CN114410646A - Gene PeARF18 for regulating and controlling development of phalaenopsis flower organ and application thereof - Google Patents

Abstract

The invention provides a gene PeARF18 for regulating and controlling the development of a butterfly orchid organ and application thereof. The invention provides a gene PeARF18 for regulating and controlling the development of a butterfly orchid organ, which has one of the following nucleotide sequences: 1) a nucleotide sequence shown as SEQ ID NO. 1; 2) a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion or addition of one or more nucleotides; 3) a nucleotide sequence having at least 80% homology with SEQ ID No. 1. The method effectively regulates and controls the development of the floral organs of the phalaenopsis by means of genetic engineering, so that the artificial lip of the phalaenopsis tricuspidata becomes a true lip, thereby facilitating pollination of the plant by insects and simultaneously providing an important basis for flower type breeding of the phalaenopsis.

Description

Gene PeARF18 for regulating and controlling development of phalaenopsis flower organ and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a gene PeaRF18 for regulating and controlling the development of a butterfly orchid organ and application thereof.

Background

Along with the improvement of living standard of people, flower plants are more and more popular with people, and people have higher requirements on flowers of different shapes. In order to provide exotic flowers and valuable flowers with more ornamental and collection values, people increasingly pay attention to research on the development mechanism of the flowers. Currently, more than 20000 orchids are one of the most varied families in the plant kingdom. The orchid flower type comprises three calyces of an outer wheel, two petals of the inner wheel and lip petals which are specially formed by the petals in various forms, and the orchid has a synanthus column formed by fusing stamens and pistils.

Disclosure of Invention

The invention provides a gene PeARF18 for regulating the development of a flower organ of phalaenopsis, which is used for providing a gene for regulating the development of the flower organ of phalaenopsis and has unique ornamental and application values.

In a first aspect, the invention provides a gene PeaRF18 for regulating and controlling the development of a flower organ of phalaenopsis, wherein the gene has one of the following nucleotide sequences:

1) a nucleotide sequence shown as SEQ ID NO. 1;

2) a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion or addition of one or more nucleotides;

3) a nucleotide sequence having at least 80% homology with SEQ ID No. 1.

In a second aspect, the invention provides a protein for regulating and controlling the development of a flower organ of phalaenopsis, wherein the protein is encoded by the gene provided by the first aspect of the invention.

In a third aspect, the present invention provides a recombinant vector comprising the nucleotide sequence provided in the first aspect of the present invention.

Further, the recombinant vector is CymMV virus.

In a fourth aspect, the present invention provides a recombinant transformant comprising the recombinant vector provided in the third aspect of the present invention.

Further, the recombinant transformant is agrobacterium.

In a fifth aspect, the invention provides the use of the gene provided in the first aspect of the invention in regulating and controlling the development of a flower organ of phalaenopsis.

In a sixth aspect, the present invention provides a method of modulating the development of a flower organ of phalaenopsis for silencing a nucleotide sequence as provided in the first aspect of the invention.

Further, the nucleotide sequence is connected to CymMV virus plasmid to obtain a recombinant vector, then the recombinant vector is transferred into agrobacterium to obtain a recombinant transformant, and the recombinant transformant is used for infecting phalaenopsis.

Further, the recombinant transformants were cultured in LB medium containing 100. mu.M acetosyringone and 50. mu.g/ml kanamycin;

after the culture is finished, subculture is carried out in LB culture medium containing 100 mu M acetosyringone and 50 mu g/ml kanamycin until OD600 reaches 0.8-1.0; and centrifuging to remove supernatant, resuspending the cell precipitate to obtain a transformation solution, and injecting the transformation solution into virus-free phalaenopsis leaves and inflorescences.

The method effectively regulates and controls the development of the floral organs of the phalaenopsis by means of genetic engineering, so that the artificial lip of the phalaenopsis tricuspidata becomes a true lip, thereby facilitating pollination of the plant by insects and simultaneously providing an important basis for flower type breeding of the phalaenopsis.

Drawings

FIG. 1 is a phenotype of a wild-type Phalaenopsis miniata provided by the prior art;

FIG. 2 is a conservative domain prediction diagram of PeaRF18 provided by an embodiment of the present invention;

FIG. 3 is a diagram of a phylogenetic analysis of the development of PeaRF18 according to an embodiment of the present invention;

FIG. 4 is a validation of the transcriptional self-activation function of PeaRF18 provided by an embodiment of the present invention;

FIG. 5 is a subcellular localization of PeaRF18 in Nicotiana benthamiana provided by an embodiment of the present invention;

FIG. 6 shows the expression level of Pearf18 at different developmental stages of the flower bud of Yulan;

FIG. 7 shows the expression level of PeaRF18 in different tissues of Phalaenopsis plantula according to the present invention;

FIG. 8 is a control and silent mutant strain phenotype of Phalaenopsis miniata provided by an embodiment of the present invention after 30 days of CymMV-PeaRF18 injection;

FIG. 9 shows the expression level of PearF18 in different sites of control and silent mutant strains of Phalaenopsis planterum after 30 days of CymMV-PearF18 injection.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The reagents and carriers used are commercially available or publicly available unless otherwise specified.

EXAMPLE 1 cloning of the PeaRF18 Gene

The wild type phalaenopsis amabilis petal total RNA shown in FIG. 1 was extracted, the extraction kit was RNAPlant (commercially available), and the total RNA was reverse-transcribed into cDNA using a reverse transcription kit (commercially available). Designing a primer according to a transcriptome sequencing result, wherein the primer sequence is shown as SEQ ID NO.3 and SEQ ID NO.4, and amplifying a 2514bp band from the Phalaenopsis miniata cDNA by adopting an RT-PCR method. Recovering PCR product to obtain gene with nucleotide sequence shown in SEQ ID No.1 and named as PeaRF 18; the amino acid sequence coded by the nucleotide sequence is shown as SEQ ID NO.2, consists of 837 amino acid residues, and has the molecular weight of 82.76 kDa.

Example 2 bioinformatic analysis of PeaRF18

1. The amino acid sequence encoded by PeARF18 was placed in the NCBI database and homologous proteins were retrieved.

2. The conserved domain between the protein encoded by the PeaRF18 and the homologous amino acid sequence was analyzed online using https:// www.genome.jp/tools-bin/clustalw, and the results are shown in FIG. 2, where the PeaRF18 has a typical N-terminal DNA Binding Domain (DBD), middle domain (MR), and C-terminal dimerization domain (CTD).

3. An amino acid sequence of an ARF gene family in Arabidopsis is selected, a MEGA5 is used for analyzing a PeARF18 phylogenetic relationship, the analysis result is shown in figure 3, and the PeARF18 and the ARF2 in Arabidopsis are clustered together and have a close relationship.

Example 3 verification of the transcriptional self-activation function of PeaRF18

1. Operably connecting the 2514bp open reading frame of the PeaRF18 gene to a pGBKT7 yeast expression vector to form a pGBKT7-PeaRF18 vector containing the gene fragment;

2. respectively transferring the pGBKT7-PeaRF18 vector, the negative control group pGBKT7 no-load vector and the positive control group Postive vector in the step 1 into yeast sensitive Y2H;

3. culturing the three yeast strains in the step 2 on a Trp selection-deficient solid medium;

4. streaking and activating the three yeast strains grown in the step 3 in a new Trp selection-deficient solid culture medium, and then shaking the strains in a Trp selection-deficient liquid culture medium;

5. and (4) respectively culturing the yeast liquid cultured in the step (4) on two Trp selection-deficient solid culture media and a Trp/His/Ade selection-deficient solid culture medium, and observing the growth state of the yeast liquid.

As shown in FIG. 4, the negative control pGBKT7 no-loaded did not grow in Trp-His-Ade selection-deficient solid medium, the positive control Positive vector had grown, and the pGBKT7-PeARF18 vector with the PeARF18 gene had not grown, indicating that PeARF18 is a non-transcriptional activator.

Example 4 subcellular localization of PeaRF18 in Nicotiana benthamiana

1. Operably connecting 2514bp open reading frame of the PeARF18 gene to a PEG104-GFP expression vector to form a PEG104-PeARF18 vector containing the gene fragment;

2. transferring the vector into agrobacterium GV 3101;

3. taking the Agrobacterium GV3101 in step 2, culturing overnight at 28 ℃ and 200rpm in 5ml LB medium containing 100. mu.M acetosyringone, 50. mu.g/ml kanamycin and 10. mu.g/ml rifampicin;

4. centrifuging the Agrobacterium liquid obtained in step 3 at 4 deg.C and 3700rpm for 10 min, discarding the supernatant and using 0.5M MgCl₂Repeatedly blowing, beating and resuspending for three times to obtain the suspension liquid of the heavy suspension bacteria of the agricultural stalks;

5. measuring OD value of the suspension liquid of the heavy suspension bacteria of the agricultural straws, and using 0.5M MgCl₂Diluting to make OD value be about 0.6;

6. adding 100 mu M AS (acetosyringone) with the total volume of 1.5 times and MES (morpholine ethanesulfonic acid) with the total volume of 20 times into the bacterial liquid obtained in the step 5, suspending the cell precipitate, and standing for 3-4h at room temperature;

7. the Agrobacterium transformation fluid of step 6 was aspirated using a 1ml syringe with a needle, injected into four-week-old grown Nicotiana benthamiana leaves, cultured for 2-3 days and the position of PEG104-PeaRF18 at the cell level was observed.

As shown in FIG. 5, fluorescence was clearly observed in the microstructure of the leaf cells of Nicotiana benthamiana, indicating that the gene of PeaRF18 was localized in the nuclear structure based on the position of the fluorescence.

Example 5 expression profiling of Phalaenopsis plantulata at different developmental stages

1. Extracting RNA of Phalaenopsis miniata at different developmental stages shown in a in FIG. 6, respectively named as B1-B5, and reverse transcribing total RNA into cDNA using a reverse transcription kit (commercially available) with the extraction kit being RNAplant (commercially available);

2. designing primers according to the sequencing data of the transcriptome, wherein the sequences of the primers are shown as SEQ ID NO.5 and SEQ ID NO. 6;

3. and (3) carrying out expression profile verification on the PeaRF18 at different development stages by taking cDNA obtained by reverse transcription of the phalaenopsis miniata at different development stages as a template.

The verification result is shown in b in fig. 6, the expression of PeARF18 is significantly induced at different developmental stages, which indicates that PeARF18 may be involved in the development of the floral organ of phalaenopsis miniata.

Example 6 validation of expression profiles of PeaRF18 in Phalaenopsis miniata of different tissues

Extracting the RNA of the phalaenopsis miniata at different tissue parts as shown in a in figure 7, wherein the extracted parts are roots, stems, leaves, sepals, petals, lipflaps, synanthus and flowering branches respectively, the extraction kit is RNAplant (sold in markets), and the total RNA is reversely transcribed into cDNA by utilizing a reverse transcription kit (sold in markets);

2. designing primers according to the sequencing data of the transcriptome, wherein the sequences of the primers are shown as SEQ ID NO.5 and SEQ ID NO. 6;

3. the cDNA obtained by reverse transcription of the phalaenopsis miniata at different tissue parts is used as a template, expression profiles of different tissue parts are verified for the PeaRF18, and the verification result is shown as b in FIG. 7. the expression levels of the PeaRF18 are different in different tissues, wherein the expression levels are lower in petals and lip flaps, which indicates that the PeaRF18 is possibly involved in the development of the organ of the phalaenopsis miniata.

Example 7CymMV Virus induces Gene silencing in Phalaenopsis miniata PeaRF18

1. Operably connecting the open reading frame 200-300bp of the PeARF18 gene to a CymMV viral vector to form a CymMV-PeARF18 vector containing the gene segment;

2. transferring the vector into agrobacterium GV 3101;

3. taking the Agrobacterium GV3101 in step 2, culturing in 5ml LB medium containing 100 μ M acetosyringone and 50 μ g/ml kanamycin at 28 deg.C and 200rpm for 16 h;

4. subjecting the Agrobacterium of step 3 to subculture in 50ml LB medium containing 100. mu.M acetosyringone and 50. mu.g/ml kanamycin, and incubating at 28 deg.C and 200rpm for 13-16h until OD600 reaches 0.8-1.0;

5. taking the agrobacterium liquid obtained in the step 4, transferring the agrobacterium liquid into a 50ml centrifugal bottle, and centrifuging the agrobacterium liquid at 4 ℃ and 3000rpm for 10 minutes;

6. after centrifugation, the supernatant was removed, 300. mu.l of MS medium containing 100. mu.M acetosyringone was added to resuspend the cell pellet, and the mixture was allowed to stand at room temperature for 0.5 h;

7. and (3) sucking the agrobacterium transformation liquid obtained in the step (6) by using a 1ml syringe with a needle, injecting the agrobacterium transformation liquid into virus-free phalaenopsis miniata leaves and inflorescences, and culturing for 30-40 days to observe the petals of the phalaenopsis miniata.

As shown in fig. 8, the artificial lip valve of the floral organ of the butterfly orchid silent mutant line of small orchid was changed into a true lip valve (position indicated by arrow in the figure) compared with the control group MOCK.

Example 8 verification of expression of the Pearf18 gene in sepals, lip flaps and synanthus in a cymbidium minitans mutant strain induced by CymMV virus

1. Selecting petals, sepals, lipflaps and synanthus of control and silent mutant strains shown in figure 8, extracting total RNA of the control and silent mutant strains, wherein an extraction kit is RNAPlant (sold in the market), and reversely transcribing the total RNA into cDNA by utilizing a reverse transcription kit (sold in the market);

2. primers are designed according to transcriptome sequencing data, and the sequences of the primers are shown as SEQ ID NO.5 and SEQ ID NO. 6.

3. cDNA obtained by reverse transcription of control and silent mutant strain petals is used as a template, and gene silencing efficiency verification is carried out on the gene of the PeaRF18 in petals, sepals, lipals and synanthus columns.

The verification result is shown in fig. 9, in the phalaenopsis miniata mutant strain, the expression of PeARF18 in petals, sepals, lip flaps and synanthus columns is obviously reduced, the higher silencing efficiency of PeARF18 is proved, and the participation of PeARF18 in the development of the organ of the phalaenopsis miniata is further demonstrated.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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Claims (10)

1. A gene PeARF18 for regulating the development of a flower organ of phalaenopsis, wherein the gene has one of the following nucleotide sequences:

1) a nucleotide sequence shown as SEQ ID NO. 1;

2) a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion or addition of one or more nucleotides;

3) a nucleotide sequence having at least 80% homology with SEQ ID No. 1.

2. A protein for regulating the development of a flower organ of phalaenopsis, wherein the protein is encoded by the gene of claim 1.

3. A recombinant vector comprising the nucleotide sequence of claim 1.

4. The recombinant vector according to claim 3, wherein the recombinant vector is a CymMV virus.

5. A recombinant transformant, characterized in that it comprises the recombinant vector of claim 3 or 4.

6. The recombinant transformant according to claim 5, wherein the recombinant transformant is Agrobacterium.

7. Use of the gene of claim 1 for regulating the development of a flower organ of phalaenopsis.

8. A method of modulating organ development of a butterfly orchid, comprising silencing said butterfly orchid with a nucleotide sequence according to claim 1.

9. The method according to claim 8, wherein the nucleotide sequence is ligated to a CymMV viral plasmid to obtain a recombinant vector, and then the recombinant vector is transferred to Agrobacterium to obtain a recombinant transformant, and the recombinant transformant is used to infect Phalaenopsis.

10. The method according to claim 9, wherein the recombinant transformant is cultured in an LB medium containing 100. mu.M acetosyringone and 50. mu.g/ml kanamycin;

after the culture is finished, subculture is carried out in LB culture medium containing 100 mu M acetosyringone and 50 mu g/ml kanamycin until OD600 reaches 0.8-1.0; and centrifuging to remove supernatant, resuspending the cell precipitate to obtain a transformation solution, and injecting the transformation solution into virus-free phalaenopsis leaves and inflorescences.

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