CN117660482A

CN117660482A - Application of PsLHCB5 gene in regulation and control of green flowers and fruit set of peony

Info

Publication number: CN117660482A
Application number: CN202311631228.6A
Authority: CN
Inventors: 舒庆艳; 郝青; 李旸; 彭丽平; 李彤彤; 鲁高杰
Original assignee: Institute of Botany of CAS; Qingdao Agricultural University
Current assignee: Institute of Botany of CAS; Qingdao Agricultural University
Priority date: 2023-12-01
Filing date: 2023-12-01
Publication date: 2024-03-08

Abstract

The invention discloses application of a PsLHCB5 gene in regulation and control of green flowers and fruits of peony, and belongs to the field of genetic engineering. The invention obtains the coding sequence of the 'green curtain cryptojade' PsLHCB5 gene, and verifies the biological function of candidate genes by transforming tobacco through the agrobacterium-mediated over-expression and virus-induced gene silencing. Compared with wild plants, the over-expression PsLHCB5 transgenic line has the phenotypic characters of increased plant height, increased crown amplitude, stronger growth vigor, increased inflorescence branch number, increased fruiting quantity, darkened leaf color and the like, but the initial color of petals is shallower than that of the wild plants, and the total chlorophyll content of petals after PsLHCB5 silencing in peony is reduced. The result shows that the PsLHCB5 positively regulates the synthesis of the peony chlorophyll, and further proves the effect of the PsLHCB5 in the formation, growth and setting of green petals of the peony 'green curtain cryptosporidium'. The invention provides theoretical basis and gene resources for peony flower color and yield breeding.

Description

Application of PsLHCB5 gene in regulation and control of green flowers and fruit set of peony

Technical Field

The invention relates to the field of genetic engineering, in particular to application of a PsLHCB5 gene in the aspect of regulating and controlling green flowers and fruits of peony.

Background

Peony (Paeonia suffruticosa Andr.) belongs to Paeonia genus of Paeoniaceae, is a well-known ornamental plant in the world, and is called "king in flower" due to its colorful petal color and unique cultural symbolism. Peony has been widely planted as an afforestation, potted flowers and cut flowers (Zhou et al, 2014 a), a history of over 2000, forming nine-high flower colors: white, pink, red, purple, black, yellow, green, blue and multiple colors (Zhang and Liu, 2018). Although there are over three thousand peony varieties worldwide, green varieties are relatively rare and varieties with unique flower colors will be more appreciated by the flower breeder (streizer et al, 2019). The peony green varieties such as 'green curtain hidden jade', 'spring willow', 'bean green', 'green fragrance ball' and the like are popular in the market due to the rare colors, and have higher commercial value. These green varieties exhibit a transition from green to white or pink during flowering. However, the current research on peony color is mainly focused on red, purple and yellow, and few researches on green peony color development mechanism and molecular bioinformatics are carried out. The research shows that the green color of petals in ornamental plants is mainly determined by chlorophyll, anthocyanin and other pigments, but the formation mechanism is not clear, so that the development of green flower breeding is limited. In order to enrich the variety of different colors of peony, it is very critical to excavate key factors and key genes of petal color development and to research the color development mechanism of other green flowers and provide effective gene resources for molecular breeding of green peony.

Disclosure of Invention

The invention aims to provide application of a PsLHCB5 gene in regulating and controlling green flowers and fruits of peony, so as to solve the problems in the prior art, the gene positively regulates and controls synthesis of chlorophyll of petals of the peony, and the gene can be over-expressed to form green flowers of the peony, so that theoretical basis and gene resources are provided for breeding of colors of the peony.

In order to achieve the above object, the present invention provides the following solutions:

the present invention provides the use of the PsLHCB5 gene in any one of the following (1) - (4):

(1) Application in regulating and controlling the formation of green flowers of peony;

(2) Application in regulating and controlling synthesis of chlorophyll of peony petals;

(3) Application in cultivating green peony varieties;

(4) The method is applied to regulation and control of peony growth and setting;

wherein, the nucleotide sequence of the PsLHCB5 gene is shown as SEQ ID NO:1 (or a gene fragment comprising the sequence shown in SEQ ID NO: 1):

ATGAGTTCTCTGGCAGCATCTAGGGCGGCCGCCTCCCTGGGCGTGTCGGAAATGCTCGGAAACCCTCTCAATTTCAGCGGTGCCGCAAGGTCCGCTCCAACAGCGTCAAGCCCC GTCACAATATTCAAGACGGTGTCGCTCTTCTCGAAGAAGAAACCGGCACCCCCACCCAAGGCGAAGGTCGTCGCTCCGGCAGATGAAGAGCTCGCCAAGTGGTATGGTCCAGACAGAAGGATCTTCTTGCCGGAAGGGCTCTTGGACCGCTCTGAGATCCCTGAATATCTTACAGGAGAAGTACCTGGAGATTACGGTTACGATCCCTTTGGACTTAGCAAGAAACCAGAAAACTTCGCTAAATATCAAGGATATGAGCTAATTCACGCCAGGTGGGCCATGCTTGGAGCGGCTGGTTTTATCATTCCTGAGGCCTTCAACAAATTCGGTGCTAACTGTGGCCCAGAGGCCGTTTGGTTCAAGACGGGTGCTCTACTGCTTGATGGGAATACGTTGAACTACTTTGGAAAGAACATCCCCATTAATCTCGTTCTTGCTGTCGTTGCTGAGGTGGTTCTTCTTGGTGGTGCAGAATATTACAGAATTACCAACGGCTTGGATTTTGAGGACAAGCTTCACCCAGGTGGTCCTTTTGATCCATTAGGGCTAGCCAAGGATCCAGACCAGGCTGCATTGCTCAAGGTGAAGGAGATTAAGAATGGTAGACTTGCCATGTTTGCCATGCTCGGTTTCTTCATCCAAGCTTACGTCACAGGACAAGGCCCCGTTGAAAATCTGGCAGCCCATCTCAGCGACCCTTTTGGCAACAACTTGCTCACTGTCATCTCCGGAACTGCTGAAAGAGCTCCAAGCCTGTGA.

the invention also provides application of the protein encoded by the PsLHCB5 gene in any one of the following (1) - (4):

(3) Application in cultivating green peony varieties;

wherein, the amino acid sequence of the protein is shown in SEQ ID NO:2 (or an amino acid fragment comprising the sequence shown in SEQ ID NO: 2):

MSSLAASRAAASLGVSEMLGNPLNFSGAARSAPTASSPVTIFKTVSLFSKKKPAPPPKAKVVAPADEELAKWYGPDRRIFLPEGLLDRSEIPEYLTGEVPGDYGYDPFGLSKKPENFAKYQGYELIHARWAMLGAAGFIIPEAFNKFGANCGPEAVWFKTGALLLDGNTLNYFGKNIPINLVLAVVAEVVLLGGAEYYRITNGLDFEDKLHPGGPFDPLGLAKDPDQAALLKVKEIKNGRLAMFAMLGFFIQAYVTGQGPVENLAAHLSDPFGNNLLTVISGTAERAPSL*.

the invention also provides the application of the expression vector comprising the PsLHCB5 gene in any one of the following (1) - (4):

(3) Application in cultivating green peony varieties;

(4) The method is applied to regulation and control of peony growth and fruiting.

The invention also provides the use of a host bacterium comprising the expression vector in any one of the following (1) - (4):

(3) Application in cultivating green peony varieties;

Preferably, the PsLHCB5 gene is overexpressed, and synthesis of chlorophyll in peony petals is positively regulated, so as to form green peony.

The invention also provides a method for regulating and controlling chlorophyll synthesis of peony petals, which comprises the steps of over-expressing or silencing a PsLHCB5 gene in the peony petals to regulate and control the chlorophyll synthesis in the peony petals, wherein the nucleotide sequence of the PsLHCB5 gene is shown as SEQ ID NO: 1.

Preferably, the PsLHCB5 gene is overexpressed, and the total content of chlorophyll in the peony petals is increased; silencing the PsLHCB5 gene, and reducing the total chlorophyll content of the peony petals.

The invention discloses the following technical effects:

according to the invention, a typical green peony variety 'green curtain cryptojade' is used as a research material, a transcriptome of a peony green flower sample is sequenced, a gene with different colors is screened out, the expression mode of the gene with different colors is detected by combining a fluorescence quantitative PCR method, a key gene PsLHCB5 is obtained, and the functions of candidate genes are verified by cloning full length, bioinformatics analysis, over-expression tobacco and transient gene silencing (VIGS), so that the role in petal chlorophyll metabolic pathway is explored. As a result, it is found that the transgenic strain of the over-expressed PsLHCB5 has phenotypic characters such as increased plant height, increased crown width, stronger growth vigor, increased inflorescence branch number, increased fruiting amount, deepened leaf color and the like, but the initial color of petals is shallower than that of a wild plant, and the total chlorophyll content of petals after the PsLHCB5 is silenced in the peony is reduced. The PsLHCB5 is shown to positively regulate and control the synthesis of peony chlorophyll, and the gene has an important regulation and control effect in the formation process of peony 'green curtain cryptosporidium' green petals. The invention provides information for deeply researching the molecular mechanism of peony flower color change, and lays a theoretical foundation for directionally cultivating color-changing peony varieties by utilizing genetic engineering.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of electrophoresis detection of petal RNA at the 'LMYY'5 period; m is a standard DNA molecule, and LMS1-LMS5 are petal RNA samples in 5 periods respectively;

FIG. 2 shows the PCR detection result of the bacterial liquid of the constructed over-expression gene vector; m is a standard DNA molecule, pSuper1300-PsLHCB5 is an over-expression gene carrier bacterial liquid;

FIG. 3 is a graph showing the prediction of the affinity/hydrophobicity, transmembrane structure, signal peptide, secondary and tertiary structure of PsLHCB 5; a: prediction analysis of hydrophilicity/hydrophobicity; b: transmembrane predictive analysis; c: predictive analysis of signal peptides; d: predicting and analyzing a secondary structure; e: three-level structure prediction analysis;

FIG. 4 shows an amino acid sequence alignment and a tree analysis of peony 'LMYY' and other known PsLHCB5 proteins of other plants; a: analyzing a PsLHCB5 evolutionary tree; b: phylogenetic tree analysis of PsLHCB5 with LHCB5 protein sequences of other species;

FIG. 5 is a tobacco genetic transformation; a: a co-cultivation stage; b: a Hyg screening stage; c: rooting; d: regenerating tobacco;

FIG. 6 is a PCR assay of PsLHCB5 transgenic tobacco; marker is a standard DNA molecule, WT is wild tobacco, psLHCB5 is a transgenic positive tobacco plant;

FIG. 7 is a fluorescent quantitative analysis of PsLHCB5 transgenic tobacco;

FIG. 8 shows the PsLHCB5 transgenic tobacco phenotype (A), L ^* a ^* b ^* Values (B) and chlorophyll content (C);

FIG. 9 is a graph showing correlation analysis of PsLHCB5 gene expression level and chlorophyll content;

FIG. 10 is a graph showing the nutritional growth index of PsLHCB5 transgenic tobacco; a: tobacco plant height at 18 weeks of age; b: a number of blades; c: internode length; d: a crown web; e: inflorescence branch number; f: inflorescence branch length; g: the weight of the individual fruits; h: number of individual fruits;

FIG. 11 shows the petal P1-P3 phenotype of transgenic tobacco at different periods;

FIG. 12 is a PCR assay for constructing a VIGS silencing vector bacterial solution; m is a standard DNA molecule, pTRV2-PsLHCB5 is a silencing gene vector bacterial solution;

FIG. 13 is a diagram of a silencing PsLHCB5 petal pair phenotype (A), L ^* a ^* b ^* Influence of the value (B), the gene expression level (C) and the chlorophyll content (D).

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

EXAMPLE 1 cloning and functional identification of the PsLHCB5 Gene

1. Cloning of the PsLHCB5 Gene

1.1 plant Material

The method is characterized in that a typical green variety 'green curtain cryptosporidium' in peony (P. Suffruticosa) is selected as a research material and planted in a peony germplasm resource garden of Qingdao agricultural university. Flowers were divided into five developmental stages according to the process of opening, S1 (tight bud stage, yellow-green petals), S2 (enlarged bud stage, light green petals), S3 (deep green petals starting to bloom), S4 (cup-shaped flowering stage, green and light pink co-existing in petals), and S5 (flowering stage, light pink petals). In 2021, 10 excellent strains of 'green curtain cryptosystem' in 5 development periods are randomly collected, each single plant selects fresh petals, one part of the petals is immediately used for flower color assessment on site, FAA is fixed for scanning electron microscope observation, and one part of the petals is packaged with tin foil paper and quickly frozen by liquid nitrogen and then stored at-80 ℃ for later-stage petal pigment content, pH value measurement, RNA extraction and other researches.

Tobacco Nc89 (Nicotiana tabacum cv.nc89) seeds were from the institute of plant study Shu Qingyan subject group of the academy of sciences of china. Wild type tobacco seeds were supplied by the subject group laboratory.

1.2 Gene cloning

Total RNA extraction and reverse transcription. Extracting total RNA from peony petals in S3 periodII 1st Strand cDNASynthesis Kit, the experiment was performed sequentially.

(1) cDNA Synthesis

And (5) reverse transcription. The RNA obtained in the previous step is used as a template, and is subjected to reverse transcription expression to prepare cDNA.

The specific operation process is as follows: first, prepare on ice16. Mu.L of the reaction system (4 XgDNA wind Mix 4. Mu.L; RNA 1. Mu.g; ddH) ₂ Supplementing O to 16 mu L), and lightly blowing and mixing the components uniformly by using a liquid-transfering gun at 42 ℃ for 2 minutes; in addition, 5X HiScript III qRT SuperMix is added, the mixture is gently whipped up and down by a liquid suction device and uniformly mixed, the liquid drops on the pipe wall are collected at the bottom of the pipe through short centrifugation, and the liquid drops are put into a PCR instrument for the following reaction: 15min at 37 ℃; 5sec at 85 ℃.

(2) Cloning of the coding region of the Gene

According to the sequencing result, the full-length sequence of PsLHCB5 can be obtained, the CDS Finder on-line software of the full-length sequence in NCBI searches the maximum open reading frame, and a pair of specific primers are designed at the 5 'end and the 3' end of the sequence of PsLHCB5 by utilizing a software primer 5.0 (see table 1).

TABLE 1 cloning of all Gene primer sequences

(3) PCR amplification of target Gene

The cDNA was used as a template, and PCR amplification was performed thereon. According toMax Master MixPCR instructions for amplification.

Then, a following procedure (cycle number 35, table 2) was performed in a PCR apparatus:

TABLE 2PCR procedure

(4) Purification and recovery of DNA gel products

After the reaction is finished, the obtained product is put on a machine, agarose gel electrophoresis is carried out, the specificity of the PCR product is detected, the prepared gel product is placed under an ultraviolet gel imager, the size of the observed band is consistent with the expected band size, the ultraviolet undercut gel is stored in a 1.5mL centrifuge tube, and the method is carried out according to the following stepsGel DNAExtraction Mini Kit purification recovery is illustrated.

(5) Connection

According to the 5min TA/Blunt-Zero Cloning Kit instructions, in ice preparation of 5 u L reaction system, and the recovered product and Blunt end Blunt-Zero Cloning reaction 5min carrier connection.

(6) Transformation competence

Transfer of ligation products to E.coli competent Fast-T ₁ Competint Cell, spread on LB solid medium containing Amp (100 mg/mL), and put the plate after sealing film in incubator at 37 deg.C for 10min, after bacterial liquid is absorbed by the medium, turn over the plate upside down, and culture overnight.

(7) Bacterial liquid PCR verification

After the plate had grown out the dispersed and larger single bacteria, single colonies were picked up again into LB liquid medium containing Amp (100 mg/mL), and the culture was expanded by shaking overnight at 37℃on a shaker, and after the bacterial liquid had clouded, the bacterial liquid was taken out for PCR verification.

(8) Sequencing

And after the reaction is finished, taking the PCR product for agarose gel electrophoresis detection, and observing the size of the band by a gel imager to determine the band consistent with the expected target band, and identifying the bacterial liquid as a positive colony. The bacterial liquid sample was sampled at 200. Mu.L and sequenced by the Protocol sequencing company. The sequencing results were aligned with PsLHCB5 (c27170. Graph_c0) by DNAMAN8.0 software.

(9) Plasmid extraction

In the sequencing result, a colony with high coincidence with the target gene sequence is found, and plasmid extraction is carried out on the colony.

1.3 bioinformatics analysis

Through prediction of the physical and chemical properties of amino acid in the coding region of the gene, prediction of the hydrophilicity and hydrophobicity of the amino acid and prediction of the secondary and tertiary structures of the coded product, the relation between the molecular biological characteristics of the coding region of the gene and the functions of the coding region of the gene is revealed. On the basis, DNAMAN and MEGA software are adopted to carry out homologous sequence comparison on amino acids of various plants, and evolutionary trees of various species are established. The analysis software is shown in table 3 below.

Table 3 on-line analysis software analysis links

1.4 construction of plant overexpression vectors

1.4.1 cloning plasmid extraction

Plasmids of pMD-18T-PsLHCB5 and pSuper1300 were extracted, respectively, using the plasmid miniprep kit from Nannofurozan.

1.4.2 recombinant insert acquisition

Primers were designed on-line on the Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /) website, and conditions were set to automatically generate amplification primers for the insert (Table 1). The inserted fragment was amplified with high fidelity enzyme, step 1.3, and recovered with DNA gel recovery kit after the amplification was detected electrophoretically.

1.4.3 preparation of linearization vectors

A suitable cleavage site on the pSuper1300 vector was found, and a pSuper1300 linearized vector was prepared using double cleavage with Hind III and Kpn I restriction enzymes.

And adding a sample at 4 ℃, uniformly mixing the bottom of the flick centrifuge tube after adding the sample, placing the mixture in a constant temperature dryer at 37 ℃, and immediately placing the mixture on ice after enzyme digestion for 3-5 hours to terminate enzyme digestion reaction.

The enzyme digestion is checked to be successful by 1% agarose gel electrophoresis, and the DNA gel recovery kit is purified and recovered.

1.4.4 ligation and transformation of the Gene of interest with the Linear vector

The following reaction system was synthesized on ice by calculating the amount of DNA required: vector double enzyme digestion recovery product 3.3. Mu.L, target gene 1.23. Mu.L, 2X Taq Plus Master Mix II 5. Mu. L, ddH ₂ O was supplemented to 10. Mu.L.

The DH5 alpha chemically competent cells were rapidly dissolved in ice, thawed to liquid state, 10. Mu.L of recombinant product was injected into competent cells by a pipette, gently flicked against the vessel wall by hand, mixed well and placed on ice for 30min.

At 42 ℃, the reacted product is placed in a constant temperature dryer, heated for 45s, and then immediately placed in ice for 2-3min. 900. Mu.L of LB liquid medium without antibiotics was placed in a reaction tube and shaken at 37℃for 1h at 200 rpm. Centrifuge in a centrifuge at 5000rpm for 5min, remove 900. Mu.L of supernatant. The cells were resuspended by pipetting with the remaining medium and then gently smeared onto plates containing kanamycin (Kan) antibiotics with a sterile spreader bar. Culturing in an inverted manner at 37deg.C for 12-16 h.

1.4.5 identification of recombinant vector plasmids

PCR detection (see FIG. 2) was performed on pSuper1300-PsLHCB5 recombinant plasmid, the dispersed and plump agrobacterium single colony was picked up by the sterilizing gun head, and the single colony was added into LB liquid medium containing 100. Mu.g/mL Kan and 50. Mu.g/mL rifampicin (Rif) together with the gun head, and mixed evenly, and placed under the culture condition of 28℃and 200rpm for shake culture for 8 hours, then the positive recombinants were detected by PCR, and the clear bands were selected and sent to the Optimagin company for sequencing and the correct bacterial liquid was stored at-80℃with 50% glycerol.

1.5 transformation of tobacco with overexpression vector

1.5.1 Agrobacterium-mediated transformation of tobacco

And (5) culturing sterile seedlings of tobacco. Taking a proper amount of wild tobacco seeds into a 1.5mL centrifuge tube, sucking 1000 mu L of 75% alcohol, adding the mixture into the centrifuge tube, soaking the mixture for 2min, shaking the mixture upside down, and discarding waste liquid; adding 1000 mu L of 2% sodium hypochlorite solution, soaking seeds for 5min, mixing up and down, and discarding waste liquid; washing seeds with sterilized water, sucking out the waste liquid with a liquid-transferring gun, repeating for 3-5 times, and placing on sterile filter paper to suck out water. The seeds are clamped by a sterilized forceps and inoculated on an MS culture medium in a tissue culture bottle, then the tissue culture bottle is placed in an incubator with the temperature of 28 ℃ and the illumination intensity of 2500Lx, the photoperiod of 16h/8h and the relative humidity of 60 percent, and when 3-5 true leaves grow out of the plants, an infection test is carried out. All seeding processes were performed in a sterile operating table.

The recombinant vector converts agrobacterium. The leaf disc method for converting tobacco comprises the following specific operation steps:

(1) Will have turnedGV3101 Agrobacterium competent activation into pSuper1300-PsLHCB5 vector according to 1:100 proportion of activated bacterial liquid is taken, added into LB liquid culture medium containing 100 mug/mL Kan and 50 mug/mL Rif, mixed evenly, placed under 28 ℃ and 180rpm culture condition for shake culture until bacterial liquid OD ₆₀₀ A value of 0.6;

(2) Centrifuging at 5000rpm for 10min at room temperature, removing supernatant, collecting bacterial precipitate, and re-suspending bacterial with sterilized MS liquid culture medium to obtain infected leaf;

(3) Cutting leaf disc of aseptic tobacco seedling with 3-5 true leaves for 3 weeks, avoiding main vein and leaf margin of leaf tip, and cutting into 1cm pieces ² Left and right small blocks;

(4) Placing the cut tobacco leaf small pieces into MS suspension, carrying out infection for 10-15 min, and then gently shaking to allow bacterial liquid to fully contact with the wound at the leaf margin;

(5) Taking out the leaves infected with the bacterial liquid, placing the leaves on sterile filter paper, and sucking the bacterial liquid above;

(6) Leaves were inoculated into solid co-culture medium (MS medium+sucrose 20 g/l+agar 7.5g/L, ph=5.8) and placed at 28 ℃ for co-culture in the dark for 3d;

(7) After co-cultivation is completed, residual agrobacterium on leaves is washed with sterile water containing 500mg/L of cephalosporin (Cef), and is then absorbed cleanly by sterile filter paper, and finally transferred to a differentiation induction and screening medium MS (MS medium +6-BA (1.0 mg/L) +NAA (0.2 mg/L) +sucrose 20 g/l+agar 7.5g/l+Kan (100 mg/L) +cef (200 mg/L), pH 5.8, 28 ℃ C., illumination intensity 2500Lx, relative humidity 50% -60%, photoperiod 16h/8h stationary cultivation, and MS medium replacement every ten days;

(8) After the regenerated seedlings are differentiated, the seedlings are separated from the callus after the differentiated seedlings grow to 2-3cm high. Transferring into MS rooting culture medium (1/2 MS culture medium+sucrose 20 g/L+agar 7.5g/L+Kan (100 mg/L) +Cef (200 mg/L), pH is 5.8), inducing to root, and rooting positive transgenic seedling after about 1-2 weeks; after the seedlings were induced, when the seedlings were grown to 2-3cm, the seedlings were separated from the calli. Transferring the plant to a rooting culture medium (1/2 MS culture medium+20 g/L of sucrose+7.5 g/L of agar+Kan (100 mg/L) +Cef (200 mg/L), and pH of 5.8) of MS, and performing induced rooting, wherein positive plants of the gene can root after 1-2 weeks;

(9) After the root system grows well, taking out the tobacco tissue culture seedling, washing agar carried by the root of the tobacco tissue culture seedling with sterile water, and planting the agar in a flowerpot containing a matrix (nutrient soil: vermiculite=1:1), uncovering a film after one week of film coating, and transferring the film into a greenhouse to bloom and fruit so as to obtain seeds;

(10) Until the seeds are fully mature, collecting and storing in a refrigerator at 4 ℃ and carrying out vernalization at a lower temperature for standby.

1.5.2 PCR identification and qPCR detection of transgenic tobacco

(1) PCR identification

And respectively taking 6 tobacco strains which are well grown and transferred into pSuper1300-PsLHCB5 genes, extracting genome DNA, and carrying out PCR identification by taking the genome DNA as a template, wherein the identification method is as the common PCR identification in the above 1.4. After the reaction was completed, tobacco plants positive for the gene band of interest were determined by 1% agarose gel electrophoresis.

(2) RT-qPCR detection

And (3) performing fluorescent quantitative PCR on the wild type transgenic strain, and verifying the relative expression quantity of the PsLHCB5 gene. Extracting total RNA, reversely transcribing the RNA into cDNA (see reagent description), designing fluorescent quantitative primers of PsLHCB5 gene on NCBI functional network (see table 4), selecting NtTubulin reference gene design internal reference primers NtTubulin-F and NtTubulin-R of tobacco gene (see table 4), and performing a fluorescent quantitative PCR reaction system as follows: cDNA 2. Mu.L, 2X ChamQ Universal SYBR qPCR Master Mix. Mu.L, forward primer 0.4. Mu.L, reverse primer 0.4. Mu. L, ddH ₂ O 9.8μL。

TABLE 4 primers for analysis of expression level

The real-time fluorescent quantitative PCR reaction conditions were as follows (cycle number 40, table 5):

TABLE 5 real-time fluorescent quantitative PCR reaction conditions

According to 2 ^-ΔΔCt The relative expression level of the PsLHCB5 gene in the transgenic tobacco plants is calculated by the method.

1.6 biological functional verification of PsLHCB5 in transgenic tobacco

And (3) performing fluorescent quantitative PCR on the transgenic line, and verifying the relative expression quantity of the PsLHCB5 gene. And measuring nutrition growth indexes of the tobacco positive plants. Transgenic tobacco lines with high PsLHCB5 expression were individually selected for phenotypic validation. And comparing the nutritional growth conditions of wild type and PsLHCB5 transgenic tobacco positive plants with the seedling age of 18 weeks, measuring the plant height, crown width, inflorescence branch length, number and the like of the wild type and PsLHCB5 transgenic tobacco positive plants, photographing and recording the tobacco appearance. Harvesting after the fruits are ripe, and counting indexes such as the length, the width, the weight and the like of the fruits.

2. Results and analysis

2.1 cloning of Gene

2.1.1RNA quality inspection

RNA from the petals of 'Green Leucomatous Cryptosporidium' was extracted and detected by agarose gel electrophoresis, and the result of the electrophoresis shows that the RNA of each of the four samples had three distinct bands of 28S, 18S and 5S, and the 28S band was most pronounced (FIG. 1). The RNA quality was acceptable and either stored at-80℃or immediately reverse transcribed and stored at-20℃for subsequent testing.

2.1.2 cloning of the coding region of the Gene

Based on the transcriptome data, the nucleotide sequence of PsLHCB5 (1340 bp) was obtained and the open reading frame of PsLHCB5 was queried in CDS Finder on-line software, wherein the CDS of PsLHCB5 was 873bp. The primer PsLHCB5-F/R (Table 1) is designed at two ends of the DNA, PCR amplification is carried out by taking cDNA of 'green curtain cryptosporidium' as a template, a single specific band is obtained near 873bp, bacterial liquid is sequenced, and the result shows that the sequence is consistent with the CDS region of the splicing result.

Bioinformatics analysis of 2.2PsLHCB5

Bioinformatics analysis is carried out on the coding region sequence of PsLHCB5, and the bioinformatics analysis comprises amino acid physicochemical property prediction analysis, amino acid hydrophilcity prediction analysis and coded product secondary and tertiary structure prediction analysis.

Protein physicochemical properties were predicted using the ProtParam on-line analysis tool, with a CDS of 873bp for PsLHCB5, encoding a total of 290 amino acids. The relative molecular mass of the coded protein is 31019.67Da, and the molecular formula is C ₁₄₂₃ H ₂₂₀₀ N ₃₆₂ O ₄₀₃ S ₆ The theoretical isoelectric point PI value is 5.99, and the total number of atoms is 4394. The instability index was 29.35 and the protein was presumed to be a stable protein. The overall average hydrophobicity index was 0.016. The maximum hydrophobicity of the PsLHCB5 protein is 2.85 and the minimum is-2.219. The protein has a very pronounced alternating arrangement of hydrophilic and hydrophobic regions, generally a hydrophobic protein. PsLHCB5 has no protein transmembrane domain and is not a membrane protein. PsLHCB5 has no signal peptide and belongs to a non-secreted protein. The PsLHCB5 secondary structure was shown to contain α -helices (39.66%), β -turns (4.83%), random coils (45.17%), extended chains (10.34%) (fig. 3).

The PsLHCB5 is compared with homologous protein sequences of plum (trunk name), amania grape (trunk name), almond (trunk dulcis), guava (Psidium guajava), grape (grape vinifera), cabbage (Brassica oleracea) and the like, phylogenetic relationship between the PsLHCB5 and other species is explored, a phylogenetic tree is constructed through Jalview, and the result shows that the PsLHCB5 is closer to the root of the phylogenetic tree, is clustered with homologous proteins in the plum into one class, and has a certain relationship (figure 4).

2.3 identification of Gene overexpression vectors

The recombinant plasmid of pSuper1300-PsLHCB5 is used as a template for PCR verification, the correct band of PsLHCB5 exists at 873bp, and the comparison and analysis of the positive plasmid sequencing result shows that the sequence identity with the original sequence reaches more than 99 percent, so that the overexpression vector pSuper1300-PsLHCB5 is successfully constructed.

2.4 transformation of tobacco

The function of the PsLHCB5 is identified by introducing the recombinant vector into tobacco, an agrobacterium-mediated method is adopted in the experiment, a genetic transformation system is established by transformation through a tobacco leaf disc method, the PsLHCB5 is transformed into the tobacco for over-expression, and the state of each stage of the tobacco genetic transformation is shown in figure 5.

2.5 transgenic tobacco plant selection and quantification

Gene transfer into tobacco leaf, T, by Agrobacterium-mediated method ₁ When the transgenic tobacco plants are subjected to antibody screening, false positive conditions of the antibody plants exist, namely, exogenous genes carried by agrobacterium are not successfully inserted into tobacco genome, so that PCR detection is required to be carried out on DNA of the resistant plants to determine positive tobacco plants (figure 6).

2.6 fluorescent quantitative PCR detection of transgenic tobacco

T ₀ Repeated resistance screening of single plant seed collection after continuous planting of tobacco plants, T ₁ Single plant seed harvest continues to select resistance Hyg to T ₂ Generations may be used to observe phenotypes. For T ₂ And extracting RNA from the transgenic lines of the generation, carrying out real-time fluorescent quantitative PCR identification, and verifying the relative expression quantity of genes. As a result, the relative expression amounts of the PsLHCB5 transgenic lines were increased as compared with those in wild type tobacco, and the relative expression amounts of the 2 nd, 5 th, 8 th, 9 th, 11 th and 16 th lines were 109-fold, 123-fold, 16-fold, 569-fold, 153-fold and 157-fold, respectively, of the wild type plants (FIG. 7).

2.7PsLHCB5 transgenic tobacco index determination

2.7.1 transgenic tobacco phenotype observations

As can be seen from the figure, tobacco over-expressed by PsLHCB5 also exhibits a green darkened phenotype (A in FIG. 8), L for three strains ^* The value was generally lower than the control group (53.16), with the 9 th strain being the lowest, 47.14.a, a ^* The values were also generally lower than the control (-20.19), with the 9 th strain being the lowest, being-20.95, followed by the 5 th strain, being-20.84. b ^* The value also shows a corresponding change trend, wherein the 9 th strain is the lowest, 29.29 (B in figure 8), the chlorophyll content of the tobacco with the over-expressed PsLHCB5 is generally higher than that of the control group (C in figure 8), and the correlation analysis result shows that the chlorophyll a content, the chlorophyll B content and the total chlorophyll contentHas a significant positive correlation with the PsLHCB5 gene expression level (FIG. 9). These results indicate that overexpression of PsLHCB5 can promote tobacco chlorophyll accumulation.

2.7.2 determination of the nutritional growth index of transgenic tobacco

Comparing the vegetative growth of the wild-type and transgenic PsLHCB5 tobacco plants, a significant difference was found between the PsLHCB5 transgenic plants and the wild-type plants. The PsLHCB5 transgenic line is significantly higher than the wild type plant (64.33.+ -. 2.49 cm) and grows stronger, with the 9 th transgenic line being 84.+ -. 2.44cm highest (A in FIG. 10). The leaf number of the PsLHCB5 transgenic line is more than that of the wild type plant (36+ -1.69), and is 45.66+ -2.86 (B in FIG. 10). The internode length of the PsLHCB5 transgenic line is 3.5.+ -. 0.24cm (C in FIG. 10) higher than that of the wild type plant (3.03.+ -. 0.26 cm). The crown of the PsLHCB5 transgenic line is 35.33.+ -. 2.49cm, which is also significantly higher than the wild type (D in FIG. 10). The PsLHCB5 transgenic line has a greater number of inflorescence branches than the wild type (E in fig. 10). The inflorescence length of the PsLHCB5 transgenic plants was significantly longer than that of the wild type plants (F in fig. 9) for the same growth time. The individual fruit weights of the PsLHCB5 transgenic plants were significantly heavier than the wild type (G in fig. 10). The PsLHCB5 transgenic plants had significantly more fruits than the wild type (H in fig. 10) in the number of fruits per plant.

2.7.3 transgenic tobacco flowering process analysis

Flowers of PsLHCB5 did not appear substantially red during period P1 and petals began to be significantly colored since P2. The red color of the petals of the transgenic strain PsLHCB5 was not much different from that of the petals of the CK strain (FIG. 11).

Example 2 transient silencing of the PsLHCB5 Gene Using VIGS

1. Experimental materials

As in example 1.

2. Construction of VIGS overexpression vectors

The cloning target fragment was cloned with the aid of the upstream and downstream primers (Table 6) designed on the basis of the sequences of PsLHCB5 in the transcriptome database, with a cloning length of 339bp. The PsLHCB5 fragment for VIGS silencing was designed with upstream and downstream primers and added to the Eco RI and Kpn I cleavage sites. The PCR amplification system and procedure were as in example 1, 1.4. The target band is recovered and connected with a PMD18-T vector, DH5 alpha escherichia coli competence is transformed, PCR sequence sequencing is carried out on the target band, and positive plasmids are extracted after confirming no errors. Simultaneously, the target fragment and pTRV2 were subjected to double cleavage reaction with the corresponding 2 restriction enzymes, respectively, and recovered, and the double cleavage reaction system (50. Mu.L): 2.5. Mu.L of 10 XM Buffer, 2.5. Mu.g of plasmid DNA, 2.5. Mu.L of restriction enzyme I, 2.5. Mu.L of restriction enzyme II, and sterilized water were added to 50. Mu.L. The reaction is carried out for 3-5h at 37 ℃. And then the gel is removed for recovery. The target gene was then ligated to the recovered product of pTRV2 using homologous recombination techniques. After the connection product is transformed and plated, a positive monoclonal colony is selected from the colony and verified by bacterial liquid PCR, a specific primer is designed according to the sequence information characteristics of pTRV2, and a recombinant vector is identified for pTRV2-F/pTRV 2-R. After sequence determination, the expression vector plasmid which is verified to be correct by sequencing is stored at-20 ℃ for standby. And (3) transforming the competent cells of the agrobacterium GV310 by a freeze thawing method to obtain positive monoclonal agrobacterium, adding 50% of glycerol into the bacterial liquid, uniformly mixing, and preserving at-80 ℃ for later use.

TABLE 6 primer sequences for all genes of VIGS

3. VIGS silencing petal

The method comprises the following specific steps:

(1) Setting TRV2-PsLHCB5 as a target gene and taking a corresponding pTRV2 empty vector as a control.

(2) Inoculating 1mL of Agrobacterium GV3101 of pTRV1, pTRV2 (negative control), TRV2-PsLHCB5 into 60mL of LB liquid medium (containing 50mg/L Rif, 100mg/L Kan, 50mg/L gentamicin (Gen), 200mmol/L acetosyringone (As), 10mmol/L MES) respectively, and shake culturing at 180rpm,28℃to different treatments OD ₆₀₀ ；

(3) Determination of OD of the shaken liquid ₆₀₀ After=1.0-2.0, 4000rpm, centrifugation at room temperature for 10min, cell collection, and supernatant removal;

(4) Resuspended in infection buffer (10 mM MgCl) ₂ 20mM AS,10mM MES (pH=5.6);

(5) Weight adjustment with an infestation liquidOD of suspension ₆₀₀ The value and the set bacterial liquid OD ₆₀₀ The values were the same, and the OD of TRV2-PsLHCB5, pTRV1 and pTRV2 bacterial solutions were the same ₆₀₀ The values are the same;

(6) Respectively mixing pTRV2 and TRV2-PsLHCB5 bacterial solutions with pTRV1 according to a volume ratio of 1:1, mixing, and performing dark treatment at room temperature for 4 hours;

(7) The VIGS system is various in inoculation technology and different in species, the adopted inoculation method also influences the gene silencing efficiency, and the adopted infection method for the test is a vacuumizing method. The petal wafer is firstly processed, and the flower in the S3 period is selected. Removing the outermost petals, and taking the middle petals, wherein each flower is 6-8 pieces; the petal discs are selected to be close to the middle upper part of the edge, each petal is beaten one, a flat disc is selected, and the positions of the discs are ensured to be basically the same as possible; and (5) carrying out suction infection on the petal discs by adopting a vacuum suction method. The petal discs are soaked in the dyeing liquid. Sucking to-0.08 for the first time, sucking to-0.07 for the second time, maintaining for 90s, slowly deflating for 5min, and infecting liquid OD ₆₀₀ =1.5. After infestation, the petals are rinsed with distilled water. The length and width of petals were measured with a tape. Shooting according to a certain sequence, and adding a ruler as a ruler into the photos. During this process, care should be taken to be as rapid as possible so as not to lose moisture and not to disrupt the petal sequence. The petals that have been photographed are placed on soaked absorbent paper to ensure that the bottoms thereof can absorb moisture. Wrapping with plastic film to prevent water loss;

(8) The mixture was left at 8℃for 3d. Taking out after 3d, measuring the size according to the last order, and taking a picture;

(9) Transferring to 23 ℃ and standing for about 10 d. Measuring, photographing and sampling according to the sequence on the sampling day;

(10) And (5) putting the petal discs after treatment into liquid nitrogen for quick freezing, and then placing the petal discs at the temperature of minus 80 ℃ for standby. With reference to the bioinformatics analysis method in example 1, the change in the expression level of the gene compared with the blank control was analyzed, and the expression level analysis of the PsLHCB5 gene was performed, assuming 9 biological and 3 technical replicates. Primer sequences are shown in Table 4.

4. Results and analysis

Cloning of the 1PsLHCB5 target fragment

The electrophoresis result shows that clear single specific bands appear at about 350bp, the sizes of the specific bands are consistent with those of target gene fragments, positive bacterial liquid obtained by recovery, connection and transformation is sequenced, and the result shows that PsLHCB5 gene interference fragments (339 bp) are obtained by successful cloning, and can be used for constructing a VIGS recombinant vector.

4.2 construction of the recombinant vector of the Gene VIGS of interest

After the target fragment and the linear carrier are subjected to enzyme digestion and connection reaction, escherichia coli is transformed, and clear single specific bands are obtained through identification of bacterial liquid PCR (figure 12).

4.3 analysis of the PsLHCB5 petal phenotype, chlorophyll content and Lx a x b x values

After a transient silencing of PsLHCB5 d, the petals showed yellowing (a in fig. 13). The silencing efficiency of PsLHCB 5-impregnated petals was 58% by fluorescent quantitative PCR detection (B in FIG. 13). Against TRV (101.19. Mu.g.g) ^-1 FW) in comparison with the total chlorophyll content of the PsLHCB5 petals (35.86. Mu.g. G) ^-1 FW) decrease (D in fig. 13), consistent with phenotypic changes. The a-value (-8.87) of PsLHCB5 petals was significantly higher than TRV (C in fig. 13). It was shown that PsLHCB5 is capable of promoting chlorophyll accumulation.

From the results of examples 1-2 above, it can be seen that silencing of the PsLHCB5 gene may result in a change in flower color in the peony 'green-curtain-cryptosystem', consistent with the conclusion of over-expression of tobacco. The invention successfully silences the PsLHCB5 gene, and the silencing efficiency is 58%. The color of the silenced PsLHCB5 petals is slightly lighter than that of the control group after 10d, and yellowing phenomenon appears, which indicates that the PsLHCB5 gene possibly plays an important role in the color formation path, the total chlorophyll amount after PsLHCB5 silencing is reduced, and further proves that the PsLHCB5 transcription factor plays a promoting role in the peony 'green curtain hidden jade' green petal formation process. The invention primarily focuses on exploring the functions of key candidate gene PsLHCB5, obtains positive tobacco plant seeds, has obvious change in phenotype, and provides theoretical guidance and effective gene resources for perfecting petal color-developing mechanisms of peony green flowers and for variety breeding with petal colors as indexes.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

Use of the pslhcb5 gene in any one of the following (1) - (4):

(1) Application in regulating and controlling the formation of green flowers of peony;

(2) Application in regulating and controlling synthesis of chlorophyll of peony petals;

(3) Application in cultivating green peony varieties;

(4) The method is applied to regulation and control of peony growth and setting;

wherein, the nucleotide sequence of the PsLHCB5 gene is shown as SEQ ID NO: 1.
2. Use of a protein encoded by the PsLHCB5 gene as claimed in claim 1 in any of the following (1) - (4):

(1) Application in regulating and controlling the formation of green flowers of peony;

(2) Application in regulating and controlling synthesis of chlorophyll of peony petals;

(3) Application in cultivating green peony varieties;

(4) The method is applied to regulation and control of peony growth and setting;

wherein, the amino acid sequence of the protein is shown in SEQ ID NO: 2.
3. Use of an expression vector comprising the PsLHCB5 gene as claimed in claim 1 in any of the following (1) - (4):

(1) Application in regulating and controlling the formation of green flowers of peony;

(2) Application in regulating and controlling synthesis of chlorophyll of peony petals;

(3) Application in cultivating green peony varieties;

(4) The method is applied to regulation and control of peony growth and fruiting.
4. Use of a host bacterium comprising the expression vector of claim 3 in any one of the following (1) to (4):

(1) Application in regulating and controlling the formation of green flowers of peony;

(2) Application in regulating and controlling synthesis of chlorophyll of peony petals;

(3) Application in cultivating green peony varieties;

(4) The method is applied to regulation and control of peony growth and fruiting.
5. The use according to any one of claims 1 to 4, wherein the PsLHCB5 gene is overexpressed, positively regulating the synthesis of chlorophyll in peony petals to form green peony.
6. A method for regulating and controlling chlorophyll synthesis of peony petals is characterized by comprising the steps of over-expressing or silencing a PsLHCB5 gene in peony to regulate and control chlorophyll synthesis in the peony petals, wherein the nucleotide sequence of the PsLHCB5 gene is shown as SEQ ID NO: 1.
7. The method of claim 6, wherein said PsLHCB5 gene is overexpressed and the total content of chlorophyll in the petals of peony is increased; silencing the PsLHCB5 gene, and reducing the total chlorophyll content of the peony petals.