Application of CsRAF2 gene or protein coded by CsRAF2 gene in regulation and control of plant type development of cucumber

Abstract

The invention discloses an application of a CsRAF2 gene or a protein coded by the CsRAF2 gene in regulating and controlling plant type development of cucumbers. It is found that the cucumber CsRAF2 gene is related to the cucumber plant type, leaves of a cucumber edited strain are remarkably reduced compared with WT, petioles of the cucumber edited strain are remarkably shortened compared with WT, and the cucumber CsRAF2 gene can be applied to improvement of cucumber plant type development.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically involving CsRAF2 Application of genes or their encoded proteins in regulating cucumber plant architecture development. Background Technology

[0002] cucumber( Cucumis sativus The above-ground plant type of cucumbers (L.) is mainly determined by plant height, stem diameter, leaf size, branching, and internode length. Different regions have significantly different cultivation and harvesting methods and target markets, resulting in varying requirements for plant type. A suitable plant type is crucial for increasing cucumber yield, reducing labor costs, and minimizing pests and diseases.

[0003] Selecting and breeding superior cucumber plant types has become an important breeding goal. Cucumber leaves are not only vital organs for photosynthesis and transpiration, but their morphology also directly influences the cucumber plant type. Leaf morphological development is closely related to photosynthesis and stress resistance. In cucumber cultivation, it is usually necessary to manually remove old and diseased leaves to reduce disease. Therefore, the growth and development of cucumber leaves is a key factor affecting yield and quality improvement.

[0004] Few genes related to cucumber leaf development have been reported. Currently, the only reported genes related to cucumber leaves are those regulating leaflet development and shape. Csll Regulating the angle between cucumber leaves and petioles CsHLS1 Genes that regulate the number of cucumber leaves and fruits CsRAXs ([1] Chunhua Wang, Jie Li, Kai Fang, et al. CsHLS1-CsSCL28 moduleregulates compact plant architecture in cucumber. Plant Biotechnol Journal , 2024, 22(6):1724-1739. [2] Luming Yang, Hanqiang Liu, Jianyu Zhao, et al. LITTLELEAF (LL) encodes a WD40 repeat domain-containing protein associated with organ size variation in cucumber. The Plant Journal , 2018, 95(9): 834-847. [3] Jiacai Chen, Liu Liu, Guangxin Chen, et al. CsRAXsNegativelyregulate leaf size and fruiting ability through auxin glycosylation incucumber. Journal of Integrative Plant Biology , 2024, 66: 1024-1037.).

[0005] But regarding genes CsRAF2 Its role in regulating cucumber plant architecture has not yet been reported. Summary of the Invention

[0006] This invention addresses the aforementioned shortcomings of the prior art by providing... CsRAF2 The application of genes or their encoded proteins in regulating cucumber plant architecture development, specifically regulating the length of cucumber leaves and petioles.

[0007] This invention first provides CsRAF2 Application of genes or their encoded proteins in regulating cucumber plant architecture development.

[0008] Preferably, the regulation is to... CsRAF2 Gene knockout may CsRAF2 The loss of function of the gene-encoded protein results in smaller cucumber leaves and shorter petioles.

[0009] Preferably, the CsRAF2 The nucleotide sequence of the gene is shown in SEQ ID NO.1. CsRAF2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0010] Preferably, CsRAF2 During gene knockout, the target sequence for gene knockout is: Target 1: GGAAATCGCGAGCAATTTCGGGG Target 2: GGTGGTGGGTTGGGGAAGAGGG.

[0011] This invention also provides a method for regulating cucumber plant architecture development, by... CsRAF2 Gene knockout may CsRAF2 The loss of function of the gene-encoded protein results in smaller cucumber leaves and shorter petioles.

[0012] Preferably, in the method for regulating cucumber plant architecture development, the CsRAF2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0013] Preferably, the method for regulating cucumber plant architecture development includes the following steps: (1) Construct a gene knockout vector, wherein the gene knockout vector is a vector containing a base sequence as shown in SEQ ID NO.1. CsRAF2 Plant expression vectors containing gene knockout sequences; (2) Introduce the gene knockout vector from step (1) into cucumber cells. CsRAF2 Gene knockout and subsequent culture yielded transgenic cucumber plants.

[0014] Preferably, the plant expression vector is pCBSG015 (Basta).

[0015] Preferably, the target sequence for gene knockout is: Target 1: GGAAATCGCGAGCAATTTCGGGG Target 2: GGTGGTGGGTTGGGGAAGAGGG.

[0016] The beneficial effects of this invention are: This invention discovered cucumber CsRAF2 The gene is associated with cucumber plant architecture. The leaves of the cucumber edited lines are significantly smaller and the petioles are significantly shorter than those of the WT line. This gene can be applied to improve cucumber plant architecture development. Attached Figure Description

[0017] Figure 1 The spectrum is for the vector pCBSG015(Basta).

[0018] Figure 2 These are the gene knockout results. WT represents wild-type. Csraf2cr-1 and Csraf2cr-1 express CsRAF2 Gene-edited strains; Figure 2 A in the text represents knockout. CsRAF2 A diagram showing sequence changes at gene target sites; Figure 2 B in the text stands for knockout. CsRAF2 Schematic diagram of gene targets; Figure 2 The 'C' in the diagram represents a schematic representation of changes detected during gene editing target sequencing.

[0019] Figure 3 To knock out CsRAF2 Phenotypic identification results of wild-type WT cucumber plants. Among them, Figure 3 In the diagram, A represents the leaf area and petiole phenotype of WT and gene-edited cucumber lines; Figure 3 B in the figure represents the leaf and petiole phenotypes at the 10th node of the leaves of the WT and gene-edited strains. Figure 3 C in the figure represents the leaf area statistics of the 10th segment; Figure 3 D in the figure represents the petiole statistics at the 10th segment. Statistical results show that compared to WT, CsRAF2The cucumber plants in the gene knockout line had significantly smaller leaf area and significantly shorter petioles. * indicates P < 0.05, ** indicates P < 0.01.

[0020] Figure 4 Knockout CsRAF2 Results of leaf and petiole analysis of wild-type WT cucumber plants. Figure 4 A in the image represents leaf lamina diagrams of mature WT and gene-edited strains, with a scale bar of 200 pixels. Figure 4 In this context, B represents the leaf lamina area of ​​the WT and gene-edited strains at maturity. Figure 4 In this context, C represents the number of leaf-laying cells at maturity for WT and gene-edited lines. Figure 4 D in the image represents a longitudinal section of thin-walled cell slices from the petiole of the mature WT and gene-edited lines, with a scale bar of 97 pixels. Figure 4 In this context, E represents the area of ​​the thin-walled cells in the longitudinal section of the petiole of the WT and gene-edited strains; Figure 4 In the figure, F represents the number of parenchyma cells in the longitudinal section of the petiole of the WT and gene-edited lines. Cytological results showed that the number of plastinated cells in the leaves of the gene-edited cucumber line was not significantly different from that of the WT line, but the cell area was significantly smaller. Similarly, the number of parenchyma cells in the petioles of the gene-edited cucumber line was not significantly different from that of the WT line, but the cell area was significantly smaller. ** indicates P < 0.01. Detailed Implementation

[0021] cucumber CsRAF2 The nucleotide sequence of the gene is shown in SEQ ID NO.1, cucumber. CsRAF2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0022] SEQ ID NO.1: ATGGCCACAACCTCATCCACCCATCTCCACCTCCACCTCCCTCTCCTCCCTCTTCCCCAACCCCACCACCGCACCTTCCTTCCCTTACCCACCACCCTCCTCCGCCGTAACCTCCGTTCCCTCACCGTTTCCGTGGGAACAGACATACTAGGAGACTTCGGCGCCAGAGACCCATTTCCGGAGGAAATCGCGAGCAATTTCGGGGAAAAAGTTATCGGATTTTCCGATACAGAGCACAAGATTCTGATCCCTAATGCCCGAGCCTTGGCTCTCTCTGAACAAGAGTGTGTTCCAGTTTCTTCATTGCAGGCTCCAATGGCAGAGGATGAAGCCAAAAAGCTCCTGAGGAAGGTTGTGGGTTGGAAATTGGTGGATGGTGGAAGAGGGGAATTAAAGCTTCAATGTTTATGGAAATTGAGGGATTTTAAATGCAGTGTTGAGCTGATTAATAGAATTTGTAAAGTTGTTGAGAATGTTGGCCATTTTCCTGATCTTCATTTGGAGCAACCCAATCAAGTTAGAGCTGAGCTTTGGACTTCATCCATTGGAGGGTTGAGCATGAATGATTATATAGTAGCTGCAAAAATTGATGAGATTAAAACATCAGATCTTGCTCCCAGGAAAAGAGCTTGGGCATGA。

[0023] SEQ ID NO.2: MATTSSTHLHLHLPLLPLPQPHHRTFLPLPTTLLRRNLRSLTVSVGTDILGDFGARDPFPEEIASNFGEKVIGFSDTEHKILIPNARALALSEQECVPVSSLQAPMAEDEAKKLLRKVVGWKLVDGGRGELKLQCLWKLRDFKCSVELINRICKVVENVGHFPDLHLEQPNQVRAELWTSSIGGLSMNDYIVAAKIDEIKTSDLAPRKRAWA*。

[0024] Example 1: CsRAF2 Transformation of cucumber with the CRISPR gene vector Designed by Weimi Biotechnology (Jiangsu) Co., Ltd. CsRAF2 Gene knockout target, sequence as follows: Target 1: GGAAATCGCGAGCAATTTCGGGG Target 2: GGTGGTGGGTTGGGGAAGAGGG.

[0025] The expression sequences corresponding to the two target sites were then transferred into the vector pCBSG015(Basta) (see map). Figure 1 The data is transferred to SG1 and SG2 of the carrier, respectively, for construction. CsRAF2 -CRISPR vector.

[0026] Select the successfully constructed vector and genetically transform cucumber '649' variety (WT) to prepare GV3101 Agrobacterium competent cells. Transform the successfully constructed CRISPR vector into the GV3101 Agrobacterium competent cells and plate them on plates containing 100 mg / L kanamycin and 50 mg / L rifampin to screen for positive colonies.

[0027] Cucumber seeds were soaked in warm water, peeled, disinfected, and germinated. Agrobacterium tumefaciens containing the target gene was prepared and cultured in large quantities at 28°C using liquid LB medium containing kanamycin at a final concentration of 100 mg / L and rifampin at 50 mg / L until OD600 = 1.2~1.6.

[0028] Agrobacterium was centrifuged at 2000 r / min for 10 min, and the bacterial cells were collected. The Agrobacterium was resuspended in MS liquid medium (OD600=0.8) to obtain the Agrobacterium resuspension. The prepared Agrobacterium suspension was used to infect cucumber cotyledons; after culturing in callus induction medium, shoot induction medium, and rooting medium, regenerated cucumber plants were obtained.

[0029] The successfully transformed plants in CsRAF2 Mutation at gene target ( Figure 2 ). Figure 2 In A of Csraf2cr-1 and Csraf2cr-2 represent CsRAF2 The editing type is indicated by the rectangle on the right, which represents premature termination of the CsRAF2 protein. The inverted triangle indicates the location of the premature termination.

[0030] Example 2: Knockout CsRAF2 Phenotypic identification of wild-type WT cucumber plants Wild-type WT and gene-edited cucumber plants (constructed in Example 1) were planted. CsRAF2 After 40 days of growth, the phenotypes of wild-type WT and gene-edited lines were observed.

[0031] First, take a picture of the cucumber plant. Figure 3 (A and B in the text) and measured and analyzed the leaf size and petiole data.

[0032] The results showed that, compared with the wild-type WT, the gene-edited lines had more leaves ( Figure 3 C in the middle), petiole ( Figure 3 D) in the middle becomes significantly shorter.

[0033] Example 3: Knockout CsRAF2 Statistical analysis of leaf and petiole angles of wild-type WT cucumber plants Take wild-type WT and CsRAF2 Edited strain (constructed in Example 1) CsRAF2 The material was the middle part of the leaves and petioles of the gene knockout plant.

[0034] Leaf sampling: Mature leaves from the middle section were selected, and holes were punched using a hole puncher. The leaf samples were then placed in cell culture dishes for destaining. After destaining, the size of the plate-forming cells on the abaxial surface of the leaves was observed using a Mingmei optical microscope (Mshot, Mingmei Technology Co., Ltd., Guangzhou, China). Figure 4 A in the image), and used ImageJ software (https: / / imagej.net / downloads) to measure cell size and number. Figure 4 (B and C in the middle).

[0035] Sampling at the base of the petiole: The middle section of a mature petiole was removed with a scalpel and fixed overnight at 4°C with FAA (70% ethanol, 10% formalin, and 5% acetic acid). After fixation, the specimen was embedded in paraffin and cut into 10 μm thick transverse sections, stained with 0.1% toluidine blue. The samples were examined and imaged using a Nikon Eclipse E100 microscope (Nikon USA, Melville, NY, USA). The cell count within a specified section was determined using ImageJ software. Figure 4 (D in the text) Cytological results showed that the number of leaf lamina cells in the gene-edited cucumber line was significantly reduced compared to WT, while the cell area remained largely unchanged; the area of ​​parenchyma cells in the petiole decreased, but the number remained largely unchanged. Figure 4 (E and F in the text).

Claims

1. CsRAF2 Application of genes or their encoded proteins in regulating cucumber plant architecture development.

2. The application according to claim 1, characterized in that, Regulation is to pass CsRAF2 Gene knockout may CsRAF2 The loss of function of the gene-encoded protein results in smaller cucumber leaves and shorter petioles.

3. The application according to claim 1, characterized in that, The CsRAF2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. CsRAF2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

4. The application according to claim 1, characterized in that, Will CsRAF2 During gene knockout, the target sequence for gene knockout is: Target 1: GGAAATCGCGAGCAATTTCGGGG Target 2: GGTGGTGGGTTGGGGAAGAGGG.

5. A method for regulating cucumber plant architecture development, characterized in that, By CsRAF2 Gene knockout may CsRAF2 The loss of function of the gene-encoded protein results in smaller cucumber leaves and shorter petioles.

6. The method for regulating cucumber plant architecture development according to claim 5, characterized in that, The CsRAF2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The method for regulating cucumber plant architecture development according to claim 5, characterized in that, Includes the following steps: (1) Construct a gene knockout vector, wherein the gene knockout vector is a vector containing a base sequence as shown in SEQ ID NO.

1. CsRAF2 Plant expression vectors containing gene knockout sequences; (2) Introduce the gene knockout vector from step (1) into cucumber cells. CsRAF2 Gene knockout and subsequent cultivation yielded transgenic cucumber plants.

8. The method for regulating cucumber plant architecture development according to claim 7, characterized in that, The plant expression vector is pCBSG015 (Basta).

9. The method for regulating cucumber plant architecture development according to claim 7, characterized in that, The target sequence for gene knockout is: Target 1: GGAAATCGCGAGCAATTTCGGGG Target 2: GGTGGTGGGTTGGGGAAGAGGG.

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