Application of cucumber CsaTRM4 gene in promoting long-distance transport between pumpkin CmoCK1 rootstock and scion

Abstract

The present invention relates to an application of a cucumber CsaTRM4 gene in promoting long-distance transport between pumpkin CmoCK1 rootstocks and scions. The cucumber CsaTRM4 gene provided by the present invention can promote the long-distance transport of pumpkin rootstock CmoCK1 mRNA. The tobacco transient transformation system is used to co-express CsaTRM4 and CmoCK1, thereby improving the transport efficiency of CmoCK1. The new function of CsaTRM4 described in the present invention has good application potential, provides gene resources and new ideas for rootstock breeding research, and the CsaTRM4 gene can promote the long-distance transport of the key low-temperature resistant gene CmoCK1 mRNA, and has broad application prospects and high use value.

Description

Technical Field

[0001] In the field of plant molecular biology, specifically, the application of the cucumber CsaTRM4 gene in promoting long-distance transport between the pumpkin rootstock CmoCK1 and the scion. Background Art

[0002] Cucumber (Cucumis sativus L.) is one of the main greenhouse cultivated vegetable crops in my country. It is a typical thermophilic vegetable with poor tolerance to low temperatures. Grafting, as a widely used cultivation method, has become one of the supporting measures for factory-based seedling cultivation and winter and spring cultivation of melon and solanaceous vegetables in greenhouses. The pumpkin rootstock can not only maintain the excellent traits of cucumber cultivars, but also improve disease resistance and stress resistance, especially low temperature tolerance. Different rootstocks regulate different characteristics of cucumber scions, so clarifying the molecular mechanism of rootstock-scion interaction has always been the research focus of greenhouse cucumber cultivation. Most of the research on grafting to improve cucumber stress resistance focuses on phenotypic changes, nutrient absorption, hormone balance, etc. With the development of molecular biology and bioinformatics, the discovery and in-depth study of macromolecules such as mRNA and protein transported between rootstock and scion as long-distance signals provides a strong basis for revealing the interaction mechanism of rootstock and scion and accurately regulating yield, quality and stress resistance. Previous studies have discovered the key gene choline kinase (CHOLINE KINASE 1, CmoCK1) in pumpkin's cold tolerance and clarified the function of CmoCK1 mRNA transport in improving the cold tolerance of cucumber scion.

[0003] The interaction between the rootstock and scion regulates the yield, disease resistance and stress resistance of the grafted plants. In recent years, bioinformatics analysis has found that there is a large amount of RNA signal exchange between the rootstock and scion of grafted plants. 5 C methylation modification is a relatively clear mRNA long-distance transport regulatory factor, regulating the transport of AtTCTP (Yang et al., 2019) mRNA in Arabidopsis. NOL1 / NOP2 / sun (Nsun) family proteins are responsible for transporting m 5 C "writes" RNA, a process in which these proteins catalyze the transfer of a methyl group from S-adenosylmethionine to cytosine.

[0004] In plants, N6-methyladenosine (m 6 A) and 5-methylcytosine (m 5 C) is the most common RNA methylation modification. These modifications play a role in many developmental processes, such as embryonic development, stem cell fate, trichome branching, leaf morphogenesis, flower transformation, stress response, fruit ripening and root development. Cucume is a database previously established by the inventors. Currently, in this database, you can query cucumber and pumpkin m 6 A and m 5 C RNA methylation sites. Summary of the invention

[0005] In view of the defects in the prior art, the object of the present invention is to provide an application of cucumber CsaTRM4 gene to promote long-distance transportation between pumpkin CmoCK1 stock and scion.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] An application of cucumber CsaTRM4 gene, characterized in that: overexpression of the cucumber CsaTRM4 gene in plants can promote long-distance transport of pumpkin CmoCK1 mRNA between rootstock and scion;

[0008] The nucleotide sequence of the cucumber CsaTRM4 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cucumber CsaTRM4 gene is shown as SEQ ID NO.2.

[0009] The primer pair used to amplify the above cucumber CsaTRM4 gene is characterized in that the nucleotide sequence of the primer pair is shown in SEQ ID NO.3-4.

[0010] A method for obtaining a cucumber CsaTRM4 gene knockout mutant, characterized by comprising the following steps:

[0011] Step 1, construction of CsaTRM4 CRISPR / Cas9 vector, using pCBC-DT1T2 plasmid as template for four-primer PCR amplification, wherein the four primer sequences are shown in SEQ ID NO.15-18;

[0012] The four primer sequences are as follows:

[0013] DT1-F 0 5'-TGATGAAATCTCTTCAAGCCGGTTTTAGAGCTA GAAATAGC-3';

[0014] DT1-R 0 5'-AACAACGTTCTCTCTGTTGTCGCAATCTCTTAGT CGACTCTAC-3';

[0015] DT1-BsF 5'-ATATATGGTCTCGATTGATGAAATCTCTTCAAG CCGGTT-3';

[0016] DT1-BsR 5'-ATTATTGGTCTCGAAACAACGTTCTCTCTGTTGTCGCAA-3'.

[0017] After amplification, the target band is cut and then recovered by gel recovery; the gel recovery product is digested and ligated with the pHSE401 plasmid;

[0018] Step 2, genetically transforming cucumber using the enzyme-digested-ligated product obtained in step 1;

[0019] Step 3, selecting the genetically transformed explants obtained in step 2 by Basta to obtain CsaTRM4 gene knockout plants, namely, cucumber Csatrm4 mutants.

[0020] The application of the cucumber CsaTRM4 gene of the present invention in promoting long-distance transportation between pumpkin CmoCK1 stock and scion has the following beneficial effects:

[0021] The CsaTRM4 gene provided by the present invention can promote the long-distance transport of CmoCK1 mRNA in pumpkin rootstock. The tobacco transient transformation system was used to co-express CsaTRM4 and CmoCK1, which improved the transport efficiency of CmoCK1; knocking out CsaTRM4 reduced the transport efficiency of CmoCK1 in rooting transformed cucumber. The new function of CsaTRM4 described in the present invention has good application potential, and provides gene resources and new ideas for rootstock breeding research. The CsaTRM4 gene can promote the long-distance transport of the key cold-resistant gene CmoCK1 mRNA, and has broad application prospects and high use value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention has the following accompanying drawings:

[0023] Figure 1 This is a schematic diagram of CsaTRM4 gene cloning;

[0024] Figure 2 This is the amino acid evolution tree analysis diagram of CsaTRM4;

[0025] Figure 3 This is the analysis diagram of the cis-acting elements of the CsaTRM4 promoter;

[0026] Figure 4 Schematic diagram of the CmoCK1 gene structure and methylation sites;

[0027] Figure 5 A schematic diagram of transient transformation of tobacco;

[0028] Figure 6 Schematic diagram of the analysis of CmoCK1 mRNA transport in tobacco;

[0029] Figure 7 This is a diagram showing the identification and phenotypic analysis results of the cucumber mutant Csatrm4;

[0030] Figure 8This is a schematic diagram of the identification of hairy root transformation positive plants;

[0031] Fig. 9 Figure 2 shows the results of CmoCK1 transport analysis in cucumber WT and Csatrm4 mutant. DETAILED DESCRIPTION

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0033] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0034] Embodiment 1: Cucumber and pumpkin culture method

[0035] Cucumber and pumpkin seedling growth conditions: Soak the seeds in 55°C warm water for 30 minutes, stir twice, and then soak in distilled water for 3-6 hours. Then place the seeds on moist filter paper, and place the filter paper in a covered culture dish to keep it moist. Keep the temperature at 28°C in a constant temperature box and avoid light for germination for 24 hours. Sow the germinated seeds in a 32-hole plug tray. The ratio of the substrate in the plug tray is straw carbon: vermiculite = 3:1 (v / v). Then cover with film to keep it moist and place it in a light incubator for cultivation. After 2-3 days, remove the film when the seeds arch the soil. The light intensity in the incubator is 600μmol·m -2 ·s -1 , 16 h light / 8 h dark, 28°C light / 18°C dark.

[0036] Greenhouse cultivation conditions: When the cucumber seedlings grow to two leaves and one heart, they are planted in a solar greenhouse and managed normally.

[0037] Example 2: Plant RNA extraction

[0038] Total RNA was extracted using TRIzol RNA extraction kit:

[0039] 1) Take less than 100 mg of fresh tissue and place it in a 2 mL round-bottom centrifuge tube. Add 4 mm diameter steel beads, then freeze it in liquid nitrogen and grind the tissue thoroughly and finely using a tissue grinder.

[0040] 2) Transfer the ground sample to a centrifuge tube, add RNA lysis solution, and let it stand; after the sample is completely melted, continue to blow until the lysis solution becomes transparent.

[0041] 3) Centrifuge at 12000 rpm and 4°C for 5 minutes and aspirate the supernatant.

[0042] 4) Add 1 / 5 volume of chloroform to the above lysate, shake vigorously for 15 seconds to form an emulsion, and let stand at 4°C for 5 minutes.

[0043] 5) Centrifuge at 12000 rpm at 4°C for 15 minutes.

[0044] 6) Carefully pipette the upper aqueous phase into a new enzyme-free centrifuge tube.

[0045] 7) Add an equal volume of pre-cooled isopropanol, mix by inverting, and let stand at 4°C for 10 minutes.

[0046] 8) Centrifuge at 12000 rpm and 4°C for 10 minutes. The white precipitate is RNA.

[0047] 9) Carefully discard the supernatant and add 1 mL of 75% ethanol (RNase-free ddH2O). Gently flick the bottom of the tube to suspend the precipitate, invert it several times, and let it stand at room temperature for 3-5 minutes.

[0048] 10) Centrifuge at 12000 rpm at 4°C for 5 minutes and discard the supernatant.

[0049] 11) Dry the precipitate in an open air at room temperature for 2-5 minutes.

[0050] 12) Add 40-60 μL of RNase-free ddH2O to dissolve the precipitate. After it is completely dissolved, take 1 μL and detect the RNA concentration using Nanodrop and store it in a -80℃ refrigerator.

[0051] Example 3: Reverse transcription of RNA into cDNA

[0052] By Novozyme IIQ RT SuperMix for qPCR (+gDNA wiper) (R223-01) kit steps.

[0053] Genomic DNA Removal:

[0054] Prepare the following system in an RNase-free centrifuge tube:

[0055]

[0056] Mix by gently pipetting. Incubate at 42°C for 2 minutes.

[0057] The reverse transcription reaction system was prepared as follows:

[0058]

[0059] Mix well by pipetting. 50℃ for 15min, 85℃ for 5s.

[0060] Example 4: Full-length cloning of cucumber CsaTRM4 and pumpkin CmoCK1 CDS

[0061] Arabidopsis thaliana 5 The sequence of C methyltransferase AtTRM4B was obtained, and its homologous gene was searched in the melon genome database and named CsaTRM4. Cloning primers were designed according to its CDS sequence.

[0062] The designed primer sequences are as follows:

[0063] Upstream primer 5′-ATGGGTGGTAAGGGTAGGGG-3′ (SEQ ID NO. 3);

[0064] Downstream primer: 5′-TTACAAATCATTTTCAAGTC-3′ (SEQ ID NO. 4).

[0065] It is known that pumpkin rootstock CmoCK1 mRNA is transported to cucumber scion under low temperature stress, and primers were designed based on its CDS sequence to clone its full length.

[0066] The designed primer sequences are as follows:

[0067] Upstream primer 5′-ATGGCAATAAAGTCAAACGG-3′ (SEQ ID NO. 5);

[0068] Downstream primer: 5′-TTAGGAGGCATTCATCATTA-3′ (SEQ ID NO. 6).

[0069] The above primers were synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0070] Use cDNA or plasmid as template and high-fidelity DNA polymerase premix for PCR. The amplification system is as follows:

[0071] Amplification system (50 μL)

[0072]

[0073] After mixing by pipetting, centrifuge to the bottom of the tube and perform PCR. The PCR procedure is as follows:

[0074] Reaction conditions

[0075]

[0076] PCR product detection: Prepare 1% agarose gel according to the target fragment size, add nucleic acid dye (1 / 10,000), 1×TAE electrophoresis buffer, electrophoresis at 110-120V voltage for about 25 minutes, and detect the size of PCR product fragment under ultraviolet light ( Figure 1 ). The target strip is cut and recycled.

[0077] Example 5: CsaTRM4 phylogenetic analysis

[0078] The genome databases used for CsaTRM4 homology comparison and sequence acquisition were Cu GenDB (http: / / cucurbitgenomics.org / ), TAIR (https: / / www.arabidopsi s.org / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ). After obtaining the homologous sequences of cucumber, Arabidopsis and humans, MEGA-X was used to perform nucleic acid and amino acid sequence comparison analysis, and TBtools software was used to draw the evolutionary tree. The results are shown in the figure. Figure 2 As shown, the analysis showed that cucumber has 5 Nsun RNA methyltransferase family genes, which belong to 5 subfamilies respectively. Among them, there is only one cucumber gene, CsaTRM4, in the same subfamily as the model plant Arabidopsis thaliana AtTRM4B.

[0079] Example 6: CsaTRM4 promoter analysis

[0080] TBtools was used to extract the gene promoter sequence (2000bp upstream of the 5' end) in the CsaTRM4 genome, and then the MEME plug-in on TBtools was used to analyze the possible cis-acting elements on the promoter and generate images. It was found that the CsaTRM4 promoter contained cis-acting elements such as light response, abscisic acid response, gibberellin response, zein metabolism regulation, salicylic acid response, low temperature response and drought induction ( Figure 3 ), suggesting that CsaTRM4 can respond to stress and participate in the regulation of various biological processes.

[0081] Example 7: CmoCK1 gene structure and m 5 C methylation site analysis

[0082] Tbtools was used to extract the exon and intron regions of pumpkin CmoCK1 gene from the gff3 file and draw the map. The methylation sites on CmoCK1 mRNA were queried on the CUCUME website and it was found that CmoCK1 contained m 5 C methylation site, located in the 3' region of CDS (906-1086 bp) ( Figure 4 ).

[0083] Example 8: Agrobacterium-mediated transient transformation of tobacco leaves

[0084] Vector construction

[0085] The present invention adopts the Gateway method to construct a plant expression vector. First, an intermediate vector containing the target gene is constructed. The experiment uses the Novozymes recombinant cloning kit:

[0086] Seamless cloning reaction system (10μL)

[0087]

[0088] Mix the system by pipetting, centrifuge at low speed, react at 37℃ for 30 minutes, and cool to 4℃ on ice to transform competent E. coli. Pick a single clone and send it for sequencing. After sequencing is correct, culture it in a large volume with shaking to extract the plasmid.

[0089] The pENTR4 vector was connected to the target gene fragment by seamless cloning to successfully construct an intermediate vector and then perform LR reaction.

[0090] LR reaction system (5 μL)

[0091]

[0092] Incubate at 25°C in a metal bath for 1 hour, add 1 μL of proteinase K, and terminate the reaction at 37°C for 10 minutes. The construction of the Gateway vector is completed.

[0093] Plasmid transformation into Agrobacterium competent cells

[0094] The experiment used the Agrobacterium competent cells GV3101 of Weidi Biotechnology, and the successfully constructed Gateway vector and GV3101 competent cells were mixed in a centrifuge tube, incubated on ice for 5 minutes, in liquid nitrogen for 5 minutes, at 37°C for 5 minutes, and on ice for 5 minutes, and then added to the non-resistant LB liquid medium for shaking culture at 28°C for 3 hours, centrifuged at 5000rpm for 1 minute to collect the bacteria, and applied them on the resistance medium plate containing kanamycin and rifampicin to screen positive clones. That is, the 35S::YFP-CsaTRM4 overexpression vector and the 35S::YFP-CmoCK1 overexpression vector were successfully constructed.

[0095] Tobacco cultivation

[0096] The seeds of Nicotiana benthamiana were sown on the substrate, covered with film, and placed in a light incubator. After about one week, the tobacco seedlings with good growth were transplanted into nutrient pots of 10 cm square for cultivation. The incubator temperature was 25℃ / 18℃, and the light intensity and photoperiod were the same as those of cucumbers.

[0097] Tobacco leaf transient transformation

[0098] When the tobacco is 4 weeks old, add the Agrobacterium GV3101 containing the target gene fragment into LB liquid medium containing the corresponding resistance and shake and culture until the OD600 =0.6-0.8, centrifuged at 4000 rpm for 8 minutes to collect the bacteria, and then inoculated with an equal volume of infection solution (10 mM MES, 100 μM AS and 10 mM MgCl 2 ) Resuspend the bacteria and incubate at 28℃ in the dark for 3 hours. Use a 1mL syringe without a needle to inject 100μL of the infection solution containing the corresponding Agrobacterium to the back of the 1st, 3rd, and 5th leaves. Be careful that the bacterial solution cannot penetrate into the main vein. Spray deionized water to wash away the excess bacterial solution. After 12 hours in the dark, grow under normal conditions for about 60 hours and observe the fluorescence ( Figure 5 ) or sampling. The 1st, 3rd, and 5th leaves were mixed and sampled as injection leaf samples, and the 2nd, 4th, and 6th leaves were mixed and sampled as test leaf samples (n≥6). 48 hours after tobacco injection, RNA from injection leaves and test leaves were taken and RT-PCR was performed. The specific transient expression treatment combinations are shown in the table below.

[0099] Transient expression of tobacco treatment combinations

[0100]

[0101] Example 9: Analysis of CmoCK1 mRNA transport in tobacco transient transformation system

[0102] The transportability of CmoCK1 was identified by transient expression in tobacco leaves under different treatment conditions. The successfully constructed 35S::YFP-CsaTRM4 overexpression vector and 35S::YFP-CmoCK1 overexpression vector were transformed into Agrobacterium and co-expressed in tobacco (CsaTRM4+CmoCK1). Compared with tobacco with only 35S::YFP-CmoCK1 transiently overexpressed, the long-distance transportability of CmoCK1 mRNA in non-injected tobacco leaves was detected by RT-PCR ( Figure 6 A). Specifically, RT-PCR was used to detect the expression of YFP-CmoCK1, YFP-CsaTRM4 and Basta R gene fragments in the injected leaves and test leaves of co-expressing (CsaTRM4+CmoCK1) tobacco, respectively; RT-PCR was used to detect the expression of YFP-CmoCK1 and Basta R gene fragments in the injected leaves and test leaves of tobacco expressing only CmoCK1. Basta R is a gene that is not transported on the vector and serves as a contamination control. NtActin is used as a control for RNA extraction quality and loading amount. The absence of Basta R gene expression in the test leaves indicates that bacterial contamination has been excluded, and the expression of YFP-CmoCK1 in the test leaves indicates that it is transported from the injected leaves. Transport efficiency = (the number of tobacco transported by YFP-CmoCK1 / the number of all tobacco treated with transient expression) × 100%. The primers used are:

[0103] YFP-CmoCK1 upstream primer 5′-CCCGACAACCACTACCTGAG-3′ (SEQ ID NO. 7);

[0104] YFP-CmoCK1 downstream primer 5′-TGGCCTTTCGTGAATCCGTT-3′ (SEQ ID NO. 8).

[0105] Basta R upstream primer 5'-AAACCCACGTCATGCCAGTT-3' (SEQ ID NO. 9);

[0106] Basta R downstream primer 5'-CATCGAGACAAGCACGGTCA-3' (SEQ ID NO. 10).

[0107] YFP-CsaTRM4 upstream primer 5′-GAGCAAAGACCCCAACGAGA-3′ (SEQ ID NO. 11);

[0108] YFP-CsaTRM4 downstream primer 5′-ACTCATTGAACGCGGGACTT-3′ (SEQ ID NO. 12).

[0109] NtActin upstream primer 5'-CTGAGAGATTCCGCTGC-3' (SEQ ID NO. 13);

[0110] NtActin downstream primer: 5′-GAGGACAATGTTTCCGTAC-3′ (SEQ ID NO. 14).

[0111] The results showed that CsaTRM4 itself could not transport, and overexpression of CsaTRM4 increased the transport efficiency of CmoCK1 ( Figure 6 Middle B). This indicates that CsaTRM4 promotes the long-distance transport of CmoCK1 mRNA.

[0112] Example 10: Construction of methyltransferase CsaTRM4 CRISPR / Cas9 vector

[0113] The CsaTRM4 gene editing target was designed using the CRISPR-P 2.0 website, and the CsaTRM4-CRISPR gene editing vector was constructed. Specifically, the target gene sequence was searched in the Cucurbita genome database (CuGenDB), and two suitable target sites were selected using the CRISPR-P 2.0 website. Two pairs of primers, DT1-BsF / R and DT1-F 0 / R 0 Four-primer PCR amplification was performed using pCBC-DT1T2 plasmid as template.

[0114] The designed primer sequences are as follows:

[0115] DT1-F 0 5'-TGATGAAATCTCTTCAAGCCGGTTTTAGAGCTA GAAATAGC-3'(SEQ ID NO.15)

[0116] DT1-R 0 5'-AACAACGTTCTCTCTGTTGTCGCAATCTCTTAGT CGACTCTAC-3'(SEQ IDNO.16)

[0117] DT1-BsF 5'-ATATATGGTCTCGATTGATGAAATCTCTTCAAG CCGGTT-3'(SEQ ID NO.17)

[0118] DT1-BsR 5'-ATTATTGGTCTCGAAACAACGTTCTCTCTGTTG TCGCAA-3'(SEQ ID NO.18)

[0119] Amplification system (50 μL)

[0120]

[0121] Mix well by pipetting, centrifuge briefly, and then perform PCR reaction.

[0122] PCR procedure

[0123]

[0124] The PCR products were run on gel for identification and recovery. The gel-recovered products were subjected to restriction digestion-ligation treatment as follows:

[0125] Enzyme digestion-ligation system (20 μL)

[0126]

[0127] After mixing the system, centrifuge and perform PCR.

[0128] PCR procedure

[0129]

[0130] The PCR product was purified and recovered.

[0131] Example 11: Cucumber genetic transformation

[0132] Preparation of relevant culture media

[0133] Germination medium (GM): Add 6-benzylaminopurine (BA) at a final concentration of 0.5 mg / L and abscisic acid (ABA) at a final concentration of 1 mg / L to MS solid medium.

[0134] Inoculation medium (IM): MS liquid medium supplemented with 0.5 mg / L BA, 1 mg / L ABA, 200 μM acetosyringone (AS) and 1.25 M morpholineethanesulfonic acid (MES) (pH 5.2).

[0135] Co-cultivation medium (COM): 0.5 mg / L BA, 1 mg / L ABA, 200 μM AS, 1.25 mM MES (pH 5.2), and 250 μM LA were added to MS solid medium.

[0136] Bud induction medium (SIM): 0.5 mg / L BA, 2 mg / L AgNO 3 , 1 mg / L ABA and 200 mg / L timentin were added to MS solid medium.

[0137] Root induction medium (RIM): 200 mg / L Tim and 2 mg / L AgNO 3 Add to MS solid medium.

[0138] Sowing and germination

[0139] Sowing: Take a certain number of cucumber seeds, soak them in 55℃ distilled water for 30 minutes, then place them at room temperature for 1-2 hours, and then peel off the outer seed coat with tweezers. Sterilize the cucumber seeds in a clean bench: add 75% alcohol and shake them upside down for 30 seconds, discard the alcohol and wash them once with distilled water, add 3% NaClO solution, shake them in a shaker for 10 minutes, and wash them 5 times with distilled water. After disinfection, place the seeds on the germination medium, about 25 seeds per plate. Incubate the plates at 28℃ in the dark for 36 hours.

[0140] Agrobacterium preparation

[0141] The successfully constructed CRISPR / Cas9 plasmid and GV3101 competent cells were mixed in a centrifuge tube, incubated on ice for 5 minutes, in liquid nitrogen for 5 minutes, at 37°C for 5 minutes, and on ice for 5 minutes. Then, non-resistant LB liquid culture medium was added and shaken at 28°C for 3 hours. The bacteria were collected by centrifugation at 5000 rpm for 1 minute and spread on resistance culture medium plates containing kanamycin and rifampicin to screen positive clones.

[0142] Pick a single clone and culture it in LB liquid medium containing the corresponding antibiotics at 28°C under shaking conditions. 600When the OD was between 0.6 and 0.8, the Agrobacterium was collected by centrifugation at 4000 rpm for 8 minutes and washed once with 10 mL of MS liquid medium. The cells were centrifuged at 4000 rpm for 8 minutes and resuspended in MS liquid and diluted to OD 600 The value should be kept between 0.2-0.3 for future use.

[0143] Explant preparation

[0144] In a clean bench, use tweezers to clean the radicle of the germinated cucumber seeds prepared earlier, taking care not to damage the surrounding tissues. Then cut off one-third of the cotyledon away from the radicle, remove the endocarp, and completely separate the two cotyledons and place them in MS liquid culture medium for later use.

[0145] Agrobacterium infection of explants

[0146] Use a syringe to vacuum infect twice, 1.5 minutes each time. After the infection, pour out the bacterial solution, spread the explants flat on the filter paper, and gently absorb the attached bacterial solution. Then place them neatly on the IMS differentiation medium. After sealing, co-culture at 25℃ in the dark for 3 days.

[0147] Explant differentiation culture

[0148] The explants were transferred to differentiation medium, approximately 9 explants per plate, and cultured in a tissue culture chamber for approximately 3 weeks.

[0149] Bud resistance screening and rooting culture

[0150] Cut off the newly grown shoots of the cucumber explants in the previous step and insert them into screening medium (differentiation medium with 0.006% Basta) for culture for three days. After three days, the shoots that grow well and do not turn yellow may be Basta-resistant. Move these shoots to rooting medium and subculture until a large number of roots are produced.

[0151] Acclimation and transplanting

[0152] When the rooted buds continue to grow and develop into new plants, open the culture bottle. After several days, take out the seedlings from the culture bottle, wash off the culture medium on the roots, transplant them into a culture pot filled with sterilized matrix, cover them with a fresh-keeping bag to keep them moist, and grow them in a light incubator until acclimation is completed. After the seedlings survive, they are transplanted in a solar greenhouse and subsequently cultivated and managed normally.

[0153] Positive seedling identification

[0154] Take fresh leaves to extract DNA, perform PCR on the sequence near the gene editing site, connect the product to the T vector and transform Escherichia coli, spread the plate and pick 10-15 single clones for sequencing to detect whether it is a positive plant and the mutation type.

[0155] Three T0 generation mutant plants were obtained by using the cucumber stable transgenic technology system. After screening and identification, two T1 generation stable homozygous mutants, T1#1 and T1#2, were obtained ( Figure 7 In A and B), the editing types are a 30-base deletion and a 1-base addition in the CDS region of CsaTRM4, respectively.

[0156] Example 12: Determination of plant height and stem diameter of Csatrm4 mutant cucumber

[0157] The T2 generation seeds obtained by self-pollination of the T1 generation were sown and cultivated in a greenhouse and managed normally to observe the phenotypic changes of the Csatrm4 mutant. The height and stem diameter of the cucumber plants were measured at the 20-leaf age. The plant height is the distance from the cotyledon to the plant growth point, measured with a tape measure, and the stem diameter is the stem diameter 1 cm below the cotyledon, measured with a vernier caliper. It was found that there was no significant difference between the mutant and the wild type in either plant height or stem diameter ( Figure 7 Middle C).

[0158] Example 13: Transformation of hairy roots of WT and Csatrm4 mutant cucumbers with CmoCK1 mRNA

[0159] The overexpression vector 35S::YFP-CmoCK1 constructed in Example 8 was transformed into K599 Agrobacterium rhizogenes. The successfully constructed K599 Agrobacterium rhizogenes was used to transform WT and Csatrm4 mutant cucumbers respectively, and the specific method was as follows:

[0160] 1) Sowing: Seed soaking and disinfection are the same as in Example 10. The disinfected seeds are then placed in tissue culture bottles containing MS culture medium, with about 25 seeds per bottle, and cultured at 25° C. in the dark.

[0161] 2) Explant acquisition: After culturing for about 4 days, when the cotyledons are about to unfold, the seedlings are taken out on a clean bench, half of the cotyledons are cut off, and the growth point and the other half of the cotyledons connected to it and the hypocotyl of about 0.5 cm are retained. This part is used as the explant for transformation.

[0162] 3) Infection: Activate Agrobacterium the day before, and when the bacterial solution is shaken at 28°C to OD600 = 0.6-0.8, collect the bacteria by centrifugation at 4000 rpm for 8 minutes, resuspend the bacteria in MS liquid medium to OD600 = 0.1, and add 200 μM acetosyringone. Place the cucumber explants in the infection solution and shake at 200 rpm for 20 minutes.

[0163] 4) Co-cultivation: After infection, place the explants on sterile filter paper to absorb excess bacterial solution, place a piece of sterile filter paper on MS solid culture medium, and co-cultivate at 25°C in the dark for 4 days.

[0164] 5) Root induction: After co-cultivation, the hypocotyls of the explants were inserted into MS solid culture medium containing 200 mg / L timentin and then placed in a tissue culture room to induce hairy roots.

[0165] After culturing for about 1 week, the fluorescence of the hairy roots was observed under the GFP channel using LUYOR-3415RG, and the fluorescent roots were transformed into transgenic positive plants ( Figure 8 ). Remove the non-fluorescent roots and transfer them to culture flasks containing new culture medium.

[0166] Example 14: Analysis of CmoCK1 mRNA transport in WT and Csatrm4 mutant cucumber hairy roots-aboveground

[0167] The roots and new leaves of the above-mentioned hairy root transformation-positive cucumbers were extracted, and RAN was extracted. CsaActin, Basta R and YFP-CmoCK1 gene fragments were amplified by RT-PCR, similar to Example 8, with CsaActin as a control and Basta R as a contamination control. The RT-PCR primer sequence of CsaActin is as follows:

[0168] CsaActin-F 5'-ATGAGCTTCGTGTTGCACCT-3' (SEQ ID NO. 19);

[0169] CsaActin-R 5'-TCACACTTCATGATTGAGTTGTAGG-3' (SEQ ID NO. 20).

[0170] In the roots of transgenic positive plants, Basta R and YFP-CmoCK1 were constitutively expressed, while in the newly grown leaves above ground, Basta R was not expressed but YFP-CmoCK1 was expressed, indicating that the aboveground part of this cucumber plant was not contaminated by bacteria and that YFP-CmoCK1 mRNA was transported from the roots to the aboveground part. The transport efficiency of YFP-CmoCK1 in WT (n=17), Csatrm4#1 (n=16) and Csatrm4#2 (n=18) was calculated, respectively. Transport efficiency = (the number of cucumber plants transported by YFP-CmoCK1 / the number of cucumber plants transformed with transgenic positive roots) × 100%. Results The average transport efficiency of YFP-CmoCK1 in WT, Csatrm4#1 and Csatrm4#2 was 40.1%, 12.3% and 11.1%, respectively. Fig. 9 ). This indicates that the CsaTRM4 mutation reduces the transport efficiency of CmoCK1, which indirectly proves that CsaTRM4 enhances the transport function of CmoCK1.

[0171] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Claims

1. An application of cucumber CsaTRM4 gene, characterized in that: Overexpression of the cucumber CsaTRM4 gene in plants can promote the long-distance transport of pumpkin CmoCK1 mRNA between rootstock and scion; The nucleotide sequence of the cucumber CsaTRM4 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cucumber CsaTRM4 gene is shown as SEQ ID NO.2.

Images