Cucumber Cold Tolerance Gene CsVQ12 and Its Application

Abstract

The invention relates to a cucumber cold-resistant gene and its application, and belongs to the field of genetic engineering. The invention finds that the cucumber CsVQ12 gene has a cold-resistant effect, and overexpression of CsVQ12 can improve plant cold resistance and advance flowering.

Description

Technical Field

[0001] The invention relates to cucumber cold-resistant genes and applications thereof, and belongs to the field of genetic engineering. Background Art

[0002] Cucumber (Cucumis sativus L.) is a plant of the Cucurbitaceae family. It is popular among consumers for its crisp and refreshing taste and is widely planted throughout my country. Cucumbers originate from tropical regions and are therefore extremely sensitive to low temperatures. When cucumbers are subjected to low temperature stress during the seedling stage, their leaves tend to turn yellow and wilt, causing the growth of the plants to slow down or even stagnate, and in severe cases, causing plant death; especially during the off-season cultivation of cucumbers, the plants are more susceptible to low temperatures, resulting in a decrease in fruit yield and quality, causing huge losses to agricultural production. Therefore, research on the key genes that respond to low temperature stress in cucumbers and their molecular mechanisms in responding to low temperature stress can not only provide important genetic resources for the stress-resistant breeding of cucumbers, but also lay a theoretical foundation for achieving a balanced year-round supply of greenhouse vegetables in my country in the future.

[0003] Proteins containing the valine-glutamine (VQ) motif were first discovered, and subsequently different numbers of VQ proteins were found in different plants. Functional studies on them revealed that they can play an important role in plant growth and development and in response to various biotic and abiotic stresses independently or by interacting with other transcription factors.

[0004] There are many VQ family gene members in cucumber, and most of them have no clear and specific functions. It is still unclear what the specific functions of these members are.

[0005] The present invention finds that the cucumber CsVQ12 gene can improve the cold tolerance of plants and can also regulate the flowering period and leaf senescence. Summary of the invention

[0006] The purpose of the invention is to provide a method for improving the cold tolerance of cucumbers.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides an application of a protein in regulating plant cold tolerance and / or flowering period and / or leaf senescence, characterized in that the amino acid sequence of the protein is as shown in SEQ ID NO.1.

[0009] The present invention also provides an application of a nucleic acid in regulating plant cold tolerance and / or flowering period and / or leaf senescence, characterized in that the nucleic acid encodes the above-mentioned protein.

[0010] In some embodiments, the nucleotide sequence of the above nucleic acid is shown as SEQ ID NO.2.

[0011] The present invention also provides a method for improving plant cold tolerance and / or early flowering, characterized in that the expression and / or activity of the above protein is increased in plants, and plant materials with improved cold tolerance and / or early flowering are selected.

[0012] In some embodiments, the above method for increasing protein expression and / or activity is to use a high-activity promoter to drive a nucleic acid molecule encoding the protein.

[0013] In some embodiments, the above-mentioned high-activity promoter sequence is shown as SEQ ID NO.3.

[0014] The present invention also provides an application of an expression cassette in improving plant cold tolerance and / or early flowering, characterized in that the expression cassette is operably connected in sequence by a double 35S promoter, a nucleic acid molecule encoding a protein of the sequence shown in SEQ ID NO.1, and an Agrobacterium nopaline synthase gene terminator nos.

[0015] In some embodiments, the double 35S promoter sequence is shown as SEQ ID NO.3, the nucleic acid molecule sequence encoding the protein shown in SEQ ID NO.1 is shown as SEQ ID NO.2, and the terminator sequence is shown as SEQ ID NO.4.

[0016] Compared with the existing technology, the beneficial effects of the present invention are: the present invention finds that CsVQ12 can not only improve the cold tolerance of plants, but also affect the flowering period and leaf senescence of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1 shows the PCR amplification of CsVQ12, the construction of the PHB-35S:CsVQ12 vector, and the RT-PCR verification of positive transgenic Arabidopsis. Figure A shows the gel image of PCR amplification of the CsVQ12 gene, where M is the marker and CsVQ12 is the amplified fragment; Figure B shows the schematic diagram of the PHB-35S:CsVQ12 recombinant vector structure; Figure C shows the gel image of RT-PCR verification of positive CsVQ12 transgenic Arabidopsis.

[0018] Figure 2 CsVQ12 improves the low temperature tolerance of CsVQ12-overexpressing Arabidopsis at the seedling stage. Figures AB are pictures of wild-type Arabidopsis (WT) and CsVQ12-overexpressing Arabidopsis under normal conditions and after low temperature stress treatment; Figures CF are the determination of survival rate, relative conductivity, POD activity, and MDA content of wild-type (WT) and CsVQ12-overexpressing Arabidopsis under normal conditions and after low temperature stress treatment; data are the mean ± SD (standard deviation) of three biological replicates; different letters indicate significant differences (P < 0.05).

[0019] Figure 3 Overexpression of CsVQ12 causes early flowering in Arabidopsis. Figures AC are natural growth diagrams of 23-day-old WT, CsVQ12-OE1, and CsVQ12-OE2; Figures DE are statistical diagrams of bolting time and flowering time of WT, CsVQ12-OE1, and CsVQ12-OE2; Figure FJ is a statistical diagram of the expression levels of flowering-related genes AP1, FT, FUL, LFY, and FLC in 23-day-old WT, CsVQ12-OE1, and CsVQ12-OE2. Data are the mean ± SD (standard deviation) of three biological replicates; different letters indicate significant differences (P<0.05)

[0020] Figure 4 Overexpression of CsVQ12 accelerated leaf senescence in Arabidopsis. Figures AB and AB are natural growth pictures of WT, CsVQ12-OE1, and CsVQ12-OE2 at 3 and 7 weeks of age, respectively; Figures CE are expanded rosette leaves of WT, CsVQ12-OE1, and CsVQ12-OE2 at 7 weeks of age, respectively. DETAILED DESCRIPTION

[0021] The following definitions and methods are provided to better define this application and guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms are understood according to the conventional usage of those of ordinary skill in the relevant field. All patent documents, academic papers, industry standards and other public publications cited herein are incorporated herein by reference in their entirety.

[0022] As used herein, "cucumber" is any cucumber plant and includes all plant varieties that can be bred with cucumber, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, complete plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction. Amino acids can be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides can be represented by commonly accepted single-letter codes. Numerical ranges include numbers that limit the range. As used herein, "nucleic acid" includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides, which hybridize with single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the term "encoding" or "encoded" is used in the context of a specific nucleic acid to refer to the nucleic acid containing the necessary information for guiding the translation of the nucleotide sequence into a specific protein. Codons are used to represent information for encoding proteins. As used herein, the "full-length sequence" of a specific polynucleotide or its encoded protein refers to the entire nucleic acid sequence or the entire amino acid sequence with a natural (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of the specific protein. The terms "polypeptide", "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. The term is also used for naturally occurring amino acid polymers. The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may include known analogs of a natural amino acid that may function in a manner similar to that of a naturally occurring amino acid.

[0023] "Plant" includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and their progeny. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. "Progeny" includes any subsequent generations of a plant.

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the inventive method, step or condition, all belong to the scope of the present application. If not otherwise specified, the embodiments are according to conventional experimental conditions, such as Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or according to the conditions of the manufacturer's instructions. If not otherwise specified, the chemical reagents used in the embodiments are conventional commercial reagents, and the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0025] Example 1 Identification of cucumber VQ genes

[0026] The present invention identified 32 VQ family genes in cucumber, and the results of gene expression pattern analysis showed that the expression of six genes, CsVQ6, CsVQ9, CsVQ12, CsVQ13, CsVQ16, and CsVQ20, were regulated by cold, drought, and salt, while the expression levels of six genes, CsVQ2, CsVQ9, CsVQ12, CsVQ13, CsVQ20, and CsVQ22, changed after treatment with downy mildew and powdery mildew (Scientia Horticulturae, 2022, 295: 110874).

[0027] In order to further clarify the specific functions of these VQ genes, the inventors cloned these genes from cucumber and constructed them into expression vectors to transform the model plant Arabidopsis thaliana.

[0028] The VQ gene was obtained from the cDNA of the leaves of the North China cucumber variety "HB" as a template. Primers were designed based on the information of the reference sequence of the CDS coding region of the VQ gene in the cucumber genome database (CuGenDB). The PCR method was used to amplify and sequence the fragments. The verified CDS fragments were connected with the PHB overexpression linear vector to form an overexpression recombinant plasmid. Overexpression used double 35S as a promoter (SEQ ID NO.3) and nos as a terminator (SEQ ID NO.4). The vector was transferred to the Agrobacterium strain GV3101 and the wild-type Arabidopsis thaliana Col-0 was infected by the floral dip method.

[0029] Example 2 Plant cold tolerance evaluation

[0030] The obtained Arabidopsis thaliana transformed with each VQ gene was subjected to cold treatment to evaluate the cold tolerance of these VQ genes.

[0031] The WT and CsVQ-overexpressing Arabidopsis plants that had grown normally for 21 days were tested for MDA content, POD activity, and relative conductivity to ensure that there were no differences in these physiological indicators between the WT and CsVQ-overexpressing Arabidopsis plants. After 30 minutes of low temperature stress (-20°C) on the WT and CsVQ-overexpressing Arabidopsis plants, most of the WT plants had wilted. The growth of the CsVQ-overexpressing Arabidopsis plants was checked, and the results showed that the growth of the Arabidopsis plants overexpressing CsVQ12 and CsVQ22 was still good, with a survival rate of about 90%, while the survival rate of the WT was below 10%, and the survival rate of the Arabidopsis plants overexpressing other CsVQs was not high. In addition, after low temperature stress, the relative conductivity and MDA content of the leaves of the CsVQ12 and CsVQ22-overexpressing Arabidopsis plants were significantly lower than those of the WT; while the POD activity was significantly higher than that of the WT (see the results for CsVQ12). Figure 2 The above experimental results show that transgenic Arabidopsis plants overexpressing CsVQ12 and CsVQ22 can improve their low temperature tolerance by increasing POD activity and inhibiting the accumulation of MDA under low temperature stress.

[0032] Example 3 Other traits of CsVQ12 plants

[0033] The inventors also found that the flowering period of CsVQ12-overexpressing Arabidopsis was earlier than that of wild-type plants. Two highly expressed CsVQ12-overexpressing strains (OE1 and OE2) were further selected to grow in a constant temperature culture room with 22°C, 16h light / 8h dark, and the bolting and flowering time of Arabidopsis were recorded. The results showed that most of the CsVQ12-overexpressing Arabidopsis began to bolt around 20 days of growth and began to bloom around 22 days. Compared with WT, the bolting time and flowering time were both advanced. Then, RNA was extracted from leaves of 23-day-old WT and CsVQ12-overexpressing Arabidopsis plants and reverse transcribed into cDNA. The expression levels of flowering-related genes were determined by qRT-PCR. Statistics showed that the expression levels of flowering-upregulated genes AP1, FT, LFY, and FUL in CsVQ12-overexpressing Arabidopsis plants were significantly upregulated compared with WT, while the expression level of flowering-downregulated gene FLC was significantly lower than that in WT ( Figure 3 ), indicating that CsVQ12 has the function of promoting plant flowering.

[0034] The phenotype of CsVQ12 overexpressing transgenic plants was further observed. At 3 weeks old, there was no significant difference between CsVQ12 overexpressing Arabidopsis and WT; at 5 weeks old, the leaves of CsVQ12 overexpressing Arabidopsis turned yellow compared with WT; at 7 weeks old, the senescence of CsVQ12 overexpressing Arabidopsis leaves was significantly stronger than that of WT, indicating that the leaves of transgenic Arabidopsis plants overexpressing CsVQ12 were prematurely aged. To verify whether overexpression of the CsVQ12 gene accelerated the leaf senescence process of Arabidopsis, this study measured the chlorophyll content and relative conductivity of transgenic Arabidopsis and WT leaves at 3 and 7 weeks of age. The results showed that there was no difference in the chlorophyll content and relative conductivity in the leaves of WT and CsVQ12 overexpressing Arabidopsis plants at 3 weeks of age; however, when grown to 7 weeks of age, the chlorophyll content of CsVQ12 overexpressing Arabidopsis leaves was significantly lower than that of WT, while its relative conductivity was significantly higher than that of WT. The above results indicate that overexpression of CsVQ12 positively regulates the senescence process of Arabidopsis leaves.

[0035] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Use of a protein in improving Arabidopsis cold tolerance and / or advancing the flowering period of Arabidopsis and / or accelerating Arabidopsis leaf senescence, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. Use of a nucleic acid in improving Arabidopsis thaliana cold tolerance and / or advancing Arabidopsis thaliana flowering period and / or accelerating Arabidopsis thaliana leaf senescence, characterized in that: The nucleic acid encodes the protein according to claim 1.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

2.

4. A method for improving the cold tolerance of Arabidopsis thaliana and / or advancing the flowering period of Arabidopsis thaliana, characterized in that: The expression and / or activity of the protein in claim 1 is increased in Arabidopsis thaliana, and Arabidopsis thaliana materials with improved cold tolerance and / or early flowering period are selected.

5. The method according to claim 4, characterized in that The method for increasing protein expression and / or activity is to use a high-activity promoter to drive a nucleic acid molecule encoding a protein.

6. The method according to claim 5, characterized in that The high-activity promoter sequence is shown in SEQ ID NO.

3.

7. Use of an expression cassette for improving Arabidopsis cold tolerance and / or advancing the flowering period of Arabidopsis, characterized in that: The expression cassette is operably connected in sequence by a double 35S promoter, a nucleic acid molecule encoding a protein of the sequence shown in SEQ ID NO.1, and a terminator of the Agrobacterium nopaline synthase gene.

8. The use according to claim 7, characterized in that: The double 35S promoter sequence is shown in SEQ ID NO.3, the nucleic acid molecule sequence encoding the sequence protein shown in SEQ ID NO.1 is shown in SEQ ID NO.2, and the terminator sequence of the Agrobacterium nopaline synthase gene is shown in SEQ ID NO.4.

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