CN103484535B - Applications of gene relating to pathogenic mechanism of xanthomonas campestris pathovar campestris - Google Patents
Applications of gene relating to pathogenic mechanism of xanthomonas campestris pathovar campestris Download PDFInfo
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- CN103484535B CN103484535B CN201310265130.3A CN201310265130A CN103484535B CN 103484535 B CN103484535 B CN 103484535B CN 201310265130 A CN201310265130 A CN 201310265130A CN 103484535 B CN103484535 B CN 103484535B
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention provides applications of a gene relating to pathogenic mechanism of xanthomonas campestris pathovar campestris in prevention of crucifer black rot. The DNA sequence of a protein represented by sequence No.2 in a coding sequence table is the DNA sequence of the gene. More specifically, the DNA sequence of the gene is represented by sequence No.1. Gene XC2038 relating to pathogenic mechanism of xanthomonas campestris pathovar campestris is capable of providing drug targets for prevention of crucifer black rot.
Description
Technical field
The present invention relates to pathogenic related gene of plant pathogenetic bacteria and uses thereof, relate in particular to pathogenic relevant gene of crucifer black rot germ and uses thereof.
Background technology
Plant diseases is one of principal element of crop production reduction, quality reduction always.Along with the enhancing of pathogenic bacteria to drug resistance, pesticide dosage is in continuous increase, and this has not only aggravated environmental pollution, drug residue, has also greatly increased agricultural cost, therefore, urgently researches and develops new prevent and cure diseases strategy and novel nuisanceless medicine.Understand fully that the genetic mechanism that phytopathogen infects host is the key of development of new medicine and diseases prevention strategy.
Crucifer black rot bacterium (Xcc, Xanthomonas campestris pv.campestris) is a kind of gram negative bacterium that can cause in the world all cress Black Rotten.This bacterium can, in any vegetative period of host by water hole, pore or wound invaded plants body, cause crucifer black rot disease, and host comprises wild cabbage, Cauliflower, Chinese cabbage, radish and rape etc.Typical Black Rotten symptom is on leaf margin, to form V font scab.Along with the expansion of scab, vein blackening, thereby be called as Black Rotten.Black Rotten is considered to the most serious disease of cress harm, and especially in subtropical and tropical zones, temperature and humidity is all suitable for the growth and breeding of this bacterium, and therefore morbidity is more serious.Due to genetic stability and hereditary operability, crucifer black rot bacterium is used as the pattern bacterium of microorganisms and plant interaction molecule mechanism always.Therefore, qualification and the pathogenic relevant gene of crucifer black rot bacterium, have significant meaning to controlling plant diseases.
At present, in the genome of the pathogenic mutation Xcc 8004 of crucifer black rot bacterium, the coded albumen annotation of open reading frame (ORF, Open Reading Frame) that discovery is numbered the gene of XC2038 is putative protein (Hypothetical Protein).Putative protein refers to because of the not albumen of certified new genes encoding of Unknown Function.The ORF that discovery annotation is putative protein in the genome of Xcc 8004 is more.At present, identified be noted as putative protein and to cause a disease relevant new gene of crucifer black rot bacterium, as XC0241 gene, be accredited as pathogenic relevant III type effector gene.Function for XC2038 gene but rarely has report at present.
Reference
1、Beringer，J.，Beynon，J.，Buchanan-Wollaston，A.，Johnston，A.Transfer?of?the?drug-resistance?transposon?Tn5to?Rhizobium.Nature.1978.276：633-634.
2、Jiang，B.L.，He，Y.Q.，Cen，W.J.，Wei，H.Y.，Jiang，G.F.，Jiang，W.，Hang，X.H.，Feng，J.X.，Lu，G.T.，Tang，D.J.The?type?III?secretion?effector?XopXccN?of?Xanthomonas?campestris?pv.campestris?is?required?for?full?virulence.Research?in?Microbiology.2008.159(3)：216-220.
3、Jiang，G.F.，Jiang，B.L.，Chen，L.G.，Liu，S.，Wei，H.Y.，Cen，W.J.，Hang，X.H.，Wen，Z.Z.，Tang，D.J.，Lu，G.T.，He，Y.Q.，Yu，D.Q.，Tang，J.L.The?T3S?effector?XopXccN?of?Xanthomonas?campestris?pv.campestris?is?involved?in?plant?defense?through?interference?with?photosystems，reactive?oxygen?species(ROS)generation，and?callose?deposition.African?Journal?of?Microbiology?Research.2012.6(15)：3673-3683.
4、Liu，Y.G.，Whittier，R.F.Thermal?asymmetric?interlaced?PCR：automatable?amplification?and?sequencing?of?insert?endfragmentsfrom?P1and?YAC?clones?for?chromosome?walking.Genomics.1995.25(3)：674-681.
5、Qian，W.，Jia，Y.，Ren，S.X.，He，Y.Q.，Feng，J.X.，Lu，L.F.，Sun，Q.，Ying，G.，Tang，D.J.，Tang，H.，Wu，W.，Hao，P.，Wang，L.，Jiang，B.L.，Zeng，S.，Gu，W.Y.，Lu，G.，Rong，L.，Tian，Y.，Yao，Z.，Fu，G.，Chen，B.，Fang，R.，Qiang，B.，Chen，Z.，Zhao，G.P.，Tang，J.L.，He，C.Comparative?and?functional?genomic?analyses?of?the?pathogenicity?of?phytopathogen?Xanthomonas?campestris?pv.campestris.Genome?Research.2005.15(6)：757-767.
6、Sharma，S.，Signer，E.Temporal?and?spatial?regulation?of?the?symbiotic?genes?of?Rhizobium?meliloti?in?planta?revealed?by?transposon?Tn5-gusA.Genes&Development.1990.4(3)：344-356.
7、Staskawicz，B.，Dahlbeck，D.，Keen，N.，Napoli，C.Molecular?characterization?of?cloned?avirulence?genes?from?race?0?and?race?1?of?Pseudomonas?syringae?pv.glycinea.Journal?of?Bacteriology.1987.169(12)：5789-5794.
Summary of the invention
Technical problem to be solved by this invention is to provide the purposes of the pathogenic relevant gene of a kind of crucifer black rot bacterium, is tested and appraised this gene and provides drug targets for preventing and treating cress Black Rotten disease.
In order to solve the problems of the technologies described above, the invention provides a kind of and the crucifer black rot germ purposes of relevant gene in control radish crucifer black rot disease of causing a disease, this gene is protein DNA sequence shown in sequence 2 in code sequence list.
Preferably, this gene is the DNA sequence dna shown in sequence 1 in sequence table.
The present invention has identified the crucifer black rot bacterium relevant gene XC2038 that causes a disease, and can provide drug targets for control cress Black Rotten disease.
Brief description of the drawings
Fig. 1 is the restriction enzyme digestion and electrophoresis collection of illustrative plates of the XC2038 gene clone identified of the present invention;
Fig. 2 is at the PCR of the XC2038 gene insertion mutation body 164H09 of the present invention's qualification checking gel figure;
Fig. 3 is at the exocellular polysaccharide of the XC2038 gene insertion mutation body 164H09 of the present invention's qualification, swimming and biological poly-film Phenotypic examination;
Fig. 4 is the pathogenicity of the XC2038 gene insertion mutation body 164H09 that identifies of the present invention.
Embodiment
Below in conjunction with accompanying drawing and preferred embodiment, technical scheme of the present invention is at length set forth.Should be appreciated that, the embodiment below enumerating is only for description and interpretation the present invention, and do not form the restriction to technical solution of the present invention.
The present invention aims to provide and the pathogenic relevant gene XC2038 of crucifer black rot bacterium.First determine the relation that XC2038 gene and crucifer black rot are pathogenic, and obtain thus the purposes of this gene aspect control plant crucifer black rot.
Provided by the present invention and the crucifer black rot bacterium relevant gene XC2038 that causes a disease is protein DNA sequence shown in sequence 2 in code sequence list.
And this gene is the DNA sequence dna shown in sequence 1 in sequence table.
At present, should be to the sequence of the pathogenic relevant gene XC2038 (hereinafter to be referred as XC2038 gene) of crucifer black rot bacterium at the American National biotechnology (NCBI of information center, National Center of Biotechnology Information) announce, its genome sequence number is NC_007086, this gene coded protein sequence number YP_243119; The plasmid pCXC2038 that carries this gene preserves with utilizing National Key Laboratory in Guangxi University's subtropics agro-ecology conservation of resources.
In sequence table, the DNA of sequence 1 is the gene of crucifer black rot bacterium 8004 bacterial strains, by 960 based compositions, containing complete XC2038 gene, from the 498th to 875 ORF that Nucleotide is this gene of 5 ' end, from the 498th to 500 initiator codon ATG that Nucleotide is this gene of 5 ' end, be terminator codon TGA from the 876th to 878 Nucleotide of 5 ' end.
In sequence table, the protein of sequence 2 is products of XC2038 genes encoding, is made up of 126 amino acid.This protein prediction molecular weight is 13926.73 dalton, and iso-electric point is 6.41.
The present invention by the following method step qualification XC2038 gene is caused a disease relevant to crucifer black rot:
(1) structure of crucifer black rot bacterium mutant library
Taking the shuttle plasmid pLAFR1 between intestinal bacteria and Xcc as carrier, Tn5gusA5 is introduced to Xcc wild type strain 8004, then drive pLAFR1 by introducing incompatible plasmids pPH1JI, by kantlex as microbiotic, resistance marker screening Xcc::Tn5gusA5 insertion mutation body, in conjunction with Xcc whole genome sequence and TAIL-PCR (Thermal Asymetric Interlaced-PCR) technology, determine the on position of Tn5gusA5 on genome, and on position is carried out to PCR checking.
By investigating pathogenic, Extracellular enzyme activity, the isophenous variation of transposon mutagenesis of mutant, screening pathogenic related gene, the present invention relates to one of them new pathogenic related gene, be XC2038 gene (coding putative protein), its insertion mutation body is numbered 164H09 (this is numbered from naming number, i.e. the numbering of the insertion mutation body of the XC2038 gene of crucifer black rot bacterium mutant library).
(2) clone and sequence of XC2038 gene
According to the gene order of XC2038, design primer (XC2038F:GGGGAATTCGCGCCAAGCAACGGACAGACG and XC2038R:GGGGGATCCAACACGACGCCGACCAGCACG), taking the total DNA of crucifer black rot bacterium 8004 bacterial strain as template, by PCR method this full length gene sequence that increases, and be cloned in cloning vector pGEM3Zf (+), on ABI 377DNA automatic sequencer, measure DNA nucleotide sequence with dideoxyribonucleoside acid system.XC2038 gene order correct sequence verification is cloned in shuttle vectors pLAFRJ, has obtained the recombinant plasmid pCXC2038 containing this gene.
EcoR I, BamH I enzyme for this plasmid are cut, except the carrier ribbon of a 22kb, also had the external source fragment of a 960bp, refer to Fig. 1, wherein, M1: λ/HindIII2 standard DNA, clip size is followed successively by from big to small: 23.1kb, 9.4kb, 6.6kb, 4.4kb, 2.4kb, 2.0kb; M2:100bp standard DNA, clip size is followed successively by from big to small: 3kb, 2kb, 1.5kb, 1.2kb, 1kb, 0.9kb, 0.8kb, 0.7kb etc.; 1 is XC2038 gene fragment (960bp).
(3) in the checking of XC2038 gene insertion mutation body 164H09
Insertion mutation body 164H09 is carried out to TAIL-PCR order-checking location, find that it is inserted in XC2038 gene.
Match and carry out PCR checking with the upper primer (CCGCCGAAGAGAACACAGATTTA) of sp1 side of transposon Tn5 and a primer (AGTTCGGAGGAATCACGCGG) of downstream gene XC2037, product is expected big or small 837bp.Refer to Fig. 2, wherein, 1 is 100bp standard DNA; 2 is XC2038 gene insertion mutation body 164H09 (837bp).
(4) exocellular polysaccharide of XC2038 gene insertion mutation body 164H09, swimming and biological poly-film Phenotypic examination
By wild type strain 8004, XC2038 mutants which had 164H09 and complementary bacterial strain C164H09 are incubated in NYGB substratum (every liter containing peptone 5.0g, yeast powder 3.0g, glycerine 20.0g, pH7.0), 28 DEG C of shaking table incubated overnight.
Use NYGB substratum by the OD of cultured bacterium liquid to be measured for the detection of exocellular polysaccharide
600value is adjusted to unanimously, accurately get the culture of 2 μ l with pipettor, be inoculated in respectively containing on NYGA (NYGB substratum adds approximately 3% the agar powder) flat board of 2% glucose, in 28 DEG C of incubators, cultivate, after 3 days, again this flat board is positioned over outside incubator, see light cultivation 2～5 days, by the form size that compares bacterium colony, the output of EPS is compared;
Use NYGB substratum by the OD of cultured bacterium liquid to be measured for the detection of swimming
600value is adjusted to unanimously, and each thalline is got 5 μ l, and o'clock on the NYGA of 0.3% agarose flat board, 28 DEG C leave standstill to cultivate after 48 hours and observe and photographic recording; Use NYGB substratum by the OD of cultured bacterium liquid to be measured for the detection of the poly-film of biology
600value is adjusted to unanimously, is forwarded in LB liquid nutrient medium according to 10% ratio, and 28 DEG C leave standstill cultivation after 5-7 days, observe and record result.
Result shows, XC2038 mutants which had 164H09 affects output, the swimming of bacterial strain and the formation of biological poly-film of exocellular polysaccharide, and its complementary strain C164H09 can well return to the level of wild-type.Because output, the swimming of bacterial strain and the formation of biological poly-film of exocellular polysaccharide are all the important pathogenic Some Circulating Factors of pathogenic bacteria, this explanation XC2038 is to pathogenic relevant; Refer to Fig. 3, wherein, object A is wild-type; Object B is XC2038 gene insertion mutation body 164H09; Object C is the complementary strain C164H09 of XC2038 gene insertion mutation body.
(5) the pathogenic detection after XC2038 gene insertion mutation body 164H09
Testing host plant used is Turnip Sprouts (planting Raphanus sativus L.var.radiculus Pers. by name), and inoculation method used is leaf-cutting method.Wild type strain 8004, XC2038 gene insertion mutation body 164H09 and complementary bacterial strain C164H09 are carried out to liquid culture 15-18 hour at 28 DEG C.
Be diluted to OD with NYGB
600=0.001, soak in bacterium liquid with sterilizing scissors after 5 seconds, cut axis place at healthy leaves apart from the direction of the vertical vein of blade tip 1-2cm, stopped for 5 seconds, the plant being vaccinated is cultivated one week rear observations at 25-30 DEG C, and positive control is selected crucifer black rot bacterium 8004 bacterial strains.
Above-mentioned pharmacosensitive test shows, the pathogenic of the insertion mutation body 164H09 of XC2038 gene is zero, and referring to Fig. 4, and the complementary strain C164H09 of mutant can be by the pathogenic level that well returns to wild-type reducing.This explanation XC2038 gene is an important pathogenic related gene.In Fig. 4, object A is wild-type; Object B is XC2038 gene insertion mutation body 164H09; Object C is the complementary strain C164H09 of XC2038 gene insertion mutation body 164H09.
In aforesaid method step of the present invention, material used comprises:
Intestinal bacteria (Escherichia coli) strain JM109;
Carrier pGEM-3Zf (+) is purchased from Promega company;
PLAFR1, pPH1JI, pLAFRJ are (referring to reference 7) coemid that this research department preserves;
The reagent such as restriction enzyme, modifying enzyme is purchased from Promega, Stratagene, QIAGEN company, Nihon Pharmaceutical Co., Ltd..
Claims (2)
1. cause a disease relevant gene in a purposes of preventing and treating in radish crucifer black rot disease to crucifer black rot germ, it is characterized in that, described gene is protein DNA sequence shown in sequence 2 in code sequence list.
2. according to purposes claimed in claim 1, it is characterized in that, described gene is the DNA sequence dna shown in sequence 1 in sequence table.
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| CN103966239B (en) * | 2014-05-20 | 2019-05-07 | 广西大学 | A kind of purposes of lipoprotein encoding gene |
| CN103993021B (en) * | 2014-05-27 | 2017-01-11 | 广西大学 | Type III effector gene of Xanthomonas campestris pathovar campestris and application of gene |
| CN106916831B (en) * | 2015-12-28 | 2020-04-14 | 广西大学 | Application of xanthomonas pathogenic related gene |
| CN111139250B (en) * | 2020-01-03 | 2023-04-07 | 广东食品药品职业学院 | Lipoic acid synthesis related gene and application thereof |
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