A gene regulating the degradation of ethylene receptor RhETR3 and its application

Abstract

It is of great significance to study the mechanism of flower opening regulated by ethylene and then use it to maintain the ornamental value of cut roses. The present invention provides a gene for regulating the degradation of ethylene receptor RhETR3 and its application. The gene and the application of the gene can provide a basis and direction for the screening and directional cultivation of early and late flowers of roses. The gene sequence is SEQ ID NO.1. By molecular regulation of the gene of the plant, the sensitivity of the plant to ethylene can be significantly increased or decreased, thereby extending or shortening the flowering time of the plant. By microscopic regulation or observation of the gene, the presence of abnormal flowering plants can be reduced by reducing the ethylene sensitivity of the plant or screening the ethylene sensitivity of the plant.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a gene for regulating the degradation of ethylene receptor RhETR3 and its application. Background Art

[0002] Flowering of plants is the process of transition from vegetative growth to reproductive growth in higher plants, and is the central link of individual development and offspring reproduction. This developmental transition is determined by the developmental conditions of the plant itself and external environmental factors. While studying artificial mutations of specific genes in model plants, people have also conducted a lot of research on natural early-flowering mutants. The study of natural early-flowering mutants has enriched the means and content of studying the mechanism of plant vernalization and the flowering process, and is of great significance for improving and promoting the study of the mechanism of plant vernalization. Physiological and genetic studies on the flowering of the model plant Arabidopsis thaliana have shown that the factors affecting plant flowering are mainly divided into four pathways: photoperiod pathway, hormone pathway, autonomous pathway and vernalization pathway.

[0003] Rose is one of the most important crops in the floriculture industry. It is widely used as cut flowers, potted flowers and garden flowers. It is a vital crop in the floriculture industry. The economic value of roses is often reflected in the quality of their flowers. If there are problems such as abnormal flowering and premature flowering during transportation, the roses will lose their value, thus causing economic losses.

[0004] In the post-harvest circulation link, ethylene in the environment and ethylene produced by the flowers themselves during transportation are often the main reasons for abnormal opening of rose flowers, resulting in serious post-harvest losses. According to statistics, quality deterioration caused by ethylene accounts for more than 70% of post-harvest physiological losses.

[0005] Ethylene plays an important role in the hormone pathway that affects plant flowering. Unlike hormones such as gibberellins that are involved in plant growth regulation, ethylene does not directly regulate the opening of flowers, but opens by allowing flowers to sense ethylene. Existing research results show that there are genes in roses that regulate flower opening: two ethylene receptors (RhETR1, RhETR3) and two CTR genes (RhCTR1, RhCTR2).

[0006] Since there are few studies on ethylene regulatory pathways in roses, we first constructed the basic pathway of ethylene signal transduction based on the screening of ethylene mutants in Arabidopsis and combined with genetic epistasis analysis. Ethylene receptors are the primary link in the ethylene signaling pathway and are negative regulators of the ethylene signaling pathway. Studies in Arabidopsis have found that the expression of family II ethylene receptor AtETR2 is induced by ethylene, but at 100 ppm, the ETR2 protein is degraded; among the six receptor genes in tomatoes, the expression levels of LeETR3 / NR, LeETR4 and LeETR6 genes increase during fruit ripening, and high concentration ethylene treatment promotes their expression. However, the content of LeETR3 / NR, LeETR4 and LeETR6 proteins continues to decrease during ripening, and ethylene treatment leads to further degradation of these three receptor proteins; 200 μM CHX treatment in tobacco promotes NTHK1 protein degradation, and this degradation can be inhibited by MG132. It is speculated that the ubiquitin-proteasome degradation system plays a key role in the ethylene-dependent regulation of ethylene receptor stability.

[0007] Family II ethylene receptor RhETR3 is the main receptor that responds to ethylene during the opening of flowers. As the flowers open, the expression level of RhETR3 gradually increases, but its protein level gradually decreases. While ethylene induces the expression of RhETR3, it accelerates the degradation of its protein, and this degradation can be inhibited by MG132. This indicates that RhETR3 is degraded through the 26S proteasome pathway, but it is not clear how it is ubiquitinated and degraded, thereby affecting the ethylene sensitivity of rose flowers.

[0008] Due to the vast territory of my country and the large import and export volume of cut roses, the fresh cut roses produced from the production site need to be transported to the consumption site by cold chain over long distances, which affects the normal opening of cut roses, especially the cut roses that leave the mother body, and the phenomenon of "dead flowers" often occurs. Therefore, it is of great significance to study the flower opening mechanism regulated by ethylene and then use it to maintain the ornamental value of cut roses. Based on this, the present invention provides a gene RhUBC32 that regulates the degradation of ethylene receptor RhETR3, which can affect the pathway of plant perception of ethylene and thus change the sensitivity of plants to ethylene. By reducing the sensitivity of plants to ethylene and increasing the influence of ethylene on plants during transportation, it is avoided that the plants are affected by their own hormones or external hormones after leaving the mother body and bloom early or abnormally.

[0009] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0010] In view of the deficiencies of the prior art, the present invention provides a gene RhUBC32 that regulates the degradation of the ethylene receptor RhETR3. The gene sequence is SEQ ID NO.1. Ethylene receptor is a key protein that senses ethylene and plays a key role in determining plant ethylene sensitivity and regulating ethylene signals. Rose cut flowers are an ornamental crop that is very sensitive to ethylene, and their growth and development as well as the opening and aging of flowers after harvest are significantly affected by ethylene. The present invention provides a gene that changes the sensitivity of plants to ethylene and explores its role in plants. Through molecular regulation of the gene in plants, it is found that the sensitivity of the regulated plants to ethylene is significantly increased or decreased, thereby extending or shortening their flowering time. Through microscopic regulation or observation of the gene, it is found that the gene can reduce the presence of abnormal flowering plants by reducing the ethylene sensitivity of plants or screening the ethylene sensitivity of plants.

[0011] The present invention provides a protein RhUBC32 for regulating the degradation of ethylene receptor RhETR3. The amino acid sequence of the protein is SEQ ID NO.2.

[0012] The present invention provides a method for cultivating a plant with a high ethylene sensitivity phenotype. The method comprises:

[0013] A 500 bp fragment is selected from the coding region containing the gene, and the target fragment is inserted into the pFGC1008 vector to obtain a silencing vector; the silencing vector is used to transform Agrobacterium to obtain an Agrobacterium genetically engineered bacterium containing the silencing vector; the Agrobacterium genetically engineered bacterium is used to infect rose embryonic callus to obtain a mutant strain with low expression of the gene.

[0014] According to a preferred embodiment, the nucleotide sequences of the primers used for cloning the target fragment in the forward direction are SEQ ID NO.37 and SEQ ID NO.38.

[0015] According to a preferred embodiment, the nucleotide sequences of the primers used for cloning the target fragment in the reverse direction are SEQ ID NO.39 and SEQ ID NO.40.

[0016] The present invention provides a Rosaceae plant with a high ethylene sensitivity phenotype. The gene is lowly expressed in the Rosaceae plant.

[0017] The invention provides a Rosaceae plant with a high ethylene sensitivity phenotype. The protein encoded by the gene is lowly expressed in the Rosaceae plant.

[0018] The present invention provides a biological material containing the gene. Preferably, the biological material is an expression vector. Preferably, the biological material is a strain.

[0019] According to a preferred embodiment, the expression vector can be a pTRV2 vector.

[0020] According to a preferred embodiment, the strain can be Agrobacterium.

[0021] According to a preferred embodiment, the plant of the Rosaceae family can be rose, apple or pear.

[0022] The present invention provides the application of the gene RhUBC32 for regulating the degradation of the ethylene receptor RhETR3 in the following aspects:

[0023] (1) Regulate the opening and aging process of plant flowers;

[0024] (2) Regulating ethylene-promoted flower opening in plants;

[0025] The plant hormone ethylene regulates the growth and development of various plants and participates in the aging of plants, such as accelerating the aging of leaves and flowers, fruit ripening and organ shedding. According to a preferred embodiment, since the gene provided by the present invention can participate in regulating the sensitivity of plants to ethylene, the growth state of plants affected by ethylene will also change due to the different expression levels of the gene provided by the present invention.

[0026] According to a preferred embodiment, in specific application, the plant expression vector containing the gene RhUBC32 that regulates the degradation of the ethylene receptor RhETR3 is introduced into rose cells or seeds to overexpress the gene in the rose, thereby obtaining transgenic plants with significantly extended flowering period of flowers.

[0027] RhETR3 is the key receptor for rose flowers to sense ethylene. The present invention uses pBT-STE-RhETR3 as bait to screen the split ubiquitination library and obtains a ubiquitin-binding enzyme RhUBC32. Yeast two-hybrid analysis, fluorescent bimolecular complementation analysis (BiFC), fluorescent colocalization analysis and co-immunoprecipitation (Co-IP) analysis show that RhUBC32 and RhETR3 are co-localized in the lipid membrane, and the binding domain is a transmembrane domain. Transient overexpression of RhUBC32 and RhETR3 genes in tobacco leaves accelerated the degradation of RhETR3 protein. Ethylene promotes its degradation and this degradation process can be inhibited by MG132. After the putative active site in the UBC domain of RhUBC32 was mutated (RhUBC32 C92-A) lost the ability to degrade RhETR3. Protein structure analysis showed that RhUBC32 had a typical UBC domain. In vitro ubiquitin activity analysis showed that RhUBC32 had ubiquitin-binding enzyme activity. The RhUBC32 gene was significantly induced by ethylene, and the expression level gradually increased with the opening of the flowers, reaching the highest expression level in level 5 flowers. In roses, the expression of the RhUBC32 gene was silenced. After ethylene treatment, 95% of the flowers in the silent plants showed ethylene hypersensitive phenotypes such as petal shedding and deformed eversion, which was significantly higher than 39% in the TRV2 control group. The ethylene sensitivity of rose flowers increased significantly. In the roses stably transgenic with RhUBC32 RNAi, the number of branches was significantly higher than that of the wild type, and the expression of the ethylene response gene RhEBF2 in level 2 flowers was significantly higher than that in the control group. After ethylene treatment of rose flowers, RhETR3 protein showed accumulation, petals fell off, and showed an ethylene hypersensitive phenotype. In this application, level 5 flowers refer to fully open flowers.

[0028] The above study reveals the role of the RhUBC32 gene in ethylene-regulated flower opening, provides new ideas and technical means for regulating the opening of plant flowers, especially the opening of rose fresh cut flowers, and has great academic value and important production and application prospects. The gene and the application of the gene provided by the present invention can provide a molecular biological basis for the study of abnormal flowering of roses. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A This is a diagram of the RhUBC32 protein sequence analysis;

[0030] Figure 1B This is a schematic diagram of the evolutionary tree of RhUBC32;

[0031] Figure 2A This is the result diagram of the split ubiquitination library;

[0032] Figure 2B This is the result diagram of RhUBC32 subcellular localization;

[0033] Figure 2C This is the result of BIFC analysis of the interaction between RhETR3 and RhUBC32;

[0034] Figure 2D This is the result of MH2Y analysis of the interaction between RhETR3 and RhUBC32;

[0035] Figure 2E This is the result diagram of the Co-IP method analysis of the interaction between RhUBC32 and RhETR3;

[0036] Figure 3A is the analysis diagram of RhUBC32 expression pattern;

[0037] Figure 3B This is a diagram for in vitro analysis of the ubiquitin conjugating enzyme activity of RhUBC32;

[0038] Figure 3C This is a semi-in vitro analysis diagram of the ubiquitin-conjugating enzyme activity of RhUBC32;

[0039] Figure 4A This is the result diagram of co-expression of RhUBC32 and RhETR3;

[0040] Figure 4B A diagram showing that MG132 inhibits RhUBC32 from degrading RhETR3;

[0041] Figure 4C This is a diagram showing the effect of RhUBC32, which has lost its enzyme active site, on RhETR3;

[0042] Figure 4D This is the LCI result diagram;

[0043] Figure 5A This is the structure diagram of the RhUBC32 silencing vector;

[0044] Figure 5B The statistical diagram of RhUBC32 expression in silenced plants and control plants;

[0045] Figure 5C The phenotypes of silenced plants and control plants are shown in Figure 2.

[0046] Figure 5D It is the statistical diagram of phenotypic data of silenced plants and control plants;

[0047] Fig. 6A The statistical diagram of RhUBC32 expression in RhUBC32-RNAi transgenic plants;

[0048] Figure 6B Schematic diagram of the plant type of RhUBC32-RNAi transgenic plants;

[0049] Figure 6C Schematic diagram of ethylene sensitivity analysis of RhUBC32-RNAi transgenic plants;

[0050] Fig.6D Schematic diagram of the expression level of RhEBF2 gene in RhUBC32-RNAi transgenic plants;

[0051] Fig. 6E Schematic diagram of the effect of RhUBC32-RNAi transgenic plants on the stability of RhETR3 protein. DETAILED DESCRIPTION

[0052] The following is a detailed description with reference to the accompanying drawings.

[0053] The invention relates to growth conditions of rose tissue culture seedlings and cultivation conditions of tobacco.

[0054] Rose tissue culture seedling growth and cultivation method:

[0055] Cut the stem segments of rose tissue culture seedlings and insert them into rose propagation medium. After growing for 4 to 6 weeks, move the strong seedlings with a length of 3 to 5 cm to the rooting medium, and continue to cut the stem segments of the remaining seedlings and place them in the new propagation medium for culture.

[0056] Tobacco cultivation methods:

[0057] Nicotiana benthamiana was selected as the breeding variety, and the seeds were preserved in this laboratory;

[0058] Sow tobacco seeds in the soil (with sufficient water), and after 3 to 4 days, the seeds will germinate;

[0059] Transplant the seedlings into pots after 5-6 days, 1 seedling per pot, and water them with nutrient solution (5-6 days / time) to ensure the normal growth of tobacco;

[0060] After about 4 to 5 weeks, the 3rd to 5th leaves can be used for protein expression injection experiments, wherein the light conditions for tobacco growth are 16h light / 8h dark cycle supply, and the optimal growth temperature is 23℃±2℃.

[0061] The present invention also relates to the following experimental operations:

[0062] 1. RhUBC32 gene cloning

[0063] Based on the partial sequence of RU05948 obtained from the screening library, the sequence fragment of the RhUBC32 gene was obtained by comparing it with the rose RNA-seq database;

[0064] The full-length gene sequence was obtained using homologous cloning.

[0065] 2. Protein sequence analysis

[0066] The full-length amino acid sequence of RhUBC32 was aligned with that of Arabidopsis thaliana UBCc using the ClustalW program. Phylogenetic analysis was performed using the neighbor-joining method with MEGA 7 replicates.

[0067] 3. qRT-PCR analysis

[0068] Total RNA was extracted from rose petals using the hot borate method;

[0069] The first-strand cDNA was synthesized using HiScript II Q RT SuperMix for qPCR reverse transcription kit with 1 μg of total RNA as template;

[0070] qRT-PCR reactions (20 μl volume containing 1 μl cDNA template) were performed using the StepOne Real-Time PCR System (Applied Biosystems) with the KAPASYBR FASTUniversal qRT-PCR kit (Kapa Biosystems), in which the rose RhUBI gene (GenBank accession number JK622648) was used as an internal reference gene;

[0071] Each reaction was performed in triplicate and verified by melting curve analysis;

[0072] All reactions were performed with at least three biological replicates.

[0073] PCR primers are listed in Table 1. The qRT-RhUBC32 gene primer sequences are SEQ ID NO.9 and SEQ ID NO.10. The qRT-RhUBI2 gene primer sequences are SEQ ID NO.11 and SEQ ID NO.12.

[0074] 4. Tobacco injection

[0075] Resuspend the Agrobacterium carrying the target gene in the infection solution and adjust the OD 600 =1.5, silencing suppressor P19 adjusted the concentration OD 600 =1.0;

[0076] According to experimental needs, mix equal volumes and let stand at room temperature for 3 h;

[0077] Select leaves that are growing well, dark green, and thick, and inject with a 1ml syringe until the entire leaf is filled with bacterial solution;

[0078] Samples were taken 3 days after injection to extract total protein or stored at -80°C for later experiments.

[0079] 5. RhUBC32 promotes RhETR3 protein degradation in vivo

[0080] RhETR3-Flag was respectively combined with RhUBC32-GFP and RhUBC32 C93A -GFP and super1300-GFP were co-injected into tobacco at different ratios and expressed for 3 d;

[0081] Sampling was performed, total protein was extracted, and the level of RhETR3-Flag protein was detected.

[0082] 6. Colocalization Experiment Methods

[0083] RhUBC32-GFP was mixed with RhETR3-mCherry and PM-mCherry in equal volumes;

[0084] Co-infection of tobacco leaves for subcellular localization;

[0085] Three days after the co-injection of tobacco leaves, the fluorescence of the leaves was observed under a fluorescence confocal microscope.

[0086] The primer sequences of RhETR3-1300 are SEQ ID NO.23 and SEQ ID NO.24.

[0087] The primer sequences of RhUBC32-1300C are SEQ ID NO.25 and SEQ ID NO.26.

[0088] The sequences of the point spike primers for Rhubc32 are SEQ ID NO.35 and SEQ ID NO.36.

[0089] 7. Bimolecular fluorescence complementation (BIFC)

[0090] RhETR3-YNE(R), RhUBC32-YCE(M), pYCE(M) vectors and silencing suppressor P19 were transformed into Agrobacterium GV3101 respectively;

[0091] Agrobacterium containing the target plasmid was cultured overnight with shaking (10 mL) at 5000 rpm for 8 min to collect the bacteria;

[0092] Adjust OD with tobacco infection solution 600 =3.0, RhETR3-YNE(R) was mixed with other YCE(M) bacteria in equal volumes, shaken at 200 rpm for 3 h at 28°C, and then injected into tobacco;

[0093] After culturing under light for 1.5 days, fluorescence was observed under a fluorescence confocal microscope.

[0094] The primer sequences of RhUBC32-BIFC are SEQ ID NO.19 and SEQ ID NO.20.

[0095] The primer sequences of RhETR3-BIFC are SEQ ID NO.21 and SEQ ID NO.22.

[0096] 8. Co-immunoprecipitation (Co-IP)

[0097] RhETR3-Flag was co-injected with RhUBC32-GFP and super1300-GFP into tobacco leaves respectively. After 3 days of expression, samples were taken, ground in liquid nitrogen, and total protein of tobacco leaves was extracted using 2×SDS protein loading buffer.

[0098] Use anti-Flag and anti-GFP antibodies to detect the expression of the two target gene proteins by WB. When the target bands can be detected for both tag proteins, it means that both proteins are expressed and the following operations can be performed.

[0099] Add 800 μl of non-denaturing protein extraction buffer to every 1 ml of material to extract total protein. Extract 3 tubes of protein for each combination;

[0100] Pipette 20 μL of ANTI-FLAG-Flag M2 Magnetic Beads into a 1.5 ml centrifuge tube and add 5 times the volume of TBS;

[0101] After thorough mixing, place the centrifuge tube on a magnetic rack, let it stand for about 15 seconds, and remove the TBS buffer;

[0102] Add 5 volumes of TBS to the beads and mix thoroughly;

[0103] Place the centrifuge tube on the magnetic rack, remove the TBS buffer, and repeat 2 to 3 times;

[0104] Add 1.2 ml of total protein to the centrifuge tube with beads (if the concentration of the target protein is low, the beads can be divided into two tubes and 1.2 ml of total protein can be added to each centrifuge tube);

[0105] Mix slowly at room temperature. If the interaction between the two proteins will lead to degradation, add 50 μM MG132 to the total protein.

[0106] After incubation at room temperature for 1 h, place the centrifuge tube on a magnetic rack and let it rest for 15 s (beads are fully adsorbed on the tube wall), then remove the supernatant with a pipette;

[0107] Wash the beads with TBS buffer to remove non-specifically bound proteins, add 20 times the volume of beads with TBS buffer, and wash three times;

[0108] Add 100 μl 2×SDS protein loading buffer and boil for 10 min to denature;

[0109] The target protein was detected using anti-Flag and anti-GFP antibodies, respectively.

[0110] 9. Yeast two-hybrid

[0111] Yeast strain MNY51 was streaked on YPDA plates and cultured at 30°C for 3 d;

[0112] Pick 4 to 5 single clones and culture them in 10 mL YPDA medium at 28°C and 200 rpm overnight;

[0113] 3000rpm, 5min to collect bacteria;

[0114] Add 10 ml of sterile water to resuspend the yeast strain;

[0115] 3000rpm, 5min; resuspend in 10ml sterile water and dispense into 1.5ml centrifuge tubes, 1ml bacterial solution per tube;

[0116] Centrifuge at 6000 rpm for 5 min;

[0117] Discard the supernatant and resuspend in 100 μl one-step buffer;

[0118] Add 6 μl salmon sperm DNA (before use, boil in boiling water for 5 minutes and ice bath for 5 minutes; repeat once before use);

[0119] Add 500 ng of each of the two plasmids containing the target gene into the competent cells, mix well, and mark well;

[0120] Incubate in 45°C water bath for 30 min, vortexing every 10 min;

[0121] The transformed products were spread on two-deficient SD-leu-Trp and four-deficient SD-Leu-Trp-His-Ade plates, cultured at 30°C for 2 to 3 days, and the growth of yeast was observed.

[0122] The primer sequences of RhETR3-STE are SEQ ID NO.13 and SEQ ID NO.14.

[0123] The primer sequence of RhETR3-STE-TM R is SEQ ID NO.15.

[0124] The primer sequence of RhETR3-STE-ΔTM F is SEQ ID NO.16.

[0125] The primer sequences of RhUBC32-PR3N are SEQ ID NO.17 and SEQ ID NO.18.

[0126] 10. Expression of recombinant proteins in prokaryotic cells

[0127] The RhUBC32 ORF containing Sac I and Sal I sites was cloned into the pET28a-His-Myc vector to obtain the pET28a-His-Myc-RhUBC32 vector for His-Myc-RhUBC32 protein purification;

[0128] The target plasmid was transferred into inducible Escherichia coli Transata, and 12 single clones were picked and tested for bacterial P to confirm that the single clone contained the target plasmid;

[0129] After the correct monoclonal strain is identified, 2.5% of the bacterial solution is added to a 2 ml centrifuge tube containing 200 ml of LB containing relevant resistance, and cultured at 37°C, 200 rpm for 3 h;

[0130] Add 0.4 mM IPTG and induce culture overnight at 16°C;

[0131] 8000 rpm, 4°C, 5 min, collect bacteria;

[0132] His protein resuspension solution Lysis buffer resuspends bacteria;

[0133] After the bacterial solution was resuspended, it was broken with an ultrasonic disruptor, 9.9s on, 9.9s off, for 15min;

[0134] After disruption, the bacterial solution was centrifuged at 4°C, 10,000 rpm, for 40 min;

[0135] Take the supernatant and proceed directly to protein purification.

[0136] The primer sequences of RhUBC32-pET28a myc are SEQ ID NO.27 and SEQ ID NO.28.

[0137] 11. Purification of His fusion protein

[0138] Rinse the gasket with 20% alcohol in advance to remove the air inside, and add a certain amount of His resin into the column containing the gasket according to 100μl His resin / 100mL bacterial liquid;

[0139] 20% alcohol was passed through 3 column volumes to clean the packing;

[0140] Pre-wash the column with binding buffer, 3 column volumes;

[0141] Resuspend the protein supernatant in resin and pour into a clean conical flask, incubate at 60rpm on ice for more than 1 hour;

[0142] Collect the resin and wash the column with wash buffer, 3 column volumes;

[0143] Elution: Add 2 ml of eluent I into the column, react for 3 to 5 minutes, and collect the eluent;

[0144] Add 2 ml of eluent II to the column, react for 3 to 5 minutes, and collect the eluent;

[0145] Add 2 ml of eluent III, react for 3 to 5 minutes, and collect the eluent;

[0146] After purification, the protein was run on a protein gel to detect the concentration and stored in aliquots at -80°C.

[0147] 12. Ubiquitin-conjugating enzyme E2 activity analysis

[0148] Add 50 ng E1 (purified Arabidopsis E1 protein), 100 ng E2 (purified protein with His and MYC tags), 1 μl 20× reaction buffer, 2 μl purified His-Ub (about 2 μg / μl) to a 1.5 ml centrifuge tube, and add ddH2O to a total volume of 20 μl;

[0149] Four tubes of each E2 were prepared simultaneously, two tubes containing Ub and two tubes without Ub, and the reaction was carried out at 37°C and 900 rpm for 5 min;

[0150] After the end, 4 μl of 5× SDS loading buffer was added, with one tube in each group containing +DTT (containing β-mercaptoethanol) and -DTT;

[0151] The mixture was boiled at 100°C for 6 min, and anti-Ub and anti-Myc antibodies were used to detect whether E2 was stained with Ub.

[0152] 13. Luciferase complementation assay

[0153] The RhETR3 ORF containing BamH I and Spe I sites was cloned into the pCAMBIA-nLUC vector to obtain the RhETR3-nLUC vector;

[0154] The RhUBC32 ORF containing BamH I and Spe I sites was cloned into the pCAMBIA-cLUC vector to obtain the RhUBC32-cLUC vector, and transformed into Agrobacterium;

[0155] Agrobacterium transformed with RhETR3-nLUC and RhUBC32-cLUC were mixed in different proportions, left to stand at room temperature for 3 hours, injected into tobacco, and cultured under light for 3 days. Then, the leaves were removed and fixed on white A4 paper with double-sided tape, with the back facing up, and fluorescein was evenly sprayed. After dark treatment for 5 minutes, fluorescence was detected by a fluorescence meter.

[0156] The primer sequences of nLUC-RhETR3 are SEQ ID NO.29 and SEQ ID NO.30.

[0157] The primer sequences of cLUC-RhUBC32 are SEQ ID NO.31 and SEQ ID NO.32.

[0158] 14. Virus-induced gene silencing

[0159] The 507 bp fragment at the 3′ end of the RhUBC32 gene was inserted into the pTRV2 vector to construct pTRV2-RhUBC32;

[0160] pTRV1, pTRV2, and pTRV2-target fragment were transformed into Agrobacterium GV3101;

[0161] Pick a single clone into a 50 ml centrifuge tube containing 5 ml of resistant LB culture medium, and shake the culture overnight at 28°C and 200 rpm;

[0162] Use the vector primers of pTRV1 and pTRV2 to detect whether the bacterial solution contains the target plasmid by PCR;

[0163] The correctly detected Agrobacterium was diluted at a ratio of 1:100 and shaken. 100 ml LB containing antibiotics and 1 ml bacterial solution were added to a 500 ml Erlenmeyer flask and cultured overnight at 28°C.

[0164] Centrifuge at 5000rpm for 8min, discard the supernatant, collect the bacterial solution, resuspend the bacteria in the infection solution, and adjust the OD 600 =1.0;

[0165] Mix pTRV1 bacterial solution with pTRV2 and pTRV2-target gene fragment in equal volumes, with about 300-500 ml of bacterial solution for each group;

[0166] Let stand in the dark for 3 to 6 hours and set aside.

[0167] The seedlings that have been rooted in the tissue culture bottle for 4 weeks are taken out of the bottle and grouped according to size, with 12 seedlings in a group, and equally distributed to each group of bacteria;

[0168] Wrap the grouped tissue culture seedlings with gauze, immerse them in the bacterial solution, and place a mortar above the seedlings to prevent the seedlings from floating on the infection solution during the vacuum extraction process;

[0169] Evacuate the air, and when the pressure reaches 0.8 atmospheres, maintain the pressure for 5 minutes and then slowly release the air.

[0170] Repeat once, and for the third time, when the pressure reaches 1.0 atmosphere, maintain the pressure for 5 minutes, and slowly release the air to allow the bacterial solution to enter the rose seedlings;

[0171] After the infection is over, rinse under running water to wash off the excess bacterial liquid;

[0172] Place the seedlings in a small pot with lines (roots in water, stems and leaves in air), wrap with plastic wrap to maintain humidity;

[0173] Place in dark at 8℃ for 3 days;

[0174] After 3 days, the tissue culture seedlings were planted in small pots containing vermiculite and nutrient soil (1:1), covered with plastic film to keep moisture for one week, and placed in a culture room for cultivation;

[0175] After one month of growth, total RNA from the leaves of the silenced plants was extracted by the hot boron method, reverse transcribed, and the CP gene was detected to determine whether the silencing vector had entered the seedlings;

[0176] The material containing the CP gene was tested for gene expression level using gene detection primers, and plants with lower expression levels were selected for subsequent experiments.

[0177] The primer sequences of RhUBC32-TRV2 are SEQ ID NO.33 and SEQ ID NO.34.

[0178] The primer sequences of TRV1 are SEQ ID NO.3 and SEQ ID NO.4.

[0179] The primer sequences of TRV2 are SEQ ID NO.5 and SEQ ID NO.6.

[0180] The primer sequences of TRV-CP are SEQ ID NO.7 and SEQ ID NO.8.

[0181] 15. RhUBC32 RNAi stable transgenic rose

[0182] The 405 bp sequence of RhUBC32 constructed pTRV2 vector was ligated into pFGC1008 vector in both forward and reverse directions through ASC I / Swa I and Pac I / Bam HI sites;

[0183] The EH105A Agrobacterium containing the RNAi plasmid was resuspended in resuspension buffer to OD 600 =0.6, 28°C, 200rpm, after 2h, put in rose embryonic callus, evacuate, when the pressure reaches 0.8 atmospheres, keep the pressure for 5min, and slowly release the air;

[0184] After repeating once, after culturing at 28℃ and 180ppm for 40min, discard the infection solution, dry the bacterial solution on the surface of embryonic callus with sterile absorbent paper, put it into co-cultivation medium, and culture it in the dark;

[0185] After 3 days, callus tissue was transferred to selective proliferation medium, and the medium was changed every two weeks. After 2 months of selection and proliferation, resistant callus tissue was transferred to selective germination medium, and the medium was changed every two weeks. After 2 months of screening, the medium was changed every 4 weeks.

[0186] After the resistant buds grow to about 3-5 cm, some of them take root in the rooting medium, and some continue to be selected for germination and propagation. After 3 weeks of rooting, the rooted seedlings are transplanted into culture pots and grown for 3 weeks at 22±1℃ with a photoperiod of 16h / 8h (light / dark). Then, DNA from the leaves is extracted to detect whether the vector is inserted;

[0187] When the flowers opened to level 5, total RNA was extracted and the expression of RhUBC32 was detected.

[0188] RhUBC32 RNAi contains two pairs of specific primers:

[0189] Forward sequence primers: SEQ ID NO.37 and SEQ ID NO.38;

[0190] Reverse sequence primers: SEQ ID NO.39 and SEQ ID NO.40.

[0191] 16. Ethylene treatment

[0192] The selected plants with reduced target gene expression and the control TRV2 plants were placed in the same large dish for cultivation. When the flower buds reached level 2 (sepals were open, petals were tightly wrapped and not loose), photos were taken.

[0193] Place in a 0.4L glass cylinder (a certain volume of 1M NaOH solution is placed in the cylinder), seal with paper tape and transparent tape, add 0.4ml ethylene (10ppm), and the control group does not add ethylene, and treat under light for 24h;

[0194] Unbox and take photos, take samples of petals, freeze in liquid nitrogen, and store at -80°C. Calculate the ethylene sensitivity of rose flowers according to the degree of flower opening.

[0195] According to a preferred embodiment, the method for preparing the culture medium for rapid propagation of rose tissue culture seedlings is as follows: MS, 1 mg / L 6-BA, 1 mg / L GA3, 0.05 mg / L NAA, 7 g / L agar powder, and 30 g / L sucrose are mixed and the pH is adjusted to 6.0.

[0196] According to a preferred embodiment, the preparation method of the rooting medium for rose tissue culture seedlings is: MS, 0.1 mg / L NAA, 7 g / L agar, and 30 g / L sucrose are mixed and the pH is adjusted to 6.0.

[0197] According to a preferred embodiment, the infection solution is prepared by mixing 10 mM MES, 20 mM AS, and 10 mM MgCl2, and adjusting the pH of the mixture to 5.6.

[0198] According to a preferred embodiment, the LB configuration method is: 50 mg / L Kan, 25 mg / L Rif, 2 mM AS mixture.

[0199] According to a preferred embodiment, the non-denaturing protein extraction buffer is prepared by mixing 50 mM Tris-HCl at pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM Na3VO4, 10 mM NaF, 50 mM β-mercaptoethanol, 10 mM DTT, 1 mM PMSF, 5% glycerol, and protease inhibitors.

[0200] According to a preferred embodiment, the reaction buffer is prepared by mixing 1M Tris-HCl at pH 7.5, 100mM ATP sigma, 50mM MgCl2, and 2mM DTT.

[0201] According to a preferred embodiment, the TBS preparation method is: 50 mM Tris-HCl and 150 mM NaCl are mixed, and the pH is adjusted to 7.4.

[0202] According to a preferred embodiment, the elution buffer is prepared by mixing 10 mM reduced glutathione and 50 mM Tris-HCl and adjusting the pH to 8.0.

[0203] According to a preferred embodiment, the wash buffer is prepared by mixing 50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole, and 5% glycerol, adjusting the pH to 8.0 with NaOH, and adding 1 mM PMSF before use.

[0204] According to a preferred embodiment, the binding buffer is prepared by mixing 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and 5% glycerol, adjusting the pH to 8.0 with HCl, and adding 1 mM PMSF for elution before use.

[0205] According to a preferred embodiment, the preparation method of eluent II is: 50 mM NaH2PO4, 300 mM NaCl, 5% glycerol, 250 mM imidazole are mixed, the pH is adjusted to 8.0 with HCl, and 1 mM PMSF is added before use.

[0206] According to a preferred embodiment, the eluent III is prepared by mixing 50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, and 5% glycerol, adjusting the pH to 8.0 with HCl, and adding 1 mM PMSF before use.

[0207] According to a preferred embodiment, the co-culture medium is configured as follows: MS, 1.0 mg L -1 2,4-D, 0.05mgL -16-BA, 60g L –1 of glucose, 2.5 g L –1 GEL, 100mg L -1 The hydrolyzed complexed protein was mixed with 100 mM AS.

[0208] According to a preferred embodiment, the proliferation medium is prepared as follows: MS, 1.0 mg L -1 2,4-D, 0.05mgL -1 6-BA, 60gL -1 Glucose, 2.5 g L –1 GEL, 20mg L -1 Hygromycin B, 300mg L -1 Cef Mix.

[0209] According to a preferred embodiment, the germination medium is configured as follows: MS, 1.0 mg L -1 6-BA, 0.05 mg L - 1 NAA, 1.0 mg L -1 GA3, 0.1mg L -1 CH, 30g L -1 Glucose, 7.0 g L -1 Agar, 20 mg L -1 Hygromycin B, 300mg L -1 Cef Mix.

[0210] According to a preferred embodiment, the preparation method of 10 ml One-Step Buffer (prepared immediately before use) is as follows: 2 ml 10xLiAc (1N), 8 ml 50% PEG3350, and 76.9 μl β-mercaptoethanol are mixed.

[0211] Example 1

[0212] This embodiment provides a method for cloning the RhUBC32 gene. The present invention provides a method for cloning the ubiquitin conjugating enzyme gene.

[0213] The present invention used RhETR3 as bait to screen the rose split ubiquitination yeast two-hybrid library, and obtained a total of 245 clones. After sequence alignment and yeast rotation verification, a total of 28 confirmed positive clones were obtained, and a ubiquitin conjugating enzyme RhUBC32 (RU05948) was obtained from them. Figure 2A shown.

[0214] Through multiple sequence alignment, the full length of RU05948 was obtained and named RhUBC32 according to gene annotation. The full length of RhUBC32 gene is 1170 bp, ORF is 912 bp, encoding 303 aa. Through SMART protein sequence analysis program, such as Figure 1A As shown, the N-terminus of RhUBC32 protein has a conserved UBCc domain (amino acids 13-128), the sequence of which is the underlined sequence in the figure. The C-terminus of RhUBC32 protein has a possible transmembrane region (amino acids 269-289), the sequence of which is the sequence marked by the gray box in the figure.

[0215] like Figure 1B As shown, the phylogenetic tree shows that RhUBC32 is closest to Arabidopsis thaliana UBC32, proving that RhUBC32 is most closely related to Arabidopsis thaliana UBC32. This result indicates that RhUBC32 belongs to the UBC family.

[0216] Example 2

[0217] This embodiment provides a method for detecting the association between the RhUBC32 gene and the RhETR3 gene.

[0218] The interaction between RhUBC32 and RhETR3 was verified by three experiments: bimolecular fluorescence complementation (BiFC), split ubiquitination yeast two-hybrid (MYTH) and Co-IP.

[0219] RhUBC32-GFP was co-expressed with RhETR3-mCherry and ER-mCherry in tobacco, and the fluorescence was observed by confocal fluorescence microscopy for co-localization experimental analysis. Figure 2B As shown, the green fluorescence of RhUBC32-GFP can completely overlap with the red fluorescence of the plasma membrane marker, indicating that RhUBC32 is localized in the plasma membrane and is a membrane-localized protein; and its green fluorescence can also overlap with the red fluorescence of RhETR3-mCherry, indicating that RhUBC32 and RhETR3 are co-localized and may be bound to each other in the plant body.

[0220] like Figure 2C As shown, the ethylene receptor RhETR3 protein has four domains, namely the N-terminal transmembrane domain (Transmembrane), the GAF domain (GAF), the histidine kinase domain (Histidine Kinase) and the receiver domain (Receiver).

[0221] like Figure 2CAs shown in the figure, after RhETR3 was truncated and divided into two parts, the transmembrane domain and the detransmembrane domain, the two parts of RhETR3 were verified. RhUBC32-PR3N and RhETR3-STE were co-transfected into yeast MNY51, and they could grow on the two-deficient plate SD-TL, which indicated that the plasmid transformation into yeast was successful. On the four-deficient plate SD-TLHA, RhETR3-STE and RhUBC32-PR3N could grow. With the dilution of the yeast solution, the plaques could still grow and could be stained blue by X-Gal, while the negative control RhETR3-STE and the empty PR3N would not grow on the four-deficient plate, that is, RhETR3 interacted with RhUBC32 in yeast, and its binding occurred in the transmembrane domain of RhETR3.

[0222] The results of BiFC in vivo validation showed that RhUBC32 and RhETR3 proteins can bind to each other in vivo. Figure 2D As shown, green fluorescence can be seen under a fluorescence confocal microscope, and the fluorescence is located at the cell membrane, while no fluorescence is seen in RhETR3-YNE (R) and empty pSPYCE (M). This result indicates that RhETR3 interacts with RhUBC32 in plants.

[0223] like Figure 2E The results of RhUBC32-GFP and RhETR3-Flag co-injection into tobacco are shown. All three proteins are expressed. After immunoprecipitation (IP) with anti-Flag beads in the RhETR3-Flag and RhUBC32-GFP co-expression samples, the GFP antibody can detect the RhUBC32-GFP protein, while no bands are detected in the negative control Super1300-GFP after IP, indicating that RhETR3 and RhUBC32 are bound in vivo. However, the amount of RhETR3-Flag protein after IP is much less than the total input protein. It may be that during the IP process, as the concentration of RhETR3 and RhUBC32 proteins increases, the degradation of RhETR3 protein is accelerated.

[0224] Example 3

[0225] This embodiment provides a method for detecting the expression level of the RhUBC32 gene.

[0226] The response of RhUBC32 to ethylene and its changes during flower opening were detected by qRT-PCR.

[0227] like Figure 3A As shown in the figure, the expression level of RhUBC32 in flowers of level 3 is higher than that in flowers of level 2. The expression level of RhUBC32 in flowers of level 5 is higher than that in flowers of level 3. During the natural opening process, the expression of RhUBC32 gradually increases. Figure 3A As shown, after 24 hours of ethylene treatment, the expression level of RhUBC32 (Eth) was significantly higher than that of the control group (Control).

[0228] like Figure 3B As shown, in the control reaction without adding ubiquitin or adding ubiquitin but with DTT in the system, no band of RhUBC32 bound to ubiquitin was detected; while in the lane with ubiquitin added but without DTT, a ubiquitinated band (the band indicated by the arrow in the figure) was detected, proving that RhUBC32 has E2 conjugating enzyme activity.

[0229] like Figure 3C As shown in the figure, the same result was obtained for the transient overexpression of RhUBC32-Flag protein in tobacco. In the control reaction without ubiquitin or with ubiquitin but DTT in the system, no bands of RhUBC32 bound to ubiquitin were detected; while ubiquitinated bands (the bands indicated by arrows in the figure) were detected in the lanes with ubiquitin but no DTT, proving that RhUBC32 has E2 conjugating enzyme activity.

[0230] Example 4

[0231] The present invention provides a method for analyzing the activity of ubiquitin conjugating enzyme E2, a method for promoting the degradation of RhETR3 protein by RhUBC32, and a method for demonstrating that RhUBC32 promotes the degradation of RhETR3 protein.

[0232] RhETR3-Flag was co-expressed with RhUBC32-GFP and Supper1300-GFP in tobacco leaves, and RhETR3-Flag protein was detected. As shown in the electrophoresis diagram on the left of 4A, in the presence of RhUBC32 and RhETR3, the brightness of the RhETR3 band was higher than that of the RhETR3 band not mixed with RhUBC32, indicating that RhUBC32 can promote the degradation of RhETR3 protein in the absence of ethylene. As shown in the electrophoresis diagram on the right of 4A, in the presence of 10ppm ethylene, in the presence of RhUBC32 and RhETR3, the brightness of the RhETR3 band was higher than that of the RhETR3 band not mixed with RhUBC32, indicating that RhUBC32 can promote the degradation of RhETR3 protein in the absence of ethylene. At the same time, the brightness of the mixed lane of RhUBC32 and RhETR3 without ethylene was higher than that of the mixed lane of RhUBC32 and RhETR3 under 10 ppm ethylene treatment, which indicated that ethylene accelerated the degradation of RhETR3 protein. The above results indicate that RhUBC32 can promote the degradation of RhETR3 protein, and ethylene treatment can accelerate the degradation rate of the receptor.

[0233] like Figure 4B As shown, with the lane in the first column as a negative control, the protein brightness of the lane in the third column with MG132 added is higher than that of the lane in the second column without MG132 added. This result shows that the phenomenon of RhUBC32 promoting RhETR3 degradation can be inhibited after adding MG132.

[0234] Figure 4C The results showed that the active site mutant of RhUBC32 UBC domain C93-A The intensity of the band in the second column mixed with RhETR3 is greater than that in the first column mixed with RhUBC32 and RhETR3. This result shows that the active site mutant of the UBC domain RhUBC32 C93-A The above results indicate that RhETR3 is degraded through the 26S proteasome ERAD pathway.

[0235] The dual luciferase results showed that Figure 4D As shown, the brightness of Figure a is higher than that of Figures b, c, and d. As the ratio of RhETR3-nLUC to RhUBC32-cLUC increases, the amount of RhUBC32-cLUC gradually increases, and the LUC fluorescence intensity gradually weakens. This result shows that RhETR3 and RhUBC32 proteins interact in vivo, and this interaction accelerates the degradation of RhETR3, causing the LUC fluorescence intensity to weaken.

[0236] Example 5

[0237] The present invention provides a method for constructing a rose plant that is highly sensitive to ethylene. The present invention provides a method for cultivating roses using ethylene. The present invention provides a phenotypic detection method for promoting the opening of rose flowers by ethylene.

[0238] The 500 bp sequence of the RhUBC32 328-3~UTR region was connected to the viral silencing vector pTRV2. The vector structure is as follows Figure 5A shown.

[0239] like Figure 5B As shown in the figure, the brightness of the RhUBC32 gene bands (1, 2, and 3 electrophoretic bands corresponding to TRV-RhUBC32) in the rose plants with RhUBC32 silenced is lower than that in the rose plants transformed with empty TRV (1, 2, and 3 electrophoretic bands corresponding to TRV). Taking RhUBI2 as the negative control, the expression level of the RhUBC32 gene in the rose plants with RhUBC32 silenced is significantly lower than that in the control TRV2 plants, and the silencing effect is obvious.

[0240] The flowers in level 2 state appear with sepals fully expanded and buds tightly closed.

[0241] After the flowers in the second state were treated with 10 ppm ethylene for 24 hours, the degree of opening of the flowers of the silent plants and the flowers of the TRV2 control plants was observed. According to the degree of opening of the flowers, they were divided into four levels: petal shedding and curling, full opening, half opening and not opening, and statistics were conducted. Figure 5C The flower phenotypes of RhUBC32 silenced plants and normal plants after ethylene treatment are shown. Figure 5D As shown, the proportion of flowers that fell off and curled after ethylene treatment in the RhUBC32-silenced plants was 95%, which was significantly higher than the 39% in the TRV2 control group, indicating that the ethylene sensitivity of the silenced group plants was increased.

[0242] The present invention also relates to a RhUBC32 RNAi vector, which is used to obtain stable transgenic plants through Agrobacterium-mediated transgenic technology. Fig. 6A As shown, two RhUBC32 low-expression strains, RhUBC32-RNAi#18 and RhUBC32-RNAi#22, were obtained.

[0243] like Figure 6B As shown, the number of branches in the low-expressing plants was higher than that in the wild type.

[0244] like Figure 6C As shown in the figure, 24 hours after ethylene treatment, the petals of flowers of RhUBC32-RNAi#18 and RhUBC32-RNAi#22 fell off to only one petal compared with the wild type (WT). This phenotype indicates that the ethylene sensitivity of RhUBC32-RNAi transgenic plants is increased, which is consistent with the RhUBC32 VIGS silenced plants.

[0245] like Fig.6D As shown in Figure 2, after 10 ppm ethylene treatment for 3 h, the ethylene response gene RhEBF2 in RhUBC32-RNAi#18 and RhUBC32-RNAi#22 increased significantly compared with the wild type. Fig. 6E As shown in the figure, after 3h of ethylene induction, the RhUBC32-RNAi#18 protein band was brighter than that of the wild-type RhETR3. The results of the wild-type indicated that RhUBC32 could promote the degradation of RhETR3 protein, and ethylene treatment could accelerate the degradation rate of the receptor. The results of RhUBC32-RNAi#18 induced the accumulation of RhETR3 protein and inhibited its degradation by ethylene.

[0246] Table 1

[0247]

[0248]

[0249]

[0250] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", all of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the feature guided by "preferably" is only an optional method and should not be understood as a must-have setting. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A method for cultivating rose plants with a high ethylene sensitivity phenotype, characterized in that: Include: In the presence of a gene that regulates the degradation of the ethylene receptor RhETR3 RhUBC32 A target fragment is selected from the coding region of the gene, and the target fragment is inserted into the pFGC1008 vector to obtain a silencing vector; the silencing vector is ASC I / Swa I, Pac I / Bam The forward and reverse sequences were ligated into the pFGC1008 vector at the HI site to obtain; Using the silencing vector to transform Agrobacterium to obtain genetically engineered Agrobacterium containing the silencing vector; The genetically engineered Agrobacterium is used to infect rose embryonic callus to obtain the RhUBC32 Mutant strains with low gene expression; It is stated RhUBC32 The nucleotide sequence of the gene is SEQ ID NO.1; the nucleotide sequences of the primers used to clone the forward target fragment are SEQ ID NO.37 and SEQ ID NO.38; the nucleotide sequences of the primers used to clone the reverse target fragment are SEQ ID NO.39 and SEQ ID NO.

40.

2. Genes that regulate the degradation of ethylene receptor RhETR3 RhUBC32 Applications in the following areas: (1) By reducing gene RhUBC32 The expression of α-hydroxy-1-hydroxy-1-4-nitropropene can increase the sensitivity of rose plants to ethylene; (2) By reducing gene RhUBC32 to increase the number of branches of rose plants.

Images