Use of wheat ta cipk19-3d gene and method for improving salt tolerance of crops

Abstract

The application discloses a use of a wheat TaCIPK19-3D gene and a method for improving salt tolerance of crops. The wheat TaCIPK19-3D gene is taken as the object, and it is found that wild type (WT), a transgenic line overexpressing the TaCIPK19-3D gene and an oscipk19 gene knockout mutant plant all show obvious phenotype differences after salt stress. The transgenic line overexpressing the TaCIPK19-3D gene shows obvious salt tolerance phenotype, and the leaves of the transgenic line overexpressing the TaCIPK19-3D gene are greener and more active than those of the wild type and the mutant after salt treatment. The leaves of the oscipk19 gene knockout mutant line of a rice homologous gene are more withered than those of the wild type after salt treatment. The results show that the TaCIPK19-3D gene can positively regulate the salt tolerance of plants, and can be applied to the breeding of wheat salt-tolerant varieties through molecular breeding and other technologies.

Description

Technical Field

[0001] This article generally relates to the field of genetic engineering technology, with a particular focus on the use of a wheat TaCIPK19-3D gene or its encoded protein in improving crop salt tolerance. Background Technology

[0002] Wheat (Triticum aestivum L.) is widely cultivated in China and around the world, and is one of the world's most important food crops. In recent years, with the increasingly severe global climate (such as drought and high temperatures), wheat production has faced more and more challenges. When wheat is subjected to abiotic stresses such as drought, salinity, and high temperatures, its chloroplast function is impaired, thereby affecting photosynthesis and growth. Wheat varieties that can adapt to abiotic stresses usually have stronger stress resistance and chloroplast stability, and can better maintain photosynthesis. Therefore, studying the molecular mechanisms of wheat's response to abiotic stresses and further exploring related genes is of great significance for improving wheat's abiotic resistance and is also one of the important directions for future wheat breeding research. Summary of the Invention

[0003] Based on this, this application provides the use of the wheat TaCIPK19-3D gene or the protein it encodes in improving crop salt tolerance.

[0004] On the other hand, this application also provides a method for improving the salt tolerance of crops, wherein the method includes: overexpressing the wheat TaCIPK19-3D gene in the crop.

[0005] On the other hand, this application also provides the use of genetically modified crops obtained by the methods described herein in crop breeding.

[0006] This application focuses on the wheat TaCIPK19-3D gene. Wild-type (WT) rice lines, transgenic rice lines overexpressing the TaCIPK19-3D gene, and rice lines with a knockout mutant of the oscipk19 gene (a homologous gene) were simultaneously treated with 125 mM NaCl for 72 hours. Significant phenotypic differences were observed among the wild-type (WT), overexpressing transgenic lines, and knockout mutant plants. The overexpressing transgenic lines exhibited a marked salt tolerance phenotype; their leaves were greener and more active after salt treatment compared to the wild-type and mutant lines. Conversely, the leaves of the oscipk19 knockout mutant were more wilted after salt treatment than those of the wild-type. These results indicate that the TaCIPK19-3D gene can positively regulate plant salt tolerance and can be applied to promote the breeding of salt-tolerant wheat varieties through molecular breeding techniques.

[0007] The function of the TaCIPK19-3D gene discovered in this application is of great significance for promoting the breeding of salt-tolerant wheat varieties. By overexpressing the TaCIPK19-3D gene through genetic engineering and other techniques, the stress resistance of wheat can be improved, thereby enabling the breeding of new wheat germplasm with better quality. The technical solution of this application is based on existing genetic engineering techniques, which have been widely used in the plant field. Therefore, this application is highly operable and widely applicable, providing an effective method for plant genetic improvement and variety breeding.

[0008] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings. Attached Figure Description

[0009] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0010] Figure 1 Phenotypic diagrams of wild-type (WT), overexpression lines, and oscipk19 gene knockout mutant plants in Example 1 of this application.

[0011] Figure 2 This is a graph showing the results of physiological indicators related to salt stress in Example 2 of this application. (A) shows the Pro content before and after salt treatment; (B) shows the SOD activity before and after salt treatment; and (C) shows the O2 content before and after salt treatment. - Content. Error bars show the mean ± SD of three analyses; t-tests were used to analyze significant differences, with different lowercase letters indicating a significant difference when the p-value is less than 0.05.

[0012] Figure 3 This study validated the expression of salt stress-related genes in wild-type and transgenic plants in Example 3 of this application using qRT-PCR. (A) shows the expression level of the OsAPX2 gene in rice before and after salt treatment; (B) shows the expression level of the P5CS gene in rice before and after salt treatment; and (C) shows the expression level of the ABA2 gene in rice before and after salt treatment. Error bars represent the mean ± SD of the three analyses. A t-test was used to analyze significant differences; different lowercase letters indicate a significant difference when the p-value is less than 0.05. Detailed Implementation

[0013] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0014] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0015] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0016] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0017] CIPKs are a class of serine / threonine protein kinases that interact with CBL proteins. The CIPK gene family in plants plays a crucial role in regulating plant responses to salt stress. The expression level of the wheat TaCIPK14 gene is significantly upregulated under cold and salt stress conditions. Studies have shown that, compared to the wild type, tobacco transgenic lines overexpressing the TaCIPK14 gene under cold and salt stress conditions have higher levels of chlorophyll, sugar, and catalase, while lower levels of other stress physiological indicators such as malondialdehyde (MDA) and hydrogen peroxide (H2O2). Compared to the wild type, tobacco transgenic lines overexpressing the TaCIPK29 gene under salt stress conditions exhibit salt-tolerant phenotypes such as higher seed germination rates, longer root lengths, and better growth status. Further studies have revealed that the H2O2 content is reduced and the activity of antioxidant enzymes is increased in the overexpressing transgenic lines. Transgenic Arabidopsis thaliana lines overexpressing the TaCIPK24 gene showed tolerance to salt stress. Further studies revealed that TaCIPK24 can enhance the salt tolerance of plants by increasing sodium ion efflux and enhancing the activity of antioxidant enzymes.

[0018] In recent years, the development of genetic engineering technology has provided new tools for crop improvement. Through biotechnology, it has been discovered that the CIPK gene family can participate in plant responses to salt stress by regulating mechanisms such as antioxidant accumulation and ion transport, thereby improving plant salt tolerance. However, the wheat TaCIPK19-3D gene and its functional application in wheat have not been reported.

[0019] In one aspect, this application provides the use of the wheat TaCIPK19-3D gene or the protein it encodes in improving crop salt tolerance.

[0020] In some implementations, the nucleic acid sequence of the wheat TaCIPK19-3D gene has at least 80% sequence identity with the sequence shown in SEQ ID NO:1; or

[0021] The amino acid sequence of the protein encoded by the wheat TaCIPK19-3D gene has at least 80% sequence identity with the sequence shown in SEQ ID NO:2.

[0022] In some embodiments, the nucleic acid sequence of the wheat TaCIPK19-3D gene used herein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:1.

[0023] In some embodiments, the amino acid sequence of the protein encoded by the wheat TaCIPK19-3D gene used herein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:2.

[0024] On the other hand, this application also provides a method for improving the salt tolerance of crops, wherein the method includes: overexpressing the wheat TaCIPK19-3D gene in crops.

[0025] In some implementations, the crop is selected from rice, wheat, corn, and barley; preferably, the crop is rice.

[0026] The wheat TaCIPK19-3D gene can be overexpressed in crops using any overexpression method available in the art. In some embodiments, the wheat TaCIPK19-3D gene overexpressed in crops may have one or more nucleotide mutations based on the wild-type wheat TaCIPK19-3D gene.

[0027] In some embodiments, the nucleic acid sequence of the wheat TaCIPK19-3D gene used herein has at least 80% sequence identity with the sequence shown in SEQ ID NO:1. In some embodiments, the nucleic acid sequence of the wheat TaCIPK19-3D gene used herein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:1.

[0028] In some implementations, the crop is rice, wheat, corn, or barley; preferably, the crop is rice.

[0029] The wheat TaCIPK19-3D gene can be overexpressed in crops using any overexpression method available in the art. In some embodiments, methods for overexpressing the wheat TaCIPK19-3D gene in crops include:

[0030] 1) By operatively linking a strong promoter to the wheat TaCIPK19-3D gene in crops; and / or

[0031] 2) By increasing the copy number of the wheat TaCIPK19-3D gene on the chromosomes of crops.

[0032] In some implementations, the wheat TaCIPK19-3D gene is linked to a constitutive promoter in crops, and the expression of the wheat TaCIPK19-3D gene is controlled by the constitutive promoter.

[0033] In some implementations, the constitutive promoter is a 35S promoter or an ubiquitin promoter; preferably, the constitutive promoter is an ubiquitin promoter. Other types of constitutive promoters can also be used to control the expression of the wheat TaCIPK19-3D gene in crops.

[0034] On the other hand, this application also provides for the use of transgenic crops obtained by the methods described herein in crop breeding. In some embodiments, the breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0035] In some implementations, homologs of the wheat TaCIPK19-3D gene can also improve certain traits of crops (e.g., rice) through overexpression, RNAi (reducing expression levels), and gene editing.

[0036] This application utilizes genetic engineering techniques to achieve overexpression of the TaCIPK19-3D gene in rice, enabling targeted editing and regulation of the gene. Furthermore, the TaCIPK19-3D gene may also have application potential in salt stress management of other crops. It may also improve the stress resistance of other crops such as maize, soybeans, and vegetables. This application involves research on the function and regulatory mechanism of the TaCIPK19-3D gene. This is of great significance for a deeper understanding of basic research in the fields of plant salt stress and plant growth and development. This application may provide a specific research object and experimental model for research in the fields of molecular biology and genetics.

[0037] This application describes several embodiments, but these descriptions are exemplary and not limiting, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with, or may replace, any feature or element of any other embodiment.

[0038] This application includes and contemplates combinations of features and elements known to those skilled in the art. The embodiments, features, and elements disclosed in this application can also be combined with any conventional features or elements to form unique inventive solutions. Any feature or element of any embodiment can also be combined with features or elements from other inventive solutions to form another unique inventive solution. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0039] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0040] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application.

[0041] Example

[0042] The materials used in the embodiments are shown below:

[0043] 1. Reagents and materials

[0044] Chinese spring wheat varieties; Japanese dry rice varieties.

[0045] 2. Instruments

[0046] Multifunctional microplate reader, six-channel real-time PCR instrument

[0047] Example 1. Construction of TaCIPK19-3D gene overexpression plants

[0048] (1) Total RNA was extracted from *Cymbidium goeringii* using a plant total RNA extraction kit. Quality was assessed by 1% agarose gel electrophoresis, and its concentration was detected using Genova Nano. The RNA was reverse transcribed into cDNA using a reverse transcription kit. Specific primers were designed based on the TaCIPK19-3D gene sequence information obtained from the *Cymbidium goeringii* transcriptome database. The primer pair mCIPK19-3D-F / R was used.

[0049] mCIPK19-3D-F:5'-GCTCTAGAATGGCGCCATCAAGCC-3 ’ (SEQ ID NO:3)

[0050] mCIPK19-3D-R:5'-CGCGGATCCCTACTCAGAAATGGTAGGTGAATTGGAAC-3(SEQ ID NO:4)

[0051] The TaCIPK19-3D gene was obtained by PCR amplification of cDNA using Biorun High Fidelity PCR Mix, and its nucleotide sequence was obtained by sequencing as shown in SEQ ID NO:1.

[0052] (2) The gene was ligated into the pBI121 vector by double enzyme digestion to construct an overexpression vector for the TaCIPK19-3D gene. The overexpression vector was introduced into the normal rice variety Nipponbare using Agrobacterium-mediated genetic transformation, and transgenic seedlings were obtained by plant tissue culture.

[0053] The obtained transgenic seedlings were cultured in nutrient solution. When they reached the three-leaf-one-heart stage, DNA was extracted from the leaves and PCR amplified using primers mCIPK19-3D-F / R. The PCR products were verified by 1% agarose gel electrophoresis to identify positive plants, which were then numbered and harvested individually for preservation after maturity (T1 generation).

[0054] (3) The harvested T1 generation seeds were hydroponically germinated and transferred to nutrient solution culture. When they reached the three-leaf-one-heart stage, they were planted in the field. After maturity, the positive single plants were harvested (T2 generation). T2 generation transgenic lines with a ratio of approximately 3:1 were selected for further propagation until T3 generation was identified as homozygous transgenic plants (three homozygous plants were identified: OE-13, OE-16 and OE-22).

[0055] (4) Construction of oscipk19 gene knockout mutant rice plants

[0056] Gene knockout vectors were constructed using the CRISPR / Cas9 method, employing a dual-target knockout strategy with two targets designed in the coding region. The gene sequence, species, and PAM were input into the CRISPR-P website (http: / / crispr.hzau.edu.cn / ) to obtain the corresponding sgRNAs, from which two sgRNAs with high specificity and low off-target rate were selected as target sequences. The Ensembl Plants database indicates that the OsCIPK19 gene encodes only one exon; therefore, the sgRNAs 5'-CCTCGTCCCGCACATCAAGCGGG-3' (SEQ ID NO:5) and 5'-CTGTTCGGCCGCGTCGCCAAGGG-3' (SEQ ID NO:6) were selected as targets. Following the instructions of the monocotyledonous gene editing vector kit (Wuhan Boyuan Biotechnology Co., Ltd.), the CRISPR / Cas9 knockout vector was constructed using the Golden Gate seamless cloning method. After the recombinant vector was constructed, it was sequenced and analyzed by the company, and plasmids were extracted after confirmation. Subsequently, the plasmid was used to construct oscipk19 gene knockout mutant rice plants using Agrobacterium-mediated genetic transformation.

[0057] (5) The overexpressing plants and seeds of Nipponbare rice were hydroponically germinated and then transferred to nutrient solution for seedling cultivation until the three-leaf stage. Wild-type (WT), overexpressing transgenic lines, and oscipk19 gene knockout mutant rice seedlings at the three-leaf-one-heart stage were simultaneously treated with 125 mM NaCl for 72 hours. Significant phenotypic differences were observed among the wild-type (WT), overexpressing lines, and oscipk19 gene knockout mutant plants. Phenotypic diagrams are shown below. Figure 1 As can be seen, there are obvious phenotypic differences between wild-type (WT), overexpressing transgenic lines, and oscipk19 gene knockout mutant plants. Overexpressing transgenic lines show obvious salt tolerance phenotype, and the leaves of overexpressing transgenic lines are greener and more active than those of wild-type and mutant after salt treatment; while the leaves of the oscipk19 gene knockout mutant in rice are more wilted than those of wild-type after salt treatment.

[0058] Example 2.

[0059] The effects of Pro and O2 on wild-type (WT), overexpressing transgenic lines and mutant plants before and after salt stress treatment. - The content and SOD activity were determined.

[0060] Proline (Pro) content determination: Following the instructions of the kit (Grace, Suzhou, China), after the reaction, the absorbance of the test solution was read at 520 nm, and the value was A. ΔA = Adetermined - Ablank. According to the standard curve y = 0.1625x - 0.0064, where x is the mass of the standard (μg) and y is ΔA, the Pro content (μg / g) = 41.03 × (ΔA + 0.0064) ÷ W (W is the sample mass 0.1g).

[0061] Superoxide anion (O2) - Assay: Following the instructions of the kit (Grace, Suzhou, China), after the reaction, read the absorbance of the test solution at 540 nm as A. ΔA = Ameasurement - Ablank. According to the standard curve y = 0.0482x - 0.001, x is NO2. - The molar mass (μmol) of O2 is given by ΔA. - Content (nmol / g) = 237.1 x (△A + 0.0011) ÷ W (W is the sample mass of 0.1g).

[0062] Superoxide dismutase (SOD) assay: Following the instructions of the kit (Grace, Suzhou, China), after the reaction, the absorbance of the test solution was read at 450 nm as A. ΔAassay = Aassay - Acontrol, ΔAblank = Ablank1 - Ablank2. The inhibition percentage was calculated as (ΔAblank - ΔAassay) ÷ ΔAblank × 100%. SOD activity (U / g) = 11.11 × inhibition percentage ÷ (1 - inhibition percentage) ÷ W (W is the sample mass 0.1g).

[0063] The results showed that TaCIPK19-3D could enhance the salt tolerance of plants by increasing Pro content and improving SOD activity to reduce the superoxide anion content in plant cells. (See figure below.) Figure 2 .

[0064] Example 3.

[0065] This study further determined the expression levels of salt stress-related genes, such as proline-5-carboxylate synthase (P5CS), xanthoxin dehydrogenase (ABA2), and ascorbate peroxidase 2 (APX2), in the leaves of wild-type (WT), overexpressing transgenic lines, and mutant plants before and after salt treatment.

[0066] Using wild-type plants as a control, the expression levels of OsAPX2, OsABA2, and OsP5CS genes were detected by qRT-PCR. The obtained cDNA was diluted to an appropriate concentration (approximately 10-20 times) as a template. The qRT-PCR experiment was performed according to the PCR mixing system and procedures outlined in the instructions for the Tiangen Talent qPCR PreMix (SYBR Green) kit. The expression levels of the target genes were calculated using the 2-ΔΔCT method.

[0067] For detailed results, please see [link to results]. Figure 3 The results showed that TaCIPK19-3D could increase the expression levels of OsP5CS, OsABA2 and OsAPX2 genes, thereby improving the salt tolerance of the plant.

[0068] In conclusion, the TaCIPK19-3D gene can improve plant salt tolerance by increasing the expression levels of salt stress-related genes.

[0069] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. Wheat TaCIPK19-3D The use of genes or their encoded proteins in improving salt tolerance in rice, among which, By overexpressing the wheat in rice TaCIPK19-3D The wheat TaCIPK19-3D gene is used to improve the salt tolerance of rice. The nucleic acid sequence of the wheat TaCIPK19-3D gene is shown in SEQ ID NO: 1; the amino acid sequence of the protein encoded by the wheat TaCIPK19-3D gene is shown in SEQ ID NO:

2.

2. A method for improving the salt tolerance of rice, wherein, The method includes: overexpressing wheat in rice. TaCIPK19- 3D The nucleic acid sequence of the wheat TaCIPK19-3D gene is shown in SEQ ID NO:

1.

3. The method according to claim 2, wherein, Overexpression of the wheat in rice TaCIPK19-3D Genetic methods include: 1) By combining strong promoters in rice with the wheat TaCIPK19-3D Genes can be operatively linked; and / or 2) By increasing the number of chromosomes on rice as described in wheat TaCIPK19-3D The copy number of a gene.

4. The use of the transgenic rice obtained by the method of claim 2 or 3 in rice breeding.

5. The use according to claim 4, wherein, Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

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