A key miRNA regulating peony plant height, stem diameter, and xylem formation and its application

Abstract

This invention discloses a key miRNA that regulates peony plant height, stem diameter, and xylem formation, and its applications. The precursor and mature sequences of this peony miRNA are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The peony miRN82 provided by this invention can be used to regulate peony growth. Overexpression of miRN82 reduces peony plant height and stem diameter, and narrows the xylem width, indicating that miRN82 is a key miRNA regulating peony plant height, stem diameter, and xylem formation. This invention regulates peony plant height, stem diameter, and xylem thickness by overexpressing or knocking out / silencing miRN82, enabling the breeding of peony varieties with different stem heights, thicknesses, and hardnesses according to specific needs. Therefore, it has significant application value in peony molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a key miRNA that regulates peony plant height, stem diameter and xylem formation and its application. Background Technology

[0002] Peony (Paeonia lactiflora Pall.) is a perennial herbaceous plant belonging to the genus Paeonia (Paeonia L.) of the family Paeoniaceae. Renowned for its beautiful and vibrant flowers, it is often referred to as the "Prime Minister of Flowers" and possesses high ornamental value. Peonies are not only a traditional Chinese flower but also a world-renowned cut flower. In recent years, peonies have gained increasing prominence in the international cut flower trade. At the Amsterdam flower auction market, the world's largest flower market, the sales growth of peony cut flowers has surpassed that of traditional cut flowers such as roses and lilies. China is a major producer of peony varieties and cut flowers. However, one of the bottlenecks currently facing China's peony cut flower industry is the problem of bent stems due to insufficient stem strength, which significantly affects the ornamental value of peony cut flowers and has seriously hindered the development of China's peony cut flower industry. Therefore, how to improve stem strength or obtain peonies with different plant heights, stem diameters, and xylem sizes through regulation has become an urgent problem to be solved for the peony cut flower industry.

[0003] miRNAs are a class of single-stranded non-coding RNAs, 20-24 nt in length, with regulatory functions. At the post-transcriptional level, they specifically recognize and bind to complementary sequences on target gene mRNAs, participating in post-transcriptional gene regulation through splicing or translation repression. Numerous studies have shown that miRNAs participate in regulating various aspects of plant growth and development, as well as biotic and abiotic stresses. Recent research has revealed that miRNAs also play a crucial role in plant secondary cell wall formation; however, there are currently few reports on miRNAs regulating peony plant height, stem diameter, and xylem formation. Summary of the Invention

[0004] Purpose of the invention: To address the shortcomings of existing technologies, the purpose of this invention is to provide a key miRN82 that regulates the height, stem diameter, and xylem formation of peony plants. By regulating the expression of this miRNA, the height, stem diameter, and xylem formation of peony plants can be controlled.

[0005] The present invention also provides a vector for expressing the key peony miRN82 and its application.

[0006] Technical solution: In order to achieve the above objectives, the present invention provides a key peony miRN82 for regulating peony plant height, stem diameter and xylem formation, the precursor sequence of which is shown in SEQ ID NO.1.

[0007] The mature sequence of peony miRN82 is shown in SEQ ID NO.2.

[0008] The overexpression vector of the peony miRN82 precursor sequence or the peony miRN82 mature sequence as described in claim 2.

[0009] The overexpression vector is a TRV2 vector containing the precursor sequence of peony miRN82.

[0010] The overexpression vector assembles a constitutive strong expression promoter CAMV35S at the 5' end of the miRN82 precursor sequence and a strong terminator NOS-ter at the 3' end.

[0011] The present invention contains the key peony miRN82 or the host cell overexpression vector.

[0012] The host cell is based on Agrobacterium as the starting strain.

[0013] The application of the peony miRN82 described in this invention in regulating peony plant height, stem diameter and xylem formation.

[0014] The application is as follows: when miRN82 is transferred into peony plants, transgenic peonies that overexpress miRN82 have reduced plant height and stem diameter, narrower stem xylem width, and smaller secondary cell wall thickness.

[0015] The application involves transferring the precursor sequence of peony miRN82 into peony plants. Transgenic peonies that overexpress the precursor sequence of peony miRN82 have reduced plant height and stem diameter, narrower stem xylem width, and smaller secondary cell wall thickness.

[0016] The peony miRN82 provided by this invention can be used to regulate peony growth. Overexpression of miRN82 reduces peony plant height and stem diameter, and narrows the stem xylem width, indicating that miRN82 is a key miRNA regulating peony plant height, stem diameter, and xylem formation. Furthermore, knocking out or silencing miRN82 increases peony plant height and stem diameter, widens the stem xylem width, and increases the thickness of the secondary cell walls of sclerenchyma cells. By overexpressing or knocking out / silencing miRN82 to regulate peony plant height, stem diameter, and xylem thickness, peony varieties with stems of different heights, thicknesses, and hardness can be bred according to specific needs, thus possessing significant application value in peony molecular breeding.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0018] This invention is the first to clone a new miRN82 gene from peony. By transferring miRN82 into peony plants, transgenic peonies that overexpress miRN82 have reduced plant height and stem diameter, and narrower stem xylem width. Therefore, by regulating the expression level of miRN82, peony varieties with different stem heights, diameters, and hardness can be bred according to requirements. Thus, it has important application value in the molecular breeding of peony. Attached Figure Description

[0019] Figure 1 This is a gel image of peony miRN82 precursor amplification;

[0020] Figure 2 The cloned miRN82 precursor sequence was compared with the sequence obtained from small RNA sequencing (a) and the miRN82 secondary structure (b);

[0021] Figure 3 It is a TRV2 vector map;

[0022] Figure 4 This represents the expression level of miRN82 in peony plants that overexpress miRN82;

[0023] Figure 5 This is the phenotype of peony plants that overexpress miRN82;

[0024] Figure 6 The figures show the plant height (a) and stem diameter (b) of peony plants overexpressing miRN82; different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences.

[0025] Figure 7 The observations are of paraffin sections of peony stems with control treatment (a) and paraffin sections of peony stems overexpressing miRN82 (b, c). Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0028] In the examples, all raw materials are known conventional materials, among which TRV1 and TRV2 are known carriers (An R2R3-MYB network modulates stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony[J].The Plant Journal,2023,113(6):1237-1258).

[0029] Example 1

[0030] Cloning miRN82

[0031] (1) Based on the peony miRNA-seq data, miRN82 was screened and primers were designed for miRN82. The forward primer (F primer) was 5'-TTAGAGGAAGGCAAAACGTGG-3', and the reverse primer (R primer) was 5'-TAGAGAAACATGACCCAGTTCATCC-3'.

[0032] (2) PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (Novizan, China). The PCR system is as follows:

[0033]

[0034] Gently mix the above mixture, briefly centrifuge at low speed, and then place it in a standard PCR instrument. Set the following program:

[0035]

[0036] Electrophoresis: The gene amplification product from the PCR instrument is retrieved, and an appropriate amount of the product is spotted onto a 1% agarose gel using an electrophoresis apparatus. After approximately 20 minutes, the product is removed and observed using an imaging system to obtain the target fragment. Figure 1 ).

[0037] (3) Ligation reaction of purified fragments and cloning vector

[0038] Following the instructions of the 5min TA / Blunt-Zero Cloning Kit (Novizan, China), the gel-recovered product was ligated into the cloning vector. The specific system is as follows:

[0039]

[0040] Mix the solutions in the system in a microtube and react at room temperature for 5 minutes. After the reaction is complete, place on ice until ready to use.

[0041] (4) Escherichia coli transformation

[0042] Referring to the Trans1-T1 Phage Resistant Chemically Competent Cell product instructions (TransGold, China), the ligated product was mixed with competent cells, and after ice bath, heat shock, and recovery, an appropriate amount was spread on LB plates, the plates were inverted, and cultured overnight at 37°C.

[0043] (5) Positive clone screening and sequencing analysis

[0044] Single colonies were selected from the screening culture plate and inoculated into LB liquid medium. The culture was incubated overnight at 37°C and 250 rpm. The recombinant transformants were directly detected by PCR using the overnight culture as a template.

[0045] Reaction system:

[0046]

[0047]

[0048] Reaction procedure:

[0049]

[0050] Clones that tested positive by bacterial culture PCR were sent to Shanghai Sangon Biotech Co., Ltd. (Shanghai) for sequencing and identification. Plasmid extraction revealed that the precursor sequence of miRN82 was 156 bp, as shown in SEQ ID NO.1, exhibiting a typical neck-loop structure. Figure 2 This conforms to the structural characteristics of miRNA. The mature sequence of miRN82 is 23 bp, as shown in SEQ ID NO.2.

[0051] Example 2

[0052] Construction of plant expression vector for TRV2:miRN82

[0053] (1) This experiment used TaKaRa QuickCut restriction enzyme (TaKaRa, Japan) TRV2 vector ( Figure 3 The precursor sequences of ) and miRN82 were subjected to enzyme digestion experiments, and the specific reaction systems are as follows:

[0054]

[0055] After mixing all solutions in the system, the mixture was briefly centrifuged and incubated in a 37°C water bath for 1 hour to end the enzyme digestion reaction. The enzyme digestion bands were observed by agarose gel electrophoresis. Subsequently, the target gene and vector fragments were digested and recovered separately for subsequent vector ligation reactions.

[0056] (2) Following the instructions for TaKaRa T4 DNALigase (TaKaRa, Japan), the expression vector recovered after the double enzyme digestion reaction was ligated with the target DNA fragment product. The system is as follows:

[0057]

[0058] The solutions in the system were mixed in a microtube and reacted in a metal bath at 16°C for 5-6 hours.

[0059] PCR detection confirmed the successful construction of the TRV2:miRN82 expression vector, which was named TRV2:miRN82. The constructed overexpression vector was equipped with the constitutive strong expression promoter CaMV35S at the 5' end of the miRN82 precursor sequence and the terminator NOS at the 3' end.

[0060] (3) Transformation of Agrobacterium tumefaciens

[0061] Following the instructions for the GV3101 / EHA105 Chemically Competent Cell product (TransGold, China), the TRV2:miRN82 expression vector plasmid constructed in step (2) was mixed with GV3101 Agrobacterium competent cells. The mixture was then incubated sequentially on ice for 30 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min before adding culture medium and shaking. An appropriate amount was spread onto LB agar plates and incubated upside down at 28°C. Single colonies were picked from the plates, and an appropriate amount of LB liquid medium was added. After incubation for 48 h, the bacterial culture was sequenced to obtain Agrobacterium containing the TRV2:miRN82 vector.

[0062] Example 3

[0063] Genetic transformation of TRV2:miRN82

[0064] 1. Preparation and transformation of bacterial culture

[0065] The obtained positive Agrobacterium tumefaciens bacterial suspension was prepared into an infection solution. An appropriate amount of Agrobacterium tumefaciens bacterial suspension stored at 4℃ or -80℃ was activated on a YEB plate (containing 50 mg / L Kamagra and 50 mg / L LRif). Single colonies were picked and incubated at 28℃ with shaking. The turbid bacterial suspension was then added to 20 mL of liquid YEB (containing 50 mg / L Kamagra and 50 mg / L LRif) and incubated at 28℃ with shaking until the OD reached 1.2. The cells were collected by centrifugation at 25℃ for 10 min, the supernatant was discarded, and 20 mL of antibiotic-free liquid YEB was added for elution. The cells were collected by centrifugation at 25℃ for 10 min, and 20 mL of antibiotic-free liquid YEB was added for elution. The cells were collected by centrifugation at 25℃ for 10 min, and resuspended in liquid (containing 10 mM MES, MgCl, and 200 μM MAS). The OD600 of the cells was 1.2. After the resuspended suspension was allowed to stand for 1 hour for activation, it could be used for infection.

[0066] Because the TRV2 vector requires the assistance of the TRV1 vector to function, and the TRV1 vector is an empty vector that does not require gene ligation, equal volumes of bacterial cultures containing empty TRV1 and empty TRV2 vectors were mixed as the control TRV. Equal volumes of bacterial cultures containing empty TRV1 vector and the target gene (TRV2:miRN82) were mixed as the experimental group. Peonies with five buds were selected. The mixed bacterial culture was added to a vacuum pump. The peony roots were washed, trimmed, and placed in a vacuum filter, ensuring the trimmed wounds were immersed in the bacterial culture. The container was sealed, and Vaseline was evenly applied. Vacuum filtration was performed at 0.06 MPa for 20 minutes. After removal, the roots were washed and planted in pots, incubated in the dark for 3 days, and then cultured under normal conditions. Subsequent experiments were conducted after 5 weeks of culture.

[0067] 2. Detection and phenotypic observation of transgenic materials

[0068] Using PrimeScript TM The Reverse Transcriptase Reagent Kit (TaKaRa, Japan) was used for real-time quantitative PCR to determine the expression level of miRN82 in transgenic plants. The results showed that the expression level of miRN82 in transgenic plants was significantly increased. Figure 4 Phenotypic observation revealed that overexpression of miRN82 significantly reduced peony plant height and stem diameter. Figure 5 , Figure 6 Further observation using histochemical staining of paraffin sections revealed that overexpression of miRN82 resulted in a narrowing of the xylem width and a decrease in the thickness of the secondary cell walls of sclerenchyma cells. Figure 7 These results indicate that miRN82 plays an important role in regulating peony plant height, stem diameter, and xylem thickness.

Claims

1. Application of Paeonia miRN82 in regulating plant height, stem diameter and xylem formation of Paeonia; the application is: introducing precursor sequence of Paeonia miRN82 into Paeonia plant, overexpressing the precursor sequence of Paeonia miRN82, and the transgenic Paeonia plant has reduced plant height and stem diameter, narrower stem xylem width and smaller secondary wall thickness of thick-walled cells; the precursor sequence of Paeonia miRN82 is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The mature sequence of Paeonia miRN82 is shown as SEQ ID NO.

2.

3. Use according to claim 1, characterized in that, Overexpression vector containing precursor sequence of Paeonia miRN82.

4. Use according to claim 3, characterized in that, The overexpression vector is assembled with constitutive strong expression promoter CAMV35S at 5' end of the miRN82 precursor sequence and strong terminator NOS-ter at 3' end.

5. Use according to claim 3, characterized in that, Host cell containing the overexpression vector; the host cell is based on Agrobacterium as the starting strain.

Images