CN113736794B

CN113736794B - Gene PeVIT for regulating blue generation of butterfly orchid petals and application thereof

Info

Publication number: CN113736794B
Application number: CN202110987244.3A
Authority: CN
Inventors: 明凤; 马成昊; 戴心悦; 杨熠; 张苏逸
Original assignee: Shanghai Normal University
Current assignee: Shanghai Normal University
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2023-08-08
Anticipated expiration: 2041-08-26
Also published as: CN113736794A

Abstract

The invention provides a gene PeVIT for regulating and controlling blue generation of butterfly orchid petals and application thereof. The first aspect of the present invention provides a gene for regulating blue production of butterfly orchid petals, the gene having 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. According to the invention, the colors of the petals of the butterfly orchid are effectively regulated and controlled by means of genetic engineering, and blue color blocks appear in the microstructure of the petals in the transient over-expression strain of the butterfly orchid, so that a theoretical basis is provided for breeding the butterfly orchid.

Description

Gene PeVIT for regulating blue generation of butterfly orchid petals and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a gene PeVIT for regulating and controlling blue generation of butterfly orchid petals and application thereof.

Background

With the rapid improvement of economic strength in China in recent years, the flower industry has also been developed unprecedentedly, and people are no longer satisfied with common flower types nowadays, but pursue special flowers and rare flowers with certain ornamental value and collection value.

Butterfly orchid is popular as the most ornamental orchid in the current generation because of its gorgeous flower color and unique flower shape, while butterfly orchid leaves are single in color and have no blue petals. There is increasing interest in how to change the color of butterfly orchid petals by genetic engineering means.

Disclosure of Invention

The invention provides a gene for regulating and controlling blue generation of butterfly orchid petals, which changes the colors of the butterfly orchid petals by means of genetic engineering, thereby having unique ornamental value.

The first aspect of the present invention provides a gene for regulating blue production of butterfly orchid petals, the gene having 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.

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

In a second aspect, the present invention provides a protein for regulating blue production of butterfly orchid petals, wherein the protein is encoded by the gene of the first aspect of the present invention.

In a third aspect, the invention provides a recombinant expression vector comprising a nucleotide sequence according to the first aspect of the invention.

Further, the recombinant expression vector is UBI1300-GFP or p416-TEF.

In a fourth aspect, the present invention provides a recombinant expression transformant comprising a recombinant expression vector according to the third aspect of the present invention.

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 butterfly orchid petals.

In a sixth aspect, the present invention provides a method of modulating the colour of petals of a butterfly orchid, the method comprising injecting a recombinant expression transformant according to the fourth aspect of the present invention into the petals of a butterfly orchid.

In a seventh aspect, the present invention provides the use of the gene provided in the first aspect for regulating the capacity of transporting iron element.

According to the invention, the colors of the petals of the butterfly orchid are effectively regulated and controlled by means of genetic engineering, and blue color blocks appear in the microstructure of the petals in the transient over-expression strain of the butterfly orchid, so that a theoretical basis is provided for breeding the butterfly orchid.

Drawings

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

FIG. 1a shows different tissue sites of butterfly orchid petals according to an embodiment of the present invention;

FIG. 1b shows the PeVIT expression levels at different tissue sites in the petals of butterfly orchid according to an embodiment of the present invention;

FIG. 2 is a phenotype diagram of petal microstructures of a large capsicum butterfly orchid experimental group UBI1300-GFP-PeVIT and a control group UBI1300-GFP-EV provided by an embodiment of the invention after 3 days;

FIG. 3 is a phenotypic chart of petal microstructures of the experimental group UBI1300-GFP-PeVIT and the control group UBI1300-GFP-EV of the striped butterfly orchid provided by an embodiment of the invention after 3 days;

FIG. 4 shows the results of detecting the expression level of PeVIT in the experimental group UBI1300-GFP-PeVIT and the control group UBI1300-GFP-EV of the butterfly orchid with large capsicum according to one embodiment of the present invention;

FIG. 5 shows the results of detecting the expression level of PeVIT in the experimental group UBI1300-GFP-PeVIT and the control group UBI1300-GFP-EV of the striped butterfly orchid according to an embodiment of the present invention;

FIG. 6 is a graph showing the iron transport activity of PeVIT in yeast strain according to an embodiment of the present invention.

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 followed by routine conditions, such as 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 used, unless otherwise specified, are commercially available or publicly available.

In the present invention, various vectors known in the art, such as commercially available vectors including plasmids and the like, may be used.

Extraction of butterfly orchid gene PeVIT

1. Extracting total RNA of wild butterfly orchid petals for RNAplant (commercially available) by using a kit, 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 762bp 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 PeVIT. The nucleotide sequence encodes a protein sequence shown in SEQ ID NO.2, which consists of 254 amino acid residues and has a molecular weight of 26.975 kilodaltons.

Expression profile verification of PeVIT in petal colors of capsicum annuum and striae butterfly orchid

1. Extracting RNA of different tissue parts of the phalaenopsis pattern plant phalaenopsis from RNAplant (commercially available) by using a kit, wherein the extracted parts are shown in figure 1a, and the total RNA is reversely transcribed 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 carrying out expression profile verification on the PeVIT genes by taking cDNA obtained by reverse transcription of the large capsicum butterfly orchid and the striped butterfly orchid at different tissue parts as templates. The expression quantity of PeVIT in different tissue parts is tested, and the test result is shown in figure 1b, wherein the expression quantity of PeVIT in petals is higher, which indicates that the PeVIT possibly participates in the formation of the butterfly orchid petal color.

UBI1300-GFP induces transient over-expression of PeVIT gene of capsicum annuum and Phalaenopsis amabilis

1. Operably linking 762bp of the open reading frame of the PeVIT gene with a UBI1300-GFP vector to form a UBI1300-GFP-PeVIT vector containing the gene fragment, and transferring the vector into agrobacterium GV3101 to obtain a recombinant expression transformant; the constructed UBI1300-GFP vector takes the pCAMBIA1300 vector as a framework to modify the original promoter element CAMV35S, and replaces the original CAMV35S promoter with a UbI promoter element.

2. The recombinant expression 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. taking the agrobacterium solution containing recombinant expression transformant in step 2, centrifuging at 3700rpm at 4 ℃ for 10 minutes, removing supernatant after centrifugation, and using 0.5M MgCl ₂ Repeatedly blowing and resuspending for three times;

4. taking a little agro-rod re-suspension bacterial liquid in the step 3, measuring the OD value of the bacterial liquid, and using 0.5M MgCl ₂ Diluting the bacterial liquid to make the OD value of the bacterial liquid 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, resuspending 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 respectively injecting the agrobacterium conversion liquid into virus-free large capsicum butterfly orchid and stripe butterfly orchid petals;

6. culturing for 2-3 days after injection, observing butterfly orchid and striped butterfly orchid petals, and treating butterfly orchid as experimental group, named UBI1300-GFP-PeVIT, and setting control group, named UBI1300-GFP-EV.

Selecting the petals of the large capsicum butterfly orchid experimental group UBI1300-GFP-PeVIT shown in the figure 2 a and the petals of the large capsicum butterfly orchid control group UBI1300-GFP-EV shown in the figure b, observing the same visual field under different excitation light conditions by using a fluorescence microscope, wherein the observation results are shown on the right side of the figure 2, and the recombinant vector is provided with GFP marks, so that the part with green fluorescence under green excitation light is the part for expressing PeVIT; bright Field is an observation diagram of cells in a Bright Field, and in the mode, no self-luminescence exists, so that the change situation of color can be observed visually, for example, in the Bright Field mode, blue color blocks can be seen in the area pointed by arrows of petals of an experimental group, but the blue color blocks cannot be related to each other, so that Merged is carried out by using corresponding software, namely GFP and Bright Field are fused, and the blue color blocks are caused by the over-expression of PeVIT.

Two petals of one petal of the striped butterfly orchid are selected, as shown in the left side of fig. 3, the upper petal is a control group UBI1300-GFP-EV petal, the lower petal is an experimental group UBI1300-GFP-PeVIT, and after sampling, observation is performed by adopting the same means as the large capsicum butterfly orchid, and it can be seen that blue color blocks are observed in the microstructure of the experimental group UBI1300-GFP-PeVIT petals compared with the control group UBI1300-GFP-EV petals.

Verification of PeVIT Gene expression in Pepper and Phalaenopsis mutant lines

1. Extracting total RNA of petals shown in figures 2-3 for RNAplant (commercially available) by using a kit, and reversely transcribing the total RNA into cDNA by using a reverse transcription kit (commercially available);

3. the gene expression efficiency of PeVIT was verified by using cDNA obtained by reverse transcription of petals as shown in FIGS. 2 to 3 as a template. As shown in FIGS. 4 to 5, it was observed that the expression level of PeVIT was significantly increased in petals of the transient over-expression mutant strain.

Expression of iron element transport Capacity of PeVIT Gene in p 416-TEF-induced Yeast Strain mutant ΔCCC1 and wild-type Yeast Strain DY150

1. Operably ligating 762bp of the open reading frame of the PeVIT gene to the p416-TEF expression vector to form a p416-TEF-PeVIT vector comprising the gene fragment;

2. transferring the p416-TEF-PeVIT Vector and the p416-TEF-Vector of the control group into a yeast strain mutant delta CCC1 and a wild type yeast strain DY150 respectively;

3. culturing the four yeast strains in the step 2 on a Ura selection defective solid culture medium, streaking the four grown yeast strains on a new Ura selection defective solid culture medium, and resuscitating the four grown yeast strains in a Ura selection defective liquid culture medium after the four grown yeast strains grow;

4. the bacterial liquid recovered in the step 3 and containing four yeast strains is cultured in Ura selection defect type solid medium and contains 7.5mM FeSO ₄ The strain variant ΔCCC1 of the yeast and the strain DY150 of the wild type were observed after culturing on a Ura selection-deficient solid medium, respectively, and transferring the strain variant into a p416-TEF-PeVIT expression Vector and a p416-TEF-Vector of a control group.

As shown in FIG. 6, the results of the experiment revealed that CCC1 transformed with the p416-TEF-PeVIT expression vector was saved in terms of Fe transport capacity so that it could be expressed in 7.5mM FeSO ₄ The Ura selection defect type solid culture medium grows on the surface PeVIT gene, so that the transport capacity of iron element can be improved.

In addition, the invention also provides nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO.8, which are nucleotide sequences derived from the nucleotide sequence shown as SEQ ID NO.1, and nucleotide sequences with at least 80% homology with the nucleotide sequence shown as SEQ ID NO.1, respectively, and have the same effect as the nucleotide sequences shown as SEQ ID NO. 1.

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

<110> Shanghai university of teachers and students

<120> Gene PeVIT for controlling blue production of butterfly orchid petals and use thereof

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<170> PatentIn version 3.3

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<212> DNA

<213> butterfly orchid (Phalaenopsis aphrodite H.G. Reichenbach)

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ccaaagctag ctggattgtt gaggtgtggg aagagttgca gacttcggtg gacgaattat 180

ctcagacctg atattaaaag agggaacttt accagggaag aagaggatgc aatcattaac 240

ttgcatcaaa tgctgggaaa cagatggtct gcaattgcag cgaagctacc tggtagaaca 300

gataatgaga tcaaaaacgt atggcacact catctgaaga aaagattaac gagaaccgat 360

aaagagaccg gacaagaacg aatcagaaaa actcaaatag aaccaaagga agagatgccc 420

acacaatcat acagcagcac tccagaacca tcttcttcct ccagtactgt agacaacagt 480

cagaactcca tggaatcttt cagccatgaa gctgaagccc aagggatcga tgagagcttc 540

tggactgaag tgctgaaaat ggatagtaat gatgagtatt actgtaactc gtcggattca 600

atggcgatgg aaggatttaa ttcctctgat ttcagttatg acaaattttg gctgtcagct 660

ggcgtgagag aagaggatga tgatttgagt ttttggttga ggatctttct gcaagctgaa 720

gaatttccac agatttga 738

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<213> butterfly orchid (Phalaenopsis aphrodite H.G. Reichenbach)

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Arg Lys Thr Gln Ile Glu Pro Lys Glu Glu Met Pro Thr Gln Ser Tyr

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aagagggcac tggagagcgc catgacaatc gccttctcct acattgcagg tgggttggtg 540

ccgctgcttc cttacatgtt catctccact gctcagaagg caatgttcat atctgttctt 600

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<212> DNA

<213> Synthesis (Artificial Sequence)

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atggtgaagg agttcgtgca ggacgaggag aagcagcggc tgctgctgga cgagcacacc 60

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ctcaccgtgc cgttcgccct cgccgccggc ctgtccggcg ccaacgcccc ctccgccctc 180

gtcctcaccg ccggcctcgc cgaggtcgcc gccggcgcca tctccatggg cctcggaggg 240

tatctggcgg cgaagagcga cgccgaccac taccaccgcg agctgcagag ggagcaggag 300

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Claims

1. The application of the gene in regulating and controlling the iron element transport capacity of yeast is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.