036] It can be Figure 3 seen that the expression level of the IbCHR10 gene was the highest in the fibrous roots of 'Jishu 25', followed by the stem and tuberous root, and the lowest in the shoot tip.

[0037] Example 3 Expression pattern of the IbCHR10 gene under different stresses and hormone treatments The stem segments of 'Jishu 25' with consistent growth and containing 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 respectively. Leaves were taken at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h after treatment, quickly frozen in liquid nitrogen and stored at -80 °C for later use.

[0038] The steps for the real-time fluorescence quantitative experiment were the same as in Example 2, and the results were as Figure 4 shown.

[0039] It can be Figure 4It can be seen that the expression level of the IbCHR10 gene first increases and then decreases after salt, drought, and ABA treatments, and reaches the highest level at 6 or 12 h after treatment; the expression level of the IbCHR10 gene gradually increases after GA3 treatment.

[0040] Example 4 Construction of plant expression vector The pCAMBIA1301-IbCHR10 recombinant vector was constructed using the Novoprotein ClonExpress® II One Step Cloning Kit (product number: C112) homologous recombination kit. The primer sequences are as follows: IbCHR10-1301-F: 5’-GGGGTACCCCGCCACCATGGAGTATAAG-3’ (as shown in SEQ ID NO.6) IbCHR10-1301-R: 5’-GCTCTAGATCACTCATGAAATCGAGT-3’ (as shown in SEQ ID NO.7) The vector plasmid was double digested with restriction endonucleases KpnⅠ and XbaⅠ, the ORF sequence of IbCHR10 was amplified using high-fidelity enzyme FS, and the target fragment was detected by agarose gel electrophoresis and recovered. The following reaction system was prepared on ice: linearized vector 0.03 pmol; inserted fragment 0.06 pmol; 5×CE II Buffer 4 μL; Exnase II 2 μL; ddH2O Up to 20 μL. React at 37℃ for 30 min and cool on ice. The recombinant plasmid was transformed into DH5α, and the positive plasmid pCAMBIA1301-IbCHR10 was obtained by sequencing.

[0041] Example 5 Transformation of Agrobacterium competent cells Take the Agrobacterium EHA105 competent cells stored at -80℃, place them at room temperature for a moment, and immediately add 10 μL of the recombinant plasmid pCAMBIA1301-IbCHR10 when the competent cells melt. Gently pipette and mix well. Incubate on ice for 5 min, in liquid nitrogen for 5 min, heat shock at 37℃ for 5 min, and let stand on ice for 5 min. Add an appropriate amount of LB liquid medium without resistance, and resuscitate at 28℃ for 2 - 3 h. Centrifuge at 6000 rpm to collect the bacteria, pour off part of the medium, resuspend the bacterial cells, and spread them on YEP solid medium supplemented with 50 mg / L Kan and 50 mg / L Rif. Incubate inverted at 28℃ for 2 - 3 d, detect positive colonies by colony PCR, and shake the bacteria for standby.

[0042] Example 6 Transgenic function verification - Sweet potato transformation and screening Sweet potato embryogenic callus culture: Healthy sweet potato seedlings were germinated from the tubers of 'Jishu 25'. Stem segments about 7 cm long were cut, and stem tips about 2 cm long were excised. The stem tips were successively washed and disinfected with 70% ethanol and 2% sodium hypochlorite solution, and rinsed 3 times with sterile water. The white meristem tissue enclosed in the stem tip was excised and placed on an MS solid plate supplemented with 2,4-D, and cultured in the dark at 26°C. After 8 weeks of culture in MS liquid medium, the embryogenic callus was filtered through a sieve to remove smaller cell clusters. The larger cell clusters (>1 mm) remaining in the sieve were gently broken with forceps and then cultured for another 3 - 5 days for Agrobacterium infection and transformation.

[0043] Agrobacterium infection of sweet potato callus: Positive colonies on the plate were picked and inoculated into YEP liquid medium for overnight culture. The overnight culture was inoculated into 100 mL of YEP liquid medium supplemented with Kan (100 mg / L) at a ratio of 1:100 (v / v), and cultured at 28°C and 200 rpm for 6 - 8 h until the OD of the bacterial solution 600 was between 0.8 and 1.2. The bacteria were collected by centrifugation at 5000 rpm for 5 min, resuspended twice with YEP liquid medium, and after centrifugation to collect the bacteria, 50 mL of infection solution (MS medium supplemented with 2 mg / L 2,4-D and 200 μM acetosyringone AS) was added to resuspend the bacteria, and the mixture was allowed to stand at 28°C for 2 h. The sweet potato embryogenic callus was resuspended with the infection solution, gently shaken at 40 rpm for 20 min, and the excess bacterial solution was removed with a pipette. The infected callus was spread on a co-culture solid medium (containing 2 mg / L 2,4-D and 200 μM AS) and co-cultured at 26°C in the dark for 3 - 5 days (depending on the growth status of the callus).

[0044] Screening and regeneration of transgenic plants: The co-cultured callus was transferred to sterile water supplemented with 300 mg / L Cef, gently shaken for 20 min to remove Agrobacterium on the surface of the callus, and this was repeated three times. Then it was transferred to an MS solid medium (containing 2 mg / L 2,4-D) and cultured for 1 week in the dark at 26°C. The callus after the delayed culture was transferred to an MS selection solid medium (containing 10 mg / L HYG and 300 mg / L Cef) and cultured under weak light for 8 weeks, with the medium changed every 2 weeks. The callus with good growth status on the selection medium was transferred to an MS solid medium for continued culture until complete plants grew from the callus. The newly grown putative transgenic plants were transferred to a tissue culture bottle containing MS solid medium, labeled, and propagated.

[0045] Example 7 Transgenic Function Verification - Phenotypic Analysis Identification of positive transgenic plants: Cut the leaves of the transgenic sweet potato plants to extract DNA and total RNA, and further identify positive lines by PCR and qRT-PCR. The results are as Figure 5 shown. The PCR detection primers are as follows: HYG-F: 5’-TTCTACACAGCCATCGGTCC-3’ (as shown in SEQ ID NO.8) HYG-R: 5’-CCCATGTGTATCACTGGCAA-3’ (as shown in SEQ ID NO.9) Identification of salt tolerance of transgenic plants: Acclimate the positive overexpressing plants and test-tube seedlings of ‘Jishu 25’ in the greenhouse for 4 weeks, transplant them to an isolated field. After two months, cut about 10 cm stem segments of the transgenic lines and WT lines, and culture them in nutrient soil (26 °C, light cycle of 16 h light and 8 h dark). Water and treat them with Hoagland nutrient solution containing 200 mM NaCl for 2 weeks, observe and record the growth status of the plants, and measure the fresh weight and root length of the plants after salt treatment. The results are shown in Table 1 (data are expressed as means±SE (n = 3), *P≤0.05) and Figure 6 (WT is the control of ‘Jishu 25’, IbCHR10-OE is the transgenic line).

[0046] Table 1 Results of phenotypic determination 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* As can be seen from Figure 6 and Table 1, after 2 weeks of treatment with 200 mM NaCl, the growth status and rooting of the IbCHR10-OE line are significantly better than those of the WT.

[0047] As can be seen from the above examples, there is no conserved DST element region fragment downstream of the 3' UTR region of the IbCHR10 gene provided by the present invention, and the translated mRNA is stable, which proves that the expression level of the said gene in the fibrous roots of sweet potato is higher than that in other tissues. The present invention successfully cloned IbCHR10 from sweet potato, transformed this gene into sweet potato by Agrobacterium-mediated method, and for the first time found that overexpression of IbCHR10 enhances the salt tolerance of sweet potato. It is further proved that the said IbCHR10 gene is related to the salt tolerance of sweet potato.

[0048] The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A salt tolerance-related gene IbCHR10, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Use of the IbCHR10 gene according to claim 1 in enhancing the salt stress tolerance of crops, characterized in that, Overexpressing IbCHR10 can improve the salt stress tolerance of crops.

3. The application according to claim 2, characterized in that, The method for overexpressing IbCHR10 comprises the following steps: Constructing the cloned IbCHR10 into a gene overexpression vector to obtain a recombinant vector; Transforming the obtained recombinant vector into Agrobacterium tumefaciens; Inoculating the Agrobacterium tumefaciens into the crop to achieve overexpression of the gene.

4. The application according to claim 2 or 3, characterized in that, The crop is sweet potato.

5. Use of the polypeptide encoded by the IbCHR10 gene according to claim 1 in improving the salt stress tolerance of crops.

6. A specific primer for detecting the salt tolerance-related gene IbCHR10 described in claim 1, characterized in that, The sequence from the 5'-end to the 3'-end of the upstream primer is shown in SEQ ID NO.2, and the sequence from the 5'-end to the 3'-end of the downstream primer is shown in SEQ ID NO.

3.

7. Use of the specific primer for detecting salt tolerance-related gene IbCHR10 according to claim 6 in crop trait-related research, characterized in that, For salt tolerance assisted breeding of crops.

8. A method for cultivating crops with high salt tolerance, characterized in that, The method comprises the following steps: transforming the crop by the vector-mediated method to obtain transgenic crops overexpressing IbCHR10.

9. The method for cultivating highly salt-tolerant crops according to claim 8, characterized in that, The vector-mediated method is the Agrobacterium-mediated method.

10. The method for cultivating high salt-tolerant crops according to claim 8, characterized in that, The method specifically comprises the following steps: Cloning IbCHR10 into a gene overexpression vector to obtain a recombinant vector; Transforming the obtained recombinant vector into Agrobacterium tumefaciens; Inoculating the Agrobacterium tumefaciens into the crop to achieve overexpression of the gene.

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