Application of wheat TaCIPK19-3D gene and method for improving salt tolerance of crops

Abstract

The invention relates to application of a wheat TaCIPK19-3D gene and a method for improving the salt tolerance of crops. According to the application, a wheat TaCIPK19-3D gene is taken as an object, a wild type (WT), a transgenic line of an over-expression TaCIPK19-3D gene and an oscipk19 gene knockout mutant plant are found to have obvious phenotype difference after being stressed by salt, the over-expression transgenic line shows an obvious salt-tolerant phenotype, leaves of the over-expression transgenic line are greener than those of the wild type and the mutant after being treated by the salt, and the salt tolerance of the leaves of the over-expression transgenic line is improved. The activity is higher; and the leaves of the homologous gene oscipk19 gene knockout mutant strain in the rice are more withered than 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 promoting the breeding of salt-tolerant wheat varieties through technologies such as molecular breeding and the like.

Description

Technical Field

[0001] The present invention generally relates to the field of genetic engineering technology, and in particular to the use of a wheat TaCIPK19-3D gene or a protein encoded by it in improving the salt tolerance of crops. Background Art

[0002] Wheat (Triticum aestivum L.) is widely planted in China and around the world and is one of the most important food crops in the world. In recent years, with the deterioration of the global climate environment (such as drought, high temperature, etc.), wheat production has faced more and more challenges. When wheat is subjected to stress such as drought, salinity, and high temperature, the chloroplast function of wheat will be damaged, thereby affecting photosynthesis and growth and development. Wheat varieties that can adapt to adverse stress usually have stronger stress resistance and chloroplast stability, and can better maintain photosynthesis. Therefore, studying the molecular mechanism of wheat in response to adverse stress and further exploring related genes are of great significance to improving wheat stress resistance, and are also one of the important directions for future wheat breeding research. Summary of the invention

[0003] Based on this, the present application provides a use of the wheat TaCIPK19-3D gene or the protein encoded by it in improving the salt tolerance of crops.

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

[0005] In another aspect, the present application also provides use of the transgenic crops obtained by the method described herein in crop breeding.

[0006] This application uses the wheat TaCIPK19-3D gene as the object. After treating the wild-type (WT) rice strain at the three-leaf and one-heart stage, the TaCIPK19-3D gene overexpressing transgenic rice strain in rice, and the rice homologous gene oscipk19 gene knockout mutant rice strain with 125mM NaCl for 72 hours, it was found that the wild-type (WT), overexpressing transgenic strain and gene knockout mutant plants showed obvious phenotypic differences. The overexpressing transgenic strain showed an obvious salt-tolerant phenotype. The leaves of the overexpressing transgenic strain were greener and more active than those of the wild type and mutant after salt treatment; while the leaves of the rice homologous gene oscipk19 gene knockout mutant strain were more wilted than the wild type after salt treatment. These results show that the TaCIPK19-3D gene can positively regulate the salt tolerance of plants and can be used to promote the breeding of salt-tolerant wheat varieties through molecular breeding and other technologies.

[0007] The function of the TaCIPK19-3D gene discovered based on this application is of great significance for promoting the breeding of salt-tolerant wheat varieties. Through genetic engineering and other technologies, the stress resistance of wheat can be improved by overexpressing the TaCIPK19-3D gene, thereby breeding new wheat germplasm with better quality. The technical solution of this application is based on existing genetic engineering technologies, which have been widely used in the field of plants. Therefore, this application has the characteristics of high operability and wide application, and provides an effective method for plant genetic improvement and variety breeding.

[0008] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0010] Figure 1 The phenotypic diagrams are of the wild type (WT), overexpression strain and oscipk19 gene knockout mutant plants in Example 1 of the present application.

[0011] Figure 2 The results of the physiological indicators related to salt stress in Example 2 of the present application are shown in FIG. Among them, (A) is the Pro content before and after salt treatment; (B) is the SOD activity before and after salt treatment; (C) is the O 2 - The error bars show the mean ± SD of three analyses; significant differences were analyzed using t-test, and different lowercase letters indicate significant differences when P values ​​were less than 0.05.

[0012] Figure 3 qRT-PCR verification of the expression of salt stress-related genes in wild type and transgenic plants in Example 3 of this application. Among them, (A) is the expression level of OsAPX2 gene in rice before and after salt treatment; (B) is the expression level of P5CS gene in rice before and after salt treatment; (C) is the expression level of ABA2 gene in rice before and after salt treatment. Error bars show the mean ± SD of three analyses; significant differences were analyzed using t-test, and different lowercase letters indicate significant differences when P value is less than 0.05. DETAILED DESCRIPTION

[0013] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the application belongs. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical 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 the range.

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

[0015] The expression "comprising" or its synonymous similar expressions "including", "containing" and "having" etc. are open-ended and do not exclude additional unrecited elements, steps or ingredients. The expression "consisting of excludes any element, step or ingredient not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or ingredients, plus the optional elements, steps or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

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

[0017] CIPKs are a class of serine / threonine protein kinases that interact with CBL proteins. The CIPKs gene family in plants plays a key role in regulating the response of plants to salt stress. The expression level of wheat TaCIPK14 gene was significantly upregulated under cold and salt stress conditions. The study found that compared with the wild type, the chlorophyll, sugar and catalase contents of tobacco overexpressing TaCIPK14 gene were higher than those of wild type under cold and salt stress conditions. Other physiological indicators of stress, such as malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) will be lower than the wild type. Compared with the wild type, the tobacco transgenic strains overexpressing the TaCIPK29 gene under salt stress conditions showed salt-tolerant phenotypes such as higher seed germination rate, longer root length and better growth status. Further studies found that the H 2 O 2The content of TaCIPK24 decreased while the activity of antioxidant enzymes increased. The transgenic strains of Arabidopsis thaliana overexpressing the TaCIPK24 gene showed tolerance to salt stress. Further studies found that TaCIPK24 can enhance the salt tolerance of plants by increasing the external transport of sodium ions 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 CIPKs gene family can participate in the plant's response to salt stress by regulating the accumulation of antioxidants and ion transport in plants, thereby improving the plant's salt tolerance. However, the wheat TaCIPK19-3D gene and its functional application in wheat have not been reported.

[0019] In one aspect, the present application provides a use of a wheat TaCIPK19-3D gene or a protein encoded thereby in improving salt tolerance of crops.

[0020] In some embodiments, the nucleic acid sequence of the wheat TaCIPK19-3D gene has at least 80% sequence identity to 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 in this article 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 in this article 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, the present application also provides a method for improving the salt tolerance of crops, wherein the method comprises: overexpressing the wheat TaCIPK19-3D gene in the crops.

[0025] In some embodiments, the crop plant is selected from the group consisting of rice, wheat, corn and barley; preferably, the crop plant is rice.

[0026] Any overexpression method available in the art can be used to overexpress the wheat TaCIPK19-3D gene in crops. 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 embodiments, the crop plant is rice, wheat, corn or barley; preferably, the crop plant is rice.

[0029] Any overexpression method available in the art can be used to overexpress the wheat TaCIPK19-3D gene in crops. In some embodiments, the method of overexpressing the wheat TaCIPK19-3D gene in crops includes:

[0030] 1) by operably 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 embodiments, in crop plants, the wheat TaCIPK19-3D gene is linked to a constitutive promoter, and the expression of the wheat TaCIPK19-3D gene is controlled by the constitutive promoter.

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

[0034] In another aspect, the present application also provides the use of the transgenic crops obtained by the methods described herein in crop breeding. In some embodiments, the breeding method includes transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0035] In some embodiments, homologous genes of the wheat TaCIPK19-3D gene can also improve some traits of crops (such as rice) through overexpression, RNAi (reducing expression level) and gene editing.

[0036] The technical solution of this application can achieve overexpression of the TaCIPK19-3D gene in rice through genetic engineering technology, and perform targeted editing and regulation of the gene. In addition, the TaCIPK19-3D gene may also have application potential in salt stress of other crops. Other crops such as corn, soybeans, vegetables, etc. may also have the effect of improving their stress resistance. The present application relates to the study of the function and regulatory mechanism of the TaCIPK19-3D gene. This is of great significance for in-depth understanding of basic research in the fields of plant salt stress and plant growth and development. The present application may provide a specific research object and experimental model for research in the fields of molecular biology and genetics.

[0037] The present application describes multiple embodiments, but the description is exemplary rather than restrictive, and it is obvious to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application. Although many possible feature combinations are shown in the drawings and discussed in the specific embodiments, 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 any other feature or element in any other embodiment, or may replace any other feature or element in any other embodiment.

[0038] The present application includes and contemplates combinations of features and elements known to those of ordinary skill in the art. The embodiments, features and elements disclosed in the present application may also be combined with any conventional features or elements to form a unique invention scheme. Any features or elements of any embodiment may also be combined with features or elements from other invention schemes to form another unique invention scheme. Therefore, it should be understood that any feature shown and / or discussed in the present application may be implemented individually or in any appropriate combination. Therefore, except for the limitations made according to the attached claims and their equivalents, the embodiments are not subject to other restrictions. In addition, various modifications and changes may be made within the scope of protection of the attached claims.

[0039] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps described herein, the method or process should not be limited to the steps of the specific order described. As will be understood by those of ordinary skill in the art, other sequences of steps are also possible. Therefore, the specific sequence of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for the method and / or process should not be limited to the steps of performing them in the order written, and those skilled in the art can easily understand that these sequences can be changed and still remain within the spirit and scope of the embodiments of the present application.

[0040] The experimental methods in the following examples without specifying specific conditions are usually measured according to national standards. The experimental materials in the following examples without specifying the source are all commercially available raw materials. The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise defined or specified, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of this application.

[0041] Example

[0042] The relevant materials used in the examples are as follows:

[0043] 1. Reagents and Materials

[0044] Chinese spring wheat variety; Japanese spring rice variety.

[0045] 2. Instruments

[0046] Multifunctional microplate reader, six-channel fluorescence quantitative PCR instrument

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

[0048] (1) The total RNA of Chinese spring was extracted using a plant total RNA extraction kit, and the quality was tested by 1% agarose gel electrophoresis and the concentration was tested by Genova Nano. The total RNA was reverse transcribed into cDNA using a reverse transcription kit. The TaCIPK19-3D gene sequence information was obtained based on the Chinese spring transcriptome database to design specific primers. 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 the nucleotide sequence obtained by sequencing was shown in SEQ ID NO:1.

[0052] (2) The overexpression vector of TaCIPK19-3D gene was constructed by connecting it to pBI121 vector through double enzyme digestion. The overexpression vector was introduced into normal rice variety Nipponbare by Agrobacterium-mediated genetic transformation method, and then transgenic seedlings were obtained through plant tissue culture.

[0053] The obtained transgenic seedlings were cultivated in nutrient solution. When they reached the three-leaf and one-heart stage, leaves were taken to extract DNA. PCR amplification was performed using primers for mCIPK19-3D-F / R. PCR products were verified by 1% agarose gel electrophoresis. Positive plants were identified, numbered, and harvested and stored as single plants after maturity (T1 generation).

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

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

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

[0057] (5) The seeds of the overexpression plants and Nipponbare were germinated in water culture and then transferred to nutrient solution culture until the three-leaf stage. The wild-type (WT), overexpression transgenic lines and oscipk19 gene knockout mutant rice seedlings at the three-leaf and one-heart stage were treated with 125 mM NaCl for 72 hours. It was found that the wild-type (WT), overexpression lines and oscipk19 gene knockout mutant plants showed obvious phenotypic differences. The phenotypes are shown in the figure. Figure 1 It can be seen that there are obvious phenotypic differences among the wild type (WT), overexpression transgenic lines and oscipk19 gene knockout mutant plants. The overexpression transgenic lines show obvious salt tolerance phenotypes. The leaves of the overexpression transgenic lines are greener and more active than those of the wild type and mutant after salt treatment; while the leaves of the oscipk19 gene knockout mutant lines, the homologous gene in rice, are more wilted than those of the wild type after salt treatment.

[0058] Example 2.

[0059] The expression of Pro, O in wild type (WT), overexpressing transgenic lines and mutant plants before and after salt stress 2 - The content and SOD activity were determined.

[0060] Proline (Pro) content determination: refer to the kit (Greis, Suzhou, China), after the reaction is completed, the absorbance of the test solution is read at 520nm as A, ΔA = A determination - A blank, according to the standard curve y = 0.1625x-0.0064, x is the mass of the standard (μg), y is ΔA. Pro content (μg / g) = 41.03 × (ΔA + 0.0064) ÷ W (W is the sample mass 0.1g).

[0061] Superoxide Anion (O 2 - ) Assay: Refer to the kit (Greis, Suzhou, China). After the reaction is completed, the absorbance of the test solution is read at 540 nm as A, ΔA = A assay - A blank, according to the standard curve y = 0.0482x - 0.001, x is NO 2 - The molar mass (μmol), y is △A. 2 - Content (nmol / g) = 237.1 x (△A + 0.0011) ÷ W (W is the sample mass 0.1 g).

[0062] Superoxide dismutase (SOD) assay: refer to the kit (Greis, Suzhou, China). After the reaction, the absorbance of the test solution at 450 nm is read as A, ΔA assay = A assay - A control, ΔA blank = A blank 1 -A empty 2 According to the inhibition percentage = (△A blank - △A assay) ÷ △A blank × 100%. SOD activity (U / g) = 11.11 × inhibition percentage ÷ (1-inhibition percentage) ÷ W (W is the sample mass 0.1 g).

[0063] The results showed that TaCIPK19-3D can enhance the salt tolerance of plants by increasing Pro content and improving SOD activity to reduce the content of superoxide anions in plant cells. Figure 2 .

[0064] Example 3.

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

[0066] Using wild-type plants as controls, qRT-PCR was used to detect the expression changes of OsAPX2, OsABA2, and OsP5CS genes. The obtained cDNA was diluted to an appropriate concentration (about 10-20 times) as a template. The qRT-PCR experiment was performed according to the PCR mixing system and steps in the instructions of Tiangen's Talent qPCR PreMix (SYBR Green) kit. The expression level of the target gene was calculated by the 2-△△CT method.

[0067] The results are shown in 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 plants.

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

[0069] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. Use of wheat TaCIPK19-3D gene or the protein encoded by it in improving the salt tolerance of crops.

2. The use according to claim 1, wherein 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 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.

3. The use according to claim 1 or 2, wherein The salt tolerance of the crop is improved by overexpressing the wheat TaCIPK19-3D gene in the crop.

4. The use according to claim 1 or 2, wherein The crop is rice, wheat, corn or sorghum; preferably, the crop is rice.

5. A method for improving the salt tolerance of crops, wherein: The method comprises: overexpressing the wheat TaCIPK19-3D gene in the crop.

6. The method according to claim 5, wherein: 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.

7. The method according to claim 5 or 6, wherein: The crop is rice, wheat, corn or sorghum; preferably, the crop is rice.

8. The method according to claim 5 or 6, wherein: The method for overexpressing the wheat TaCIPK19-3D gene in the crop comprises: 1) by operably linking a strong promoter to the wheat TaCIPK19-3D gene in the crop; and / or 2) By increasing the copy number of the wheat TaCIPK19-3D gene on the chromosome of the crop.

9. Use of the transgenic crops obtained by the method according to any one of claims 5 to 8 in crop breeding.

10. The use according to claim 9, wherein Breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

Images