CN104561030A

CN104561030A - Eggplant fusarium wilt-resistant gene segments or gene markers and application thereof

Info

Publication number: CN104561030A
Application number: CN201310524876.1A
Authority: CN
Inventors: 钱孝英; 钱春桃
Original assignee: Changshu Zhilin Fruits And Vegetables Professional Cooperatives
Current assignee: Changshu Zhilin Fruits And Vegetables Professional Cooperatives
Priority date: 2013-10-28
Filing date: 2013-10-28
Publication date: 2015-04-29

Abstract

The invention mainly relates to eggplant fusarium wilt-resistant gene segments or gene markers and application thereof. Three eggplant fusarium wilt-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 483bp, 501bp and 495bp. According to the eggplant fusarium wilt-resistant gene segments or gene markers, the excavating period of an eggplant fusarium wilt-resistant gene is effectively shortened; rapid identification of a wilt gene is facilitated; corresponding co-separation functional markers (SNP, SCAR and the like) are developed through identified wilt gene; the gene segments or gene markers can also be applied to assistant selection of molecular markers of the fusarium wilt-resistant gene; the accuracy is high; creation of multi-resistance breeding materials can be carried out by combining 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 eggplant fusarium wilt-resistant gene.

Description

Eggplant anti-blight gene fragment or genetic marker and application

Technical field

The present invention utilizes disease resistant gene homologous sequence method, clone eggplant anti-blight gene fragment or be translated into molecule marker etc., relate generally to eggplant conventional cross-breeding, molecular mark, the location of anti-blight gene and Molecular Mapping, the aspects such as the clone of anti-blight gene and the Regulation Mechanism analysis of disease-resistant gene, belong to Plant Biotechnology scientific domain.

Background technology

Blight is that one of upper most important disease produced by eggplant.The bottom-up flavescence gradually of wilt of eggplant diseased plant blade withers, and on present one or the two layers of branch of illness multilist, same blade is only half of sometimes turns yellow, and second half is sound as usual.Cross-sectional sick stem, sick portion vascular bundle is brown.This is sick easily obscures with verticillium, need detect cause of disease differentiation.Embrace son with invalid body in soil or be attached on seed and survive the winter with mycelium or thick wall, can saprogenesis be sought.Generally invade host from young root or wound, enter vascular bundle, obstruction conduit, and output toxic substance fusarine, diffusion is come and is caused diseased plant blade yellow withered and dead.Germ by current or irrigation water its spread in china, soil temperature 28 DEG C, soil moisture, continuous cropping ground, transplant or intertillage time to hinder root many, the morbidity weight that plant strain growth gesture is weak.In addition, acid soil and nematode take food and cause wound to be beneficial to the generation of this disease.Less than 21 DEG C or the expansion of more than 33 DEG C state of an illness are slowly.Blight has had a strong impact on the normal growth and development process of eggplant plant, brings great financial loss to production.The utilization of disease-resistant gene and breeding for disease resistance are the most effective meanss of eggplant 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 wilt of eggplant gene fragment or be translated into molecule marker and application.Result can be used for the breeding of eggplant anti-blight molecular marker assisted selection on the one hand, on the other hand also for the clone of anti-blight gene provides reference frame.

Technical scheme

The present invention carries out disease resistance screening to a large amount of Eggplant Germplasm Resources early stage, on the basis obtaining high resistance wilt of eggplant Resistant gerplasm, application resistant geneanalogus clone anti-blight gene fragment, by confirming with the method such as Known resistance gene homology and sequence alignment analysis; 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 eggplant anti-blight gene, and obtain anti-blight gene fragment or genetic marker 3, clip size is respectively 483bp, 501bp and 495bp.Based on this gene fragment order, develop corresponding molecule marker, the breeding of eggplant anti-blight 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 eggplant anti-blight 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 eggplant anti-blight gene, for being laid the foundation by the disease resistance of genetically engineered improvement eggplant; Also for final molecule mechanism of resolving eggplant anti-blight 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 wilt of eggplant 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 eggplant anti-blight gene, be conducive to the Rapid identification of blight 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 eggplant anti-blight gene fast, not only can shorten the time, blight gene identification efficiency can also be improved.

3) initiative of multiresistance breeding material is conducive to.Based on the functional molecular marker of the blight 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 eggplant anti-blight molecular mechanism.The eggplant anti-blight gene of qualification, by transgenic technology, RNAi, virus induced gene silencing (virus induced gene silencing, VIGS) molecular mechanism of the Effect of Anti blight such as technology provides genetic resources, is conducive to setting forth the antiviral mechanism of action of eggplant fast.

Accompanying drawing explanation

Fig. 1 is eggplant S65 anti-blight gene order;

Fig. 2 is eggplant S78 anti-blight gene order;

Fig. 3 is eggplant S91 anti-blight gene order;

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 eggplant anti-blight 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 eggplant, 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 eggplant 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 on the LB solid medium containing IPTG and X-Gal (containing 100mg/LAMP), 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 eggplant with Parents, containing 118 individual plants; With these individual plants for test materials extracts DNA, according to the anti-blight gene order design special primer obtained, carry out pcr amplification, according to specific DNA fragment separation case in colony's individual plant, according to wilt of eggplant Disease Resistance Identification result, obtain and the closely linked DNA fragmentation of eggplant anti-blight.

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 blight gene, pcr amplification is carried out to the eggplant genomic dna of above-mentioned acquisition, there is the hybrid material of DNA specific fragment in every amplified production, anti-blight hybrid plant, or the carrier of anti-blight 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 blight 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 eggplant anti-blight gene, obtain the genetic map of the disease-resistant section of blight, 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 anti-blight parent blade total serum IgE, utilizes cDNA synthetic agent box to synthesize cDNA first chain; By the eggplant anti-blight 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 eggplant anti-blight full length gene, for providing genetic resources by the disease resistance of genetically engineered improvement Eggplant Varieties.

Claims

1. eggplant anti-blight 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 wilt of eggplant 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 fusarium wilt disease resistance gene can be obtained.

2) anti-blight fundamental research: molecular marker analysis is carried out to 3 blight 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) anti-blight transgenic research: utilize 3 genes given by claim 1 to carry out transgenic research.