CN104561027A - Rapid Identification of pear powdery mildew resistance gene - Google Patents

Abstract

The invention relates to rapid identification of a pear powdery mildew resistance gene, and relates to the subject knowledge of plant comparative genomics, genetics and bioinformatics, belonging to the field of science of biotechnology. The rapid identification of the pear powdery mildew resistance gene comprises the following main steps of: 1) downloading of a complete genome sequence of a pear and collection of an MLO gene; 2) identification of the MLO gene; 3) MLO gene phylogenetic relationship; and 4) comparison of an MLO powdery mildew gene. The rapid identification of the pear powdery mildew resistance gene, provided by the invention, has the advantages of effectively shortening the pear powdery mildew gene mining period, and being favorable for the rapid identification of the powdery mildew gene; corresponding co-segregation functional markers (SNP, SCAR and the like) can be developed through the identification of candidate powdery mildew genes, and the co-segregation functional markers can be rapidly used for the molecular marker-assisted selection of the powdery mildew resistance gene, so that the accuracy is high; by combining other powdery mildew resistance gene molecular markers, a multi-resistance breeding material can be created, the breeding period can be shortened, and the breeding efficiency can be improved; a foundation is laid for explaining a pear powdery mildew resistance molecular mechanism.

Description

Pears mildew-resistance gene Rapid identification

Technical field

The present invention is by means of pears order-checking whole genome sequence, utilize the method Rapid identification pears powdery mildew genes such as plant comparative genomics, genetics, information biology and candidate gene strategy, be mainly concerned with the download of pears whole genome sequence, the qualification of candidate gene, the comparison of gene, the means such as cluster, and then identify powdery mildew gene, belong to Plant Biotechnology scientific domain.

Background technology

Pear Rosaceae pear, China is one of source region, Pyrus L center.The operatic circle is delicious, the crisp succulence of meat, sweet and sour taste, and local flavor fragrance is graceful.Be rich in sugar, protein, fat, carbohydrate and multivitamin, HUMAN HEALTH is played an important role.The all right processing and fabricating pears of the operatic circle are dry, preserved pears, pear syrup, pear juice, canned pear etc., also can be used to wine brewing, vinegar processed.The operatic circle also has medical value, can aid digestion, moistening lung clear away heart-fire, effect of stopping coughing and removing phlegm, carbuncle of bringing down a fever, separate, also has diuresis, profit effect just.Pear wood is careful, and soft or hard appropriateness, be the raw material of engraving seal and noble furniture, the sugar degree of pears is below 15%, and the usage quantity of every day can at 300 ~ 500 grams, and diabetic subject is edible also.Powdery Mildew is one of the Major Diseases during pears are produced.At present aborning, the control of pear tree Powdery Mildew is mainly by spraying fungicide, but life-time service medicine can cause environmental pollution and Biological Strains of The Pest variation, and therefore promoting disease-resistant variety is method the most safely and effectively.But there is no the bibliographical information of pears Powdery Mildew research aspect at present, this seriously inhibits the paces of pears Powdery Mildew research.

Mlo gene is the gene that a class is relevant to plant broad-spectrum high efficacy resistance, is first disease-resistant gene that it is found that, in the research of plant wide spectrum mildew-resistance Theory and applications, have important value.First Mlo gene is found in barley, and its sudden change can cause barley to the resistance of wide spectrum of Powdery Mildew (Blumeria.graminisf.sp.hexlei Bgh).20 century 70 people have found again the mlo of spontaneous mutation, therefore, can find out that the wild-type of Mlo is the negative regulatory factor of barley mildew-resistance, and recessive mutation mlo could induce resistance of wide spectrum.Have obviously different compared with the resistance that mlo and general disease-resistant gene mediate, people find that this gene does not have function in mildew-resistance kind under study for action, and when making it not play function its silence of plan, this kind can produce resistance to Powdery Mildew.The resistance mechanism of Mlo to plant plays negative regulation effect, is equivalent to " susceptible " gene of plant to a certain extent, and the recognition reaction of similar non-host can occur for its sudden change or disappearance, causes the resistance of wide spectrum of plant generation to all known microspecies of pathogenic bacteria.Be mainly manifested in: the nearly all Powdery Mildew physiological strain of mlo gene pairs all has resistance.Recently, investigator finds that the mildew-resistance gene of a lot of plant is all Mlo type Gene Handling, as tomato, and pea, Arabidopis thaliana, rose, capsicum, Root or stem of Littleleaf Indianmulberry etc.Therefore the Mlo type disease-resistant gene excavated in plant has important effect to Biology of Plant-Powdery Mildew Interaction breeding.Existing research shows, Mlo gene in familial form, has multiple family member, wherein has 15 family members in Arabidopis thaliana in many crops.In corn, find to have 9 at least.In paddy rice, there are 12 family members, but on other crop, delayed due to genome sequencing work, seldom have Mlo family member to be found.Therefore, this genoid has very large potentiality and application prospect improving in plant resistance to environment stress.At present, the conventional method excavating disease-resistant gene conventional has map based cloning, the methods such as transposon tagging.Because the fundamental research of pears is not deep enough, not only the time is grown but also is difficult to clone these genes exactly therefore to utilize these methods.Therefore, the Mlo type disease-resistant gene how in Rapid identification pears will become the important prerequisite of Kidney bean mildew-resistance breeding.

Plant comparative genomics (comparative genomics) is also referred to as comparative genetics (comparative genetics), be on comparative map and DNA sequencing basis to the quantity of known, to put in order and genome structure compares, understand an emerge science of gene function, expression mechanisms and spore.The molecular basis of comparative genomics is exactly the conservative property of DNA sequence dna especially encoding sequence between species, and concrete method comprises physical mapping and genetic mapping two class.Main application genetic mapping method in plant at present, namely probe is made with a set of common RFLP molecule marker, build the genetic linkage maps of species, by comparing the distribution situation of molecule marker in genome, disclose the existence of different plant species karyomit(e) or chromosome segment colinearity (synteny) and collinearity (collinearity), thus species gene group structure, function and Evolution History are analyzed.So-called colinearity (synteny) refers to that the two or more marks on a species karyomit(e) or chromosome segment are located on the homologous chromosomes of another species, but the relative ranks between these marks changes sometimes; Collinearity (collinearity) then refers to that mark on homologous chromosomes or chromosome segment and putting in order therebetween are all conservative.Between Land use models Plant Genome and other Plant Genome on coded sequence and structural homology, clone's other plant gene, discloses gene function and molecular mechanism, illustrates spore relation and genomic immanent structure.The importance of plant comparative genomics is that it links together different subjects, different biological species, has erected the bridge between fundamental research and applied research.Across kind, across belonging to, genome comparison transboundary understands the relation of gene and genomic structure, gene structure and function and DNA to us and changes and how to cause species diversity etc. to be of great significance.

The method that this patent adopts and thinking: the research of model plant arabidopsis gene group has disclosed the function of MLO type gene, utilize the homology clone pears MLO type disease-resistant gene in its order of gene, according to the feature of the superiority in model plant Arabidopis thaliana experimental system and known MLO type disease-resistant gene, " seizure " pears mildew-resistance gene fast.This patent describes premised on pears whole genome sequence, in conjunction with knowledge such as comparative genomics, genetics, genomics, information biology and candidate gene strategy, excavates powdery mildew gene fast.

Summary of the invention

Technical problem

The object of this invention is to provide a kind of by conjunction with knowledge such as plant comparative genomics, plant genetics, genomics and information biology, excavate pears mildew-resistance gene fast.Its result can be used for the exploitation of pears powdery mildew gene compact linkage molecule mark on the one hand, carries out molecular marker assisted selection breeding, on the other hand also for other crop powdery mildew gene identification provide reference frame.

Technical scheme

Cardinal principle: first Biology of Plant-Powdery Mildew Interaction gene (MLO) is cloned from barley, research finds that this gene is the special disease-resistant gene of a class, is different from most of NBS (nucleotide-binding site) the type disease-resistant gene of previously clone; Subsequently, investigator has cloned powdery mildew gene in succession from the plants such as tomato, Arabidopis thaliana, pea, capsicum, Root or stem of Littleleaf Indianmulberry, and what research found these genes encodings is all MLO type disease-resistant gene.Subsequently, by test of many times, numerous investigator confirms that MLO type disease-resistant gene has become the distinctive class mildew-resistance gene of plant.Further discovery, plant MLO type gene is a gene family; And Phylogenetic Relationships analysis discovery is carried out to the MLO gene family deriving from different plant species, in different plant species mildew-resistance gene always cluster together, become a class, this class MLO gene all has the characteristic feature of mildew-resistance gene sequence.Completing of pears gene order-checking provides a convenient approach for excavating powdery mildew gene.Therefore, can by means of the Phylogenetic Relationships of MLO gene family in the pears full-length genome checked order and the MLO powdery mildew gene of having cloned and for maintaining the conservation of amino acids of powdery mildew gene MLO critical function to identify pears powdery mildew gene.

Key step is as follows:

1) download of pears whole genome sequence and the collection of MLO type gene thereof

First pears whole genome sequence is downloaded from pears sequenced genes group database (http://peargenome.njau.edu.cn:8004/); Use " DNATOOLS " software to the pears full-length genome amino acid sequence data building database obtained, then pfam database (protein family database is used, http://pfam.janelia.org/search/sequence) in hidden Markov model (HMM) Blastp (E-value=0.001) sequence alignment is carried out to the aminoacid sequence of MLO structural domain and the pears full-length genome amino acid sequence database to have set up, preliminary screening goes out candidate gene sequence.Secondly, utilize the MLO type gene order announced, BLAST comparison is carried out to pears genome database, obtain candidate gene sequence.

2) qualification of pears MLO type gene family

By the candidate gene of homologous amino acid sequence obtained in the above results, analyzed by Pfam (E-value=1.0), remove the gene order (Fig. 1) without ' MLO ' structural domain.The ClustalW instrument (Multiple Sequence Alignment program) Candidate Disease Resistant Genes sequence provided by MEGA3.1 software again carries out Multiple Sequence Alignment, removes tumor-necrosis factor glycoproteins.

3) by the pears MLO type powdery mildew gene of the Phylogenetic Relationships qualification candidate of plant MLO type gene

Because previous research is verified, dicotyledons MLO type powdery mildew gene is positioned at plant MLO Phylogenetic Tree same district group, therefore in Phylogenetic Relationships research, we are MLO type mildew-resistance gene with pears MLO type gene together with the cluster analysis of the MLO type gene family of Arabidopis thaliana with some other crops, to obtain the pears mildew-resistance gene (Fig. 2) of candidate.

4) comparison of pears powdery mildew gene and known plant MLO powdery mildew gene

BioXM2.6 software is utilized to convert the aminoacid sequence of the MLO powdery mildew gene of the MLO type powdery mildew gene of pears candidate and Arabidopis thaliana, tomato, pea, barley the file of Fasta form to, these files are imported BioEdit7.0 software, use Clustal software in this software to carry out Multiple Sequence Alignment, disclose the conservative property in candidate's powdery mildew gene important amino acid residue and region.Thus identify the powdery mildew gene (Fig. 3) of pears candidate further.

Positively effect of the present invention:

1) shorten pears powdery mildew gene and excavate the cycle, be conducive to the Rapid identification of powdery mildew gene.Mildew-resistance gene not only takes time and effort, efficiency is low to adopt ordinary method (map based cloning, transposon tagging etc.) to excavate, and is difficult to successfully.The present invention is based on plant comparative genomics, genetics, bioinformatics method and excavate pears powdery mildew gene fast, not only can shorten the time, powdery mildew gene determination rates can also be improved.

2) pears are one of important fruit trees in the whole world.Because pears hereditary basis is narrow, Germplasm Resources Diversity is low, therefore more difficult by conventional molecule marker (RAPD, ISSR, SSR, AFLP etc.) qualification pears powdery mildew gene.The candidate's powdery mildew gene exploitation be tested and appraised is divided into from functional indicia (SNP, SCAR etc.) accordingly, can fast for the molecular marker assisted selection of disease-resistant gene, and accuracy is high.

3) initiative of multiresistance breeding material.Based on the functional molecular marker of the powdery mildew gene exploitation of new qualification, ins conjunction with the molecule marker of other disease-resistant genes after positioning, carry out the initiative of multiresistance breeding material, can shortening the breeding cycle, raising breeding efficiency.

4) lay a good foundation for setting forth pears mildew-resistance molecular mechanism.The qualification of pears mildew-resistance gene, by transgenic technology, RNAi, virus induced gene silencing (virus induced gene silencing, VIGS) molecular mechanism of the Effect of Anti Powdery Mildew such as technology provides genetic resources, is conducive to the mechanism of action setting forth pears mildew-resistance fast.

Accompanying drawing explanation

The qualification of Fig. 1 pears MLO gene;

What this figure showed is 25 MLO type gene identification results, and each gene is containing ' MLO ' conserved domain.

The Phylogenetic Relationships analysis of Fig. 2 plant MLO gene family and the qualification of pears MLO type powdery mildew gene thereof;

Arabidopis thaliana is the model plant of plant science research, in phylogenetic tree construction, the powdery mildew gene (PsMLO) of 15 MLO type genes (wherein 3 genes are powdery mildew genes: AtMLO02, AtMLO06 and AtMLO12) of Arabidopis thaliana, Gene against Powdery Mildew in Tomato (SlMLO), barley powdery mildew gene (HvMLO) and pea is selected to and the analysis of pears MLO type gene clusters.Identify the MLO type powdery mildew gene of 6 pears candidates altogether.In figure, the gene of italic mark is exactly candidate's pears powdery mildew genes.

The compare of analysis of Fig. 3 pears MLO type powdery mildew gene;

6 pears powdery mildew genes and barley (HvMLO), tomato (SlMLO), pea (PsMLO), Arabidopis thaliana powdery mildew gene (AtMLO02, AtMLO06 and AtMLO12) to compare, qualification powdery mildew infects the conservative type in amino-acid residue and the region played an important role.In figure, TM1-TM7 represents 7 revolving die regions of pears MLO type powdery mildew gene; Black round dot represents that powdery mildew infects important amino-acid residue; CaMBD represents calmodulin CaM binding region; I and II represents and infects important amino acid region to powdery mildew.

Embodiment

The qualification of disease-resistant gene has important effect in the research of crop disease-resistant theory of heredity and disease-resistant variety seed selection.Present method Rapid identification can go out pears powdery mildew gene.Specific implementation process is as follows:

1) collection of pears MLO type gene and qualification

In order to obtain the whole MLO type gene family member of pears, we are first with the MLO type gene of Arabidopis thaliana, the mildew-resistance MLO gene order of tomato, pea, capsicum, rose, capsicum, Root or stem of Littleleaf Indianmulberry builds HMM model, receives rope MLO type gene from pears genome sequence, secondly the MLO gene order delivered in Different Crop as target sequence (from DFCI database: TC171015, TC267529, DFCI:TC327983, TC289653, TC312087, TC132500, TC133436, TC317623, TC317025, TC315947, TC325903, TC315944, TC315912, TC322759, TC322059, TC330654, TC282713, TC293173, TC281861, TC283253, TC283383, TC285032, TC290021, TC302716, TC283487, TC282866, TC283441, TC281428, TC285118, TC285090, from GenBank database: AY967408, AF384145, AF384144, AY029312-AY029315, AY029317-AY029319, Z95352, AF369563-AF369565, AF369567, AF369569-AF369576, Z83834, Z95496, AY581255), BLAST comparison is carried out to pears database (http://www.phytozome.net/search.php), select the highest sequence of similarity to download, obtain the MLO type gene (Pbr025598.1 of 25 candidates altogether, Pbr041839.1, Pbr008324.1, Pbr011748.1, Pbr023914.1, Pbr034551.1, Pbr037345.1, Pbr015088.1, Pbr008325.1, Pbr041838.1, Pbr016202.1, Pbr040713.1, Pbr007426.1, Pbr022047.1, Pbr026609.1, Pbr019309.1, Pbr034555.1, Pbr015086.1, Pbr036362.1, Pbr017451.1, Pbr018561.1, Pbr025602.1, Pbr023322.2, Pbr026643.1, Pbr026539.1).

2) qualification of pears MLO type gene family

In order to verify these MLO gene accuracys further, we have carried out the qualification of conserved domain " MLO " to the MLO gene of these 25 candidates.With the aminoacid sequence of the MLO type gene of each candidate for benchmark, PFAM (http://pfam.sanger.ac.uk/) website is carried out the qualification of ' MLO ' conserved domain, and concrete outcome is shown in Fig. 1.

3) the Phylogenetic Relationships analysis of pears MLO type gene

In previous research, find that dicotyledons powdery mildew gene aggregates into district's group; Therefore, in phylogenetic tree construction, we have selected 15 MLO type genes (wherein 3 genes are powdery mildew genes: AtMLO02, AtMLO06 and AtMLO12) of model plant Arabidopis thaliana, the powdery mildew gene of Gene against Powdery Mildew in Tomato, barley powdery mildew gene and pea analyze with pears MLO type gene clusters together with cluster analysis.Pears MLO type gene and other crop powdery mildew gene protein sequences are carried out multisequencing connection to join (adopting Clustal X1.83 software to carry out), and utilize Genedoc software (http://www.nrbsc.org/gfx/genedoc/index.html) to show multisequencing to join the result of joining.Clustal multisequencing is joined the result of joining output in MEGA4.0 software, and utilize this software to construct adjacent tree (neighbor-joining, NJ) respectively, utilize Bootstrapping method to assess these evolutionary trees.Found that in dicotyledons mildew-resistance gene district group, there is the MLO type powdery mildew gene (see Fig. 2) of 6 pears candidates.

4) comparison of pears MLO type disease-resistant gene

In the research of barley MLO type powdery mildew gene, in succession found some important areas and single amino acids in research, they infect large wheat powdery mildew irreplaceable effect.In order to identify in the pears powdery mildew gene of 6 candidates, these important areas and amino acid whether high conservative, we have carried out compare of analysis to from 3 mildew-resistance genes (AtMLO02, AtMLO6 and AtMLO12) of Arabidopis thaliana, tomato powdery mildew gene (S1MLO), powdery mildew of pea gene (PsMLO).Find the powdery mildew gene of pears 6 candidates and known MLO type powdery mildew gene 7 cross-film districts, 30 important amino acid, 1 calmodulin CaM binding region (CaMBD) and two important region (I and II) high conservatives (Fig. 3).

Claims (2)

1. pears mildew-resistance gene, it is characterized in that being selected from following 6 genes or its one of:

(1)Pbr041839.1

Amino acid:

MAGATSGRSLEQTPTWAVAVVCFVLVLISIIIEHIIELIAKWLKKKHKRALYEALEKIKSELMLLGFISLLLTVGQGPISNICISQKIGNTWHPCGKKQENKLNKEENVEHEDEIPGRRLLSLFNETGGGARRVLAAASTDKCAAKGKVSFISADGIHQLHIFIFVLAVFHVLYCILTMALGRAKMRSWKRWEKETRTDEYQFSHDPERFRFARDTSFGRRHMSYWTKTPLLMWMVCFFRQFVRSVPKVDYLTLRHGFIMAHLAPQSHKKFNFQKYINRSLEEDFKVVVGISPPIWFFAVIFLLFNTHGWYSYLWLPFVPLIIILLVGTKLQVIITKMGLRIQERGEVVKGLLVVQPGDDLFWFNRPRLILYLINFVLFQNAFQLAFFAWSWYEFSLKSCFHEHTEDVIIRVSMGVLIQILCSYVTLPLYALVTQMGSTMKPTIFNERVAAALRNWHHTARKHIKQNKGSVTPMSSRPATPSHHMSPVHLLRNYRSEVDSFHTSPRKSNLEGERWANESPSPSHHLHVDDASSSYHHHESVDVNEKISVHWEATTTNTSTTTMEPAQTHVINMGRSKDFSFDKRESI

Nucleotide:

ATGGCAGGGGCAACGTCAGGAAGGTCATTAGAGCAAACGCCAACATGGGCTGTTGCCGTAGTCTGTTTTGTTTTGGTCTTAATTTCTATAATCATTGAACATATAATCGAACTTATAGCAAAGTGGTTAAAGAAGAAGCACAAAAGAGCTCTTTATGAAGCACTGGAAAAGATCAAATCAGAGCTTATGCTATTGGGGTTCATATCCTTGCTCTTAACTGTGGGACAAGGACCAATATCAAACATATGCATATCACAGAAAATTGGCAACACTTGGCATCCATGCGGCAAGAAGCAAGAAAACAAATTGAATAAGGAAGAGAATGTGGAGCATGAGGATGAAATCCCGGGTAGGAGATTGCTCTCACTGTTTAACGAGACTGGTGGTGGCGCACGCCGTGTTCTGGCGGCTGCTAGCACCGATAAATGTGCAGCAAAGGGTAAAGTATCTTTTATATCTGCGGATGGTATTCATCAACTGCACATTTTTATCTTTGTGTTGGCTGTTTTCCATGTCCTTTATTGCATCCTCACCATGGCTTTAGGAAGAGCCAAGATGAGAAGTTGGAAGCGGTGGGAAAAGGAAACAAGGACTGATGAATATCAATTTTCGCATGACCCAGAGAGATTCAGGTTTGCAAGGGACACATCTTTTGGGAGGAGGCATATGAGTTACTGGACTAAAACACCTCTCCTTATGTGGATGGTTTGTTTTTTCAGACAATTTGTTAGGTCGGTTCCGAAAGTGGATTACTTAACCTTGCGGCATGGGTTTATCATGGCACATCTGGCACCCCAGAGCCATAAGAAGTTCAATTTCCAAAAGTACATAAACAGATCACTTGAAGAGGATTTCAAGGTGGTTGTGGGAATCAGTCCCCCAATTTGGTTCTTTGCTGTGATATTCCTACTCTTTAATACTCACGGCTGGTATTCTTATCTTTGGCTACCGTTTGTCCCGTTGATTATCATTCTTTTGGTGGGGACCAAGCTACAGGTGATCATCACAAAAATGGGTCTTCGAATTCAAGAAAGAGGAGAGGTTGTGAAGGGGCTTCTTGTGGTTCAACCTGGTGATGATCTCTTTTGGTTTAACCGCCCCCGTCTCATTCTCTATCTCATCAACTTTGTTCTCTTTCAGAATGCCTTCCAGCTTGCTTTCTTTGCATGGAGTTGGTATGAATTTAGCTTGAAATCTTGTTTTCACGAGCACACTGAGGACGTGATCATCAGAGTTTCAATGGGGGTCCTCATACAAATTCTCTGCAGTTATGTCACTCTCCCCCTCTATGCCCTTGTCACACAGATGGGCTCAACCATGAAACCAACCATATTCAATGAAAGAGTAGCCGCAGCATTACGGAACTGGCATCACACGGCCAGGAAACACATAAAGCAGAACAAAGGCTCCGTAACCCCAATGTCTAGCAGACCAGCCACTCCATCCCACCACATGTCCCCCGTCCACCTCCTCCGGAACTATCGGAGCGAAGTGGATAGTTTCCATACATCACCAAGAAAATCAAATTTGGAGGGTGAACGTTGGGCCAACGAATCGCCCTCCCCCTCACACCACCTCCATGTAGATGACGCTTCATCCTCTTACCACCACCATGAGAGCGTTGATGTCAATGAAAAAATCTCGGTTCATTGGGAAGCTACTACAACTAACACTAGTACTACTACCATGGAACCTGCTCAAACACATGTAATTAACATGGGACGGTCGAAAGACTTCTCATTTGACAAGAGGGAAAGTATATAA

(2)Pbr023914.1

Amino acid:

MAELAKERTLEETPTWAVAVVCFVLLAVSIFIEHTIHLIGKWLARKDKRALVEALEKIKSELMLLGFLSLLLTVLQEPISDICISKSIGATWHPCKYNKSSKSGKNGRKLLEFFNPRRRLAVKGYDYCTGKGKVAFVSSYGIHQLHIFIFVLAVFHVLYCILTLGLGRYKMRTWKVWEKETKSIEYRQFYNDSEKFRFARDTSFGRRHLNFWSKSPVFLWIVCFFRQFFHSVTKVDYLTLRHGFIMAHLAPGSESTFDFQKYINRSVEEDFKVIVGISPIIWFSAVLFLLSNTYGWYSYIWLPTIPLIIVLMVGTKLQVIITKMGLRIQESGHVVKGAPLVQPSDHLFWFGSPHFMLYLLHFVVFQNAFQMAFFAWSTYEFGITSCFHRSTEDIVVRLSFGVIVQVLCSYVTLPLYALVTQMGSTMKPTVFNKGVAAALKDWHHKAKKNTKHSRHSPSTTPVHLLHNYNYRSNDDEFYVSPSRANLEMGHWGTEGSSHSPNNNVGPSDGDEIRQMTNVRDLDQEPGSTELPRSPLPVRRTQSQHEVAISSSDFSFGRAN

Nucleotide:

ATGGCTGAACTCGCTAAAGAGCGTACACTAGAGGAAACACCGACATGGGCTGTTGCTGTGGTCTGTTTTGTGTTGCTTGCAGTTTCAATCTTCATTGAACATACGATCCACCTCATAGGAAAGTGGCTAGCAAGGAAGGACAAGAGAGCTCTAGTTGAAGCACTTGAGAAGATCAAATCCGAGCTTATGCTGTTGGGATTTTTATCCTTGCTCCTAACTGTGCTGCAAGAACCAATCTCTGATATATGCATATCAAAAAGTATTGGAGCCACTTGGCATCCCTGCAAATATAATAAGTCATCAAAATCTGGAAAGAATGGTAGGAAACTTCTTGAGTTCTTCAATCCCAGGCGCAGATTAGCAGTAAAAGGATATGATTACTGCACAGGGAAGGGAAAAGTAGCCTTTGTGTCTTCATATGGGATTCACCAACTCCATATTTTTATCTTTGTGTTAGCAGTTTTTCATGTGCTCTACTGCATCCTTACCTTGGGTTTGGGAAGATACAAGATGAGAACGTGGAAGGTTTGGGAGAAAGAGACAAAATCAATTGAATACCGCCAGTTCTATAATGATTCAGAAAAGTTTAGGTTTGCAAGGGATACATCATTTGGACGAAGACATTTGAACTTCTGGAGCAAGTCACCAGTCTTCCTTTGGATTGTCTGTTTCTTTAGACAATTTTTCCACTCAGTTACAAAGGTTGATTACTTAACTCTGAGGCATGGATTTATCATGGCACATTTGGCACCAGGAAGTGAGTCAACATTTGATTTTCAGAAGTACATAAACAGATCAGTTGAAGAGGATTTCAAAGTTATTGTGGGGATTAGTCCAATTATATGGTTCTCTGCAGTGCTGTTCTTGCTGTCCAACACTTATGGATGGTATTCTTATATTTGGCTACCAACCATCCCTTTAATTATAGTCCTAATGGTTGGAACCAAACTACAAGTGATTATAACAAAGATGGGGCTGAGAATTCAGGAGAGCGGGCATGTGGTAAAGGGTGCACCTCTTGTTCAACCCAGTGATCACCTCTTTTGGTTCGGAAGCCCCCACTTCATGCTCTATCTCCTCCACTTTGTTGTCTTTCAGAATGCATTTCAAATGGCCTTCTTTGCATGGAGTACATATGAGTTTGGGATAACTTCTTGCTTCCACCGGAGCACCGAAGACATTGTCGTCAGGCTCTCATTCGGGGTCATTGTACAAGTTTTATGCAGTTATGTGACTCTGCCTCTCTATGCTCTAGTAACTCAGATGGGTTCGACCATGAAACCTACCGTTTTCAATAAGGGAGTGGCAGCAGCACTGAAAGATTGGCACCACAAAGCCAAAAAGAACACAAAACACAGCCGCCATTCACCTTCAACCACCCCAGTTCATCTGCTTCACAACTACAACTACCGCAGCAATGACGATGAATTTTACGTATCTCCGAGCCGAGCAAACCTTGAAATGGGTCATTGGGGCACTGAAGGGTCCTCACATTCACCAAACAACAATGTTGGACCTAGTGACGGGGATGAGATTAGGCAAATGACCAACGTTAGGGATCTGGATCAAGAGCCTGGCTCAACGGAATTGCCTCGATCTCCGCTTCCAGTTCGTCGTACCCAGTCTCAGCATGAAGTTGCCATTAGTTCATCTGACTTTTCATTTGGAAGAGCCAACTGA

(3)Pbr034551.1

Amino acid:

MAGGKEGRSLEQTPTWAVAVVCFVLVLISILIEYFIHLIAKWLTKRNKRALYEALEKIKAELMLLGFLSLLLTVGQGPISNICISKAVGATWHPCSKKHEVKSDKGEDKSSDSDNNARRRLLSALDSNGGGRRVLAASGYDKCAAKNKVPFVSFYGVHQLHILIFVLAVFHVLYCITTLVLGRAKMRRWKIWELETKTADYQFSHDPERFRFARDTSFGRRHLSFWSRSPISLWIVCFFRQFFRSVPKVDYLTLRHGFIAAHLAPQSQTKFDFQKYINRSLEEDFKVVVGIRWGPTLWLFSVLFLMSNTHGSRSYLWLPFVPLVMILMVGTKLQVIITKMGLKLSERGEVVKGTPLVEPGDHLFWFNNPRLLLYIIHFVLFQNAFALAFFAWTWVRFHLSSCLQYEFGLKSCFHDKLEDVVLRISMGVVIQILCSYVTLPLYALVTQMGSTMKPVIFNDRVATALKKWHITAKKHIKHNKNASPASASGTSSHAMSPVHLLRNYQSEQDTESVRTLPRMSYFDNEGSDSPFHHQDDLTWSQQGTNLQGQEEEISAHGHNLESNALGGYGSIIQHEIQVQSAKLTFEESDRS

Nucleotide:

ATGGCAGGAGGAAAAGAAGGAAGATCTTTGGAGCAAACACCAACTTGGGCAGTTGCCGTTGTTTGTTTCGTTTTGGTTTTGATTTCGATCCTCATCGAATATTTCATTCATTTAATTGCAAAGTGGTTAACAAAGAGAAACAAAAGAGCTCTCTATGAAGCTCTTGAGAAGATCAAAGCGGAGCTTATGCTATTAGGGTTCCTATCTTTGCTTCTAACAGTAGGACAAGGCCCCATTTCAAATATTTGTATATCGAAGGCTGTTGGAGCAACTTGGCATCCATGCAGTAAGAAGCACGAGGTCAAATCAGACAAGGGCGAAGATAAGAGTAGTGATTCAGATAACAACGCCCGCAGAAGGCTTCTCTCAGCCTTGGATTCCAATGGGGGTGGACGACGTGTTTTAGCAGCCAGCGGATACGACAAATGTGCAGCCAAGAACAAAGTTCCATTTGTGTCATTTTATGGAGTCCACCAGCTTCACATACTCATCTTCGTGCTAGCCGTCTTTCACGTGCTTTACTGCATAACAACCCTAGTGTTGGGCAGAGCAAAGATGAGAAGATGGAAAATATGGGAACTGGAAACAAAGACAGCTGACTACCAATTCTCACATGACCCGGAAAGATTCAGATTTGCCAGAGACACATCCTTTGGAAGAAGACACTTGAGCTTTTGGAGCCGCTCTCCCATTAGCTTATGGATTGTTTGCTTCTTCAGGCAGTTCTTTAGATCTGTGCCCAAAGTTGATTACCTAACTCTGCGCCATGGGTTTATTGCTGCACATTTGGCTCCTCAAAGTCAAACTAAGTTTGACTTCCAGAAGTACATTAACAGGTCACTTGAAGAGGATTTCAAGGTTGTCGTAGGGATCAGGTGGGGCCCAACTCTTTGGTTATTTTCTGTATTATTCCTCATGTCTAATACCCACGGCTCACGTTCATACCTGTGGCTCCCATTTGTTCCCCTAGTTATGATCTTAATGGTAGGGACAAAGCTGCAAGTTATAATAACGAAGATGGGACTGAAATTATCAGAAAGAGGTGAAGTGGTTAAGGGGACCCCGCTGGTTGAGCCTGGTGACCATCTCTTCTGGTTCAACAACCCTCGCCTGCTTCTTTACATCATCCACTTTGTTCTCTTCCAGAATGCTTTTGCGCTTGCTTTCTTTGCATGGACTTGGGTAAGATTCCATCTTTCTTCTTGTTTGCAGTATGAATTTGGGTTGAAGTCTTGCTTCCATGACAAGTTAGAAGATGTCGTTTTAAGAATATCAATGGGAGTCGTTATACAAATCCTGTGCAGCTATGTAACTCTTCCACTTTATGCGTTGGTAACACAGATGGGTTCAACTATGAAGCCTGTAATCTTCAATGACCGAGTGGCGACCGCACTCAAAAAATGGCACATCACAGCGAAGAAACACATAAAACACAACAAGAATGCAAGTCCAGCCAGTGCATCGGGCACTTCCTCGCATGCCATGTCCCCTGTTCATCTACTACGCAACTATCAATCCGAACAGGACACTGAAAGCGTTCGAACGTTACCAAGAATGTCTTATTTTGATAACGAAGGTTCAGACTCACCGTTCCATCACCAAGACGATTTGACTTGGTCTCAGCAAGGTACAAACCTGCAGGGTCAGGAGGAAGAGATTAGTGCTCATGGGCATAACTTGGAGAGCAATGCTTTAGGCGGTTATGGTTCGATAATTCAACATGAGATTCAAGTTCAATCGGCGAAATTGACATTTGAGGAATCAGATAGGAGTTGA

(4)Pbr008325.1

Amino acid:

MAGGKKGRSLEQTPTWAVAVVCFVLVLISILIEYFIHLIGKWLKRRNKRALYEALEKIKSELMLLGFLSLLLTVGQGPISNICISKAVGSTWHPCSKKQEVKSDKNEDKSSASDDNARRRLLSALDSSGGGRRVLAAAGYDKCAAKMRKWKAWELETKTAAYQFSHDPERFRFARDTSFGRRHLSFWSRSPISLWIVCFFRQFVRSVPKVDYLTLRHGFIAAHLAPQSQTKFDFQKYTNRALEEDFKVVVGISPTIWLFAVLFLMSNTHGSRSYLWLPFVPLVMILMVGTKLQVIITKMGLQLSERGEVVRGTPLVEPGDHLFWFNNPRLLLYIIHFVLFQNAFALAFFAWTWYEFGLKSCFHEKLEDVVLRISMGVIIQILCSYVTLPLYALVTQMGSTMKPIIFNDRVATALKKWHITAKKHLKHKNASPASAPGTPLHSMSPVHLLHNYRYEQDIDSIRTSPRISYFDNEGSDSPFHHQDNLTWSQQGTNMEGQKKEISAHGPNSESNALGAYGSIIQHEIQIHSAALTFEKSERS

Nucleotide:

ATGGCAGGAGGAAAAAAAGGAAGATCTTTGGAGCAAACACCAACTTGGGCAGTTGCCGTTGTTTGTTTCGTTTTGGTTTTGATTTCGATCCTCATCGAATATTTCATTCATTTAATTGGAAAGTGGTTGAAAAGGAGAAACAAAAGAGCTCTCTATGAAGCACTTGAGAAGATCAAGTCGGAGCTTATGCTATTAGGGTTCCTATCCTTGCTTCTAACAGTAGGACAAGGCCCCATTTCAAATATTTGTATATCCAAGGCTGTTGGATCAACTTGGCATCCATGCAGTAAGAAGCAAGAGGTCAAATCAGACAAGAACGAAGATAAGAGTAGTGCTTCAGATGACAACGCCCGCAGAAGGCTTCTCTCAGCCTTGGATTCCAGTGGGGGTGGCCGACGTGTTTTAGCAGCTGCTGGATATGACAAATGTGCTGCCAAGATGAGAAAATGGAAAGCATGGGAACTGGAAACAAAAACAGCTGCTTACCAATTCTCACATGACCCGGAAAGATTCAGATTTGCCAGAGACACATCCTTTGGGAGAAGACACTTGAGCTTTTGGAGCCGTTCTCCCATTAGTTTATGGATTGTTTGCTTCTTCAGACAGTTTGTAAGATCTGTGCCCAAAGTTGATTACTTGACTCTGCGACATGGGTTTATTGCTGCACATTTGGCTCCTCAAAGTCAAACTAAGTTTGACTTCCAGAAGTACATTAACAGGGCACTTGAAGAGGATTTCAAGGTTGTCGTAGGGATCAGCCCAACTATTTGGTTATTTGCTGTATTATTCCTCATGTCAAACACCCACGGCTCACGTTCTTATCTGTGGCTCCCATTTGTTCCCCTAGTTATGATCTTAATGGTAGGGACAAAGCTGCAAGTTATAATAACAAAGATGGGGCTCCAATTATCAGAAAGAGGTGAAGTGGTTAGGGGAACCCCACTGGTTGAGCCTGGTGACCATCTCTTCTGGTTCAATAACCCCCGACTCCTGCTTTATATCATCCACTTTGTTCTCTTTCAGAATGCTTTTGCACTTGCTTTCTTTGCATGGACTTGGTATGAATTTGGCTTGAAGTCTTGCTTCCATGAGAAGTTGGAAGATGTCGTTTTAAGAATATCAATGGGGGTGATCATACAAATTCTGTGCAGCTATGTAACTCTTCCTCTTTATGCATTGGTAACACAGATGGGTTCAACTATGAAGCCTATAATCTTCAACGACAGAGTGGCGACTGCACTCAAAAAATGGCACATCACAGCGAAGAAGCACCTAAAACACAAGAATGCAAGTCCAGCCAGTGCACCAGGCACTCCTTTGCACTCTATGTCCCCTGTTCATCTACTACACAACTATAGATATGAACAGGACATTGACAGCATTCGGACATCACCAAGAATATCTTATTTTGATAACGAAGGTTCAGACTCTCCATTTCATCACCAAGACAATTTGACTTGGTCCCAGCAAGGTACAAACATGGAGGGTCAGAAGAAAGAGATTAGTGCTCATGGACCTAACTCAGAGAGCAATGCTTTAGGCGCTTATGGTTCGATAATTCAACATGAGATTCAAATTCACTCGGCAGCACTCACATTTGAGAAATCAGAAAGGAGTTGA

(5)Pbr022047.1

Amino acid:

MAELAKERSLEETPTWAVAVVCFVLLAVSIVIERMIHLIGKWLARKHKRALVEALEKIKSELMLLGFLSLLLTVLQGTISDICISQSVGATWHPCKYNKSSKSGSNGRKLLEFFNHRRRLAVKGNDYCTEKGKVAFLSAYAIHQLHIFIFVLAVFHVLYCILTLGFGRYKMRKWKVWEEETRSIEYQCHNVTKVDYLTLRHGFIMAHLEPGSESTFDFRKYITRSLEEDFKVIVEISPVIWFSAVLFMLSNTYVIITKMGLRIQERGDVVKGAPLVQPGDHLFWFGSPRLMLFLIHFVLFQNAFQLAFFAWSTYQFGIKSCFHQRTEDIVIRLSIGIMVQVLCSYVTLPLYALVTQMGSTMKPTIFNEEVAAAVKNCHHKAKKNAKHSHHSPSTTPFSTMPPTPTQGMSPVHLLHTHNYRSNDDGFITSPRGSNLEMDHRSTEGSSHSPNNNVGPSDGDEIMQITDIGDLDQEPGSAELARAPRRTRSQHEIEIISSNFSFGKGS

Nucleotide:

ATGGCTGAACTCGCTAAAGAGCGTTCCCTGGAGGAAACACCGACATGGGCTGTTGCTGTGGTCTGTTTTGTGTTGCTTGCAGTTTCGATCGTCATTGAACGAATGATCCACCTCATAGGAAAGTGGCTAGCAAGGAAACACAAGAGAGCTCTAGTTGAAGCGCTTGAGAAGATTAAATCTGAGCTTATGCTGCTGGGATTTTTATCCTTGCTCCTAACTGTGCTGCAAGGAACAATCTCTGATATATGCATATCACAAAGTGTTGGAGCCACTTGGCATCCCTGCAAATATAATAAGTCATCAAAATCTGGAAGTAATGGTAGGAAACTTCTTGAGTTCTTCAATCACAGGCGCAGATTAGCAGTAAAAGGAAATGATTACTGCACAGAGAAGGGAAAAGTAGCCTTTCTGTCTGCATATGCGATTCACCAACTGCATATTTTTATCTTTGTGTTAGCAGTTTTTCATGTGCTCTACTGCATTCTTACCTTGGGATTTGGAAGATACAAGATGAGGAAATGGAAGGTTTGGGAGGAGGAGACAAGATCAATTGAATACCAGTGCCATAATGTTACTAAGGTTGATTACTTAACTTTGAGGCATGGATTTATCATGGCACATTTGGAACCAGGAAGCGAGTCAACGTTTGATTTTCGGAAGTACATAACCAGATCACTTGAAGAGGATTTCAAAGTTATTGTGGAGATCAGTCCAGTTATATGGTTCTCTGCAGTGTTGTTCATGCTGTCCAACACATACGTGATTATAACAAAGATGGGGCTGAGGATTCAGGAGAGAGGGGATGTGGTGAAGGGTGCACCTCTTGTTCAACCCGGCGATCACCTCTTCTGGTTCGGAAGCCCCCGTCTCATGCTCTTTCTCATCCACTTTGTTCTCTTTCAGAATGCATTTCAACTGGCCTTCTTTGCATGGAGTACGTATCAGTTTGGGATAAAATCTTGCTTCCACCAGCGCACTGAAGACATTGTCATCAGGCTCTCAATCGGGATCATGGTACAAGTTTTATGCAGTTATGTGACTCTGCCTCTCTATGCTCTAGTAACACAGATGGGTTCGACCATGAAACCTACCATTTTCAATGAGGAAGTGGCAGCAGCTGTGAAAAATTGCCACCACAAAGCCAAAAAGAACGCGAAACACAGCCACCATTCACCGTCAACCACCCCATTTTCAACCATGCCACCAACTCCCACGCAAGGCATGTCCCCAGTTCATCTGCTGCACACCCACAACTACCGCAGCAATGACGATGGATTTATCACATCTCCGAGGGGATCAAACCTTGAAATGGATCACCGGAGCACTGAAGGGTCCTCACATTCACCAAACAACAATGTTGGACCTAGCGACGGGGATGAGATTATGCAAATCACCGACATTGGGGATCTGGATCAAGAGCCTGGCTCAGCAGAACTGGCTCGCGCGCCGCGTCGTACTCGGTCTCAGCATGAAATTGAGATTATTTCATCTAATTTTTCATTTGGGAAGGGCAGCTGA

(6)Pbr025602.1

Amino acid:

MAAATSGRSLEQTPTWAVAVVCFVLVLISIIIEHIIELIAKWLKKKHKSALYEALEKIKSELMLLGFISLLLTVGQGPISNICISQKIGNTWHPCGKKQEYKLNKEEDLEYVDETPSRKLLSLFNETGGGSRRVLAAAGTDKCSAKGKVPFISADGIHQLHIFIFVLAVFHVLYCILTMALGRAKMGSWKRWEKETRTAEYQFSHGCPPIWFFAVIFLLFNTHGWYSYLWLPFIPLIIILLVGTKLQVIITKMGLRIQESGEVVKGLPVVQPGDHLFWFNRPRLILYLINFVLFQNAFQLAFFAWSWYEFSLKSCFHEHTEDVIIRVSMGILIQILCSYVTLPLYALVTQMGSTMKPTIFNERVAAALRNWHHTARKHIKQNKGSVTPMSSRPATPSHHTSPAYLLRNYRSEVDSFHASPRRSNFEGERWANESPSPSHHLHVDGTSSSYHHHIEMGDVDRERVDVNEQNSVHGKTTITNTSITTMEPARTQQEINMERSKDFSFDKRQRIQ

Nucleotide:

ATGGCAGCGGCAACGTCTGGAAGATCGTTAGAGCAAACGCCGACGTGGGCTGTTGCTGTAGTCTGTTTTGTTTTGGTCTTGATTTCAATAATCATTGAACACATAATCGAACTTATAGCAAAGTGGTTAAAGAAGAAACACAAAAGCGCCCTTTACGAAGCACTAGAAAAGATCAAATCAGAGCTTATGTTATTGGGGTTCATATCCTTGCTCTTAACTGTGGGACAAGGACCAATATCAAACATATGCATATCACAGAAAATTGGCAACACTTGGCATCCGTGTGGCAAGAAGCAAGAATACAAATTGAATAAGGAAGAGGATTTGGAGTATGTGGATGAAACCCCGAGCAGGAAACTGCTCTCTCTGTTTAACGAGACTGGTGGTGGCTCACGCCGCGTTCTGGCTGCTGCTGGCACCGACAAATGTTCAGCAAAGGGTAAAGTACCTTTTATATCTGCGGATGGTATTCATCAACTGCACATTTTTATCTTCGTGTTGGCTGTTTTCCATGTCCTTTATTGCATTCTCACAATGGCTTTAGGAAGAGCCAAGATGGGAAGTTGGAAGCGGTGGGAAAAGGAAACAAGAACAGCTGAATATCAATTTTCGCATGGTTGTCCTCCAATTTGGTTCTTTGCTGTGATATTCCTACTTTTTAATACTCACGGCTGGTATTCTTATCTTTGGCTACCGTTCATCCCATTGATCATCATTCTCTTGGTGGGTACCAAGCTACAGGTGATCATCACAAAAATGGGTCTTCGAATTCAAGAAAGTGGAGAGGTTGTAAAGGGGCTTCCTGTGGTTCAACCTGGTGATCATCTCTTTTGGTTCAACCGCCCTCGTCTCATTCTCTACCTCATCAACTTTGTTCTCTTTCAGAATGCCTTCCAGCTTGCTTTCTTTGCATGGAGTTGGTATGAATTTAGCTTGAAATCTTGTTTTCACGAGCACACTGAGGACGTGATCATCAGAGTTTCAATGGGGATCCTCATACAGATTCTCTGCAGTTATGTCACTCTCCCCCTCTATGCCCTTGTCACACAGATGGGCTCAACCATGAAACCAACCATATTCAATGAAAGAGTAGCCGCAGCTTTACGCAACTGGCATCACACCGCCAGGAAGCACATAAAGCAGAACAAAGGCTCCGTAACCCCAATGTCTAGCAGACCAGCCACTCCATCCCACCACACGTCCCCCGCCTACCTCCTCCGGAACTATCGGAGCGAAGTGGATAGTTTCCATGCATCACCAAGAAGATCAAATTTCGAGGGTGAACGTTGGGCCAACGAATCACCCTCCCCCTCACACCACCTCCATGTAGATGGCACTTCATCCTCTTACCACCACCALATTGAAATGGGAGATGTAGACCGTGAGAGGGTTGATGTCAATGAACAAAATTCGGTTCATGGGAAAACTACAATAACTAATACTAGTATTACTACCATGGAACCTGCTCGAACACAACAAGAAATTAACATGGAGCGGTCGAAAGACTTCTCATTTGACAAGAGGCAACGTATACAATAG

2. the application of Rapid identification pears powdery mildew gene described in claim 1, comprising:

1) initiative or the new variety initiative of the breeding material of mildew-resistance MLO gene is carried: utilize the MLO GENE SOURCES given by right 1 to be parent material, in hybridization, backcross progeny, the breeding material that transformation has MLO resistant gene can be obtained.

2) mildew-resistance fundamental research: molecular marker analysis is carried out to the MLO type mildew-resistance gene of right 1; Molecular Mapping or the assignment of genes gene mapping are carried out to this gene; This gene is cloned and Interaction among genes analysis.

3) mildew-resistance transgenic research: utilize the MLO type powdery mildew gene given by claim 1 to carry out transgenic research.