CN104560968A - Loofah downy mildew-resistant gene segments or gene markers and application thereof - Google Patents
Loofah downy mildew-resistant gene segments or gene markers and application thereof Download PDFInfo
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- CN104560968A CN104560968A CN201310524880.8A CN201310524880A CN104560968A CN 104560968 A CN104560968 A CN 104560968A CN 201310524880 A CN201310524880 A CN 201310524880A CN 104560968 A CN104560968 A CN 104560968A
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Abstract
The invention mainly relates to loofah downy mildew-resistant gene segments or gene markers and application thereof. Three loofah downy mildew-resistant gene segments or gene markers are rapidly separated by mainly adopting an anti-disease gene homologous sequencing method; and through sequencing, the lengths of nucleotides of these gene segments or gene markers are respectively 498bp, 504bp and 498bp. According to the loofah downy mildew-resistant gene segments or gene markers, the excavating period of a loofah downy mildew-resistant gene is effectively shortened; rapid identification of a downy mildew gene is facilitated; corresponding co-separation functional markers (SNP, SCAR and the like) are developed through identified downy mildew; the gene segments or gene markers can also be applied to assistant selection of molecular markers of the downy mildew-resistant gene; the accuracy is high; creation of multi-resistance breeding materials can be carried out by combining the loofah downy mildew-resistant gene markers with molecular markers for other anti-disease genes; the breeding period is shortened; the breeding efficiency is improved; and a foundation is laid for elaboration of molecular mechanism of the loofah downy mildew-resistant gene.
Description
Technical field
The present invention utilizes disease resistant gene homologous sequence method, clone's sponge gourd downy mildew resistance gene fragment or conversion are molecule marker etc., relate generally to sponge gourd conventional cross-breeding, molecular mark, the location of downy mildew resistance gene and Molecular Mapping, the aspects such as the clone of downy mildew resistance gene and the Regulation Mechanism analysis of disease-resistant gene, belong to Plant Biotechnology scientific domain.
Background technology
Oidium is that one of upper most important disease produced by sponge gourd.Main harm blade, sick leaf first occurs irregular faint yellow to aureus scab, after expand as the brown scab of polygon, time moist there is the mould layer of atropurpureus in the scab back side, and later stage scab is linked to be sheet, and blade is withered, Severe Reduction.Sponge gourd oidium cause of disease and cucumber downy mildew belong to a kind of germ together, and be called Pseudoperonospora cubensis fungi, germ survives the winter on invalid body, low temperature overcast and rainy day, and atmospheric moisture is high, and day and night temperature is large, and field drainage is bad, are all conducive to morbidity.Field production germ borrows wind and rain to propagate.Oidium has had a strong impact on the normal growth and development process of sponge gourd plant, brings great financial loss to production.The utilization of disease-resistant gene and breeding for disease resistance are the most effective meanss of sponge gourd disease control.
Nineteen forties, Flor proposes classical " gene-for-gene " theory, namely there is a dominant gene separately in plant and cause of disease thalline, the disappearance of plant disease resistance genes or germ nontoxicity (Avr) gene or change can cause the generation of disease.This model is applicable to most of biotroph pathogenic bacteria, comprises virus, bacterium, fungi and threadworms." gene-for-gene " disease resistance is generally acceptor by as model: plant disease-resistant albumen direct or indirect identification can be derived from the Avr product of germ.Once this identification occurs, defensive raction will be caused.Most of disease-resistant gene (Resistance gene; R) resistance triggered is relevant to quick defensive raction, is called anaphylaxis (hypersensitive response; HR).And at present, the product of most of disease-resistant gene all contains the structural domain of albumen identification, as the structural domain of the structural domain or coiled coil (CC) that are rich in leucic repeating unit (LRR); Signal transduction structural domain, as nucleotide-binding site (NBS) or protein kinase (PK) structural domain.Some disease-resistant gene products are predicted to be cytoplasm protein, and other are by membrane spaning domain (TM) transmembrane.These domain combinations can produce dissimilar R albumen.At least six kinds of dissimilar R genes have been identified: 1) NBS-LRR, 2 according to the characteristic of structure) LRR-TM-PK, 3) LRR-TM, 4) CC-TM, 5) PK, 6) PK-PK.Wherein, NBS-LRR class disease-resistant gene is the maximum class disease-resistant gene existed in plant materials; These disease-resistant genes conservative property is structurally that the disease-resistant gene that we utilize resistant geneanalogus to clone other crops brings potential possibility.Be separated in the disease-resistant gene obtained known, wheat leaf rust resistance gene Lr10 and potato anti-potato virus X gene Rx2 is exactly the classical example utilizing resistant geneanalogus to clone disease-resistant gene, illustrates that it is feasible for utilizing this method to clone disease-resistant gene.
Summary of the invention
Technical problem
The object of the invention is utilize resistant geneanalogus Rapid identification sponge gourd oidium gene fragment or be translated into molecule marker and application.Result can be used for the breeding of sponge gourd downy mildew resistance molecular marker assisted selection on the one hand, on the other hand also for the clone of downy mildew resistance gene provides reference frame.
Technical scheme
The present invention carries out disease resistance screening to a large amount of sponge gourd germ plasm resource early stage, on the basis obtaining high resistance sponge gourd oidium Resistant gerplasm, application resistant geneanalogus clone downy mildew resistance gene fragment, by confirming with the methods analyst such as Known resistance gene homology and sequence alignment; For the total length developing corresponding molecule marker and utilize cDNA end rapid amplifying technology to obtain disease-resistant gene lays the foundation.
In order to realize above-mentioned target, the present invention adopts resistant geneanalogus to clone sponge gourd downy mildew resistance gene, and obtain downy mildew resistance gene fragment or genetic marker 3, clip size is respectively 498bp, 504bp and 498bp.Based on this gene fragment order, develop corresponding molecule marker, the breeding of sponge gourd downy mildew resistance molecular marker assisted selection can be carried out; Utilize gene fragment or genetic marker to carry out the innovation of new germ plasm resource simultaneously; The Molecular Mapping of sponge gourd downy mildew resistance gene can be realized in conjunction with existing molecule marker; Utilizing these gene fragments, by design gene specific primer, adopting cDNA end rapid amplifying technology separation sponge gourd downy mildew resistance gene, for being laid the foundation by the disease resistance of genetically engineered improvement sponge gourd; Also for final molecule mechanism of resolving sponge gourd downy mildew resistance gene provides genetic resources.
Positively effect of the present invention:
1) fast Development and the closely linked molecule marker of disease-resistant gene is conducive to.The sponge gourd oidium gene be tested and appraised, exploitation 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.
2) shorten the excavation cycle of sponge gourd downy mildew resistance gene, be conducive to the Rapid identification of oidium 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 disease resistant gene homologous sequence method and excavate sponge gourd downy mildew resistance gene fast, not only can shorten the time, oidium gene identification efficiency can also be improved.
3) initiative of multiresistance breeding material is conducive to.Based on the functional molecular marker of the oidium Data mining 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 sponge gourd downy mildew resistance molecular mechanism.The sponge gourd downy mildew resistance gene of qualification, by transgenic technology, RNAi, virus induced gene silencing (virus induced gene silencing, VIGS) molecular mechanism of the Effect of Anti oidium such as technology provides genetic resources, is conducive to setting forth the antiviral mechanism of action of sponge gourd fast.
Accompanying drawing explanation
Fig. 1 is sponge gourd L09 downy mildew resistance gene sequence;
Fig. 2 is sponge gourd L32 downy mildew resistance gene sequence;
Fig. 3 is sponge gourd L47 downy mildew resistance gene sequence;
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 sponge gourd downy mildew resistance gene.Specific implementation process is as follows:
1, the extraction of DNA: grind to form powdery after getting the young leaflet tablet silica dehydrator of appropriate sponge gourd, extracts total genomic dna by CTAB method.Extract DNA UV spectrophotometer measuring, choose OD260/OD280 value 1.8 ~ 2.0 sample save backup in-20 DEG C.
2, the amplification of disease-resistant gene homologous fragment and clone: according to aminoacid sequence GG (L/I/V/M/S) GKTT and G (L/N) PL (A/T/S) (L/V) of the conserved regions of known NBS-LRR type disease-resistant gene, devise 1 pair of degenerated primer: NBS15 '-GKTT GGIGGIWSIGGIAARACIACG-3 ' and NBS25 '-GCIGCIIARIGGRTTICC-3 '.Getting sponge gourd genomic dna 50ng is template, carries out pcr amplification reaction by follow procedure: 94 DEG C of denaturation 3min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, totally 30 circulations; Last 72 DEG C extend 5min.PCR primer detects through agarose gel electrophoresis, cut the sepharose block containing target DNA fragment, reclaim test kit with the glue of Tian Gen biotech firm, reclaim purifying target DNA fragment by operation instructions, reclaim product pMD-18T CloningKit (TAKARA company) and connect.Connection product is gone to DH5 α competent cell, be applied to (containing 100mg/L AMP) on the LB solid medium containing IPTG and X-Gal, 37 DEG C of overnight incubation, the single bacterium colony of picking white, is detected by PCR and determines containing object fragment.After screening, random selecting 10 clones carry out check order (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).
3, sequencing result is removed PMD18-T carrier sequence by sequential analysis, obtains the DNA sequence dna of Insert Fragment.The open reading frame of these sequences is analyzed with ORFfinder, then the sequence by registering in internet BLAST software (network address: http://www.ncbi.nlm.nih.gov) and GenBank carries out tetraploid rice, then infers that whether these amplified productions are relevant to known disease-resistant gene.：
Be below the enforcement example that contriver provides, but be not limited to these examples.
Implement example 1:
We cooperate with foreign agriculture colleges and universities, scientific research institutions, construct the F2 colony of sponge gourd with Parents, containing 118 individual plants; With these individual plants for test materials extracts DNA, according to the downy mildew resistance gene sequences Design special primer obtained, carry out pcr amplification, according to specific DNA fragment separation case in colony's individual plant, according to sponge gourd oidium Disease Resistance Identification result, obtain and the closely linked DNA fragmentation of sponge gourd downy mildew resistance.
Implement example 2:
In germ plasm resource initiative process, the offspring individuals obtained with Susceptible parent and disease-resistant parents is for object, extract respective genomic dna, what obtain according to enforcement example 1 designs primer with the closely linked DNA fragmentation of oidium gene, pcr amplification is carried out to the sponge gourd genomic dna of above-mentioned acquisition, there is the hybrid material of DNA specific fragment in every amplified production, downy mildew resistance hybrid plant, or the carrier of downy mildew resistance gene, these material scientific researches are further used for parent and the breeding kind matter of disease-resistant variety cultivation, achieve molecular marker assisted selection.
Implement example 3:
We adopt artificial inoculation and Field inoculation to identify the method combined, inoculated identification is carried out to above-mentioned recombinant inbred lines, according to the DNA fragmentation closely linked with oidium implementing example 1 acquisition, design special primer, carries out pcr amplification to above-mentioned individual plant individuality; In conjunction with molecule marker widely as AFLP, SRAP etc., analyze these and be marked at separation case in individual plant colony, in conjunction with artificial inoculation and Field inoculation qualification result, MAPMARKER software is utilized to carry out the Molecular Mapping of sponge gourd downy mildew resistance gene, obtain the genetic map of the disease-resistant section of oidium, for follow-up clone's disease-resistant gene lays the foundation.
Implement example 4:
Application Beijing Tian Gen company RNA extracts test kit and extracts downy mildew resistance parent blade total serum IgE, utilizes cDNA synthetic agent box to synthesize cDNA first chain; By the sponge gourd downy mildew resistance gene fragment DNA sequence dna that enforcement example 1 obtains, design special primer, in conjunction with cDNA end rapid amplifying technology (RACE technology) test kit with primer combine, carry out 5'RACE and 3'RACE pcr amplification respectively, PCR primer obtains 5'RACE and 3'RACE full length sequence at the agarose gel electrophoresis of 1% respectively.Reclaim PCR primer respectively, carry out T clone, after obtaining full length sequence, carry out the connection of full length gene; Thus cloned sponge gourd downy mildew resistance gene total length, for providing genetic resources by the disease resistance of genetically engineered improvement variety of luffa.
Claims (2)
1. sponge gourd downy mildew resistance gene fragment or genetic marker, is characterized in that being selected from one of following gene fragment or genetic marker:
2. the application of Rapid identification sponge gourd oidium described in claim 1, comprising:
1) initiative or the new variety initiative of the breeding material of above-mentioned 3 genes is carried: utilize the GENE SOURCES given by right 1 to be parent material, in hybridization, backcross progeny, the breeding material that transformation has downy mildew resistance gene can be obtained.
2) downy mildew resistance fundamental research: molecular marker analysis is carried out to 3 oidium genes 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) downy mildew resistance transgenic research: utilize 3 genes given by claim 1 to carry out transgenic research.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102703439A (en) * | 2012-03-31 | 2012-10-03 | 常熟市支塘镇新盛技术咨询服务有限公司 | Calabash gourd anti-plague gene segment or gene marker and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102703439A (en) * | 2012-03-31 | 2012-10-03 | 常熟市支塘镇新盛技术咨询服务有限公司 | Calabash gourd anti-plague gene segment or gene marker and application |
Non-Patent Citations (3)
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| WAN,H.ET AL.: "JN230641.1", 《GENBANK》 * |
| WAN,H.ET AL.: "JN230642.1", 《GENBANK》 * |
| WAN,H.ET AL.: "JN230643.1", 《GENBANK》 * |
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Application publication date: 20150429 |