A gene for controlling the size of plant petals and its application

Abstract

The present invention relates to the field of plant genetic engineering, and discloses a gene for controlling the size of plant petals and its application. The present invention provides a GhPS1 gene for controlling the size of plant petals, and its nucleotide sequence is shown in SEQID NO.1. The present invention also provides a recombinant vector and an engineered bacterial strain for controlling the size of plant petals. The present invention also provides a recombinant expression vector and an engineered bacterial strain. The GhPS1 gene, recombinant vector or engineered bacterial strain for controlling the size of plant petals provided by the present invention controls the size of petals by controlling cell expansion, thereby improving flower production. The present invention cloned a GhPS1 gene from African chrysanthemum, and verified that the gene has the function of controlling the size of plant petals. It has been experimentally verified that overexpression or silencing of the gene can obtain transgenic plants with smaller or larger petals than wild-type plants. The gene of the present invention can provide a theoretical basis and gene source for breeding new flower varieties.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to a gene for controlling the size of plant petals and an application thereof. Background Art

[0002] Petal size is one of the important traits that affect the ornamental value of horticultural plants. From a cellular level, petal size is mainly affected by two factors: cell division and cell expansion. Among them, cell expansion is particularly important for the later morphological construction of petals. Therefore, mining and obtaining genetic resources related to controlling petal cell expansion is crucial for molecular breeding of flowers.

[0003] At present, although a small number of genetic resources related to the regulation of petal size have been discovered, such as the petal growth inhibition gene bHLH-type transcription factor BIGPETALp (BPEp) and the WIP-type zinc finger protein gene GhWIP2, many genes come from the model plant Arabidopsis thaliana, and it is not clear whether these genes function in other flowers. Therefore, it is of great significance to discover genetic resources that regulate petal size for the refined breeding of new varieties of related flowers. Summary of the invention

[0004] The invention provides a gene for controlling the size of plant petals and its application.

[0005] In a first aspect, the present invention provides a GhPS1 gene for controlling the size of plant petals, and the nucleotide sequence thereof is shown as SEQ ID NO.1.

[0006] In a second aspect, the present invention also provides a recombinant vector for controlling the size of plant petals, the recombinant vector comprising an overexpression vector for overexpressing the GhPS1 gene and / or a gene silencing vector for interfering with the expression of the GhPS1 gene, the overexpression vector being obtained by connecting a recombinant gene fragment and a pCanG fragment, and the gene silencing vector being obtained by connecting an antisense gene fragment and a pTRV2 fragment.

[0007] In a third aspect, the present invention further provides an engineered bacterial strain expressing a recombinant vector, which is obtained by transforming Agrobacterium with the above-mentioned overexpression vector or gene silencing vector.

[0008] In a fourth aspect, the present invention provides a GhPS1 gene for controlling the size of plant petals, and the use of the above-mentioned recombinant vector or the above-mentioned engineered bacteria in the improvement of flower production.

[0009] The GhPS1 gene for controlling the size of plant petals provided by the present invention is selected from African chrysanthemum. A large amount of African chrysanthemum whole genome data was sequenced in the previous work, and the GhPS1 gene with the function of controlling the size of petals was found. The present invention constructs a recombinant vector (overexpression vector and gene silencing vector) and an engineering strain, and conducts stable transformation and transient transformation experiments of African chrysanthemum to overexpress or silence the GhPS1 gene, so that transgenic plants with smaller or larger petals than wild-type plants can be obtained, which verifies that the gene has the function of controlling the size of plant petals. In addition, an Arabidopsis transformation experiment is conducted, and transgenic plants with smaller or larger petals than wild-type plants are also obtained, indicating that the gene has the potential and application prospects for improving flower production on other plants. The gene of the present invention can provide a theoretical basis and gene source for breeding new flower varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0011] Figure 1 Figure a shows the petal size phenotype of the GhPS1 gene overexpressed in the petals of gerbera (GhPS1-OE) and the control group (mock). Figure 1 Middle b is the petal size phenotype of the GhPS1 gene silenced in the petals of Gerbera (pTRV2-GhPS1) and the control group (mock);

[0012] Figure 2 Figure a shows the expression of GhPS1 gene in the petals of gerbera overexpressed (GhPS1-OE) and the petals of the control group (mock). Figure 2 Middle b shows the expression of GhPS1 gene in the gene silencing (pTRV2-GhPS1) and control group (mock) in the petals of Gerbera;

[0013] Figure 3 Figure a is a comparison of the petal lengths of the petals of the gerbera flower when the GhPS1 gene was overexpressed (GhPS1-OE) and the petals of the control group (mock). Figure 3 Middle b is a comparison of the petal lengths in the gerbera petals after the GhPS1 gene was silenced (pTRV2-GhPS1) and the control group (mock);

[0014] Figure 4Figure a shows the cell size and morphology of the petals of the gerbera with GhPS1 gene overexpressed (GhPS1-OE) and the control group (mock). Figure 4 Middle b is a diagram showing the cell size and morphology of the petals of the gerbera chrysanthemum in which the GhPS1 gene was silenced (pTRV2-GhPS1) and the control group (mock).

[0015] Figure 5 The figure is a comparison of the cell elongation rate of the GhPS1 gene overexpression (GhPS1-OE) and gene silencing (pTRV2-GhPS1) in the petals of gerbera and the control group (mock);

[0016] Figure 6 The figure is a comparison of the cell area between the GhPS1 gene overexpression (GhPS1-OE) and gene silencing (pTRV2-GhPS1) in the petals of gerbera and the control group (mock);

[0017] Figure 7 Figure a is a comparison of flower organs of three GhPS1 transgenic Arabidopsis lines (#23-3, #30-2, and #14-14) and the wild type (Col). Figure 7 Middle b is a comparison of the petal size and morphology of three GhPS1 transgenic Arabidopsis lines (#23-3, #30-2, and #14-14) and the wild type (Col);

[0018] Figure 8 Comparison of cell size and morphology between three GhPS1 transgenic Arabidopsis lines (#23-3, #30-2, and #14-14) and the wild type (Col);

[0019] Fig. 9 Figure a is a comparison of the petal lengths of three GhPS1 transgenic Arabidopsis lines (#23-3, #30-2, and #14-14) and the wild type (Col). Fig. 9 Middle b is a comparison of the petal area between GhPS1 transgenic Arabidopsis and the wild type;

[0020] Fig.10 The expression of GhPS1 in the petals of three transgenic Arabidopsis lines (#23-3, #30-2 and #14-14) and the wild type (Col). DETAILED DESCRIPTION

[0021] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The present invention is specifically introduced below in conjunction with the specific embodiments.

[0022] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available, unless otherwise specified, and the techniques not described in detail are all performed according to standard methods well known to those skilled in the art. The cell lines, reagents, vectors, etc. mentioned in this application are all commercially available or available to the public through other channels. They are only used as examples and are not exclusive to the present invention. They can be replaced by other suitable tools or biological materials.

[0023] The embodiment of the present invention provides a GhPS1 gene for controlling the size of plant petals, and the nucleotide sequence thereof is shown in SEQ ID NO.1:

[0024] .

[0025] Specifically, the GhPS1 gene comes from Gerbera.

[0026] The embodiment of the present invention further provides a polypeptide for controlling the size of plant petals, the polypeptide is encoded by the GhPS1 gene, and its amino acid sequence is shown in SEQ ID NO.2:

[0027] MSTSEGAANGAIITTQRQQPPAAGVLTVKKPPAKDRHSKVDGRGRRIRMPIICAARVFQLTRELGHKSDGQTIEWLLRQAEPSIIAATGTGTTPASFSTVSVALRNANVNPSLSAALDQKSSAQSQHHQH LISPTPFILGKRLRSEEDGLDGGGKEDNNHMSSATVVSAAGAGGFWALPARPDFGQVWSFAAAPPEMVVSSPTSMASQQARFMHHQQPMGEASAARVGNYLPMTQGHLNLLASLSGHPPQSSGGKDDDTR.

[0028] An embodiment of the present invention also provides a recombinant vector for controlling the size of plant petals, wherein the recombinant vector includes an overexpression vector for overexpressing the GhPS1 gene and / or a gene silencing vector for interfering with the expression of the GhPS1 gene, wherein the overexpression vector is obtained by connecting a recombinant gene fragment and a pCanG fragment, and the gene silencing vector is obtained by connecting an antisense gene fragment and a pTRV2 fragment.

[0029] Understandably, the construction of a recombinant gene expression vector is the process of combining the target gene with the carrier, and it is also the core of genetic engineering. The construction of a recombinant expression vector enables the target gene to exist stably in the recipient cell and can be inherited to the next generation, while enabling the target gene to be expressed and function. The process of combining the target gene with the carrier is actually the process of recombining DNA from different sources. If a plasmid is used as a carrier, the plasmid must first be cut with a certain restriction enzyme to make a gap in the plasmid and expose the sticky end. Then the target gene is cut with the same restriction enzyme to produce the same sticky end (some restriction endonucleases can cut out flat ends and have the same effect). The cut target gene fragment is inserted into the incision of the plasmid, and the bases are first complementary and paired, and the two sticky ends are matched together, and hydrogen bonds are formed between the bases. Then an appropriate amount of DNA ligase is added to catalyze the formation of a phosphodiester bond between the two DNA chains, thereby connecting adjacent deoxyribonucleic acids to form a recombinant expression vector. The overexpression vector provided in the embodiment of the present invention is obtained by connecting the recombinant gene fragment GhPS1 and the linear vector pCanG fragment, and the gene silencing vector is obtained by connecting the antisense gene fragment and the pTRV2 fragment. The overexpression vector or the gene silencing vector can control the expression of GhPS1 in the receptor cells, thereby controlling the size of the petals.

[0030] It can be understood that the recombinant gene fragment contains the complete gene fragment of the GhPS1 gene, which can enhance the expression of the GhPS1 gene by infection and integration into the genome of flower plants, thereby making the petals of flower plants smaller; the antisense gene fragment only contains a part of the GhPS1 gene, and is an antisense fragment, which can interfere with the expression of the GhPS1 gene by infection and integration into the genome of flower plants containing the GhPS1 gene, thereby making the petals of flower plants larger.

[0031] Specifically, the 5' end of the recombination primer of the recombinant gene fragment introduces restriction sites XhoI and BamHI, and the recombination primer of the recombinant gene fragment includes:

[0032] Primer 1-F:5'-CTCGAGATGTCAACATCGGAGGG-3'; and,

[0033] Primer 1-R:5'-GGATCCTCAGCGAGTATCGTC-3';

[0034] The 5' end of the recombination primer of the antisense gene fragment introduces restriction sites EcoRI and BamHI, and the recombination primer of the antisense gene fragment includes:

[0035] Primer 2-F:5'-GAATTCGACTTCGGTCAGGTGTGGAG-3'; and,

[0036] Primer 2-R:5'-GGATCCTGCTGGTGGTGCATAAACCT-3'.

[0037] Understandably, the restriction enzyme cutting site is a specific sequence of bases on the DNA. The restriction endonuclease can recognize this sequence and cut the DNA sequence into two segments. Restriction endonucleases are a type of enzyme that can recognize and attach to a specific nucleotide sequence and cut the phosphodiester bond between two deoxyribonucleotides at a specific position in each chain. Restriction endonucleases recognize palindromic sequences in DNA sequences. Some enzymes have cutting sites on one side of the palindrome (such as EcoR I, BamH I, Hind, etc.), thus forming sticky ends. Other class II enzymes such as AluI, BsuR I, Bal I, HalⅢ, HPa I, Sma I, etc. have cutting sites in the middle of the palindrome sequence, forming blunt ends.

[0038] Understandably, the recombination primer includes an upstream primer (Forward primer, F) and a downstream primer (Reverse primer, R). Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It is a special DNA replication outside the body. The biggest feature of PCR is that it can greatly increase trace amounts of DNA.

[0039] Understandably, the 5' ends of the recombination primers of the recombinant gene fragment and the recombination primers of the antisense gene fragment are both introduced with restriction sites, which can be digested with the same restriction endonuclease as the vector, thus producing the same sticky ends, and the two are connected under the catalysis of an appropriate amount of DNA ligase to form a recombinant expression vector.

[0040] Specifically, after the recombinant gene fragment and the antisense gene fragment are respectively connected to the T vector, Escherichia coli is transformed to obtain a positive transformant plasmid containing the recombinant gene fragment and a positive transformant containing the antisense gene fragment, respectively; the positive transformant plasmid containing the recombinant gene fragment is double-enzyme-cut and connected to the pCanG fragment to obtain an overexpression vector; the positive transformant plasmid containing the antisense gene fragment is double-enzyme-cut and connected to the pTRV2 fragment to obtain a gene silencing vector.

[0041] Understandably, the T vector is a universal vector. The recombinant gene fragment or antisense gene fragment amplified using a recombinant primer can be directly connected to the T vector, and the T vector has been commercialized. After the recombinant gene fragment and the antisense gene fragment are respectively connected to the T vector, they can be sequenced using universal primers, which is more convenient for sequencing. If the T vector is not used, the recombinant gene fragment or the antisense gene fragment must be connected to the T vector using a special ligase (without a polyA tail), and special primers must be designed for sequencing, which is a more cumbersome process.

[0042] Understandably, transformation refers to the process of introducing a plasmid or a recombinant DNA constructed with a plasmid as a vector into bacteria. The transformed bacteria can acquire new genetic characteristics and phenotypes. After the bacteria are treated with some special methods (such as electric shock, CaCl2 and other chemical reagents), the permeability of the cell membrane will change, making it easier for foreign DNA molecules to enter the cell. The DNA molecules that enter the cell achieve the transfer of genetic information through replication and expression, so that the recipient cell has new genetic traits.

[0043] The embodiment of the present invention further provides an engineered bacterial strain expressing a recombinant vector, which is obtained by transforming Agrobacterium with the above-mentioned overexpression vector or gene silencing vector.

[0044] Understandably, Agrobacterium transformation is the most commonly used method for introducing target genes into plant cells. Agrobacterium tumefaciens and Agrobacterium rhizogenes contain Ti (Tumour inducing) plasmids and Ri plasmids, respectively, on which there is a section of T-DNA (Transferring DNA). After Agrobacterium enters the cell by infecting the plant wound, T-DNA can be inserted into the plant genome, and can be stably inherited to offspring through meiosis. This characteristic becomes the theoretical basis of plant transgenic by Agrobacterium-mediated method. The principle of Agrobacterium transformation is to construct a binary expression vector system, which is composed of two plasmids, one is a shuttle plasmid located in Escherichia coli, and the other is an auxiliary plasmid located in Agrobacterium. After the recombinant plasmid is electrotransformed into Agrobacterium competent cells, the Agrobacterium containing the recombinant plasmid is used to infect the plant root incision, and the trans-action of the expression protein in the Vir region of the auxiliary plasmid and the recombinant plasmid T-DNA region (target fragment) activates the transfer of T-DNA, thereby integrating the target fragment into the plant cell genome. With the formation of callus, the new cells are made to contain the gene of the target fragment.

[0045] It can be understood that the engineered bacteria carrying the overexpression vector can increase the expression level of the GhPS1 gene in Gerbera or other plants by infecting the Gerbera containing the GhPS1 gene or other plants (such as Arabidopsis thaliana) that do not contain the GhPS1 gene. The engineered bacteria carrying the gene silencing vector can reduce the expression level of the GhPS1 gene in Gerbera or other plants by infecting the Gerbera containing the GhPS1 gene or other plants containing the GhPS1 gene (if other plants containing the GhPS1 gene are found).

[0046] Specifically, the engineered bacteria are positive for kanamycin and streptomycin or rifampicin resistance.

[0047] Understandably, the purpose of adding kanamycin (Kan) and streptomycin (Str) or rifampicin (Rif) to the culture medium is to prevent the growth of foreign bacteria and screen Agrobacterium. The plasmid transformation rate of Agrobacterium with positive kanamycin (Kan) and streptomycin (Str) or rifampicin (Rif) is high. The plasmid contains kanamycin resistance gene as a screening label. Too high kanamycin concentration is not conducive to the growth of Agrobacterium and will reduce its growth rate and transformation efficiency. In one embodiment of the present invention, Agrobacterium tumefaciens C58C1 strain is used. When transforming African chrysanthemum, the C58C1 strain contains the Str gene carried by itself and the Kan resistance screening label carried in the recombinant plasmid to give the C58C1 strain Kan and Str resistance, which is suitable for the transgenic operation of African chrysanthemum; when transforming other plants (such as Arabidopsis), the Kan resistance gene contained in the C58C1 strain and the screening label of the rifampicin Rif gene give the C58C1 strain Kan and Rif resistance, which is suitable for the transgenic operation of other plants (such as Arabidopsis).

[0048] Specifically, the overexpression vector or gene silencing vector is transformed into Agrobacterium tumefaciens using the heat shock method to obtain the engineered strain.

[0049] Understandably, introducing a plasmid containing a target gene into bacteria is a basic experimental operation in molecular biology. Conventional methods include heat shock and electroporation. Competence refers to a physiological state in which a recipient cell is most susceptible to exogenous DNA. Competence is generally determined by the genetic shape of the recipient bacteria. However, to a large extent, competence is also affected by the external environment. The permeability of the cell membrane of cells in the competent state is greatly increased, allowing exogenous DNA to enter the cell through the gap on the membrane surface. The heat shock method is to use heat shock treatment to treat cells in a competent state, and the plasmid containing the target gene enters the cell from the gap on the membrane surface of the competent cell to achieve the purpose of obtaining new genetic characteristics and phenotypes.

[0050] The embodiments of the present invention also provide a GhPS1 gene for controlling the size of plant petals, and the use of the above-mentioned recombinant vector or the above-mentioned engineered bacteria in improving flower production.

[0051] Specifically, the GhPS1 gene, the recombinant vector or the engineered bacteria improves flower production by controlling the size of petals, and the flower is gerbera or Arabidopsis.

[0052] Specifically, the controlling of petal size is achieved by controlling cell expansion.

[0053] The present invention is further described below by way of examples.

[0054] Example 1 Cloning and transformation of GhPS1 gene

[0055] Amplification of recombinant gene fragments

[0056] Using the 'Shenzhen No. 5' African chrysanthemum ray flowers as the material, RNA was extracted using Trizol (Invitrogen), and the obtained RNA was further synthesized into the first chain of cDNA using a reverse transcription kit from TakaRa Company, with a reaction system of 20 μL.

[0057] The following two pairs of gene-specific primers (Primer 1-F / R and Primer 2-F / R) were designed and synthesized. To facilitate subsequent cloning and vector construction, restriction endonucleases XhoI, BamHI, EcoRI, and BamHI recognition sites (underlined parts) were introduced at the 5' end of the primers:

[0058] Primer 1-F:5'- CTCGAG ATGTCAACATCGGAGGG-3'(XhoI)

[0059] Primer 1-R:5'- GGATCC TCAGCGAGTATCGTC-3'(BamHI)

[0060] Primer 2-F:5'- GAATTC GACTTCGGTCAGGTGTGGAG-3'(EcoRI)

[0061] Primer 2-R:5'- GGATCC TGCTGGTGGTGCATAAACCT-3'(BamHI)

[0062] The components and contents of the PCR system are shown in Table 1.

[0063] Table 1 Components and contents of PCR reaction system

[0064] Element content Template DNA 500ng 5×Primer STAR Buffer 5μL dNTP Mixture (2.5 mM) 2μL Primer 1-F or Primer 2-F 0.5μL Primer 1-R or Primer 2-R 0.5μL HS DNA Polymerase 0.25μL <![CDATA[ddH2O]]> Make up to 25 μL

[0065] PCR reaction procedure: pre-denaturation at 94°C for 3 min, then 94°C for 30 sec, 60°C for 30 sec, 72°C for 45 sec, 30 cycles, and finally 72°C for 5 min.

[0066] After the PCR reaction was completed, 25 μL of the amplified product was taken for 1.2% agarose gel electrophoresis, the target band was cut out under ultraviolet light, and the DNA purification kit was used for recovery to obtain target fragment 1 (the primers used in amplification were Primer1-F / R) and target fragment 2 (the primers used in amplification were Primer 2-F / R), respectively.

[0067] Transformed E. coli, PCR identification and sequencing of bacterial solution

[0068] After the recovered target fragment 1 and target fragment 2 were connected to the T vector (pMD18-T, TakaRa Company), they were transformed into Escherichia coli DH5α competent cells, and cultured on a solid LB plate medium containing 100 mg / L ampicillin (Amp) at 37°C for 14 hours to culture a single colony. Pick a single colony and add it to 1mL of liquid LB medium containing Amp. After 37°C and 220rpm shaking culture for 5 hours, use this bacterial liquid as a template for PCR reaction. After the reaction, the PCR product was electrophoresed on a 1% agarose gel, photographed and analyzed using a gel imaging system to obtain positive transformants.

[0069] The positive transformants were sent to Shanghai Bioengineering Company for sequencing, and the sequencing results were compared with the target sequence to obtain positive clone transformants with completely correct target sequence.

[0070] Example 2 Construction of vector and transformation of Agrobacterium

[0071] Construction of overexpression vectors and gene silencing vectors

[0072] 5 μL of the positive bacterial solution containing positive clone transformants was added to 5 mL of liquid LB medium containing Amp, and cultured at 37°C and 220 rpm for 12 hours to expand the culture. According to the instructions for use in the plasmid DNA micro-extraction kit produced by BIOMIGA Biotechnology Co., Ltd., the positive transformant plasmid was extracted by alkaline lysis method, and double enzyme digestion was performed with XhoI and BamHI. The bacterial solution containing the expression vector was also expanded and cultured to extract the plasmid and double enzyme digestion to obtain the pCanG fragment and the pTRV2 fragment. The enzyme digestion products were separated by gel electrophoresis, and the target fragments, pCanG fragments and pTRV2 fragments were cut out and recovered by the kit respectively. The target fragment 1 was connected to the pCanG fragment, and the target fragment 2 was connected to the pTRV2 fragment, and the expression vector pCanG-GhPS1 and the gene silencing vector pTRV2-GhPS1 were constructed at 4°C overnight.

[0073] Transformation of Agrobacterium

[0074] The constructed overexpression vector pCanG-GhPS1 and gene silencing vector pTRV2-GhPS1 were transformed into Agrobacterium tumefaciens C58C1 by heat shock method, and Agrobacterium carrying overexpression vector pCanG-GhPS1 and Agrobacterium carrying gene silencing vector pTRV2-GhPS1 were obtained respectively. The positive bacteria obtained by transformation were expanded and cultured, and PCR and restriction enzyme digestion were performed for identification. The correct strains were packaged and stored at -80℃ for later use.

[0075] Example 3 Stable transformation of the GhPS1 gene into Gerbera

[0076] After cutting a single bud from the sterile tissue culture seedling of Gerbera chrysanthemum, cut the single bud longitudinally into two petioles as explants, lay them flat on the pre-culture medium, and culture them under light for 5-6 days.

[0077] Take out 5 μL of Agrobacterium tumefaciens carrying the overexpression vector pCanG-GhPS1 from a -80°C low-temperature refrigerator and inoculate it into 5mL YEB liquid medium supplemented with 50mg / L kanamycin (Kan) and 100mg / L streptomycin (Str). After shaking and culturing at 28°C and 220rpm for 24h, take out 1mL of the bacterial solution and add it into 40mL YEB liquid medium. Shake and culture at 28°C and 220rpm for 12h until the bacterial solution concentration approaches the logarithmic growth phase. Pour the bacterial solution into a 10mL eppendorf tube and centrifuge (5000rpm, 5min), remove the supernatant, and retain the precipitate. Suspend the precipitate with plant liquid induction medium and dilute to OD600 of 0.4-0.5, and then add 20mg / L acetosyringone. Place the bacterial solution in an incubator at 28°C and 220rpm for shaking and culture for 0.5-2h, and then it can be used to transform plant explants.

[0078] The explants were taken out under aseptic conditions, placed in sterilized culture bottles, and poured into the activated Agrobacterium solution for infection for 20 minutes. The explants were taken out and the excess solution was quickly absorbed with filter paper, and the single buds or petioles with leaves removed were placed flat on the solid co-culture medium and cultured at 25±1℃ in the dark for about 3 days. The dark culture was terminated when the Agrobacterium colonies visible to the naked eye grew on the plate, and the explants were taken out in the clean bench, washed 5 times with sterile water containing antibacterial antibiotics (400mg / L carb) and then dried on filter paper.

[0079] The explants treated as above were transferred to a selection medium (MS+BA 2mg / L+NAA 0.2mg / L, pH 5.8, agar 0.8%, sucrose 3%) supplemented with 400mg / L Carb and 25mg / L Kan for screening, and the screening culture conditions were 25±1°C. One month later, green resistant buds were picked out and transferred to a proliferation medium containing 400mg / LCarb and 25mg / L Kan for culture, and after molecular identification, they were used for subsequent phenotypic analysis and functional verification.

[0080] Example 4 GhPS1 gene transient transformation of gerbera petals

[0081] Referring to the method established by our research group (Tang, Y., Li, LF & Wang, XJ Establishment of transient gene expression and virus-induced gene silencing (VIGS) system in Gerbera hybrida petals. Plant Physiol. J. 2017, 53: 505 512), more than 10 Gerbera chrysanthemum inflorescences with basically the same growth and development to stage 3 were selected, and the outer ligulate flower petals were peeled off and used as explants.

[0082] The overnight cultured Agrobacterium culture carrying the overexpression vector pCanG-GhPS1 and the Agrobacterium culture carrying the gene silencing vector pTRV2-GhPS1 were centrifuged at 4000×g for 10 min at 4°C, and then the Agrobacterium was suspended in infection solution (10 mmol / L 2-morpholineethanesulfonic acid + 40 mg / L acetosyringone + 10 mmol / L magnesium chloride, pH 5.6) to a concentration of OD600 = 0.8-1.0.

[0083] Fresh gerbera petals were randomly divided into several equal parts (20 or more per part as a treatment), wrapped with gauze and immersed in mixed infection solution of Agrobacterium, with Agrobacterium infection of empty vector as control (mock), and each treatment was placed under negative pressure of -0.10~-0.09MPa for 5~6min, and then slowly restored to normal atmospheric pressure (the process was about 2min); each group of materials was taken out and gently rinsed with tap water, then rinsed with distilled water 3 times, and then placed in sterile culture dishes (9cm in diameter) with sterile filter paper and 5mL of sterile double distilled water, and cultured in the dark at 8℃ for 3d. 0d was counted from the time of taking out, and the petals were used for petal phenotype analysis after 9d of culture at 25℃.

[0084] Example 5 GhPS1 gene transformation of Arabidopsis thaliana

[0085] Agrobacterium carrying the overexpression vector pCanG-GhPS1 was used to transform Arabidopsis thaliana (Columbiaecotype) by the floral dipping method. The operation is as follows: 100 μL of bacterial solution was taken from the activated Agrobacterium and added to 5 mL of YEB liquid medium containing rifampicin (Rif) and Kan, and cultured at 28°C, 200 rpm, and shaken until the final OD600 of the bacterial solution was 0.7-0.8; at room temperature, 10,000 rpm, 1 min, the bacterial cells were collected in a 1.5 mL centrifuge tube, and resuspended in an infiltration buffer (1 / 2MS macro, 5% sucrose, 0.02%-0.05% Silwet L-77), and the Agrobacterium bacterial solution was sucked with a pipette and dropped on the white flower buds, and the transformed plants were infected twice within a week, and the first batch of fruit pods after about two or three weeks can be harvested for screening.

[0086] After the infected T0 generation seeds were harvested, they were sown on MS medium plates containing 50 mg / L Kan (Kan resistance plates). The plates were wrapped with double layers of newspaper and placed in a 4°C refrigerator for vernalization for 2 days and then cultured under light for 8 days. Green seedlings (plants that failed to be successfully transformed could not grow normally on Kan resistance plates, and the cotyledons were white) were selected and transplanted into soil for culture. After they matured, T1 generation seeds were harvested from individual plants, and resistance screening was continued until homozygous T3 generation seeds were obtained for phenotypic analysis. Arabidopsis thaliana that was not transformed as described above was used as the control group (col).

[0087] Observation of transgenic plant phenotype and cell morphology

[0088] The petal measurement was performed using Image J software (http: / / rsb.info.nih.gov / ij / , NIH, MD, USA). Petal measurement: After the petals of the transgenic plants and the wild-type control were picked, no less than 20 petals were randomly selected from each inflorescence, fixed on white paper with double-sided tape, and placed on a scanner (EPSON-G850A) together with a ruler for scanning. The scanned images were measured using Image J software. The scanned images of Examples 3 and 4 are shown in Figure 1 The petal length measurement results of Examples 3 and 4 are shown in Figure 3 The scan results of the flower organs and petals of Example 5 are shown in Figure 7 .

[0089] The cell morphology and size were observed by laser confocal scanning microscopy. The middle of the upper, middle and lower parts of the petals of the ray-shaped flowers of Gerbera or the petals of Arabidopsis were cut into pieces about 1 mm 2The area of ​​​​the size of the sample was quickly placed in 0.1 mg / mL PI and stained for 5 minutes at room temperature, and then washed with distilled water 3 times to completely remove the residual PI. The stained sample was placed on a slide and the epidermal cells on it were observed using a laser confocal scanning microscope. Finally, the cell size was measured using Image J software. The cell morphology measurement results of Examples 3 and 4 are shown in Figure 4 The cell elongation measurement results of Examples 3 and 4 are shown in Figure 5 The cell area measurement results of Examples 3 and 4 are shown in Figure 6 The cell morphology measurement results of Example 5 are shown in Figure 8 The petal length and area size results of Example 5 are shown in Fig. 9 The GhPS1 transgenic expression of Example 5 is shown in Fig.10 .

[0090] qRT-PCR (real-time fluorescence quantitative PCR, Quantitative Real-time PCR) was used to measure the expression level of GhPS1 gene in Examples 3 and 4. The results are shown in Figure 2 .

[0091] Depend on Figure 2 As shown in Figure a, the expression level of the GhPS1 gene in the overexpression group (GhPS1-OE) was significantly higher than that in the control group (mock) (P < 0.01). Figure 2 As can be seen from Figure b, the expression level of the GhPS1 gene in African chrysanthemum in the gene silencing group (pTRV2-GhPS1) was significantly lower than that in the control group (P < 0.01), indicating that the expression level of the GhPS1 gene in African chrysanthemum transformed by Agrobacterium carrying the overexpression vector pCanG-GhPS1 increased, while the expression level of the GhPS1 gene in African chrysanthemum transformed by Agrobacterium carrying the gene silencing vector pTRV2-GhPS1 decreased.

[0092] Depend on Figure 1 A or Figure 3 As shown in a, the petals of the overexpression group (GhPS1-OE) are significantly smaller than those of the control group (mock), and the length of the petals is significantly shorter than that of the control group (mock) (P < 0.01). Figure 1 Medium b or Figure 3 As shown in Figure b, the petals of the gene silencing group (pTRV2-GhPS1) were larger than those of the control group (mock), and the length of the petals was greater than that of the control group (mock) (P < 0.01), indicating that increased expression of the GhPS1 gene can make the petals of gerbera smaller, and vice versa.

[0093] Depend on Figure 4As shown in a, the cells in the upper, middle and lower parts of the petals of the overexpression group (GhPS1-OE) are smaller than those in the control group (mock). Figure 4 As shown in Figure b, the cells in the upper, middle and lower parts of the petals of the gene silencing group (pTRV2-GhPS1) are larger than those in the control group (mock). Figure 5 It can be seen that the cell elongation rate of the three different parts of the petals of the gene silencing group (pTRV2-GhPS1) is greater than that of the control group (mock) and greater than that of the overexpression group (GhPS1-OE). Figure 6 It can be seen that the cell areas in the upper, middle and lower parts of the petals of the gene silencing group (pTRV2-GhPS1) are greater than the cell areas in the upper, middle and lower parts of the petals of the control group (mock) and greater than the cell areas in the upper, middle and lower parts of the petals of the overexpression group (GhPS1-OE), indicating that the GhPS1 gene makes the petals of gerbera smaller by controlling cell expansion.

[0094] Depend on Fig.10 It can be seen that the GhPS1 gene is expressed in three GhPS1 transgenic Arabidopsis lines (#23-3, #30-2 and #14-14), but not in the wild type (Col), indicating that the GhPS1 gene was successfully transferred into the Arabidopsis genome and can be expressed in Arabidopsis. Figure 7 As shown in a and b, the floral organs and petals of the three GhPS1 transgenic Arabidopsis lines (#23-3, #30-2, and #14-14) are smaller than those of the wild type (Col). Figure 8 It can be seen that the cells of the three GhPS1 transgenic Arabidopsis lines (#23-3, #30-2, and #14-14) are smaller than those of the wild type (Col). Fig. 9 As shown in a and b, the length and area of ​​the petals of the three GhPS1 transgenic Arabidopsis lines (#23-3, #30-2 and #14-14) are greater than those of the wild type, indicating that the GhPS1 gene is expressed in Arabidopsis, controlling cell expansion and making petal cells smaller, thereby reducing the length and area of ​​the petals. This suggests that this gene has the potential and application prospects for improving flower production in other plants.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A GhPS1 gene for controlling the size of plant petals, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A polypeptide for controlling the size of plant petals, characterized in that: The polypeptide is encoded by the GhPS1 gene, and its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector for controlling the size of plant petals, characterized in that: The recombinant vector is an overexpression vector for overexpressing the GhPS1 gene, and the overexpression vector is obtained by connecting the recombinant gene fragment and the pCanG fragment.

4. The recombinant vector according to claim 3, characterized in that The 5' end of the recombination primer of the recombinant gene fragment introduces restriction sites XhoI and BamHI, and the recombination primer of the recombinant gene fragment includes: Primer 1-F:5'-CTCGAGATGTCAACATCGGAGGG-3'; and, Primer 1-R:5'-GGATCCTCAGCGAGTATCGTC-3'.

5. An engineered bacterial strain expressing a recombinant vector, characterized in that: The engineered strain is obtained by transforming Agrobacterium using the overexpression vector described in claim 3.

6. A recombinant vector for controlling the size of plant petals, characterized in that: The recombinant vector is a gene silencing vector that interferes with the expression of the GhPS1 gene. The gene silencing vector is obtained by connecting the antisense gene fragment and the pTRV2 fragment.

7. The recombinant vector according to claim 6, characterized in that The 5' end of the recombination primer of the antisense gene fragment introduces restriction sites EcoRI and BamHI, and the recombination primer of the antisense gene fragment includes: Primer 2-F:5'-GAATTCGACTTCGGTCAGGTGTGGAG-3'; and, Primer 2-R:5'-GGATCCTGCTGGTGGTGCATAAACCT-3'.

8. An engineered bacterial strain expressing a recombinant vector, characterized in that: The engineered strain is obtained by transforming Agrobacterium using the gene silencing vector described in claim 6.

9. Use of the GhPS1 gene for controlling the size of plant petals according to claim 1, the recombinant vector according to claim 3, the engineered strain according to claim 5, the recombinant vector according to claim 6 or the engineered strain according to claim 8 in the production and improvement of gerbera or Arabidopsis thaliana, wherein the GhPS1 gene, the recombinant vector according to claim 3, the engineered strain according to claim 5, the recombinant vector according to claim 6 or the engineered strain according to claim 8 improves the production of gerbera or Arabidopsis thaliana by controlling the size of petals.

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