CN102703439A - Calabash gourd anti-plague gene segment or gene marker and application - Google Patents

Abstract

The invention mainly relates to a calabash gourd anti-plague gene segment or gene marker and application. Three calabash gourd anti-plague gene segments or gene markers are rapidly separated by adopting an anti-disease gene homologous sequence method, and by sequencing, the nucleotide lengths of the gene segments or gene markers are 510bp, 498bp and 501bp. According to the calabash gourd anti-plague gene segment or gene marker and application provided by the invention, the excavation period of the calabash gourd anti-plague gene is effectively shortened, and the rapid identification of plague gene is facilitated; the identified plague gene can be used for developing corresponding coseparation functional markers (SNP (single nucleotide polymorphism), SCAR (sequence-characterized amplified region) and the like), and can also be rapidly used for assisted selection of a molecule marker of the anti-plague gene, and the accuracy is high; development of multi-resistance breeding materials can be carried out by combining with other anti-disease gene molecule markers, the breeding time is shortened and the breeding efficiency is improved; and foundation is established for expounding the molecular mechanism of the anti-plague gene.

Description

The anti-eqpidemic disease gene fragment of bottle gourd or genetic marker and application

Technical field

The present invention is by means of the disease resistant gene homologous sequence method; The anti-eqpidemic disease gene fragment of clone's bottle gourd perhaps is converted into molecule marker etc.; Relate generally to the breeding of bottle gourd conventional hybridization, utilize molecular mark, the location of anti-eqpidemic disease gene and molecule mapping; Aspects such as the Regulation Mechanism analysis of the clone of anti-eqpidemic disease gene and disease-resistant gene belong to the Plant Biotechnology scientific domain.

Background technology

Eqpidemic disease is one of most important disease in the bottle gourd production.Mainly cause harm stem, leaf and the fruit of bottle gourd.Stem catches an illness: how at basal part of stem or the water stain shape spot of tender stipes portion's generation sap green, back deliquescing is hung and is contracted, and causes the above blade wilting of disease portion or withered.Blade is caught an illness: produce the circular extremely big scab of the water stain shape of irregular shape, the soft corruption of blade is sagging, is Bluish white to light brown thin paper shape under the drying conditions, is prone to break.Fruit is caught an illness: produce the dark green color spot of water stain shape, the puncture of hanging gradually falls into, and grows thin pearl mustiness thing under wet weather or the high humidity condition continuously, i.e. sporangiophore of germ and sporocyst.This disease propagation rate is exceedingly fast, and sick portion is perishable, gives out stench flavor.Germ survives the winter in soil or seed with invalid body with mycelia, oospore, chlamydospore, produces spore when next year, condition was suitable, borrows on the basal part of stem or subaerial fruit that wind, rain, irrigation water propagate into bottle gourd etc.; Carry out just infecting, sick portion produces a large amount of sporocysts again after several days, discharges zoospore behind the chance water and repeatedly infects again; Eqpidemic disease is expanded rapidly; Temperature 20-30 ℃, rain day is many, rainfall is big, longer duration, or very easy this disease that causes of field moisture delay is very popular.The normal growth and development process that eqpidemic disease has had a strong impact on the bottle gourd plant brings great financial loss to production.The utilization of disease-resistant gene and breeding for disease resistance are the effective meanss of bottle gourd disease control.

Nineteen forties; Flor has proposed classical " gene pairs gene " theory; Be to have a dominant gene in plant and the cause of disease thalline separately, the disappearance of plant disease resistance genes or germ nontoxicity (Avr) gene or change can both cause the generation of disease.This model is applicable to most of biotroph pathogenic bacterias, comprises virus, bacterium, fungi and threadworms." gene pairs gene " disease resistance generally is that acceptor is used as model: plant disease-resistant albumen can direct or indirect identification be derived from the Avr product of germ.In case this identification takes place, and will cause defensive raction.The resistance that most of disease-resistant genes (R) trigger is called anaphylaxis (hypersensitive response with defensive raction is relevant fast; HR).And at present, the product of most of disease-resistant genes all contains the structural domain of albumen identification, as is rich in the structural domain of leucic repeating unit (LRR) or the structural domain of coiled coil (CC); The signal transduction structural domain is like nucleotide-binding site (NBS) or protein kinase (PK) structural domain.Some disease-resistant gene products are predicted to be cytoplasm protein, and other are through a membrane spaning domain (TM) transmembrane.These structural domain combinations can produce dissimilar R albumen.Characteristic according to structure has identified at least six kinds of dissimilar R genes: 1) NBS-LRR, 2) LRR-TM-PK, 3) LRR-TM, 4) CC-TM, 5) PK, 6) PK-PK.Wherein, NBS-LRR class disease-resistant gene is the one type of disease-resistant gene of maximum that exists in the plant materials; These disease-resistant genes conservative property has structurally been brought potential possibility for we utilize the disease-resistant gene of other crops of resistant geneanalogus clone.In the disease-resistant gene that known separation obtains, the anti-potato virus X gene of wheat leaf rust resistance gene Lr10 and yam Rx2 is exactly the classical example that utilizes resistant geneanalogus clone disease-resistant gene, explains that it is feasible utilizing this method clone disease-resistant gene.

Summary of the invention

Technical problem

The purpose of this invention is to provide a kind of through resistant geneanalogus Rapid identification bottle gourd eqpidemic disease gene fragment or molecule marker and application.The result can be used for the breeding of the anti-eqpidemic disease molecular marker assisted selection of bottle gourd on the one hand, also for the clone of anti-eqpidemic disease gene reference frame is provided on the other hand.

Technical scheme

The present invention carries out the disease resistance screening to a large amount of bottle gourd germ plasm resources early stage; Obtain on the basis of the disease-resistant germplasm of high anti-bottle gourd eqpidemic disease; Use resistant geneanalogus and clone anti-eqpidemic disease gene fragment, through with known disease-resistant gene homology analysis, methods such as sequence alignment are confirmed; Lay the foundation with the total length of utilizing the terminal rapid amplifying technology of cDNA to obtain disease-resistant gene for developing corresponding molecule marker.

In order to realize above-mentioned target, the present invention adopts resistant geneanalogus that the anti-eqpidemic disease gene of bottle gourd is cloned, and has obtained 3 of anti-eqpidemic disease gene fragment or genetic markers, and clip size is respectively 510bp, 498bp and 501bp.Be the basis with this gene fragment order, develop corresponding molecule marker, can carry out the breeding of the anti-eqpidemic disease molecular marker assisted selection of bottle gourd; Utilize gene fragment or genetic marker to carry out the innovation of new germ plasm resource simultaneously; Can realize the molecule mapping of the anti-eqpidemic disease gene of bottle gourd in conjunction with existing molecule marker; Utilize these gene fragments, through the design gene specific primer, adopt the terminal rapid amplifying technical point of cDNA, for the disease resistance through genetically engineered improvement bottle gourd lays the foundation from the anti-eqpidemic disease gene of bottle gourd; Also genetic resources is provided for final molecule mechanism of resolving the anti-eqpidemic disease gene of bottle gourd.

Positively effect of the present invention:

1) utilization exploitation and the closely linked molecule marker of disease-resistant gene are fast arranged.The bottle gourd eqpidemic disease gene that is tested and appraised, exploitation 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.

2) shorten the anti-eqpidemic disease gene excavating cycle of bottle gourd, helped the Rapid identification of eqpidemic disease 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 the anti-eqpidemic disease gene of disease resistant gene homologous sequence method fast mining bottle gourd, not only can shorten the time, can also improve eqpidemic disease gene identification efficient.

3) initiative of multiresistance breeding material.Based on the functional molecular marker of the eqpidemic disease 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 the anti-eqpidemic disease molecular mechanism of bottle gourd.The anti-eqpidemic disease gene of identifying of bottle gourd; Gene silencing (virus induced gene silencing through transgenic technology, RNAi, virus induction; VIGS) the antilemic molecular mechanism of research such as technology provides genetic resources, helps setting forth fast the antiviral mechanism of action of bottle gourd.

Description of drawings

Fig. 1 is the anti-eqpidemic disease gene order of bottle gourd A54;

Fig. 2 is the anti-eqpidemic disease gene order of bottle gourd A59;

Fig. 3 is the anti-eqpidemic disease gene order of bottle gourd A64.

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 the Root or stem of Littleleaf Indianmulberry powdery mildew gene.The practical implementation process is following:

1, the extraction of DNA: grind to form powdery after getting the young leaflet tablet silica dehydrator of an amount of bottle gourd, extract total genomic dna with the CTAB method.The DNA that extracts uses UV spectrophotometer measuring, and it is subsequent use in-20 ℃ of preservations at 1.8 ~ 2.0 sample to choose the OD260/OD280 value.

2, the amplification of disease-resistant gene homologous fragment and clone: according to aminoacid sequence GG (L/I/V/M/S) GKTT of the conserved regions of known NBS-LRR type disease-resistant gene and G (L/N) PL (A/T/S) (L/V), designed 1 pair of degenerated primer: NBS1 5'-GKTT GGIGGIWSIGGIAARACIACG-3' and NBS2 5'-GCIGCIIARIGGRTTICC-3'.Getting bottle gourd genomic dna 50 ng is template, carries out pcr amplification reaction by follow procedure: 94 ℃ of preparatory sex change 3 min; 94 ℃ of 30 s, 55 ℃ of 30 s, 72 ℃ of 30 s, totally 30 circulations; Last 72 ℃ are extended 5 min.The PCR product detects through agarose gel electrophoresis; Cutting-out contains the sepharose piece of target DNA fragment; Reclaim test kit with day glue of root biotech firm, reclaim the purifying target DNA fragment, reclaim product and connect with pMD-18T CloningKit (TAKARA company) by operation instructions.To connect product and go to DH5 α competent cell, and be applied on the LB solid medium that contains IPTG and X-Gal and (contain 100 mg/L AMP), 37 ℃ of overnight cultures, the single bacterium colony of picking white detects through PCR and to confirm to contain the purpose fragment.Screening back 10 clones of picked at random check order (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).

3, sequential analysis is removed PMD18-T carrier sequence with sequencing result, obtains inserting segmental dna sequence dna.Analyze the ORFs of these sequences with ORF finder, then through the internet with BLAST software (network address:

Http:// www.n cbi.nlm.nih.gov) with GenBank in the sequence registered carry out homology relatively, infer then whether these amplified productions relevant with known disease-resistant gene.:

Below be the enforcement example that the contriver provides, but be not limited to these examples.

Implement example 1:

We cooperate with foreign agriculture colleges and universities, scientific research institutions, and the recombinant inbred lines so that good parent has made up bottle gourd contains 165 individual plants; With these individual plants is that test materials extracts DNA; Anti-eqpidemic disease gene order according to obtaining designs specially primer, carries out pcr amplification, according to specific DNA fragment separation case in colony's individual plant; According to bottle gourd eqpidemic disease disease resistance qualification result, obtain and the closely linked dna fragmentation of the anti-eqpidemic disease of bottle gourd.

Implement example 2:

In germ plasm resource initiative process; Offspring's individuality of hybridizing acquisition with susceptible parent and disease-resistant parent is an object, extracts genomic dna separately, according to implement example 1 that obtain with the closely linked dna fragmentation design of eqpidemic disease gene primer; Bottle gourd genomic dna to above-mentioned acquisition carries out pcr amplification; The hybrid material of DNA specific fragment appears in every amplified production, anti-eqpidemic disease hybrid plant, or the carrier of anti-eqpidemic disease gene; These material scientific researches are further used for parent and the breeding germplasm that disease-resistant variety is cultivated, and have realized molecular marker assisted selection.

Implement example 3:

We adopt artificial inoculation and field inoculation to identify the method that combines; Recombinant inbred lines to above-mentioned is inoculated evaluation; According to implement example 1 that obtain with the closely linked dna fragmentation of eqpidemic disease, the design special primer carries out pcr amplification to above-mentioned individual plant individuality; In conjunction with molecule marker such as AFLP, SRAP etc. widely; Analyze these and be marked at the separation case in the individual plant colony; In conjunction with artificial inoculation and field inoculation qualification result; Utilize MAPMARKER software to carry out the molecule mapping of the anti-eqpidemic disease gene of bottle gourd, obtain the genetic map of the disease-resistant section of eqpidemic disease, for follow-up clone's disease-resistant gene lays the foundation.

Implement example 4:

Use sky, Beijing RNA of root company extraction test kit and extract the total RNA of anti-eqpidemic disease parent's blade, utilize cDNA synthetic agent box to synthesize cDNA first chain; With implementing the anti-eqpidemic disease gene fragment of the bottle gourd dna sequence dna that example 1 obtains; The design special primer; In conjunction with the terminal rapid amplifying technology of cDNA (RACE technology) test kit with primer combine; Carry out 5 ' RACE and 3 ' RACE pcr amplification respectively, the PCR product obtains 5 ' RACE and 3 ' RACE full length sequence at 1% agarose gel electrophoresis respectively.Reclaim the PCR product respectively, carry out the T clone, carry out the connection of full length gene behind the acquisition full length sequence; Thereby cloned the anti-eqpidemic disease full length gene of bottle gourd, for the disease resistance through genetically engineered improvement bottle gourd kind provides genetic resources.

Claims (4)

1. anti-eqpidemic disease gene fragment of bottle gourd or genetic marker is characterized in that being selected from one of following gene fragment or genetic marker:

1 GGGGGGATGG GTAAGACGAC TATAGCAAAA GCCATCTTTG ATACTCTATC TTGTCAATTT

61 AAAGCTTCTT GTTTTCTAGC AGATGTTAAA GAAAATGCAA ATTATAATCA ACTGCATTCT

121 TTACAAAATT CCCTTCTCTC TGAACTGTTA AGAAAGAAAG ATGATTACGT CAATAATAAG

181 TATGATGGAA AGTACATGAT TCAGAGTAGA CTTTGTTCTA TGAAGGTGCT AATTGTGCTT

241 GATGACATAG ATCATGGTGA TCATTTGGAG TATTTAGCAG GTGATGTTGG TTGGTTTGGT

301 AATGGCAGCA GAATTGTTGT AACGACTAGA AATAAACATT TGGTAGAGAA GGATGATCCA

361 ATATACGAAG TGTCTACACT ATGTGATCAA GAAGCTATTC AATTGTTCAA TCGACATGCT

421 TTCAGAAAAG AAATTCCAGA TGAACGTTTT ATGAAGTTCT CGTTGGAGGT AGTAAATCAT

481 GCTAAAGGCC TTCCTTTAGC CCTC

1 GGTGGTGTTG GGAAGACGAC AATAGCAAGG GCAATTTTTG ACACACTCTC GTATCAGTTT

61 GAAGCTGCTT GTTTCATTGA GGATGTTAAA GAAAACAGAT TTGGAATGCA TTCTTTGCAA

121 AATATCCTTC TCTCAGAGTT GTTAAGGGAA AAAGATAGCT ACGTGAATAA TAAGGAGGAC

181 GGAAAGCACA TGATAGCTCG TAGACTTCCT TTTAAGAAGG TTATAGTTGT GCTTGATGAC

241 ATAGATCATA GAGACCATTT GGATTACCTA GCAGGGAATC CTAGTTGGTT TGGTGATGGA

301 AGTCGAATTA TTACAACAAC TAGAGATAAG CATTTGATTG GGAAGAATGA TGTAGTATAT

361 GAAGTGTCTA CACTTGTTGA TCGTCATGCT ATTAAATTGT TCAACCAATA TGCTTTCAAA

421 GAAGAAGTTC CCGATGAATG TTTTGAGAAG CTCTCATTGG AGGTTATACG TCATGCTAAA

481 GGCCTTCCTT TAGCCCTC

1 GGGGGGGTAG GCAAGACGAC TATAGCTAGA GCTATTTTTG ATTTACTCTC ATCTAAATTT

61 AAATTTGACG GGGCTTGTTT CCTTCCGTAC AATAAAGAAA ACAAGTATGA AATACATTCT

121 CTGCAAAGTA TTCTTCTCTC TAAACTGGTT GGGGAAAAAG AAAGTGTGCA TGATAAGGAG

181 GAAGGGAGGC ACCTGATGGC TCGTAGACTT CGTTTGAAGA AGGTTCTAGT TGTGCTTGAT

241 AACATAGATC ATGAAGACCA ATTGGATTAC CTTGCAGGGG ATCTTGATTG GTTTGGTAAT

301 GGCAGCAGAA TTATTGCAAC AACTAGGGAC AAACATTTCA TAGGAAAAAA CGATGCTGTA

361 TATCCTATGA CTACACTACT TGAACATGAT GCTATTCAGT TGTTCAACCA ATATGCTTTC

421 AAAAATGAAG TTCCAGATAA GTGTTTCGAG GAGATAACGT TGGAGGTAGT AAGTCATGCA

481 GAAGGCCTTC CTTTAGCCCT C。

2. the application of the said Rapid identification bottle gourd of claim 1 eqpidemic disease comprises:

1) initiative of breeding material.

3.2) antilemic breeding practice.

4.3) anti-eqpidemic disease fundamental research.

Images