A gene CsSUS3 for regulating waterlogging stress-induced adventitious root formation in cucumber and its application

Abstract

The present invention discloses a gene CsSUS3 for regulating waterlogging stress-induced adventitious root formation in cucumbers and its application. The present invention identifies a new cucumber adventitious root formation regulating gene CsSUS3, whose edited mutant has weakened adventitious root formation ability, while the overexpressed mutant has significantly enhanced adventitious root formation ability. The gene CsSUS3 of the present invention can be used to guide the breeding and improvement of cucumber waterlogging-resistant germplasm resources, and provide technical support for the breeding of cucumber waterlogging-resistant varieties. The present invention uses two transgenic technologies to jointly explore the application of CsSUS3 in inducing adventitious root formation in cucumbers under waterlogging stress, and provides new gene resources for molecular genetic improvement of cucumber waterlogging resistance. The present invention provides a gene CsSUS3 for inducing adventitious root formation in cucumbers under waterlogging stress, which has important application value in studying how to improve waterlogging resistance in cucumbers.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene CsSUS3 for regulating waterlogging stress-induced adventitious root formation in cucumber and an application thereof. Background Art

[0002] Cucumber (Cucumis sativus L.) is a widely cultivated vegetable that is popular among consumers. However, cucumber production in summer and autumn is often threatened by rain and waterlogging. Because cucumber taproots are not well developed, they are aerobic and difficult to form aeration tissues, the oxygen content of the plant drops sharply after waterlogging, making it very vulnerable. After waterlogging, cucumbers will produce stem-derived adventitious roots. The newly formed adventitious roots replace the damaged primary roots and promote the plant to resume growth. Therefore, exploring the key genes that regulate adventitious root formation is the basis for achieving genetic improvement of cucumber waterlogging resistance.

[0003] Prior art has reported that in the cucumber flooding-tolerant line Zaoer-N, the CsARN6.1 gene promotes cucumber's adaptation to flooding by increasing the production of adventitious roots. This effect of increasing adventitious roots may be caused by a non-synonymous SNP mutation in the coiled-coil domain of the CsARN6.1 protein in Zaoer-N. The regulation of the number of adventitious roots by the CsARN6.1 gene depends on the activity of its ATPase. Heterologous expression of the CsARN6.1 gene in Arabidopsis thaliana resulted in significantly faster growth of the main root after seed germination than in the control line, and earlier lateral root formation. After the CsARN6.1 gene from Zaoer-N was transferred into a line with the same genetic background as the Pepino allele of the flood-intolerant parent, it showed an increase in the number of adventitious roots compared to the control under flooding conditions. Summary of the invention

[0004] Purpose of the invention: In view of the deficiencies in the prior art, the present invention provides a gene CsSUS3 that regulates waterlogging stress-induced adventitious root formation in cucumbers. The present invention identifies a new cucumber adventitious root formation regulating gene CsSUS3, whose edited mutant has weakened adventitious root formation ability, while the overexpressed mutant has significantly enhanced adventitious root formation ability. The gene CsSUS3 of the present invention can be used to guide the breeding and improvement of cucumber waterlogging-resistant germplasm resources, and provide technical support for the breeding of cucumber waterlogging-resistant varieties.

[0005] The present invention also provides application of the gene CsSUS3 for regulating waterlogging stress and inducing adventitious root formation in cucumber.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses a gene CsSUS3 for regulating waterlogging stress-induced adventitious root formation in cucumber, and the nucleotide sequence of the gene CsSUS3 is shown in SEQ ID NO.1.

[0007] The primer pair used to amplify the gene CsSUS3 is CsSUS3-F: 5'-CCTGTGGATTGCCTACCTTT-3'; CsSUS3-R: 5'-TGGGTCTTCCTTGCTCTTCT-3'.

[0008] The amino acid sequence of the protein expressed by the gene CsSUS3 for regulating waterlogging stress-induced adventitious root formation in cucumber is shown in SEQ ID NO.2.

[0009] The present invention discloses a gene knockout vector CRISPR-CsSUS3 based on the gene CsSUS3 that regulates waterlogging stress and induces adventitious root formation in cucumber.

[0010] The gene knockout vector CRISPR-CsSUS3 was constructed by designing the target gene sequences sgRNA1 and sgRNA2 of CsSUS3, sgRNA1: 5'-CGATCAGTTCATGATGTTGCAGG-3'; sgRNA2: 5'-CCCCATCAGCTAGTTTC CTTCGG-3'; using pCBC-DT1T2 (Cm) as a template to amplify the double targets, connecting the formed double-stranded sgRNA1 / 2 to the pkSE402 vector, and finally obtaining the gene knockout vector CRISPR-CsSUS3 by Escherichia coli transformation and Agrobacterium transformation, extracting the plasmid.

[0011] The invention discloses an overexpression vector OE-CsSUS3 containing the gene CsSUS3 for regulating waterlogging stress-induced adventitious root formation in cucumber.

[0012] The overexpression vector OE-CsSUS3 is constructed by designing primers for vector construction based on the CDS region of the CsSUS3 gene.

[0013] OE-CsSUS3-F:gagaacacgggggacggatccATGGCAGAACGAGTTCTCAACC;

[0014] OE-CsSUS3-R:atggtctttgtagtcggatccCTCATCCACAGCCGGTGGC;

[0015] The gene was amplified, restriction enzyme-cut and connected to the pCAMBIA1305.4 vector, and then transformed into Escherichia coli and Agrobacterium tumefaciens, and the plasmid was extracted to finally obtain the overexpression vector OE-CsSUS3.

[0016] The invention discloses an application of the gene CsSUS3 for regulating waterlogging stress-induced adventitious root formation in cucumbers in inducing adventitious root formation in waterlogging stress-induced cucumbers.

[0017] Among them, by editing the cucumber CsSUS3 gene through CRISPR / Cas9, its ability to induce adventitious root formation under waterlogging stress was reduced; by overexpressing the cucumber CsSUS3 gene, its ability to induce adventitious root formation under waterlogging stress was increased.

[0018] The invention discloses an application of the gene CsSUS3 for regulating waterlogging stress-induced adventitious root formation in cucumber in the cultivation of waterlogging-resistant cucumber germplasm.

[0019] The present invention uses cucumber "CCMC" strain as material, constructs dual targets through CRISPR / Cas9 technology, obtains stable CRISPR-CsSUS3 gene editing material, floods the CRISPR-CsSUS3 transgenic material and a control material (WT), observes plant phenotypic changes, and detects changes in gene expression levels of hypocotyls of cucumbers under waterlogging stress at different times after knocking out the CsSUS3 gene through real-time fluorescence quantitative technology. The results show that after knocking out the CsSUS3 gene, the gene expression level is significantly lower than that of the control and can inhibit the formation of adventitious roots of cucumbers under waterlogging stress. The cucumber "XTMC" strain was used as the material, and the OE-CsSUS3 overexpression vector was constructed using pCAMBIA1305.4 as the vector to obtain a stable OE-CsSUS3 overexpression material. The OE-CsSUS3 transgenic material and the control material (WT) were flooded, and the phenotypic changes of the plants were observed. The changes in the gene expression of the hypocotyls of cucumbers at different times of waterlogging stress after overexpression of the gene were detected by real-time fluorescence quantitative technology. The results showed that the gene expression level after overexpression of the CsSUS3 gene was significantly higher than that of the control and could promote the formation of adventitious roots of cucumbers under waterlogging stress. Through two transgenic technologies, the application of CsSUS3 in inducing adventitious root formation in cucumbers under waterlogging stress was explored together, providing new gene resources for molecular genetic improvement of cucumber waterlogging resistance. The present invention provides a gene CsSUS3 that induces adventitious root formation in cucumbers under waterlogging stress, which has important application value in studying how to improve waterlogging resistance in cucumbers.

[0020] The invention discloses an application of a new cucumber waterlogging-resistant germplasm based on overexpression of CsSUS3. The transgenic plants obtained by overexpression of the CsSUS3 gene in the invention have a significantly higher adventitious root formation ability than the negative control plants, and a significantly stronger waterlogging resistance than the control plants. The new cucumber waterlogging-resistant germplasm created by the invention has important application value in the research of cucumber waterlogging-resistant genetic breeding.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] The present invention identifies a new gene that regulates adventitious root formation, providing a new gene resource for molecular genetic improvement of cucumber waterlogging resistance. Further, the present invention obtains a new cucumber germplasm with enhanced waterlogging resistance. The present invention obtains cucumber CsSUS3 gene-edited mutants and overexpression mutants through genetic transformation. Compared with the wild type, the gene-edited mutant has a weakened adventitious root formation ability, while the overexpression mutant has an enhanced adventitious root formation ability.

[0023] The present invention clones a gene CsSUS3 using molecular biological methods and proves its function of regulating adventitious root formation. The gene mining and waterlogging-tolerant plant acquisition method of the present invention has clear operation steps and strong replicability. The gene can be applied to other crops with mature transgenic systems to obtain waterlogging-tolerant germplasm, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To use real-time fluorescence PCR to detect the tissue specificity of CsSUS3 in different organs of cucumber and at different stages of flooding treatment. Figure 1 (a) is the expression level of CsSUS3 in different organs of cucumber, Root: root, Stem: stem, Leaf: leaf, MaleFlower: female flower, FemaleFlower: male flower, Fruit: fruit; Figure 1 (b) and Figure 1 (c) The expression levels of different tissues of cucumber seedlings treated with flooding for 3 days and 7 days, respectively. Root: root, Hypocotyl: hypocotyl, Leaf: leaf. Variance is expressed as ±SDs, and biological statistics were repeated three times.

[0025] Figure 2 The expression characteristics of CsSUS3 in cucumber hypocotyls under waterlogging stress at different times. Variance is expressed as ±SDs, and biological statistics were performed three times.

[0026] Figure 3 Analysis of the expression level of the CsSUS3 gene in cucumber after it was knocked out by CRISPR / Cas9 technology. (a) Schematic diagram of CRISPR / Cas9-CsSUS3 targeting sites and genotyping; (b) Identification of gene expression levels in gene-edited plants (cr-1, cr-2, cr-3) and control plants under waterlogging stress. WT is the control plant, cr-1, cr-2, cr-3 are gene-edited plants. Variance is expressed as ±SDs, and biological statistics were performed three times.

[0027] Figure 4The phenotypes of transgenic plants under waterlogging stress after the cucumber CsSUS3 gene was knocked out by CRISPR / Cas9 technology. (a) Statistics of adventitious roots of gene-edited plants (cr-1, cr-2, cr-3) and control plants under waterlogging stress; (b) Phenotypic observation of gene-edited plants (cr-1, cr-2, cr-3) and control plants under waterlogging stress; (c) Partial diagram of phenotypic observation. WT is the control plant, cr-1, cr-2, cr-3 are gene-edited plants. Variance is expressed as ±SDs, and biological statistics are repeated three times.

[0028] Figure 5 Expression level analysis and phenotypic observation of CsSUS3 gene in cucumber plants overexpressing CsSUS3 gene. (a) Identification of gene expression in overexpressed plants (oe-1, oe-2, oe-3) and control plants under waterlogging stress. (b) Statistical analysis of adventitious roots in overexpressed plants (oe-1, oe-2, oe-3) and control plants under waterlogging stress; (c) Phenotypic observation in overexpressed plants (oe-1, oe-2, oe-3) and control plants under waterlogging stress; (d) Partial diagram of phenotypic observation. WT is the control plant, oe-1, oe-2, oe-3 are overexpressed plants. Variance is expressed as ±SDs, and biological statistics are repeated three times.

[0029] Figure 6 Anatomical structure and morphological observation of adventitious root formation in CsSUS3 gene-edited cucumber plants.

[0030] Figure 7 This is the anatomical structure and morphological observation of adventitious root formation in cucumber plants overexpressing the CsSUS3 gene. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0032] The present invention uses the cultivated cucumber variety "Zaoer-N" for relevant experiments. This variety comes from the germplasm resource bank of the Cucumber Genetic Breeding and Molecular Innovation Team of the College of Horticulture and Landscape Architecture of Yangzhou University (Sugar enhances waterlogging-induced adventitious root formation in cucumber by promoting auxin transport and signaling. Plant Cell and Environment, 2020, 43, 1545-1557.).

[0033] The transformed receptor material was "CCMC" which is a known receptor material (A CsEIL3-CsARN6.1 module promotes waterlogging-triggered adventitious root formation in cucumber by activating the expression of CsPrx5, The Plant Journal, 2023, 114, 824-835).

[0034] pCBC-DT1T2 (Cm), pkSE402, pCAMBIA1305.4, etc. are all well-known or directly purchased vectors, among which pCAMBIA1305.4 (The exocyst subunit CsExo70B promotes both fruit length and disease resistance via regulating receptor kinase abundance at plasmamembrane in cucumber. Plant Biotechnology Journal, 2024, 22: 347–362).

[0035] The cloning method of CsSUS3 gene comprises the following steps:

[0036] (1) Cucumber tissue cDNA synthesis: Extract RNA from cucumber "Zaoer-N" hypocotyl tissue and reverse transcribe to obtain the first-strand cDNA;

[0037] (2) PCR amplification of CsSUS3 gene: Primers (CsSUS3-F: 5'-CCTGTGGATTGCCTACCTTT-3'; CsSUS3-R: 5'-TGGGTCTTCCTTGCTCTTCT-3') were designed and PCR amplification was performed using cucumber tissue cDNA as a template. The PCR amplification products were recovered and purified, and sequenced.

[0038] The nucleotide sequence encoded by the cucumber CsSUS3 gene of the present invention is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2.

[0039] Example 1

[0040] The total RNA of each tissue was extracted from different tissues (roots, stems, leaves, female flowers, male flowers, and fruits) of 45-day-old cucumber "Zaoer-N" strains and different tissues (roots, hypocotyls, and leaves) of 21-day-old cucumber "Zaoer-N" seedlings treated with flooding for 3 and 7 days using the MiniBEST Plant RNA Extraction Kit (Takara). The Prime Script RT kit was used to reverse transcribe cDNA. After quantitative gene amplification using the ChamQ SYBR qPCR Master Mix kit, the expression of the target gene CsSUS3 was detected using Real-Time PCR, with actin (CsaV3_6G041900) of cucumber as the internal reference gene. Specific quantitative primers were designed based on the gene sequence. Using the cDNA of the above-mentioned different tissues as templates, the specificity of CsSUS3 in different cucumber tissues was detected by fluorescent quantitative PCR.

[0041] Cucumber actin (CsaV3_6G041900) was used as the reference gene:

[0042] actin-F:5'-GCTGGATTCTGGTGATGGTG-3';

[0043] actin-R:5'-AGCAAGGTCCAAACGGAGAA-3';

[0044] Primer sequences designed for target gene CsSUS3:

[0045] CsSUS3-F:5'-CCTGTGGATTGCCTACCTTT-3';

[0046] CsSUS3-R:5'-TGGGTCTTCCTTGCTCTTCT-3';

[0047] like Figure 1 As shown, CsSUS3 is expressed in all tissues of cucumber "Zaoer-N", and is dominantly expressed in the root. In cucumber seedlings, CsSUS3 was highly expressed in the hypocotyl at 3 days of flooding treatment, followed by the root; at 7 days of flooding treatment, CsSUS3 was still highly expressed in the hypocotyl, followed by the adventitious root, and finally the root. This shows that the gene is dominantly expressed in the primary root system of mature plants, and is dominantly expressed in the new root formation site and new root system of cucumber seedlings treated with flooding.

[0048] Example 2

[0049] The cucumber strain "Zaoer-N" was used as the material, and the seeds were soaked and germinated at 28°C. When the buds grew 0.5 cm, they were sown in a 50-hole plug tray and cultured in an artificial climate culture room until the seedlings were 21 days old. The cucumber seedlings were cut along the holes and placed in a transparent plastic cup, and water was added to the lower end of the cotyledons of the plants for a flooding experiment. The cDNA of the hypocotyls at 0d, 1d, 2d, 3d, and 7d of flooding treatment was used as a template, and the changes of CsSUS3 with flooding time were detected by fluorescent quantitative PCR, and the method was the same as in Example 1.

[0050] Cucumber actin (CsaV3_6G041900) was used as the reference gene:

[0051] actin-F:5'-GCTGGATTCTGGTGATGGTG-3';

[0052] actin-R:5'-AGCAAGGTCCAAACGGAGAA-3';

[0053] Primer sequences designed for target gene CsSUS3:

[0054] CsSUS3-F:5'-CCTGTGGATTGCCTACCTTT-3';

[0055] CsSUS3-R:5'-TGGGTCTTCCTTGCTCTTCT-3';

[0056] like Figure 2 The results show that CsSUS3 was highly induced to express in the hypocotyls of cucumber seedlings treated with waterlogging at 0d, 1d, 2d, 3d, and 7d, and the gene expression level first increased and then decreased, with the highest expression level at 2d, further indicating that the CsSUS3 gene plays an important role in the induction of adventitious roots under waterlogging stress.

[0057] Example 3

[0058] CRISPR / Cas9 vector construction using cucumber CsSUS3 gene.

[0059] (1) Construction of CsSUS3 gene knockout vector: Design two CsSUS3 target gene primer sequences sgRNA1 and sgRNA2. The designed target gene sequences are:

[0060] sgRNA1:5'-CGATCAGTTCATGATGTTGCAGG-3';

[0061] sgRNA2:5'-CCCCATCAGCTAGTTTCCTTCGG-3';

[0062] Using pCBC-DT1T2 as a template, PrimerSTAR high-fidelity enzyme was used for PCR amplification to obtain dual-target products. The reaction system was 50 μL, including 5× PrimeSTAR Buffer 10 μL, dNTP Mixture 4 μL, sgRNA1 primer 1 μL, sgRNA2 primer 1 μL, ddH2O 32.5 μL, pCBC-DT1T2 (Cm) plasmid 1 μL, PrimerSTAR high-fidelity enzyme 0.5 μL. The reaction program was 95°C for 5 min; 95°C for 15 sec, 55°C for 15 sec, 72°C for 15 sec, 35 cycles; 72°C for 5 min.

[0063] The amplified dual-target product sgRNA1 / 2 was connected to the pkSE402 vector. The reaction system was 15 μL, including 1.2 μL pkSE402 vector, 2 μL dual-target product, 1 μL Bsal, 1 μL T4 Liguse, 1.5 μL 10×NEB T4 Buffer, and 8.3 μL ddH2O. The reaction procedure was 37°C for 5 min; 16°C for 5 min, 60°C for 5 min, and 12°C insulation. 15 μL of the ligation product was added to 50 μL of E. coli competent cells, and the two were mixed evenly and frozen on ice for 30 min, heat-shocked at 42°C for 30 sec, and then left to stand on ice for 2 min. 500 μL LB liquid culture medium was added, mixed evenly, and cultured in a 37°C constant temperature oscillator for 1 h. 100 μL of the cells were spread on Kan+LB solid culture medium, and the culture medium was cultured in a 37°C incubator for 12-16 h. Single colonies were picked for positive detection and sent to Qingke Biotechnology Co., Ltd. for sequencing. The correct single clone was selected for expansion and Plasmid MiniKit plasmid DNA extraction kit was used to extract plasmid. The plasmid was transferred into Agrobacterium competent EHA105, and competent EHA105 was placed on ice to melt, and 50 μL competent and 2 μL plasmid were taken, mixed, and placed on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C for 5 minutes, and ice for 5 minutes, and 700 μL LB liquid culture medium was added, mixed, and cultured in a 28°C shaker for 2-3 hours, 100 μL of bacteria was spread on Kan+Rif LB solid culture medium, and cultured in a 28°C constant temperature incubator for 2-3 days, and the monoclonal strain was selected for detection and preservation to obtain Agrobacterium with gene knockout vector CRISPR-CsSUS3 for subsequent genetic transformation.

[0064] (2) Acquisition and positive detection of CRISPR-CsSUS3 transgenic plants

[0065] Transgenic plants were obtained by Agrobacterium-mediated genetic transformation, and the transformation receptor material was "CCMC". The basic method was as follows: "CCMC" seeds were soaked in distilled water and placed in a 55°C water bath for 30 minutes, the seed coat was peeled off, rinsed with sterile water 3 to 4 times, soaked in 75% alcohol for 30 seconds, soaked in 2% sodium hypochlorite solution for 10 minutes, and rinsed with sterilized ddH2O 4 to 5 times. The seeds were sown on SGM culture medium, wrapped in tin foil and placed in a 28°C constant temperature incubator for 36 hours; Agrobacterium carrying the CRISPR-CsSUS3 gene knockout vector was cultured in LB solid culture medium containing Kan and Rif antibiotics for 12 hours, and the bacteria were added to 40mL IM liquid culture medium, mixed and cultured, and the OD was measured. 600 , so that OD 600 =0.2, and placed in a constant temperature incubator at 28°C; skim off the buds, cut off 1 / 3 of the seeds and divide them into two, soak them in IM liquid culture medium, and after the seeds are processed, transfer them together to IM liquid culture medium containing bacterial solution, use KQ5200DE CNC ultrasonic cleaner to ultrasonicate for 20sec, vacuum infiltrate for 90sec (twice in total), and finally place the explants on IM solid culture medium with tweezers, wrap them with tin foil and place them in a constant temperature incubator at 25°C for 3d; transfer the explants to SRM culture medium and culture them in an environment of 26°C with 16h light / 8h darkness for 2-3 weeks; observe and select explants with green fluorescence under a fluorescence microscope, cut off the putative transformants with a length of about 1cm, and place the remaining parts in RM culture medium to induce rooting, transfer the rooted tissue culture seedlings to the substrate for acclimatization and culture, and finally move them to the greenhouse for cultivation.

[0066] CRISPR-CsSUS3 transgenic positive plants were screened by PCR, and the gene editing type of the knockout plants was further determined by sequencing. Specific primers were designed near the CsSUS3 target gene sequence to detect the gene knockout fragment sequence. The primer sequences are as follows:

[0067] CRISPR-CsSUS3-F:5'-CAACCGTATTCATAGCC-3';

[0068] CRISPR-CsSUS3-R:5'-CTGGTTGGGTTCTGATA-3'.

[0069] Using the leaf DNA of CRISPR-CsSUS3 transgenic plants as a template, PrimerSTAR high-fidelity enzyme was used for PCR amplification and sequencing to obtain the editing site. The reaction system was 50 μL, including 5× PrimeSTAR Buffer 10 μL, dNTPMixture 4 μL, CRISPR-CsSUS3-F primer 1 μL, CRISPR-CsSUS3-R primer 1 μL, ddH2O 32.5 μL, DNA template 1 μL, PrimerSTAR high-fidelity enzyme 0.5 μL. The reaction program was 95℃5min; 95℃15sec, 55℃15sec, 72℃15sec, 35 cycles; 72℃5min. The results are as follows Figure 3 (a) There are three types of editing, and sequencing analysis revealed that cr-1, cr-2 and cr-3 had base deletions of 51bp, 3bp and 25bp, respectively.

[0070] Example 4

[0071] Detection of gene expression and phenotypic observation of CRISPR-CsSUS3 transgenic cucumber plants

[0072] (1) Detection of gene expression in CRISPR-CsSUS3 transgenic cucumber plants

[0073] After the control plants (WT) and CRISPR-CsSUS3 transgenic plants (cr-1, cr-2, cr-3) were flooded for 12h, 24h, 48h, and 72h, the hypocotyls were removed to extract RNA for reverse transcription, and the expression levels of CsSUS3 in the control plants and the three types of edited transgenic plants were detected by real-time fluorescence PCR (CsSUS3-qRT-F: 5'-CCTGTGGATTGCCTACCTTT-3', CsSUS3-qRT-R: 5'-TGGGTCTTCCTTGCTCTTCT-3'). Figure 3 As shown in (b), with the increase of flooding time, the expression level of CsSUS3 gene gradually increased, and the expression levels of transgenic plants of the three editing types were significantly lower than those of the control plants at each time point.

[0074] (2) Phenotypic observation of CRISPR-CsSUS3 transgenic cucumber plants

[0075] In order to confirm the function of CsSUS3 gene in inducing adventitious roots, the control plants (WT) and CRISPR-CsSUS3 transgenic plants (cr-1, cr-2, cr-3) were flooded for 12h, 24h, 48h, and 72h and then observed. Figure 4As shown in (a)(b), the CRISPR-CsSUS3 transgenic plants grew well, and the number of adventitious roots of the three edited types of transgenic plants was significantly lower than that of the control plants at each time point.

[0076] Example 5

[0077] The pCAMBIA1305.4 overexpression vector was constructed using the cucumber CsSUS3 gene.

[0078] Primers for vector construction were designed based on the CDS region of the CsSUS3 gene (excluding the stop codon). The designed primer sequences are:

[0079] OE-CsSUS3-F:gagaacacgggggacggatccATGGCAGAACGAGTTCTCAACC;

[0080] OE-CsSUS3-R:atggtctttgtagtcggatccCTCATCCACAGCCGGTGGC.

[0081] First, the CsSUS3 gene was used as a template and PrimerSTAR high-fidelity enzyme was used for PCR amplification to obtain the product. The reaction system was 50 μL, including 10 μL of 5× PrimeSTAR Buffer, 4 μL of dNTP Mixture, 1 μL of OE-CsSUS3-F primer, 1 μL of OE-CsSUS3-R primer, 32.5 μL of ddH2O, 1 μL of template, and 0.5 μL of PrimerSTAR high-fidelity enzyme. The reaction procedure was 95°C for 5 min; 95°C for 15 sec, 55°C for 15 sec, and 72°C for 15 sec, for 35 cycles; 72°C for 5 min. The homologous recombination kit ( ⅡOne Step Cloning Kit) was used to recombinantly connect the purified PCR product with the linear vector. 2μL of 5×CE II Buffer, 1μL of purified PCR product, 1μL of pCAMBIA1305.4 vector fragment, 1μL of recombinase Exnase II, and 5μL of sterile water were added to 10μL of the recombination connection system. After mixing, the mixture was connected at 37°C for 30min. The heat shock method was used to transform Escherichia coli DH5a (Quanshijin, Beijing), and a single clone was picked. PCR amplification detection and sequencing were performed. The correct single clone was selected for propagation and plasmid was extracted to obtain the overexpression vector OE-CsSUS3. The plasmid was transferred into Agrobacterium competent EHA105, and the single clone strain was picked for detection and preservation to obtain the Agrobacterium overexpressing the vector OE-CsSUS3, which was used for subsequent genetic transformation (the method was the same as that in Example 3 (1)).

[0082] (2) Obtaining and positive detection of OE-CsSUS3 transgenic plants

[0083] The transgenic plants were obtained by Agrobacterium-mediated genetic transformation, with the transformation recipient material being "CCMC" and the method being the same as in Example 3(2).

[0084] The overexpressed transgenic plants were positively detected, and the OE-CsSUS3 transgenic positive plants were screened by PCR. The primer sequences were as follows:

[0085] OE-CsSUS3-F:5'-GTCACTTTATTGTGAAGATAGTGGA-3';

[0086] OE-CsSUS3-R:5'-atggtctttgtagtcggatCCCTCATCCACAGCCGGTGGC-3'.

[0087] Using the leaf DNA of the overexpressed transgenic plant as a template, PCR amplification was performed using Taq PCR MasterMixⅡ. The reaction system was 20μL, including 2×Taq PCR MasterMixⅡ10μL, OE-CsSUS3-F primer 1μL, OE-CsSUS3-R primer 1μL, ddH2O 7μL, and DNA template 1μL. The reaction procedure was 98℃5min; 98℃30sec, 55℃30sec, 72℃3min, 35 cycles; 72℃10min; positive strains of OE-CsSUS3 transgenic plants (oe-1, oe-2, oe-3) were screened out after agarose gel electrophoresis.

[0088] Example 6

[0089] Detection of gene expression and phenotypic observation of OE-CsSUS3 transgenic cucumber plants

[0090] (1) Detection of gene expression in OE-CsSUS3 overexpressing transgenic cucumber plants

[0091] After waterlogging for 0h, 12h, 24h, 48h, 72h and 168h, the hypocotyls of the control plants (WT) and the OE-CsSUS3 transgenic plants (oe-1, oe-2, oe-3) were removed to extract RNA for reverse transcription, and the expression levels of CsSUS3 in the control plants and overexpressing transgenic plants were detected by real-time fluorescence PCR (CsSUS3-qRT-F: 5'-CCTGTGGATTGCCTACCTTT-3', CsSUS3-qRT-R: 5'-TGGGTCTTCCTTGCTCTTCT-3'). Figure 5As shown in (a), with the increase of flooding time, the expression level of CsSUS3 gene gradually increased, and the expression level of overexpressing transgenic plants was significantly higher than that of control plants at each time point.

[0092] (2) Phenotypic observation of OE-CsSUS3 transgenic cucumber plants

[0093] In order to confirm the function of CsSUS3 gene in inducing adventitious roots, the control plants (WT) and OE-CsSUS3 transgenic plants (oe-1, oe-2, oe-3) were flooded for 0h, 12h, 24h, 48h, 72h and 168h and observed. Figure 5 As shown in (b) and (c), the OE-CsSUS3 overexpressing transgenic plants grew well, and the number of adventitious roots of the overexpressing transgenic plants at each time point was significantly higher than that of the control plants. The number, area and growth state of new leaves of the overexpressing plants flooded for 168 hours were better than those of the control, indicating that their waterlogging tolerance was stronger than that of the control.

[0094] Example 7

[0095] Observation on the anatomical structure of hypocotyls of CRISPR-CsSUS3 transgenic cucumber plants

[0096] In order to confirm the formation process of adventitious roots in control plants and CRISPR-CsSUS3 transgenic plants (cr-1, cr-2, cr-3) after flooding, their hypocotyls were cross-sectioned and observed. Fresh hypocotyl samples were taken from plants treated with flooding for 12h, 24h, and 48h, and the cross-sections of the hypocotyls in the flooded parts were observed using a manual small plant tissue slicer MTH-1, 0.1M toluidine blue stain, and a stereo microscope. CRISPR-CsSUS3 transgenic plants had almost no adventitious roots at 12h and 24h of flooding, and adventitious root primordia began to differentiate from the cambium of the vascular bundles 48h after flooding; while in control plants, adventitious root primordia began to differentiate from the cambium of the vascular bundles 24h after flooding, and adventitious root primordia broke through the epidermis 48h after flooding to form adventitious roots visible to the naked eye. It can be seen that the formation of adventitious roots in CRISPR-CsSUS3 transgenic plants is significantly slower than that in control plants. Figure 6 shown.

[0097] Example 8

[0098] Observation on the anatomical structure of hypocotyls of OE-CsSUS3 transgenic cucumber plants

[0099] In order to confirm the formation process of adventitious roots in control plants and OE-CsSUS3 transgenic plants (oe-1, oe-2, oe-3) under waterlogging stress, their hypocotyls were cross-sectioned and observed. Fresh hypocotyl samples were taken from plants treated with waterlogging for 12h, 24h, and 48h, and the cross-sections of the hypocotyls in the waterlogged parts were observed using a manual small plant tissue slicer MTH-1, 0.1M toluidine blue stain, and a stereo microscope. There was almost no adventitious root formation in the control plants and OE-CsSUS3 transgenic plants after 12h of waterlogging. The adventitious root primordium of the OE-CsSUS3 transgenic plants began to differentiate from the cambium in the vascular bundle and even broke through the epidermis after 24h of waterlogging; adventitious roots visible to the naked eye were formed after 48h of waterlogging. In contrast, in the control plants, adventitious root primordia began to differentiate from the cambium of the vascular bundle 24 hours after flooding, but did not break through the epidermis until 48 hours after flooding. This shows that the formation of adventitious roots in the OE-CsSUS3 transgenic plants was significantly faster than that in the control plants. Figure 7 shown.

Claims

1. A gene that regulates waterlogging stress to induce adventitious root formation in cucumber CsSUS3 CRISPR-based gene knockout vector CsSUS3 , characterized in that, The gene knockout vector CRISPR- CsSUS3 The construction method is: design CsSUS3 The target gene sequences sgRNA1 and sgRNA2, sgRNA1: 5'-CGATCAGTTCATGATGTTGCAGG-3'; sgRNA2: 5'-CCCCATCAGCTAGTTTCCTTCGG-3'; pCBC-DT1T2 (Cm) was used as a template to amplify the double-stranded sgRNA1 / 2, and the formed double-stranded sgRNA1 / 2 was connected to the pkSE402 vector, and the plasmid was extracted through Escherichia coli transformation and Agrobacterium transformation, and finally the gene knockout vector CRISPR-CsSUS3 was obtained; the gene CsSUS3 The nucleotide sequence is shown in SEQ ID NO.

1.

2. A gene that regulates waterlogging stress-induced adventitious root formation in cucumber CsSUS3 Overexpression vector OE- CsSUS3 , characterized in that, The overexpression vector OE- CsSUS3 The construction method is: CsSUS3 Primers for designing vector construction in the CDS region of the gene: OE- CsSUS3 -F: gagaacacgggggacggatccATGGCAGAACGAGTTCTCAACC; OE- CsSUS3 -R: atggtctttgtagtcggatccCTCATCCACAGCCGGTGGC; The gene was amplified, digested and connected to the pCAMBIA1305.4 vector, and then transformed with E. coli and Agrobacterium tumefaciens to extract the plasmid, and finally the overexpression vector OE- CsSUS3 The gene CsSUS3 The nucleotide sequence is shown in SEQ ID NO.

1.

3. A gene that regulates waterlogging-induced adventitious root formation in cucumber CsSUS3 Application of the gene in inducing adventitious root formation in cucumber under waterlogging stress CsSUS3 The nucleotide sequence is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that: Editing cucumbers via CRISPR / Cas9 CsSUS3 gene, which reduces its ability to induce adventitious root formation under waterlogging stress; by overexpressing cucumber CsSUS3 Gene, which increases its ability to induce adventitious root formation under waterlogging stress.

5. A gene that regulates waterlogging-induced adventitious root formation in cucumber CsSUS3 Application of the gene in cultivating waterlogging-resistant cucumber germplasm CsSUS3 The nucleotide sequence is shown in SEQ ID NO.1.

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