Application of cucumber CsTOR gene in cucumber grafted seedling

Abstract

The invention relates to the technical field of biology, and discloses application of a cucumber CsTOR gene in cucumber grafted seedlings, and the cucumber CsTOR gene has high expression level in tender tissues of root tips, stem tips and male flower buds of cucumbers; subcells of the cucumber CsTOR gene are localized in a cell nucleus and a cell membrane; the cucumber CsTOR gene is used for regulating and controlling the survival rate of cucumber grafted seedlings. The cucumber-pumpkin grafted seedlings treated by a control group and the TOR inhibitor are further subjected to transcriptome and metabolome analysis, and it is found that after the TOR kinase activity is inhibited, the content of plant hormone metabolism related genes and hormones is remarkably changed, especially auxin. The CsTOR-mediated auxin-regulated grafting interface healing mechanism research is preliminarily disclosed, and while the research on the heterologous grafting interface healing mechanism is enriched, theoretical support is provided for creating a rapid healing material and optimizing a grafted cucumber production technology in the future.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to cucumber CsTOR Application of genes in cucumber grafted seedlings. Background Art

[0002] cucumber( Cucumis sativus L. ), also known as cucumber and cucumber, is one of the important greenhouse vegetable crops in my country. According to research, cucumbers are native to tropical humid areas such as the south side of the Himalayas. At present, its planting areas have covered the world. China's cucumber cultivation area is about 1.31 million hectares, and the total output is about 77.3 million tons, accounting for 60.3% and 81.6% of the world's total cultivation area and total output respectively (FAO 2022), and the cultivation area and output rank first in the world. In the cultivation and production of cucumbers, it is often affected by biological and abiotic stresses. Grafting technology has the advantages of improving cucumber yield and fruit quality, preventing continuous cropping obstacles, resisting soil-borne diseases and stress resistance, and has been widely used in greenhouse cucumber production. In the production of grafted cucumber seedlings in my country, Chinese pumpkin is often used as the rootstock. According to statistics in 2011, the area of ​​grafted cucumber seedlings in my country was about 7.6625 million mu, accounting for about 30% of the total cultivation area, and more than 95% of seedlings in greenhouse production were grafted seedlings. Therefore, the development of the grafted cucumber industry is conducive to driving local economic development, increasing farmers' income, and promoting the process of agricultural and rural modernization.

[0003] The healing of grafting interface mainly includes three processes: initial tissue adhesion and isolation layer formation between scion and rootstock, callus formation, and reconnection of scion and rootstock vascular bundles. The healing of grafting interface is a prerequisite for grafting affinity. The factors that currently limit the development of my country's grafted cucumber seedling industry are: on the one hand, there is a lack of high-yield, high-quality and multi-resistant self-bred cucumber rootstock varieties; on the other hand, the slow healing speed and instability of the grafting interface cause high management costs. Therefore, improving the healing rate and stability of the grafting interface can significantly improve the grafting efficiency and reduce the cost of grafting seedlings, which is conducive to the development and utilization of low-affinity high-quality rootstock germplasm resources and promotes the intensive production of cucumber grafted seedlings. Callus formation is a key developmental event in the healing of grafting interface, and its molecular mechanism is still unclear. Cucurbitaceae crops, as double-tough vascular bundle crops, are model crops for studying vascular bundle development and function, providing material advantages for studying grafting interface healing.

[0004] Due to the blockage of vascular bundles, auxin accumulates above the grafting interface, which may induce cells to enter mitosis and promote the formation of callus. However, studies have shown that auxin can also be transported upward from the rootstock or passively diffused to the interface, accelerating the formation of callus by activating genes such as Histone H4 and PERX. Overexpression of iaaM in the rootstock can produce more callus and promote rapid healing of the grafting interface. The increase of auxin can also induce the rootstock and scion at the interface to produce more ethylene, which jointly promotes callus formation. The wound gene WIND1 can activate cytokinin signals at the wound to promote callus formation. The application of exogenous BA can promote callus formation at the interface and thus improve the grafting survival rate, but cytokinin synthesis or signal mutants do not affect the grafting survival rate in Arabidopsis homologous grafting. In general, compared with other plant hormones (gibberellins, abscisic acid, salicylic acid, ethylene, jasmonic acid, etc.), auxin and cytokinin have been studied more in the formation of callus at the grafting interface of plants.

[0005] TOR, the target of rapamycin, is a central regulator in the growth and development of plants and plays a core role in the growth and metabolism of eukaryotic organisms. TOR, the target of rapamycin, is a highly conserved serine / threonine protein kinase in eukaryotic organisms and belongs to the PIKK family. It is a very large and highly conserved protein with a protein structure that includes an N-terminal HEAT repeat domain, a FAT domain, a FRB domain, a kinase domain, and a C-terminal FAT domain. Recent studies have shown that sugar activates TOR kinase to phosphorylate and stabilize E2Fa protein, thereby upregulating S-phase-related genes in the cell cycle, promoting tissue dedifferentiation to form callus, and this process is coupled with auxin signals to promote cell proliferation. In the division and growth of root and shoot tip cells, TOR kinase phosphorylates the EIN2-Thr657 site to inhibit the cytoplasmic-nuclear shuttle of EIN2 protein, promote the expression of the key cell cycle factor E2Fa, and promote cell proliferation in meristems. Auxin export carrier protein PIN2 is also a key downstream messenger of TOR signaling. Sugar stabilizes PIN2 by activating TOR phosphorylation, thereby affecting its distribution in different root zones of Arabidopsis. The auxin concentration gradient established subsequently promotes the elongation of cells in the elongation zone. In addition, studies have found that the phosphorylation substrates of TOR kinase are mostly plant hormone-related proteins, including transcription factors E2Fa / b, ABA receptors PYLs, auxin export protein PIN2, and ethylene signaling protein EIN2. The above studies show that TOR regulates callus formation and cell growth, and this process is inseparable from plant hormones.

[0006] Therefore, cucumber CsTOR Whether genes play an important role in the healing of cucumber grafted interfaces needs further exploration. Summary of the invention

[0007] The present invention aims to solve the above problems. Specifically, the technical solutions provided by this application are as follows: cucumber CsTOR Application of gene in cucumber grafted seedlings, the cucumber CsTOR The gene is expressed at a high level in the root tip, stem tip and young tender tissues of male flower buds of cucumber; the cucumber CsTOR The gene subcellular localization is in the cell nucleus and cell membrane; the cucumber CsTOR Genes are used to regulate the survival rate of grafted cucumber seedlings.

[0008] Further, by silencing or downregulating the cucumber CsTOR Gene, TOR silencing causes yellowing of the scion cotyledons and withering of the true leaves of the grafted seedlings.

[0009] Furthermore, the cucumber CsTOR After the gene is inhibited, the plant hormones respond actively at the cucumber-pumpkin grafting interface, which has a positive effect on the healing of the grafting interface.

[0010] Based on the above technical solution, the TOR inhibition can affect the transport and signal response of auxin in the reconnection of vascular tissue of cucumber-squash grafted seedlings.

[0011] Based on the above technical solution, TOR inhibitors were used to treat cucumber CsTOR Gene suppression treatment.

[0012] Further preferably, the TOR inhibitor is AZD8055.

[0013] Furthermore, the dosage of the TOR inhibitor is 10-100 µM.

[0014] Based on the above technical solution, by overexpression CsTOR Genes to improve the survival rate of grafted cucumber seedlings.

[0015] On the basis of the above technical scheme, the cucumber grafted seedling is a grafted seedling obtained by grafting cucumber 'R1461' as scion and pumpkin 'Zhongshuzhen No. 1' as rootstock by using sleeve grafting method.

[0016] The present invention has the following advantages / benefits: The present invention regulates cucumber CsTOR Expression levels of genes silenced and / or overexpressed in cucumber CsTOR Genes, explore cucumber CsTOR The application of genes in the survival rate of cucumber and pumpkin grafted seedlings. In addition, tissue expression specificity analysis found CsTOR May be involved in cell differentiation and cell growth in young cucumber tissues, and CsTORResponds to plant hormone signals. Found through subcellular localization experiments CsTOR It is localized to the cell nucleus and cell membrane. CsTOR The graft survival rate decreased significantly after CsTOR Participate in regulating the healing process of grafted interface of cucumber-pumpkin grafted seedlings.

[0017] Further transcriptome and metabolome analysis of the cucumber-squash grafted seedlings in the control group and those treated with TOR inhibitors revealed that after inhibiting TOR kinase activity, there were significant changes in plant hormone metabolism-related genes and hormone contents, especially auxin.

[0018] The present invention initially discloses CsTOR Research on the mechanism of grafting interface healing mediated by auxin regulation will help enrich the research on the healing mechanism of heterologous grafting interfaces, while providing theoretical support for the future creation of rapid healing materials and optimization of grafted cucumber production technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0020] Figure 1 : The present invention CsTOR Analysis of expression characteristics in cucumber tissue and after hormone spraying; A is CsTOR Expression patterns in various cucumber tissues, B is after hormone treatment CsTOR Expression in cucumber, C is after self-grafting CsTOR The expression at the graft interface of cucumber, D is the expression of CsTOR at the graft interface of cucumber after heterologous grafting; Figure 2 : The present invention CsTOR Subcellular localization in tobacco lower epidermal cells; Figure 3 :The present invention utilizes PDS Analysis of the infection efficiency of cucumber VIGS; A is PDS Phenotype of silenced plants (bar = 2 cm), B PDS Quantitative analysis after silencing; Figure 4 : The present invention CsTOR Phenotype (A) (bar = 2 cm) and quantitative (B) and survival rate (C) analysis of silent plants after grafting; Figure 5: The number of differentially expressed genes (A) and cluster analysis (B) of the effects of TOR inhibitors at different times on the healing of cucumber-squash grafting interfaces of the present invention; Figure 6 : Weighted gene co-expression network analysis (WGCNA) and the most relevant module KEGG analysis among different groups of the present invention; wherein A and B are the co-expression network analysis of the healing of the cucumber-squash graft interface at different time points after TOR inhibition, and C and D are the KEGG analysis of the MEtan module of the control group and the MEcyan module of the treatment group at 5DAG; Figure 7 : Cluster analysis of differentially expressed genes at each stage of grafting interface healing of the present invention; Figure 8 : Clustering of hormone metabolism-related differentially expressed genes (A), statistics of the number of differentially expressed genes (B) and protein interaction network analysis (C) of the present invention; Fig. 9 : Analysis of plant hormone metabolism at the interface healing of cucumber-squash grafted seedlings after treatment with 100 μM TOR inhibitor of the present invention. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0022] Example 1: Materials and Methods 1.1 Experimental materials and growth conditions Cucumber variety 'R1461' and pumpkin variety 'Zhongshuzhen No. 1' were used to construct the cucumber-pumpkin grafting combination. 'R1461' cucumber germplasm was provided by Zhejiang Academy of Agricultural Sciences, and 'Zhongshuzhen No. 1' pumpkin was purchased from Zhongshu Seed Technology (Beijing) Co., Ltd. The cucumber VIGS transient transformation material was cucumber '9930', which was preserved in the school laboratory.

[0023] 1.2 Grafting methods and management methods Plant seedling raising and management were carried out in the climate chamber of the College of Horticultural Science, Zhejiang A&F University. Select complete and full cucumber seeds, wrap them with clean gauze, and soak them in warm water at 55 ℃ for 2-3 hours. Then transfer the seeds to wet filter paper and germinate them in a petri dish at 28 ℃ in the dark for 12-24 hours. Pumpkin seeds are germinated 2 days later than cucumber seeds. Soak the seeds at the same temperature for 5-6 hours and germinate them at 28 ℃ in the dark for 24-36 hours. When most of the seeds are white, sow them in a 50-hole tray filled with substrate (peat: vermiculite = 2:1) and place them in an artificial climate chamber for cultivation.

[0024] Culture conditions: 12 h light / 12 h dark, light intensity of 50,000 Lux, day temperature of 28±1°C / night temperature of 18±1°C, relative air humidity maintained at 65–85%.

[0025] The grafting was performed by sleeve grafting. Using 11-day-old cucumber seedlings as scions and 7-day-old pumpkin seedlings as rootstocks, a sharp new blade was used to make a 30° oblique cut from the rootstock hypocotyl along the vertical direction of the cotyledons, and the rootstock above the incision was completely removed (including cotyledons, growth points, and part of the hypocotyl). Then, a blade was used to make a 30° oblique cut along the cotyledon direction 2-2.5 cm below the cotyledons of the scion. After cutting, the two were closely attached and fixed with a grafting clamp.

[0026] After grafting, a transparent plastic tray cover was used to seal the seedlings to maintain high humidity to prevent the grafted seedlings from being damaged by wind in the early stage. At the same time, the sealed tray was moved into the artificial climate room for management measures such as heat preservation and moisture retention, light conditions, and wind protection. This day was defined as 0 Days After Grafting (0 DAG). The specific measures for grafting management are as follows: Light management: From 0 to 3 DAG, the grafted seedlings were kept in the dark; from 3 to 5 DAG, they were managed under weak light (5000 Lux); from 6 to 7 DAG, they were gradually put into normal light (8000 Lux) conditions. Ventilation management: The humidity was kept above 95% in the first 4 days (0-3 DAG), and the vents of the transparent plastic tray cover were opened for 2-3 h from the 5th to the 8th day (4-7 DAG), and the ventilation time was gradually increased to gradually reduce the humidity to about 75%. After 7 DAG, the grafted seedlings survived and were managed normally.

[0027] 1.3 Treatment with TOR inhibitor AZD8055, auxin and 6-BA Cucumber-squash grafted seedlings were uniformly sprayed with different concentrations of TOR inhibitor AZD8055 (0, 10, 25, 50 and 100 µM) at the same time. The spraying site was the cotyledons of the grafted seedlings, and the spraying time was 0, 1, 3, and 5 days after the grafting of the cucumber-squash grafted seedlings, with 50-100 plants in each treatment. The spraying method of auxin NAA (5 µM) and 6-BA (0.1 mM) was the same as that of TOR inhibitor AZD-8055.

[0028] 1.4 Survival rate statistics The survival rate of cucumber-pumpkin grafted seedlings was calculated on 8 DAG. Survival rate = (number of grafted seedlings with good growth in each treatment ÷ total number of grafted seedlings in each treatment) × 100%.

[0029] 1.5 RNA extraction and reverse transcription Tissue specificity: Each tissue sample (R: root tip; S: stem; L: leaf; ST: stem tip; FF: female flower bud; MF: male flower bud; F: fruit) was taken from 30-day-old cucumber plants, with 3 biological replicates for each sample. The samples were placed in 2 ml enzyme-free centrifuge tubes filled with high-temperature sterilized steel beads, labeled with their names, and then placed in liquid nitrogen. After all samples were taken, they were transferred to a -80 ℃ refrigerator for storage.

[0030] Plant hormone response: Cucumber plants sprayed with auxin and 6-BA were sampled at 0, 6, 12, 24, and 48 HAG (Hour After Grafting) from the hypocotyls of the cucumbers, with three biological replicates for each sample. The sampling and storage methods were the same as above.

[0031] Response of the healing site of scion and stock: Cucumbers were grafted autologously (CC: cucumber-cucumber) and heterologously (CP: cucumber-pumpkin; PC: pumpkin-cucumber) and samples were taken at 0, 1, 2, 3, and 4 days after grafting. The sampling sites were all the healing sites of the cucumber interface, and each sample was biologically replicated three times. The sampling and storage methods were the same as above.

[0032] VIGS experiment: The sample was a single true leaf of cucumber scion. The sampling and preservation methods were the same as above.

[0033] Before RNA extraction, prepare 1000 ml enzyme-free pipette tips, 200 ml enzyme-free pipette tips, and 1.5 ml enzyme-free centrifuge tubes, and place 75% ethanol and isopropanol in a 4 ℃ refrigerator. Set the grinder to 45 frequency for 30 seconds to grind the sample into powder in the centrifuge tube, then quickly add 1 ml RNAiso Plus (Takara, Code No.9109) and vortex and shake, then let it stand at room temperature for 5 minutes. The subsequent RNA extraction steps are carried out according to the instructions. Finally, the centrifuge tube containing RNA precipitate is dried in a fume hood for 10-15 minutes, and 30 µl RNase-free water is added to dissolve the precipitate.

[0034] After RNA concentration measurement, reverse transcription was performed immediately. All-in-one RT SuperMix Perfectfor qPCR kit (Novozyme, R333) was used to calculate the total amount of RNA. After adding reagents according to the instructions, the sample was mixed thoroughly by gently pipetting 8-10 times and centrifuged briefly.

[0035] The reaction system is as follows:

[0036] The PCR instrument reaction program was set as follows:

[0037] After the reaction was completed, the product was placed in a -20 °C refrigerator.

[0038] 1.6 Analysis of CsTOR subcellular localization Vector construction: Using 1305.4-35S-GFP as the expression vector, construct CsTOR Or its domains and GFP fusion genes for subcellular localization analysis. Download from the cucumber gene library CsTOR The full-length CDS (coding sequence) was identified and cloning primers were designed according to the experimental requirements. The GXL DNA Polymerase high-fidelity enzyme kit (Takara, R050Q) was used to amplify the target gene.

[0039] The reaction system is as follows:

[0040] The PCR program was set up as follows:

[0041] Add 5 µl 10× DNA loading buffer (Novozyme, P022) to the PCR product for electrophoresis monitoring. Use 10000 Maker as a control to cut out the fragment at the correct position and recover it by gel. Prepare the in-fusion recombination system (Takara, 638947) with the gel-recovered product and linearized vector in proportion. Recombinate at 50°C for 15 minutes to obtain the final product.

[0042] E. coli transformation: 5 µl of the final product was added to a 1.5 ml sterile centrifuge tube containing 25 µl of DH5α competent E. coli (Qingke, TSC-C01). The centrifuge tube was placed in ice for 30 min, then heat-shocked at 42 °C for 60 s, and then placed in ice again for 2 min. 500 µl of LB culture medium without resistance was drawn from the clean bench and added to the centrifuge tube, and then cultured at 37 °C 200 r / min for 45 min. Centrifuge at 7 000rpm for 1 min, take 50 µl of the supernatant and resuspend the precipitate, apply all of it to the corresponding resistance LB culture plate (containing 50 mg / L Kan), and culture at 37 °C for 12-16 h. When the monoclonal strain grows to 1 mm in size, use a sterile toothpick to pick a single bacterium and place it in LB culture medium containing 50 mg / L kanamycin, culture it at 37 °C 200 r / min for 3-5 hours, and perform PCR positive detection, and then send the positive clone to Qingke for sequencing. After successful sequencing, the bacterial solution was shaken again, and the glycerol culture (50% glycerol: bacterial solution = 1:1) was stored in a -80 ℃ refrigerator and the plasmid was extracted.

[0043] Plasmid extraction: The FastPure Plasmid Mini Kit (Novozyme, DC201-01) was used for plasmid extraction. Please refer to the instructions for specific methods. The final plasmid was tested for concentration using Nanodrop and stored in a -20°C refrigerator.

[0044] Agrobacterium transformation: The Agrobacterium strain GV3101 (Qingke, TSC-A01) was selected, and all sample addition operations were carried out in a clean bench. The specific transformation method was carried out with reference to the instructions. When the monoclonal strain on the bacterial plate grew to 1 mm in size after being cultured at 28 °C for 36-48 h, a single bacterium was picked up with a sterilized toothpick and placed in 200 µl LB culture solution containing 50 mg / L Kan and 50 mg / L Rif, cultured at 28 °C and 200 r / min for 12-16 h, and colony PCR positive identification was performed. The identified positive single bacteria were subjected to glycerol bacteria (50% glycerol: bacterial solution = 1:1) preservation operation and placed in a -80 °C refrigerator for long-term storage.

[0045] Tobacco transient transformation: Subcellular localization GV3101 containing expression vectors 35S::GFP empty control, 35S::CsTOR(1-7410 bp)-GFP, 35S::CsTOR(1-4311 bp)-GFP, 35S::CsTOR(4312-6240 bp)-GFP, 35S::CsTOR(6241-7410bp)-GFP plasmids was used as the infection Agrobacterium, the nuclear Maker was used as the positive control, and 4-week-old Nicotiana benthamiana with 5-8 flat leaves was used as the infection material. The specific steps are as follows: 50 ul of glycerol bacteria stored in a -80 ℃ refrigerator were inoculated into 25 ml of LB culture medium containing 50 mg / L Kan and 50 mg / L R. If resistance, and cultured at 28 ℃ 200 r / min for 12-16 h until OD600=0.8-1.2. The bacteria were collected by centrifugation at 4500 rpm for 15 min. During this period, the infection solution (10 mM MES; 10 mM MgCl2; 200 μM AS) was prepared, and the pH of the infection solution was about 5.6. After the centrifugation, the supernatant was discarded, and an equal volume of infection solution was added to resuspend the bacteria, and the bacteria were collected by centrifugation at 4500 rpm for 20 min again. Add the infection solution and adjust the infection solution with bacteria to OD600=0.5-0.6. The infection solution containing the expression vector and the Maker infection solution were mixed in a 1:1 ratio. After standing for 3 h in the dark, inject it into the back of the flat Nicotiana benthamiana leaf with a 2 ml syringe (without a needle) and mark it. After injection, the tobacco leaves were protected from light for 48 h, and the injected tobacco leaves were cut off and the fluorescence was observed and photographed under a laser confocal microscope (Zeiss, Germany).

[0046] 1.7 Cucumber VIGS transient transformation Vector construction: The CDS sequence of the CsTOR kinase domain was entered into the VIGS primer online design website (https: / / vigs.solgenomics.net / ), and a 300 bp specific fragment primer was designed using the website and sent to the company for primer synthesis. Fragment cloning, gel recovery, E. coli transformation, plasmid mini-extraction, and Agrobacterium transformation were the same as in 3.1.4 subcellular localization. The single clone that was correctly detected by PCR in the Agrobacterium culture was selected and stored at -80 ℃ for later use.

[0047] The experimental bacterial solution mixture is as follows:

[0048] VIGS plate preparation: Mix the desired strain with sterilized water (100 ul water + 20 ul glycerol bacteria) and apply it to LB culture medium containing corresponding resistance (containing 50 mg / L Kan and 50 mg / L Rif) for activation. Prepare multiple copies of TRSV1 according to the experimental needs. Culture in a 28 ℃ incubator for 36-40 hours.

[0049] sowing: Soak the plump cucumber seeds in 55 ℃ water for 30 minutes to sterilize, place them at room temperature for 1-2 hours, and remove the outer seed coat. Rinse the cucumber seeds without seed coat with pure water 2-3 times and then move them to the clean bench. Soak them in 75% alcohol for 30 seconds, rinse them with sterilized water, and then keep them in 5% sodium hypochlorite solution for 15 minutes (shake gently during this period, or place them in a shaker), and rinse them with sterilized water 5 times. Pour the seeds onto filter paper. When there is almost no water on the surface of the seeds, sow them on the sowing medium and culture them in the dark at 28 ℃ for 36 hours. When the radicle of the seeds grows to about 2 cm long, they are used for subsequent infection.

[0050] Preparation of infection solution: Add 7-8 ml of MS infection solution to each bacterial plate, scrape the bacteria off and pour the mixed solution into a 50 ml sterile centrifuge tube. Adjust the concentration of the infection solution to a final OD600 of 1.0. At the same time, make a 1:1 mixed infection solution according to the above combination.

[0051] Pre-infection treatment: Take the seeds that meet the standards and place them on a culture dish containing filter paper, which is soaked with MS infection culture solution. Make a wound on the cucumber cotyledon along the 1 / 2 of the longitudinal main vein to separate the outer ends of the two cotyledons and place them in a conical flask containing infection solution for subsequent infection.

[0052] Agrobacterium infection: Put a vacuum pump into the conical flask opening and keep the vacuum state at -0.09 pressure for 10 minutes. It is best if the hypocotyl becomes transparent after infection. Co-cultivation: After the infection is complete, pour the seeds and spread them on sterilized filter paper. After drying until there is no obvious water stain on the surface, place the seeds in the co-cultivation medium and culture them in the dark at 28°C for 5 days until obvious bacteria appear on the medium.

[0053] Transplanting: Prepare Hoagland hydroponic nutrient solution (Coolaber, NS10205) and pour it into the hydroponic box. Use tweezers to gently move the plant into the sponge and place it in the hydroponic box. Cultivate it in the incubator. After about 15 days, the PDS treatment group showed phenotypes, and the experimental group was grafted at the same time.

[0054] 1.8 Real-time fluorescence quantitative PCR detection Extract RNA from the leaves of mutant materials. The specific RNA extraction and reverse transcription steps are the same as 3.1.3.

[0055] The Taq Pro Universal SYBR qPCR Master Mix (Novagen, Q712-02) was used according to the instructions. CsTIP41 is an internal reference gene (Miao et al., 2019c).

[0056] The reaction system is as follows:

[0057] The qRT-PCR procedure is as follows:

[0058] Each sample was repeated three times. The relative expression of genes was calculated using 2 -ΔΔCt formula.

[0059] Example 2: Tissue expression and plant hormone response patterns First, the expression level of CsTOR in various cucumber tissues was detected. Figure 1 As shown in Figure 2, the analysis found that CsTOR was expressed at a high level in the root tip, stem tip, and young male flower bud tissues ( Figure 1 -A, where R, root tip; S, stem; L, leaf; ST, stem tip; FF, female flower bud; MF, male flower bud; F, fruit), which suggests that CsTOR may be involved in cell differentiation and cell growth in young cucumber tissues.

[0060] Then, by spraying auxin NAA and cytokinin 6-BA respectively, it was found that the expression level of CsTOR at the grafting interface of cucumber changed significantly ( Figure 1 -B), indicating that CsTOR responds to plant hormone signals.

[0061] By detecting the expression level of CsTOR in the healing process of cucumber-cucumber, cucumber-squash, and pumpkin-cucumber grafting interfaces at different time periods, it was found that the expression level of CsTOR in the rootstock changed significantly in cucumber-cucumber self-grafting ( Figure 1 -C, where CCS, cucumber-cucumber scion; CCR, cucumber-cucumber rootstock), while in heterologous grafting, CsTOR changes in both scion and rootstock ( Figure 1 -D, where CPS, cucumber-squash scion; PCR, pumpkin-cucumber rootstock), therefore CsTOR is responsive in the healing of the graft interface.

[0062] Embodiment 3: CsTOR Gene subcellular localization Using the Arabidopsis AtTOR protein sequence, we blasted the cucumber genome database to obtain CsTOR (Csa7G070760) gene sequence, the gene is located in the genome position 4373608-4410770, the CsTOR protein is 2470aa (7413 bp) long, composed of 5 conserved domains, including HEAT domain 1437 aa, FAT domain 472 aa, FRB domain 171 aa, kinase domain 359 aa, FATC domain 31 aa. In order to determine the signal position of CsTOR and its domains, and in Arabidopsis thaliana, the kinase domain has a nuclear entry signal.

[0063] In the present invention, except for the HEAT domain of CsTOR amplified separately, the other four CsTOR domains are adjacently combined to form two combinations (FAT domain / FRB domain; Kinase domain / FATC domain), and are constructed on the pCAMBIA1305.4 vector containing 35S::GFP with the full length of CsTOR, and the GV3101 bacterial solution containing the above plasmids is injected into the lower epidermis of tobacco, with 35S::GFP as a control. In the lower epidermal cells of tobacco, each domain of CsTOR has fluorescent signals in the nucleus and cell membrane, indicating that CsTOR has nuclear and cell membrane localization ( Figure 2 ).

[0064] Embodiment 4: CsTOR Gene VIGS transient silencing by PDS A preliminary experiment was conducted as a positive control. After successfully silencing the PDS gene, the cucumber plants showed an albino phenotype. PDS Phenotypic observation and quantitative analysis of silenced plants showed that VIGS technology can silence cucumber genes ( Figure 3 ).

[0065] Subsequently, primers were designed and amplified for the silencing region of the CsTOR kinase domain using an online website (https: / / vigs.solgenomics.net / ), and the successfully sequenced plasmids were transformed into Agrobacterium for subsequent infection.

[0066] The experiment was conducted with cucumber germplasm 9930 as the infection material, and WT (uninfected plants) and TRSV1 (plants infected with no expression vector) as the control group. Quantitative analysis showed that TRSV1 (empty vector) infection did not affect the normal expression of TOR. On the contrary, the expression of TOR in TRSV-Kin (kinase domain silenced) plants was downregulated to varying degrees, and was downregulated by about 40% compared with WT ( Figure 4-B). The plants with kinase domain silencing were grafted with pumpkin on the 14th day after transplanting, and the phenotype was observed on the 5th day after grafting. It was found that the WT and TRSV1 scion leaves were phenotypically vigorous, while TOR silencing caused the scion cotyledons to turn yellow and the true leaves to wilt ( Figure 4 -A).

[0067] The survival rate of grafted plants after TOR silencing was also analyzed. It was found that the survival rate of grafted seedlings using scions infected with TRSV1 or TRSV-Kin was significantly reduced, especially the survival rate of grafted seedlings using TRSV-Kin (kinase domain silenced) cucumber plants as scions was as low as 31.8% ( Figure 4 -C). Therefore, it was further proved that the loss of CsTOR would lead to poor growth of cucumber and significantly affect the survival of cucumber-squash grafted seedlings.

[0068] Example 5: Effect of TOR inhibitors on the number of differentially expressed genes and cluster analysis of cucumber-squash graft interface healing Cucumber-squash grafted seedlings treated with 100 µM TOR inhibitor and the control group (WT) were sampled at 0, 1, 3, and 5 days after grafting, and transcriptome sequencing and analysis were performed on the 24 materials. It was found that compared with the control (WT), the number of differentially expressed genes (DEGs) in the healing of the cucumber-squash graft interface at different times of TOR inhibitor treatment, including the number of up-regulated (Up) and down-regulated (Down) genes, was statistically analyzed. The results showed that at 5 days (5 DAG) after grafting, the total number of DEGs in the interface tissue after TOR inhibitor treatment and the control group was very different. The number of up-regulated and down-regulated differentially expressed genes between the two treatments at this time was 1206 and 1785, respectively ( Figure 5 -A, WT0dvsTOR0d, refers to the control group and the 100µM TOR inhibitor-treated group grafted for 0 days; WT1dvsTOR1d, refers to the control group and the 100µM TOR inhibitor-treated group grafted for 1 day; WT3dvsTOR3d, refers to the control group and the 100µM TOR inhibitor-treated group grafted for 3 days; WT0dvsTOR5d, refers to the control group and the 100µM TOR inhibitor-treated group grafted for 5 days;).

[0069] To clarify the metabolic pathways associated with the 2991 differentially expressed genes treated with TOR inhibitors at 5 DAG, KEGG enrichment analysis was performed and it was found that the 2991 differentially expressed genes were mainly enriched in pathways such as zeatin synthesis, plant hormone signal transduction, photosynthesis, carotenoid synthesis, and phenylpropionic acid synthesis ( Figure 5 -B).

[0070] Example 6: WGCN analysis of TOR inhibitors on the healing of cucumber-squash graft interfaces To further clarify the response of the healing of the cucumber-squash graft interface at different time points after TOR inhibition, the genes between different samples were analyzed by WGCNA (weighted gene co-expression network analysis), and the weighted value of the correlation coefficient was used to obtain a hierarchical clustering tree. Then, based on the weighted correlation coefficient of the gene, the module was identified according to the genes with high connectivity in the co-expression network. One most relevant module was found in the control group and 5DAG treated with 100 µM TOR inhibitor, and the correlation coefficients were close to 1, namely, MEtan module, MEcyan module ( Figure 6 -A, B).

[0071] KEGG enrichment analysis was performed on the two modules respectively, and it was found that the differentially expressed genes in the MEtan module of the control group samples analyzed on the 5th day after grafting mainly played a role in 48 pathways of cellular processes, environmental information processes, genetic information processes, metabolic pathways and organic system pathways, and were mainly enriched in pathways such as plant hormone signal transduction, phenylpropanoid synthesis, phosphoinositide metabolism, starch and sucrose metabolism ( Figure 6 -C). The differentially expressed genes in the MEcyan module of the samples treated with 100 µM TOR inhibitor on the 5th day after grafting mainly played a role in 49 pathways of cellular processes, environmental information processes, genetic information processes, metabolic pathways and organic system pathways, and were mainly enriched in pathways such as plant hormone signal transduction, protein processing in the endoplasmic reticulum, and plant-pathogen interaction ( Figure 6 -D). Comparing the differential gene enrichment pathways in the MEcyan module with the MEtan module, it was found that the enrichment pathways between the two modules had the same hormone and sugar metabolism pathways, including plant hormone signal transduction, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, and zeatin biosynthesis. Comparing the number of differential genes in the above four pathways, it was found that the number of differential genes in the samples treated with 100 µM TOR inhibitor was higher than that in the control group, especially in the plant hormone signal transduction pathway, the number of differential genes between the two groups was 4 times different. According to the above analysis, after CsTOR was inhibited, plant hormones responded positively at the cucumber-pumpkin grafting interface, and plant endogenous hormones played a positive role in the healing of the grafting interface.

[0072] Example 7: Cluster analysis of genes related to grafting interface healing and vascular bundle development Through transcriptome data, we analyzed the genes related to each stage of graft interface healing (protovascular bundle formation stage, cambium formation stage, phloem formation stage, xylem formation stage, and cell division stage), and found that 23 related genes in these five stages after TOR inhibitor treatment at 5 DAG were significantly upregulated or downregulated compared with the control group. Two protovascular bundle-related genes, three cambium-related genes, five xylem-related genes, and three cell division-related genes were all upregulated to varying degrees at 5 DAG, especially VND6.1 , CESA9 , REV1.1 , PHB1.2 and PHB1.3 The expression levels of the five xylem-related genes were upregulated to the greatest extent at 5 DAG. On the contrary, the five protovascular bundle-related genes, two phloem-related genes, and three xylem-related genes showed a downregulation trend ( Figure 7 ). It is speculated that when CsTOR is inhibited, the slow healing or non-healing of the cucumber-squash graft interface will stimulate a significant upregulation or downregulation of the expression of genes related to the graft healing stage, which also proves the importance of CsTOR in the graft interface healing process.

[0073] Example 8: Effects of TOR inhibitors on plant hormone metabolism during graft interface healing To further study the effect of plant hormones on the healing of grafted interfaces after TOR inhibition, we clustered the genes related to the six plant hormones (auxin, cytokinin, gibberellin, abscisic acid, jasmonic acid, and ethylene) and found that the expression levels of different hormone-related genes were up-regulated or down-regulated to varying degrees at different times ( Figure 8 -A). At the same time, the number of differentially expressed genes under different time periods of the six hormones was counted, and it was found that the number of differentially expressed genes under different hormones treated with CK (0A) and 100 µM TOR inhibitor was significantly different at 5DAG, and except for gibberellin-related genes, the other plant hormone-related genes had differential genes in each time period ( Figure 8 -B).

[0074] A protein interaction network was drawn for TOR and six hormone-related genes, and it was found that TOR interacted with seven genes, namely, auxin-related gene PIN2.1, jasmonic acid-related genes JAZ10 / JAS1 and JMT, ethylene-related genes ERS and ETR2, and cytokinin-related genes CKI1 and AHK2.1. However, it may also interact with AUX2-11, WIP2, WRKY40, and GA2ox1 ( Figure 8 , C). This proves that there is an inseparable relationship between TOR and plant hormones, especially auxin, cytokinin, jasmonic acid and ethylene.

[0075] Example 9: Plant hormone metabolomics analysis of TOR inhibitors on the healing of cucumber-squash graft interface After finding the expression changes of plant hormone-related genes in cucumber-pumpkin grafted seedlings through transcriptome analysis, we also conducted a metabolome analysis of plant hormones at the cucumber-pumpkin graft interface, including auxin, cytokinin, abscisic acid, jasmonic acid, salicylic acid, ethylene, and trichololactone. First, through the analysis of differential metabolites (DMs) at different healing times, it was found that there were 11 differential metabolites in 1 DAG, 19 differential metabolites in 3 DAG, 22 differential metabolites in 5 DAG, and 19 differential metabolites in 7 DAG. The Venn diagram analysis of differential metabolites in each healing time period showed that the number of differential metabolites between 3 DAG and 5 DAG was the largest, with a total of 13 ( Fig. 9 -A). KEGG analysis of 3 DAG and 5 DAG differential metabolites revealed the same enriched pathways, including plant hormone signal transduction, zeatin biosynthesis, metabolic pathways, secondary metabolite synthesis, etc., which were also included in the transcriptome analysis ( Fig. 9 -B).

[0076] According to the above analysis, we mainly analyzed the different plant hormone-related metabolites at 3 DAG and 5 DAG and found that 19, 13, and 7 related metabolites of cytokinin, auxin, and jasmonic acid had changes in content, while only 1-2 related metabolites of salicylic acid, abscisic acid, ethylene, and strigolactone changed. Among them, the content of auxin changed significantly from 3 DAG to 5DAG, and the expression changes of CK and 100A samples with and without TOR inhibitors were significantly different, while the content of plant hormone-related metabolites such as cytokinin did not change significantly ( Fig. 9 -C). Therefore, it can be seen that the transport of auxin and the signal response after TOR inhibition play a significant role in the reconnection of vascular tissues in cucumber-squash grafted seedlings.

[0077] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. Cucumber CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: The cucumber CsTOR The gene is expressed at a high level in the root tip, stem tip and young tender tissues of male flower buds of cucumber; the cucumber CsTOR The gene subcellular localization is in the cell nucleus and cell membrane; the cucumber CsTOR Genes are used to regulate the survival rate of grafted cucumber seedlings.

2. The cucumber according to claim 1 CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: By silencing or downregulating the cucumber CsTOR gene, TOR silencing caused the cotyledons of the scions of grafted seedlings to turn yellow and the true leaves to wither.

3. The cucumber according to claim 1 CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: The cucumber CsTOR After the gene is inhibited, the plant hormones respond actively at the cucumber-pumpkin grafting interface, which has a positive effect on the healing of the grafting interface.

4. The cucumber according to claim 1 CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: The TOR inhibition can affect the transport and signaling response of auxin in the reconnection of vascular tissue of cucumber-squash grafted seedlings.

5. The cucumber according to any one of claims 3 or 4. CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: Using TOR inhibitors on cucumbers CsTOR Gene suppression treatment.

6. The cucumber according to claim 5 CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: The TOR inhibitor is AZD8055.

7. The cucumber according to claim 5 CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: The dosage of the TOR inhibitor is 10-100 μM.

8. The cucumber according to claim 1 CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: Through overexpression CsTOR Genes to improve the survival rate of grafted cucumber seedlings.

9. The cucumber according to claim 1 CsTOR The application of the gene in cucumber grafted seedlings is characterized in that: The cucumber grafted seedling is obtained by using cucumber 'R1461' as scion and pumpkin 'Zhongshuzhen No. 1' as rootstock through a sleeve grafting method.

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