Gene PeVIT for regulating and controlling blue generation of butterfly orchid petals and application thereof
阅读说明：本技术 一种调控蝴蝶兰花瓣蓝色生成的基因PeVIT及其应用 (Gene PeVIT for regulating and controlling blue generation of butterfly orchid petals and application thereof ) 是由 明凤 马成昊 戴心悦 杨熠 张苏逸 于 2021-08-26 设计创作，主要内容包括：本发明提供一种调控蝴蝶兰花瓣蓝色生成的基因PeVIT及其应用。本发明第一方面提供一种调控蝴蝶兰花瓣蓝色生成的基因,所述基因具有以下核苷酸序列中的一种：1)SEQ ID NO.1所示的核苷酸序列；2)SEQ ID NO.1所示的核苷酸序列通过一个或几个核苷酸的取代、缺失、或添加衍生产生的核苷酸序列；3)与SEQ ID NO.1具有至少80％同源性的核苷酸序列。本发明通过基因工程的手段有效调控了蝴蝶兰花瓣的颜色,在蝴蝶兰瞬时过表达株系中,其花瓣颜色在显微结构中出现蓝色色块,为蝴蝶兰的育种提供了理论基础。(The invention provides a gene PeVIT for regulating and controlling butterfly orchid petal blue generation and application thereof. The invention provides a gene for regulating and controlling the generation of blue color of butterfly orchid petals, 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 color of the butterfly orchid petal by means of genetic engineering, and provides a theoretical basis for breeding of the butterfly orchid, wherein the color of the butterfly orchid petal appears as a blue color block in a microstructure in a transient overexpression strain of the butterfly orchid.)
1. A gene for regulating and controlling blue generation of phalaenopsis petals, which is characterized by having 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. The gene as claimed in claim 1, wherein the phalaenopsis is phalaenopsis capsici or phalaenopsis striata.
3. A protein for regulating the blue color production of petals of a butterfly orchid, which is encoded by the gene of claim 1.
4. A recombinant expression vector comprising the nucleotide sequence of claim 1.
5. The recombinant expression vector of claim 4, wherein the recombinant expression vector is UBI1300-GFP or p 416-TEF.
6. A recombinant expression transformant comprising the recombinant expression vector according to claim 4 or 5.
7. The recombinant expression transformant according to claim 6, wherein the recombinant expression transformant is Agrobacterium.
8. The use of the gene of claim 1 for modulating the petal color of phalaenopsis.
9. A method for controlling the color of petals of moth orchid, which comprises injecting the recombinant expression transformant according to claim 6 or 7 into petals of moth orchid.
10. Use of the gene of claim 1 for regulating the transport capacity of iron element.
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a gene PeVIT for regulating and controlling butterfly orchid petal blue generation and application thereof.
Background
With the rapid improvement of the economic strength of China in recent years, the flower industry has also been developed unprecedentedly, and nowadays, people no longer satisfy common flower varieties, but pursue peculiar flowers and rare flowers with certain ornamental value and collection value.
Butterfly orchid is the most ornamental orchid plant in the modern time, and is favored by people due to its gorgeous flower color and unique flower type, but the butterfly orchid has a single leaf color and does not have blue petals. How to change the color of the petals of the phalaenopsis by genetic engineering means is receiving more and more attention.
Disclosure of Invention
The invention provides a gene for regulating and controlling the blue generation of phalaenopsis petals, which changes the color of the phalaenopsis petals by means of genetic engineering, thereby having unique ornamental value.
The invention provides a gene for regulating and controlling the generation of blue color of butterfly orchid petals, 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.
Further, the phalaenopsis is a large hot pepper phalaenopsis or a striped phalaenopsis.
In a second aspect, the invention provides a protein for regulating and controlling the blue generation of phalaenopsis petals, wherein the protein is encoded by the gene of the first aspect of the invention.
In a third aspect, the present invention provides a recombinant expression vector comprising a nucleotide sequence according to the first aspect of the present invention.
Further, the recombinant expression vector is UBI1300-GFP or p 416-TEF.
In a fourth aspect, the present invention provides a recombinant expression transformant comprising the recombinant expression vector according to 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.
In a sixth aspect, the invention provides a method for regulating and controlling the color of petals of phalaenopsis, which comprises injecting the recombinant expression transformant according to the fourth aspect into the petals of phalaenopsis.
The seventh aspect of the invention provides the use of the gene provided in the first aspect in regulating the transport capacity of iron element.
The method effectively regulates and controls the color of the butterfly orchid petal by means of genetic engineering, and provides a theoretical basis for breeding of the butterfly orchid, wherein the color of the butterfly orchid petal appears as a blue color block in a microstructure in a transient overexpression strain of the butterfly orchid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1a illustrates various tissue portions of a petal of a butterfly orchid according to an embodiment of the present invention;
FIG. 1b shows the PeVIT expression levels of different tissue regions in a petal of Phalaenopsis according to an embodiment of the present invention;
FIG. 2 is a phenotypic graph of petal microstructures of an experimental group UBI1300-GFP-PeVIT and a control group UBI1300-GFP-EV of Phalaenopsis capsici after 3 days according to an embodiment of the invention;
FIG. 3 is a phenotypic plot of petal microstructures after 3 days for the experimental group UBI1300-GFP-PeVIT and the control group UBI1300-GFP-EV of striped butterfly orchid according to an embodiment of the present invention;
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 Phalaenopsis capsici according to an embodiment of the present invention;
FIG. 5 shows the result of detecting the expression level of PeVIT in the experimental group UBI1300-GFP-PeVIT and the control group UBI1300-GFP-EV of Phalaenopsis striata according to an embodiment of the present invention;
FIG. 6 is a graph showing the expression of iron transport activity of PeVIT in a yeast strain according to an embodiment of the present invention.
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 used are commercially available or publicly available reagents unless otherwise specified.
In the present invention, various vectors known in the art, such as commercially available vectors, including plasmids and the like, can be used.
Extraction of butterfly orchid gene PeVIT
1. Extracting the wild phalaenopsis petal total RNA for RNAblant (sold in the market) by using a kit, 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 762bp 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, wherein the gene is named as PeVIT. The protein sequence coded by the nucleotide sequence is shown as SEQ ID NO.2, consists of 254 amino acid residues, and has the molecular weight of 26.975 kilodaltons.
Expression spectrum verification of PeVIT on big pepper and striped butterfly orchid petal colors
1. Extracting RNA of different tissue parts of a butterfly orchid model plant butterfly orchid by using a kit (commercially available) for RNAplantt, wherein the extracted parts are shown in figure 1a, and reverse transcribing 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 PeVIT gene by taking cDNA (complementary deoxyribonucleic acid) obtained by reverse transcription of the large hot pepper butterfly orchid and the striped butterfly orchid at different tissue parts as a template. The expression level of PeVIT in different tissue parts is tested, the test result is shown in figure 1b, the expression level of PeVIT in petals is higher, and the PeVIT is probably involved in the formation of the petal color of phalaenopsis.
UBI1300-GFP (Green fluorescent protein) induced transient overexpression of PeVIT genes of capsicum annuum and phalaenopsis striata
1. Operably connecting 762bp of an open reading frame of a PeVIT gene to a UBI1300-GFP vector to form a UBI1300-GFP-PeVIT vector containing the gene segment, and then transferring the vector into agrobacterium GV3101 to obtain a recombinant expression transformant; the constructed UBI1300-GFP vector takes pCAMBIA1300 vector as a framework to transform the original promoter element CAMV35S, and the original CAMV35S promoter is replaced by a UbI promoter element.
2. The recombinant expression 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, centrifuging for 10 minutes at 4 ℃ and 3700rpm, removing the supernatant after centrifugation, and using 0.5M MgCl2Repeating blowingResuspending for three times;
4. taking a little of the agrobacterium tumefaciens re-suspended bacteria liquid obtained in the step 3, measuring the OD value of the bacteria liquid, and using 0.5M MgCl2Diluting the bacterial liquid to make OD value about 0.6, adding 100 μ M acetosyringone 1.5 times of total volume and MES (morpholine ethanesulfonic acid) 20 times of total volume, resuspending the cell precipitate, and standing at room temperature for 3-4 hr;
5. sucking the standing agrobacterium transformation liquid by using a 1ml syringe with a needle head, and respectively injecting the agrobacterium transformation liquid into virus-free large capsicum phalaenopsis and striated phalaenopsis petals;
6. culturing for 2-3 days after injection, observing petals of large hot pepper butterfly orchid and striped butterfly orchid, taking the treated butterfly orchid as an experimental group and naming the treated butterfly orchid as UBI1300-GFP-PeVIT, and setting a control group and naming the treated butterfly orchid as UBI 1300-GFP-EV.
Selecting a large hot pepper butterfly orchid experimental group UBI1300-GFP-PeVIT petals shown as a in figure 2 and a large hot pepper butterfly orchid control group UBI1300-GFP-EV petals shown as b, observing the same visual field by using a fluorescence microscope under the condition of different exciting lights, wherein the observation result is shown as the right side of figure 2, and the recombinant vector is marked with GFP, so that the part with green fluorescence under the green exciting lights is the part expressed by PeVIT; bright Field is an observation image of cells under Bright Field, no self-luminescence exists in the Bright Field, and the change of color can be visually observed, for example, in the Bright Field mode of petals of an experimental group, a blue color block can be seen in a region indicated by an arrow, but the blue color block cannot be related to the white color block to indicate that the blue color block is caused by overexpression of PeVIT, so that Merged is carried out by using corresponding software, namely GFP and the Bright Field are fused to indicate that the blue color block is caused by the overexpression of PeVIT.
Two petals of one petal of the striped phalaenopsis are selected, as shown in the left side of the figure 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, the observation is carried out by adopting the same method as that of the big pepper phalaenopsis, so that the experimental group UBI1300-GFP-PeVIT petal is observed with a blue color block in a microstructure compared with the control group UBI1300-GFP-EV petal.
Expression verification of PeVIT gene in large hot pepper and striped butterfly orchid mutant strain
1. Extracting total RNA of petals shown in FIGS. 2-3 with kit RNAPlant (commercially available), and reverse transcribing the total RNA into cDNA with 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. PeVIT was subjected to gene expression efficiency verification using cDNA obtained by reverse transcription of petals shown in FIGS. 2 to 3 as a template. As shown in FIGS. 4-5, it was observed that the expression level of PeVIT was significantly increased in the petals of the transient overexpression mutant line.
Expression of iron element transport ability of PeVIT gene in p 416-TEF-inducible yeast strain mutant delta CCC1 and wild-type yeast strain DY150
1. Operably connecting 762bp open reading frame of PeVIT gene to p416-TEF expression vector to form p416-TEF-PeVIT vector containing the gene segment;
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 defect type solid culture medium, streaking the four grown yeast strains on a new Ura selection defect type solid culture medium, and recovering the four grown yeast strains in a Ura selection defect type liquid culture medium after the yeast strains grow out;
4. the bacterial liquid recovered in the step 3 and containing the four yeast strains is subjected to Ura selection defect type solid culture medium and contains 7.5mM FeSO4The above-mentioned Ura selection-deficient solid medium was cultured separately, and yeast strain mutants Δ CCC1 transformed with p416-TEF-PeVIT expression Vector and control p416-TEF-Vector and wild-type yeast strain DY150 were observed.
As shown in FIG. 6, it was found that the Fe transport ability of CCC1 transferred into p416-TEF-PeVIT expression vector was rescued to make it possible to obtain a Fe transport level of 7.5mM FeSO4The surface PeVIT gene can be extracted by growing on the Ura selection defect type solid culture mediumTransport capacity of high-iron element.
In addition, the invention also provides nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO.8, which are respectively a nucleotide sequence derived from the nucleotide sequence shown as SEQ ID NO.1 and a nucleotide sequence with at least 80% homology with the nucleotide sequence shown as SEQ ID NO.1, and have the same effect as the sequence shown as SEQ ID NO. 1.
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
<110> Shanghai university of Master
<120> gene PeVIT for regulating and controlling generation of butterfly orchid petal blue and application thereof
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 738
<212> DNA
<213> Phalaenopsis aphrodite (Phalaenopsis aphrodite H.G. Reichenbach)
<400> 1
atggtgagag ctccatgctg cgagaagatg gggctaaaga aggggccatg gactgcagag 60
gaggaccaga ttctgatctc ttatatacaa aaccatggcc atggaaactg gagagctctc 120
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
<210> 2
<211> 245
<212> PRT
<213> Phalaenopsis aphrodite (Phalaenopsis aphrodite H.G. Reichenbach)
<400> 2
Met Val Arg Ala Pro Cys Cys Glu Lys Met Gly Leu Lys Lys Gly Pro
1 5 10 15
Trp Thr Ala Glu Glu Asp Gln Ile Leu Ile Ser Tyr Ile Gln Asn His
20 25 30
Gly His Gly Asn Trp Arg Ala Leu Pro Lys Leu Ala Gly Leu Leu Arg
35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Thr Asn Tyr Leu Arg Pro Asp
50 55 60
Ile Lys Arg Gly Asn Phe Thr Arg Glu Glu Glu Asp Ala Ile Ile Asn
65 70 75 80
Leu His Gln Met Leu Gly Asn Arg Trp Ser Ala Ile Ala Ala Lys Leu
85 90 95
Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Val Trp His Thr His Leu
100 105 110
Lys Lys Arg Leu Thr Arg Thr Asp Lys Glu Thr Gly Gln Glu Arg Ile
115 120 125
Arg Lys Thr Gln Ile Glu Pro Lys Glu Glu Met Pro Thr Gln Ser Tyr
130 135 140
Ser Ser Thr Pro Glu Pro Ser Ser Ser Ser Ser Thr Val Asp Asn Ser
145 150 155 160
Gln Asn Ser Met Glu Ser Phe Ser His Glu Ala Glu Ala Gln Gly Ile
165 170 175
Asp Glu Ser Phe Trp Thr Glu Val Leu Lys Met Asp Ser Asn Asp Glu
180 185 190
Tyr Tyr Cys Asn Ser Ser Asp Ser Met Ala Met Glu Gly Phe Asn Ser
195 200 205
Ser Asp Phe Ser Tyr Asp Lys Phe Trp Leu Ser Ala Gly Val Arg Glu
210 215 220
Glu Asp Asp Asp Leu Ser Phe Trp Leu Arg Ile Phe Leu Gln Ala Glu
225 230 235 240
Glu Phe Pro Gln Ile
245
<210> 3
<211> 24
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 3
atggtgagag ctccatgctg cgag 24
<210> 4
<211> 24
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 4
tcaaatctgt ggaaattctt cagc 24
<210> 5
<211> 20
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 5
gcgaagctac ctggtagaac 20
<210> 6
<211> 22
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 6
attcgttctt gtccggtctc tc 22
<210> 7
<211> 750
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 7
atggcgttag aacaaggcca attttcggcg aacgaaagtt tgtcagagaa gtttctggcc 60
caacacaagg agaagcattt caccgccggt gaaattgtcc gcgacgtcat catcggcgtc 120
tctgatggcc ttactgtccc cttcgccctc gccgccggtc tgtccggcgc cgccgcacct 180
tcctctctca tcctcaccgc cggactcgcc gaggtcgccg ccggcgccat ctccatggga 240
ctcggcgggt atctagcggc gaagagcgag gcggaccatt actggcgcga atacaagcgg 300
gagcaggagg agatcgttag tgttcctgac actgaggaag ctgaagtatg cgagatactc 360
gcggagtatg gtctgcagcc ccacgagtac gcccccctag ttgcagcgct ccgcaggaat 420
cctcaagcct ggctggactt tatgatgaaa tttgagctgg gattagagaa gccagagcca 480
aagagggcac tggagagcgc catgacaatc gccttctcct acattgcagg tgggttggtg 540
ccgctgcttc cttacatgtt catctccact gctcagaagg caatgttcat atctgttctt 600
gtcactattg ctgcgctcct tttctttggc tacgtcaaag gccatttcac aggcaaccgc 660
ccgctctata gcgctttcca gacagctttt atcggcgcga tcgcgtctgc tgccgcattt 720
gggatggcga aggccgtgca ggccctctga 750
<210> 8
<211> 741
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 8
atggtgaagg agttcgtgca ggacgaggag aagcagcggc tgctgctgga cgagcacacc 60
gagaagcact tcaccgccgg cgaggtggtc cgcgacatca tcatcggcgt ctccgacggc 120
ctcaccgtgc cgttcgccct cgccgccggc ctgtccggcg ccaacgcccc ctccgccctc 180
gtcctcaccg ccggcctcgc cgaggtcgcc gccggcgcca tctccatggg cctcggaggg 240
tatctggcgg cgaagagcga cgccgaccac taccaccgcg agctgcagag ggagcaggag 300
gagatcgaca ccgtgccgga cacagaggcc gcggagatcg cggacatact gtcgcagtat 360
gggctgggcc cagaggagta tgggcccgtt gttaactccc tccgcagcaa ccccaaggcc 420
tggctcgaat tcatgatgaa gtttgagttg ggactggaga agccggaacc aaggagggcg 480
ctgatgagcg cgggaacgat cgcgctggct tatgtggtgg gtgggctggt gccactccta 540
ccctacatgt ttgtgccgac ggccgaccga gccatggcca cctcggtcgt cgtcacgctc 600
gccgcacttc tcttcttcgg ctatgtcaag ggccggttta ctggcaaccg gcccttcatc 660
agcgccttcc agaccgctgt tatcggcgct ctcgcctccg ccgccgcctt cggcatggcc 720
aaggccgtgc agtccatcta a 741