Application of RhALEU protein and coding gene thereof in senescence regulation and control of Chinese rose petals

Abstract

The invention discloses an application of RhALEU protein and a coding gene thereof in senescence regulation and control of Chinese rose petals. According to the invention, the RhALEU gene is screened from a Chinese rose transcriptome database, and research finds that the expression of RhALEU is induced in the whole petal development stage. A VIGS technology is utilized to verify that the RhALEU gene has a function of promoting the aging of Chinese rose petals. The silence of the RhALEU gene slows down the speed of petals from level 5 to loss of ornamental value. In petals with the silent RhALEU gene, the expression of the senescence-related gene SAG12 is remarkably reduced. Research results of the invention reveal the effect of the RhALEU gene in natural aging of petals, a new thought and technical means are provided for regulating and controlling plant flower aging, especially post-harvest preservation of fresh Chinese rose flowers, a new flower regulation and control research field is developed, and the RhALEU gene has great academic value and important production and application prospects.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and specifically relates to the application of RhALEU protein and its encoding gene in regulating the senescence of rose petals. Background Art

[0002] The flower industry is an important part of large-scale agriculture and a green industry that integrates economic, social and ecological benefits. Fresh cut flowers are the most important products in the flower industry, and roses rank first among the four major cut flowers in the world. Cut roses are extremely intolerant to long-distance transportation, and the loss in post-harvest circulation usually exceeds 30%, sometimes even as high as 50%. The large loss in post-harvest storage and transportation has become a bottleneck problem restricting the quality and efficiency of the cut rose industry. Therefore, cultivating new varieties that are resistant to storage and transportation and developing efficient post-harvest preservation technologies are effective ways to break through the industry bottleneck of "high post-harvest loss of cut roses", and the excavation of key genes for petal aging and the analysis of their biological mechanisms are the theoretical basis for variety improvement and technological innovation.

[0003] From the external morphology, petal aging is mainly manifested as wilting, shedding and color change; the physiological essence is a series of processes such as decreased cell turgor pressure and enhanced hydrolysis activity. Among them, the change in protein content can best reflect the aging process. As the aging process progresses, the catalytic metabolic hydrolase increases, the soluble protein in the petals continues to decrease, and the free amino acid content increases, resulting in an increase in the pH value of the petals, and the petals turn brown and blue. Although the aging process is very important, the proteases that play a role in the aging process of petals have not yet been fully identified and characterized.

[0004] Cysteine ​​proteases are a large class of proteolytic enzymes. Analysis of gene expression during natural or induced senescence in different plant species shows that CPs are one of the most abundantly expressed proteases during plant senescence. Among all plant cysteine ​​proteases, papain-like cysteine ​​proteases (PLCPs) have been studied most thoroughly. PLCPs are the main enzymes involved in the degradation of a large number of proteins during senescence. SAG2 of Arabidopsis encodes PLCPs and is used as a standard for measuring leaf senescence. ALEU encodes a protease associated with senescence, but the research mechanism is not yet clear, and it is unclear whether it plays a role in the senescence process. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to identify that the expression of the RhALEU gene in rose petals increases with the occurrence of petal aging, and further discover that the RhALEU gene is an important protease in regulating petal aging.

[0006] Technical solution: In order to solve the above technical problems, the present invention provides the use of RhALEU protein in regulating the senescence of rose petals, and the amino acid sequence of the RhALEU protein is shown in SEQ ID NO.3.

[0007] The present invention also provides an application of the RhALEU gene in regulating rose petal senescence, wherein the sequence of the RhALEU gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] The present invention also provides a RhALEU gene silencing vector and a RhALEU gene silencing strain for use in delaying rose petal senescence. The sequence of the RhALEU gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0009] The silencing vector is obtained by constructing a specific fragment of the RhALEU gene into a vector pTRV2, and the sequence of the specific fragment of the RhALEU gene is shown in SEQ ID NO.6.

[0010] The RhALEU gene silenced strain or knockdown strain is obtained by introducing a specific fragment silencing vector or knockdown vector of the RhALEU gene into Agrobacterium, and the sequence of the specific fragment of the RhALEU gene is shown in SEQ ID NO.6.

[0011] The present invention also provides a method for regulating the aging of rose petals, the method comprising inhibiting or down-regulating the expression or activity of the rose RhALEU gene to delay the occurrence of the aging process or prolong the flower opening time or the viewing period, or promoting the occurrence of the aging process by promoting the expression of the RhALEU protein, the amino acid sequence of the RhALEU protein is shown in SEQ ID NO.3.

[0012] Among them, the down-regulating expression or activity of the rose RhALEU gene includes: knocking out or silencing the RhALEU gene in the rose genome; or transferring a down-regulator that down-regulates RhALEU gene transcription, polypeptide expression or polypeptide activity into the rose, or hybridizing a rose variety with a RhALEU gene deletion with an ordinary rose variety, and the RhALEU gene sequence is shown in SEQ ID NO.1 or 2.

[0013] The knockout or silencing of the RhALEU gene in the rose genome is to introduce a specific fragment of the RhALEU gene into a silencing vector, and the specific fragment of the RhALEU gene is shown in SEQ ID NO.6.

[0014] The present invention also provides a method for identifying the lifespan of the rose obtained by the method, by observing the aging phenotypes of knockout or silenced plants and control plants, taking wilting, fading and falling off as indicators, observing the effect of RhALEU on the aging of rose petals, dividing the flower cycle into levels 1 to 6, and measuring the speed of petal aging by the duration of flowers at levels 5 and 6.

[0015] The silenced plant is a plant obtained by introducing the TRV-RhALEU vector into Agrobacterium GV3101 and then infecting rose tissue culture seedlings.

[0016] Among them, the down-regulator is an interfering molecule that specifically interferes with the expression of the RhALEU gene, and the interfering molecule is a dsRNA, antisense nucleic acid, small interfering RNA, microRNA, sgRNA that inhibits or silences the RhALEU gene or its transcript, or a construct that can express or form the dsRNA, antisense nucleic acid, small interfering RNA, microRNA.

[0017] The present invention also achieves the delay by selecting the 3' end of the RhALEU gene to silence the RhALEU gene, or at least partially silence the RhALEU gene; or achieves the promotion by overexpressing the RhALEU gene.

[0018] The present invention also provides a method for identifying the lifespan of the rose obtained by the method, by observing the aging phenotypes of knockout or silenced plants and control plants, taking wilting, fading and falling off as indicators, observing the effect of RhALEU on the aging of rose petals, dividing the flower cycle into 1 to 6 levels, and measuring the speed of petal aging by the duration of the flower from level 5 to level 6.

[0019] The silenced plant is a plant obtained by introducing the TRV-RhALEU vector into Agrobacterium GV3101 and then infecting rose tissue culture seedlings.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention cloned the RhALEU gene from the rose petals, and it was found through research that the expression of the RhALEU gene was induced throughout the petal development stage, and the silencing of the RhALEU gene slowed down the speed at which the petals lost their ornamental value from level 5. In the petals where the RhALEU gene was silenced, the expression of the aging-related gene SAG12 was significantly downregulated. The research results of the present invention reveal the role of the RhALEU gene in the natural aging of petals, provide new ideas and technical means for regulating the aging of plant flowers, especially the post-harvest preservation of fresh cut roses, open up a new field of flower regulation research, and have great academic value and important production application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 For the acquisition of RhALEU gene.

[0022] Figure 2 The expression detection of RhALEU gene in the flower opening stage is shown. The expression pattern of RhALEU gene in different opening stages of rose petals. RhUBI2 gene was used as internal control. All values ​​are mean ± SD of 5 biological replicates. The figure shows the difference after Tukey-Kramer test, and letters indicate significant difference (P < 0.05).

[0023] Figure 3 This is the agarose gel electrophoresis diagram of the specific gene fragment of the RhALEU gene in Example 2.

[0024] Figure 4 Figure 1 shows that silencing the RhALEU gene affects the petal senescence process. (A) The expression of RhALEU in RhALEU-silenced plants (TRV-RhALEU) and TRV control plants was detected by qRT-PCR. (B) The flowering phenotypes of the control (TRV) and silenced plants (TRV-RhALEU) were recorded and photographed at different time points. (C) The number of days from the opening of flowers to the loss of ornamental value. (D) The expression of the senescence marker gene RhSAG12 in RhALEU-silenced plants (TRV-RhALEU) and TRV control plants. RhUBI2 was used as an internal reference. The results are the mean ± SD of at least 5 biological replicates. The results of the Student's t-test are shown in the figure, where asterisks indicate statistically significant differences (***P<0.001). DETAILED DESCRIPTION

[0025] Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing technology. As an effective reverse genetics technology, it has been widely used in the research of plant genetic engineering. Among them, the more commonly used viral vector is tobacco rattle virus (TRV). TRV is jointly functioned by two vectors pTRV1 and pTRV2. The vectors pTRV-1 and pTRV-2 of the present invention were donated by Professor Liu Yule of Tsinghua University. In the present invention, the combination of the control pTRV-1 and pTRV-2 is named pTRV, and the combination of pTRV-1 and pTRV-2-RhALEU is named pTRV-RhALEU.

[0026] Example 1 Acquisition of the gene RhALEU and the expression pattern of the RhALEU gene at different opening stages of rose petals

[0027] 1. Gene cloning and sequence analysis

[0028] The present invention uses the ALEU gene in Arabidopsis thaliana, compares it with the rose (Rosa hybrida'Samantha') genome data (https: / / lipmbrowsers.toulouse.inra.fr / pub / RchiOBHm-V2 / ) using Blast, and combines it with the annotations in the NCBI (National Center for Biotechnology Information) database, and finally screens out 13 ALEU genes ( Figure 1 ). The gene RchiOBHm_Chr2g0095151, which is most closely related to the Arabidopsis ALEU gene, was selected as a candidate gene and named RhALEU (see SEQ ID NO.1 for full length). The ORF length of RchiOBHm_Chr2g0095151 is 1074 bp (see SEQ ID NO.2), which can encode a protein containing 357 amino acids (see SEQ ID NO.3).

[0029] 2. Cultivation of rose seedlings

[0030] Rose 'Samantha' tissue culture seedlings were cultured on MS medium containing 1.0 mg / L 6-benzylaminopurine (6-BA), 0.05 mg / L α-naphthylacetic acid (NAA) and 3 mg / L gibberellin (GA3) at 22±1°C and long day (16 h light / 8 h dark). Four-week-old seedlings were transferred to 1 / 2 MS medium containing 0.1 mg / L NAA for rooting. Next, the rooted seedlings were transplanted into a culture soil with a 1:1 ratio of vermiculite and peat moss at 22±1°C, relative humidity ~60%, and long day (16 h light / 8 h dark conditions) to obtain rooted seedlings through tissue culture.

[0031] 3. RNA extraction and fluorescence quantitative RT-PCR analysis

[0032] The expression changes of RhALEU at different levels were detected by real-time fluorescence quantitative PCR (qRT-PCR). The specific evaluation criteria of different levels refer to the previous study of the inventors (Ma, N., Cai, L., Lu, W. et al. Exogenous ethylene influences flower opening of cut roses (Rosa hybrida) by regulating the genes encoding ethylene biosynthesis enzymes. Sci. China Ser. C.-Life Sci. 48, 434–444 (2005). https: / / doi.org / 10.1360 / 062004-37.). The specific method is as follows: Total RNA was extracted from rose petals using the hot borate method (Ma, N., Tan, H., Liu, X., Xue, J., Li, Y., & Gao, J. (2006). Transcriptional regulation of ethylene receptor and CTR genes involved inethylene-induced flower opening in cut rose (Rosa hybrida) cv. Samantha. Journal of experimental botany, 57 (11), 2763–2773). cDNA was obtained from 1 μg of total RNA using HiScript III reverse transcriptase (Vazyme). Fluorescence quantitative RT-PCR (qRT-PCR) was performed using ChamQ SYBR qPCR Master Mix (Low ROX Premixed) reagent (Vazyme) in an Applied Biosystems StepOnePlusTM real-time quantitative PCR system. The RhUBI2 gene was used as an internal control, with at least 3 biological replicates. 2 -ΔΔCT The relative gene expression was calculated.

[0033] qRT-PCR reaction system

[0034]

[0035] qRT-PCR reaction procedure

[0036]

[0037] The results showed that the transcription level of RhALEU gene increased with the increase of flower opening level ( Figure 2 ), which suggests that the RhALEU gene may be involved in the petal senescence process and play a major role in the flower senescence stage. Figure 2 S3 is when the outer petals of the flower are loose, S4 is when the outer petals of the flower are flat but the pistil is not exposed, and S5 is when the flower is fully open with the pistil exposed.

[0038] Example 2 Virus-induced gene silencing

[0039] The experimental method refers to the previous study of the present inventors (Tian, ​​J., Pei, H., Zhang, S., Chen, J., Chen, W., Yang, R., Meng, Y., You, J., Gao, J., & Ma, N. (2014). TRV-GFP: a modified Tobaccorattle virus vector for efficient and visualizable analysis of gene function. Journal of experimental botany, 65 (1), 311-322.). Specifically, a 300 bp RhALEU specific fragment (ORF region 38 bp and 3' untranslated region 262 bp, see SEQ ID NO. 6) was inserted into the pTRV2-RhALEU vector using the restriction vector containing EcoRI and BamH I restriction sites. The specific steps are as follows:

[0040] (1) PCR amplification of 38 bp of the ORF region and 262 bp of the 3' untranslated region

[0041] Primer design: For the sequence of specific fragment primer RhALEU-TRV-F, see SEQ ID NO.4; for the sequence of specific fragment primer RhALEU-TRV-R, see SEQ ID NO.5; for the sequence of RhALEU quantitative primer RhALEU-qRT-F, see SEQ ID NO.7; for the sequence of RhALEU quantitative primer RhALEU-qRT-R, see SEQ ID NO.8; for the sequence of RhUBI2 quantitative primer RhUBI2-qRT-F, see SEQ ID NO.9; for the sequence of RhUBI2 quantitative primer RhUBI2-qRT-R, see SEQ ID NO.10.

[0042] PCR amplification:

[0043] PCR amplification system

[0044]

[0045] Place the PCR tube in the PCR instrument and perform the PCR amplification reaction program as follows:

[0046]

[0047] The amplification results were analyzed by agarose gel electrophoresis. Figure 3 .from Figure 3 It can be seen that the specific gene fragment amplified by the present invention is 300 bp in size. The target fragments were recovered using Gel DNA Extraction Mini Kit.

[0048] (2) Vector construction

[0049] Use 2×Seamless Cloning Mix kit to set up the following reaction system in a 0.2mL PCR tube, mix gently and centrifuge briefly, and keep warm in a PCR instrument (50°C, 15min). After the reaction is completed, put the centrifuge tube on ice to obtain the ligation product pTRV2-RhALEU, which is ready for use in the transformation experiment. If you are not doing a transformation experiment for the time being, freeze the ligation product at -20°C.

[0050] Homologous recombination reaction system

[0051]

[0052] (3) Obtaining Agrobacterium tumefaciens strain GV3101 carrying pTRV2-RhALEU vector

[0053] (3.1) Transformation of E. coli competent cells

[0054] (3.1.1) Thaw T1 Phage Resistant cells in an ice bath for 1-2 min, add the vector, mix gently, and let stand in an ice bath for 5 min.

[0055] (3.1.2) Heat the centrifuge tube in a 42°C water bath for 60 seconds, then quickly transfer the centrifuge tube to an ice bath and cool the cells for 2-3 minutes, keeping the centrifuge tube still.

[0056] (3.1.3) Add 500 μL of sterile LB medium (without antibiotics) to each centrifuge tube, mix well, and culture at 37°C on a shaker at 180 rpm for 60 min.

[0057] (3.1.4) Pipette an appropriate volume of transformed competent cells onto LB solid agar medium containing the corresponding antibiotics, and spread the competent cells evenly with a spreader.

[0058] (3.1.5) After the liquid is absorbed, invert the plate and incubate at 37°C for 16 h.

[0059] (3.2) Plasmid extraction

[0060] Use the RapidLyse Plasmid Mini Kit and follow these steps:

[0061] (3.2.1) Before the experiment, please place Buffer QLB (please check whether RapidLyse Mix has been added) on ice and pre-cool it to 0-4℃.

[0062] (3.2.2) Take a 2 mL centrifuge tube and add 1 mL of overnight cultured bacterial solution. Centrifuge at 12000 rpm for 1 min and discard the supernatant as much as possible (if the bacterial solution is large, centrifuge several times to collect the bacterial precipitate into one centrifuge tube).

[0063] (3.2.3) Take 600 μL of pre-cooled Buffer QLB and add it to the centrifuge tube containing the bacterial pellet. After use, store Buffer QLB at 2-8°C.

[0064] (3.2.4) Please use a vortex machine to vortex for 30 seconds immediately to thoroughly resuspend the bacterial pellet and incubate at room temperature for 3 minutes.

[0065] (3.2.5) Transfer all the solution in (3.2.4) to the adsorption column RapidLyse DNA Mini Columns (make sure the adsorption column has been placed in the collection tube), centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0066] (3.2.6) Place the adsorption column into a collection tube, add 600 μL of Buffer QWB along the wall of the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0067] (3.2.7) Place the adsorption column into a collection tube and centrifuge at 12,000 rpm for 1 min until the residual liquid in the adsorption column is completely removed.

[0068] (3.2.8) Take a new clean 1.5 mL centrifuge tube, place the adsorption column in the centrifuge tube, take 50 μL of Buffer QEB and add it to the central part of the adsorption column membrane. Centrifuge at 12000 rpm for 30-60 seconds. After centrifugation, collect the plasmid DNA solution and store it at -20℃ or use it directly in subsequent experiments.

[0069] 3.3 Agrobacterium transformation:

[0070] (3.3.1) Take the GV3101 (pSoup-p19) Agrobacterium competent cells stored at -70°C and bring them to room temperature. Wait for the cells to thaw slightly and be in an ice-water mixture state, then place them in an ice bath for storage.

[0071] (3.3.2) Add 1 μg of plasmid DNA per 100 μL GV3101 (pSoup-p19) competent cells, flick the bottom of the tube to promote mixing, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0072] (3.3.3) Add 800 μL of LB medium without antibiotics and culture at 28°C with shaking for 2 to 3 h.

[0073] (3.3.4) Collect the bacteria by centrifugation at 5000 rpm for 1 min. Collect about 100 μL of supernatant and gently blow to resuspend the bacteria. Spread the bacteria evenly on LB plates containing the corresponding antibiotics. Invert the plates and place them in a 28°C incubator for 2-3 days.

[0074] According to the same method, the pTRV-1 vector and pTRV-2 were respectively transformed into the Agrobacterium tumefaciens strain GV3101 to obtain the corresponding Agrobacterium.

[0075] (4) Infection of rose seedlings with Agrobacterium tumefaciens strain GV3101 carrying pTRV2-RhALEU vector

[0076] Agrobacterium tumefaciens strain GV3101 carrying pTRV-1, pTRV-2, and pTRV2-RhALEU vectors were grown in Luria-Bertani (LB) medium supplemented with 50 μg / mL kanamycin and 50 μg / mL rifampicin, respectively, and then shaken in liquid medium at 28°C, 200 rpm for 14 h. Agrobacterium cells were collected by centrifugation at 4000 rpm and resuspended in infiltration buffer (10 mM 2-(N-Morpholino)ethanesulfonic acid (MES), 200 mM acetosyringone, and 10 mM magnesium chloride (MgCl2), pH 5.6, and the final concentration of bacteria was OD 600 =0.8-1.0.

[0077] The experimental group was a mixture of Agrobacterium carrying pTRV-1 vector and pTRV2-RhALEU vector at a ratio of 1:1 (v / v) (the control group was a mixture of Agrobacterium carrying pTRV-1 and pTRV-2 vector at a ratio of 1:1 (v / v)) (Virus-induced gene silencing in tomato reference doi: https: / / doi.org / 10.1046 / j.1365-313X.2002.01394.x ), cultured in the dark at room temperature for 3-4 hours, soaked the rose seedlings cultured in step 1 in the infiltration buffer containing the above-mentioned Agrobacterium solution, and placed in a -25KPa vacuum for transformation. Rinse the test tube seedlings with deionized water, and then culture them in the dark at 8°C for 3 days. Transplant them into culture soil and grow them at 22±1°C, relative humidity 60%, and long daylight (16h light / 8h dark). After 40 days of culture, the flower phenotype of level 1 flowers was continuously monitored, that is, the flowers were photographed and recorded at fixed time points every day.

[0078] The reaction was carried out in the Applied Biosystems StepOnePlusTM real-time quantitative PCR system using ChamQ SYBR qPCR Master Mix (Low ROX Premixed) reagent (Vazyme). The reaction system and reaction procedure were the same as in Example 1. The expression levels of the RhALEU gene and the aging marker gene RhSAG12 were calculated by real-time fluorescence quantitative RT-PCR (qRT-PCR). The RhUBI2 gene was used as an internal control, and at least 3 biological replicates were performed. -ΔΔCT The results are shown in Figure 2. Figure 4 Compared with the TRV control, the expression of RhALEU in the petals of RhALEU-silenced plants was reduced by 56.6%, indicating that the RhALEU-silenced plants can be used for subsequent studies ( Figure 4 A). The difference in flowering process between RhALEU silenced plants and TRV control plants ranges from level 5 (fully open bud stage) to loss of ornamental value (obvious petal drop in flowers) ( Figure 4 B), the control plants extended from 3.8±0.4 days to 5.9±0.8 days in the RhALEU-silenced plants, an increase of about 55.3% ( Figure 4 C). Compared with the TRV control, SAG12 expression in petals of level 5 RhALEU silencing was reduced by 51.2% ( Figure 4 D) These results indicate that RhALEU plays a significant positive regulatory role in petal senescence.

Claims

1. The application of RhALEU protein in regulating the senescence of rose petals, characterized in that: The amino acid sequence of the said RhALEU protein is as shown in SEQ ID NO.

3.

2. Encoding the protein according to claim 1 RhALEU The application of the gene in regulating the senescence of rose petals is characterized in that: Said RhALEU The sequence of the gene is shown as SEQ ID NO.1 or SEQ ID NO.

2.

3. RhALEU Gene silencing vector or RhALEU The application of gene-silenced strains in delaying the senescence of rose petals is characterized in that: Said RhALEU The sequence of the gene is shown as SEQ ID NO.1 or SEQ ID NO.

2.

4. The use according to claim 3, characterized in that: The silencing vector comprises the RhALEU The specific fragment of the gene was constructed into the vector pTRV2 to obtain RhALEU The sequence of the specific fragment of the gene is shown in SEQ ID NO.

6.

5. The use according to claim 3, characterized in that: Said RhALEU The gene-silencing strain is a strain in which the RhALEU The silencing vector of the specific fragment of the gene is introduced into Agrobacterium to obtain RhALEU The sequence of the specific fragment of the gene is shown in SEQ ID NO.

6.

6. A method for regulating the senescence of rose petals, characterized in that: The method includes inhibiting or downregulating the RhALEU The expression or activity of genes can delay the occurrence of aging process or prolong the flower opening time or the ornamental flowering period, or promote the RhALEU The expression of the gene promotes the occurrence of the aging process. The amino acid sequence of the RhALEU protein is shown in SEQ ID NO.

3.

7. The method for regulating rose petal senescence according to claim 6, characterized in that: The downward adjustment of rose RhALEU Gene expression or activity including knockout or silencing in the rose genome RhALEU Gene; or down-regulation RhALEU Down-regulators of gene transcription, polypeptide expression or polypeptide activity are introduced into roses, or RhALEU The rose varieties with gene deletion are hybridized with ordinary rose varieties. RhALEU The gene sequence is shown in SEQ ID NO.1 or 2.

8. The method for regulating rose petal senescence according to claim 6, characterized in that: The knockout or silencing of RhALEU Genes are RhALEU The specific fragment of the gene is introduced into the silencing vector, RhALEU The specific fragment of the gene is shown in SEQ ID NO.

6.

9. A method for identifying the lifespan of a rose obtained by the method according to any one of claims 6 to 8, characterized in that: By observing the senescence phenotypes of knockout or silenced plants and control plants, with wilting, fading and shedding as indicators, RhALEU To study the impact of rose petals aging, the flower cycle is divided into levels 1 to 6, and the duration of flowers at levels 5 and 6 is used to measure the speed of petal aging.

10. The method for identifying the life span of a rose obtained according to claim 9, characterized in that: The silenced plants are TRV- RhALEU The vector was introduced into Agrobacterium GV3101 and then infected into rose tissue culture seedlings to obtain plants.

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