CN114410647B

CN114410647B - Gene PeNHX1 for regulating and controlling petal color of butterfly orchid and application thereof

Info

Publication number: CN114410647B
Application number: CN202111581137.7A
Authority: CN
Inventors: 明凤; 徐庆于; 李东世; 何国仁; 孟扬
Original assignee: Shanghai Normal University
Current assignee: Shanghai Normal University
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2023-11-14
Anticipated expiration: 2041-12-22
Also published as: CN114410647A

Abstract

The invention provides a gene PeNHX1 for regulating and controlling the petal color of butterfly orchid and application thereof. The first aspect of the invention provides a gene PenHX1 for regulating and controlling the petal color of butterfly orchid, which has one of the following nucleotide sequences: 1) A nucleotide sequence shown as SEQ ID NO. 1; 2) The nucleotide sequence shown in SEQ ID NO.1 is derived by substitution, deletion or addition of one or several nucleotides; 3) A nucleotide sequence having at least 80% homology with SEQ ID No. 1. The butterfly orchid with blue petal color is cultivated by means of genetic engineering, so that the variety of the butterfly orchid petal color is enriched, the butterfly orchid has unique ornamental value, and theoretical basis and new thought are provided for directionally improving flower color in future.

Description

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

Background

Butterfly orchid is one of the most popular flowers in the flower market as an important cash crop. Butterfly orchid petals have rich colors, such as red to blue-violet, whereas truly blue butterfly orchid is rare. It is found that the pH of the vacuole is an important factor influencing the color blue bias, and as the pH of the vacuole is increased, partial organs of the flower can be changed from pink to purple blue, and the absorption spectrum of anthocyanin substances in different states has different red shift or blue shift effects. Based on the current research, the butterfly orchid is bred by searching a proper genetic means, and the butterfly orchid with blue petals is cultivated by the continuous attention of the person skilled in the art.

Disclosure of Invention

The invention provides a gene PeNHX1 for regulating and controlling the petal color of the butterfly orchid, which is used for cultivating the butterfly orchid with blue petal color by means of genetic engineering, so that the variety of the petal color of the butterfly orchid is enriched, the butterfly orchid has unique ornamental value, and theoretical basis and new thought are provided for directionally improving the flower color in the future.

The first aspect of the invention provides a gene PeNHX1 for regulating and controlling the color of butterfly orchid petals, wherein the gene has one of the following nucleotide sequences:

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

2) The nucleotide sequence shown in SEQ ID NO.1 is derived by substitution, deletion or addition of one or several nucleotides;

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

In a second aspect, the present invention provides a protein for regulating the colour of butterfly orchid petals, said protein being encoded by a gene provided in the first aspect of the present invention.

In a third aspect, the present invention provides a recombinant expression vector comprising a 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.

Further, the recombinant expression transformant is Agrobacterium.

In a fifth aspect, the present invention provides the use of the gene provided in the first aspect for regulating the colour of a butterfly orchid petal.

In a sixth aspect, the present invention provides a method of controlling the colour of petals of a butterfly orchid, the method comprising: the recombinant transformant provided in the fifth aspect of the present invention is injected into the petals of the butterfly orchid.

Further, the butterfly orchid is a small orchid butterfly orchid or a large capsicum butterfly orchid.

Further, when the butterfly orchid is a small orchid butterfly orchid, the method specifically includes:

connecting the nucleotide sequence to a CymMV virus plasmid to obtain a recombinant vector; then transferring the recombinant vector into agrobacterium to obtain recombinant transformant; and infecting the recombinant transformant into the small orchid butterfly orchid.

Further, when the butterfly orchid is a large capsicum butterfly orchid, the method specifically includes:

ligating the nucleotide sequence to the over-expression vector of UBI1300 to obtain a recombinant vector; then transferring the recombinant vector into agrobacterium to obtain recombinant transformant; and infecting the recombinant transformant into the large capsicum butterfly orchid.

The butterfly orchid with blue petals is cultivated by means of genetic engineering, so that the variety of the butterfly orchid petal colors is enriched, the butterfly orchid has unique ornamental value, and theoretical basis and new thought are provided for directionally improving flower colors in future.

Drawings

FIG. 1 shows the expression level of PeNHX1 in different developmental stages of flower buds of Phalaenopsis amabilis according to an embodiment of the present invention;

FIG. 2 shows the expression levels of PeNHX1 in different tissues of Phalaenopsis amabilis according to an embodiment of the present invention;

FIG. 3 shows petal phenotypes of control and mutant groups 1-2 after 30-40 days after injection of CymMV-PeNHX1 in small orchid butterfly orchid according to an embodiment of the invention;

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

FIG. 5 shows petal phenotypes of the control group and mutant group of the butterfly orchid of the large capsicum according to an embodiment of the present invention after 3 days of injection of UBI1300-GFP-PeNHX 1;

FIG. 6 shows the expression level of PeNHX1 in petals of a control group and a mutant group after 3 days of injection of UBI1300-GFP-PeNHX1 in the butterfly orchid with big capsicum according to an embodiment of the present invention;

FIG. 7 shows petal extracts of a control group and a mutant group of the butterfly orchid of the present invention after 3 days of injection of UBI1300-GFP-PeNHX 1;

fig. 8 shows pH values of petal extracts of a control group and a mutant group of the butterfly orchid of the large capsicum according to an embodiment of the present invention after 3 days of injection of UBI1300-GFP-PeNHX 1.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The experimental procedure, which does not specify specific conditions in the examples below, is generally carried out according to conventional conditions, such as, for example, molecular cloning by Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The reagents and carriers used, unless otherwise specified, are commercially available or publicly available.

EXAMPLE 1 extraction of butterfly orchid Gene

1. Extracting total RNA of wild type butterfly orchid petals by using a kit RNAplant (commercially available), and reversely transcribing the total RNA into cDNA by using a reverse transcription kit (commercially available);

2. designing a primer according to a transcriptome sequencing result, wherein the primer sequences are shown as SEQ ID NO.3 and SEQ ID NO.4, amplifying a 1632bp band from the butterfly orchid cDNA by adopting an RT-PCR method, recovering a PCR product, and obtaining a gene with a nucleotide sequence shown as SEQ ID NO.1, which is named as PeNHX1. 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 a molecular weight of 60.05 kilodaltons.

Example 2 verification of the expression profile of PenHX1 in various stages of the Phalaenopsis from Small orchid

The small orchid butterfly orchid is taken as a model plant of orchid, and the gene is verified through the small orchid butterfly orchid, which comprises the following steps:

1. extracting RNA of buds of the small orchid butterfly orchid at different development stages shown in the graph A of FIG. 1 by using a kit RNAplant (commercially available), and reversely transcribing the total RNA into cDNA by using a reverse transcription kit (commercially available);

2. designing a primer according to transcriptome sequencing data, wherein the primer sequences 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 obtained by reverse transcription of the small orchid butterfly orchid in different development periods as a template.

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

Example 3 verification of expression profile of PeNHX1 at different tissue sites of Phalaenopsis parvum

1. Extracting RNA of different tissue parts of the small orchid butterfly orchid shown in the graph A of FIG. 2 by using a kit (commercially available), and reversely transcribing the total RNA into cDNA by using a reverse transcription kit (commercially available);

3. and (3) carrying out expression profile verification on the PeNHX1 gene by taking cDNA obtained by reverse transcription of the small orchid butterfly orchid at different tissue parts as a template.

As shown in fig. 2B, the PeNHX1 gene was expressed in different flower organs, and the expression amount of the labial flap was higher compared with that of the petals and sepals, which may be due to the redness of the petals and sepals of the wild-type small orchid butterfly orchid, which indicates that the expression of the PeNHX1 gene affects the petal color.

Example 4 CymMV Virus induces PeNHX1 Gene silencing in Phalaenopsis parvum

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

2. the recombinant transformant was cultured in 5ml of LB medium containing 100. Mu.M acetosyringone and 50. Mu.g/ml kanamycin at 28℃for 16 hours at 200 rpm; subsequently, subculturing was continued in 50ml of LB medium containing 100. Mu.M acetosyringone and 50. Mu.g/ml kanamycin, and incubation was performed at 28℃and 200rpm for 13-16 hours until OD600 reached 0.8-1.0;

3. the Agrobacterium solution containing the recombinant transformant in step 2 was transferred to a 50ml centrifuge bottle and centrifuged at 3000g for 10 minutes at 4℃and after centrifugation, the supernatant was removed. Adding 300 μl of MS culture medium containing 100 μM acetosyringone, resuspending the cell pellet, and standing at room temperature for 0.5h;

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

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

Example 5 verification of expression of PeNHX1 Gene in petals in CymMV Virus-induced mutant strain of Phalaenopsis parvula

1. Selecting petals of a control group and a mutant group as shown in figure 3, extracting total RNA of the calyx and the petals, wherein an extraction kit is RNAplant (commercially available), and performing reverse transcription on the total RNA into cDNA by using a reverse transcription kit (commercially available);

3. and (3) taking cDNA as a template, and carrying out gene silencing efficiency verification on PeNHX1.

As shown in FIG. 4, the expression of the PeNHX1 gene is obviously reduced in the mutant group, the silencing efficiency of the PeNHX1 is higher, and the PeNHX1 is further verified to participate in the formation of the petal color of the Phalaenopsis amabilis.

Example 6 CymMV Virus induces petal color CIE value in Dutch Phalaenopsis silencing lines

CIE (Commission International Eclairage) is a color pattern established according to the measured color criteria by which the color change of petals is expressed.

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

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

2. As shown in table one, in Lip (labial lobe) the L increases compared to petals of the control group, indicating an increase in labial lobe color brightness; an increase, indicating an increase in lip color red; increasing b indicates a darkening of the lip color yellow. In Petal (petals), L decreases, indicating a decrease in lip color brightness; a, decrease, indicating a red lightening of the lip color; increasing b indicates a darkening of the lip color yellow. The above results demonstrate that the PenHX1 gene has a significant effect on the color change of the Phalaenopsis minor.

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 variety and content of anthocyanin in the small orchid butterfly orchid are less than those of other butterfly orchids, we find that the petal color can be blue through over-expression of the PeNHX1 gene by taking the large capsicum butterfly orchid as an experimental material, and the method is specifically described as follows:

example 7 UBI1300 vector induces transient overexpression of PeNHX1 Gene in Phalaenopsis Capsici

1. Operably linking 1632bp of the open reading frame of the PeNHX1 gene with a UBI1300-GFP expression vector to form a UBI1300-GFP-PeNHX1 vector containing the gene fragment, and transferring the vector into agrobacterium GV3101 to obtain a recombinant transformant;

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

3. transferring the Agrobacterium solution containing the recombinant expression transformant in step 2 into a centrifuge bottle, centrifuging at 3700rpm at 4deg.C for 10 min, removing supernatant, and using 0.5M MgCl ₂ Repeatedly blowing and resuspending for three times;

4. taking agrobacterium liquid containing recombinant expression transformant after re-suspension in step 3, using 0.5M MgCl ₂ Diluting the bacterial liquid, and controlling the OD value of the bacterial liquid to be about 0.6. Then adding 100 mu M acetosyringone with the total volume of 1.5 times and MES (morpholinoethanesulfonic acid) with the total volume of 20 times, re-suspending the cell sediment, and standing for 3-4h at room temperature;

5. sucking the standing agrobacterium conversion liquid by using a 1ml syringe with a needle, and injecting the agrobacterium conversion liquid into the petals of the butterfly orchid in the large capsicum;

6. after 2-3 days of culture after injection, petals of the large capsicum butterfly orchid are observed, as shown in fig. 5, the treated large capsicum butterfly orchid is a mutation group, named UBI1300-NHX1, and blue deposition appears in petal color structures of the mutation group.

Example 8 transient overexpression of PeNHX1 Gene in butterfly orchid Capsici, verification of PeNHX1 Gene expression in petals

1. Extracting total RNA of petals newly grown in the mutant group and the control group in the example 7 by using a kit (commercially available), and reversely transcribing the total RNA into cDNA by using a reverse transcription kit (commercially available);

3. and (3) carrying out gene expression efficiency verification on the PeNHX1 by taking the cDNA obtained by reverse transcription in the step (1) as a template.

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

Example 9 determination of petal color CIE value after transient overexpression of PeNHX1 Gene of Phalaenopsis amabilis

1. The color of the petals of the butterfly orchid in the capsicum after transient overexpression was measured as shown in the CIE value measurement method of example 6. The petals of the mutant group and the control group provided in example 8 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 an average value was obtained as a standard curve.

2. As shown in Table II, the petals of the experimental group are less red and blue than those of the control group, which shows that the PeNHX1 gene has a significant effect on the change of the petal color of the butterfly orchid with big capsicum.

Petal CIE measurements after 3 days for Table two experimental and control groups

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

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

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

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Sequence listing

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Claims

1. GenePeNHX1The application of the gene in regulating the color of butterfly orchid petals is characterized in that the genePeNHX1The nucleotide sequence of (2) is shown as SEQ ID NO. 1.

2. A method of controlling the color of butterfly orchid petals, comprising: injecting a recombinant transformant comprising the recombinant vector intoIn the butterfly orchid petals, the recombinant vector comprises a genePeNHX1The genePeNHX1The nucleotide sequence of (2) is shown as SEQ ID NO. 1.

3. A method according to claim 2, wherein the butterfly orchid is a capsicum butterfly orchid.

4. A method according to claim 3, wherein when the butterfly orchid is a large capsicum butterfly orchid, the method specifically comprises:

5. A method for regulating and controlling the color of petals of phalaenopsis amabilis, which is characterized by comprising the following steps:

genes are addedPeNHX1The open reading frame 200-300bp of (B) is connected to the CymMV virus plasmid to obtain a recombinant vector; then transferring the recombinant vector into agrobacterium to obtain recombinant transformant; infecting the recombinant transformant with phalaenopsis amabilis;

the genePeNHX1The nucleotide sequence of (2) is shown as SEQ ID NO. 1.