CN113789318B - Gene PeFtsH5 and application thereof in improvement of pathogenic bacteria infection resistance of butterfly orchid - Google Patents

Abstract

The application discloses a gene PeFtsH5, which is characterized in that the protein sequence coded by the gene PeFtsH5 is shown as SEQ ID NO. 2; the application also provides application of the gene PeFtsH5 in improving pathogenic bacteria infection resistance of the butterfly orchid, which is characterized in that the application comprises down-regulating the expression of the PeFtsH5 gene in the butterfly orchid.

Description

Gene PeFtsH5 and application thereof in improvement of pathogenic bacteria infection resistance of butterfly orchid

Technical Field

The application relates to the field of biological variety cultivation, in particular to a gene PeFtsH5 and application thereof in improving pathogenic bacteria infection resistance of butterfly orchid.

Background

Orchid is one of the largest families in flowering plants, and orchid has peculiar and changeable flowers and rich colors, and is deeply favored by people. Molecular biology research on orchid is one of the hot spots of research on orchid at present. Butterfly orchid (Phalaenopsis aphrodite Rchb.F.) is a species of the genus Phalaenopsis of the family Orchidaceae, also known as butterfly orchid, named as butterfly, and is native to subtropical rainforest areas. The butterfly orchid is called as "the ocean orchid queen", especially in the new spring, the leaf axilla draws out long pedicel, and the flower is shaped like the butterfly fly, the flower shape is beautiful and unique, the color is gorgeous, and the flowering period is long; while the butterfly orchid has thick, flat, elliptic and oblong blades, which are generally wider, and a base part is more or less narrowed, and has a sheath for joints and stems, and the butterfly orchid stays or withers in dry season. Butterfly orchid is popular with the public by virtue of its big flower and leaf.

The Ftsh gene was originally independently found by 4 different study groups when screening for different e.coli phenotypes. Santos and de Almeida (1975) when studying cell division, a temperature sensitive fibrillogenesis (filamentation temperature-positive) mutant ftsh1 was identified and designated the ftsh gene. Protease FtsH an ATP and Zn encoded by the FtsH gene ²⁺ Depending on the protein, hexamer complexes are composed of homotype or heterotype subunits with molecular weight of 70-80 kDa, and individual subunits have no biological activity. In Arabidopsis, ftsH, a conserved membrane-bound metalloprotease, forms a hexameric ring structure by oligomerization, embedding the proteolytic active site in the center of the hexameric complex cavity.

FtsH was found in prokaryotes such as bacillus subtilis and lactococcus lactis, and in a variety of eukaryotes such as yeast, arabidopsis thaliana, tobacco, alfalfa, etc., indicating that this gene is widely distributed in the genome of organisms. In prokaryotes, the FtsH gene is part of a monocistronic or polycistronic operon and the FtsH protein is localized on the cytoplasmic membrane; in eukaryotes, however, the known FtsH localizes to either the chloroplast or mitochondrial membranes. At present, the expression condition and the function of the PeFtsH5 gene in the butterfly orchid are not clear.

With the improvement of living standard, plants and flowers are paid more attention to, and butterfly orchids have wide market prospect and economic value as ornamental plants with wide audiences; however, various plant diseases seriously affect the cultivation cost and even bring about serious losses, and thus, a person skilled in the art has been dedicated to providing a method for improving the resistance of butterfly orchid against pathogenic bacteria infection.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present application is how to improve the capability of butterfly orchid to resist pathogenic bacteria infection.

In one aspect, the application provides a gene PeFtsH5, which is characterized in that the protein sequence encoded by the gene PeFtsH5 is shown as SEQ ID NO. 2.

In another aspect, the application also provides the use of the gene PeFtsH5 for increasing resistance of butterfly orchid to pathogenic bacterial infection, wherein the use comprises down-regulating expression of the PeFtsH5 gene in butterfly orchid.

In certain embodiments, the down-regulation comprises gene silencing or gene knockout.

In certain embodiments, the gene silencing reduces the expression level of the PeFtsH5 gene by 60% to 70% in the post-silencing plant as compared to the wild-type plant.

In certain embodiments, the protein sequence encoded by the PeFtsH5 gene is set forth in SEQ ID No. 2.

In certain embodiments, the coding sequence of the PeFtsH5 gene is set forth in SEQ ID No. 1.

In certain embodiments, the pathogenic bacteria include botrytis cinerea and/or Phytophthora capsici.

In certain embodiments, the pathogenic bacteria is botrytis cinerea.

In certain embodiments, the pathogenic bacteria is phytophthora capsici.

In another aspect, the application provides a method for preventing or treating gray mold of butterfly orchid, wherein the use comprises down-regulating the expression of the PeFtsH5 gene in butterfly orchid.

In certain embodiments, the down-regulation comprises gene silencing or gene knockout.

In certain embodiments, the gene silencing reduces the expression level of the PeFtsH5 gene by 60% to 70% in the post-silencing plant as compared to the wild-type plant.

In certain embodiments, the protein sequence encoded by the PeFtsH5 gene is set forth in SEQ ID No. 2.

In certain embodiments, the coding sequence of the PeFtsH5 gene is set forth in SEQ ID No. 1.

In another aspect, the application provides a method for increasing resistance of butterfly orchid to pathogenic bacterial infection, comprising down-regulating expression of the PeFtsH5 gene in butterfly orchid.

In certain embodiments, the down-regulation comprises gene silencing or gene knockout.

In certain embodiments, the gene silencing reduces the expression level of the PeFtsH5 gene by 60% to 70% in the post-silencing plant as compared to the wild-type plant.

In certain embodiments, the protein sequence encoded by the PeFtsH5 gene is set forth in SEQ ID No. 2.

In certain embodiments, the coding sequence of the PeFtsH5 gene is set forth in SEQ ID No. 1.

In certain embodiments, the pathogenic bacteria include botrytis cinerea and/or Phytophthora capsici.

In certain embodiments, the pathogenic bacteria is botrytis cinerea.

In certain embodiments, the pathogenic bacteria is phytophthora capsici.

In another aspect, the application also provides a method for improving tobacco resistance to phytophthora capsici infection, characterized in that the application comprises down-regulating the expression of the NbFtsH5 gene in tobacco.

In certain embodiments, the down-regulation comprises gene silencing or gene knockout.

In certain embodiments, the gene silencing reduces the expression level of the NbFtsH5 gene by 60% -70% in post-silencing plants compared to wild-type plants.

In certain embodiments, the coding sequence of the NbFtsH5 gene is set forth in SEQ ID No. 3.

In another aspect, the application also provides a method for preventing or treating phytophthora capsici leonian in tobacco, characterized in that said application comprises the expression of the NbFtsH5 gene in the following tobacco.

In certain embodiments, the down-regulation comprises gene silencing or gene knockout.

In certain embodiments, the gene silencing reduces the expression level of the NbFtsH5 gene by 60% -70% in post-silencing plants compared to wild-type plants.

In certain embodiments, the coding sequence of the NbFtsH5 gene is set forth in SEQ ID No. 3.

In another aspect, the application also provides the use of the NbFtsH5 gene in increasing resistance to phytophthora capsici infection in tobacco, the use comprising down-regulating expression of the NbFtsH5 gene in tobacco.

In certain embodiments, the down-regulation comprises gene silencing or gene knockout.

In certain embodiments, the gene silencing reduces the expression level of the NbFtsH5 gene by 60% -70% in post-silencing plants compared to wild-type plants.

In certain embodiments, the coding sequence of the NbFtsH5 gene is set forth in SEQ ID No. 3.

In another aspect, the application also provides a nucleic acid molecule NbFtsH5, which is characterized in that the coding sequence of the nucleic acid molecule NbFtsH5 is shown in SEQ ID NO. 3.

In another aspect, the application also provides a primer pair, the sequences of which are shown in SEQ ID NO.5 and 6, and the primer pair is used for silencing a tobacco gene NbFtsH5.

According to the application, the expression of the PeFtsH5 gene of the butterfly orchid is reduced through virus-induced gene silencing (virus induced gene silencing, VIGS), and the resistance of the PeFtsH5 silencing strain and the wild strain to botrytis cinerea is analyzed, so that the reduction of the expression of the PeFtsH5 of the butterfly orchid plant can obviously improve the capability of resisting botrytis cinerea infection; in addition, the expression of the homologous gene NbFtsH5 of the butterfly orchid PeFtsH5 gene in tobacco is reduced through VIGS, the resistance of NbFtsH5 silent strains and wild type strains to phytophthora capsici is analyzed, and the resistance of NbFtsH5 silent tobacco strains and corresponding control plants to phytophthora capsici infection is analyzed, so that the reduction of the expression of the NbFtsH5 gene of the tobacco plants and the reduction of the expression of the butterfly orchid PeFtsH5 can obviously improve the resistance of the butterfly orchid plants to phytophthora capsici infection. The application provides an effective method for cultivating new disease-resistant varieties, preventing and treating the gray mold of the butterfly orchid and preventing and treating the phytophthora capsici of the tobacco or the butterfly orchid, reduces the cultivation cost and improves the economic benefit of the market. The conception, specific structure, and technical effects of the present application will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present application.

Drawings

FIG. 1 is a graph showing the results of analysis of the expression pattern of PeFtsH5 in different organs of Phalaenopsis minor according to the present application.

FIG. 2 shows the phenotype of tobacco leaves after NbFtsH5 silencing in the present application.

FIG. 3 is a graph showing the analysis results of the variation of the expression level of NbFtsH5 in the NbFtsH5 silent strain of the present application and the control strain.

FIG. 4 is a graph showing analysis results of changes in expression levels of salicylic acid synthesis-related genes and WRKY transcription factors in NbFtsH5 silent strains and control strains according to the present application.

FIG. 5 is a graph showing the analysis of lesion area variation in NbFtsH5 silent strains and control strains according to the application.

FIG. 6 shows the phenotype of the leaves of Phalaenopsis amabilis after PeFtsH5 silencing in accordance with the present application.

FIG. 7 is a graph showing the analysis of changes in the expression level of PeFtsH5 in the PeFtsH5 silent strain of the present application as compared with the control strain.

FIG. 8 is a graph showing analysis results of changes in the expression level of the transcription factor WRKY in the PeFtsH 5-silenced strain of the present application and in the control strain.

FIG. 9 is a graph showing the analysis of lesion area variation in PeFtsH5 silent strains versus control strains according to the present application.

Detailed Description

The application will be further described with reference to examples, which are to be understood as being illustrative only, which may be embodied in many different forms and should not be construed as limiting the scope of the application to the examples set forth herein.

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

EXAMPLE 1 cloning of the PeFtsH5 Gene of Phalaenopsis parvula

Extracting total RNA of tender leaves of the phalaenopsis amabilis by utilizing a commercially available extraction kit RNAplant, and reversely transcribing the total RNA into cDNA by utilizing a commercially available reverse transcription kit. A2109 bp band was amplified from the small orchid butterfly orchid cDNA by RT-PCR using the reverse transcribed cDNA as a template. And (3) recovering the PCR product to obtain a PeFtsH5 gene coding sequence (CDS), wherein the nucleotide sequence is shown as SEQ ID NO.1, the amino acid sequence coded by the nucleotide sequence is shown as SEQ ID NO.2, and the nucleotide sequence consists of 702 amino acid residues and has the molecular weight of 76.1 kilodaltons.

Example 2 expression profiles of different organs of the Phalaenopsis PeFtsH5 Gene

q-PCR primers (shown as SEQ ID NO.7 and 8) are designed according to CDS sequences (shown as SEQ ID NO. 1) of the PeFtsH5 genes of the butterfly orchid of the small orchid, RNA of different organs (roots, petals and leaves) of the butterfly orchid of the small orchid is extracted, the RNA is reversely transcribed into cDNA, and q-PCR is carried out by taking the cDNA as a template to verify the expression mode of the PeFtsH5 genes of the butterfly orchid of the small orchid on the different organs.

As shown in FIG. 1, the expression analysis of the organs of the small orchid butterfly orchid PeFtsH5 gene by q-PCR shows that the small orchid butterfly orchid PeFtsH5 gene is expressed in different organs (root, petal and leaf), but the expression in the root and leaf is dominant.

Example 3 VIGS Down-regulating tobacco homologous Gene NbFtsH5 to increase disease resistance in tobacco leaves

The homologous gene of PeFtsH5 in Nicotiana benthamiana is NbFtsH5, and the coding sequence of the homologous gene is shown as SEQ ID NO. 3. The coding protein sequence is shown as SEQ ID NO. 4. It is known in the art that the WRKY40, 46, 51, 60, 63, 75 transcription factors in the arabidopsis thaliana AtFtsH4 mutant are up-regulated and combined with the Salicylic Acid (SA) synthesis-related gene promoter, so that the Salicylic Acid (SA) synthesis gene expression is up-regulated and the Salicylic Acid (SA) level is increased; salicylic Acid (SA) is a hormone of plants against biotic stress, the homologous gene of AtFtsH4 in the butterfly orchid of small orchid is PeFtsH5, and the down regulation of the homologous gene NbFtsH5 in Nicotiana benthamiana also has the result of being consistent with Arabidopsis thaliana, which shows that the function of the gene is conserved among different species (see "The Arabidopsis Mitochondrial Protease FtSH Is Involved in Leaf Senescence via Regulation of WRKY-Dependent Salicylic Acid Accumulation and Signaling", shengchun Zhang et al.2017, plant Physiology).

(1) Selecting a homologous gene sequence NbFtsH5 of the butterfly orchid PeFtsH5 gene in tobacco, taking the sequence as a template, designing a VIGS primer (shown as SEQ ID NO.5 and 6), and connecting the partial sequence to a TRV2 virus vector (purchased from Shanghai Rui Chu biotechnology Co., ltd.) to form a recombinant vector.

(2) Recombinant vector Agrobacterium tumefaciens GV3101 (purchased from Shanghai Weidi Biotechnology Co., ltd.) was transformed into Agrobacterium tumefaciens GV3101 as well as viral TRV1 vector (purchased from Shanghai Rui Biotechnology Co., ltd.), two GV3101 bacterial solutions were obtained by culturing, and then tobacco was co-injected to silence the tobacco NbFtsH5 gene by means of virus-induced gene silencing (Virus induced gene silence, VIGS).

(3) After 2 weeks of injection, phenotype observation and q-PCR are carried out to verify the expression changes of the tobacco NbFtsH5 gene, the tobacco salicylic acid synthesis related genes NbPR1 and NbEDS1 and the disease resistance related WRKY transcription factors WRKY40, 1, 3 and 8, and the primers are shown as SEQ ID NO. 9-22.

(4) TRV2-NbFtsH5 and TRV2-GFP tobacco leaves were inoculated with Phytophthora capsici (Phytophthora capsici) (available from Shanghai Ruicha Biotechnology Co., ltd.) and cultured on water agar plates at 28℃for 2 d.

The steps (2), (3) and (4) are all performed by using TRV2-GFP as a parallel control.

The results were as follows: as shown in FIG. 3, nbFtsH5 gene expression in tobacco was smoothly reduced by VIGS, and expression of the silent strain was down-regulated by 60% -70%. As shown in FIG. 2, after the expression of the NbFtsH5 gene in tobacco is reduced by VIGS, the growth condition is basically consistent with that of a control, which indicates that the down regulation of the gene has no obvious influence on the growth and development of plants. As shown in FIG. 4, after the expression of tobacco NbFtsH5 is downregulated by VIGS, the salicylic acid synthesis related genes NbPR1 and NbEDS1 of the tobacco are increased, wherein NbPR1 is a marker gene of a plant Salicylic Acid (SA) signal pathway, nbEDS1 is an accumulation gene of the plant Salicylic Acid (SA) signal pathway, and in addition, WRKY transcription factors related to plant disease resistance and WRKY40, 1, 3 and 8 are also upregulated to different degrees. Thus, when plant FtsH5 is down-regulated, the synthesis of the plant hormone Salicylic Acid (SA) is increased to resist the stress environment. As shown in fig. 5, after NbFtsH5 gene was down-regulated, VIGS down-regulated tobacco NbFtsH5 homologous gene tobacco inoculated with phytophthora capsici, the lesion area was significantly smaller than that of the control group, compared to TRV2-GFP, indicating that decreasing NbFtsH5 gene could increase resistance of tobacco to phytophthora capsici infection.

Example 4 ViGS Down-regulating Phalaenopsis PeFtsH5 to enhance disease resistance of Phalaenopsis leaves

The coding sequence of the PeFtsH5 is shown as SEQ ID NO. 1. The coding protein sequence is shown as SEQ ID NO. 2.

(1) Selecting CDS sequence of small orchid butterfly orchid PeFtsH5 gene, using the sequence as template, designing VIGS primer (shown as SEQ ID NO.23 and 24), and connecting the partial sequence to CymMv virus vector (purchased from Shanghai Ruichu Biotechnology Co., ltd.) to form recombinant vector.

(2) The recombinant vector is transferred into agrobacterium tumefaciens GV3101 (purchased from Shanghai Weidi biotechnology Co., ltd.) and simultaneously the virus CymMv vector is also transformed into the agrobacterium tumefaciens GV3101, two GV3101 bacterial liquids are obtained by culture, then the small orchid butterfly orchid leaves are respectively injected, and the small orchid butterfly orchid PeFtsH5 gene is silenced by a virus induced gene silencing (Virus induced gene silence, VIGS) mode.

(3) After 5 weeks of injection, phenotype observation and q-PCR are carried out to verify the expression changes of the PeFtsH5 gene, the salicylic acid synthesis related genes PePR1 and PeNPR1 and the disease resistance related WRKY transcription factors WRKY46 and 75 of the small orchid butterfly orchid, and the primers are shown as SEQ ID NO. 25-32.

(4) Botrytis cinerea is inoculated to cymMv-PeFtsH5 and cymMv-Ev small orchid butterfly orchid leaves, and the leaves are cultured on a water agar plate at 28℃for 4d and then observed.

The steps (2), (3) and (4) are all performed with CymMv-Ev as parallel control.

The results were as follows: as shown in FIG. 7, peFtsH5 gene expression in Phalaenopsis parvula was reduced by VIGS, and expression of the silent strain was down-regulated by 60% -70%. As shown in FIG. 6, after the expression of the PeFtsH5 gene in the butterfly orchid of the small orchid was reduced by the VIGS, the leaf growth rate was higher than that of the control group, indicating that the downregulation of the gene did not adversely affect the growth and development of the plant. As shown in FIG. 8, after the expression of the PeFtsH5 of the Phalaenopsis amabilis was down-regulated by the VIGS, the salicylic acid synthesis related genes PePR1 and PeNPR1 of the Phalaenopsis amabilis were elevated, and PR1 and NPR1 were marker genes of plant Salicylic Acid (SA) signal pathways, and in addition, WRKY transcription factors such as WRKY46 and 75 related to plant disease resistance were up-regulated to different extents. Thus, when plant FtsH5 is down-regulated, the synthesis of the plant hormone Salicylic Acid (SA) is increased to resist the stress environment. As shown in fig. 9, after the PeFtsH5 gene was down-regulated, VIGS down-regulated the small orchid butterfly orchid PeFtsH5 and inoculated with botrytis cinerea compared to the control group, indicating that decreasing the PeFtsH5 gene can increase the resistance of the small orchid butterfly orchid to botrytis cinerea infection.

The foregoing describes in detail preferred embodiments of the present application. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the application without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Sequence listing

<110> Shanghai university of teachers and students

<120> gene PeFtsH5 and application thereof in improvement of pathogenic bacteria infection resistance of butterfly orchid

<130> CN015-21009PICN

<160> 32

<170> PatentIn version 3.5

<210> 1

<211> 2109

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeFtsH5 coding sequence

<400> 1

atggcttgga ggcaactcct tacgcaggtt ggtaagaggc acttaaagtg ccctatttct 60

cacaaattaa gtggtcatcc atctcctaac ttgtacaagg tcgacaatgg aagtcatctt 120

ggaaattatc atgaaagatg gcaatcaagt tatgctggtg ctttttctcg taggctacat 180

gatgcagatt cacctagcga tatgactcta cttaaagaac tttatagaag agatccagaa 240

ggggttattc ggctatttga aagtcggcct tcattacatt ctaaccctgc tgcccttgct 300

gagtatgtta aagcgttagt taaggttgac aggctcgatg aaagctcatt gctaaaaaca 360

ctacaaagag gtatggctga ttcgttgagg aacgaggaga gcatcagcaa tattccaggc 420

ttgaagaaac ggggtttagc aactaaagag ggggttcttg ggactgcaaa ttctccgatt 480

cacatggtga cagcagaaac cagtaatttt aaagagcaat tatggcgcac atttcgcact 540

attgcactta cctttcttgt gatttctggg gttggtgcgc ttattgagga tagaggaatt 600

agcaaagggc ttggactgca tgaggaggtg cagccaatca tggattctag cacaaaattt 660

cttgatgtaa agggagttga tgaggctaaa gctgaattag aggaaattgt ccattacctt 720

cgggatccta tgcgtttcac ttgcttaggt gggaagcttc caaaaggcgt cctccttgtt 780

ggtccccctg gcactggaaa gaccatgtta gctcgggcta ttgcaggaga agctggagtt 840

ccctttttct cttgtagtgg aagcgagttt gaagagatgt ttgttggcgt aggagcaagg 900

agggttaggg accttttcgc cgcagctaaa aagcggtctc cctgtataat ttttattgat 960

gagatcgatg caattggtgg aagtcgtaat ccaaaggatc agcagtacat gaagatgaca 1020

cttaatcaat tgcttgttga acttgatggt tttaagcaaa acgaggggat tattgtcatc 1080

gccgcaacaa actttccaga gtcattggac aaggcactgg tgaggcctgg gcgtttcgat 1140

cgtcatattg ttgtcccgaa tccagatgtc gagggtcgaa gacagattat ggaatcccat 1200

atgtctaaga tcttgaaggc agatgacgtc gacttgtcaa tcatagcaag aggcacgccc 1260

ggtttctcgg gcgccgacct tgcaaacctg gttaacatgg ctgctctcaa ggctgccatg 1320

gatggggcca aagctgtcac catggctgat cttgaatacg caaaagacaa gataatgatg 1380

ggtagcgagc gcaaatctgc cgtgatttcc gacgagtcga agaagctaac tgcataccac 1440

gaaggcggtc atgctcttgt cgccattcat accgatggcg ctctccccgt ccacaaggct 1500

acgatcgtgc cccgtggaat ggctcttggt atggtcgccc agttgcccga caaggatgag 1560

acgagcctct ctctaaagca aatgctcgca cggcttgatg tctgcatggg tggacgcgtt 1620

gcggaagagc ttatatttgg ggagaacgaa atcacttctg gagcttcgtc ggatcttcaa 1680

caagcgacct ccattgctag agccatggtg acaaagtatg ggatgagcaa ggaggtggga 1740

gttgttgcgc ataactatga tgataatggg aagagcatga gcacagagac gaggcttttg 1800

attgagaaag aggtgaagca gttcctggag cgggcgtatc acaatgcaaa aactattctg 1860

tctaatcata acaaagagtt gcatgcttta gccaatgctc tattggagaa ggaaacttta 1920

actggagctc agatcaagca tttgcttgct caggttaact cgcagcagca agcggcggta 1980

attccgttgc agcagaattc aacgatgccg gcagcatctt cgcaaccttc agctgcggcg 2040

gcggcggtgg cagctgcttc tgcagcggtg aaagcgaagg gggttgcaca accagtagtg 2100

ggatcttag 2109

<210> 2

<211> 702

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeFtsH5 encoded protein sequence

<400> 2

Met Ala Trp Arg Gln Leu Leu Thr Gln Val Gly Lys Arg His Leu Lys

1 5 10 15

Cys Pro Ile Ser His Lys Leu Ser Gly His Pro Ser Pro Asn Leu Tyr

20 25 30

Lys Val Asp Asn Gly Ser His Leu Gly Asn Tyr His Glu Arg Trp Gln

35 40 45

Ser Ser Tyr Ala Gly Ala Phe Ser Arg Arg Leu His Asp Ala Asp Ser

50 55 60

Pro Ser Asp Met Thr Leu Leu Lys Glu Leu Tyr Arg Arg Asp Pro Glu

65 70 75 80

Gly Val Ile Arg Leu Phe Glu Ser Arg Pro Ser Leu His Ser Asn Pro

85 90 95

Ala Ala Leu Ala Glu Tyr Val Lys Ala Leu Val Lys Val Asp Arg Leu

100 105 110

Asp Glu Ser Ser Leu Leu Lys Thr Leu Gln Arg Gly Met Ala Asp Ser

115 120 125

Leu Arg Asn Glu Glu Ser Ile Ser Asn Ile Pro Gly Leu Lys Lys Arg

130 135 140

Gly Leu Ala Thr Lys Glu Gly Val Leu Gly Thr Ala Asn Ser Pro Ile

145 150 155 160

His Met Val Thr Ala Glu Thr Ser Asn Phe Lys Glu Gln Leu Trp Arg

165 170 175

Thr Phe Arg Thr Ile Ala Leu Thr Phe Leu Val Ile Ser Gly Val Gly

180 185 190

Ala Leu Ile Glu Asp Arg Gly Ile Ser Lys Gly Leu Gly Leu His Glu

195 200 205

Glu Val Gln Pro Ile Met Asp Ser Ser Thr Lys Phe Leu Asp Val Lys

210 215 220

Gly Val Asp Glu Ala Lys Ala Glu Leu Glu Glu Ile Val His Tyr Leu

225 230 235 240

Arg Asp Pro Met Arg Phe Thr Cys Leu Gly Gly Lys Leu Pro Lys Gly

245 250 255

Val Leu Leu Val Gly Pro Pro Gly Thr Gly Lys Thr Met Leu Ala Arg

260 265 270

Ala Ile Ala Gly Glu Ala Gly Val Pro Phe Phe Ser Cys Ser Gly Ser

275 280 285

Glu Phe Glu Glu Met Phe Val Gly Val Gly Ala Arg Arg Val Arg Asp

290 295 300

Leu Phe Ala Ala Ala Lys Lys Arg Ser Pro Cys Ile Ile Phe Ile Asp

305 310 315 320

Glu Ile Asp Ala Ile Gly Gly Ser Arg Asn Pro Lys Asp Gln Gln Tyr

325 330 335

Met Lys Met Thr Leu Asn Gln Leu Leu Val Glu Leu Asp Gly Phe Lys

340 345 350

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

355 360 365

Leu Asp Lys Ala Leu Val Arg Pro Gly Arg Phe Asp Arg His Ile Val

370 375 380

Val Pro Asn Pro Asp Val Glu Gly Arg Arg Gln Ile Met Glu Ser His

385 390 395 400

Met Ser Lys Ile Leu Lys Ala Asp Asp Val Asp Leu Ser Ile Ile Ala

405 410 415

Arg Gly Thr Pro Gly Phe Ser Gly Ala Asp Leu Ala Asn Leu Val Asn

420 425 430

Met Ala Ala Leu Lys Ala Ala Met Asp Gly Ala Lys Ala Val Thr Met

435 440 445

Ala Asp Leu Glu Tyr Ala Lys Asp Lys Ile Met Met Gly Ser Glu Arg

450 455 460

Lys Ser Ala Val Ile Ser Asp Glu Ser Lys Lys Leu Thr Ala Tyr His

465 470 475 480

Glu Gly Gly His Ala Leu Val Ala Ile His Thr Asp Gly Ala Leu Pro

485 490 495

Val His Lys Ala Thr Ile Val Pro Arg Gly Met Ala Leu Gly Met Val

500 505 510

Ala Gln Leu Pro Asp Lys Asp Glu Thr Ser Leu Ser Leu Lys Gln Met

515 520 525

Leu Ala Arg Leu Asp Val Cys Met Gly Gly Arg Val Ala Glu Glu Leu

530 535 540

Ile Phe Gly Glu Asn Glu Ile Thr Ser Gly Ala Ser Ser Asp Leu Gln

545 550 555 560

Gln Ala Thr Ser Ile Ala Arg Ala Met Val Thr Lys Tyr Gly Met Ser

565 570 575

Lys Glu Val Gly Val Val Ala His Asn Tyr Asp Asp Asn Gly Lys Ser

580 585 590

Met Ser Thr Glu Thr Arg Leu Leu Ile Glu Lys Glu Val Lys Gln Phe

595 600 605

Leu Glu Arg Ala Tyr His Asn Ala Lys Thr Ile Leu Ser Asn His Asn

610 615 620

Lys Glu Leu His Ala Leu Ala Asn Ala Leu Leu Glu Lys Glu Thr Leu

625 630 635 640

Thr Gly Ala Gln Ile Lys His Leu Leu Ala Gln Val Asn Ser Gln Gln

645 650 655

Gln Ala Ala Val Ile Pro Leu Gln Gln Asn Ser Thr Met Pro Ala Ala

660 665 670

Ser Ser Gln Pro Ser Ala Ala Ala Ala Ala Val Ala Ala Ala Ser Ala

675 680 685

Ala Val Lys Ala Lys Gly Val Ala Gln Pro Val Val Gly Ser

690 695 700

<210> 3

<211> 2163

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbFtsH5 coding sequence

<400> 3

atggctttaa tgcgccttct cagacaggtt gaaaggcagc aatctcatct aaggcagcta 60

aacaattttc tcaatagaac ctatcttacc tctggaaagg cgataggagg tggagtactt 120

ggggctgcaa gaaccaaggg aaggtttcga tcaagttatg tgggcagcct ggcccgccga 180

gtacgtgata cagaagaagc tactgatgcg gcatatctca gagagctata tcataagaac 240

gatccagagg cagtcataag gttgtttgaa ggtcaacctt cacttcattc taatccagca 300

gcactctcgg agtatgttaa ggcactggtt aaggttgata gactagatga aagtgagttg 360

ctccggacat tgcagagagg gattggtggt actgctagct ctcatgcgga agaagcaaat 420

ttaggagctc tgtcagcctt taggaatgtt gggaaagcta tgaaggatgg cgttcttggt 480

acttccagtg ctcctataca tatggtgact gttgaagggg ctcatttcaa agaacagtta 540

tggcgtacat tccgtgctct tggtttggcc tttctcttga tttcaggtgt tggtgcgctc 600

attgaagata gaggaatcag caaaggactt ggtctaaatg aggaagtgca gccgactatg 660

gaaaccaata caagatttgc tgatgtgaag ggtgttgatg aggcgaaagg ggagctggaa 720

gagattgtgc actatcttcg agaccctaag cgattcactc gtcttggtgg caagctccca 780

aagggtgtgt tacttgttgg tccacctgga actggtaaga ccatgttagc aagagccata 840

gcaggagaag ctggggttcc ctttttctca tgcagtggca gtgaatttga agaaatgttt 900

gttggtgttg gagcccggag agtaagagac ctttttgcag ctgctaagaa acgatcaccg 960

tgcattatat tcattgatga aattgatgcc attggaggta gccgtaaccc aaaggaccag 1020

caatacatga ggatgacttt gaatcagttg cttgttgagc tggatggttt caaacagaac 1080

gatggaatca ttgtgattgc ggccactaac ttccctgagt cattggataa ggcactggtg 1140

agacctgggc gttttgatcg caacattgtt gtcccaaatc ctgatgttga aggtcggaag 1200

cagattctag aatctcacat gtcgaaggtt gttaaagccg atgatgccga ccttatgatc 1260

attgctagag ggactcctgg attttctggt gctgaccttg ccaatttggt taatattgct 1320

gctgtgaagg ccgcgatgga tggtgccaaa gctgtaagct tggctgatct agagtatgct 1380

aaggacaaga tcatgatggg aagtgagcgc aaatcagctt tcatatccaa agagacgaaa 1440

aagcttacag cctaccatga aggcggccat gctcttgttg ctattcatac tgatggggca 1500

cttccagttc acaaggcaac cattgttcca cgtggaatgg ccctcggtat ggttgctcaa 1560

ttacctgaga aggatgagac tagtatgtcc cgtaagcaga tgcttgctcg gcttgatgtt 1620

gctatgggtg ggcgtgtcgc tgaagagctc atttttggag aaagtgaagt aacttctggt 1680

ccatctagcg atctccagca agcaaccaat ctcgccaggg ccatggttac caagtggggt 1740

atgagcaaag aagttgggct cgtcacccac aactatgatg ataatgggaa gagtatgagc 1800

actgaaacga ggcttctcat tgagaaggaa gtaagagaat tgcttgaaaa ggcttacaac 1860

aatgcgaaaa cgattctgac cacccacaac aaagagcttc atgctcttgc caatgcatta 1920

cttgagcagg aaacgctgac tggaaaccag ataaaggccc tgcttacaca ggtcaattct 1980

caacatacac aacagaagca acctcaatta gtgaccgagg agagcacatc accatccaat 2040

ccagctcctc cgtcaagtcc aactgcagca gcagctgctg cggccgctgc agcagcagct 2100

ggcgctgcag ctgcagccac tgccgccgcc aaaaccaaag gcattgctcc tgtaggacct 2160

tag 2163

<210> 4

<211> 720

<212> PRT

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbFtsH5 encoded protein sequence

<400> 4

Met Ala Leu Met Arg Leu Leu Arg Gln Val Glu Arg Gln Gln Ser His

1 5 10 15

Leu Arg Gln Leu Asn Asn Phe Leu Asn Arg Thr Tyr Leu Thr Ser Gly

20 25 30

Lys Ala Ile Gly Gly Gly Val Leu Gly Ala Ala Arg Thr Lys Gly Arg

35 40 45

Phe Arg Ser Ser Tyr Val Gly Ser Leu Ala Arg Arg Val Arg Asp Thr

50 55 60

Glu Glu Ala Thr Asp Ala Ala Tyr Leu Arg Glu Leu Tyr His Lys Asn

65 70 75 80

Asp Pro Glu Ala Val Ile Arg Leu Phe Glu Gly Gln Pro Ser Leu His

85 90 95

Ser Asn Pro Ala Ala Leu Ser Glu Tyr Val Lys Ala Leu Val Lys Val

100 105 110

Asp Arg Leu Asp Glu Ser Glu Leu Leu Arg Thr Leu Gln Arg Gly Ile

115 120 125

Gly Gly Thr Ala Ser Ser His Ala Glu Glu Ala Asn Leu Gly Ala Leu

130 135 140

Ser Ala Phe Arg Asn Val Gly Lys Ala Met Lys Asp Gly Val Leu Gly

145 150 155 160

Thr Ser Ser Ala Pro Ile His Met Val Thr Val Glu Gly Ala His Phe

165 170 175

Lys Glu Gln Leu Trp Arg Thr Phe Arg Ala Leu Gly Leu Ala Phe Leu

180 185 190

Leu Ile Ser Gly Val Gly Ala Leu Ile Glu Asp Arg Gly Ile Ser Lys

195 200 205

Gly Leu Gly Leu Asn Glu Glu Val Gln Pro Thr Met Glu Thr Asn Thr

210 215 220

Arg Phe Ala Asp Val Lys Gly Val Asp Glu Ala Lys Gly Glu Leu Glu

225 230 235 240

Glu Ile Val His Tyr Leu Arg Asp Pro Lys Arg Phe Thr Arg Leu Gly

245 250 255

Gly Lys Leu Pro Lys Gly Val Leu Leu Val Gly Pro Pro Gly Thr Gly

260 265 270

Lys Thr Met Leu Ala Arg Ala Ile Ala Gly Glu Ala Gly Val Pro Phe

275 280 285

Phe Ser Cys Ser Gly Ser Glu Phe Glu Glu Met Phe Val Gly Val Gly

290 295 300

Ala Arg Arg Val Arg Asp Leu Phe Ala Ala Ala Lys Lys Arg Ser Pro

305 310 315 320

Cys Ile Ile Phe Ile Asp Glu Ile Asp Ala Ile Gly Gly Ser Arg Asn

325 330 335

Pro Lys Asp Gln Gln Tyr Met Arg Met Thr Leu Asn Gln Leu Leu Val

340 345 350

Glu Leu Asp Gly Phe Lys Gln Asn Asp Gly Ile Ile Val Ile Ala Ala

355 360 365

Thr Asn Phe Pro Glu Ser Leu Asp Lys Ala Leu Val Arg Pro Gly Arg

370 375 380

Phe Asp Arg Asn Ile Val Val Pro Asn Pro Asp Val Glu Gly Arg Lys

385 390 395 400

Gln Ile Leu Glu Ser His Met Ser Lys Val Val Lys Ala Asp Asp Ala

405 410 415

Asp Leu Met Ile Ile Ala Arg Gly Thr Pro Gly Phe Ser Gly Ala Asp

420 425 430

Leu Ala Asn Leu Val Asn Ile Ala Ala Val Lys Ala Ala Met Asp Gly

435 440 445

Ala Lys Ala Val Ser Leu Ala Asp Leu Glu Tyr Ala Lys Asp Lys Ile

450 455 460

Met Met Gly Ser Glu Arg Lys Ser Ala Phe Ile Ser Lys Glu Thr Lys

465 470 475 480

Lys Leu Thr Ala Tyr His Glu Gly Gly His Ala Leu Val Ala Ile His

485 490 495

Thr Asp Gly Ala Leu Pro Val His Lys Ala Thr Ile Val Pro Arg Gly

500 505 510

Met Ala Leu Gly Met Val Ala Gln Leu Pro Glu Lys Asp Glu Thr Ser

515 520 525

Met Ser Arg Lys Gln Met Leu Ala Arg Leu Asp Val Ala Met Gly Gly

530 535 540

Arg Val Ala Glu Glu Leu Ile Phe Gly Glu Ser Glu Val Thr Ser Gly

545 550 555 560

Pro Ser Ser Asp Leu Gln Gln Ala Thr Asn Leu Ala Arg Ala Met Val

565 570 575

Thr Lys Trp Gly Met Ser Lys Glu Val Gly Leu Val Thr His Asn Tyr

580 585 590

Asp Asp Asn Gly Lys Ser Met Ser Thr Glu Thr Arg Leu Leu Ile Glu

595 600 605

Lys Glu Val Arg Glu Leu Leu Glu Lys Ala Tyr Asn Asn Ala Lys Thr

610 615 620

Ile Leu Thr Thr His Asn Lys Glu Leu His Ala Leu Ala Asn Ala Leu

625 630 635 640

Leu Glu Gln Glu Thr Leu Thr Gly Asn Gln Ile Lys Ala Leu Leu Thr

645 650 655

Gln Val Asn Ser Gln His Thr Gln Gln Lys Gln Pro Gln Leu Val Thr

660 665 670

Glu Glu Ser Thr Ser Pro Ser Asn Pro Ala Pro Pro Ser Ser Pro Thr

675 680 685

Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Ala Ala

690 695 700

Ala Ala Thr Ala Ala Ala Lys Thr Lys Gly Ile Ala Pro Val Gly Pro

705 710 715 720

<210> 5

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbFtsH5-VIGS-F

<400> 5

tgagtaaggt taccgaattc tctggaaagg cgataggagg tggag 45

<210> 6

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbFtsH5-VIGS-R

<400> 6

gacatgcccg ggcctcgagt gctgctggat tagaatgaag tgaa 44

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbFtsH5-qPCR-1F

<400> 7

aagaacagtt atggcgtaca ttccg 25

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbFtsH5-qPCR-1R

<400> 8

catcaacacc cttcacatca gcaaa 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbFtsH5-qPCR-2F

<400> 9

ttgctcggct tgatgttgct atggg 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbFtsH5-qPCR-2R

<400> 10

gcttgctgga gatcgctaga tggac 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbPR1-F

<400> 11

gacggctgct aaggctgttg agatg 25

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbPR1-R

<400> 12

acacgaaccg agttacgcca aacca 25

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbEDS1-F

<400> 13

aactgggctc gttacttcat acatt 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbEDS1-R

<400> 14

atgatcttac aactacctcg tgctg 25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbWRKY40-F

<400> 15

ctataagtgt tcttttgcac catcg 25

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbWRKY40-R

<400> 16

ggcactgtta cttgagcttg ggatg 25

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbWRKY1-F

<400> 17

ccacctacta agtcccatag tgaac 25

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbWRKY1-R

<400> 18

attgagcatc tgtagtaact ccttg 25

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbWRKY3-F

<400> 19

gtatccaagg tgttacttta ggtgc 25

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbWRKY3-R

<400> 20

ccaaagtatg tggtctggta aatga 25

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbWRKY8-F

<400> 21

attcctcctg gtttaagccc tactg 25

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> NbWRKY8-R

<400> 22

ttgtcttcct gtttgacacc ctgat 25

<210> 23

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeFtsH5-VIGS-F

<400> 23

ggggacaagt ttgtacaaaa aagcaggctg ccatggatgg ggccaaagct gtca 54

<210> 24

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeFtsH5-VIGS-R

<400> 24

ggggaccact ttgtacaaga aagctgggta gtttttgcat tgtgatacgc ccgc 54

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PePR1-F

<400> 25

cagccttccc actcaattct tagcc 25

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PePR1-R

<400> 26

atttgcagtc ggtttgtcgt cgtag 25

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeNPR1-F

<400> 27

tactgtgctg catgttgctg ctatg 25

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeNPR1-R

<400> 28

ggcttccctt cttctgtcgc cttac 25

<210> 29

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeWRKY46-F

<400> 29

gaagcgacaa gacaggtgca gaagt 25

<210> 30

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeWRKY46-R

<400> 30

gtttggtgcc attgacggcg ttaga 25

<210> 31

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeWRKY75-F

<400> 31

aataattgaa gagggaagaa aggag 25

<210> 32

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial sequence)

<220>

<223> PeWRKY75-R

<400> 32

aaccatcgtc gagaatatca acctg 25

Claims (7)

1. The application of the gene PeFtsH5 in improving pathogenic bacteria infection resistance of the butterfly orchid is characterized by comprising the step of down-regulating the expression of the PeFtsH5 gene in the butterfly orchid, wherein the protein sequence coded by the PeFtsH5 gene is shown as SEQ ID NO. 2; the pathogenic bacteria are phytophthora capsici and/or botrytis cinerea.

2. The use of claim 1, wherein down-regulation comprises gene silencing or gene knockout.

3. The use of claim 2, wherein the gene silencing results in a 60% -70% decrease in the expression of the PeFtsH5 gene in the post-silencing plant as compared to the wild-type plant.

4. The use according to claim 1, wherein the coding sequence of the PeFtsH5 gene is shown in SEQ ID No. 1.

5. A method for improving tobacco resistance to phytophthora capsici infection, which is characterized by comprising the step of down-regulating the expression of NbFtsH5 genes in tobacco, wherein the protein sequence coded by the NbFtsH5 genes is shown as SEQ ID No. 4.

6. The method of claim 5, wherein down-regulating comprises gene silencing or gene knockout.

7. The method of claim 5, wherein the NbFtsH5 gene has a coding sequence set forth in SEQ ID No. 3.