CN104561253A - Rapid identification of corn powdery mildew resistant genes - Google Patents

Rapid identification of corn powdery mildew resistant genes Download PDF

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CN104561253A
CN104561253A CN201310517530.9A CN201310517530A CN104561253A CN 104561253 A CN104561253 A CN 104561253A CN 201310517530 A CN201310517530 A CN 201310517530A CN 104561253 A CN104561253 A CN 104561253A
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powdery mildew
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钱孝英
钱春桃
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Nau (changshu) New Rural Development Research Institute Co Ltd
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Abstract

The invention provides a rapid identification method of corn powdery mildew resistant genes and relates to plant comparative genomics. Such subject knowledge as genetics and bioinformatics belong to the field of plant biotechnology science. The method provided by the invention comprises the following main steps: 1) downloading a corn whole genome sequence and collecting an MLO gene; 2) verifying the MLO gene; 3) researching the phylogenetic relationship of the MLO gene; and 4) comparing MLO type powdery mildew genes. By adopting the invention, the mining cycle of the corn powdery mildew genes is effectively shortened to facilitate the rapid identification of the powdery mildew genes; corresponding co-separation functional markers (SNP, SCAR and the like) are developed via the identified powdery mildew genes, and the identified powdery mildew genes can also be quickly applied to the auxiliary selection of the molecular markers of the powdery mildew resistant genes, and the accuracy is high; in combination with other anti-disease gene molecular markers, a multi-resistance breeding material can be cultured to shorten the breeding period and improve the breeding efficiency; a foundation is laid for illustrating the corn powdery mildew resistant molecular mechanism.

Description

Corn mildew-resistance gene Rapid identification
Technical field
The present invention is by means of corn order-checking whole genome sequence, utilize the method Rapid identification corn powdery mildew genes such as plant comparative genomics, genetics, information biology and candidate gene strategy, be mainly concerned with the download of corn 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
Powdery Mildew is a kind of global Plant diseases, and majority of plant all can infect this disease.It occurs in leaf, tender stem, anthocaulus and the position such as bud, petal.Initial stage is the irregular stigma of yellow-green colour, and edge is not obvious.Soon scab constantly expands, and white powder spot is born on surface, and finally this director goes out countless stain affected part grizzle, and cover its surface in flakes, blur margin is clear, in dirty white or light canescence.When being injured serious, leaf-shrinkage diminishes, tender tip distortion deformity, thus affects vine growth and development.
In recent years, due to hybrid maize establishing in large scale, and the increase of continuous cropping, Powdery Mildew is serious in some summer autumn corn plantation area morbidities of China, and loss is serious.At present aborning, spraying fungicide and to promote disease-resistant variety be main prevention and controls, but life-time service medicine can cause environmental pollution and Biological Strains of The Pest variation, therefore promoting disease-resistant variety is method the most safely and effectively.And the qualification of powdery mildew disease-resistant gene, location and clone, be the important foundation of cultivating disease-resistant variety.
MLO is the distinctive class disease-resistant gene of plant, the albumen of its coding 7 cross-films, directly can cause disease resistance response, the expression product of dominant gene may be the negative regulatory factor of programmed cell death and defensive raction, the disease resistance of wide spectrum can be showed when it is in recessiveness, there is great application prospect.At present, in corn, identified 9 MLO family members abroad, but the domestic research data about the differentiation of corn Powdery Mildew bacterial classification and its genetics of resistance rule is less, there is no bibliographical information at present, this seriously inhibits the paces of corn Powdery Mildew research.
At present, the elementary tactics of clone gene is generally divided into two kinds, i.e. forward genetics approach and reverse genetrics approach.Forward genetics be the proterties or gene expression product that show with target gene for starting point, finally determine target gene by phenotypic mutation or qualification Product characteristics; Reverse genetics is then take gene as starting point, and the methods involving verified by gene function finally determines target gene.Can specifically be divided into several as follows according to different Strategies For The Clonings and experimental technique: positional cloning is also known as map based cloning, sequence clone, functional cloning, phenotypic cloning etc.But under normal circumstances, the target sequence obtained by above-mentioned technology is all shorter, be generally not easy directly to obtain full length gene sequence.Along with the assignment of genes gene mapping and Plant Genome order-checking and the development of information biology, the sequencing result screening how fast utilization has obtained obtains the focus that target gene becomes research at present.Nearest completing of Maize genome order-checking provides condition for we excavate corn powdery mildew gene fast.This patent describes premised on corn whole genome sequence, in conjunction with knowledge such as genetics, genomics and information biology, excavates the method for 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 corn mildew-resistance gene fast.Its result can be used for the exploitation of corn 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 Maize genome 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 corn 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 corn powdery mildew gene.
Key step is as follows:
1) download of corn whole genome sequence and the collection of MLO type gene thereof
First corn whole genome sequence is downloaded from corn sequenced genes group database (http://www.phytozome.net/search.php); Use " DNATOOLS " software to the corn full-length genome amino acid sequence data building database obtained, then with pfam number pick storehouse (protein family database, 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 corn 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 Maize genome database, obtain candidate gene sequence.
2) qualification of corn MLO type gene family
By the candidate gene of homologous nucleotide 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 corn 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 corn MLO type gene together with the cluster analysis of the MLO type gene family of Arabidopis thaliana with some other crops, to obtain the corn mildew-resistance gene (Fig. 2) of candidate.
4) comparison of corn 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 corn 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 corn candidate further.
Positively effect of the present invention:
1) shorten corn 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 corn powdery mildew gene fast, not only can shorten the time, powdery mildew gene determination rates can also be improved.
2) corn (Zea mays) is one of whole world food crop.Because maize genetic basis is narrow, Germplasm Resources Diversity is low, therefore more difficult by conventional molecule marker (RAPD, ISSR, SSR, AFLP etc.) qualification corn powdery mildew gene.The 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, carry out the initiative of multiresistance breeding material, can shortening the breeding cycle, improve breeding efficiency.
3) lay a good foundation for setting forth corn mildew-resistance molecular mechanism.The qualification of corn 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 corn mildew-resistance fast.
Accompanying drawing explanation
The qualification of Fig. 1 corn MLO gene;
What this figure showed is 15 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 corn 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 (SIMLO), barley powdery mildew gene (HvMLO and HvMLO02) and pea is selected to and the analysis of corn MLO type gene clusters.Identify 1 corn MLO type powdery mildew gene altogether.In figure, the gene of italic mark is exactly corn powdery mildew gene.
The compare of analysis of Fig. 3 corn MLO type powdery mildew gene;
1 corn powdery mildew gene 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 corn 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 corn powdery mildew gene.Specific implementation process is as follows:
1) collection of corn MLO type gene
In order to obtain the whole MLO type gene family member of corn, 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, retrieves MLO type gene from maize genomic 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 maize database (http://www.phytozome.net/search.php), select the highest sequence of similarity to download, obtain the MLO type gene (GRMZM2G032219 of 15 candidates altogether, GRMZM2G040441, GRMZM2G051974, GRMZM2G031331, GRMZM2G471142, GRMZM2G033665, GRMZM5G830747, GRMZM2G089259, GRMZM2G325653, GRMZM2G000376, GRMZM2G117608, GRMZM5G881803, GRMZM2G062042, GRMZM2G110739, GRMZM2G416887, ).
2) qualification of corn MLO type gene conserved domain
In order to verify these MLO gene accuracys further, we have carried out the qualification of conserved domain " MLO " to these 15 MLO genes.With the aminoacid sequence of each MLO type gene for benchmark, PFAM (http://pfam.sanger.ac.uk/) website is carried out the qualification of ' MLO ' conserved domain, result shows, each MLO type gene has one or more ' MLO ' structural domain (Fig. 1).
3) the Phylogenetic Relationships analysis of corn MLO type gene
In previous research, find that dicotyledons powdery mildew gene aggregates into district's group; Therefore, in phylogenetic tree construction, the powdery mildew gene of 15 MLO type genes (wherein 3 genes are powdery mildew genes: AtMLO02, AtMLO06 and AtMLO12) of our selection mode plant Arabidopsis thaliana, Gene against Powdery Mildew in Tomato, barley powdery mildew gene and pea analyze with corn MLO type gene clusters together with cluster analysis.Corn 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 1 corn MLO type powdery mildew gene (see Fig. 2).
4) comparison of corn 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 1 corn powdery mildew gene, these important areas and amino acid whether high conservative, we have carried out amino acid alignment 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 corn 1 powdery mildew gene and known MLO type powdery mildew gene 7 cross-film districts, 30 important amino-acid residue, 1 calmodulin CaM binding region (CaMBD) and two regions played an important role with powdery mildew identification (I and II) high conservative (Fig. 3).Show that this gene is exactly corn MLO type mildew-resistance gene.

Claims (2)

1. corn mildew-resistance gene, is characterized in that being selected from following 1 gene:
GRMZM2G032219
Aminoacid sequence:
MAGGGGGRDLPSTPTWAVALVCAVIVLVSVAMEHGLHKLGHWFHTRQKKAMREALEKIKAEL
MLMGFISLLLAVGQTPISKICIPAKAGSIMLPCKPPKGAAAAADDDKSDGRRRLLWYPPYPGYD
EPGHHRRFLAGAAPDDNYCSDQGKVSLISSAGVHQLHIFIFVLAVFHIVYSVATMALGRLKMRK
WKKWESETNSLEYQYANDPSRFRFTHQTSFVKRHLGLSSTPGVRWVVAFFRQFFASVTKVDYL
TMRQGFINYHLSPSTKFNFQQYIKRSLEDDFKVVVGISLPLWFVAIFTLLIDIKGFGTLVWISFVPL
VILLLVGAKLEVVIMEMAKEIQDKATVIKGAPVVEPSDRFFWFNRPGWVLFLIHLTLFQNAFQM
AHFVWTLLTPDLKKCYHERLGLSIMKVAVGLVLQVLCSYITFPLYALVTQMGSHMKKTIFEEQT
AKAVMKWRKTAKDKVRQREAAGFLDVLTSADTTPSHSRATSPSRGNSPVHLLHKYRGRSEEPQ
SGPASPGRELGDMYPVADQHRLHRLDPERMRPASSTAVNIDIADADFSFSMR
Nucleotide sequence:
ATGGCGGGGGGCGGGGGCGGCCGGGACCTGCCGTCGACGCCGACGTGGGCGGTGGCCCTG
GTGTGCGCCGTCATCGTGCTCGTCTCCGTCGCCATGGAGCATGGCCTCCACAAGCTCGGCCA
CTGGTTCCATACGCGGCAGAAGAAGGCCATGCGGGAGGCCCTGGAGAAGATCAAAGCAGA
GTTGATGCTGATGGGCTTCATCTCGCTGCTCCTCGCCGTGGGGCAGACGCCCATCTCCAAGA
TATGCATCCCGGCCAAGGCTGGCAGCATCATGCTGCCGTGCAAGCCGCCGAAAGGCGCCGC
CGCCGCCGCCGACGACGACAAGAGCGACGGCCGCCGGAGACTCCTCTGGTACCCGCCGTA
CCCTGGATACGATGAGCCCGGGCACCACCGCCGTTTCCTCGCCGGCGCGGCTCCGGACGAC
AACTACTGCAGTGACCAAGGCAAGGTGTCCCTCATCTCCTCGGCCGGCGTCCACCAGCTGC
ACATCTTCATCTTCGTGCTCGCGGTGTTCCATATCGTCTACAGCGTCGCCACCATGGCGCTGG
GGCGTCTCAAAATGAGGAAATGGAAGAAATGGGAATCGGAGACCAACTCCCTGGAATACCA
GTACGCAAACGACCCTTCACGGTTCCGGTTCACGCACCAGACGTCGTTCGTGAAGCGGCAC
CTGGGCCTCTCGAGCACCCCTGGAGTGAGATGGGTCGTGGCGTTCTTCAGGCAGTTCTTCG
CGTCCGTGACCAAGGTGGATTACCTGACCATGCGGCAGGGGTTCATCAACTACCATCTGTCG
CCCAGCACCAAGTTCAACTTCCAGCAGTACATCAAGCGGTCCTTGGAGGACGACTTCAAAG
TCGTCGTTGGCATCAGTCTCCCGCTGTGGTTCGTCGCCATCTTCACTCTCTTGATCGATATCA
AGGGATTCGGCACGCTTGTCTGGATCTCTTTTGTCCCGCTCGTTATACTCCTGCTAGTTGGGG
CCAAGCTGGAGGTTGTCATCATGGAGATGGCCAAGGAGATACAGGACAAGGCGACGGTCAT
CAAGGGGGCGCCTGTGGTGGAGCCAAGTGACAGGTTCTTCTGGTTTAACCGCCCTGGCTGG
GTCCTCTTCCTCATCCACCTCACGCTCTTCCAGAACGCCTTCCAGATGGCGCATTTCGTTTGG
ACACTGCTCACCCCAGACCTGAAGAAATGCTACCACGAGAGGCTGGGCCTGAGCATCATGA
AAGTTGCGGTGGGGCTGGTTCTCCAGGTCCTCTGCAGCTACATCACCTTCCCGCTCTACGCG
CTCGTCACGCAGATGGGGTCGCACATGAAGAAGACCATCTTCGAGGAGCAGACGGCCAAG
GCGGTGATGAAGTGGCGCAAGACGGCCAAGGATAAGGTGCGGCAGCGGGAGGCGGCAGGC
TTCCTCGACGTGCTGACGAGCGCCGACACCACGCCGAGCCACAGCCGCGCGACGTCGCCG
AGCCGGGGCAACTCGCCGGTGCACCTGCTCCACAAGTACAGGGGCAGGTCGGAGGAACCG
CAGAGCGGGCCGGCGTCGCCGGGGCGGGAGCTCGGGGACATGTACCCGGTGGCTGACCAG
CATCGCCTGCACAGGCTGGACCCCGAGAGGATGAGGCCCGCCTCGTCCACCGCCGTCAACA
TTGACATCGCTGATGCCGATTTTTCTTTTAGCATGCGGTGA。
2. the application of Rapid identification corn 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.
CN201310517530.9A 2013-10-28 2013-10-28 Rapid identification of corn powdery mildew resistant genes Pending CN104561253A (en)

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