Gene RBOHB and application thereof in regulating and controlling formation of cucumber adventitious roots induced by waterlogging stress

Abstract

The invention discloses a gene RBOHB and application of the gene RBOHB in regulating and controlling waterlogging stress to induce cucumber adventitious root formation, a novel gene RBOHB for inducing cucumber adventitious root formation is identified, the adventitious root formation capability of an edited mutant of the gene RBOHB is weakened, the extension of the adventitious root is obviously inhibited, and the adventitious root formation capability of an overexpressed mutant is enhanced. The gene RBOHB disclosed by the invention is used for regulating and controlling the waterlogging tolerance of cucumbers, and has important application value in screening and improving waterlogging-tolerant germplasm resources of the cucumbers and breeding waterlogging-tolerant varieties of the cucumbers. The cucumber is subjected to transgenic genetic transformation through a gene editing technology and a gene overexpression technology, the cucumber waterlogging tolerance regulation gene RBOHB and application of the cucumber waterlogging tolerance regulation gene RBOHB in waterlogging tolerance cucumber varieties are provided, and the cucumber waterlogging tolerance regulation gene RBOHB has important application value in the field of breeding of resistant cucumbers.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and molecular biology, and in particular to a gene RBOHB and an application thereof in regulating waterlogging stress to induce adventitious root formation in cucumber. Background Art

[0002] Cucumber (Cucumissativus L.; 2n=2X=14) is a vine crop of the Cucurbitaceae family and one of the main vegetable crops in China. According to statistics from the Food and Agriculture Organization of the United Nations, China's cucumber planting area in 2022 was about 1.311 million hectares, and the output reached 77.301 million tons, accounting for 60.32% and 81.62% of the world's total, respectively, both ranking first in the world (2022FAO data). Cucumber is a shallow root distribution plant, extremely sensitive to water, requiring a large amount of water during planting but not tolerant to flooding. Due to seasonal rainfall, poor drainage systems, and rising groundwater levels in the middle and lower reaches of the Yangtze River and its southern regions in my country, cucumbers in the summer and autumn are facing long-term waterlogging stress, resulting in production losses and reduced cucumber quality. Therefore, discovering genes that regulate the growth of adventitious roots of cucumbers under waterlogging stress is the basis for breeding cucumber waterlogging-resistant germplasm resources.

[0003] Rice OsRBOHA is the first plant RBOH gene isolated in the world (Groom et al., 1996). Since then, the gene has been isolated and identified in plants such as Arabidopsis and tobacco. There are 9 and 10 RBOH family genes in rice and Arabidopsis, respectively, and different genes have different biological functions (Xia et al., 2015). For example, Arabidopsis At-RBOHB regulates seed maturation (Müller et al., 2009), At-RBOHC regulates root hair development (Foreman et al., 2003), At-RBOHE regulates cell programmed death in the tapetum (Foreman et al., 2003), and At-RBOHD and At-RBOHF regulate immune responses (Torres et al., 2002, 2005). Studies on cucumber have found that RBOH genes are involved in regulating stress resistance after grafting (Li et al., 2014; Niu et al., 2018), brassinolide-induced stress response (Xia et al., 2009) and cadmium stress response (Jakubowska et al., 2015). However, to date, there have been no reports on the involvement of RBOH family genes in cucumber in adventitious root formation induced by waterlogging stress. Summary of the invention

[0004] Purpose of the invention: In view of the shortcomings of the prior art, the present invention provides a gene RBOHB that regulates waterlogging stress-induced adventitious root formation in cucumbers. The present invention identifies a new cucumber adventitious root formation regulating gene RBOHB, whose edited mutant has weakened adventitious root formation ability, and the adventitious root formation ability of the overexpressed material is enhanced. The gene RBOHB 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 RBOHB for regulating waterlogging stress and inducing adventitious root formation in cucumber.

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

[0007] Among them, the primer pair used to amplify the gene RBOHB is

[0008] RBOHB-F1:5'-AGCTAAATGGCCAACAACCC-3';

[0009] RBOHB-R1:5'-GTGCCTCCCGCATGTAAAAGG-3';

[0010] RBOHB-F2:5'-AGTACAACCCCATCAAGCGG-3';

[0011] RBOHB-R2:5'-TTCAACAAAGATTTGTATGCTGT-3'.

[0012] The primer pair used to amplify the CDS sequence of the gene RBOHB is

[0013] RBOHB-F3:5'-TCATGACCAAAAGCGTCGGA-3';

[0014] RBOHB-R4:5'-CAACACTCGACTGTCCGTGA-3'.

[0015] The amino acid sequence of the protein expressed by the gene RBOHB for regulating waterlogging stress-induced adventitious root formation in cucumber is shown in SEQ ID NO.3.

[0016] The present invention discloses a gene knockout vector CRISPR-RBOHB or an overexpression vector OE-RBOHB based on the gene RBOHB for regulating waterlogging stress to induce adventitious root formation in cucumber.

[0017] Among them, the gene knockout vector CRISPR-RBOHB construction method is to design the target gene sequences sgRNA1 and sgRNA2 of RBOHB, sgRNA1: 5'-TCCTCCTCTGATGTTCTGAACGG-3'; sgRNA2: 5'-CGATTCATGACCAAAAGCGTCGG-3', use pCBC-DT1T2 (Cm) as a template to amplify the double targets, connect the formed double-stranded sgRNA1 / 2 to the pkSE402 vector, transform Escherichia coli and Agrobacterium tumefaciens, extract the plasmid, and finally obtain the gene knockout vector CRISPR-RBOHB.

[0018] The overexpression vector OE-RBOHB is constructed by designing the primer sequence of RBOHB:

[0019] OE-RBOHB-F:

[0020] 5'-gagaacacgggggacggatccATGGAGAGTCAAGAAAACAAACCTATG-3';

[0021] OE-RBOHB-R:

[0022] 5'-atggtctttgtagtcggatccTATTTGCTGAAGCACTCAATGCA-3'

[0023] The CDS gene was amplified as a template, homologously recombined into the pCAMBIA1305.4 vector, and then transformed and the plasmid was extracted to finally obtain the overexpression vector OE-RBOHB.

[0024] The host cell of the present invention uses Agrobacterium as the starting strain, and contains the gene knockout vector CRISPR-RBOHB or the overexpression vector OE-RBOHB.

[0025] The invention discloses an application of the gene RBOHB for regulating waterlogging stress-induced adventitious root formation in cucumbers in inducing adventitious root formation in cucumbers under waterlogging stress.

[0026] Furthermore, the cucumber RBOHB gene was edited by CRISPR / Cas9 to reduce its ability to induce adventitious root formation under waterlogging stress; or the RBOHB gene was overexpressed to increase its ability to induce adventitious root formation under waterlogging stress.

[0027] The protein expressed by the gene RBOHB, the gene knockout vector CRISPR-RBOHB or the overexpression vector OE-RBOHB or the host cell of the present invention are used in inducing adventitious root formation of cucumber under waterlogging stress.

[0028] The invention discloses an application of the gene RBOHB for regulating waterlogging stress-induced adventitious root formation in cucumber, the protein expressed by the gene RBOHB, the gene knockout vector CRISPR-RBOHB or the overexpression vector OE-RBOHB or the host cell in cultivating waterlogging-resistant cucumber germplasm.

[0029] The present invention uses cucumber "CCMC" strain as material, constructs dual targets through CRISPR / Cas9 technology, obtains stable CRISPR-RBOHB gene editing material, floods the CRISPR-RBOHB transgenic material and a control material (WT), observes plant phenotypic changes, and detects changes in gene expression levels of hypocotyls of cucumbers at different times under waterlogging stress after knocking out the RBOHB gene through real-time fluorescence quantitative technology. The results show that the gene expression level after knocking out the RBOHB gene is significantly lower than that of the control, indicating that knocking out the gene can inhibit the formation of adventitious roots of cucumbers under waterlogging stress. The cucumber "CCMC" strain was used as the material, and the OE-RBOHB overexpression vector was constructed using pCAMBIA1305.4 as the vector to obtain a stable OE-RBOHB overexpression material. The OE-RBOHB transgenic material and the control material (WT) were flooded, and the phenotypic changes of the plants were observed. The changes in the gene expression levels 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 RBOHB 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 RBOHB in inducing adventitious root formation of 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 RBOHB for inducing adventitious root formation under waterlogging stress in cucumbers, which has important application value in studying how to improve waterlogging resistance in cucumbers.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0031] The present invention identifies a new gene that regulates the formation of adventitious roots under waterlogging stress, providing a new gene resource for the molecular genetic improvement of waterlogging resistance in cucumbers. Further, the present invention obtains a new cucumber germplasm with enhanced waterlogging resistance. The present invention obtains cucumber RBOHB gene editing mutants and overexpression mutants through genetic transformation. Compared with the control, the gene editing mutant has a weakened adventitious root formation ability, while the overexpression mutant has an enhanced adventitious root formation ability.

[0032] The present invention clones a gene RBOHB 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

[0033] Figure 1 Real-time fluorescence PCR was used to detect the expression of RBOHB in cucumber at different stages of flooding treatment. (a) Expression in hypocotyls; (b) Expression in different organs. Variance is expressed as ±SDs, and three biological statistics were performed. p≤0.001, **

[0034] Figure 2 Analysis of the expression level of the RBOHB gene in cucumber after it was knocked out by CRISPR / Cas9 technology. (a) Schematic diagram of CRISPR / Cas9-RBOHB targeting sites and genotyping; (b) Identification of gene expression levels in gene-edited plants (Csrbohb#1, Csrbohb#2) and control plants under waterlogging stress. WT is the control plant, Csrbohb#1 and Csrbohb#2 are gene-edited plants. (c) Identification of gene expression levels in overexpression plants (OE#1, OE#2) and control plants under waterlogging stress. WT is the control plant, OE#1 and OE#2 are overexpression plants. Variance is expressed as ±SDs, and biological statistics were repeated three times.

[0035] Figure 3 The phenotypes of transgenic plants under waterlogging stress after the RBOHB gene of cucumber was knocked out by CRISPR / Cas9 technology. (a) Statistics of the number and length of adventitious roots of gene-edited plants (Csrbohb#1, Csrbohb#2) and control plants under waterlogging stress; (b) Phenotypic observation of gene-edited plants (Csrbohb#1, Csrbohb#2) and control plants under waterlogging stress; (c) Partial diagram of phenotypic observation. WT is the control plant, Csrbohb#1 and Csrbohb#2 are gene-edited plants. Variance is expressed as ±SDs, 3 biological statistics; p≤0.01, *; p≤0.001, **.

[0036] Figure 4The phenotypes of cucumber RBOHB gene overexpression plants under waterlogging stress obtained by transgenic technology. (a) Statistics of adventitious root number and length of overexpression plants (OE#1, OE#2) and control plants under waterlogging stress; (b) Phenotypic observation of overexpression plants (OE#1, OE#2) and control plants under waterlogging stress; (c) Partial diagram of phenotypic observation. WT is the control plant, OE#1 and OE#2 are overexpression plants. Variance is expressed as ±SDs, 3 times biological statistics; p≤0.01, *; p≤0.001, **.

[0037] Figure 5 Anatomical structure and morphological observation of adventitious root formation in RBOHB gene-edited cucumber plants and RBOHB gene-overexpressing cucumber plants. (a) Partial view of hypocotyl cross section observation. WT is the control plant, Csrbohb#1 and Csrbohb#2 are gene-edited plants. (b) Partial view of hypocotyl cross section observation. WT is the control plant, OE#1 and OE#2 are overexpressed plants. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0039] The present invention uses the cultivated cucumber variety "Zaoer-N" for relevant experiments. The 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.).

[0040] 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).

[0041] The cloning method of RBOHB gene comprises the following steps:

[0042] (1) Cucumber tissue cDNA synthesis: Extract RNA from cucumber "Zaoer-N" hypocotyl tissue and reverse transcribe to obtain the first-strand cDNA;

[0043] (2) PCR amplification of RBOHB gene and its CDS sequence:

[0044] Primers (RBOHB-F1: 5'-AGCTAAATGGCCAACAACCC-3'; RBOHB-R1: 5'-GTGCTCCCGCATGTAAAAGG-3'; RBOHB-F2: 5'-AGTACAACCCCATCAAGCGG-3'; RBOHB-R2: 5'-TTCAACAAAGATTTGTATGCTGT-3') were designed, and cucumber tissue DNA was used as a template. PCR amplification of the RBOHB gene product was performed with primers RBOHB-F1 / R1 and RBOHB-F2 / R2, respectively. PCR amplification products 1 and 2 were recovered and purified, and sequenced. The full-length RBOHB gene was obtained by truncating at the Kpn1 restriction site and splicing.

[0045] Primers (RBOHB-F3: 5'-GGCTTCAGCGGTCCATTA-3'; RBOHB-R3: 5'-GAACAACTCACCGGCAAA-3') were designed, and PCR was performed to amplify the CDS of the RBOHB gene using cucumber tissue cDNA as a template. The PCR amplification product was recovered, purified, and sequenced.

[0046] The nucleotide sequence encoded by the cucumber RBOHB gene of the present invention is shown as SEQ ID NO.1, the CDS sequence thereof is shown as SEQ ID NO.2, and the amino acid sequence encoded by the gene is shown as SEQ ID NO.3.

[0047] Example 1

[0048] The cucumber strain "Zaoer-N" was used as the material. The seeds were soaked and germinated at 28°C. When the buds grew 0.5 cm, they were sown in 50-hole plug trays and cultured in an artificial climate culture room until the seedlings were 21 days old. The cucumber seedlings were cut off from the holes and placed in a transparent plastic cup. Water was added to the lower end of the cotyledons of the plants for flooding experiments. The hypocotyls of the cucumber "Zaoer-N" strain at different time points of flooding treatment at 21 days old were used as samples. The total RNA of each tissue was extracted using the MiniBEST Plant RNA Extraction Kit (Takara), and the Prime Script RT kit was reverse transcribed to obtain cDNA. After quantitative gene amplification using the ChamQ SYBR qPCR Master Mix kit, the expression of the target gene RBOHB was detected by Real-Time PCR, and the cucumber actin (CsaV3_6G041900) was used as the internal reference gene. Specific quantitative primers were designed according to the gene sequence. Using the cDNA of hypocotyls treated with flooding for 0, 4, 8, 12, 24, 48 and 168 h as template, fluorescence quantitative PCR was used to detect the changes of RBOHB with flooding time.

[0049] Cucumber actin (CsaV3_6G041900) was used as the reference gene:

[0050] actin-F:5'-GCTGGATTCTGGTGATGGTG-3';

[0051] actin-R:5'-AGCAAGGTCCAAACGGAGAA-3'.

[0052] Primer sequences designed for target gene RBOHB:

[0053] RBOHB-F:5'-TCATGACCAAAAGCGTCGGA-3';

[0054] RBOHB-R:5'-CAACACTCGACTGTCCGTGA-3'.

[0055] like Figure 1 (a) shows that RBOHB was induced to express in the hypocotyls of cucumber seedlings treated with waterlogging for 0, 4, 8, 12, 24, 48 and 168 h, and the gene expression level increased with time, with the most obvious increase after 24 h, further indicating that the RBOHB gene plays an important role in the induction of adventitious roots under waterlogging stress.

[0056] 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 RBOHB 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 and organs as templates, the specificity of the gene RBOHB in different cucumber tissues was detected by fluorescence quantitative PCR.

[0057] Cucumber actin (CsaV3_6G041900) was used as the reference gene:

[0058] actin-F:5'-GCTGGATTCTGGTGATGGTG-3';

[0059] actin-R:5'-AGCAAGGTCCAAACGGAGAA-3';

[0060] Primer sequences designed for target gene RBOHB:

[0061] RBOHB-F:5'-TCATGACCAAAAGCGTCGGA-3';

[0062] RBOHB-R:5'-CAACACTCGACTGTCCGTGA-3'.

[0063] like Figure 1 (b) RBOHB is expressed in all tissues and organs of cucumber "Zaoer-N", and is dominantly expressed in roots and hypocotyls. This indicates 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. Example 2

[0064] CRISPR / Cas9 vector construction using cucumber RBOHB gene.

[0065] (1) Construction of RBOHB gene knockout vector: Design two RBOHB target gene primer sequences sgRNA1 and sgRNA2. The designed target gene sequences are:

[0066] sgRNA1:5'-TCCTCCTCTGATGTTCTGAACGG-3';

[0067] sgRNA2:5'-CGATTCATGACCAAAAGCGTCGG-3'.

[0068] 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 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.

[0069] 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 was extracted using Plasmid MiniKit plasmid DNA extraction kit. The plasmid was transferred into Agrobacterium competent EHA105, and competent EHA105 was thawed on ice. 50 μL competent and 2 μL plasmid were aspirated, mixed, and placed on ice for 5 min, liquid nitrogen for 5 min, 37°C for 5 min, and ice for 5 min, and 700 μL LB liquid culture medium was added. After mixing, the mixture was cultured in a shaker at 28°C for 2-3 h, 100 μL of the bacteria was spread on Kan+Rif LB solid culture medium, and cultured in a constant temperature incubator at 28°C for 2-3 days, and the monoclonal strain was selected for detection and preservation for subsequent genetic transformation, and the plasmid was extracted to obtain the gene knockout vector CRISPR-RBOHB.

[0070] (2) Acquisition and positive detection of CRISPR-RBOHB transgenic plants

[0071] 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 45-55°C water bath for 30 minutes, the seed coat was peeled off, rinsed with sterile water 3-4 times, soaked in 75% alcohol for 30 seconds, soaked in 2% sodium hypochlorite solution for 12 minutes, and rinsed with sterilized ddH2O 4-5 times. The seeds were sown on SGM medium, wrapped in tin foil and placed in a 28°C constant temperature incubator for 36 hours; the bacterial solution carrying the CRISPR-RBOHB gene was cultured in LB solid medium containing Kan and Rif antibiotics for 12 hours, and the bacteria were added to 40mL IM liquid medium, mixed and measured OD 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.

[0072] CRISPR-RBOHB 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 RBOHB target gene sequence to detect the gene knockout fragment sequence.

[0073] CRISPR-RBOHB-F:5'-GGCTTCAGCGGTCCATTA-3';

[0074] CRISPR-RBOHB-R:5'-GAACAACTCACCGGCAAA-3'.

[0075] Using the leaf DNA of CRISPR-RBOHB 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-RBOHB-F primer 1 μL, CRISPR-RBOHB-R primer 1 μL, ddH2O 32.5 μL, DNA template 1 μL, PrimerSTAR high-fidelity enzyme 0.5 μL. The reaction procedure was 95℃5min; 95℃15sec, 55℃15sec, 72℃15sec, 34 cycles; 72℃5min. The results are as follows Figure 2 (a) shows that there are two types of editing, and sequencing analysis found that Csrbohb#1 and Csrbohb#2 had 10bp and 3bp base deletions, respectively. Transgenic plants Csrbohb#1 and Csrbohb#2 were screened and obtained.

[0076] Example 3

[0077] Detection of gene expression and phenotypic observation of CRISPR-RBOHB transgenic cucumber plants

[0078] (1) Detection of gene expression in CRISPR-RBOHB transgenic cucumber plants

[0079] The control plants (WT) and CRISPR-RBOHB transgenic plants (Csrbohb#1, Csrbohb#2) were cultured for 21 days until the two-leaf and one-heart stage and then flooded. The hypocotyls were removed 12, 24, and 48 hours after treatment to extract RNA for reverse transcription. The expression levels of RBOHB in the control plants and the three types of edited transgenic plants were detected by real-time fluorescence PCR (RBOHB-qRT-F: 5'-TCATGACCAAAAGCGTCGGA-3'; RBOHB-qRT-R: 5'-CAACACTCGACTGTCCGTGA-3'). Figure 2 As shown in (b), with the increase of flooding time, the expression level of RBOHB gene gradually increased, and the expression levels of transgenic plants of the two editing types were significantly lower than those of the control plants at each time point.

[0080] (2) Phenotypic observation of CRISPR-RBOHB transgenic cucumber plants

[0081] To confirm the function of RBOHB gene in inducing adventitious roots, control plants (WT) and CRISPR-RBOHB transgenic plants (Csrbohb#1, Csrbohb#2) were cultured for 21 days until the two-leaf and one-heart stage and then flooded. The plants were observed 48h and 168h after treatment. Figure 3As shown, the CRISPR-RBOHB transgenic plants were in good growth condition. The adventitious root lengths of the three edited types of transgenic plants were significantly lower than those of the control plants at each time point, and there was no significant difference in the number of adventitious roots.

[0082] Example 4

[0083] The pCAMBIA1305.4 overexpression vector was constructed using the cucumber RBOHB gene.

[0084] Primers for vector construction were designed based on the CDS region of the RBOHB gene (excluding the stop codon). The designed primer sequences are:

[0085] OE-RBOHB-F:

[0086] 5'-gagaacacgggggacggatccATGGAGAGTCAAGAAAACAAACCTATG-3';

[0087] OE-RBOHB-R:

[0088] 5'-atggtctttgtagtcggatccTATTTGCTGAAGCACTCAATGCA-3'.

[0089] First, the CDS sequence of the RBOHB 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-RBOHB-F primer, 1 μL of OE-RBOHB-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. 5×CE II Buffer 2μL, purified PCR product 1μL, BamHⅠ single enzyme digested pCAMBIA1305.4 vector fragment 1μL, recombinase Exnase II 1μL, and 5μL 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 expansion and plasmid extraction to obtain the overexpression vector OE-RBOHB. The plasmid was transferred into Agrobacterium competent EHA105, and the single clone strain was picked for detection and preservation to obtain Agrobacterium overexpressing the vector OE-CsSUS3, which was used for subsequent genetic transformation (the method was the same as in Example 2 (1)).

[0090] (2) Obtaining and positive detection of OE-RBOHB transgenic plants

[0091] 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 2(2).

[0092] The overexpressed transgenic plants were positively detected. OE-CsRBOHB transgenic positive plants were screened by PCR. The detection primer sequences were:

[0093] OE-RBOHB-F:

[0094] 5′-TCCGACATCGATCTCCTGGT-3′;

[0095] OE-RBOHB-R:

[0096] 5′-CAGGGTGAGGACCACATTCC-3′.

[0097] 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-RBOHB-F primer 1μL, OE-RBOHB-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 OE-Cs RBOHB transgenic plants (OE#1, OE#2) were screened out after agarose gel electrophoresis.

[0098] Example 5

[0099] (1) Detection of gene expression in OE-RBOHB overexpressing cucumber plants

[0100] The control plants (WT) and OE-RBOHB overexpressing plants (OE#1, OE#2) were cultured for 21 days until the two-leaf one-heart stage and then flooded. The hypocotyls were removed 12, 24, and 48 hours after treatment to extract RNA for reverse transcription. The expression levels of RBOHB in the control plants and the two edited transgenic plants were detected by real-time fluorescence PCR (RBOHB-qRT-F: 5'-TCATGACCAAAAGCGTCGGA-3'; RBOHB-qRT-R: 5'-CAACACTCGACTGTCCGTGA-3'). Figure 2 As shown in (c), with the increase of flooding time, the expression level of RBOHB gene gradually increased, and the expression levels of transgenic plants of the two editing types were significantly higher than those of the control plants at each time point.

[0101] (2) Phenotypic observation of cucumber plants overexpressing OE-RBOHB

[0102] To confirm the function of RBOHB gene in inducing adventitious roots, the control plants (WT) and OE-RBOHB overexpressing plants (OE#1, OE#2) were cultured for 21 days until the two-leaf and one-heart stage and then flooded. The plants were observed 48h and 168h after treatment. Figure 4 As shown, the OE-RBOHB overexpressing plants grew well, the adventitious root lengths of the two editing types of transgenic plants were higher than those of the control plants at each time point, and there was no significant difference in the number of adventitious roots.

[0103] Example 6

[0104] (1) Observation of the anatomical structure of the hypocotyl of CRISPR-RBOHB transgenic cucumber plants

[0105] In order to confirm the formation process of adventitious roots in control plants and CRISPR-RBOHB transgenic plants (Csrbohb#1, Csrbohb#2) after flooding, the plants were flooded at the two-leaf and one-heart stage after 21 days of cultivation, and the cucumber hypocotyls were cross-sectioned and observed at 0h, 24h, and 48h after treatment. Fresh hypocotyl samples were taken from plants treated with flooding for 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-RBOHB transgenic plants had almost no adventitious roots at 24h of flooding, and adventitious root primordia began to differentiate from the cambium of the vascular bundles 48h after flooding; while the adventitious root primordia of the control plants began to differentiate from the cambium of the vascular bundles 24h after flooding, and the 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-RBOHB transgenic plants is significantly slower than that in control plants. Figure 5 (a) shown.

[0106] (2) Observation of the anatomical structure of the hypocotyl of cucumber plants overexpressing OE-RBOHB

[0107] In order to confirm the formation process of adventitious roots in the control plants and OE-RBOHB overexpressing plants (OE#1, OE#2) after flooding, the plants were flooded at the two-leaf and one-heart stage after 21 days of cultivation, and the cucumber hypocotyls were cross-sectioned and observed at 0h, 24h, and 48h after treatment. Fresh hypocotyl samples were taken from the plants treated with flooding for 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. The control plants had almost no adventitious roots at 24h of flooding, and the adventitious root primordium began to differentiate from the cambium of the vascular bundle 48h after flooding; while the adventitious root primordium of the overexpressing plants began to differentiate from the cambium of the vascular bundle 24h after flooding, and the adventitious root primordium 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 OE-RBOHB overexpressing plants was significantly faster than that in control plants. Figure 5 (b) as shown.

Claims

1. A gene RBOHB for regulating waterlogging stress to induce adventitious root formation in cucumber, characterized in that: The nucleotide sequence of the gene RBOHB is shown in SEQ ID NO.1, and the CDS sequence thereof is shown in SEQ ID NO.

2.

2. The gene RBOHB for regulating waterlogging stress and inducing adventitious root formation in cucumber according to claim 1, characterized in that: The primer pair used to amplify the gene RBOHB is preferably RBOHB-F1:5'-AGCTAAATGGCCAACAACCC-3'; RBOHB-R1:5'-GTGCCTCCCGCATGTAAAAGG-3'; RBOHB-F2:5'-AGTACAACCCCATCAAGCGG-3'; RBOHB-R2:5'-TTCAACAAAGATTTGTATGCTGT-3'. The primer pair used to amplify the CDS sequence of the gene RBOHB is RBOHB-F3: 5'-TCATGACCAAAAGCGTCGGA-3'; RBOHB-R4:5'-CAACACTCGACTGTCCGTGA-3'.

3. A protein for regulating the expression of the gene RBOHB for inducing adventitious root formation in cucumber under waterlogging stress according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

4. A gene knockout vector CRISPR-RBOHB or an overexpression vector OE-RBOHB of the gene RBOHB for regulating waterlogging stress-induced adventitious root formation in cucumber according to claim 1.

5. The gene knockout vector CRISPR-RBOHB according to claim 4, characterized in that The gene knockout vector CRISPR-RBOHB is constructed by designing the target gene sequences sgRNA1 and sgRNA2 of RBOHB: sgRNA1:5'-TCCTCCTCTGATGTTCTGAACGG-3'; sgRNA2:5'-CGATTCATGACCAAAAGCGTCGG-3'. pCBC-DT1T2 (Cm) was used as a template to amplify dual targets, and the formed double-stranded sgRNA1 / 2 was connected to the pkSE402 vector. After macro-transformation, the plasmid was extracted to finally obtain the gene knockout vector CRISPR-RBOHB.

6. The overexpression vector OE-RBOHB according to claim 4, characterized in that: The overexpression vector OE-RBOHB is constructed by designing the primer sequence of RBOHB: OE-RBOHB-F: 5'-gagaacacgggggacggatccATGGAGAGTCAAGAAAACAAACCTATG-3'; OE-RBOHB-R: 5'-atggtctttgtagtcggatccTATTTGCTGAAGCACTCAATGCA-3' The CDS gene of the RBOHB gene was amplified as a template, homologously recombined into the pCAMBIA1305.4 vector, and then transformed and the plasmid was extracted to finally obtain the overexpression vector OE-RBOHB.

7. A host cell, characterized in that The host cell uses Agrobacterium as a starting strain, and contains the gene knockout vector CRISPR-RBOHB or the overexpression vector OE-RBOHB according to claim 5.

8. Use of the gene RBOHB according to claim 1 or the protein expressed by the gene RBOHB according to claim 2 or the gene knockout vector CRISPR-RBOHB according to claim 5 or the overexpression vector OE-RBOHB or the host cell according to claim 7 in inducing adventitious root formation of cucumber under waterlogging stress and improving waterlogging resistance.

9. The use according to claim 8, characterized in that: By editing the cucumber RBOHB gene through CRISPR / Cas9, the ability of cucumber to induce adventitious root formation under waterlogging stress was reduced; by overexpressing the RBOHB gene, the ability of cucumber to induce adventitious root formation under waterlogging stress was improved.

10. Use of the gene RBOHB according to claim 1 or the protein expressed by the gene RBOHB according to claim 2 or the gene knockout vector CRISPR-RBOHB or the overexpression vector OE-RBOHB according to claim 5 or the host cell according to claim 7 in cultivating waterlogging-resistant cucumber germplasm.

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