Rose gene RhJMJ25 and its application

Abstract

The present invention provides a rose gene RhJMJ25, the gene sequence of which is SEQ ID NO.1. The present invention also provides the application of the gene in the development of plant flower organs and in low temperatures. Previous research found that the RhJMJ25 gene plays an important role in low-temperature-induced rose flower organ deformities. Based on this, in view of the gap in the RhJMJ25 gene in regulating roses, the present invention provides the application of the rose gene RhJMJ25 in regulating the development of rose low-temperature flower organs. Furthermore, the gene can be applied to the development of plant flower organs, such as increasing the number of petals, petalization of stamens, and regulation of low-temperature tolerance.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a rose gene RhJMJ25 and an application thereof. Background Art

[0002] Flowers are the reproductive organs of angiosperms, and in life, flowers or flowers are an important part of human life in all aspects. In a broad sense, flowers can refer to all plants with ornamental value (or artificially planted bonsai), while in a narrow sense, they refer to all flowering plants. Flowers have always been widely loved and used by people. Flowers can be used for viewing, as well as as food or raw materials for extraction.

[0003] There are more than 250,000 species of flowering plants, which have obvious advantages in the ecosystem. Floral organs are important functional organs in the reproductive process of plants. Based on the cloning and analysis of characteristic genes of floral organs, predecessors have established the "ABCE" model to explain the functions of A, B, C and E genes. In this model, flowers contain four regions called floral organ "whorls", and the combination of A-, B-, C- and E-class homologous genes can regulate floral organ development. In the first whorl, class A genes (APETALA1, AP1; APETALA2, AP2) regulate sepal development; in the second whorl, class A and class B (APETALA3, AP3; PISTILLATA, PI) jointly regulate petal development; in the third whorl, class B and class C (AGAMOUS, AG) regulate stamen development; in the fourth whorl, class C genes regulate carpel development. In Arabidopsis, except for APETALA2 (AP2) as a class A gene, all ABCE functions are encoded by MADS-box genes. In eukaryotes, cells in the body contain a common set of genetic information, but at different stages of growth and development, these cells differentiate into different structures with different functions. This process that exists stably in mitosis and amitosis and affects gene expression is called epigenetic inheritance. Epigenetic inheritance mainly includes chromatin conformational changes, histone modification, DNA methylation and non-coding RNA. Histone modification refers to covalent modifications that occur on the N-terminus of histones, such as acetylation, methylation, phosphorylation, ubiquitination, SUMOylation and ADP ribosylation. Existing studies have shown that histone modification plays a key role in regulating chromatin structure, gene expression and genome stability. Histone methylation mainly occurs on the lysine and arginine residues at the N-terminus of histones. It has been found that at least 11 sites of lysine or arginine at the end of histones can be methylated. These sites are lysine at the 4th, 9th, 27th and 36th positions at the N-terminus of H3 and the 20th position at the N-terminus of H4. Arginine at the 2nd, 8th, 17th and 26th positions at the N-terminus of H3 and the third position at the N-terminus of H4. Histone lysine can be monomethylated, dimethylated, and trimethylated. Histone arginine can be monomethylated and dimethylated, and different methylation states can produce different biological functions. Histone methylation modification has long been considered an irreversible process. It was not until the discovery of the first histone demethylase LSD1 (Lysine-Specific Demethylase1) that researchers accepted the theory that the methylation state of histones exists in a dynamic equilibrium. LSD1 is a member of the amine oxidase family and can specifically remove monomethyl and dimethyl modifications on H3K4 through oxidation reactions. In addition, the protein family containing the JmjC (Jumonji-C) domain has also been found to have histone demethylase activity, belonging to the second type of histone demethylase discovered so far.

[0004] There are many members of the JmjC protein family. Currently, 20 JmjC proteins have been found in rice, and they are divided into 5 subfamilies based on phylogenetic analysis. 21 JmjC proteins have been found in Arabidopsis, and they play an important role in the regulation of plant gene transcription. ELF6 / JMJ11 is a suppressor of the photoperiod pathway, which can delay the flowering of Arabidopsis; REF6 / JMJ12, as a suppressor of FLC, can inhibit the expression of FLC and promote flowering. JMJ14 can inhibit the expression of FT, AP, SOC1 and LFY, inhibiting the early flowering of Arabidopsis during the vegetative growth stage. Overexpression of JMJ15 will inhibit the expression of FLC and promote flowering. Overexpression of JMJ18 will also promote flowering of Arabidopsis. JMJ16 can regulate leaf senescence. In summary, there are many studies on the JmjC family proteins in plant floral organ development and flowering time regulation, but there are few reports on their mechanism of action in floral organ development under low temperature, which needs further exploration.

[0005] Temperature is one of the key factors affecting plant growth and development, and plant growth and development must be carried out within a certain temperature range. The fixation of plants determines that plants cannot avoid environmental stress and can only resist and adapt to environmental changes through a series of physiological and biochemical reactions. Among them, low temperature stress is one of the important abiotic adversities that affect plant growth and development. At the same time, low temperature also controls the formation and development of floral organs. The study found that under low temperature conditions, the overall spatial distribution of RhAG transcripts in flower buds was significantly reduced, and the RhAG gene was silenced, resulting in an increase in the number of rose petals.

[0006] The research on the effect of low temperature on floral organs is also ongoing. The Chinese patent with patent number CN109576282B relates to the rose transcription factor RhMYB4 and its application in the regulation of floral organ development. The gene comprises a coding region nucleotide sequence as shown in SEQID NO.23, 263-1039 bp. The invention also provides a protein encoded by the gene or having an amino acid sequence as shown in SEQ ID NO.24. Based on the function of the transcription factor RhMYB4 discovered so far, the gene can be used to regulate the floral organ development of plants, such as petal doubling, petal heteromorphism and stamen abortion.

[0007] In the annual production of rose cut flowers, the demand for roses reaches the maximum during the traditional festivals of the Spring Festival and Valentine's Day, but rose production in winter is most susceptible to low temperature stress. The impact of low temperature (<10℃) on the yield and quality of cut roses is becoming more and more prominent, such as the extension of the growth period and the malformation of flowers caused by low temperature stress. The impact of low temperature on rose growth has caused huge economic losses in rose production. Early studies have found that roses are over-petaled at low temperatures, flower bud differentiation is delayed, and flower organ development is slow in low temperature environments. In winter production, low temperatures can cause malformation of rose flower organs and as the temperature decreases, the number of petals in rose flower organs will increase and the number of stamens will decrease. In short, low temperatures can seriously reduce the quality of cut flowers and bring huge economic losses.

[0008] In the work of breeding double-petal roses, the RhJMJ25 gene proposed in this application also has an important influence on the petal development of roses.

[0009] Previous research found that RhJMJ25 plays an important role in low temperature-induced rose organ deformities. Based on this, the present invention provides a rose gene RhJMJ25 and the regulatory role of the gene in rose low temperature flower organ development in view of the blank of the RhJMJ25 gene in regulating roses. The gene and the application of the gene provided by the present invention can provide a basis and direction for the directional cultivation of rose development.

[0010] 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

[0011] In view of the deficiencies of the prior art, the present invention provides a rose gene RhJMJ25, the gene sequence of which is SEQ ID NO. 1. Preferably, the present invention provides a rose histone demethylase RhJMJ25 and its application in flower development regulation.

[0012] The present invention provides a method for improving the low temperature resistance of rose. The method comprises: reducing or blocking the expression of the RhJMJ25 gene of rose; and / or reducing or blocking the expression of the RhJMJ25 protein of rose, wherein the RhJMJ25 gene has a sequence as shown in SEQ ID NO.1, and the RhJMJ25 protein is encoded by the RhJMJ25 gene. Preferably, the low temperature refers to 4 to 16°C.

[0013] The present invention provides a method for reducing the number of stamens in roses, comprising: reducing or blocking the expression of a RhJMJ25 gene of roses; and / or reducing or blocking the expression of a RhJMJ25 protein of roses, wherein the RhJMJ25 gene has a sequence as shown in SEQ ID NO.1, and the RhJMJ25 protein is encoded by the RhJMJ25 gene.

[0014] According to a preferred embodiment, the rose is a fresh cut rose. The rose is 'Samantha'.

[0015] The present invention provides biological materials containing RhJMJ25 gene, which are expression vectors and strains.

[0016] The present invention provides an application of the RhJMJ25 gene in regulating the development of rose organs. The regulation of the development of rose organs comprises increasing the number of rose petals and / or reducing the number of rose stamens.

[0017] The present invention provides the use of RhJMJ25 gene in cultivating double-petal roses.

[0018] According to a preferred embodiment, the use of the RhJMJ25 gene in the cultivation of double-petal roses comprises achieving the increase in petals by inhibiting the RhJMJ25 gene or the RhJMJ25 protein encoded by the RhJMJ25 gene.

[0019] According to a preferred embodiment, the use of the RhJMJ25 gene in breeding double-petal roses comprises achieving the increase in petals by at least partially silencing the RhJMJ25 gene.

[0020] According to a preferred embodiment, the RhJMJ25 gene can affect the development of plant flower organs by regulating the biological level of plants in a low temperature environment. Preferably, the low temperature environment can refer to 1° C. to 15° C. Flower organs refer to petals and / or stamens.

[0021] The present application can construct a rose RhJMJ25 gene knockout plant by means of technical means at the molecular biology level, and the probability of flower organ deformity in the plant is reduced.

[0022] According to a preferred embodiment, the expression vector can be a TRV vector connected to the RhJMJ25 gene.

[0023] Low temperature seriously reduces the ornamental quality and yield of cut roses, leading to excessive double petals of roses. The present invention found that JmjC epigenetic modifier genes play a certain role in bud development under low temperature, so this application uses RhJMJ25, whose expression level is greatly reduced under low temperature stress, as a candidate gene for further research.

[0024] After transient silencing of the RhJMJ25 gene, the rose flower organs showed a decrease in the number of stamens, severe stamen petalization, and an increase in the number of petals. The AG gene is a C-type gene in the ABCE model that controls the development of stamens, and AP2 is an A-type gene in the ABC model that antagonizes AG and controls the development of petals. Therefore, the JMJ25 protein may regulate AG / AP2 by binding to related proteins, thereby regulating the development of rose flower organs.

[0025] Based on the screening library, two candidate genes were finally obtained in this example for further verification. RhMYB17 is a R2R3 MYB transcription factor, and it is speculated that it mediates the response of rose to the environment under low temperature stress through interaction with RhJMJ25 protein. RhDNAJ1 protein may be related to DNA methylation. There are no relevant reports on the genes encoding RhDNAJ1 protein and RhMYB17 protein in rose, and there are no proteins encoded by homologous genes of RhDNAJ1 protein and RhMYB17 protein in other species that interact with RhJMJ25 protein.

[0026] In summary, the present application found that transient silencing of the rose histone demethylase RhJMJ25 caused the rose stamens to become petalized; RhJMJ25 has demethylase activity, interacts with RhMYB17 and RhDNAJ1, respectively, and jointly regulates the abnormal development of rose floral organs caused by low temperature.

[0027] This application identifies the histone demethylase RhJMJ25 and explores its involvement in the epigenetic molecular mechanism of low temperature affecting the development of rose flower organs, in order to provide a theoretical basis for rose cold-resistant molecular breeding, and at the same time provide a basis for the development of new rose cold-resistant cultivation technologies. The RhJMJ25 gene provided in this application provides a genetic resource for new rose varieties that prevent flower organ deformities, and has good potential application value. Through transgenic technology, the double petals of roses can be controlled to provide more rose varieties with ornamental value, or the number of roses that grow normally in winter can be increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the normal temperature (NT) / low temperature (LT) phenotype of the rose 'Samantha';

[0029] Figure 2 This is a statistical chart of the petal and stamen quantity of the rose 'Samantha' at normal temperature (NT) / low temperature (LT);

[0030] Figure 3 This is a schematic diagram of the expression level of in situ hybridization-RhJMJ25 in normal temperature / low temperature flower buds;

[0031] Figure 4This is a schematic diagram of the expression level of RhJMJ25 in flower buds at 0h, 12h, 16h, and 24h after low temperature treatment;

[0032] Figure 5 Schematic diagram of the TRV-RhJMJ25 silencing phenotype;

[0033] Figure 6 This is a schematic diagram of the expression level of the RhJMJ25 gene in the TRV-RhJMJ25 silenced plants;

[0034] Figure 7 This is a statistical diagram of the number of petals and stamens in TRV-RhJMJ25 silenced plants;

[0035] Figure 8 Schematic diagram of subcellular localization of RhJMJ25;

[0036] Fig. 9 Schematic diagram of yeast hybrid self-activation of pGBKT7-RhJMJ25-3' and pGBKT7-RhJMJ25-5';

[0037] Fig.10 Schematic diagram of the fluorescence localization of RhJMJ25 histone demethylase in tobacco;

[0038] Fig.11 Schematic diagram of yeast hybridization to verify whether pGBKT7-RhJMJ25-5' and pGBKT7-RhJMJ25-3' are toxic and self-activated;

[0039] Fig.12 Schematic diagram of the binding effect of yeast binders;

[0040] Fig.13 This is a schematic diagram of the screening results of RhJMJ25 interacting proteins;

[0041] Fig.14 Schematic diagram of the bimolecular fluorescence complementation (BiFC) results to verify the full-length interacting protein of RhJMJ25. DETAILED DESCRIPTION

[0042] The following is a detailed description with reference to the accompanying drawings.

[0043] This application uses the rose 'Samantha' (Rosa hybrida 'samantha') as the experimental material, and the material processing method of the rose 'Samantha' subculture seedlings is:

[0044] Insert a stem segment of about 2-3 cm with at least one leaf bud into the subculture medium. 1L of subculture medium contains 4.74g MS, 1mg 6-BA, 0.05mg NAA, 30g sucrose, and 6.8g agar. The pH of the subculture medium is between 5.85 and 5.95. After the subculture medium is sterilized by high temperature and high pressure, 1mg / L GA is added. 3 (Sterilized by suction filtration).

[0045] When the rose seedlings grow to 9-10 cm in height, they are transferred to the rooting medium. 1L of rooting medium contains 2.37g MS powder, 0.01mg NAA, 30g sucrose, and 7.5g agar. The pH of 1L of rooting medium is between 5.85 and 5.95.

[0046] After 15-20 days, the rose seedlings will have roots of about 2-4 cm and can be taken out of the tissue culture bottle. Wash the roots with water, and pay attention to maintaining the humidity of the seedlings during the process. Plant the rooted rose seedlings in a nutrient pot with a ratio of nutrient soil to vermiculite of 2:1, cover with a fresh-keeping cover to moisturize and cultivate for 15-20 days to accelerate the seedlings. After removing the cover, keep it for 16h / 8h (light / dark), keep the air humidity at 40%-60%, and the temperature at 22±2℃.

[0047] Treatment of Nicotiana benthamiana:

[0048] Evenly spread the seeds of Nicotiana benthamiana on the soil surface with a ratio of 1:1 between nutrient soil and vermiculite, transplant the seedlings after 7-10 days, and do subsequent tests when the tobacco leaves are 4-5. The temperature is 23℃-26℃, and the humidity is 50%-80%.

[0049] Example 1

[0050] This example relates to the identification and cloning of the RhJMJ25 gene.

[0051] (1) Establishment of a transcriptome database of rose buds in the early stage of flower bud differentiation under low temperature treatment.

[0052] The 'Samantha' variety of rose was used as the test material and cultured at room temperature (22℃ / 15℃ day / night) and low temperature (4℃ / 4℃ day / night) respectively. Figure 1The flowering state of roses of the varieties 'Samantha' cultivated at normal temperature and low temperature is shown. The first line in the figure shows the flower state of roses cultivated at normal temperature. The second line shows the flower state of roses cultivated at low temperature. 1 corresponds to the flower picture on the first day of flowering. 3 corresponds to the flower picture on the third day of flowering. 5 corresponds to the flower picture on the fifth day of flowering. 8 corresponds to the flower picture on the eighth day of flowering. 10 corresponds to the flower picture on the tenth day of flowering. From the pictures of low temperature and normal temperature flowers on the eighth and tenth days, it can be seen that the number of petals of rose flowers under low temperature treatment increases and the number of deformed petals increases. The flower buds at the early stage of flower bud differentiation at two growth temperatures were taken as materials for transcriptome sequencing (RNA Sequencing), and each treatment was repeated three times. Through the comparative analysis of differential genes of the above two groups of data, epigenetic modification-related genes with different expression levels were screened out. The total RNA of roses was extracted using the NoviZan RNA extraction kit in this process, and RNASequencing was completed by Novo. The RNA-seq data were processed, assembled, and annotated and stored in the SRA database of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov) with gene numbers SRR18440429-SRR18440424.

[0053] Based on the comparative analysis of the transcriptome of the rose 'Samantha' at the early stage of flower bud differentiation under low temperature, it was found that there were 28 JmjC genes with significant differences, including the RhJMJ25 gene, as shown in Table 1.

[0054] The rooted tissue culture seedlings of 'Samantha' were taken out of the tissue culture bottle, planted in nutrient soil, and grown in a 22℃ culture room. The seedlings were allowed to grow slowly for the first two weeks. Half of the seedlings were selected and treated with low temperature at 4℃ for 7 days before the differentiation of flower bud pistil primordium. Then, they were kept at room temperature (22℃) to observe the phenotype. The control was the seedlings that were always grown at room temperature. The number of petals and stamens of the plants was counted. Figure 2 As shown, the number of petals of the seedlings after low temperature treatment increased significantly and the number of stamens decreased significantly compared with the control. The number of petals of a single rose treated at normal temperature was 15, and the number of petals of a single rose treated at low temperature was 18. The number of petals of a single rose treated at low temperature increased by 3 compared with the number of petals of a single rose treated at normal temperature. The number of stamens of a single rose treated at normal temperature was 49. The number of stamens of a single rose treated at low temperature was 34. The number of stamens of a single rose treated at low temperature decreased by 15 compared with the number of stamens of a single rose treated at normal temperature.

[0055] (2) Semi-quantitative RT-PCR analysis.

[0056] Total RNA from rose petals was extracted using the hot borate method. The first-strand cDNA was synthesized using HiScript II Q RT SuperMix for qPCR (Cat. R123-01, Vazyme) reverse transcription kit with 1 μg of total RNA as a template. qRT-PCR reactions (20 μL volume containing 1 μL cDNA template) were performed using the StepOne Real-Time PCR System (Applied Biosystems) and the KAPA SYBR FAST Universal qRT-PCR Kit (Kapa Biosystems).

[0057] The rooted tissue culture seedlings 'Samantha' were taken out of the tissue culture bottle, planted in nutrient soil, and grown in a 22℃ culture room for the first two weeks. After low temperature (4℃) treatment for 0h, 12h, 16h, and 24h, samples were taken and fixed, RNA was extracted and reverse transcribed, and the expression of RhJMJ25 gene under normal temperature and low temperature conditions was detected by qRT-PCR and in situ hybridization.

[0058] Figure 3 The slice pictures of flowers under low temperature conditions and flowers under normal temperature conditions after in situ hybridization are shown. The two left pictures are slice pictures of antisense RNA in situ hybridization, and the two right pictures are slice pictures of sense RNA in situ hybridization. With sense RNA in situ hybridization as a negative control, it can be seen from the slice pictures of antisense RNA in situ hybridization that the color of the normal temperature slice is darker than that of the low temperature slice, which shows that the expression level of RhJMJ25 gene in normal temperature flower buds is higher than that in low temperature flower buds. The in situ hybridization results show that the expression level of RhJMJ25 gene is reduced compared with normal temperature conditions, especially in the petals, stamens and pistils.

[0059] Figure 4 The expression level of RhJMJ25 gene in low temperature flower buds is shown, among which the expression level at 0h is the highest. As time goes by, the expression level of RhJMJ25 gene decreases. The qRT-PCR results show that compared with the low temperature 0h, the expression level of RhJMJ25 gene decreases under low temperature conditions of 12h, 16h, and 24h. Figure 4 As shown, this is basically consistent with the results of the low-temperature transcriptome library. The above results prove that the RhJMJ25 gene responds to low temperature signals and may be involved in the regulation of low temperature effects on rose organ development.

[0060] (3) Virus-induced gene silencing.

[0061] In order to construct the VIGS silencing vector, a specific fragment of about 400 bp of the RhJMJ25 gene was constructed into the pTRV vector, and then transferred into Agrobacterium GV3101, and mixed with Agrobacterium carrying pTRV1 at a ratio of 1:1 to infect the newly rooted rose tissue culture seedlings. At the same time, the plants infected with the pTRV empty vector were used as controls. After growing for one month, young leaves were taken to extract RNA and detect the silencing efficiency. The flower development phenotype of the VIGS transient transformation strain of the RhJMJ25 gene was further observed, and the number of sepals, normal petals, deformed petals, stamens and pistils was specifically counted.

[0062] In order to explore the function of RhJMJ25 gene in rose organ development, VIGS silencing technology was used to transiently silence RhJMJ25 in rose. The 3' end and part of the CDS region of the RhJMJ25 gene were inserted into TRV to construct the TRV-RhJMJ25 vector. At the same time, TRV empty vector was used as a negative control and transferred into Agrobacterium GV3101, and the rose 'Samantha' tissue culture seedlings were infected by vacuum suction. The treated tissue culture seedlings grew in the culture room for about 40 days and began to bud. The new leaves of TRV and TRV-RhJMJ25 were sampled and tested by qRT-PCR. Plants with low expression of the RhJMJ25 gene were selected for phenotypic observation. Figure 5 The flower phenotypes of roses transformed with TRV empty vector and TRV-RhJMJ25 vector are shown. Figure 5 From the complete flower pictures in the figure, it can be seen that the number of petals of the rose flowers transformed with the TRV-RhJMJ25 vector is increased compared with the number of petals of the rose flowers transformed with the TRV empty vector. Figure 5 As can be seen from the petal number display graph, the number of petals of the rose flowers transformed with the TRV-RhJMJ25 vector increased by 5 compared to the number of petals of the rose flowers transformed with the TRV empty vector.

[0063] Depend on Figure 6 It can be seen that the expression level of RhJMJ25 in the rose transformed with the TRV-RhJMJ25 vector is lower than the expression level of RhJMJ25 in the rose transformed with the TRV empty vector.

[0064] When the plants were fully bloomed, the growth and development of flowers in the silent group and the control group were statistically observed. Figure 7As shown, the number of petals of the rose transformed with the TRV-RhJMJ25 vector is 19. The number of petals of the rose transformed with the TRV empty vector is 14. The number of petals of the rose transformed with the TRV-RhJMJ25 vector is 5 more than the number of petals of the rose transformed with the TRV empty vector. The number of stamens of the rose transformed with the TRV-RhJMJ25 vector is 45. The number of stamens of the rose transformed with the TRV empty vector is 54. The number of stamens of the rose transformed with the TRV-RhJMJ25 vector is 9 less than the number of stamens of the rose transformed with the TRV empty vector. The rose transformed with the TRV-RhJMJ25 vector can silence the RhJMJ25 gene in the rose. It can be seen that silencing the RhJMJ25 gene can reduce the stamen petals of the rose and increase the total number of petals.

[0065] (4) Cloning of the RhJMJ25 gene.

[0066] Obtain the full-length gene sequence.

[0067] The gene coding region sequence was obtained from the rose transcriptome database, and PCR amplification was performed using cDNA from the leaves of rose 'Samantha' tissue culture seedlings as a template. The sequencing results showed that the full length of the rose RhJMJ25 gene coding region was 2763 bp, encoding 921 amino acids, and the gene sequence was shown in SEQ ID NO.1 in Table 3. Analysis using NCBI software showed that the sequence had a conserved functional domain as the JmjC family.

[0068] Example 2

[0069] This example relates to the functional identification of the RhJMJ25 protein.

[0070] (1) Protein property detection of RhJMJ25

[0071] Subcellular localization of RhJMJ25 protein was determined by fluorescence co-localization method:

[0072] The CDS sequence of the RhJMJ25 gene was fused with the reporter gene GFP to construct the overexpression vector Super1300-GFP, and the positive clone with the correct sequence was obtained after sequencing identification. The Super1300-RhJMJ25-GFP plasmid and P19 were co-injected into tobacco leaves, cultured normally for 48 to 72 hours, observed under a laser confocal microscope, and finally stained with DAPI to obtain the subcellular localization results of the RhJMJ25 protein.

[0073] The ORF of RhJMJ25 gene was constructed into pSuper1300C-GFP and used for tobacco infection. Figure 8The fluorescence photo of RhJMJ25-GFP is shown, in which the yellow dots displayed in the bright area by red and green colors are located at their expression locations (Merge). The results show that the negative control pSuper1300C-GFP empty vector is bright in the nuclear membrane of tobacco, while pSuper1300C-GFP-RhJMJ25 is only bright in the nucleus in tobacco, indicating that RhJMJ25 is localized in the nucleus.

[0074] (2) Detection of histone demethylase activity of RhJMJ25 in plant cells

[0075] The RhJMJ25 protein overexpression vector Super1300-RhJMJ25-GFP was constructed and the overexpression vector was transferred into tobacco leaves by Agrobacterium through tobacco leaf injection. After 48-72 hours of infection, 4-6 injected leaves were cut into small pieces and fixed with 4% paraformaldehyde. Then the tissue was completely minced to extract the cell nucleus, which was smeared on a glass slide and dried at room temperature, and then fixed once with 4% paraformaldehyde. After removing the fixative, two drops of Image-iT TM FX signal enhancer, block at room temperature for 30 minutes. Then add primary antibody to each slide, overnight at 4°C. Then add fluorescent secondary antibody (AlexaFluor 555goat anti rabbit), at room temperature for 1 hour. Finally, add one to two drops of VECTASHIELD TM mounting mediumwith DAPI (Vector Laboratory), and the sections were sealed and observed under a fluorescence microscope.

[0076] As a histone demethylase, RhJMJ25 has another biochemical characteristic, in addition to its nuclear localization, that it has H3K9 demethylase activity. In order to detect whether RhJMJ25 has the function of modifying histone methylation, the demethylase activity of RhJMJ25 was detected in tobacco.

[0077] Fig.10 A schematic diagram of the demethylase activity of RhJMJ25 is shown. When RhJMJ25 is overexpressed, the level of H3K9me2 modification is reduced (less enriched in the nucleus).

[0078] The results showed that it has H3K9me2 demethylase activity.

[0079] (3) Screening of RhJMJ25 interacting proteins and verification of their functions

[0080] (3.1) Screening of RhJMJ25 interacting proteins (yeast two-hybrid screening library method)

[0081] (3.1.1) Preparation of competent yeast and plasmid transformation:

[0082] Y2H Gold strain was cultured on YPDA plate at 30℃ for 2-3 days. Centrifuge at 3000rpm for 5min, remove supernatant; wash with 25mL sterile water, mix with pipette tip; centrifuge again at 3000rpm for 5min, remove supernatant; wash with 10mL sterile water, mix with pipette tip; aliquot 1mL into 1.5mL tube; centrifuge at 6000rpm for 5min; suspend with 100uL one-step buffer; add 6uL salmon sperm DNA (carrier DNA) (boil and heat shock for 20min before use, and keep on ice for later use); add RhJMJ25 carrier DNA (empty, fusion or reporter), mix; incubate at 45℃ for 30min, vortex every 10min; plate on -Trp, -Trp / -His deficiency medium, and culture at 30℃ for 3d.

[0083] One step buffer (10mL)

[0084] 50% PEG3350 8mL 1M LiAc(PH 7.5) 2mL β-Mercaptoethanol 76.9μL

[0085] -Trp deficiency board

[0086] -Trp / -Leu solid / liquid) pH 5.8 YNB 100mL 200mL Yeast extract 0.67g 1.34g -Trp 0.064g 0.128g glucose 2g 4g water 100mL 200mL Solid agar 2g 4g

[0087] Sterilization, 115℃, 15min (liquid without agar)

[0088] -Trp / -His deficiency plate

[0089] -Trp / -Leu / -His / -Ade solid / liquid) pH 5.8 YNB 100mL 200mL Yeast extract 0.67g 1.34g Trp / -His 0.06g 0.12g glucose 2g 4g water 100mL 200mL Solid agar 2g 4g

[0090] Sterilization, 115℃, 15min (liquid without agar)

[0091] Thaw the AD bacterial solution in a water bath at room temperature, then dilute 10 μL and apply it to 100x, 1000x, and 10000x SD / –Leu plates. If there are more than 200 clones on the 10000×SD / –Leu plate, it proves that the vitality of the library bacteria meets the screening conditions.

[0092] The full-length RhJMJ25 is difficult to express in the yeast system, so JMJ25 was shortened into two segments for library screening, namely pGBKT7-RhJMJ25-5' and pGBKT7-RhJMJ25-3'.

[0093] Yeast transformed with S23 (positive control), empty BD (negative control), RhJMJ25-5', and RhJMJ25-3' were cultured in SD / -Trp-deficient medium. Fig.11As shown in the first row of pictures, there are more than 200 colonies in the culture medium in the first row.

[0094] Yeast transformed with S23 (positive control), empty BD (negative control), RhJMJ25-5', and RhJMJ25-3' were cultured in SD / -Leu / -Trp dual-deficient medium (DDO / X / A) containing Aureobasidin A and Xa-Gal.

[0095] like Fig. 9 As shown in the second column of pictures, fewer colonies grew in the culture medium transformed with RhJMJ25-3' in the second column compared with the control. This result proves that RhJMJ25-3' has self-activation.

[0096] In order to ensure that the efficiency of the screening library is high enough, AbAi is used to inhibit the transcriptional activation activity of RhMYB4. Therefore, a gradient of AbAi of 0 ng / mL, 100 ng / mL, 200 ng / mL, 250 ng / mL, and 300 ng / mL is used for screening. Fig. 9 As shown, the culture medium in the third column of images from left to right does not grow yeast at the beginning, wherein the third row corresponds to an AbAi concentration of 100 ng / mL. The results show that 100 ng / mL AbAi can inhibit the self-activation of RhJMJ25.

[0097] Transfer pGBKT7-RhJMJ25-5' and pGBKT7-RhJMJ25-3' to Y2H Gold and AD library bacterial solution Y187 for mating. After 20 to 24 hours, observe the hybridization solution at 40x to see if Mickey heads appear. Fig.12 As shown in the left picture, the bound yeast solution was spread on SD / -Trp-deficient medium, SD / -Leu-deficient medium and SD / -Leu / -Trp-deficient medium with gradient dilutions of 1 / 10, 1 / 100, 1 / 1000 and 1 / 10000. Fig.12 The right picture shows that pGBKT7-RhJMJ25-5' and pGBKT7-RhJMJ25-3' were expressed in different concentrations of one or both deficiencies.

[0098] (3.1.2) Toxicity and self-activation of bait vectors

[0099] Testing the toxicity of bait vectors:

[0100] The empty BD and pGBKT7-RhJMJ25 recombinant plasmids were transferred into the Y2H Gold strain and plated again according to the above method. After culturing at 30°C for 3-5 days, the growth (size and number) of single clones of the empty BD and recombinant plasmids on the SD / -Trp plate was observed.

[0101] Detection of self-activation of bait vector:

[0102] The empty BD and pGBKT7-RhJMJ25 were transferred into the Y2H Gold strain by the above method and then coated on SD / -Trp and SD / -Trp / X-α-gal plates. After culturing at 30°C for 3 to 5 days, observe whether the recombinant plasmid has changed color on the SD / -Trp / X-α-gal plate. If it has turned blue, pick a spot from the SD / -Trp plate and then coat it on the SD / -Trp / X-α-gal / AbAi plate containing different concentration gradients to screen out a suitable AbAi concentration for subsequent screening.

[0103] Mating method two-hybrid library screening:

[0104] After the bait vector was transformed into the Y2H Gold strain, the toxicity and self-activation were tested. The strain containing Bait was cloned into 50mL SD / -Trp liquid medium and cultured overnight at 30℃. Centrifuge, resuspend the cells with 4-5mL SD / –Trp, melt the AD bacterial solution in a water bath at room temperature, take 10uL of 100x, 1000x, and 10000x dilutions to coat SD / –Leu plates. Add 1mL AD bacterial solution and 4-5mL pGBKT7-RhJMJ25 bacterial solution to a 2L conical flask. Add 45mL 2x YPDA liquid medium (containing 50ug / mL Kan), culture at 30℃ for 20h, and observe the hybridization solution at 40x to see if Mickey heads appear. Centrifuge and resuspend the cells in 0.5x YPDA liquid medium (containing 50ug / mL Kan), and then dilute to 1 / 10, 1 / 100, 1 / 1000, and 1 / 10000, and then apply SD / -Trp, SD / -Leu, SD / -Leu / -Trp, SD / -Leu / -Trp / X / A. The remaining mixture was spread on a 150mm diameter primary screening QDO / X / A plate, and the positive clones on QDO / X / A were further sequenced and verified one-to-one.

[0105] 1xYPDA yeast culture medium (2xYPDA, 0.5xYPDA are doubled or reduced by half based on 1xYPDA)

[0106] YPDA (solid / liquid) pH 5.8 YPDA 100mL 200mL Yeast extract 1g 2g Peptone 2g 4g glucose 2g 4g Adesult 0.004g 0.008g Solid agar 2g 4g

[0107] Sterilization, 115℃, 15min (liquid without agar)

[0108] (3.1.3) Yeast plasmid extraction and interaction verification

[0109] The selected yeast plasmids were extracted using TIANGEN's TIANprep Yeast Plasmid DNA Kit according to the instructions, and the plasmid concentration was determined using Nano Drop. The interaction verification was performed using yeast rotation verification. The candidate plasmid and the pGBKT7-RhJMJ25 plasmid were first co-transformed into the Y2H Gold strain, and then the candidate gene was verified to be positive. If it was positive, a one-to-one verification was performed, the coding region of the candidate plasmid was cloned, and the candidate plasmid pGBKT7-RhJMJ25 plasmid containing the complete coding region was co-transformed into the Y2H Gold strain, and then the candidate gene was verified to be positive.

[0110] (3.2) Verification of RhJMJ25 interacting proteins

[0111] BiFC: The CDS sequence of the RhJMJ25 gene was constructed onto the BiFC vector YNE-173 containing GFP-N terminus, and the selected interacting proteins were constructed onto the BiFC vector YCE-M containing GFP-C terminus. After sequencing and identification, they were co-injected into tobacco leaves through Agrobacterium and observed under a laser confocal microscope for 2 to 4 days to determine whether there was interaction.

[0112] The rose flower bud library was used to screen for interacting proteins of RhJMJ25. The yeast monoclones that turned blue on the SD-Trp-Leu-His-Ade / X-α-gal four-deficient medium were shaken, the plasmids were extracted and primers were designed for PCR detection, the positive clones were selected for sequencing, and the sequencing results were compared with the NCBI, Arabidopsis library and rose library to confirm that the target protein was screened. In this experiment, a total of about 384 yeast monoclones were screened, and 97 blue yeast monoclones were found.

[0113] From the above screened proteins, the interacting proteins were selected for further verification according to the protein function. First, the candidate genes were verified by transfection, and the screened genes were co-transfected with pGBKT7-RhJMJ25 into yeast Y2H Gold. Fig.13 As shown, the positive control pGBAD-T / pGBKT7-53 grew normally in SD-Trp-Leu-His-Ade / X-α-gal and showed obvious color. pGBKT7-Lam and pGADT7-T were hybridized as negative controls. The results showed that there were blue colonies on the QDO / X / A plate in the positive control, but not in the negative control, indicating that the QDO / X / A plate can be used for subsequent yeast two-hybrid screening. pGBKT7-RhJMJ25 / pGADT7-T-SD-Trp-Leu-His-Ade / X-α-gal (pGBKT7-RhJMJ25 was added with 100 ng / mL AbAi to inhibit self-activation) could not grow normally ( Fig.13The pGBKT7-RhJMJ25 interacting protein grew normally in SD-Trp-Leu-His-Ade / X-α-gal and showed obvious color ( Fig.13 The fifth to thirteenth rows in the figure show that they all grew normally in QDO / X / A medium and produced blue plaques).

[0114] In order to select proteins that interact with the full-length pGBKT7-RhJMJ25, we further cloned the proteins that were verified to interact with the above-mentioned round-robin and co-transformed them into yeast with pGBKT7-RhJMJ25. We found that AD-5'-20 / AD-5'-110 / AD-5'-108 / AD-5'-125 interacted with the full-length CDS of pGBKT7-RhJMJ25.

[0115] At the same time, BiFC was used to verify the above, and the results showed that some proteins interacted with RhJMJ25. Fig.14 As shown in the Merge diagram, the second to seventh rows show the expression position of each protein interacting with RhJMJ25. The results of yeast and BiFC were comprehensively analyzed, and the functions of the proteins were linked. AD-5'-110 / AD-5'-108 was selected for subsequent experiments. The preliminary annotation results are shown in Table 2. Based on the screening and analysis of the above interacting proteins, it was further proved that the RhJMJ25 gene is involved in the regulatory pathway of rose organ development, and can induce the development of rose organs when the rose is in a low temperature environment.

[0116] Table 1

[0117]

[0118]

[0119] Table 3

[0120]

[0121]

[0122]

[0123] 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 rose gene RhJMJ25 , characterized in that, Said RhJMJ25 The sequence of the gene is shown in SEQ ID NO.

30.

2. A method for improving the low temperature resistance of roses, characterized in that: The method comprises: Reduce or block the growth of roses RhJMJ25 Gene expression; and / or Reduce or block the expression of RhJMJ25 protein in roses, Among them, the RhJMJ25 The sequence of the gene is shown in SEQ ID NO.30, and the RhJMJ25 protein is RhJMJ25 Genetic coding.

3. The method according to claim 2, characterized in that The rose in question is 'Samantha'.

4. A method for reducing the number of rose stamens, characterized in that: The method comprises: Reduce or block the growth of roses RhJMJ25 Gene expression; and / or Reduce or block the expression of RhJMJ25 protein in roses, Among them, the RhJMJ25 The sequence of the gene is shown in SEQ ID NO.30, and the RhJMJ25 protein is RhJMJ25 Genetic coding.

5. The method according to claim 4, characterized in that The rose in question is 'Samantha'.

6. Containing the composition according to claim 1 RhJMJ25 Gene expression vector.

7. Containing the composition according to claim 1 RhJMJ25 Gene strains.

8. The use of histone demethylase RhJMJ25 in regulating rose organ development, characterized in that: RhJMJ25 The sequence of the gene is shown as SEQ ID NO.30, wherein the regulation of rose organ development includes increasing the number of rose petals and / or reducing the number of rose stamens.

9. RhJMJ25 The use of the gene in cultivating double-petal roses is characterized in that: Said RhJMJ25 The sequence of the gene is shown in SEQ ID NO.30, wherein by inhibiting the RhJMJ25 Gene or RhJMJ25 The gene encodes the RhJMJ25 protein to achieve petal enlargement.

10. The use according to claim 9, characterized in that By at least partially silencing the RhJMJ25 Gene to achieve petal increase.

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