CN113755503B

CN113755503B - Gene PeGRF6 for regulating and controlling butterfly orchid leaf development and application thereof

Info

Publication number: CN113755503B
Application number: CN202110974455.3A
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: 2021-08-24
Filing date: 2021-08-24
Publication date: 2023-09-01
Anticipated expiration: 2041-08-24
Also published as: CN113755503A

Abstract

The application provides a gene PeGRF6 for regulating and controlling the leaf development of butterfly orchid and application thereof. The first aspect of the application provides a gene PeGRF6 for regulating and controlling the leaf development 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 gene provided by the application can effectively regulate and control the development of the small orchid butterfly orchid leaves, reduce the leaf area of the new small orchid butterfly orchid leaves, improve the anthocyanin and chlorophyll content in the leaves, and provide a theoretical basis for breeding the small orchid butterfly orchid.

Description

Gene PeGRF6 for regulating and controlling butterfly orchid leaf development and application thereof

Technical Field

The application relates to the technical field of genetic engineering, in particular to a gene PeGRF6 for regulating and controlling the leaf development of butterfly orchid and application thereof.

Background

With the rapid improvement of economic strength in China in recent years, the flower industry has also been developed unprecedentedly, people are no longer satisfied with common flower types, but pursue special flowers and rare flowers with certain ornamental value and collection value, and people prefer to combine potted flowers, so that the flower pot is attractive and small, and can be placed in any places needing decoration such as desktops. The small orchid butterfly orchid is popular as the most ornamental orchid plant in the current generation due to the gorgeous flower color and unique flower shape, and the small orchid butterfly orchid has long and wide leaves, which brings operational difficulties to flower assembly and greatly reduces the ornamental value due to disorder. There is increasing interest in how to obtain a small and attractive orchid.

Disclosure of Invention

The application provides a gene PeGRF6 for regulating and controlling the leaf development of butterfly orchid, which changes the leaf development of small orchid by means of genetic engineering, thereby having unique ornamental value.

The first aspect of the application provides a gene PeGRF6 for regulating and controlling the leaf development of phalaenopsis amabilis, 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 application provides a protein for regulating the leaf development of butterfly orchid, said protein being encoded by the gene according to the first aspect of the present application.

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

Further, the recombinant expression vector is a cymv virus.

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

Further, the recombinant expression transformant is Agrobacterium.

In a fifth aspect, the present application provides the use of the gene provided in the first aspect for reducing the leaf area of phalaenopsis amabilis.

In a sixth aspect, the present application provides a method for reducing the leaf area of phalaenopsis amabilis, comprising injecting the recombinant expression transformant according to the fourth aspect of the present application into phalaenopsis amabilis leaves.

In a seventh aspect, the present application provides an application of the gene provided in the first aspect in increasing chlorophyll and anthocyanin contents in phalaenopsis leaves.

According to an eighth aspect of the present application, there is provided a method for increasing chlorophyll and anthocyanin levels in leaves of phalaenopsis amabilis, the method comprising injecting the recombinant expression transformant according to the fourth aspect of the present application into leaves of phalaenopsis amabilis.

The application effectively regulates and controls the development of the small orchid butterfly orchid leaves by means of genetic engineering, reduces the area of new leaves of the small orchid butterfly orchid, improves the anthocyanin and chlorophyll contents in the leaves, and provides a theoretical basis for breeding the small orchid butterfly orchid.

Drawings

In order to more clearly illustrate the embodiments of the present application 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 application, and that other drawings can be obtained according to these drawings without inventive effort to a person skilled in the art.

FIG. 1a shows the sizes of the leaves of Phalaenopsis amabilis at different developmental stages according to the embodiment of the present application;

FIG. 1b shows the amount of PeGRF6 expressed in the leaves of Phalaenopsis amabilis at different developmental stages according to the examples of the present application;

FIG. 2 shows the whole plant development conditions of experimental group pCymMV-PeGRF6 and control group cymMV-EV small orchid butterfly orchid at 0d and 35d provided by the embodiment of the application;

FIG. 3 shows the single leaf development of the experimental group pCymMV-PeGRF6 and the control group cymMV-EV small orchid butterfly orchid at 35d according to the embodiment of the application;

FIG. 4a is a view of the experimental group of pCymMV-PeGRF6 leaf cells provided in the examples of the present application under a 20-fold microscope;

FIG. 4b is a 20-fold microscopic view of a control CymMV-EV leaf cell according to an embodiment of the application;

FIG. 5 shows the number of cells in the leaves of the experimental group pCymMV-PeGRF6 and the control group cymMV-EV small orchid butterfly orchid provided in the example of the present application;

FIG. 6 shows cell volumes in leaves of experimental group pCymMV-PeGRF6 and control group cymMV-EV small orchid butterfly orchid according to an embodiment of the present application;

FIG. 7 shows the expression levels of PeGRF6 in the leaves of experimental group pCymMV-PeGRF6 and control group CymMV-EV provided in the examples of the present application;

FIG. 8 shows the expression levels of cyclin in the leaves of experimental group pCymMV-PeGRF6 and control group CymMV-EV provided in the examples of the present application;

FIG. 9a is a view of 40-fold microscope of the experimental group pCymMV-PeGRF6 leaf cells provided in the examples of the present application;

FIG. 9b is a view of a control CymMV-EV leaf cell according to an embodiment of the present application under a 40-fold microscope;

FIG. 10 shows anthocyanin levels in the leaves of experimental group pCymMV-PeGRF6 and control group CymMV-EV provided in the examples of the present application;

FIG. 11 shows chlorophyll content in leaves of experimental group pCymMV-PeGRF6 and control group CymMV-EV provided in examples of the present application.

Detailed Description

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

With the rapid improvement of economic strength in China in recent years, the flower industry has also been developed unprecedentedly, people are no longer satisfied with common flower types, but pursue special flowers and rare flowers with certain ornamental value and collection value, and people prefer to combine potted flowers, so that the flower pot is attractive and small, and can be placed in any places needing decoration such as desktops. The small orchid butterfly orchid is popular as the most ornamental orchid plant in the current generation due to the gorgeous flower color and unique flower shape, and the small orchid butterfly orchid has long and wide leaves, which brings operational difficulties to flower assembly and greatly reduces the ornamental value due to disorder. Therefore, there is increasing interest in how to provide a small and attractive orchid. The following describes the technical scheme of the present application and how the technical scheme of the present application solves the above technical problems in detail with specific embodiments.

Extraction of small orchid butterfly orchid gene

1. Extracting total RNA of leaves of the phalaenopsis amabilis by using a kit (commercially available) for RNAplant, 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 1746bp band from the 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 PeGRF6. The amino acid sequence coded by the nucleotide sequence is shown as SEQ ID NO.2, consists of 581 amino acids and has a molecular weight of 63.061 kilodaltons.

Verification of PeGRF6 expression profile at different stages of the leaf development of Phalaenopsis amabilis

1. Extracting RNA of leaves of the small orchid butterfly orchid at different stages of development by using a kit (commercially available), and reversely transcribing the total RNA into cDNA by using a reverse transcription kit (commercially available);

2. designing fluorescent real-time quantitative primers according to transcriptome sequencing data (the amplified fragments are 100-300bp according to experimental requirements, so that an optimal pair of primers is screened to amplify partial fragments of PeGRF6 to reflect the change condition of the relative expression quantity of the PeGRF6 in each period of leaf development), wherein the primer sequences are shown in SEQ ID NO.5 and SEQ ID NO. 6;

3. the expression profile of the PeGRF6 gene was verified using cDNA obtained by reverse transcription of the small orchid butterfly orchid (five sets of leaves shown in FIG. 1 a) at different developmental stages as a template. The results of observing the expression quantity of PeGRF6 in different stages of the leaf development of the butterfly orchid in the small orchid show in FIG. 1b, and the expression of PeGRF6 in the leaf 0 is higher, which indicates that the PeGRF6 may be involved in the formation of tender new leaves of the butterfly orchid in the small orchid.

Cymv virus-induced PeGRF6 gene silencing of phalaenopsis parvula

1. Operably linking 200-300bp of an open reading frame of a PeGRF6 gene with a CymMV virus vector to form a CymMV-PeGRF6 vector containing the gene fragment, and transferring the vector into agrobacterium (GV 3101) to obtain a recombinant expression transformant;

2. culturing recombinant expression transformant in 5ml LB culture medium containing 100 mu M acetosyringone and 50 mu g/ml kanamycin at 28 deg.C and 200rpm for 16 hr to activate agrobacterium thallus to propagate and reach optimal plant infection concentration; subsequently, the culture was continued in 5ml of LB medium containing 100. Mu.M acetosyringone and 50. Mu.g/ml kanamycin at 28℃and 200rpm for 13-16 hours, and the recombinant expression transformant obtained by the culture was subjected to subculture until OD600 reached 0.8-1.0;

3. the Agrobacterium solution containing the recombinant expression 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 transformation liquid by using a 1ml syringe with a needle, and injecting the agrobacterium transformation liquid into the leaves of the phalaenopsis amabilis;

5. culturing for 30-40 days after injection, observing the leaves of the phalaenopsis amabilis, and taking the treated phalaenopsis amabilis as an experimental group, named pCymMV-PeGRF6, and setting a control group, named pCymMV-EV.

As shown in FIGS. 2-3, leaves of the experimental group pCymMV-PeGRF6 grew slowly compared to the control group, and showed a tendency of "slimming".

Observation of cell number and volume of cymv virus-induced small orchid butterfly orchid leaf

1. Selecting newly grown leaves of the experiment group and the control group shown in fig. 2, cutting part of epidermal tissues on the epidermis of the leaves by using a cutter, then placing the leaves on a glass slide, adding water, covering the glass slide, and manufacturing simple plant tissue slices;

2. placing the plant tissue slice on a microscope stage, observing the slice by using a 40X microscope, and preserving pictures, wherein the number of cells in the new leaves of the experimental group pCymMV-PeGRF6 is obviously more than that in the new leaves of the control group, and the cell volume is obviously lower than that of the control group as shown in the figures 4a-4 b;

3. the pictures were subjected to detailed calculation of cell number and volume, and the summary data were made by prism's drawing software, resulting in the results shown in fig. 5-6, which showed the same law as in fig. 4a-4 b.

Expression verification of PeGRF6 gene and cyclin gene in cymMV virus-induced small orchid butterfly orchid mutant line

1. Extracting total RNA of newly grown leaves of the experimental group and the control group shown in FIG. 2 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 transcriptome sequencing data, wherein the primer sequences are shown as SEQ ID NO.5 and SEQ ID NO. 6;

3. the gene expression efficiency of PeGRF6 was verified using cDNA obtained by reverse transcription of the leaf as shown in FIG. 2 as a template. The expression levels of both the PeGRF6 and cyclin family genes (genes of the PeCycs family are indicated on the abscissa) were observed to decrease in the leaves of the VIGS silent mutant line, as shown in FIGS. 7 to 8.

Determination of anthocyanin content and chlorophyll content of cymv virus-induced small orchid butterfly orchid leaves

1. The newly grown leaves of the experimental group and the control group shown in fig. 2 were selected, and 6 groups each of 0.2g were weighed. The cell status was observed under a 40X microscope as shown in FIGS. 9a-9 b.

In order to further verify the chlorophyll and anthocyanin contents in cells, in the embodiment, the materials are equally divided, and half of new leaves of an experimental group and new leaf materials of a control group strain are placed in a chlorophyll extracting solution (70% acetone+20% ethanol+10% water) so that the leaves are all immersed in the extracting solution, and a shaking table is gently shaken overnight at 4 ℃ until all chlorophyll in the leaves enters the extracting solution; placing new leaf materials of half of small orchid butterfly orchid VIGS silent strains and control strains in a water bath at a high temperature of 40 ℃ and leaching for 24h, wherein 5ml of anthocyanin extracting solution (90% (V/V) ethanol: 0.4 mol.L-1 hydrochloric acid=1:1) is added;

2. measuring absorbance of chlorophyll extract of the material on a spectrophotometer at corresponding wavelengths (663 nm and 645 nm); shaking the anthocyanin extracting solution of the material uniformly, and measuring absorbance at 519nm wavelength;

3. taking the absorbance of chlorophyll extract of the material as data, and taking the absorbance into the following formula to calculate the total chlorophyll content, wherein the formula is as follows:

Chla(mg/L)＝12.7×A663-2.69×A645

Chlb(mg/L)＝22.9×A645-4.68×A663

total chlorophyll Chl (mg/L) =chla+chlb=20.2×a645+8.02×a663

Chlorophyll content in sample (mgChl/g) =chlorophyll content in cuvette x dilution fold/sample amount (g)

A645 and a663 are absorbance at 645nm and 663nm, respectively;

taking the absorbance of anthocyanin extracting solution of the material as data, and taking the absorbance into the following formula to calculate the total anthocyanin content, wherein the formula is as follows: anthocyanin content (μg) =a519×n×v/0.0772

A519 is absorbance at 519nm, n is dilution factor, V is total volume of anthocyanin extract, and 0.0772 is absorption coefficient;

5. the data were finally presented as icon data by prism's drawing software, and as can be seen from fig. 10-11, cymv virus induced significant increases in chlorophyll and anthocyanin content in new leaves of the mutant strain of cymv butterfly orchid.

In addition, the application 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 application, and not for limiting the same; although the application 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 application.

Sequence listing

<110> Shanghai university of teachers and students

<120> Gene PeGRF6 for regulating and controlling butterfly orchid leaf development and use thereof

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 1746

<212> DNA

<213> little orchid butterfly orchid (Phalaenopsis equestris)

<400> 1

atggacttgg gttgttccct gagcacggat ggcttggtcc ctgtctcgtc tgaccccaat 60

gttctcatcc cctccttttt tcccttggac tccgaaccca gcaagcaaag ggggctgttt 120

caatccagtt tctccaagca tggaagatct gtcgtggttg aggaggatga tgagtactgg 180

actcgtttga agacggctag aggggatcca atgctcacat caacaagcaa ggcggcaact 240

tgcctccttg gatccggctc tcactccctc ttccctgatg gagagcaaat gctgaacttt 300

tcttcatgga aatcagatgc tctggtgctc aataatcatt ggaactcacc ttccttcaac 360

catccttcat catcttcagc ttaccctact ttcaggaatg cagcgtgctc tggcaatcca 420

acccttaaca ttcaaggagc tgtgtctgag atcagtggac ccttcaccac ttctcagtgg 480

tcagaactgg agcaacagac tctgatctac aaatatattg atgcaaaagt tccattacca 540

tctaatttgc tcttgtcaat cagacgaagc atcaaccctt ctgggttgtc ttccttcatt 600

gctggaactt atagaccagc aggcacattg ggatggggaa atttccatct gggtttggct 660

ggaagtgctg atccggagcc tggcagatgc cgtcgaacag atgggaaaaa atggcggtgt 720

tcgagggatg cagttgtgga tcagaagtac tgcgagcgac acatgaaccg tggtcgccat 780

cgttcaagaa agcgtgtgga aggccacaat ggccatgctg ccaaggctgc tgtgcctgct 840

gccattactt cgtcgcaatc ggcatcggct ctttccagtg gtggttcttc taataatctc 900

gccgtggctc agcagcagag caaaaacata caaccaagct tcacggaacc ttcatctctg 960

caattggata ggttactgat gaacagagac aatacaaatg tacaaagaca agatgccaaa 1020

ggcctctccg tgctgactga tgtaaactca aaatccacag gtaccttgtt gcccatgtcc 1080

aaacaactaa atccctttga agaaacttca acccgtgtag acttgggact tgtatcaact 1140

gattctgtac ttaataatga agggaactgc tcctcagaga atctcagttt tgttttttat 1200

cactctgaaa tgaatgatca gcacactcga tcccatccct ttcccttccg acattttatt 1260

gatgactggt ccaaaacacc atctgatggg atggaaaaga agcaatcaaa cagaactgaa 1320

ctctccattt caatccccat ctcctcctcc gccaaccaag acaagctgtc attttcccct 1380

ctcaggctat cttgtgagtt caatcctacg aatgatggca cccaaagggg tttaagttgg 1440

atacccattt cgtgggagcc aaacattggc ggacctcttg gggaggtttt gaccaacacg 1500

agcagtacta ccccaaagga tcttagtagc aatttatcag cttcatctct gaacctcatg 1560

actgatgggt ggaactccaa ggctagattt gaatcatcgc ccacaggagt gttgcaggag 1620

tctggctttg gttcgttgtc cagcagtgca ggaagcagcc cccgggcaga gaaccataag 1680

gctcacgaaa gtgatgacct acttggctca acctttgtga atttatcaac gaacccctcg 1740

ctctaa 1746

<210> 2

<211> 581

<212> PRT

<213> little orchid butterfly orchid (Phalaenopsis equestris)

<400> 2

Met Asp Leu Gly Cys Ser Leu Ser Thr Asp Gly Leu Val Pro Val Ser

1 5 10 15

Ser Asp Pro Asn Val Leu Ile Pro Ser Phe Phe Pro Leu Asp Ser Glu

20 25 30

Pro Ser Lys Gln Arg Gly Leu Phe Gln Ser Ser Phe Ser Lys His Gly

35 40 45

Arg Ser Val Val Val Glu Glu Asp Asp Glu Tyr Trp Thr Arg Leu Lys

50 55 60

Thr Ala Arg Gly Asp Pro Met Leu Thr Ser Thr Ser Lys Ala Ala Thr

65 70 75 80

Cys Leu Leu Gly Ser Gly Ser His Ser Leu Phe Pro Asp Gly Glu Gln

85 90 95

Met Leu Asn Phe Ser Ser Trp Lys Ser Asp Ala Leu Val Leu Asn Asn

100 105 110

His Trp Asn Ser Pro Ser Phe Asn His Pro Ser Ser Ser Ser Ala Tyr

115 120 125

Pro Thr Phe Arg Asn Ala Ala Cys Ser Gly Asn Pro Thr Leu Asn Ile

130 135 140

Gln Gly Ala Val Ser Glu Ile Ser Gly Pro Phe Thr Thr Ser Gln Trp

145 150 155 160

Ser Glu Leu Glu Gln Gln Thr Leu Ile Tyr Lys Tyr Ile Asp Ala Lys

165 170 175

Val Pro Leu Pro Ser Asn Leu Leu Leu Ser Ile Arg Arg Ser Ile Asn

180 185 190

Pro Ser Gly Leu Ser Ser Phe Ile Ala Gly Thr Tyr Arg Pro Ala Gly

195 200 205

Thr Leu Gly Trp Gly Asn Phe His Leu Gly Leu Ala Gly Ser Ala Asp

210 215 220

Pro Glu Pro Gly Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys

225 230 235 240

Ser Arg Asp Ala Val Val Asp Gln Lys Tyr Cys Glu Arg His Met Asn

245 250 255

Arg Gly Arg His Arg Ser Arg Lys Arg Val Glu Gly His Asn Gly His

260 265 270

Ala Ala Lys Ala Ala Val Pro Ala Ala Ile Thr Ser Ser Gln Ser Ala

275 280 285

Ser Ala Leu Ser Ser Gly Gly Ser Ser Asn Asn Leu Ala Val Ala Gln

290 295 300

Gln Gln Ser Lys Asn Ile Gln Pro Ser Phe Thr Glu Pro Ser Ser Leu

305 310 315 320

Gln Leu Asp Arg Leu Leu Met Asn Arg Asp Asn Thr Asn Val Gln Arg

325 330 335

Gln Asp Ala Lys Gly Leu Ser Val Leu Thr Asp Val Asn Ser Lys Ser

340 345 350

Thr Gly Thr Leu Leu Pro Met Ser Lys Gln Leu Asn Pro Phe Glu Glu

355 360 365

Thr Ser Thr Arg Val Asp Leu Gly Leu Val Ser Thr Asp Ser Val Leu

370 375 380

Asn Asn Glu Gly Asn Cys Ser Ser Glu Asn Leu Ser Phe Val Phe Tyr

385 390 395 400

His Ser Glu Met Asn Asp Gln His Thr Arg Ser His Pro Phe Pro Phe

405 410 415

Arg His Phe Ile Asp Asp Trp Ser Lys Thr Pro Ser Asp Gly Met Glu

420 425 430

Lys Lys Gln Ser Asn Arg Thr Glu Leu Ser Ile Ser Ile Pro Ile Ser

435 440 445

Ser Ser Ala Asn Gln Asp Lys Leu Ser Phe Ser Pro Leu Arg Leu Ser

450 455 460

Cys Glu Phe Asn Pro Thr Asn Asp Gly Thr Gln Arg Gly Leu Ser Trp

465 470 475 480

Ile Pro Ile Ser Trp Glu Pro Asn Ile Gly Gly Pro Leu Gly Glu Val

485 490 495

Leu Thr Asn Thr Ser Ser Thr Thr Pro Lys Asp Leu Ser Ser Asn Leu

500 505 510

Ser Ala Ser Ser Leu Asn Leu Met Thr Asp Gly Trp Asn Ser Lys Ala

515 520 525

Arg Phe Glu Ser Ser Pro Thr Gly Val Leu Gln Glu Ser Gly Phe Gly

530 535 540

Ser Leu Ser Ser Ser Ala Gly Ser Ser Pro Arg Ala Glu Asn His Lys

545 550 555 560

Ala His Glu Ser Asp Asp Leu Leu Gly Ser Thr Phe Val Asn Leu Ser

565 570 575

Thr Asn Pro Ser Leu

580

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggacttgg gttgttccct 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ttagagcgag gggttcgttg 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

tccgaaccca gcaagcaaag 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tgagagccgg atccaaggag 20

<210> 7

<211> 1749

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atggacttgg gttgttccct gagcacggat ggcttggtcc ctgtctcgtc tgaccccaat 60

gttctcatcc cctccttttt tcccttggac tccgaaccca gcaagcaaag ggggctgttt 120

caatccagtt tctccaagca tggaagatct gtcgtggttg aggaggatga tgagtactgg 180

actcgtttga agacggctag aggggatcca atgctcacat caacaagcaa ggcggcaact 240

tgcctccttg gatccggctc tcactccctc ttccctgatg gagagcaaat gctgaacttt 300

tcttcatgga aatcagatgc tctggtgctc aataatcatt ggaactcacc ttccttcaac 360

catccttcat catcttcagc ttaccctact ttcaggaatg caggagcgtg ctctggcaat 420

ccaaccctta acattcaagg agctgtgtct gagatcagtg gacccttcac cacttctcag 480

tggtcagaac tggagcaaca gactctgatc tacaaatata ttgatgcaaa agttccatta 540

ccatctaatt tgctcttgtc aatcagacga agcatcaacc cttctgggtt gtcttccttc 600

attgctggaa cttatagacc agcaggcaca ttgggatggg gaaatttcca tctgggtttg 660

gctggaagtg ctgatccgga gcctggcaga tgccgtcgaa cagatgggaa aaaatggcgg 720

tgttcgaggg atgcagttgt ggatcagaag tactgcgagc gacacatgaa ccgtggtcgc 780

catcgttcaa gaaagcgtgt ggaaggccac aatggccatg ctgccaaggc tgctgtgcct 840

gctgccatta cttcgtcgca atcggcatcg gctctttcca gtggtggttc ttctaataat 900

ctcgccgtgg ctcagcagca gagcaaaaac atacaaccaa gcttcacgga accttcatct 960

ctgcaattgg ataggttact gatgaacaga gacaatacaa atgtacaaag acaagatgcc 1020

aaaggcctct ccgtgctgac tgatgtaaac tcaaaatcca caggtacctt gttgcccatg 1080

tccaaacaac taaatccctt tgaagaaact tcaacccgtg tagacttggg acttgtatca 1140

actgattctg tacttaataa tgaagggaac tgctcctcag agaatctcag ttttgttttt 1200

tatcactctg aaatgaatga tcagcacact cgatcccatc cctttccctt ccgacatttt 1260

attgatgact ggtccaaaac accatctgat gggatggaaa agaagcaatc aaacagaact 1320

gaactctcca tttcaatccc catctcctcc tccgccaacc aagacaagct gtcattttcc 1380

cctctcaggc tatcttgtga gttcaatcct acgaatgatg gcacccaaag gggtttaagt 1440

tggataccca tttcgtggga gccaaacatt ggcggacctc ttggggaggt tttgaccaac 1500

acgagcagta ctaccccaaa ggatcttagt agcaatttat cagcttcatc tctgaacctc 1560

atgactgatg ggtggaactc caaggctaga tttgaatcat cgcccacagg agtgttgcag 1620

gagtctggct ttggttcgtt gtccagcagt gcaggaagca gcccccgggc agagaaccat 1680

aaggctcacg aaagtgatga cctacttggc tcaacctttg tgaatttatc aacgaacccc 1740

tcgctctaa 1749

<210> 8

<211> 1779

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atggatttag gttgttctct gagctctgat ggcttggtct ctgtctattc tgaccccaat 60

attcgtatgc cctcctcttt gtttcccttg gaatctgaac ccagcaagca aaggggactg 120

ttccaatcca gtttctccaa gcatggaagg tccattgtgg ttgaggacaa tgatgaatac 180

tggagtcgtt tgaagacggc tagatgtgat acaatgctca cagcaacaag caaggcggca 240

gcttgcctcc ttagatccgg ctctcatacc cttttccctg atggagagca aatgctgaac 300

ttttcttcat ggaaaccaga agctttggtg ctcaataatc attggaactt acctttcttc 360

aaccatccat catcatcttc atgctcttca gcttacccta ccttcaggaa cgcaggagtg 420

tgctctgaca actcaaccct taacatgcat ggagctgtgt ctgggatcag aggacccttc 480

accacttcgc agtggttaga actggagcac caggctctga tctacaagta tatagatgca 540

aaaatgtcaa taccatcaaa tttgctcatg acaatcagac gaagcaacaa ccaatctggg 600

ttctcatcct tcactgctgg atcttttaga caaacaggca cattgggatg gggaaatttc 660

catccaggtt tctctggagg tgctgaccca gagcctggca gatgccgtcg aaccgatgga 720

aaaaagtggc ggtgttcgag ggatgcagtt gttgatcaga agtactgcga gcgacacatg 780

aaccgcggtc gccatcgttc aagaaagcgt gtggaaggcc accttggcca tgctgcaaag 840

gcagctgtgc ctgccatcac ttcttcacaa tccgcatcag ctgtttccag tggtggttct 900

tctaacaacc ttaccactgc tcagcagcag agcgaaaact tacaatcaag tgggacagaa 960

ccttcatccc tgcaattgga taggttgcta ataaacaaag acaatacagg cgaccaaaga 1020

cgaaattccc aaggtctctc catgatgact gatttaatat ccaaatccac aggtaccttg 1080

ttacctattt ccaagcaaca aaatcccttt gaagaaacat cgacccgtgt agacttggga 1140

ctgatatcta ctgattccct acctaacagt caaggaaact gctcctcaga gaaccttagt 1200

tttgttttct ataaccctga aatgaatgat caacatatta gatcccatcc ctttcgacat 1260

tttattgatg attggtcgaa aaacccttct gatgctgatg tggccgataa gatgcaatca 1320

tacagaaccg aactctctat ttcgatcccc atctcttctt tgggcttctc atcatcccct 1380

tgctccccca tccaagacaa actgtcattt ccccccttca gattatcttc tgagttggat 1440

cctctgaatg ctggcagcca aagaggttta agttgggtac caatttcttg ggaaccaacc 1500

attggaggac ctcttggcga ggttttgact aacacaagca ccaccccgaa ggacctgggt 1560

agtaatttat cgacttcatc gctgaacttc atgactgatg gttggaacat gaggacaaga 1620

tttgaatcat caccaacagg agtgttgcag gattctggat ttggttcaat gtccagtagt 1680

acaggaagta gtccccgggc agagaaccat aaggcgcacg aaagtgatga cctccttggc 1740

acaacctttg tgaatctatc ggtgaatccc tctctctaa 1779

Claims

1. GenePeGRF6The application of silencing in reducing the leaf area of phalaenopsis amabilis is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. A method for reducing the leaf area of phalaenopsis amabilis, comprising injecting a recombinant expression transformant comprising a recombinant expression vector which is a CymMV-PeGRF6 vector comprising the nucleotide sequence as set forth in SEQ ID No.1 into phalaenopsis amabilis leaves.

3. GenePeGRF6The application of silencing in improving chlorophyll and anthocyanin contents in leaves of Phalaenopsis amabilis, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

4. A method for increasing chlorophyll and anthocyanin content in leaves of phalaenopsis amabilis, comprising injecting a recombinant expression transformant into leaves of phalaenopsis amabilis, wherein the recombinant expression transformant comprises a recombinant expression vector which is a CymMV-PeGRF6 vector comprising the nucleotide sequence shown in SEQ ID No.1 of claim 1.