CN114410647A - Gene PeNHX1 for regulating and controlling butterfly orchid petal color and application thereof - Google Patents

Abstract

The invention provides a gene PeNHX1 for regulating and controlling the petal color of phalaenopsis and application thereof. The invention provides a gene PeNHX1 for regulating and controlling the petal color of phalaenopsis, 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 phalaenopsis with the blue petal color is obtained by means of genetic engineering cultivation, so that the variety of the phalaenopsis petal color is enriched, the phalaenopsis has unique ornamental value, and theoretical basis and new thought are provided for directional improvement of flower color in future.

Description

Gene PeNHX1 for regulating and controlling butterfly orchid petal color and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a gene PeNHX1 for regulating and controlling the petal color of phalaenopsis and application thereof.

Background

Butterfly orchid is one of the most popular flowers in the flower market as an important economic crop. Moth orchid petals have rich flower colors, such as a red to bluish-purple color change, whereas very few moth orchids are truly bluish. The research shows that the vacuole pH is an important factor influencing the bluish flower color, and as the vacuole pH is increased, part of flower organs can be changed from pink to purple blue due to different red-shift or blue-shift effects of absorption spectra of anthocyanin substances in different states. Based on the current research, the continuous attention of technicians in the field is paid to the culture of the phalaenopsis with bluish petal colors by searching a suitable genetic means to breed the phalaenopsis.

Disclosure of Invention

The gene PeNHX1 for regulating the petal color of the phalaenopsis is cultured by means of genetic engineering to obtain the phalaenopsis with the petal color being blue, so that the variety of the petal color of the phalaenopsis is enriched, the phalaenopsis has unique ornamental value, and theoretical basis and new thought are provided for directionally improving the flower color in the future.

The invention provides a gene PeNHX1 for regulating and controlling the petal color of phalaenopsis, 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.

In a second aspect, the invention provides a protein for regulating and controlling the petal color 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.

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 expression transformant is agrobacterium.

The fifth aspect of the invention provides the application of the gene provided by the first aspect in regulating and controlling the color of petals of butterfly orchid.

The sixth aspect of the invention provides a method for regulating and controlling the color of petals of phalaenopsis, which comprises the following steps: the recombinant transformant provided by the fifth aspect of the present invention is injected into moth orchid petals.

Further, the butterfly orchid is a small orchid butterfly orchid or a large hot pepper butterfly orchid.

Further, when the phalaenopsis is phalaenopsis miniata, the method specifically comprises the following steps:

connecting the nucleotide sequence to a CymMV virus plasmid to obtain a recombinant vector; then transferring the recombinant vector into agrobacterium to obtain a recombinant transformant; infecting the recombinant transformant with Phalaenopsis plantula.

Further, when the phalaenopsis is a large hot pepper phalaenopsis, the method specifically comprises the following steps:

connecting the nucleotide sequence to an over-expression vector of UBI1300 to obtain a recombinant vector; then transferring the recombinant vector into agrobacterium to obtain a recombinant transformant; infecting the recombinant transformant with large hot pepper phalaenopsis.

The phalaenopsis with blue petal colors is obtained by means of genetic engineering cultivation, so that the variety of the phalaenopsis petal colors is enriched, the phalaenopsis has unique ornamental value, and theoretical basis and new thinking are provided for directional improvement of flower colors in future.

Drawings

Fig. 1 shows the expression level of PeNHX1 in different developmental stages of a phalaenopsis plantula flower bud according to an embodiment of the present invention;

fig. 2 shows the expression level of PeNHX1 in different tissues of phalaenopsis miniata according to an embodiment of the present invention;

FIG. 3 shows the petal phenotype of Phalaenopsis miniata in the control and mutant groups 1-2 after CymMV-PeNHX1 injection for 30-40 days according to an embodiment of the present invention;

FIG. 4 shows the expression level of PeNHX1 in the control group and the mutant group 1-2 after CymMV-PeNHX1 injection for 30-40 days in Phalaenopsis parviflora according to an embodiment of the present invention;

FIG. 5 shows the petal phenotype of the control and mutant phalaenopsis capsici after 3 days of UBI1300-GFP-PeNHX1 injection;

FIG. 6 shows the expression level of PeNHX1 in petals of a control group and a mutant group after 3 days of UBI1300-GFP-PeNHX1 injection of Phalaenopsis capsici provided by an embodiment of the invention;

FIG. 7 shows petal extracts of Phalaenopsis capsici after 3 days of UBI1300-GFP-PeNHX1 injection;

FIG. 8 shows the pH of petal extracts of Phalaenopsis capsici after 3 days of UBI1300-GFP-PeNHX1 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.

The experimental procedures, which are not specified in the following examples, are generally carried out according to 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 extraction of moth orchid Gene

1. Extracting the total RNA of wild butterfly orchid petals by using a kit RNAPlant (sold in the market), and reversely transcribing the total RNA into cDNA by using a reverse transcription kit (sold in the market);

2. designing a primer according to a transcriptome sequencing result, wherein the sequence of the primer is shown as SEQ ID NO.3 and SEQ ID NO.4, amplifying a 1632bp strip from butterfly orchid cDNA by adopting an RT-PCR method, recovering a PCR product, and obtaining a gene with the nucleotide sequence shown as SEQ ID NO.1, which is named as PeNHX 1. The protein coded by the nucleotide sequence has an amino acid sequence shown as SEQ ID NO.2, consists of 543 amino acid residues, and has the molecular weight of 60.05 kilodaltons.

Example 2 expression profiling of PeNHX1 at different stages of butterfly orchid development

The gene is verified by the phalaenopsis miniata as a model plant of the orchidaceae, and the method specifically comprises the following steps:

1. extracting RNA of the buds of the Phalaenopsis plantula shown in the part A in the figure 1 at different development stages by using a kit RNAPlaland (commercially available), and performing reverse transcription on the total RNA into cDNA by using a reverse transcription kit (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 PeNHX1 gene by taking cDNA (complementary deoxyribonucleic acid) obtained by reverse transcription of the phalaenopsis miniata at different development stages as a template.

As shown in B in FIG. 1, the expression level of PeNHX1 was different at different developmental stages, and the expression level was higher when all buds were full (B5).

Example 3 expression profiling of PeNHX1 at different tissue sites of Phalaenopsis miniata

1. Extracting RNA of different tissue parts of Phalaenopsis miniata shown in A in figure 2 by using a kit for RNAplant (commercially available), and performing reverse transcription on the total RNA into cDNA by using a reverse transcription kit (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 PeNHX1 gene by taking cDNA (complementary deoxyribonucleic acid) obtained by reverse transcription of the phalaenopsis miniata at different tissue parts as a template.

The verification result is shown as B in FIG. 2, the PeNHX1 gene is expressed in different flower organs, and the expression level is higher in the lip valve compared with the petals and the sepals, which is probably related to that the wild type Phalaenopsis miniata petals and sepals are reddish and the lip valve is yellowish, which indicates that the PeNHX1 gene expression influences the petal color.

Example 4 CymMV Virus induces Gene silencing in Phalaenopsis miniata PeNHX1

1. Operably connecting the open reading frame 200-300bp of the PeNHX1 gene to a CymMV virus vector to form a CymMV-NHX1 vector containing the gene segment, and then transferring the vector into agrobacterium GV3101 to obtain a recombinant transformant;

2. culturing the recombinant transformant in 5ml LB culture medium containing 100 μ M acetosyringone and 50 μ g/ml kanamycin at 28 ℃ for 16h at 200 rpm; then, subculture was continued in 50ml LB medium containing 100. mu.M acetosyringone and 50. mu.g/ml kanamycin, and incubation was carried out at 28 ℃ and 200rpm for 13-16 hours until OD600 reached 0.8-1.0;

3. and (3) taking the agrobacterium liquid containing the recombinant transformant in the step (2), transferring the agrobacterium liquid into a 50ml centrifugal bottle, centrifuging the agrobacterium liquid at 4 ℃ and 3000g for 10 minutes, and removing a supernatant after centrifugation. Adding 300 μ l MS culture medium containing 100 μ M acetosyringone, resuspending the cell precipitate, standing at room temperature for 0.5 h;

4. sucking the standing agrobacterium transformation liquid by using a 1ml syringe with a needle head, and injecting the agrobacterium transformation liquid into the petals of the butterfly orchid;

5. and culturing for 30-40 days after injection, observing petals of the butterfly orchid of the small orchid, wherein the treated butterfly orchid of the small orchid is a mutation group and is named as pCymMV-PeNHX1, and the observation result is shown in figure 3, wherein the color of the petals of the silent mutation strain of the butterfly orchid of the small orchid is obviously lightened.

Example 5 verification of expression of the PeNHX1 gene in petals in a mutant strain of Phalaenopsis miniata induced by CymMV virus

1. Selecting petals of a control group and a mutation group shown in the figure 3, extracting total RNA of calyx and petals, wherein the extraction kit is RNAplant (commercially available), and performing reverse transcription on the total RNA into cDNA by using a reverse transcription kit (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 gene silencing efficiency verification on the PeNHX1 by using the cDNA as a template.

The verification result is shown in fig. 4, the expression of the PeNHX1 gene is obviously reduced in a mutation group, the silencing efficiency of PeNHX1 is high, and the PeNHX1 is further verified to participate in the formation of the petal color of the phalaenopsis miniata.

Example 6 CymMV Virus Induction of petal color CIE values in Phalaenopsis miniata silencing lines

Cie (commission International eclairage) is a color pattern established according to a color standard, and a color change of petals is expressed by the color pattern.

One element in the CIE is the luminance (L), a and b are the two color channels. a colors are from dark green (low brightness value) to gray (medium brightness value) to bright pink red (high brightness value); the colors b are from bright blue (low brightness value) to gray (medium brightness value) to yellow (high brightness value).

1. The petals of the experimental group and the mutant group as shown in fig. 3 were selected, the control group was used as a standard sample, the petals of the mutant group were used as a treatment sample, and the standard sample and the treatment sample were sampled three times, wherein each sample was measured for three biological replicates, and finally the average value was obtained as a standard curve.

2. As shown in table one, L increases in Lip (Lip) of the experimental group compared to the petals of the control group, indicating increased Lip color intensity; a increases, indicating an increase in the lip color red; b increases, indicating a deepening of the yellow lip color. In Petal (petals), L decreases, indicating a decrease in lip color intensity; a decreases, indicating a red decrease in the lipped color; b increases, indicating a deepening of the yellow lip color. The above results indicate that the PeNHX1 gene has a significant effect on the change in the color of butterfly orchid of the small orchid.

TABLE-petal CIE measurements after 30-40d days for the experimental and control groups

RHSCC Royal Horticultural Society color chart；L*lightness；a*,b*chromatic component；C*brightness；h(hue angle)＝arctan(b*/a*)

Because the types and the content of anthocyanin of the phalaenopsis miniata are less than those of other phalaenopsis, the excessive expression of the PeNHX1 gene by taking the phalaenopsis maxima as an experimental material can enable the color of petals to be blue, and the specific description is as follows:

example 7 UBI1300 vector Induction of transient overexpression of the Phalaenopsis cayensis PeNHX1 Gene

1. The open reading frame 1632bp of the PeNHX1 gene is operably connected with the UBI1300-GFP expression vector to form a UBI1300-GFP-PeNHX1 vector containing the gene segment, and then the vector is transferred into agrobacterium GV3101 to obtain a recombinant transformant;

2. the recombinant transformants were cultured overnight at 28 ℃ at 200rpm in 5ml of LB medium containing 100. mu.M acetosyringone, 50. mu.g/ml kanamycin and 10. mu.g/ml rifampicin;

3. taking the agrobacterium liquid containing the recombinant expression transformant in the step 2, transferring the agrobacterium liquid into a centrifugal bottle, centrifuging the agrobacterium liquid at 4 ℃ and 3700rpm for 10 minutes, removing supernatant after centrifugation, and using 0.5M MgCl₂Repeatedly blowing, beating and resuspending for three times;

4. taking the agrobacterium liquid containing the recombinant expression transformant after being resuspended in the step 3, and using 0.5M MgCl₂And (4) diluting the bacterial liquid, and controlling the OD value of the bacterial liquid to be about 0.6. Then adding 100 μ M acetosyringone 1.5 times of the total volume and MES (morpholine ethanesulfonic acid) 20 times of the total volume to resuspend the cell precipitate, and standing at room temperature for 3-4 h;

5. sucking the standing agrobacterium transformation liquid by using a 1ml syringe with a needle head, and injecting the agrobacterium transformation liquid into the petals of the large hot pepper phalaenopsis;

6. and (3) culturing for 2-3 days after injection, observing petals of the large hot pepper phalaenopsis, wherein the treated large hot pepper phalaenopsis is a mutation group named as UBI1300-NHX1 as shown in figure 5, and showing that blue deposition appears in the petal color structure of the mutation group.

Example 8 transient overexpression of PeNHX1 Gene and verification of PeNHX1 Gene expression in petals in Phalaenopsis capsici

1. Extracting total RNA of petals newly grown in a mutation group and a control group in example 7 by using a kit (commercially available), and performing reverse transcription on the total RNA into cDNA by using a reverse transcription kit (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 gene expression efficiency verification on the PeNHX1 by using the cDNA obtained by reverse transcription in the step 1 as a template.

As a result of the verification, as shown in FIG. 6, the expression level of PeNHX1 was significantly increased in petals of the mutant strain.

Example 9 measurement of petal color CIE value after transient overexpression of PeNHX1 Gene in Phalaenopsis LARGE CALIA

1. The petal color of phalaenopsis capsici after transient overexpression was measured as described in example 6CIE value measurement method. The petals of the mutant and control groups provided in example 8 were selected, the control group was used as the standard sample, the petals of the mutant group were used as the treatment sample, and the standard and treatment samples were sampled three times, wherein three biological replicates were measured for each sample, and finally the mean was obtained as the standard curve.

2. As shown in Table II, the petals of the experimental group are less red and more blue than those of the control group, which indicates that the PeNHX1 gene has a significant influence on the change of the petal color of the large hot pepper phalaenopsis.

CIE measurement of petals of experimental group and control group after 3 days

RHSCC Royal Horticultural Society color chart；L*lightness；a*,b*chromatic component；C*brightness；h(hue angle)＝arctan(b*/a*)

Example 10 determination of pH value and significance analysis of petal extract after transient overexpression of PeNHX1 Gene in Phalaenopsis capsici

1. Petals provided as in example 8 mutant and control groups were selected for direct milling, centrifuged at 13000rpm for 15 minutes, and the supernatant was immediately measured using a pH electrode. Three biological replicates of petals of the control group and the experimental group were taken for measurement, and the measured pH values were subjected to significance analysis by the graphic analysis software GraphPad Prism.

2. As shown in fig. 7-8, the petal extract of the mutant group was significantly bluish and darker in color and increased in pH value compared to the petal extract of the control group, indicating that the PeNHX1 gene may affect the petal color by increasing the pH value of the vacuole of phalaenopsis amabilis of capsicum.

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.

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Claims (10)

1. A gene PeNHX1 for regulating the petal color of phalaenopsis, 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.

2. A protein for regulating the petal color of phalaenopsis, which is encoded by the gene of claim 1.

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

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

5. The recombinant transformant according to claim 4, wherein the recombinant expression transformant is Agrobacterium.

6. The use of the gene of claim 1 for modulating the petal color of phalaenopsis.

7. A method of modulating the color of petals of a butterfly orchid, comprising: the recombinant transformant according to claim 4 or 5 is injected into petals of moth orchid.

8. The method of claim 7, wherein the phalaenopsis is phalaenopsis miniata or phalaenopsis capsici.

9. The method according to claim 8, wherein when the phalaenopsis is phalaenopsis miniata, the method specifically comprises:

connecting the nucleotide sequence to a CymMV virus plasmid to obtain a recombinant vector; then transferring the recombinant vector into agrobacterium to obtain a recombinant transformant; infecting the recombinant transformant with Phalaenopsis plantula.

10. The method according to claim 8, wherein when the phalaenopsis is phalaenopsis maxima, the method specifically comprises:

connecting the nucleotide sequence to an over-expression vector of UBI1300 to obtain a recombinant vector; then transferring the recombinant vector into agrobacterium to obtain a recombinant transformant; infecting the recombinant transformant with large hot pepper phalaenopsis.

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