CN104593481A - Rapid identification of soybean anti-powdery mildew gene by using candidate gene strategy - Google Patents
Rapid identification of soybean anti-powdery mildew gene by using candidate gene strategy Download PDFInfo
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Abstract
The invention provides rapid identification of soybean anti-powdery mildew gene, relates to knowledge in the disciplines of plant comparative genomics, genetics and bioinformatics, and belongs to the scientific field of plant biotechnology. The invention includes the steps of: 1) downloading the soybean whole genome sequence and acquiring MLO type gene; 2) identifying the MLO genotype; 3) identifying MLO gene phylogenetic relationship; and 4) comparing MLO-type powdery mildew gene. The invention effectively shortens the gene mining cycle for soybean powdery mildew, and is conducive to the rapid identification of anti-powdery mildew gene; corresponding coseparation functional markers (SNP, scar, etc.) are developed through the identified candidate powdery mildew genes, and the identified candidate powdery mildew genes can also be used for fast molecular marker assisted selection of anti-powdery mildew genes and have high accuracy; and combined with other resistant gene molecular markers, multi-resistance breeding material can be created, so as to shorten the breeding period and improve breeding efficiency. Therefore, the invention lays foundation for the explanation of powdery mildew resistant molecular mechanism of soybean.
Description
Technical field
The present invention is by means of soybean order-checking whole genome sequence, utilize the method Rapid identification soybean powdery mildew genes such as plant comparative genomics, genetics, information biology and candidate gene strategy, be mainly concerned with the download of soybean 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
Soybean Powdery Mildew is that one has fungus-caused disease, is one of Major Diseases affecting world soybean production, is also a kind of Common Diseases threatening soybean in China.Powdery Mildew main harm soybean leaves, beanpod of not causing harm, petiole and stem stalk are seldom fallen ill.At the susceptible initial stage, leaf occurs spot is circular, the dark green haloing of tool.Cover with white powder thing gradually afterwards, later stage white powder thing occurs chocolate spherical particle thing, last sick leaf turns yellow and comes off, and has a strong impact on vine growth and development.Facts have proved, control soybean Powdery Mildew method that is most economical, effective, environmental protection is the kind of cultivating mildew-resistance.And the qualification of soybean 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 product of its coding directly can cause disease resistance response, but up to the present only in barley, find to there is such gene, the albumen of its coding 7 cross-films, 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.Therefore the MLO type disease-resistant gene excavated in soybean has great importance to the breeding of Soybean Resistance Powdery Mildew.
MLO type disease-resistant gene is the special class disease-resistant gene of plant.Investigator find the earliest MLO (
mildew resistance
locus
o) gene pairs powder mildew resistance starts from 1937-1938, acquired the barley of a lot of kind in Ethiopia by fritz, two strains wherein all have efficient resistance to Powdery Mildew (Blumeria graminis f.sp.hordei) all known physiological strains.Further research shows, in barley, the recessive mutation mlo of MLO gene can make barley produce resistance that is lasting, wide spectrum to the physiological strain of nearly all known barley powdery mildew bacteria.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.
At present, the conventional method excavating disease-resistant gene conventional has map based cloning, the methods such as transposon tagging.But because the fundamental research of soybean 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 soybean will become the important prerequisite of Soybean Resistance Powdery Mildew breeding.
Plant comparative genomics (Comparative Genomics) is based on gene order-checking basis, compares, understand the subject of the function of gene, expression mechanisms and spore to known gene and genome structure.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 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 soybean 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 " Soybean Resistance powdery mildew gene fast.In recent years, completing of soybean gene group order-checking provides condition for we excavate soybean powdery mildew gene fast.This patent describes premised on soybean 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 Soybean Resistance powdery mildew gene fast.Its result can be used for the exploitation of soybean 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 soybean gene group 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 soybean 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 soybean powdery mildew gene.
Key step is as follows:
1) download of soybean whole genome sequence and the collection of MLO type gene thereof
First soybean whole genome sequence is downloaded from soybean sequenced genes group database (http://www.phytozome.net/search.php); Use " DNATOOLS " software to the soybean full-length genome amino acid sequence data building database obtained, then pfam database (protein family database is used, http://pfamjanelia.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 soybean 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 soybean gene group database, obtain candidate gene sequence.
2) qualification of soybean 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 soybean 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 soybean MLO type gene together with the cluster analysis of the MLO type gene family of Arabidopis thaliana with some other crops, to obtain the Soybean Resistance powdery mildew gene (Fig. 2) of candidate.
4) comparison of soybean 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 soybean 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 soybean candidate further.
Positively effect of the present invention:
1) shorten soybean 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 soybean powdery mildew gene fast, not only can shorten the time, powdery mildew gene determination rates can also be improved.
2) soybean (Glycine max) belongs to Papilionaceae Glycine, is one of whole world important crops of extensively planting.Because soybean heredity basis is narrow, Germplasm Resources Diversity is low, therefore more difficult by conventional molecule marker (RAPD, ISSR, SSR, AFLP etc.) qualification soybean 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, carry out the initiative of multiresistance breeding material, can shortening the breeding cycle, improve breeding efficiency.
3) lay a good foundation for setting forth Soybean Resistance Powdery Mildew molecular mechanism.The qualification of Soybean Resistance powdery mildew 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 Soybean Resistance Powdery Mildew fast.
Accompanying drawing explanation
The qualification of Fig. 1 soybean MLO gene;
What this figure showed is 24 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 soybean 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 (S1MLO), barley powdery mildew gene (HvMLO and HvMLO02) and pea is selected to and the analysis of soybean MLO type gene clusters.Identify the MLO type powdery mildew gene of 1 soybean candidate altogether.In figure, the gene of italic mark is exactly candidate soybean powdery mildew gene.
The compare of analysis of Fig. 3 soybean MLO type powdery mildew gene;
1 soybean powdery mildew gene and barley (HvMLO), tomato (S1MLO), 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 soybean 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 soybean powdery mildew gene.Specific implementation process is as follows:
1) collection of soybean MLO type gene
In order to obtain the whole MLO type gene family member of soybean, we are 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 builds HMM model, receives rope MLO type gene from soybean 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 soybean database (http://www.phytozome.net/search.php), select the highest sequence of similarity to download, obtain the MLO type gene (Glyma01g36170 of 24 candidates altogether, Glyma01g37000, Glyma03g22960, Glyma03g33660, Glyma04g00370, Glyma04g01710, Glyma04g01730, Glyma06g00440, Glyma06g01800, Glyma06g01820, Glyma06g38140, Glyma08g20120, Glyma09g32920, Glyma11g09270, Glyma11g15920, Glyma12g29080, Glyma12g35160, Glyma13g30760, Glyma13g35390, Glyma15g08530, Glyma15g13070, Glyma16g08900, Glyma16g21510, Glyma19g36370, ).
2) qualification of soybean MLO type gene conserved domain
In order to verify these MLO gene accuracys further, we to the MLO gene of these 24 candidates carry out conserved domain " MLO ' qualification.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, result shows, each MLO type gene has one or more ' MLO ' structural domain (Fig. 1).
3) the Phylogenetic Relationships analysis of soybean 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 soybean MLO type gene clusters together with cluster analysis.Soybean 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 1 soybean candidate.
4) comparison of soybean 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 soybean powdery mildew gene of 1 candidate, 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 the powdery mildew gene of soybean 1 candidate 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 1 gene is soybean MLO type mildew-resistance gene.
Claims (2)
1. Soybean Resistance powdery mildew gene, it is characterized in that being selected from following 1 gene or its one of:
Glyma11g15920
Aminoacid sequence:
MAGGSGGRNLEETPTWAVSAVCFVLILISIIIEHIIHLIGKWLKKKHKTALYESLEKIKSELMLLGFISLLLTVGQGPISRICISEKVAGTWHPCDDSSSIHESDTEESENVNGTNSRRLLAAFYGSDDVNPRRVLAGGGTDKCREGKVPFVSSDGIHQLHIFIFVLAVFHVLYCILTMALGRAKMKRWKRWEEETKTPEYQFSHDPERFRFARETSFGRRHLSFWTKNPVLMWIVCFFRQFVRSVPKVDYLTLRHGFIMAHLAPQSHSKFDFRKYIKRSLDEDFKVVVGISPPFWFFAVLFLLLNTHGKQLRPFLIYILTMMCLNYAWTGWYSYLWLPFIPLIIILLVGTKLQVIITEMGLRIQQRGEVLKGVPLVQPGDHLFWFNRPGLILYLINFVLFQNAFQLAFFAWSALQFGIKSCFHSHTEDVVIRITMGVLIQILC SYVTLPLYALVTQMGSTMKPTIFNDRVAVALRNWHQTAKKHIRQNRVGPLSLSGTPTSSRPTTPSHHLSPVHLLRYYRSEIDSFPASPRRSNFDDNNMDSPSPSYSLHQLPPQTTATTNDSANDNNATTHHEIVVVHSKEFSFDKRPTSSSGNT
Nucleotide sequence:
ATGGCAGGAGGAAGCGGAGGAAGAAACTTGGAGGAAACGCCTACATGGGCTGTCTCCGCTGTTTGTTTTGTTTTGATCTTAATCTCTATAATCATCGAGCACATCATCCATCTCATAGGAAAGTGGTTGAAGAAGAAGCACAAAACAGCTCTGTACGAGTCACTTGAAAAGATCAAATCAGAGCTTATGTTATTGGGGTTCATATCGTTGCTTCTAACGGTAGGACAAGGTCCAATATCAAGAATATGCATTTCAGAAAAAGTTGCAGGGACATGGCACCCCTGCGACGATAGTAGCAGCATCCACGAGTCAGATACTGAAGAATCAGAAAACGTAAACGGAACCAATAGCCGCAGATTATTGGCGGCGTTCTATGGTTCAGACGACGTAAATCCACGACGTGTTTTGGCGGGAGGAGGAACCGACAAGTGTCGTGAGGGTAAAGTCCCGTTTGTGTCATCGGATGGCATTCATCAACTCCATATATTTATCTTCGTGCTGGCTGTTTTTCATGTCCTTTACTGCATTCTCACTATGGCTCTGGGCAGAGCAAAGATGAAAAGGTGGAAGCGATGGGAAGAGGAAACCAAGACACCAGAATACCAATTTTCACACGATCCTGAGAGATTCAGATTTGCGAGGGAGACATCTTTTGGAAGAAGACATTTGAGTTTCTGGACCAAAAATCCTGTCCTCATGTGGATTGTTTGTTTCTTTAGGCAATTTGTGCGGTCAGTTCCTAAAGTGGATTACTTGACCTTAAGACATGGATTTATCATGGCACATTTGGCTCCTCAAAGTCACTCGAAGTTTGATTTTCGGAAATATATAAAACGATCACTGGACGAGGATTTCAAAGTCGTAGTTGGAATCAGTCCTCCATTCTGGTTCTTCGCAGTGCTGTTTCTGCTCTTAAACACTCATGGTAAGCAACTACGACCCTTTTTAATTTATATACTTACTATGATGTGTCTCAATTATGCATGGACAGGGTGGTACTCTTATCTGTGGCTGCCATTCATTCCTTTGATTATAATCTTGTTAGTGGGAACCAAGCTTCAAGTGATCATAACCGAGATGGGTCTAAGAATTCAACAAAGAGGAGAGGTCCTCAAGGGTGTGCCCTTGGTTCAGCCAGGGGATCATTTGTTCTGGTTTAACCGACCTGGCCTTATTCTTTACCTTATCAATTTTGTGCTCTTTCAGAATGCTTTTCAGCTGGCTTTCTTCGCATGGTCTGCGCTTCAGTTTGGGATTAAATCCTGTTTCCATTCGCACACGGAGGACGTGGTGATCAGAATCACAATGGGGGTCCTTATTCAAATCCTCTGCAGCTACGTTACTCTTCCTCTCTACGCTCTAGTGACACAGATGGGTTCAACAATGAAGCCAACGATATTCAACGATAGAGTAGCGGTAGCGTTGCGGAATTGGCACCAGACAGCGAAGAAGCACATAAGACAGAACCGTGTGGGGCCGCTGTCGTTGTCGGGGACTCCGACGTCGAGCAGGCCCACAACCCCAAGTCACCACCTGTCCCCGGTGCACCTCTTGCGATACTACCGCTCCGAAATCGACAGCTTCCCCGCGTCTCCGCGAAGGTCAAACTTCGACGATAATAATATGGATTCTCCTTCTCCTTCCTATTCGCTCCACCAATTGCCACCACAAACAACAGCAACAACCAATGACTCCGCCAATGATAATAACGCCACCACACACCATGAGATTGTCGTGGTGCACAGCAAGGAGTTTTCGTTTGATAAAAGACCTACCAGTAGTAGTGGTAATACGTAA。
2. the application of Rapid identification soybean 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.
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| CN108690883A (en) * | 2018-08-17 | 2018-10-23 | 华南农业大学 | A kind of molecular labeling RMD7 of the soybean powder mildew resistance of auxiliary identification soybean to be measured |
| CN109022449A (en) * | 2018-07-25 | 2018-12-18 | 沈阳农业大学 | Cucumber CsMLO1 gene and its silencing expression vector establishment method, application |
| CN111996275A (en) * | 2020-08-14 | 2020-11-27 | 华南农业大学 | Molecular marker RMD16 for assisting in identifying powdery mildew resistance of soybean to be detected |
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| CN109022449B (en) * | 2018-07-25 | 2021-03-23 | 沈阳农业大学 | Cucumber CsMLO1 gene and construction method and application of silent expression vector thereof |
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| CN108690883B (en) * | 2018-08-17 | 2020-09-15 | 华南农业大学 | Molecular marker RMD7 for assisting in identifying soybean powdery mildew resistance of soybean to be detected |
| CN111996275A (en) * | 2020-08-14 | 2020-11-27 | 华南农业大学 | Molecular marker RMD16 for assisting in identifying powdery mildew resistance of soybean to be detected |
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