Application of rhaleu protein and its encoding gene in petunia petal senescence regulation

Abstract

The application discloses RhALEU protein and an application of an encoding gene thereof in regulation of old age of rose petals. RhALEU The application screens out a gene from a rose transcriptome database RhALEU , and finds that expression of the gene is induced in the whole petal development stage. RhALEU The application verifies that the gene has a function of promoting old age of rose petals by using VIGS technology. RhALEU Silencing of the gene slows down the speed of the petal from the 5th grade to losing ornamental value. RhALEU In the petal with the silenced gene, expression of the old age related gene SAG12 is significantly down-regulated. RhALEU The application discloses the function of the gene in natural old age of the petal, provides a new thought and technical means for regulating old age of a plant flower, especially postharvest preservation of fresh cut rose, opens up a new research field of flower regulation, and has great academic value and important production application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology technology, and particularly relates to application of RhALEU protein and a coding gene thereof in petunia petal senescence regulation. BACKGROUND

[0002] Flower industry is an important part of large-scale agriculture, and is a green industry integrating economic, social and ecological benefits. Fresh-cut flowers are the most important product in the flower industry, and petunia ranks first among the four major cut flowers in the world. Petunia cut flowers are extremely intolerant to long-distance transportation, and the postharvest loss during circulation is usually more than 30%, and sometimes even as high as 50%. The large postharvest storage and transportation loss has become a bottleneck problem restricting the quality and efficiency of the petunia cut flower industry. Therefore, breeding new varieties resistant to storage and transportation and developing efficient postharvest preservation technology are effective ways to break through the industrial bottleneck of high postharvest loss of petunia cut flowers, and the mining of petal senescence key genes and the analysis of their biological mechanisms are the theoretical basis for variety improvement and technological innovation.

[0003] From the external morphology, petal senescence mainly shows wilting, shedding and flower color change; the physiological essence is a series of processes such as the decrease of cell turgor pressure and the increase of hydrolytic activity. Among them, the change of protein content best reflects the senescence process, with the progress of senescence, the catalytic metabolic hydrolase increases, the soluble protein in petal continuously decreases, the free amino acid content increases, leading to the increase of petal PH value, petal browning and blue change. Although this process is very important, the proteases that play a role in petal senescence have not been fully identified and characterized.

[0004] Cysteine proteases (CPs) 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 abundant proteases in the process of plant senescence. Among all plant cysteine proteases, papain-like cysteine proteases (PLCPs) have been most thoroughly studied. PLCPs are the main enzymes related to a large amount of protein degradation during the process of senescence. SAG2 of Arabidopsis encodes PLCPs and is used as a standard to measure leaf senescence. ALEU encodes a senescence-related protease, but the mechanism has not been clear, and whether it plays a role in the process of senescence is not clear. SUMMARY

[0005] The technical problem to be solved by the present application is to identify that the expression of RhALEU gene in petunia petals increases with the occurrence of petal senescence process, and then find that RhALEU gene is an important protease in the regulation of petal senescence.

[0006] Technical solution: In order to solve the above technical problems, the application provides application of RhALEU protein in petunia petal senescence regulation, and the amino acid sequence of the RhALEU protein is shown as SEQ ID NO. 3.

[0007] The application also provides application of RhALEU gene in petunia petal senescence regulation, and the sequence of the RhALEU gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

[0008] The application also provides application of a RhALEU gene silencing vector and a RhALEU gene silencing strain in delaying petunia petal senescence, and the sequence of the RhALEU gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

[0009] The silencing vector comprises a specific fragment of the RhALEU gene constructed into a vector pTRV2, and the sequence of the specific fragment of the RhALEU gene is shown as SEQ ID NO. 6.

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

[0011] The application also provides a method for regulating petunia petal senescence, which comprises inhibiting or down-regulating the expression or activity of a petunia RhALEU gene to delay the occurrence of the senescence process or prolong the flower opening time or ornamental flowering period, or promoting the expression of the RhALEU protein to promote the occurrence of the senescence process, and the amino acid sequence of the RhALEU protein is shown as SEQ ID NO. 3.

[0012] The down-regulation of the expression or activity of the petunia RhALEU gene comprises knocking out or silencing the RhALEU gene in the genome of the petunia, or introducing a down-regulation agent of RhALEU gene transcription, polypeptide expression or polypeptide activity into the petunia, or crossing a petunia variety with RhALEU gene deletion with a common petunia variety, and the sequence of the RhALEU gene is shown as SEQ ID NO. 1 or 2.

[0013] The RhALEU gene is introduced into a silencing vector, and the specific fragment of the RhALEU gene is shown as SEQ ID NO. 6.

[0014] The application also provides a method for identifying the lifespan of the Rosa chinensis obtained by the method, wherein the influence of RhALEU on the petal senescence of Rosa chinensis is observed by observing the senescence phenotype of the knockout or silenced plant and the control plant, taking wilting, discoloration and shedding as indexes, the flower period is divided into 1-6 levels, and the petal senescence speed is measured by the time duration of the 5th and 6th levels of the flower.

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

[0016] The down-regulation agent is an interference molecule for specifically interfering with the expression of the RhALEU gene, and the interference molecule is dsRNA, antisense nucleic acid, small interfering RNA, microRNA, sgRNA for inhibiting or silencing the RhALEU gene or its transcript, or a construct capable of expressing or forming the dsRNA, antisense nucleic acid, small interfering RNA, microRNA.

[0017] The application also realizes the delay by selecting the 3' end of the RhALEU gene to silence the RhALEU gene, and realizes the promotion by overexpressing the RhALEU gene.

[0018] The application also provides a method for identifying the lifespan of the Rosa chinensis obtained by the method, wherein the influence of RhALEU on the petal senescence of Rosa chinensis is observed by observing the senescence phenotype of the knockout or silenced plant and the control plant, taking wilting, discoloration and shedding as indexes, the flower period is divided into 1-6 levels, and the petal senescence speed is measured by the time duration of the 5th and 6th levels of the flower.

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

[0020] Beneficial effects: Compared with the prior art, the application has the following obvious advantages: the RhALEU gene is cloned from the petal of Rosa chinensis, and research shows that the expression of the RhALEU gene is induced in the whole petal development stage, and the silencing of the RhALEU gene slows down the speed of the petal from the 5th level to the loss of ornamental value. In the petal with the silenced RhALEU gene, the expression of the senescence-related gene SAG12 is significantly down-regulated. The research results of the application reveal the role of the RhALEU gene in the natural senescence of the petal, provide a new idea and technical means for regulating the senescence of the flower of the plant, especially the postharvest preservation of the fresh-cut flower of Rosa chinensis, open up a new research field of flower regulation, and have great academic value and important production and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 RhALEU gene is obtained.

[0022] Figure 2 Expression of RhALEU gene was detected in different stages of flower opening. Expression pattern of RhALEU gene in different stages of petal opening in Rosa hybrida. RhUBI2 gene was used as internal control. All values are the average of 5 biological replicates ± SD. The figure shows the results of the Tukey-Kramer test, where letters indicate significant differences (P < 0.05).

[0023] Figure 3 Agarose gel electrophoresis map of the specific gene fragment of RhALEU gene in Example 2.

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

[0025] Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing technique that has been widely used in plant genetic engineering research as an effective reverse genetics technique. The commonly used viral vector is tobacco rattle virus (TRV). TRV is functioned by two vectors pTRV1 and pTRV2. The vectors pTRV-1 and pTRV-2 of the present application were donated by Professor Liu Yule of Tsinghua University. In the present application, the combination of 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 Obtaining of gene RhALEU and expression pattern of RhALEU gene in different stages of petal opening in Rosa hybrida

[0027] 1. Gene cloning and sequence analysis

[0028] The application utilizes ALEU gene in Arabidopsis thaliana, carries out alignment using Blast in rose (Rosa hybrida 'Samantha') genome data (https: / / lipmbrowsers.toulouse.inra.fr / pub / RchiOBHm-V2 / ) and combines with annotation in the NCBI (National Center for Biotechnology Information) database, and finally 13 ALEU genes are screened (https: / / lipmbrowsers.toulouse.inra.fr / pub / RchiOBHm-V2 / ). Figure 1 ) is selected as a candidate gene, and is named as RhALEU (the full length is referred to SEQ ID NO. 1). The ORF length of RchiOBHm_Chr2g0095151 is 1074 bp (referred to SEQ ID NO. 2), and can encode a protein containing 357 amino acids (referred to SEQ ID NO. 3).

[0029] 2. Culture of rose seedlings

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

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

[0032] The expression level of RhALEU in different levels is detected by real-time fluorescence quantitative PCR (qRT-PCR). The specific evaluation criteria of different levels refer to the previous research of the present 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 is extracted from rose petal using the heat borate method (Ma, N., Tan, H., Liu, X., Xue, J., Li, Y., & Gao, J. (2006). Transcriptional regulation of ethylene receptor and CTR genes involved in ethylene-induced flower opening in cut rose (Rosa hybrida) cv. Samantha. Journal of experimental botany, 57(11), 2763-2773). The cDNA is obtained from 1 μg of total RNA using HiScript III reverse transcriptase (Vazyme). The fluorescence quantitative RT-PCR (qRT-PCR) is 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 is used as an internal control, and at least 3 biological replicates are used. The expression of related genes is calculated using 2 -ΔΔCT

[0033] qRT-PCR reaction system

[0034]

[0035] qRT-PCR reaction program

[0036]

[0037] The results show that the transcription level of RhALEU gene increases with the increase of flower opening stage Figure 2 ), which indicates that the RhALEU gene may be involved in the petal senescence process and plays a major role in the flower senescence stage. Figure 2 S3 is the loose state of the outer petals of the flower, S4 is the flat but not exposed core of the flower, and S5 is the fully open flower with the core exposed.

[0038] Example 2 Virus-induced gene silencing

[0039] ​The experimental method refers to the previous study of the inventor (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 Tobacco rattle virus vector for efficient and visualizable analysis of gene function. Journal of experimental botany, 65(1), 311-322.). Specifically, the pTRV2-RhALEU vector is constructed by inserting the 300bp RhALEU specific fragment (38bp of ORF region and 262bp of 3' untranslated region, see SEQ ID NO. 6) into the pTRV2 enzyme cutting vector containing EcoRI and BamH I enzyme cutting sites. The specific steps are as follows:

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

[0041] Design of primers: the sequence of the specific fragment primer RhALEU-TRV-F is shown in SEQ ID NO. 4; the sequence of the specific fragment primer RhALEU-TRV-R is shown in SEQ ID NO. 5; the sequence of the RhALEU quantitative primer RhALEU-qRT-F is shown in SEQ ID NO. 7; the sequence of the RhALEU quantitative primer RhALEU-qRT-R is shown in SEQ ID NO. 8; the sequence of the RhUBI2 quantitative primer RhUBI2-qRT-F is shown in SEQ ID NO. 9; the sequence of the RhUBI2 quantitative primer RhUBI2-qRT-R is shown in SEQ ID NO. 10.

[0042] PCR amplification:

[0043] PCR amplification system

[0044]

[0045] The PCR tube is placed in the PCR instrument, and the PCR amplification reaction program is as follows:

[0046]

[0047] The agarose gel electrophoresis result of the amplification result is shown in Figure 3 It can be seen from Figure 3 that the specific gene fragment of 300bp is amplified by the present application. The Gel DNA Extraction Mini Kit to recover the target fragment.

[0048] (2) Vector construction

[0049] Using 2x Seamless Cloning Mix kit, the following reaction system was established in a 0.2 mL PCR tube, mixed gently and centrifuged briefly, and incubated in a PCR instrument (50°C, 15 min). After the reaction, the centrifugal tube was placed on ice to obtain the ligation product pTRV2-RhALEU, ready for use in transformation experiments. If not used for transformation, the ligation product was stored at -20°C.

[0050] Homologous recombination reaction system

[0051]

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

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

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

[0055] (3.1.2) Place the centrifugal tube in a 42°C water bath for 60 sec, then quickly transfer the centrifugal tube to an ice bath, cool the cells for 2-3 min, and keep the centrifugal tube stationary during this period.

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

[0057] (3.1.4) Take an appropriate volume of transformed competent cells and add them to LB solid agar medium containing the corresponding antibiotic, 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 miniprep

[0060] Using RapidLyse Plasmid Mini Kit, the operation steps are as follows:

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

[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 there is a large amount of bacterial solution, centrifuge several times to collect the bacterial precipitate into a centrifuge tube).

[0063] (3.2.3) Take 600 μL of pre-cooled Buffer QLB and add it to a centrifuge tube containing bacterial precipitate. After use, please store Buffer QLB at 2-8℃.

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

[0065] (3.2.5) Transfer all the solution from (3.2.4) to the RapidLyse DNA Mini Columns (make sure the adsorption column is in the collection tube), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0066] (3.2.6) Place the adsorption column into the collection tube, add 600 μL of Buffer QWB along the wall of the adsorption column, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0067] (3.2.7) Place the adsorption column into the 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, add 50 μL of Buffer QEB to the center of the adsorption column membrane, centrifuge at 12000 rpm for 30-60 seconds, collect the plasmid DNA solution after centrifugation and store it at -20℃ or use it directly for subsequent experiments.

[0069] 3.3 Agrobacterium-mediated transformation:

[0070] (3.3.1) Take GV3101(pSoup-p19) Agrobacterium competent cells stored at -70℃ and place them at room temperature. Wait for the cells to thaw slightly and be in an ice-water mixed state before placing them in an ice bath for storage.

[0071] (3.3.2) Add 1 μg of plasmid DNA to every 100 μL of GV3101 (pSoup-p19) competent cells, gently tap the bottom of the tube to promote mixing, and incubate 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 in sequence.

[0072] (3.3.3) Add 800 μL of antibiotic-free LB medium and incubate at 28°C with shaking for 2–3 h.

[0073] (3.3.4) 5000 rpm centrifugation for 1 min to collect bacteria, about 100 μL supernatant was taken, the bacterial pellet was resuspended by gently blowing, and then evenly spread on LB plates containing the corresponding antibiotics, and the plates were inverted and cultured in a 28 °C incubator for 2-3 days.

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

[0075] (4) Agrobacterium tumefaciens strain GV3101 carrying pTRV2-RhALEU vector infected Rosa hybrida seedlings

[0076] The Agrobacterium tumefaciens strain GV3101 carrying pTRV-1, pTRV-2, and pTRV2-RhALEU vectors was respectively grown in Luria-Bertani (LB) medium supplemented with 50 μg / mL kanamycin and 50 μg / mL rifampicin, followed by 14 h of liquid culture at 28 °C and 200 rpm. The Agrobacterium cells were collected by centrifugation at 4000 rpm, resuspended in the 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 the 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 vectors at a ratio of 1:1 (v / v)) (Virus-induced gene silencing in tomato, doi: https: / / doi.org / 10.1046 / j.1365-313X.2002.01394.x ), and the seedlings were cultured in the dark at room temperature for 3-4 h. The Rosa hybrida seedlings cultured in step 1 were soaked in the infiltration buffer containing the Agrobacterium bacterial solution, and then transformed in a vacuum of -25 KPa. The test tube seedlings were rinsed with deionized water, and then cultured in the dark at 8 °C for 3 days. Transplanting was performed in the culture soil, and the seedlings were grown at 22±1 °C, 60% relative humidity, and long-day photoperiod (light 16 h / dark 8 h). The flower phenotype of the first grade flowers was continuously monitored at a fixed time point every day, and the flowers were photographed and recorded.

[0078] The reaction was carried out in an Applied Biosystems StepOnePlusTM real-time quantitative PCR system using ChamQ SYBR qPCR Master Mix (Low ROX Premixed) reagent (Vazyme), and the reaction system and reaction program were the same as in Example 1. The expression of RhALEU gene and senescence marker gene RhSAG12 was calculated by real-time fluorescent quantitative RT-PCR (qRT-PCR). RhUBI2 gene was used as an internal control, and at least 3 biological replicates were used. The expression of related genes was calculated by 2 -ΔΔCT The results are shown in Figure 4 Compared with the TRV control, the expression of RhALEU in the petal of RhALEU silenced plant was reduced by 56.6%, indicating that the RhALEU silenced plant could be used for subsequent study Figure 4 A) The difference between RhALEU silenced plant and TRV control plant in the process of flowering was from 5 grade (full open bud stage) to lose ornamental value (flower obvious petal drop) Figure 4 B), the control plant was prolonged from 3.8 ± 0.4 days to 5.9 ± 0.8 days of RhALEU silenced plant, which was prolonged by about 55.3% Figure 4 C) Compared with the TRV control, the expression of SAG12 in the petal of RhALEU silenced plant at 5 grade was reduced by 51.2% Figure 4 D) These results indicated that RhALEU played a significant positive regulatory role in petal senescence.

Claims

1. The use of inhibiting or down-regulating RhALEU protein in delaying the senescence of rose petals, characterized in that, The amino acid sequence of the RhALEU protein is shown as SEQ ID NO.

3.

2. A gene encoding the protein of claim 1. RhALEU Knockout or silencing of the gene in delaying petunia petal senescence, characterized in that, The RhALEU The sequence of the gene is shown in SEQ ID NO. 1 or SEQ ID NO.

2.

3. RhALEU a silencing vector of a gene or RhALEU application of a silencing strain of a gene in delaying petunia petal senescence, characterized in that, The RhALEU The sequence of the gene is shown in SEQ ID NO. 1 or SEQ ID NO.

2.

4. Use according to claim 3, characterized in that, The silencing vector comprises a specific fragment of the RhALEU gene constructed into vector pTRV2, and the RhALEU sequence of the specific fragment of the gene is shown as SEQ ID NO.

6.

5. Use according to claim 3, characterized in that, The RhALEU Gene-silencing strains are those that... RhALEU The gene-specific silencing vector was introduced into Agrobacterium to obtain the gene. RhALEU The sequence of the gene-specific fragment is shown in SEQ ID NO.

6.

6. A method of retarding senescence of rose petals, characterized by, The method comprises inhibiting or down-regulating the expression or activity of the Rosa hybrida RhALEU Gene to delay the occurrence of aging process or prolong the flower opening time or ornamental flowering period, the amino acid sequence of the RhALEU protein is shown in SEQ ID NO.

3.

7. The method for delaying petunia petal senescence according to claim 6, characterized in that, The down-regulation of the Rose RhALEU The expression or activity of the gene includes: knocking out or silencing RhALEU The gene RhALEU The gene sequence is shown as SEQ ID NO. 1 or 2.

8. The method of delaying petal senescence in Rosa chinensis according to claim 6, wherein, Knocking out or silencing in the genome of Rosa hybrida RhALEU A gene is a segment of DNA that codes for RhALEU A specific segment of the gene is introduced into a silencing vector, which is RhALEU A specific segment of the gene is shown in SEQ ID NO. 6.

Images