A tomato ubiquitin conjugating enzyme gene and its application

Abstract

The present invention provides a tomato ubiquitin conjugating enzyme gene and the hormone regulating effect of the gene in tomato ripening. The gene and the application of the gene provided by the present invention can provide a basis and direction for tomato breeding and directional cultivation. The gene sequence is SEQ ID NO.1. Based on the tomato ubiquitin conjugating enzyme gene provided by the present invention, on the one hand, it can increase screening conditions for molecular identification of tomato breeding; on the other hand, it can provide more variable conditions for tomato molecular breeding.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a tomato ubiquitin conjugating enzyme gene and application thereof. Background Art

[0002] Tomato fruits tend to over-ripen and become soft, which results in poor pressure resistance, shortened storage period, and reduced resistance to pathogens, seriously affecting the quality of production, storage, transportation and processing.

[0003] Tomato is a respiratory climacteric fruit, which produces a large amount of ethylene, namely system II ethylene, to ripen the fruit. System II ethylene has an autocatalytic mechanism, which makes the tomato ripening process difficult to control, causing the fruit to overripe, soften, rot and deteriorate. In the process of fruit ripening, ethylene as the main plant hormone has been studied in depth in many species. At present, tomato has become the main model for vegetable fruit development research in my country and is widely used in ethylene ripening experiments.

[0004] As an endogenous hormone in plants, ethylene has the physiological functions of promoting fruit and cell enlargement, seed ripening, and leaf, flower, and fruit abscission. It also has the functions of inducing flower bud differentiation, breaking dormancy, promoting germination, inhibiting flowering, organ abscission, dwarfing plants, and promoting adventitious root formation.

[0005] Climax fruits, such as tomatoes, are characterized by increased respiration at the onset of ripening, and a concomitant increase in ethylene biosynthesis. These fruits and vegetables need to sense and produce ethylene during their growth. Unlike the problem of over-ripening during transportation, when the production or perception of ethylene is blocked, the fruits of these fruits and vegetables cannot ripen. Ethylene acts on different growth stages of tomatoes. Yang et al. (1987) stated that "exposure of unripe fruit to ethylene leads to system I ethylene synthesis."

[0006] In contrast, ripe fruit exhibits system II ethylene synthesis, which contains a large number of genes that induce the process of autocatalysis of ethylene. Yang et al. published a paper in 1987 stating that "although the application of ethylene does not initiate the ripening of immature fruit, it does significantly accelerate the onset of ripening." The more ethylene an immature fruit is exposed to, the earlier it ripens, and this cumulative effect also exists in bananas.

[0007] The results of receptor gene analysis in tomato and Arabidopsis in existing studies show that plant ethylene receptors act as negative regulators in the ethylene response pathway. In the absence of ethylene, active receptors inhibit ethylene response. When plants enter the period of maturity, they produce ethylene by themselves or exogenously supplemented ethylene. Ethylene binds to plant ethylene receptors, the ethylene signaling pathway opens, and the next step of the reaction begins. There are currently six known ethylene receptors in tomatoes, namely LeETR1, LeETR2, NR, LeETR4, LeETR5, and LeETR6. Based on the protein structure of ethylene receptors, ethylene receptors are divided into two subfamilies: subfamily I has histidine kinase activity, and subfamily II has serine / threonine kinase activity.

[0008] The Chinese patent with patent number CN112322644B discloses an application of a tomato SlSPY gene in controlling the ripening process of tomato fruit. The expression level of the tomato SlSPY gene is increased by gene overexpression technology. Tomato SlSPY overexpression plants and gene knockout plants are constructed by genetic means, and the expression level of the gene SlSPY is regulated to study its regulatory mechanism for tomato fruit ripening. The results show that SlSPY overexpression can increase the sensitivity of green-ripe tomato fruit to ethylene, accelerate the color change of tomato fruit, promote fruit softening and the accumulation of carotenoids such as lycopene. In SlSPY gene knockout plants, the color change and softening process of tomato fruit are inhibited, and the accumulation of carotenoids such as lycopene is less than that of wild-type fruits of the same period. The regulatory mechanism of tomato ripening, especially the regulatory mechanism of ethylene-catalyzed ripening, is explored in the prior art.

[0009] Existing studies have shown that the loss of the tomato receptor LeETR4 leads to increased ethylene sensitivity. Tieman et al. published a paper in 2000 stating that LeETR4 RNAi plants exhibited a constitutive ethylene response phenotype, including significantly earlier fruit ripening, and that the phenotype of this mutant could be restored to the wild type by overexpressing the subfamily I receptor NR.

[0010] In the prior art, the family II ethylene receptor LeETR4 is the main regulator of ethylene response and ripening in tomato fruit. Ethylene promotes the degradation of LeETR4 protein, and its mutants show early maturity and increased ethylene sensitivity. The receptor signal transduction model shows that the receptor negatively regulates the ethylene response. Ciardi et al. published a paper in 2000 stating that "a decrease in receptor content increases ethylene sensitivity, while an increase in receptor content has the opposite effect." NR and LeETR4 transcripts are upregulated during fruit ripening. Contrary to RNA data, protein blot analysis shows that the receptor protein level is highest in the unripe stage of the fruit and decreases significantly in the early stage of ripening; at the same time, ethylene treatment causes rapid degradation of the receptor protein, which can be inhibited by MG132. Therefore, LeETR4 may be degraded through the 26S proteasome pathway, but its specific degradation mechanism is still unknown.

[0011] LeETR4 is an important information transmission factor involved in the physiological pathway of tomato response to ethylene. Substances that regulate its production and degradation in the pathway can significantly increase or decrease the sensitivity of tomatoes to ethylene. Based on this, in view of the gap in the ethylene receptor regulation mechanism in the prior art, the present invention provides a tomato ubiquitin conjugating enzyme gene and the hormone regulation effect of the gene in tomato ripening. The gene provided by the present invention and the application of the gene can provide a basis and direction for tomato breeding and directional cultivation.

[0012] 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

[0013] In view of the deficiencies of the prior art, the present invention provides a tomato ubiquitin conjugating enzyme gene (SlUBC32), the gene sequence of which is SEQ ID NO. 1. The present invention also provides the use of the SlUBC32 gene or protein in regulating the process of ethylene-induced flower opening. Preferably, the use is the use of the SlUBC32 gene or the SlUBC32 protein in regulating the ethylene sensitivity of fruits in tomatoes.

[0014] According to a preferred embodiment, the SlUBC32 protein sequence is substituted, deleted and / or one or more amino acids are added and the protein derived from the SlUBC32 protein with equivalent activity also has ethylene regulation function in plants.

[0015] Preferably, the S1UBC32 can be applied to the regulation of ethylene sensitivity of Solanaceae plants. Preferably, the Solanaceae plants can be tomatoes.

[0016] The present invention also provides a vector for constructing ethylene low-sensitive tomatoes, wherein the vector is constructed as described in the examples.

[0017] According to a preferred embodiment, the low expression of the tomato ubiquitin conjugating enzyme gene in the tomato can reduce the sensitivity of the tomato to ethylene.

[0018] The invention provides a tomato ubiquitin conjugating enzyme for regulating the degradation of ethylene receptor S1ETR4 protein. The amino acid sequence of the tomato ubiquitin conjugating enzyme is SEQ ID NO.2.

[0019] Cultivating Solanaceae plants with low ethylene sensitivity by gene editing. Specifically, the present invention provides a method for cultivating tomatoes with a high ethylene sensitivity phenotype, comprising the following steps:

[0020] Selecting at least one target fragment in the coding region of the tomato ubiquitin conjugating enzyme gene to construct a CRISPR / Cas9 vector;

[0021] Constructing genetically engineered Agrobacterium containing the CRISPR / Cas9 vector;

[0022] Transforming tomato cotyledons with the genetically engineered Agrobacterium to obtain a homozygous mutant strain that does not contain exogenous Cas9 protein and is stably inherited;

[0023] The nucleotide sequence of the tomato ubiquitin conjugating enzyme gene is shown in SEQ ID NO.1.

[0024] According to a preferred embodiment, the nucleotide sequence of the primer for constructing the first target fragment is such as SEQ ID NO. 19 and SEQ ID NO. 20. Preferably, blocking or reducing the expression of the SlUBC32 gene or the SlUBC32 protein in plants is achieved by gene editing technology.

[0025] According to a preferred embodiment, the nucleotide sequences of the primers for constructing the second target fragment are such as SEQ ID NO.21 and SEQ ID NO.22.

[0026] The present invention constructs a tomato SlUBC32 gene knockout plant by means of technical means at the molecular biology level, and confirms the regulatory mechanism of tomato sensitivity to ethylene by exploring the expression level of the gene SlUBC32 and the plant phenotype. The results show that tomatoes with low or no expression of SlUBC32 will increase the sensitivity of tomato fruits to ethylene, promote the color change of tomato fruits, reduce the accumulation of carotenoids and the hardness of fruits, and thus promote the low maturity of fruits. Therefore, the tomato SlUBC32 gene promotes the maturity of tomato fruits by increasing the sensitivity of tomato fruits to ethylene. The SlUBC32 gene provided by the present invention provides a gene resource for new varieties of early-maturing or late-maturing tomatoes, has good potential application value, and can adjust the time to market of tomato fruits through transgenic technology, and reduce the accumulation and rot of mature fruits.

[0027] The SlUBC32 gene or the protein is induced by ethylene, and the expression level increases with increasing maturity. Based on this, the present invention can affect the function of ethylene in plants by changing the expression of the SlUBC32 gene or the SlUBC32 protein in plants. Increasing the expression of the SlUBC32 gene or the SlUBC32 protein in plants weakens the response of tomato fruit to ethylene. Preferably, the present invention provides a tomato with a low ethylene sensitivity phenotype. The SlUBC32 gene is highly expressed in the tomato. The present invention provides a tomato with a low ethylene sensitivity phenotype, and the protein encoded by the SlUBC32 gene is highly expressed in the tomato.

[0028] According to a preferred embodiment, the regulation of plant biological levels caused by the SlUBC32 gene can affect the performance of plant functions by affecting the response of the plant to ethylene.

[0029] The invention provides an application of the SlUBC32 gene as a negative regulatory factor in regulating the ethylene-induced tomato ripening process.

[0030] The present invention provides a biological material containing SlUBC32 gene, which is an expression vector and a strain.

[0031] According to a preferred embodiment, the expression vector can be a pDIRECT-22C vector connected to the SlUBC32 gene.

[0032] The present invention provides an application of a SlUBC32 gene as a negative regulatory factor in regulating the ethylene-induced tomato fruit ripening process. The expression amount of the gene in a plant is increased by a genetic engineering method, thereby reducing the sensitivity of the plant to ethylene.

[0033] The present invention provides an application of a protein encoded by a SlUBC32 gene as a negative regulatory factor in regulating the ethylene-induced tomato fruit ripening process. The expression amount of the protein encoded by the gene in a plant is increased by a genetic engineering method, thereby reducing the sensitivity of the plant to ethylene.

[0034] The protein encoded by the SlUBC32 gene provided by the present invention is used as a negative regulatory factor in regulating the process of ethylene-induced flower opening. The amino acid sequence of the protein is shown in SEQ ID NO.2. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A This is a schematic diagram of the sequence comparison of tomato ethylene receptor proteins;

[0036] Figure 1B This is a schematic diagram of the evolutionary tree of tomato ethylene receptors;

[0037] Figure 2A This is a comparison result diagram of SlUBC32 protein;

[0038] Figure 2B This is a diagram showing the results of the SlUBC32 protein characteristic analysis;

[0039] Figure 3 This is a demonstration diagram of the interaction between SlETR4 and SlUBC32;

[0040] Figure 4A This is the expression diagram of SlUBC32 in tomatoes after 3 h of ethylene treatment by qRT-PCR analysis;

[0041] Figure 4B The expression profile of SlUBC32 in different developmental stages of tomato fruit was analyzed by qRT-PCR;

[0042] Figure 4C This is a diagram for in vitro analysis of the ubiquitin conjugating enzyme activity of SlUBC32;

[0043] Figure 5 This is a detection diagram of SlUBC32 degrading SlETR4 through the 26S proteasome pathway;

[0044] Fig. 6A Schematic diagram of SlUBC32 CRSPR transgenic typing;

[0045] Figure 6B This is a plant type analysis diagram of SlUBC32 CRSPR transgenic plants;

[0046] Figure 6C This is the phenotype diagram of SlUBC32 CRSPR transgenic tomato ethylene sensitivity detection;

[0047] Fig.6DThis is a statistical chart of the ethylene sensitivity test results of SlUBC32 CRSPR transgenic tomatoes;

[0048] Fig. 6E This is a statistical chart of the ripening time of SlUBC32 CRSPR transgenic tomatoes;

[0049] Fig. 6F This is the detection diagram of SlETR4 protein expression. DETAILED DESCRIPTION

[0050] The following is a detailed description with reference to the accompanying drawings.

[0051] The gene number of SlETR4 is Solyc06g053710.

[0052] The gene number of SlUBC32 is Solyc12g099310.

[0053] The invention adopts TCA method to extract tomato total protein.

[0054] Here’s how:

[0055] Tomato fruit samples were ground after adding liquid nitrogen, and about 1 ml was placed in a 2 ml centrifuge tube, filled with 10% TCA / acetone, extraction buffer, and shaken to mix;

[0056] Incubate on ice for about 30 min, centrifuge at 16000 g for 4 min at 4 °C, and discard the supernatant;

[0057] Add 80% methanol and 0.1% NH 4 AC, fill up, shake and mix;

[0058] Centrifuge at 16000g for 4 min at 4°C and discard the supernatant;

[0059] Add 80% acetone to the centrifuge tube, fill it up, and shake to mix;

[0060] Centrifuge at 16000g for 4 min at 4°C and discard the supernatant;

[0061] Place in a fume hood for 15 to 25 minutes until all the acetone evaporates;

[0062] Add 800 μl SDS buffer / Tris-phenol (1:1), flick the centrifuge tube until all the precipitate is suspended, and shake for 5 min;

[0063] Centrifuge at 16000g for 6 min at 4°C and transfer the supernatant to a new centrifuge tube;

[0064] Add anhydrous methanol and 0.1 M ammonium acetate to the centrifuge tube, fill it up, mark it and mix well;

[0065] -20℃ precipitation for more than 1h;

[0066] Centrifuge at 16000g for 5 min at 4°C and discard the supernatant;

[0067] Fill with anhydrous methanol, shake to mix, and centrifuge at 16000g for 4 min at 4°C;

[0068] The supernatant was discarded, anhydrous acetone was added to the top, the mixture was shaken and mixed, and centrifuged at 16000g for 4 min at 4°C;

[0069] Discard the supernatant and place in a fume hood for 15 to 25 minutes until the acetone evaporates completely;

[0070] According to the volume of the precipitate, add a certain volume of protein dissolution buffer-2×SDS protein loading buffer;

[0071] The extracted total protein was denatured in boiling water at 100°C for 6-7 min, and then ice-bathed for 2 min. The protein level was detected by WB or stored at -20°C for later use.

[0072] According to a preferred embodiment, the infection solution is prepared as follows: 10 mM MES, 20 mM AS, 10 mM MgCl 2 Mix and adjust the pH of the mixture to 5.6.

[0073] According to a preferred embodiment, the His protein resuspension lysis buffer is prepared as follows: 50 mM NaH 2 PO 4 , 300 mM NaCl, adjust the pH of the mixture to 8.0 with HCl, and add 1 mM PMSF before use.

[0074] According to a preferred embodiment, the binding buffer is configured as follows: 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, and 5% glycerol were mixed, the pH of the mixture was adjusted to 8.0 with NaOH, and 1 mM PMSF was added before use.

[0075] According to a preferred embodiment, the wash buffer is prepared as follows: 50 mM NaH 2 PO 4 , 300 mM NaCl, 30 mM imidazole, and 5% glycerol were mixed, the pH of the mixture was adjusted to 8.0 with NaOH, and 1 mM PMSF was added before use.

[0076] According to a preferred embodiment, the eluent I is prepared as follows: 50 mM NaH 2 PO 4, 300 mM NaCl, 100 mM imidazole, and 5% glycerol were mixed, the pH of the mixture was adjusted to 8.0 with NaOH, and 1 mM PMSF was added before use.

[0077] According to a preferred embodiment, the eluent II is prepared as follows: 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, and 5% glycerol were mixed, the pH of the mixture was adjusted to 8.0 with NaOH, and 1 mM PMSF was added before use.

[0078] According to a preferred embodiment, the eluent III is prepared as follows: 50 mM NaH 2 PO 4 , 300 mM NaCl, 500 mM imidazole, and 5% glycerol were mixed, the pH of the mixture was adjusted to 8.0 with NaOH, and 1 mM PMSF was added before use.

[0079] According to a preferred embodiment, E1 refers to Arabidopsis thaliana E1 purified protein, and E2 refers to purified protein with His and Myc tags.

[0080] According to a preferred embodiment, the reaction buffer is prepared as follows: 1 M Tris-HCl at pH 7.5, 100 mM ATP sigma, 50 mM MgCl 2 , 2 mM DTT mixed.

[0081] According to a preferred embodiment, the seed germination culture medium is prepared by mixing MS, 30 g / L sucrose and 2.4 g / L plant gel.

[0082] According to a preferred embodiment, the resuspension solution is prepared by mixing MS, 2.1 g / L MES, 30 g / L glucose, 0.05 MAS, 1 mg / L NAA and 1 mg / L 6-BA.

[0083] According to a preferred embodiment, the co-cultivation culture medium is prepared by mixing MS, 2.1 g / L MES, 30 g / L glucose, 0.05 M AS, 1 mg / L NAA, 1 mg / L 6-BA and 2.4 g / L plant gel.

[0084] According to a preferred embodiment, the screening culture medium is configured as follows: a mixture of MS, 2 mg / L ZT, 0.1 mg / L IAA, 300 mg / L Tim, 100 mg / L Kan and 2.4 g / L plant gel.

[0085] According to a preferred embodiment, the rooting medium is configured as follows: 1 / 2MS, 2 mg / L IBA, 300 mg / L Tim, 100 mg / L Kan, and 2.4 g / L plant gel.

[0086] According to a preferred embodiment, the protein dissolution buffer is prepared by mixing 50 mM HEPES at pH 7.4, 137 mM NaCl, 10% glycerol, 1 mg / L Pepstatin A, 1 mg / L Leupetin and 1 mM protease inhibitor (PMFS).

[0087] Example 1

[0088] This embodiment provides a method for cloning the S1UBC32 gene.

[0089] According to the RhUBC32 protein sequence, the homologous gene Solyc12g099310 was obtained by comparison in the tomato database, and its gene sequence was obtained. The full-length gene sequence SEQ ID NO.1 was obtained by homologous cloning method;

[0090] The full-length amino acid sequence of SlUBC32 was aligned with the UBCc domains in rose, Arabidopsis, yeast, and human using the ClustalW program;

[0091] Phylogenetic analysis was performed using the neighbor-joining method using MEGA7.0.

[0092] The protein sequence of rose RhETR3 was used to perform multiple sequence alignment and phylogenetic tree analysis with six known receptor proteins in tomato, such as Figure 1A As shown, the sequence overlap between RhETR3 and SlETR4 reached 67.97%, therefore, the sequence homology between RhETR3 and SlETR4 was the highest.

[0093] like Figure 1B As shown, phylogenetic tree analysis was performed using MEGA7.0 software. The positions of SlETR4 and RhETR3 were closest in the phylogenetic tree, and the results showed that SlETR4 and RhETR3 had the highest homology.

[0094] The protein sequence of rose RhUBC32 was used for comparison in the tomato database.

[0095] The results are as follows Figure 2AAs shown, the sequence SEQ ID NO.2 is closest to the amino acid sequence of Solyc12g099310, indicating that the two have high homology, based on which the sequence SEQ ID NO.2 is named SlUBC32. The phylogenetic tree analysis in the figure shows that SlUBC32 is most closely related to the rose RhUBC32, and SlUBC32 is relatively conserved in eukaryotes.

[0096] Figure 1A and 2B The figure shows that the SlUBC32 gene is 906 bp in length and encodes 301 aa. The SMART protein sequence analysis program revealed that the SlUBC32 protein has a conserved UBCc domain (amino acids 14 to 174) at the N-terminus. The sequence of this domain is shown in Figure 2B The underlined sequence is shown. There is a transmembrane domain (amino acids 267 to 284) at the C-terminus. The sequence of this domain is as follows Figure 2B The grey boxed sequences are shown.

[0097] Example 2

[0098] This embodiment provides a method for detecting the association between the S1UBC32 gene and the S1ETR4 gene.

[0099] The method is a bimolecular fluorescence complementation (BiFC) experiment used to determine whether SlETR4 and SlUBC32 interact with each other.

[0100] SlETR4-YNE(R), SlUBC32-YCE(M), pYCE(M) vectors and silencing suppressor P19 were transformed into Agrobacterium GV3101 respectively;

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

[0102] Use infection solution to adjust the bacterial solution to OD 600 was 3.0, SlETR4-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 2 days, fluorescence was observed under a fluorescence confocal microscope.

[0104] The forward and reverse primers of SlETR4-YNE are shown in Table 1 as SEQ ID NO. 3 and SEQ ID NO. 4.

[0105] The forward and reverse primers of SlUBC32-YCE(M) are shown in Table 1 as SEQ ID NO. 5 and SEQ ID NO. 6.

[0106] like Figure 3 The green fluorescence shown is localized on the lipid membrane. Green fluorescence can be observed in the two-color merge image where SlETR4-nYFP and empty cYFP are overlapped. Figure 3 The results shown indicate that SlETR4 interacts with SlUBC32 in plants.

[0107] Example 3

[0108] This embodiment provides a method for detecting the expression level of the SlUBC32 gene. This embodiment provides a method for cultivating tomatoes using ethylene. This embodiment provides a method for detecting the phenotype of tomatoes ripened by ethylene.

[0109] The protein extraction method in tomato fruit is:

[0110] Green ripe tomatoes with basically the same fruit setting days, fruit setting positions and sizes were collected and treated with 20 ppm ethylene for 3 h. The tomatoes were cut horizontally in the middle, and about 0.5 mm of flesh was cut from each of the upper and lower sections. Samples were taken, frozen in liquid nitrogen, and stored at -80°C pending protein extraction experiments.

[0111] Cultivation method for ripe tomato fruits: Select green ripe tomatoes with basically the same fruit setting days, fruit setting positions and sizes. After treating with 100ppm ethylene for 24 hours, cultivate them under light and take photos regularly every day to record the status of the tomatoes.

[0112] Tomato total RNA was extracted using the Novozyme RNA extraction kit;

[0113] HiScriptⅡQ RT SuperMix for qPCR reverse transcription kit was used to synthesize the first-strand cDNA with 1 μg of total RNA as template;

[0114] qRT-PCR reactions were performed using the StepOne Real-Time PCR System with the KAPA SYBR FAST Universal qRT-PCR Kit. The reactions used a 10-μl reaction system containing 1 μl of cDNA template and the tomato SlACTIN gene as an internal reference gene;

[0115] Each reaction was performed in triplicate and verified by melting curve analysis;

[0116] All reactions were performed at least three times biologically replicated.

[0117] The PCR primers of the reference gene and cDNA used in the above experiment are listed in Table 1, wherein the forward and reverse primers of the reference gene S1ACTIN are SEQ ID NO. 7 and SEQ ID NO. 8, respectively. The forward and reverse primers used to detect the expression of the S1UBC32 gene in tomatoes are SEQ ID NO. 9 and SEQ ID NO. 10, respectively.

[0118] like Figure 4A As shown in Figure 2, after ethylene treatment, the expression level of SlUBC32 was significantly higher than that of the control group. Figure 4B As shown, the expression of SlUBC32 gradually increased during fruit ripening.

[0119] The response of SlUBC32 to ethylene and the changes in tomato ripening were detected by qRT-PCR, thereby analyzing some potential biological functions of the SlUBC32 gene.

[0120] Based on this, the present invention provides a method for applying ethylene, which specifically comprises the following steps: continuously applying ethylene to plants with low SlUBC32 expression for at least 5 to 7 days.

[0121] Example 4

[0122] This embodiment provides a method for expressing S1UBC32 recombinant protein in prokaryotic cells. This embodiment provides a method for purifying S1UBC32 recombinant protein. This embodiment provides a method for analyzing the activity of ubiquitin conjugating enzyme E2.

[0123] The method steps are:

[0124] The ORF sequence of the SlUBC32 gene containing SacⅠ and SalⅠ sites was cloned into the pET28a-His-Myc vector to obtain the pET28a-His-Myc-SlUBC32 vector for His-Myc-SlUBC32 protein purification;

[0125] The target plasmid was transferred into inducible E. coli BL21 or Transata, 12 single clones were picked, and the SlUBC32 gene in the bacterial colony was detected to confirm that the single clone contained the target plasmid;

[0126] Pipette 2.5% of the bacterial solution containing the correctly identified monoclonal strain into a 2 ml centrifuge tube containing 200 ml of LB containing relevant resistance, and culture at 37°C, 200 rpm for 3 h;

[0127] Add 0.4 mM IPTG and induce culture overnight at 16°C;

[0128] The cells were collected by centrifugation at 8000 rpm for 5 min at 4 °C;

[0129] Resuspend the bacteria using His protein resuspension Lysis buffer;

[0130] After the bacterial solution was resuspended, it was broken with an ultrasonic disruptor, with the cycle of 9.9s on and 9.9s off for 15min.

[0131] The disrupted bacterial solution was centrifuged at 10000 rpm for 40 min at 4 °C;

[0132] Take the supernatant and proceed directly to protein purification.

[0133] The primers used to construct the vector containing the SlUBC32 gene are SEQ ID NO.11 and SEQ ID NO.12 respectively.

[0134] The purification method of His fusion protein is as follows:

[0135] Wash 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 solution;

[0136] 20% alcohol was passed through 3 column volumes to clean the packing;

[0137] Pre-wash the column with 3 column volumes of binding buffer;

[0138] Resuspend His resin in the protein supernatant and pour into a clean conical flask, and place on ice at 60 rpm for more than 1 hour;

[0139] Collect His resin and wash the column with 3 column volumes of wash buffer;

[0140] Add 2 ml of eluent I to the column, react for 3 to 5 minutes, and collect the eluent;

[0141] Add 2 ml of eluent II to the column, react for 3 to 5 minutes, and collect the eluent;

[0142] Add 2 ml of eluent III, react for 3 to 5 minutes, and collect the eluent;

[0143] After purification, the protein was run on a protein gel to detect the concentration and stored at -80°C after aliquoting.

[0144] The method for analyzing the activity of ubiquitin conjugating enzyme E2 is as follows:

[0145] Add 50ng E1, 100ng E2, 1μl 20× reaction buffer, 2μl purified His-Ub (about 2μg / μl), and ddHO to a 1.5ml centrifuge tube. 2 O to 20 μl;

[0146] E2 was divided into 4 tubes, of which two tubes contained Ub and two tubes did not contain Ub, and reacted at 37°C and 900 rpm for 5 min;

[0147] After the end, add 4 μl 5×SDS loading buffer, one tube for each group. + DTT (containing β-mercaptoethanol) and - DTT;

[0148] 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.

[0149] The His-Myc-SlUBC32 tagged protein was purified in vitro and subjected to in vitro ubiquitin conjugation enzyme activity assay. Figure 4C As shown in the figure, in the control reaction without adding ubiquitin or adding ubiquitin but with DTT in the system, no band of SlUBC32 binding to ubiquitin was detected; while the ubiquitination band was detected in the lane with ubiquitin added but without DTT. This result proves that SlUBC32 has E2 conjugating enzyme activity.

[0150] Example 5

[0151] This embodiment provides a method for promoting SlUBC32 to degrade SlETR4 protein. This embodiment also provides a method for displaying SlUBC32 to promote SlETR4 protein degradation. The present invention also provides a method for expressing SlUBC32 gene in tobacco.

[0152] 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, mix equal volumes and let stand at room temperature for 3h;

[0153] 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;

[0154] Samples were taken 3 days after injection to extract total protein or stored at -80°C for subsequent verification of protein content or type.

[0155] The primers used for cloning the SlETR4 gene are shown in Table 1 as SEQ ID NO.13 and SEQ ID NO.14.

[0156] Specifically, the method for SlUBC32 to promote SlETR4 protein degradation in vivo is as follows: SlETR4-Flag is co-injected into tobacco with SlUBC32-GFP and super1300-GFP, respectively, and after 3 days of expression, samples are taken, total protein is extracted, and the level of SlETR4-Flag protein is detected.

[0157] The primers for SlETR4-Flag used for binary expression are SEQ ID NO.15 and SEQ ID NO.16 respectively.

[0158] The primers for dual expression of SlUBC32-GFP are SEQ ID NO. 17 and SEQ ID NO. 18 respectively.

[0159] The infection solution consisted of 10 mM MES, 20 mM AS and 10 mM MgCl 2 Configuration. The pH of the infection solution is 5.6.

[0160] like Figure 5 As shown in the results, SlUBC32 can promote the degradation of SlETR4 protein, and this degradation can be partially inhibited when MG132 is added. This result indicates that SlETR4 is degraded through the 26S proteasome ERAD pathway.

[0161] Example 6

[0162] The present invention provides a method for constructing a SlUBC32 CRSPR stable transgenic tomato. The present invention provides a method for constructing a CRISPR-Cas9 plant transgenic vector containing a SlUBC32 gene.

[0163] Two target sites of the SlUBC32 gene were designed using the CRISPR program, where the target site length was 20 bp;

[0164] The target sequence was constructed into the pDIRECT-22C vector;

[0165] The pDIRECT-22C vector was sequenced, and the successfully constructed plasmid was transformed into Agrobacterium GV3101 and used to transform the cotyledons of M82;

[0166] Sterilize M82 seeds with 5% NaClO for 5 minutes, wash with sterile water 3-5 times, place in seed germination medium, and culture under light until tomato cotyledons are fully expanded;

[0167] pDIRECT-22C-SlUBC32 Agrobacterium, collect bacteria, resuspend the strain in resuspension solution, and adjust OD 600 =0.4-0.5, infect tomato cotyledons, centrifuge at 60 rpm for 30 min at room temperature, discard the infection solution, transfer the tomato cotyledons to the co-cultivation medium, and culture in the dark for 48 h;

[0168] After the co-cultivation, the cotyledons were transferred to the screening medium for screening, cultured under light, and the medium was changed every 2 weeks;

[0169] When the regenerated plants grow to 3-5 cm, cut off the callus tissue and transfer them to the rooting medium. After culturing for 3-4 weeks until the tomato takes root, transplant them to the soil.

[0170] Take the leaves of transgenic materials, extract total DNA by CTAB method, design detection primers according to the target site, use the positive material DNA as template, perform PCR, send the product for testing, and detect the Cas9 plant positive material by identifying whether the gene is edited;

[0171] Select positive materials and transplant them into a solar greenhouse, harvest them, and select homozygous edited transgenic materials without Cas9.

[0172] like Fig. 6A As shown, the two transgenic plants are mutants edited at different sites of the SlUBC32 gene, wherein slubc32-69 is a mutant with a 70bp deletion between the first target site and the second target site of the SlUBC32 gene, and slubc32-109 is a mutant with a 2bp deletion at the first target site of the SlUBC32 gene. The primer sequences of the slubc32-69 and slubc32-109 target sites are shown in Table 1 as SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22. The editing type detection primer sequences of slubc32-69 and slubc32-109 are shown in Table 1 as SEQ ID NO.23 and SEQ ID NO.24.

[0173] like Figure 6B As shown, the number of branches of SlUBC32 CRISPR transgenic plants was significantly increased and shorter than that of the wild type, and the mutant showed a 'dwarf' ethylene-sensitive phenotype.

[0174] like Figure 6C As shown, mutant and wild-type green-ripe tomatoes were treated with 100 ppm ethylene for 24 h, and photos were taken to observe the fruit ripening process. Figure 6C Shown are the fruit color changes of mutant and wild-type green-ripe tomatoes under ethylene treatment. Fig.6D The figure shows the statistical graph of fruit change days of mutant and wild-type green-ripe tomatoes under ethylene treatment. Figure 6C and 6DAs shown in the figure, without ethylene treatment, the slubc32-69 mutant strain turned red at about 6.3 days, 4 days earlier than the wild type at 10.3 days; after ethylene treatment, the ripening of the fruit was accelerated, and the slubc32-69 mutant strain turned red at about 6 days, 2.6 days earlier than the wild type at 8.6 days. This result shows that the ethylene sensitivity of SlUBC32 CRISPR transgenic fruit is increased.

[0175] like Fig. 6E The days to maturity of tomato fruits are shown in Figure 1. The slubc32-69 mutant turns red in about 43.4 days, and the slubc32-109 mutant turns red in about 44 days, which is 4 days and 3.3 days earlier than the wild type (47.2 days). The phenotype statistics of ethylene-treated and untreated fruits showed that SlUBC32 is involved in the ethylene response of tomatoes and the fruit ripening process.

[0176] like Fig. 6F The protein bands shown indicate that ethylene-induced degradation of SlETR4 protein was suppressed in SlUBC32 CRISPR transgenic fruits. The results suggest that SlUBC32 alters the ethylene response of tomato by mediating the stability of SlETR4 protein.

[0177] The SlUBC32 CRSPR stable transgenic tomatoes slubc32-69 and slubc32-109 showed a 'dwarf, bushy' plant type, more branches, early fruit maturation, and faster response to ethylene. Compared with the wild type, the mutant SlETR4 protein accumulated after ethylene treatment, showing an ethylene hypersensitive phenotype.

[0178] SlETR4 is the main receptor for ethylene response in tomatoes, and its mutants show early maturity and ethylene-sensitive phenotypes. After ethylene treatment, the inactivated SlETR4 protein in the fruit with low expression of SlUBC32 will not be degraded and will remain on the lipid membrane. The plant cannot sense the reduction of SlETR4 protein, and there will be no new receptor protein synthesis. The ethylene signaling pathway is always in an open state, and the plant shows a phenotype with enhanced ethylene sensitivity.

[0179] Table 1

[0180]

[0181]

[0182]

[0183] 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 tomatoes with a high ethylene sensitivity phenotype, characterized in that: The following steps are involved: Selecting at least one target fragment in the coding region of a tomato ubiquitin conjugating enzyme gene, inserting the target fragment or its complementary sequence into a CRISPR vector to obtain a CRISPR / Cas9 vector; Using the CRISPR / Cas9 vector to transform Agrobacterium to obtain genetically engineered Agrobacterium containing the CRISPR / Cas9 vector; Transfecting tomato cotyledons with the genetically engineered Agrobacterium to obtain a homozygous mutant strain that does not contain exogenous Cas9 protein and is stably inherited; The nucleotide sequence of the tomato ubiquitin conjugating enzyme gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that The nucleotide sequences of the primers used for cloning or detecting the first target fragment are SEQ ID NO.19 and SEQ ID NO.20 respectively.

3. The method according to claim 1, characterized in that The nucleotide sequences of the primers used for cloning or detecting the second target fragment are SEQ ID NO.21 and SEQ ID NO.22 respectively.

4. Application of tomato ubiquitin conjugating enzyme gene as a negative regulatory factor in regulating ethylene-induced fruit ripening, characterized in that: The expression level of the gene is reduced through genetic engineering to increase the sensitivity of the plant to ethylene. The nucleotide sequence of the tomato ubiquitin conjugating enzyme gene is shown in SEQ ID NO.

1.

5. Application of a protein encoded by a tomato ubiquitin conjugating enzyme gene as a negative regulatory factor in regulating ethylene-induced fruit ripening, characterized in that: The expression level of the protein encoded by the gene is reduced through genetic engineering to increase the sensitivity of the plant to ethylene. The nucleotide sequence of the tomato ubiquitin conjugating enzyme gene is shown in SEQ ID NO.1.

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