A rose ubiquitin ligase gene and its application

Abstract

The present invention provides a rose ubiquitin ligase gene. The gene and the application of the gene can provide a basis and direction for early and late flower screening and directional cultivation 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 rose ubiquitin ligase gene and application thereof. Background Art

[0002] Flowers are an indispensable organ of flowering plants. The regulation of flower opening is an important aspect of plant physiology research, especially in the physiology of ornamental plants with flowers as ornamental parts. Whether the flowers can open successfully and fully depends mainly on the growth state of the plant.

[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 opening process of rose flowers is an important part of the evaluation of roses as ornamental plants and an important part of their ornamental quality. With the coordinated expansion and orderly opening of the petals, the rose flowers can form the unique flower shape of their variety and finally reveal the pistil.

[0004] However, due to the gradual circulation of the ornamental value of roses, large quantities of fresh-cut roses will open abnormally during the post-harvest circulation due to ethylene in the environment and ethylene produced by the flowers themselves during transportation. This process will cause serious post-harvest losses. According to statistics, quality deterioration caused by ethylene accounts for more than 70% of post-harvest physiological losses.

[0005] Ethylene is an important hormone that affects the growth rate of plants. It also affects the opening speed and quality of rose flowers. Unlike hormones involved in 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 that regulate the opening of rose flowers: 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] Existing research results show that ethylene promotes the degradation of RhETR3 protein, and this degradation is inhibited by ethylene. Based on this, in view of the blank of ethylene receptor regulation mechanism in the prior art, the present invention provides a rose ubiquitin ligase gene and the hormone regulation effect of the gene in rose flowering. The gene provided by the present invention and the application of the gene can provide a molecular biological basis for the study of abnormal flowering of roses.

[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 shortcomings of the prior art, the present invention provides a rose ubiquitin ligase gene, the gene sequence of which is SEQ ID NO.1.

[0011] Ethylene receptors are key proteins that sense ethylene and play 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. By molecular regulation of the gene in plants, the sensitivity of plants to ethylene can be significantly increased or decreased, thereby extending or shortening the flowering time of plants. By microscopic regulation or observation of the gene, the presence of abnormal flowering plants can be reduced by reducing the ethylene sensitivity of plants or screening the ethylene sensitivity of plants.

[0012] The present invention provides a rose ubiquitin ligase for regulating the degradation of ethylene receptor RhETR3 protein. The amino acid sequence of the rose ubiquitin ligase gene is SEQ ID NO. 2. Preferably, the biological function of the protein is to regulate the stability of the rose ethylene receptor RhETR3 protein.

[0013] The present invention provides an application of a rose ubiquitin ligase in regulating an ethylene receptor RhETR3 protein. The ubiquitin ligase acts on sites C62, C65, C78, ​​C81, H88, C91, H94, C104, C107, and H108 of an amino acid sequence SEQ ID NO.2 to reduce the activity of the rose ubiquitin ligase. By reducing the activity of the rose ubiquitin ligase in this method, a plant with a high ethylene sensitivity phenotype is obtained.

[0014] The present invention provides a method for cultivating a plant with a high ethylene sensitivity phenotype. The method comprises:

[0015] Selecting a target fragment from the coding region of the rose ubiquitin ligase gene, inserting the target fragment into the pFGC1008 vector to obtain a silencing vector;

[0016] Using the silencing vector to transform Agrobacterium to obtain genetically engineered Agrobacterium containing the silencing vector;

[0017] The genetically engineered Agrobacterium is used to infect rose embryonic callus to obtain a mutant strain with low expression of the RhRED gene.

[0018] According to a preferred embodiment, the nucleotide sequence of the primer used to clone the target fragment is one of the following groups:

[0019] (1) SEQ ID NO. 69 and SEQ ID NO. 70;

[0020] (2) SEQ ID NO.71 and SEQ ID NO.72.

[0021] According to a preferred embodiment, the nucleotide sequences of the primers used for cloning or detecting the silencing efficiency of the target fragment are as shown in SEQ ID NO.21 and SEQ ID NO.22.

[0022] The present invention provides a Rosaceae plant with a high ethylene sensitivity phenotype. The rose ubiquitin ligase gene is lowly expressed in the Rosaceae plant.

[0023] According to a preferred embodiment, the method for reducing gene expression in plants can be virus-induced gene silencing technology.

[0024] The present invention provides biological materials containing the gene, which are expression vectors and strains.

[0025] According to a preferred embodiment, the expression vector can be a pFGC1008 vector.

[0026] According to a preferred embodiment, the strain can be Agrobacterium.

[0027] The invention provides a Rosaceae plant with a high-ethylene-sensitive phenotype, wherein the protein encoded by the rose ubiquitin ligase gene is lowly expressed in the Rosaceae plant.

[0028] According to a preferred embodiment, the Rosaceae plant can be a rose.

[0029] The invention provides an application of a rose ubiquitin ligase in regulating the process of ethylene-induced flower opening.

[0030] According to a preferred embodiment, the expression vector containing the rose ubiquitin ligase gene is introduced into rose embryonic callus to overexpress the rose ubiquitin ligase gene in the rose. The transgenic plant with an obvious longer flowering period phenotype is obtained by the above method.

[0031] RhETR3 is a key receptor for rose flowers to sense ethylene. The present invention is based on the exploration of the decomposition and production process of this key receptor to discover the gene RhRED that plays a key role in RhETR3. Using pBT-STE-RhETR3 as bait, a split ubiquitination library was screened to obtain a ubiquitin ligase RhRED. The results of yeast two-hybrid analysis, fluorescent bimolecular complementation analysis and fluorescent co-localization analysis showed that RhRED and RhETR3 were co-localized in the lipid membrane, and the binding domain was a transmembrane domain. RhRED contains a typical RINGv domain. In vitro ubiquitination analysis shows that RhRED has typical ubiquitin ligase activity. After the active site in the RINGv domain is mutated, the RhRED ligase activity disappears, and C80 and C96 do not affect its activity. The yeast two-hybrid results show that RhUBC32 interacts with RhRED, and RhUBC32 can transfer ubiquitin to RhRED in vitro. In vitro substrate ubiquitination analysis shows that RhETR3 is a substrate of RhRED. RhRED and RhETR3 were co-expressed in tobacco. With the increase of RhRED, RhETR3 protein gradually decreased. LCI analysis showed that with the increase of RhRED-cLUC, the fluorescence gradually weakened, indicating that RhRED interacted with RhETR3 and degraded RhETR3 in a dose-dependent manner. The RhRED gene was significantly induced by ethylene, and the expression level gradually increased with the opening of the flowers, reaching the highest expression level in the 5th grade flowers. The expression of the RhRED gene was silenced in roses. After ethylene treatment, 75% of the flowers in the silenced plants showed petal shedding and abnormal eversion, which was significantly higher than the 20% in the TRV2 control group, indicating that RhRED silencing led to increased ethylene sensitivity in rose plants. In the RhRED RNAi stable transgenic roses, the plants were thin and weak, and the stem diameter was significantly thinner. The expression of the ethylene response gene RhHB6 in the 2nd grade flowers of WT was significantly higher than that in the control group. The rose flowers were treated with ethylene and the petals fell off, showing an ethylene hypersensitive phenotype. At the same time, the senescence process of the RhRED RNAi stable transgenic rose flowers was significantly slowed down. In this application, grade 5 flowers refer to fully open flowers.

[0032] In the present invention, different states of reproductive organs of angiosperms are classified, wherein:

[0033] Level 0: sepals are tightly clasped and the organ is in the bud stage;

[0034] Level 1: petals are exposed from sepals, and organs are in the bud stage;

[0035] Level 2: sepals droop, outer petals begin to loosen, and organs are in the budding stage;

[0036] Level 3: Initial opening, the outer petals are unfolded, and the organs are in the initial opening stage;

[0037] Level 4: in full bloom, with multiple layers of petals unfolding and organs in full bloom;

[0038] Level 5: late bloom, flowers are showing their cores, organs are at the late bloom stage;

[0039] Level 6: Petals turn over or begin to wilt, turn blue, or have burnt edges, and the organs are in the wilting stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the RhRED protein sequence analysis diagram;

[0041] Figure 2A This is the result diagram of MH2Y analyzing the interaction between RhETR3 and RhRED;

[0042] Figure 2B This is the result diagram of RhRED subcellular localization;

[0043] Figure 2C This is the result diagram of BIFC analysis of the interaction between RhETR3 and RhRED;

[0044] Figure 3A This is a diagram of the RhRED expression pattern analysis;

[0045] Figure 3B This is a diagram for analyzing the activity of RhRED ubiquitin ligase;

[0046] Figure 3C This is the RhRED active site analysis diagram;

[0047] Figure 4A This is a graph showing the analysis of RhRED ligase activity in the presence of RhUBC32;

[0048] Figure 4B This is a diagram of RhRED in vitro ubiquitination analysis of RhETR3;

[0049] Figure 4C The results of RhUBC32 and RhRED ubiquitinating RhETR3 in vitro;

[0050] Figure 4D This is the LCI verification diagram;

[0051] Figure 4E Schematic diagram of RhETR3 degradation by RhRED;

[0052] Figure 5A This is the structure diagram of the RhRED silencing vector;

[0053] Figure 5B The statistical diagram of RhRED expression in silenced plants and control plants;

[0054] Figure 5C The phenotypes of silenced plants and control plants are shown in Figure 2.

[0055] Figure 5D It is a statistical diagram of the phenotypic data of silenced plants and control plants;

[0056] Fig. 6A It is a statistical diagram of RhRED expression in RhRED-RNAi transgenic plants;

[0057] Figure 6B Schematic diagram of the expression level of RhHB6 gene in RhRED-RNAi transgenic plants;

[0058] Figure 6C Schematic diagram of ethylene sensitivity analysis of RhRED-RNAi transgenic plants;

[0059] Fig.6D Schematic diagram of flower senescence in RhRED-RNAi transgenic plants;

[0060] Fig. 6E This is a statistical chart of flower senescence in RhRED-RNAi transgenic plants. DETAILED DESCRIPTION

[0061] The following is a detailed description with reference to the accompanying drawings.

[0062] The invention relates to growth conditions of rose tissue culture seedlings and cultivation conditions of tobacco.

[0063] Rose tissue culture seedling growth and cultivation method:

[0064] 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 cultivation;

[0065] After rooting for 3 to 4 weeks, the plants are planted in soil mixed with vermiculite in equal proportions and cultured at 23°C in a cross-cycle lighting environment of 16 h light / 8 h dark.

[0066] Tobacco cultivation methods:

[0067] Nicotiana benthamiana was selected as the breeding variety, and the seeds were preserved in this laboratory;

[0068] Sow tobacco seeds in the soil (with sufficient water), and after 3 to 4 days, the seeds will germinate;

[0069] 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;

[0070] 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℃.

[0071] The present invention also relates to the following experimental operations:

[0072] 1. RhRED gene cloning

[0073] Based on the partial sequence of RU18315 obtained from the screening library, the sequence fragment of the RhRED gene was obtained by comparing it with the rose RNA-seq database;

[0074] The full-length gene sequence was obtained using homologous cloning.

[0075] 2. Protein sequence analysis

[0076] The full-length amino acid sequence of RhRED was analyzed using the SMART protein sequence analysis program (http: / / smart.embl-heidelberg.de / ).

[0077] 3. qRT-PCR analysis

[0078] Total RNA from rose petals was extracted using the hot borate method;

[0079] The HiScript II Q RT SuperMix for qPCR reverse transcription kit was used to synthesize the first-strand cDNA with 1 μg of total RNA as a template;

[0080] qRT-PCR reactions (20 μl volume containing 1 μl cDNA template) were performed using the StepOne Real-Time PCR System (Applied Biosystems) with the KAPA SYBR FASTUniversal qRT-PCR Kit (Kapa Biosystems), in which the rose RhUBI gene (GenBank accession number JK622648) was used as an internal reference gene;

[0081] Each reaction was performed in triplicate and verified by melting curve analysis;

[0082] All reactions were performed with at least three biological replicates.

[0083] The PCR primers are listed in Table 1. The sequences of the primers for the RhRED Q gene are SEQ ID NO.7 and SEQ ID NO.8.

[0084] The primer sequences of RhUBI Q gene are SEQ ID NO.9 and SEQ ID NO.10.

[0085] 4. Tobacco injection

[0086] 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;

[0087] According to experimental needs, mix equal volumes and let stand at room temperature for 3 h;

[0088] 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;

[0089] Samples were taken 3 days after injection, and total protein was extracted or stored at -80°C for later experiments.

[0090] 5. RhRED promotes RhETR3 protein degradation in vivo

[0091] RhETR3-Flag and RhRED-GFP were co-injected into tobacco at different ratios and expressed for 3 days;

[0092] Sampling was performed, total protein was extracted, and the level of RhETR3-Flag protein was detected.

[0093] 6. Colocalization Experiment Methods

[0094] RhRED-GFP was mixed with RhETR3-mCherry and PM-mCherry in equal volumes;

[0095] Co-infection of tobacco leaves for subcellular localization;

[0096] Three days after the co-injection of tobacco leaves, the fluorescence of the leaves was observed under a fluorescence confocal microscope.

[0097] The primer sequences of RhETR3-1300 are SEQ ID NO.29 and SEQ ID NO.30.

[0098] The primer sequences of RhRED-1300C are SEQ ID NO.31 and SEQ ID NO.32.

[0099] 7. Bimolecular fluorescence complementation (BIFC)

[0100] RhETR3-YNE(R), RhRED-YCE(M), pYCE(M) vectors and silencing suppressor P19 were transformed into Agrobacterium GV3101 respectively;

[0101] Agrobacterium containing the target plasmid was cultured overnight in a shaking chamber (10 ml) at 5000 rpm for 8 min to collect the bacteria;

[0102] 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;

[0103] After culturing under light for 1.5 days, fluorescence was observed under a fluorescence confocal microscope.

[0104] The primer sequences of RhRED-BIFC are SEQ ID NO.25 and SEQ ID NO.26.

[0105] The primer sequences of RhETR3-BIFC are SEQ ID NO.27 and SEQ ID NO.28.

[0106] 8. Yeast two-hybrid

[0107] Yeast strain MNY51 was streaked on YPDA plates and cultured at 30°C for 3 d;

[0108] Pick 4 to 5 single clones and culture them in 10 ml YPDA medium at 28°C and 200 rpm overnight;

[0109] 3000rpm, 5min to collect bacteria;

[0110] Add 10 ml of sterile water to resuspend the yeast strain;

[0111] 3000rpm, 5min; resuspend in 10ml sterile water and dispense into 1.5ml centrifuge tubes, 1ml bacterial solution per tube;

[0112] Centrifuge at 6000 rpm for 5 min;

[0113] Discard the supernatant and resuspend in 100 μl one-step buffer;

[0114] 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);

[0115] Add 500 ng of each of the two plasmids containing the target gene into the competent cells, mix well, and mark them;

[0116] Incubate in 45°C water bath for 30 min, vortexing every 10 min;

[0117] 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.

[0118] The primer sequences of RhETR3-STE are SEQ ID NO.11 and SEQ ID NO.12.

[0119] The primer sequence of RhETR3-STE-TM R is SEQ ID NO.13.

[0120] The primer sequence of RhETR3-STE-ΔTM F is SEQ ID NO.14.

[0121] The primer sequences of RhUBC32-STE are SEQ ID NO.15 and SEQ ID NO.16.

[0122] The primer sequences of RhRED-PR3N are SEQ ID NO.17 and SEQ ID NO.18.

[0123] 9. Expression of recombinant proteins in prokaryotic cells

[0124] The RhRED ORF containing Bam HI and Xho I sites was cloned into the pGEX4T-2-GST vector and the pMalC2-MBP vector to obtain the pGEX4T-2-GST-RhRED and pMalC2-MBP-RhRED vectors for GST-RhRED and MBP-RhRED protein purification;

[0125] The target plasmid was transferred into inducible E. coli BL21 or Transata, and 12 single clones were selected for bacterial P test to confirm that the single clone contained the target plasmid;

[0126] 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;

[0127] Add 0.2 mM IPTG and induce culture overnight at 16°C;

[0128] 8000 rpm, 4°C, 5 min, collect bacteria;

[0129] Resuspend the bacteria in their respective resuspension buffers; after the bacterial solution is resuspended, use an ultrasonic disruptor to disrupt it, start at 9.9s, stop at 9.9s, and last for 15min;

[0130] After disruption, the bacterial solution was centrifuged at 4°C, 10,000 rpm, for 40 min;

[0131] Take the supernatant and proceed directly to protein purification.

[0132] The primer sequences of RhUBC32-pET28a myc are SEQ ID NO.33 and SEQ ID NO.34.

[0133] The primer sequences of RhRED are SEQ ID NO.35 and SEQ ID NO.36.

[0134] The primer sequences of RhETR3 are SEQ ID NO.37 and SEQ ID NO.38.

[0135] The primer sequences of RhRED T1 are SEQ ID NO.39 and SEQ ID NO.40.

[0136] The primer sequences of RhRED T2 are SEQ ID NO.41 and SEQ ID NO.42.

[0137] The primer sequences of RhRED T3 are SEQ ID NO.43 and SEQ ID NO.44.

[0138] The primer sequences of RhRED T4 are SEQ ID NO.45 and SEQ ID NO.46.

[0139] The primer sequences of RhRED T5 are SEQ ID NO.47 and SEQ ID NO.48.

[0140] The primer sequences of RhRED T6 are SEQ ID NO.49 and SEQ ID NO.50.

[0141] The primer sequences of RhRED T7 are SEQ ID NO.51 and SEQ ID NO.52.

[0142] The primer sequences of RhRED T8 are SEQ ID NO.53 and SEQ ID NO.54.

[0143] The primer sequences of RhRED T9 are SEQ ID NO.55 and SEQ ID NO.56.

[0144] The primer sequences of RhRED T10 are SEQ ID NO.57 and SEQ ID NO.58.

[0145] The primer sequences of RhRED T11 are SEQ ID NO.59 and SEQ ID NO.60.

[0146] The primer sequences of RhRED T12 are SEQ ID NO.61 and SEQ ID NO.62.

[0147] 10. GST fusion protein purification

[0148] Prepare the syringe and gasket. Soak the gasket in 20% alcohol before use. After removing the gas inside, place the gasket in the matching syringe tube.

[0149] Pipette a certain amount of Glutathione-sepharose 4B (about 200 μl / 200 ml bacterial solution) and place it on the gasket;

[0150] After washing with 20% alcohol for 3 column volumes, wash with GGB buffer for 3 column volumes;

[0151] The treated GST filler was resuspended with the supernatant of the broken protein, poured into a clean conical flask, incubated at 60 rpm, in an ice bath, and allowed to bind for more than 1 hour;

[0152] The bound protein supernatant was passed through the column to collect the filler. This process can be repeated several times until all the filler is collected. Then, 5 column volumes of GGB buffer were used to wash the column.

[0153] Add elution buffer to the column with GST filler to elute the protein. Add 1 ml of elution buffer for the first time and collect the eluate after reacting for 15 minutes.

[0154] After that, each reaction should last at least 3 to 5 minutes, for a total of 10 times;

[0155] After purification, the protein was run on a protein gel to detect the concentration and stored in aliquots at -80°C.

[0156] 11. Purification of MBP fusion protein

[0157] The MBP-RhRED bacterial solution was resuspended in 30 ml MBP resuspension buffer and disrupted by ultrasonication at 9.9 s on and 9.9 s off for 15 min.

[0158] 10000rpm, 4℃, 40min;

[0159] Pipette 100 μl of MBP beads, wash twice with 500 ml of MBP resuspension buffer, mix with the supernatant after centrifugation, incubate at 60 rpm, room temperature for 1 h or 4 °C for 3 h;

[0160] Collect beads, wash beads 2-3 times with 500μl MBP buffer, add 100μl MBP buffer and 50mM maltose to elute MBP protein, increase elution volume and collect protein;

[0161] After purification, the protein was run on a protein gel to detect the concentration and stored in aliquots at -80°C.

[0162] 12. Ubiquitin ligase activity analysis

[0163] Add 50ng E1 (purified Arabidopsis E1 protein), 100ng human E2 (purified protein with His and MYC tags), 1μl 10× reaction buffer, MBP-RhRED protein (500ng), 2μl purified His-Ub (about 2μg / μl) to a 1.5ml centrifuge tube, and add ddH2O to a total volume of 30μl;

[0164] In order to ensure the accuracy of the experiment, five tubes, -E1, -E2, -E3, -Ub and all, were used for reaction at 30°C and 900 rpm for 90 min;

[0165] Add 6 μl 5× SDS loading buffer, boil at 100°C for 6 min, and use anti-Ub and anti-GST / MBP antibodies to detect whether E3 has been added with Ub.

[0166] 12. Substrate ubiquitination labeling analysis

[0167] Add 50 ng E1 (purified Arabidopsis E1 protein), 100 ng human E2 or His-Myc-RhUBC32, 1 μl 10× reaction buffer, MBP-RhRED (500 ng), 2 μl purified His-Ub (about 2 μg / μl), 500 ng GST-RhETR3 to a 1.5 ml centrifuge tube, and add ddH2O to a total volume of 30 μl;

[0168] In order to ensure the accuracy of the experiment, five tubes, -E1, -E2, -E3, -Ub and all, were used for reaction at 30 °C and 900 rpm for 2.5 h;

[0169] Add 6 μl of 5× SDS loading buffer, boil at 100°C for 6 min, and use anti-Ub and anti-GST antibodies to detect whether the substrate is modified by ubiquitin.

[0170] The primer sequences of nLUC-RhETR3 are SEQ ID NO.63 and SEQ ID NO.64.

[0171] The primer sequences of cLUC-RhRED are SEQ ID NO.65 and SEQ ID NO.66.

[0172] 13. Luciferase complementation assay

[0173] The RhETR3 ORF containing Bam HI and Spe I sites was cloned into the pCAMBIA-nLUC vector to obtain the RhETR3-nLUC vector;

[0174] The RhRED ORF containing Bam HI and Spe I sites was cloned into the pCAMBIA-cLUC vector to obtain the RhRED-cLUC vector, and transformed into Agrobacterium;

[0175] Agrobacterium transformed with RhETR3-nLUC and RhRED-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.

[0176] The primer sequences of RhRED-TRV2 are SEQ ID NO.67 and SEQ ID NO.68.

[0177] 14. Virus-induced gene silencing

[0178] In order to construct the silencing vector, the 5' end 507 bp fragment of the RhRED gene was inserted into the pTRV2 vector to construct pTRV2-RhRED. The primers for constructing the vector are listed in Table 2.

[0179] The target fragments of pTRV1, pTRV2, and pTRV2-RhRED were transformed into Agrobacterium GV3101;

[0180] 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;

[0181] Use the vector primers of pTRV1 and pTRV2 to detect whether the bacterial solution contains the target plasmid by PCR;

[0182] 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.

[0183] 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;

[0184] Mix pTRV1 bacterial solution with pTRV2 and pTRV2-RhRED target gene fragments in equal volumes, about 300-500 ml of bacterial solution for each group; let stand in the dark for 3-6 hours for later use;

[0185] 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;

[0186] 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;

[0187] Evacuate the air, and when the pressure reaches 0.8 atmospheres, maintain the pressure for 5 minutes and then slowly release the air.

[0188] 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;

[0189] After the infection is over, rinse under running water to wash off the excess bacterial liquid;

[0190] Place the seedlings in a small pot with lines (roots in water, stems and leaves in air), wrap with plastic wrap to maintain humidity;

[0191] Place in dark at 8℃ for 3 days;

[0192] After 3 days, plant the tissue culture seedlings in a small pot containing vermiculite and nutrient soil (1:1), cover with plastic film to keep moisture for one week, and place in a culture room for cultivation;

[0193] 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;

[0194] The material containing CP gene was tested for gene expression level using gene detection primers, and plants with lower expression were selected for subsequent experiments. The primer sequences of RhUBI2 are SEQ ID NO.5 and SEQ ID NO.6. The primer sequences of RhRED ex are SEQ ID NO.3 and SEQ ID NO.4.

[0195] The TRV-CP primer sequences used to detect viruses are SEQ ID NO. 23 and SEQ ID NO. 24. The primer sequences for detecting TRV1 are SEQ ID NO. 19 and SEQ ID NO. 20. The primer sequences for detecting TRV2 are SEQ ID NO. 21 and SEQ ID NO. 22.

[0196] 15. RhRED RNAi stable transgenic rose

[0197] The 500 bp sequence of RhRED 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;

[0198] 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;

[0199] 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;

[0200] 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.

[0201] 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;

[0202] When the flowers opened to level 5, total RNA was extracted and the expression of RhRED was detected.

[0203] RhRED RNAi contains two pairs of specific primers:

[0204] Forward sequence primers: SEQ ID NO.69 and SEQ ID NO.70;

[0205] Reverse sequence primers: SEQ ID NO.71 and SEQ ID NO.72.

[0206] 16. Ethylene treatment

[0207] 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.

[0208] 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;

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] According to a preferred embodiment, the LB configuration method is: 50 mg / L Kan, 25 mg / L Rif, 2 mM AS mixture.

[0214] 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.

[0215] 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.

[0216] According to a preferred embodiment, the GST-RhRED resuspension buffer-GGB buffer is prepared by mixing 50 mM Tris-HCl, 120 mM NaCl, 1 mM DTT, 0.5% NP-40, and 1 mM PMSF at pH 8.0.

[0217] According to a preferred embodiment, the MBP-RhRED resuspension buffer is prepared by mixing 20 mM Tris-HCl, 20 mM NaCl at pH 8.0, and 1 mM DTT.

[0218] 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.

[0219] Example 1

[0220] This embodiment provides a method for cloning the RhRED gene.

[0221] RhRED is a RING-type ubiquitin ligase.

[0222] The present invention uses RhETR3 as bait to screen the rose split ubiquitination yeast two-hybrid library, and obtains a total of 245 clones. After sequence alignment and yeast rotation verification, a total of 28 confirmed positive clones are obtained, and a ubiquitin ligase RhRED (RU18315) is obtained from them.

[0223] Through multiple sequence alignment, the full length of RU18315 was obtained and named RhRED according to gene annotation. The full length of RhRED gene is 1123 bp, ORF is 849 bp, encoding 282 aa. Through SMART protein sequence analysis program, the following was obtained: Figure 1 The RhRED protein sequence is shown in Figure 1 The protein linear structure diagram shown shows that the RhRED protein has a conserved RINGv domain at the N-terminus (corresponding to amino acids 26-57 in the sequence diagram) and two possible transmembrane regions at the C-terminus (corresponding to amino acids 176-198 and 213-235 in the sequence diagram). This result indicates that the RhRED protein belongs to the RING type ubiquitin ligase.

[0224] Example 2

[0225] This embodiment provides a method for detecting the association between the RhRED gene and the RhETR3 gene.

[0226] The interaction between RhRED and RhETR3 was verified by three experiments: bimolecular fluorescence complementation (BiFC), split ubiquitination yeast two-hybrid (MYTH) and co-localization.

[0227] RhRED-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 RhRED-GFP can completely overlap with the red fluorescence of the plasma membrane marker, indicating that RhRED is localized in the plasma membrane and is a membrane-localized protein. Its green fluorescence can also overlap with the red fluorescence of RhETR3-mCherry, indicating that RhRED and RhETR3 are co-localized and may be bound to each other in plants.

[0228] like Figure 2A As shown, the ethylene receptor RhETR3 protein has four domains, namely the N-terminal transmembrane domain (corresponding to Figure 2A Transmembrane in), GAF domain (corresponding to Figure 2A GAF in ), histidine kinase domain (corresponding to Figure 2AHistidineKinase) and the receiving domain (corresponding to Figure 2A Receiver in the .

[0229] After RhETR3 was truncated and divided into two parts, the transmembrane domain and the detransmembrane domain, the two parts of RhETR3 were verified, and two truncated sequences, RhETR3-TM and RhETR3-ΔTM, were designed. RhRED-PR3N was co-transfected with RhETR3-STE and RhUBC32-STE into yeast MNY51, and could grow on the two-deficient plate SD-TL, indicating that the plasmid transformation of yeast was successful. On the four-deficient plate SD-TLHA, RhUBC32-PR3N, RhETR3-STE and RhUBC32-STE 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 RhRED-PR3N and the empty pBT3-STE would not grow on the four-deficient plate, that is, RhRED interacted with RhETR3 and RhUBC32 in yeast, and its binding occurred in the transmembrane domain of RhETR3.

[0230] The results of BiFC in vivo validation showed that RhRED interacted with RhETR3 in vivo. Figure 2C As shown, green fluorescence can be seen under a fluorescence confocal microscope, and the fluorescence is located at the cell membrane, while RhETR3-YNE (R) and empty pSPYCE (M) do not show fluorescence. This result indicates that RhETR3 interacts with RhRED in plants.

[0231] Example 3

[0232] This embodiment provides a method for detecting the expression level of the RhRED gene.

[0233] The response of RhRED to ethylene and its changes during flower opening were detected by qRT-PCR.

[0234] like Figure 3A As shown in the figure, during the natural opening process, the expression of RhRED gradually increased. After ethylene treatment, the expression of RhRED was significantly higher than that of the control group.

[0235] like Figure 3B As shown, compared with the lanes lacking AtE1 and Human E2, in the presence of AtE1, Human E2, RhRED and Ub, some larger bands were obviously additional above the main band, and these bands could be detected by Ub antibody, indicating that RhRED can bind ubiquitin and has ubiquitin ligase activity.

[0236] Sequence analysis of the RhRED protein showed that there are 12 possible active sites in its RING domain. The C and H amino acids corresponding to the 12 sites were mutated to A through experiments, and the ubiquitin ligase activity of the mutants was analyzed. Figure 3C As shown in Figure 2, it was confirmed that each active site affects the activity of RhRED. Figure 3C As shown, each column corresponds to an activity analysis reaction, in which RhREDm04A and RhREDm09A appear in the same multi-band image of RhRED, while other corridors do not appear. This result also proves that RhREDm04A and RhREDm09A have ubiquitin ligase activity. The 12 active sites of RhRED are C62, C65, C78, ​​C80, C81, H88, C91, H94, C96, C104, C107, and H108. The above numbers represent their positions in the gene amino acid sequence, and the English letters represent the C and H molecules corresponding to the position in the amino acid sequence.

[0237] Mutants of various active sites of RhRED connected to the pMalC2-MBP vector were constructed, among which RhREDm04A corresponds to the C80 mutant and RhREDm09A corresponds to the C96 mutant. The C80 and C96 mutants have ubiquitin ligase activity, while the mutants at other sites have no ligase activity, indicating that C80 and C96 have no effect on the RhRED ligase active site, and other sites are essential for RhRED activity.

[0238] Example 4

[0239] The present invention provides a method for analyzing the activity of ubiquitin ligase E3, a method for promoting the degradation of RhETR3 protein by RhRED, and a method for demonstrating that RhRED promotes the degradation of RhETR3 protein.

[0240] like Figure 4A As shown in the figure, in the presence of AtE1, His-Myc-RhUBC32, GST-RhRED and Ub, some larger bands appeared above the main band, and these bands could be detected by Ub antibody. This result indicates that RhUBC32 can transfer ubiquitin to RhRED, and RhRED has ubiquitin ligase activity.

[0241] Figure 4B and 4C The results showed that in the presence of Human E2 or His-Myc-RhUBC32, MBP-RhRED, some larger bands appeared above the main band, and these bands could be detected by Ub antibody. This result indicated that RhETR3 could be ubiquitinated and RhETR3 was the substrate of RhRED.

[0242] RhETR3-Flag and RhRED-GFP were co-expressed in tobacco leaves at different ratios. Figure 4E It was determined that as the ratio of RhRED-GFP increased, the amount of RhRED protein detected by GFP gradually increased, while the amount of RhETR3-Flag protein gradually decreased, and the internal reference protein Actin had no significant change. This result shows that RhRED can affect the stability of RhETR3 protein in a dose-dependent manner and accelerate its degradation.

[0243] The dual luciferase results showed that Figure 4D As shown in the figure, with the increase of the ratio of RhETR3-nLUC to RhRED-cLUC, the amount of RhRED-cLUC gradually increased and the fluorescence intensity of LUC gradually decreased when RhETR3-nLUC was quantified. This result shows that RhETR3 and RhRED proteins interact with each other in vivo, and this binding increases with the increase of the ratio of RhETR3-nLUC to RhRED-cLUC, and this interaction accelerates the degradation of RhETR3. Figure 4D The fluorescence images in the figure show that the LUC fluorescence intensity becomes weaker.

[0244] Example 5

[0245] 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 method for detecting the phenotype of roses ripened by ethylene.

[0246] The 500 bp sequence of the RhRED 3'UTR-250 region was ligated to the viral silencing vector TRV2. Figure 5A As shown, the RhRED gene was inserted into the multiple cloning site (MCS) of the TRV2 vector using restriction enzyme sites XbaⅠ and KpnⅠ.

[0247] like Figure 5B As shown, with RhUBI2 as the control, the brightness of the electrophoresis band of TRV2-RhRED was lower than that of the electrophoresis band of TRV2. The results showed that the expression level of RhRED gene in the rose plants with RhRED silenced was significantly lower than that in the control TRV2 plants, and the silencing effect was obvious.

[0248] The flowers in level 2 state appear with sepals fully expanded and buds tightly closed.

[0249] After treating the flowers in the second state 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 5CAs shown in the figure, the picture corresponding to TRV2 is a picture of normal rose organ development, and TRV2-RhRED is a picture of silent rose organ development. The upper row of the picture shows the falling flowers, and the lower row of the picture shows the curling flowers. According to the statistical results Figure 5D It can be seen that after ethylene treatment, the flowers that fell off and curled up accounted for 75% in the RhRED-silenced plants, which was significantly higher than the 20% in the TRV2 control group, that is, the ethylene sensitivity of the silenced group plants increased.

[0250] The present invention also relates to a RhRED RNAi vector, which is used to obtain stable transgenic plants through Agrobacterium-mediated transgenic technology. Fig. 6A As shown in the figure, the RhRED expression levels of RhRED-RNAi#3 and RhRED-RNAi#5 were lower than those of the wild type (WT), indicating that two RhRED low-expression strains, RhRED-RNAi#3 and RhRED-RNAi#5, were obtained. Figure 6B As shown, the expression levels of RhHB6 in RhRED-RNAi#3 and RhRED-RNAi#5 were higher than that in the wild type (WT).

[0251] like Figure 6C As shown, the first column shows the phenotypes of the wild type, RhRED-RNAi#3, and RhRED-RNAi#5 after ethylene treatment. The second column shows the phenotypes of the wild type, RhRED-RNAi#3, and RhRED-RNAi#5 24 hours after ethylene treatment. As shown in 6C, the petals of RhRED-RNAi#3 and RhRED-RNAi#5 flowers fell off 24 hours after ethylene treatment, while the petals of wild type flowers did not fall off. This phenotype indicates that the ethylene sensitivity of RhRED-RNAi transgenic plants is increased, which is consistent with the RhRED VIGS silenced plants.

[0252] Fig.6D The figure shows the daily wilting status of wild type and RhRED-RNAi#3 under normal open state. Fig.6D As shown in Figure 2, the number of days from flower level 5 to flower shedding and wilting in low-expressing plants under normal opening conditions was significantly greater than that in the wild type. Fig.6D It can be seen that the flowering duration of flowers of RhRED-RNAi#3 plants is 4 days longer than that of the wild type.

[0253] like Fig. 6E As shown, under normal opening state, the flowers of low-expressing plants RhRED-RNAi#3 wilted significantly on the 15th day, and RhRED-RNAi#5 wilted significantly on the 13th day, while the wild type wilted significantly on the 6th day, the same as the above low-expressing plants.

[0254] Table 1

[0255]

[0256]

[0257]

[0258]

[0259] 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. An application of rose ubiquitin ligase in regulating ethylene receptor RhETR3 protein, characterized in that: The rose ubiquitin ligase is prepared by replacing the C62, C65, C78, ​​C81, H88, C91, H94, C104, C107 or H108 sites with amino acid A based on the amino acid sequence SEQ ID NO.2, and the replacement reduces the activity of the rose ubiquitin ligase.

2. A method for cultivating rose plants with a high ethylene sensitivity phenotype, characterized in that: Include: A target fragment is selected from the coding region of the rose ubiquitin ligase gene, and the target fragment is inserted into the pFGC1008 vector to obtain a silencing vector, wherein the nucleotide sequence of the rose ubiquitin ligase gene is SEQ ID NO.1, and the silencing vector is obtained by connecting the forward sequence and the reverse sequence into the pFGC1008 vector through the ASC I / Swa I and Pac I / Bam HI sites, and the amplification primers of the forward sequence are SEQ ID NO.69 and SEQ ID NO.70; the amplification primers of the reverse sequence are SEQ ID NO.71 and SEQ ID NO.72; 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 a mutant strain with low expression of the rose ubiquitin ligase gene.

3. Application of the method described in claim 2 in prolonging the duration of flowering of Chinese rose.

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