CN102719445A

CN102719445A - Rapid identification of powdery mildew gene of Medicago truncatula by utilizing comparative genomics

Info

Publication number: CN102719445A
Application number: CN2012100949972A
Authority: CN
Inventors: 钱孝英; 俞锞
Original assignee: CHANGSHU ZHITANG TOWN XINSHENG TECHNICAL CONSULTATION SERVICE CO LTD
Current assignee: CHANGSHU ZHITANG TOWN XINSHENG TECHNICAL CONSULTATION SERVICE CO LTD
Priority date: 2012-03-31
Filing date: 2012-03-31
Publication date: 2012-10-10

Abstract

The present invention is rapid identification of a powdery mildew gene of Medicago truncatula, relates to discipline knowledge of plant comparative genomics, genetics and bioinformatics and belongs to the technical field of plant biotechnology. The invention mainly comprises the following steps: 1) download of Medicago truncatula genome sequence and collection of an MLO gene; 2) identification of MLO type gene; 3) MLO gene phylogenetic relationship; and 4) comparison of MLO type powdery mildew gene. The invention effectively shortens a mining cycle of the powdery mildew gene of Medicago truncatula and facilitates the rapid identification of the powdery mildew gene. The identified candidate powdery mildew genes can be used to develop corresponding coseparation functional markers (SNP and SCAR, etc.), and can also be used quickly for molecular marker assisted selection of powdery mildew resistant gene, and has high accuracy. Combined with other disease resistant gene molecular markers, the identified candidate powdery mildew genes can be used in development of multiresistance breeding material, so as to shorten the breeding period and improve breeding efficiency. The invention lays foundation to elaborate molecular mechanism of powdery mildew resistance of Medicago truncatula.

Description

Utilization comparative genomics Rapid identification puncture vine Alfalfa Powdery Mildew Disease gene

Technical field

The present invention is by means of puncture vine clover order-checking whole genome sequence; Utilize method Rapid identification puncture vine Alfalfa Powdery Mildew Disease genes such as plant comparative genomics, genetics, information biology and candidate gene strategy, be mainly concerned with the download of puncture vine clover whole genome sequence, the evaluation of candidate gene; The comparison of gene; Means such as cluster, and then identify powdery mildew gene, belong to the Plant Biotechnology scientific domain.

Background technology

Alfalfa Powdery Mildew Disease mainly by pulse family internal thread powdery mildew ( Leveillula leguminosarumGolov.) and the pea powdery mildew ( Erysiphe pisiDC.) cause that germ survives the winter on the residual body of diseased plant with cleistothecium, or survives the winter with resting mycelium, carry out just infecting with thecaspore spring next year after clover turns green growth.Germ is repeatedly infected with conidium again in the season of growth, and conidium borrows wind-force to propagate distance quite far away.Behind the pathogen infection plant, white mustiness spot all can appear in positions such as the two sides of clover blade, stem, petiole, pod, and the initial stage scab is little and rounded, enlarges then and converges mutually, covers whole blade faces gradually, forms the mould layer of one deck felted.Back side mildew is obviously more than the front on blade.Later stage produces faint yellow, orange to the black point in mould layer.The cleistothecium of pulse family internal thread powdery mildew is buried and is born in the mould layer of felted; The cleistothecium hypergene of pea powdery mildew is on the mycelium of spread.These two kinds of pathogenic bacterias can mix sometimes and be born on the same diseased plant.Alfalfa Powdery Mildew Disease is one of common disease of dry clover, falls ill especially serious in arid and warm area.China in Xinjiang, the central and west regions in Gansu and the Inner Mongol mainly are the microbial Powdery Mildews of internal thread white powder, the microbial Alfalfa Powdery Mildew Disease of pea white powder all has report on east Gansu, Beijing, Hebei, Shanxi, Guizhou, Sichuan and Xinjiang and other places.Brought great influence for the yield and quality of clover in the infringement of these producing region Powdery Mildews.Selecting the mildew-resistance alfalfa variety, excavating mildew-resistance gene is the important means that this type of disease of control takes place all the time.

MLO type disease-resistant gene is one type of special disease-resistant gene of plant.The investigator find the earliest MLO ( MIldew resistance lOcus o ) the gene pairs powder mildew resistance is to start from 1937-1938, gathered the barley of a lot of kinds by the fritz in Ethiopia, two strains systems wherein to the white powder germ ( Blumeria graminisF. sp. Hordei) physiological strains that all oneself know all have efficient resistance.Further research shows, the recessive mutation mlo of MLO gene can make barley that the physiological strain of nearly all oneself knowledge barley powdery mildew bacteria is produced resistance lasting, wide spectrum in the barley.Recently, the investigator finds that the mildew-resistance gene of a lot of plants all is a MLO type Gene Handling, like tomato, and pea, Arabidopis thaliana, rose, capsicum, Root or stem of Littleleaf Indianmulberry or the like.Therefore breeding has important effect to the plant mildew-resistance to excavate MLO type disease-resistant gene in the plant.

At present, the disease-resistant gene method commonly used of excavating commonly used has map based cloning, methods such as transposon tagging.But because the fundamental research of puncture vine clover is not deep enough, not only the time is long but also very difficult these genes of cloning exactly therefore to utilize these methods.Therefore, how the MLO type disease-resistant gene in the Rapid identification puncture vine clover will become the important prerequisite of puncture vine clover mildew-resistance breeding.

Plant comparative genomics (Comparative Genomics) is based on Genome Atlas and the order-checking basis, and known gene and genome structure are compared, and understands the subject of function, expression mechanism and the spore of gene.Utilize between model plant genome and other Plant Genome on the coded sequence and structural homology, clone's other plant gene discloses gene function and molecular mechanism, illustrates spore relation and genomic immanent structure.Method that this patent adopted and thinking: the research of model plant arabidopsis gene group has disclosed the function of MLO type gene; Utilize the homology clone puncture vine clover MLO type disease-resistant gene on its order of gene; According to the characteristics of meliority on the model plant Arabidopis thaliana experimental system and known MLO type disease-resistant gene, quick " seizure " puncture vine clover mildew-resistance gene.In recent years, the completion of puncture vine clover gene order-checking provides condition for our fast mining puncture vine Alfalfa Powdery Mildew Disease gene.It is prerequisite that this patent has been introduced with puncture vine clover whole genome sequence, in conjunction with knowledge such as comparative genomics, genetics, genomics, information biology and candidate gene strategy, fast mining powdery mildew gene.

Summary of the invention

Technical problem

The purpose of this invention is to provide a kind of through combining knowledge such as plant comparative genomics, plant genetics, genomics and information biology, fast mining puncture vine clover mildew-resistance gene.Its result can be used for the exploitation of puncture vine Alfalfa Powdery Mildew Disease gene compact linkage molecule mark on the one hand, carries out the molecular marker assisted selection breeding, also for other crop powdery mildew gene identification reference frame is provided on the other hand.

Technical scheme

Cardinal principle: first plant mildew-resistance gene (MLO) is cloned from barley, discovers that this gene is one type of special disease-resistant gene, is different from most of NBS (nucleotide-binding site) type disease-resistant gene of previous clone; Subsequently, the investigator has cloned powdery mildew gene in succession from plants such as tomato, Arabidopis thaliana, pea, capsicum, Root or stem of Littleleaf Indianmulberry, and what discover these genes encodings all is MLO type disease-resistant gene.Subsequently, numerous investigators confirm that through test of many times MLO type disease-resistant gene has become one type of mildew-resistance gene of plant specific.Find that further plant MLO type gene is a gene family; And the MLO gene family that derives from different plant species is carried out the Phylogenetic Relationships analysis finds, in the different plant species mildew-resistance gene always cluster become one type together, this type MLO gene all has the characteristic feature of mildew-resistance gene sequence.The completion of puncture vine clover gene order-checking provides a convenient for excavating powdery mildew gene.Therefore, can identify puncture vine Alfalfa Powdery Mildew Disease gene by means of the Phylogenetic Relationships of MLO gene family in the full genome of puncture vine clover that has checked order and the MLO powdery mildew gene of having cloned and for the amino acid conservative property of keeping powdery mildew gene MLO critical function.

Key step is following:

1) collection of the download of puncture vine clover whole genome sequence and MLO type gene thereof

At first download puncture vine clover whole genome sequence from puncture vine clover sequenced genes group DB (http://www.phytozome.net/search.php); Use " DNATOOLS " software that the full genome amino acid sequence data of puncture vine clover that obtains is set up DB; Use pfam DB (protein family DB then; Http:// pfam.janelia.org/search/sequence) hidden Markov model in (HMM) carries out Blastp (E-value=0.001) sequence alignment to the aminoacid sequence of MLO structural domain with the full genome amino acid sequence database of having set up of puncture vine clover, and preliminary screening goes out candidate gene sequence.Secondly, utilize the MLO type gene order of having announced, puncture vine clover genome database is carried out the BLAST comparison, obtain candidate gene sequence.

2) evaluation of puncture vine clover MLO type gene family

With the candidate gene of the homologous nucleotide sequence that obtains in the The above results, analyze through Pfam (E-value=1.0), remove the gene order (Fig. 1) of not having ' MLO ' structural domain.The ClustalW instrument that again candidate's disease-resistant gene sequence is provided through MEGA3.1 software (multisequencing comparison program) carries out the multisequencing comparison, removes Tumor-necrosis factor glycoproteins.

3) Phylogenetic Relationships through plant MLO type gene is identified candidate's puncture vine clover MLO type powdery mildew gene

Because previous research is verified; Dicotyledons MLO type powdery mildew gene is positioned at the same district of plant MLO Phylogenetic Tree group; Therefore in Phylogenetic Relationships research; We are MLO type mildew-resistance gene and the cluster analysis together of puncture vine clover MLO type gene of the MLO type gene family of Arabidopis thaliana and some other crops, to obtain candidate's puncture vine clover mildew-resistance gene (Fig. 2).

4) comparison of puncture vine Alfalfa Powdery Mildew Disease gene and known plant MLO powdery mildew gene

Utilize BioXM 2.6 softwares the aminoacid sequence of the MLO powdery mildew gene of puncture vine clover candidate's MLO type powdery mildew gene and Arabidopis thaliana, tomato, pea, barley to be converted to the file of Fasta form; These files are imported BioEdit 7.0 softwares; Use in this software Clustal software to carry out the multisequencing comparison, disclose the conservative property in candidate's powdery mildew gene important amino acid residue and zone.Thereby further identify puncture vine clover candidate's powdery mildew gene (Fig. 3).

Positively effect of the present invention:

1) shortens the puncture vine Alfalfa Powdery Mildew Disease gene excavating cycle, helped the Rapid identification of powdery mildew gene.Employing ordinary method (map based cloning, transposon tagging etc.) excavation mildew-resistance gene not only takes time and effort, efficient is low, and is difficult to successfully.The present invention is based on plant comparative genomics, genetics, bioinformatics method fast mining puncture vine Alfalfa Powdery Mildew Disease gene, not only can shorten the time, can also improve powdery mildew gene and identify efficient.

2) the puncture vine clover ( Medicago truncatula) belong to the pulse family Medicago, be one of pulse family mode crop.Because the hereditary basis of puncture vine clover is narrow, the germ plasm resource variety is low, therefore identifies puncture vine Alfalfa Powdery Mildew Disease genetic comparison difficulty through conventional molecule marker (RAPD, ISSR, SSR, AFLP etc.).The candidate's powdery mildew gene exploitation that is tested and appraised is divided into from functional mark (SNP, SCAR etc.) accordingly, can be used for the molecular marker assisted selection of disease-resistant gene fast, and accuracy is high.

3) initiative of multiresistance breeding material.Based on the functional molecular marker of the powdery mildew gene of new evaluation exploitation, combine the molecule marker of localized other disease-resistant genes, carry out the initiative of multiresistance breeding material, can shortening the breeding cycle, the raising breeding efficiency.

4) lay a good foundation for setting forth puncture vine clover mildew-resistance molecular mechanism.The evaluation of puncture vine clover mildew-resistance gene; Gene silencing (virus induced gene silencing through transgenic technology, RNAi, virus induction; VIGS) molecular mechanism of research mildew-resistance such as technology provides genetic resources, helps setting forth fast the mechanism of action of puncture vine clover mildew-resistance.

Description of drawings

The evaluation of Fig. 1 puncture vine clover MLO gene;

That this figure shows is 15 MLO type gene identification results, and each gene all contains ' MLO ' conserved domain.

The evaluation of the Phylogenetic Relationships analysis of Fig. 2 plant MLO gene family and puncture vine clover MLO type powdery mildew gene thereof;

Arabidopis thaliana is the model plant of plant science research; Grow in the tree at constructing system; The powdery mildew gene (PsMLO) of 15 MLO type genes of Arabidopis thaliana (wherein 3 genes are powdery mildew genes: AtMLO02, AtMLO06 and AtMLO12), tomato mildew-resistance gene (SlMLO), barley powdery mildew gene (HvMLO and HvMLO02) and pea is selected and is used for and the cluster analysis of puncture vine clover MLO type gene.Identify 1 puncture vine clover candidate's MLO type powdery mildew gene altogether.The gene of grey mark is exactly a candidate puncture vine Alfalfa Powdery Mildew Disease gene among the figure.

The compare of analysis of Fig. 3 puncture vine clover MLO type powdery mildew gene;

1 puncture vine Alfalfa Powdery Mildew Disease gene and barley (HvMLO), tomato (SlMLO), pea (PsMLO), Arabidopis thaliana powdery mildew gene (AtMLO02; AtMLO06 and AtMLO12) compare, identify that powdery mildew infects the amino-acid residue and regional conservative type that plays an important role.TM1-TM7 representes 7 revolving die zones of puncture vine clover MLO type powdery mildew gene among the figure; The black round dot representes that powdery mildew infects important amino-acid residue; CaMBD representes the calmodulin land; I and II represent powdery mildew is infected important amino acid region.

Embodiment

The evaluation of disease-resistant gene has important effect in research of crop disease-resistant theory of heredity and disease-resistant variety seed selection.Present method can Rapid identification go out puncture vine Alfalfa Powdery Mildew Disease gene.The practical implementation process is following:

1) collection and the evaluation of puncture vine clover MLO type gene

In order to obtain the whole MLO type gene family member of puncture vine clover; We are at first with the MLO type gene of Arabidopis thaliana; The mildew-resistance MLO gene order of tomato, pea, rose, capsicum, Root or stem of Littleleaf Indianmulberry makes up the HMM model, from puncture vine clover genome sequence, receives rope MLO type gene; Secondly with the MLO gene order delivered in the Different Crop as target sequence (from DFCI DB: 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 DB: AY967408, AF384145, AF384144; AY029312-AY029315, AY029317-AY029319, Z95352; AF369563-AF369565, AF369567, AF369569-AF369576; Z83834, Z95496, AY581255); Puncture vine clover DB (http://www.phytozome.net/search.php) is carried out the BLAST comparison, select the highest sequence of similarity to download, obtained 15 candidates' MLO type gene (AC202568_19 altogether; AC202568_22; AC225519_22; AC225519_24; Medtr2g006370; Medtr2g034050; Medtr2g110820; Medtr3g160010; Medtr3g160040; Medtr3g163760; Medtr4g108740; Medtr4g108950; Medtr5g024650; Medtr5g022330; Medtr8g087610).

2) evaluation of puncture vine clover MLO type gene family

In order further to verify these MLO gene accuracys, we have carried out the evaluation of conserved domain " MLO " to these 15 candidates' MLO gene.Aminoacid sequence with each candidate's MLO type gene is a benchmark, on PFAM (http://pfam.sanger.ac.uk/) website, carries out the evaluation of ' MLO ' conserved domain, and concrete outcome is seen Fig. 1.

3) the Phylogenetic Relationships analysis of puncture vine clover MLO type gene

In the research formerly, find that the dicotyledons powdery mildew gene aggregates into district's group; Therefore; Grow in the tree at constructing system; We have selected the powdery mildew gene and the puncture vine clover MLO type gene cluster analysis cluster analysis together of 15 MLO type genes (wherein 3 genes are powdery mildew genes: AtMLO02, AtMLO06 and AtMLO12), tomato mildew-resistance gene, barley powdery mildew gene and the pea of model plant Arabidopis thaliana.Puncture vine clover MLO type gene and other crop powdery mildew gene protein sequences are carried out the multisequencing couplet join (adopting Clustal X 1.83 softwares to carry out), and utilize Genedoc software (http://www.nrbsc.org/gfx/genedoc/index.html) to show that multisequencing joins the result who joins.The Clustal multisequencing is joined the result who joins output in MEGA 4.0 softwares, and (neighbor-joining NJ), utilizes the Bootstrapping method that these evolutionary trees are assessed to utilize this software to make up the adjacency tree respectively.The result finds in dicotyledons mildew-resistance gene district group, to have 1 puncture vine clover candidate's MLO type powdery mildew gene (see figure 2).

4) comparison of puncture vine clover MLO type disease-resistant gene

In the research of barley MLO type powdery mildew gene, found some important areas and single amino acids in the research in succession, they infect big wheat powdery mildew has irreplaceable effect.In the puncture vine Alfalfa Powdery Mildew Disease gene of identifying 1 candidate; Whether these important areas and amino acid high conservative, and we have carried out compare of analysis to 3 mildew-resistance genes (AtMLO02, AtMLO6 and AtMLO12), tomato powdery mildew gene (SlMLO), the powdery mildew of pea gene (PsMLO) from Arabidopis thaliana.1 candidate's of discovery puncture vine clover powdery mildew gene and 7 of known MLO type powdery mildew genes are striden the film district, 30 important amino acid, 1 calmodulin land (CaMBD) and two important zone (I and II) high conservatives (Fig. 3).

Claims

1. puncture vine clover mildew-resistance gene is characterized in that being selected from following 1 gene:

1）Medtr2g110820

Amino acid:

MAGGSVGRSLTETPTWAVAVVCFVILSISIFIEHIFHIIEKWLKKKHKSALYESLEKIKSELMLLGFISLLLTVGEGLISRICISEKVAATWHPCSNNANIESDDEELIDHETGGSRRLLAALLASQGDNHHRILAGGGGDKCAEEGKVAFVSAGAIHQLHIFIFVLAVFHILYCILTLALGRAKMRRWKRWEEATKTPEYQFSHDPERFRFANETSFGRRHLSFWTKNPVLIWIVCFFRQFVRSVPEVDYLTLRHGFMMAHLAPQSHLKFDFRQYIKRCLEEDFKVVVGISPPIWFITVFFLLFHTHGWHSYLWLPFLPLIIVLLVGTKLQVIITQMGLRIQKQGMVVKGEPVVQPRDDLFWFNKPRLILFLINFVLFQNAFQLAFFSWTALQFGVTSCYNSRKDGVVIRICMGVFVQILCSYVTLPLYALVTQMGSTMKPTIFNERVATALRNWHHTARKNIKHNRGSGSQTPFSSRSITPARSMSPAQILRHYRNQMDTPTRLNFETSHHYESYSPSPSNSHHHKVEINVASSSSTHLHEMEMGHLAHVEQQEVIKPNSISVGSGRPQFEIDIQQSDELSFSTMPTNQLE

Nucleotide:

ATGGCAGGAGGAAGCGTTGGAAGAAGCTTAACTGAAACACCTACTTGGGCCGTTGCAGTTGTTTGCTTTGTTATACTTTCTATTTCTATCTTCATTGAACACATTTTCCACATCATAGAAAAGTGGTTGAAGAAGAAGCATAAAAGTGCCTTGTATGAGTCACTTGAAAAGATCAAATCAGAGCTAATGTTACTAGGGTTCATATCATTGCTCCTAACAGTAGGAGAAGGTTTAATATCAAGAATATGTATATCAGAAAAAGTTGCAGCCACATGGCATCCATGTAGCAACAATGCAAATATTGAATCAGATGATGAAGAGTTAATAGACCATGAAACCGGTGGCAGCCGGAGATTACTAGCCGCGTTGCTTGCTTCTCAAGGCGACAATCACCACCGTATTTTGGCTGGTGGAGGAGGTGACAAATGTGCAGAAGAGGGAAAAGTAGCATTTGTATCAGCAGGGGCCATTCATCAACTCCATATATTTATATTTGTGCTTGCTGTTTTTCATATCCTCTACTGCATACTTACTCTGGCTCTAGGTAGAGCAAAGATGAGAAGGTGGAAAAGATGGGAAGAGGCAACCAAGACACCTGAGTACCAATTTTCACACGATCCTGAAAGATTCAGATTCGCTAACGAGACTTCGTTTGGAAGAAGACACTTAAGTTTCTGGACCAAAAATCCTGTCCTCATTTGGATTGTATGTTTTTTTAGGCAATTTGTAAGGTCAGTTCCTGAAGTGGATTACTTGACCTTAAGGCATGGATTTATGATGGCACATTTGGCTCCTCAAAGTCACCTGAAGTTTGACTTTAGACAATACATCAAAAGATGTTTGGAAGAAGACTTCAAAGTTGTTGTTGGAATCAGTCCTCCAATTTGGTTCATCACAGTGTTCTTCCTCCTGTTCCATACTCATGGGTGGCACTCTTATCTATGGCTACCATTTCTTCCTTTGATTATTGTCCTATTAGTTGGAACAAAGCTGCAAGTGATCATAACTCAAATGGGTCTTAGAATTCAAAAACAAGGAATGGTGGTAAAGGGTGAGCCAGTGGTGCAACCTAGGGATGACCTCTTTTGGTTTAACAAACCTAGACTTATTCTCTTCCTTATTAATTTTGTACTCTTTCAGAATGCCTTCCAGCTTGCTTTCTTTTCATGGACTGCTCTTCAATTTGGGGTGACATCCTGTTACAATTCACGTAAAGATGGTGTTGTCATTAGAATTTGCATGGGAGTCTTCGTTCAAATCCTTTGCAGCTACGTCACTCTCCCTCTCTATGCTCTCGTGACGCAGATGGGTTCGACCATGAAACCAACCATATTCAACGAAAGAGTAGCTACAGCTCTACGGAATTGGCACCACACCGCAAGGAAGAACATAAAGCACAACCGTGGATCGGGCTCTCAAACCCCATTTTCAAGTCGGTCCATAACTCCGGCACGTTCAATGTCTCCGGCCCAAATCCTTCGTCATTACCGCAACCAAATGGACACTCCAACAAGACTCAATTTTGAGACCAGTCACCACTATGAATCTTATTCACCCTCACCTTCCAACTCACACCACCACAAGGTTGAGATCAATGTTGCTTCTTCCAGCTCAACTCATCTTCATGAAATGGAAATGGGTCACCTAGCCCATGTTGAACAACAAGAAGTCATTAAGCCCAATAGTATTTCTGTGGGCTCAGGCCGGCCTCAATTTGAAATTGATATCCAACAGAGTGATGAACTCTCATTTTCAACAATGCCCACAAATCAATTAGAATGA。

2. the application of the said Rapid identification puncture vine of claim 1 Alfalfa Powdery Mildew Disease gene comprises:

1) initiative of breeding material.

3.2) breeding practice of mildew-resistance.

4.3) mildew-resistance fundamental research.