CN105969778A - Haynaldia villosa gene NAM-V1 and molecular marker and application thereof - Google Patents
Haynaldia villosa gene NAM-V1 and molecular marker and application thereof Download PDFInfo
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Abstract
The invention relates to a Haynaldia villosa gene NAM-V1 and a molecular marker and application thereof. The gene NAM-V1 provided by the invention is derived from a genome V of Haynaldia villosa and has a nucleotide sequence shown in SEQ ID NO: 1. The gene NAM-V1 plays a significant role in the aspect of accumulation of grain protein of wheat, is co-separate from a powdery mildew resistant gene Pm21 of the wheat and has a potential application value in the breeding of new varieties, i.e., high-protein and powdery mildew resistant wheat. In addition, the invention further provides the molecular marker CauNAM-V1 for specifically detecting the gene NAM-V1 and an application of the molecular marker CauNAM-V1. The marker can be used for specifically detecting whether a wheat variety has the gene NAM-V1 or not, can also be used for detecting whether a wheat material contains the powdery mildew resistant gene Pm21 or not and can simultaneously be used as the molecular marker for screening the high-protein and powdery mildew resistant wheat.
Description
Technical field
The present invention relates to genetic engineering and biology field, specifically, relate to haynaldia villosa
NAM-V1 gene and molecular marker thereof and application.
Background technology
Semen Tritici aestivi is currently global first generalized grain crop, and the whole world there are about the population of 35%～40%
As main food source, accounting for the 20% of whole world calorie total quantity consumed, the whole world is little
Wheat annual production about 700,000,000 tons, it is provided that the protein of about 70,000,000 tons.Therefore, Semen Tritici aestivi seed is increased
The protein content of grain can not only improve the quality of Semen Tritici aestivi, and contributes to increasing substantially edible
The total amount of protein.Wheat seed protein content (grain protein concentration, GPC)
And composition decides processing quality and the nutritional quality of Semen Tritici aestivi.The grain protein content of Semen Tritici aestivi is
Typical quantitative trait, and it is easily subject to the impact (Groos et al., 2003) of environmental condition.
2006, Uauy etc. obtained the gene NAM-B1 controlling protein content by map based cloning,
This gene is positioned on Semen Tritici aestivi 6BS chromosome, a kind of NAC transcription factor of coding (Uauy et al.,
2006).Haynaldia villosa (Haynaldia villosa, 2n=2x=14, V genome) belongs to Tribe Triticeae,
For annual or perennial different strain pollination diplont.Haynaldia villosa gene is cold-resistant, tillering ability is strong,
The good characteristics such as disease-resistant, salt tolerant, drought resisting and grain protein content are high, are genetic improvement of wheat
Important gene resource (2006).Wheat-Haynaldia villosa translocation lines is to utilize tuft
The important channel of wheat favorable genes resource, such as, 6VS 6AL translocation line carries mildew-resistance base
Because of Pm21, to wheat powdery mildew performance resistance, anti-spectrum extensively, the kind of incubation is accumulative to be promoted
Area reaches 3,400,000 hectares (Cao et al., 2011).Except mildew-resistance gene, at tuft
Secondary sulfur alpha-alcohol soluble protein (Sulfur-richa-prolamins) is also located on wheat 6VS chromosome
The excellent character such as gene Gli-V2, Rust resistance (Stripe rust) gene Yr26 (Blanco et al.,
1991；Ma et al.,2001).But there is presently no control grain protein content in haynaldia villosa
The gene report of shape.
Powdery mildew is a kind of worldwide disease having a strong impact on yield and quality of wheat, little in various countries
All there is generation in wheat producing region.Recent two decades comes, and scope and area that wheat powdery mildew occurs constantly expand
Greatly, the extent of injury increases the weight of day by day, and at present, wheat powdery mildew has become the weight affecting wheat yield
Want limiting factor.The pathogen of wheat powdery mildew is grass family dlumeria graminis Semen Tritici aestivi specialized form,
Belonging to Ascomycotina fungus, this pathogenic bacteria has multiple biological strain, in different geographical ecological environmenies
In fast with speed of mutation in host's Interaction, the kind of some large area production applications is at present
Through or losing resistance, therefore powdery mildew harm is in the serious situation of pandemic at any time.
Pesticide control serves certain effect to prevention and control powdery mildew, but can expend substantial amounts of manpower and
Financial resource, does not utilize ecological environment.Utilize modern biotechnology, constantly identify and excavate little
Mildew-resistance gene in wheat or QTL site, pass through to divide by mildew-resistance gene or QTL site
Sub-genetic engineering or molecular marker assisted selection are cultivated and promote disease-resistant variety, are preventing and treating white wheats
Powder disease is the safest, economically and efficiently approach.The most identified wheat anti-powdery mildew base
Cause/site has more than 70, along with the application on wheat breeding of these genes and popularization, cause of disease
The biological strain of bacterium also has a very large change.Current most mildew-resistance has been out
Resistance.Powdery mildew resistance gene Pm 21 derives from haynaldia villosa, and Pm21 resists domestic all of white lead
Pathogenic bacteria strain and 120 the European biological strains detected, be that the most anti-spectrum is the widest, anti-at present
The most stable of mildew-resistance gene of property (Cao et al., 2011), therefore, develops chain with Pm21
Molecular marker significant for wheat anti-powdery mildew breeding for disease resistance.At present, existing
In document, the molecular marker report of wheat powdery mildew resistance gene Pm 21 is less, and most of
The molecular marker of Pm21 is labelling based on SDS-PAGE electrophoresis, in-convenience in use, limit
Powdery mildew resistance gene Pm 21 application in wheat breeding for disease resistance.
Summary of the invention
It is an object of the invention to provide haynaldia villosa NAM-V1 gene, and NAM-V1 gene is increasing
Add the effect in Protein Content of Wheat Kernel, and prove this gene and anti-white lead in Semen Tritici aestivi
Ospc gene Pm21 be divided into from.
It is a further object of the present invention to provide haynaldia villosa NAM-V1 gene molecule marker
Whether CauNAM-V1 and application thereof, utilize in this labelling specific detection wheat breed and exist
NAM-V1 gene, is additionally operable to detect in wheat lines whether contain powdery mildew resistance gene Pm 21,
It is used simultaneously as Semen Tritici aestivi high protein and the molecular marker of mildew-resistance screening.
In order to realize the object of the invention, the haynaldia villosa NAM-V1 gene that the present invention provides, it is
Control the gene of Protein Content of Wheat Kernel, utilize this gene can improve the albumen of wheat seed
Content.
The nucleotides sequence of haynaldia villosa NAM-V1 gene is classified as:
I) nucleotide sequence shown in SEQ ID NO:1；Or
Ii) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases by one
Or multiple nucleotide and the nucleotide sequence of expression identical function protein；Or
Iii) under strict conditions with sequence hybridization shown in SEQ ID NO:1 and express identical function egg
The nucleotide sequence of white matter, described stringent condition is at 0.1 × SSPE containing 0.1%SDS or to contain
In 0.1 × SSC solution of 0.1%SDS, hybridize at 65 DEG C, and wash film with this solution；Or
Iv) with i), ii) or nucleotide sequence iii) there is more than 90% homology and express phase
The nucleotide sequence of congenerous protein.
The present invention also provides for by the albumen of above-mentioned haynaldia villosa NAM-V1 gene code, its aminoacid
Sequence is as shown in SEQ ID NO:2.
The present invention also provides for the expression cassette containing described haynaldia villosa NAM-V1 gene, restructuring carries
Body, recombinant bacterium or transgenic cell line.
The present invention also provides for described haynaldia villosa NAM-V1 gene, containing described NAM-V1 gene
Expression cassette, recombinant vector, recombinant bacterium or transgenic cell tie up to improve in crop protein content
Application.
The crop related in the present invention include but not limited to Semen Tritici aestivi, Fructus Hordei Vulgaris, rye (Secale cereale L.), Herba bromi japonici, bunch
Dirty wheat.
The expression vector carrying described genes of interest can be by using Ti-plasmids, plant virus
The standard biologic technical methods such as carrier, directly delivered DNA, microinjection, electroporation import plants
In thing cell (Weissbach, 1998, Method for Plant Molecular Biology VIII,
Academy Press, New York, the 411-463 page；Geiserson and Corey, 1998,
Plant Molecular Biology, 2nd Edition)。
Such as, can be by the expression cassette containing described NAM-V1 gene, recombinant vector, recombinant bacterium
Or transgenic cell line is imported by agriculture bacillus mediated, pollen tube or the method for particle gun proceeds to make
In thing, cultivate the genetically modified crops of high grain protein content.
The present invention also provides for the molecular marker CauNAM-V1 of described haynaldia villosa NAM-V1 gene,
The primer expanding this molecular marker for specific PCR is: forward primer
F5'-TCCCCGGTATGCCATGTC-3' and reverse primer
R5'-AAGATACCGCTAGACCGTGA-3'。
The present invention also provides for described molecular marker CauNAM-V1 NAM-V1 in detection crop
Application in gene and/or Pm21 gene,
The present invention also provide for described molecular marker CauNAM-V1 at Semen Tritici aestivi high protein content and/or
Application in the molecular mark of mildew-resistance.
The present invention also provides for described molecular marker CauNAM-V1 and has high egg concurrently in screening and qualification
Application in the new variety of wheat of Bai Hanliang and mildew-resistance.
The present invention also provides for for detecting drawing of NAM-V1 gene in crop and/or Pm21 gene
Thing, including forward primer F5'-TCCCCGGTATGCCATGTC-3' and reverse primer
R5'-AAGATACCGCTAGACCGTGA-3'。
The present invention further provides the detection kit containing described primers F and R.
The present invention clones from haynaldia villosa V chromosome and obtains a Semen Tritici aestivi NAM family gene, life
Entitled NAM-V1, this full length gene 1528bp, have complete open reading frame, including 3
Individual exon and 2 introns, in Semen Tritici aestivi 6VS 6AL translocation line, NAM-V1 gene is replaced
Improve the content of seed protein after NAM-A1 gene, illustrate that NAM-V1 gene can improve little
The protein content of wheat seed.According to the nucleotide sequence of NAM-V1 gene, develop special
The molecular marker CauNAM-V1 of detection NAM-V1 gene, at Semen Tritici aestivi 6VS 6AL translocation line
Segregating population proving, this labelling and wheat powdery mildew resistance gene Pm 21 are divided into from, it was demonstrated that this mark
Note can detect NAM-V1 gene and Pm21 gene simultaneously.Therefore, NAM-V1 gene energy is utilized
Enough improve the protein content of wheat seed.Utilize CauNAM-V1 labelling can detect Semen Tritici aestivi simultaneously
Powdery mildew resistance gene Pm 21, this mark is for the molecular breeding of Semen Tritici aestivi, it is possible to screens and obtains
Have the new variety of wheat of high protein and mildew-resistance concurrently.
The invention have the advantages that
(1) Semen Tritici aestivi is one of the most most important cereal crops, and whole world Semen Tritici aestivi annual production is about
700000000 tons, if wheat seed protein content increases by 1%, be equivalent to whole world wheat protein content and increase
Add 7,000,000 tons.The contribution rate of the NAM-V1 gene pairs Protein Content of Wheat Kernel of the present invention is
Height has exceeded 1%, and therefore, NAM-V1 gene is for increasing the edible protein supply tool in the whole world
Significant.
(2) wheat powdery mildew resistance gene Pm 21 be that at present the most anti-spectrum is the widest, resistance
Stable mildew-resistance gene, the labelling of the powdery mildew resistance gene Pm 21 developed before this is mostly
Labelling based on SDS-PAGE electrophoretic techniques, glue, staining procedure complexity in operating process,
And acrylamide, TEMED etc. have the highest toxicity to human body.The present invention's
CauNAM-V1 labelling is labelling based on sepharose electrophoresis, and detection method is relatively simple, can
Novel nucleic acid dye is used to dye, more safe, practical.For making full use of Pm21
Mildew-resistance gene resource, promotes that wheat breeding for disease resistance is significant.
(3) the CauNAM-V1 labelling that the present invention provides can detect control wheat seed simultaneously
The gene NAM-V1 of protein content and powdery mildew resistance gene Pm 21, i.e. one labelling selects simultaneously
Two kinds of character, for improving the efficiency of Wheat Molecular Breeding, it is achieved the pyramiding breeding tool of multiple characters
There is important meaning.
Accompanying drawing explanation
Fig. 1 is the electrophoresis in the embodiment of the present invention 1 to the haynaldia villosa NAM-V1 gene that clone obtains
Testing result and genomic organization result；Wherein, a:NAM-V1 genome and cDNA
Clone；B:NAM-V1 genomic organization；C:NAM-V1 gene guards territory prediction.
Fig. 2 is the phyletic evolution of the haynaldia villosa NAM-V1 gene built in the embodiment of the present invention 2
Tree；Wherein, species abbreviation: goatweed (Aet), durum wheat (Tt), Fructus Hordei Vulgaris (Hv), water
Rice (Os), arabidopsis (At), carry is it possible that tie up Semen Tritici aestivi (G).
Fig. 3 be in the embodiment of the present invention 3 NAM family gene sequence alignment result and
The position of CauNAM-V1 molecular marker.
Fig. 4 is that the embodiment of the present invention 4 Middle molecule labelling CauNAM-V1 is in Wheat Cultivars
Amplification situation.
Fig. 5 is the phenotype of wheat powdery mildew resistance gene Pm 21 segregating population in the embodiment of the present invention 5
Analysis result.
Fig. 6 is that the embodiment of the present invention 5 Middle molecule labelling CauNAM-V1 separates group at Semen Tritici aestivi Pm21
Amplification situation in body.
Fig. 7 is the effect of NAM-V1 gene regulation wheat seed protein content in the embodiment of the present invention 6
Really.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not
Specializing, following example are all cloned according to conventional laboratory conditions, such as Sambrook equimolecular
Institute in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989)
The operating technology code stated, or according to the experiment condition proposed by manufacturer；Raw materials used all
For commercial goods.
The clone of embodiment 1 haynaldia villosa NAM-V1 gene
According on NCBI announce NAM-A1 (DQ869672), NAM-D1 (DQ869675),
NAM-B1(DQ869673)、NAM-B2(DQ869676)、NAM-D2(DQ869677)
The sequence information of gene, the design two primer to clone NAM gene complete coding region
NAMORF1 and NAMORF2, forward primer includes that start codon, reverse primer include
Termination codon.Respectively with the genomic DNA of haynaldia villosa and cDNA as template, with primer pair
NAMORF1 and NAMORF2 carries out PCR amplification.PCR reaction system is: template DNA 3
μ l, each 1 μ l of forward and reverse primer, dNTP (10mM) 0.4 μ l, high-fidelity Taq DNA are polymerized
Enzyme (5U/ μ l) 0.2ul, 10 × PCR reaction buffer 2 μ l, adds ddH2After O supplies 20 μ l
Mixing.PCR reaction condition is: 94 DEG C of 3min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 2min,
Totally 33 circulations；72 DEG C of 7min, 16 DEG C are terminated reaction.After PCR reaction terminates, with 1% fine jade
Lipolysaccharide electrophoresis detection amplification.Electrophoresis result shows, divides from genomic DNA and cDNA
Kuo Zeng not obtain purpose band (Fig. 1 a) of a length of about 1.5kb and 1.2kb.By purpose bar
Band is connected to pMD18-T carrier after reclaiming, convert competent escherichia coli cell.Cut glue to reclaim
Target DNA, is connected to carry out on cloning vehicle (pMD18-T) mensuration of DNA sequence.
Known (albumen) in sequencing result and NCBI is carried out BlastN (BlastXP) comparison
Analyzing, result shows this gene code NAC transcription factor, is the wild emmer reported
NAM-B1 gene, the homologous genes of NAM-A1, D1, D2, B2 of common wheat, but
Sequence differences is relatively big, owing to deriving from the V dyeing group of haynaldia villosa, the most named NAM-V1
Gene.NAM-V1 full length gene 1528bp, including 3 exons and 2 introns (Fig. 1 b).
Open reading frame total length 1224bp, encodes 407 aminoacid, it was predicted that molecular weight is 43kDa,
Isoelectric point, IP is 8.39.The CDD prediction of NCBI shows that the N end of NAM-V1 albumen encodes one
The conserved domain (Fig. 1 c) of NAM_superfamily.
Table 1 primer sequence information
The phylogenetic analysis of embodiment 2 haynaldia villosa NAM-V1 gene
Utilize comparison software ClustalW 1.83 to the NAM-V1 of haynaldia villosa and Semen Tritici aestivi, arabidopsis,
The NAC albumen of the plants such as Oryza sativa L. carries out multisequencing homology comparison, and the file of output passes through
BOXSHADE 3.21 colours.Use the ortho position phase connection in Mega 4 software
(Neighbor-joining) constructing system cladogram.Encoding proteins to haynaldia villosa NAM-V1
Carrying out Phylogenetic analysis with homology NAC albumen in other species, result is as shown in Figure 2.
The NAC albumen of separate sources divide into 4 classes (Group).Wherein haynaldia villosa NAM-V1 belongs to
Group I, Group I also includes the NAC4 of Oryza sativa L., and (6B contaminates the TtNAM-B1 of durum wheat
Colour solid), TtNAM-B2 (2B chromosome) and TtNAM-A1 (6A chromosome), goatweed
AetNAM-D2 (2D chromosome) and AetNAM-D1 (6D chromosome), the NAM-1 of Fructus Hordei Vulgaris
With NAM-2 (H gene group), carry is it possible that tie up the NAM-G (G genome) of Semen Tritici aestivi.Wherein come
Come from the NAM-V1 of haynaldia villosa and derive from subgenomic No. 6 chromosome of Semen Tritici aestivi A, D, B
TtNAM-A1, AetNAM-D1 and TtNAM-B1 sibship nearest, secondly for deriving from
Semen Tritici aestivi A, D, B subgenomic No. 2 chromosome TtNAM-B2, AetNAM-D2.This table
Bright one side, NAM-V1 may have identical life with NAM-B1, A1, D1, B2, D2
Thing function, i.e. improves protein, ferrum, the content of zinc in seed.On the other hand, derive from
TtNAM-B2, AetNAM-D2 of No. 2 chromosome of B and D sub-gene group are derived from No. 6
The paralog gene of chromosome NAM-B1, A1 and D1, is probably derived from a gene replication
Event (Gene duplication events).Genetic evolution model according to Tribe Triticeae, in advance
Survey this gene duplication event and be likely to occur in A genome ancestors kind and B (S) D genome ancestral
First planting after separating, B (S) genome ancestors plant before planting separation with D genome ancestors, i.e.
About between 5.8 ± 1MYA to 2.6 ± 0.8MYA；Only 3 genes in Group II,
It both is from arabidopsis, is AtNAC2, AtNAC18 and AtNAC20 respectively.Wherein AtNAC2
Corresponding salt stress, and relevant with lateral root development；Group III comprises 3 genes, respectively
It is OsABA91266, OsABA95705 and the TaNAC69 of Semen Tritici aestivi of Oryza sativa L..TaNAC69 phase
Answer low temperature, arid and salt stress, relevant to the adaptability under Semen Tritici aestivi adverse circumstance；Group IV wraps
Containing 3 genes, it is the TaNAC2 of Semen Tritici aestivi, the OsNP 912423 of Oryza sativa L. and arabidopsis respectively
AtNAC3, wherein TaNAC2 with AtNAC3 is also relevant to plant stress-resistance.
The sequence alignment of embodiment 3 haynaldia villosa NAM-V1 gene and molecular markers development
Gene NAM-V1 and NAM-A1, NAM-B1, NAM-B2, NAM-D1, NAM-D2
Nucleotide sequence comparison result show there is one in the position of the 247th base of NAM-V1
The insertion of " ATGTC ", has the SNP difference of one " G/T " at the 785th base.Root
According to the flanking sequence in the two site, design the labelling for specific detection NAM-V1 gene
CauNAM-V1 (Fig. 3).Primer for specific PCR amplification label CauNAM-V1 is shown in
Table 1.
Embodiment 4 utilizes in the different wheat lines of molecular marker CauNAM-V1 detection
NAM-V1 gene
In order to detect the specificity of CauNAM-V1 primer, and also to prove that NAM-V1 comes
Come from the 6VS chromosome of haynaldia villosa, respectively at China spring (CS), aegilops tauschii (Aegilops
Tauschii), Uralensis Fisch (T.urartu), einkorn wheat (T.mononcoccum),
Chinese spring nullisomic four system 2B/2D and the susceptible individual plant of 6A/6B, Pm12, Pm21 and Pm21
Disease-resistant individual plant expands.Result shows, at the disease-resistant list of Pm21 containing 6V the short arm of a chromosome
Strain (6VS 6AL translocation line) can amplify and expect the identical special purpose band of size,
And in other materials to be tested, all do not expand corresponding specific band.Prove CauNAM-V1
Primer can the existence of specific detection NAM-V1 gene.This result also demonstrates NAM-V1
Gene source is in the 6VS chromosome (Fig. 4) of haynaldia villosa.
Embodiment 5 molecular marker CauNAM-V1 answering in detection Semen Tritici aestivi Pm21 disease-resistant gene
With
Build the segregating population of Semen Tritici aestivi 6VS 6AL translocation line, the offspring's individual plant warp to segregating population
Physiological Races of Powdery Mildew E09 carries out Disease Resistance Identification after infecting, and disease-resistant phenotype is as shown in Figure 5.
Select 10 disease-resistant individual plants and 10 susceptible individual plants, carry out the PCR of CauNAM-V1 labelling
Amplification, amplification shows, CauNAM-V1 labelling present mildew-resistance characteristic be divided into from
Phenomenon, i.e. all for examination disease-resistant individual plants in specific purpose band all can be detected,
And all for trying susceptible individual plant is all not detected by corresponding purpose band (Fig. 6).Result
Confirm that CauNAM-V1 is can be as the molecular marker of Pm21 disease-resistant gene detection.
The application in increasing wheat seed protein content of the embodiment 6NAM-V1 gene
In order to prove the effect in Protein Content of Wheat Kernel of the haynaldia villosa NAM-V1 gene,
Utilize Semen Tritici aestivi 6VS 6AL translocation line, construct 4 segregating populations of NAM-V1 gene, respectively
It is W50200, W50175, W50156 and W50176.Then divide with CauNAM-V1 labelling
Not have detected the genotype of segregating population offspring, and individual plant determines the protein of wheat seed
Content (Fig. 7).Result shows, containing NAM-V1 gene with without NAM-V1 gene
In the case of, the grain protein content in 4 segregating populations is respectively 13.94%/13.42%,
17.99%/16.88%, 13.33%/13.31%, 15.41%/14.33%, result show containing
Grain protein content in the progeny material of NAM-V1 gene all increased, Qi Zhong
In W50175 and W50176 segregating population, grain protein content adds 1.11% He respectively
1.08%.Result above shows, the NAM-V1 gene of haynaldia villosa can improve the egg of wheat seed
White matter content.
Although, the most with a general description of the specific embodiments the present invention has been made in detail
Most description, but on the basis of the present invention, it can be made some modifications or improvements, this is to this
It is apparent from for skilled person.Therefore, on the basis without departing from spirit of the present invention
Upper these modifications or improvements, belong to the scope of protection of present invention.
List of references
1、Groos,C.,Robert,N.,Bervas,E.and Charmet,G.(2003)Genetic analysis of grain protein-content,
grain yield and thousand-kernel weight in bread wheat.Theor Appl Genet,106,1032-1040.
2、Cao,A.,Xing,L.,Wang,X.,Yang,X.,Wang,W.,Sun,Y.,Qian,C.,Ni,J.,Chen,Y.and Liu,D.
(2011)Serine/threonine kinase gene Stpk-V,a key member of powdery mildew resistance gene Pm21,
confers powdery mildew resistance in wheat.Proceedings of the National Academy of Sciences,108,
7727-7732.
3、Uauy,C.,Distelfeld,A.,Fahima,T.,Blechl,A.and Dubcovsky,J.(2006)A NAC Gene Regulating
Senescence Improves Grain Protein,Zinc,and Iron Content in Wheat.Science,314,1298-1301.
4、A.(2006)The genus Dasypyrum––part 1.The taxonomy and relationships within
Dasypyrum and with Triticeae species.Euphytica,152,429-440.
5、Blanco,A.,Resta,P.,Simeone,R.,Parmar,S.,Shewry,P.R.,Sabelli,P.and Lafiandra,D.(1991)
Chromosomal location of seed storage protein genes in the genome ofDasypyrum villosum(L.)
Candargy.Theoretical and Applied Genetics,82,358-362.
6、Ma,J.,Zhou,R.,Dong,Y.,Wang,L.,Wang,X.and Jia,J.(2001)Molecular mapping and detection
of the yellow rust resistance gene Yr26in wheat transferred from Triticum turgidum L.using
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Claims (10)
1. haynaldia villosa NAM-V1 gene, it is characterised in that its nucleotides sequence is classified as:
I) nucleotide sequence shown in SEQ ID NO:1；Or
Ii) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases by one
Or multiple nucleotide and the nucleotide sequence of expression identical function protein；Or
Iii) under strict conditions with sequence hybridization shown in SEQ ID NO:1 and express identical function egg
The nucleotide sequence of white matter, described stringent condition is at 0.1 × SSPE containing 0.1%SDS or to contain
In 0.1 × SSC solution of 0.1%SDS, hybridize at 65 DEG C, and wash film with this solution；Or
Iv) with i), ii) or nucleotide sequence iii) there is more than 90% homology and express phase
The nucleotide sequence of congenerous protein.
2., by the albumen of haynaldia villosa NAM-V1 gene code described in claim 1, its feature exists
In, its aminoacid sequence is as shown in SEQ ID NO:2.
3. contain the expression cassette of haynaldia villosa NAM-V1 gene, recombinant vector described in claim 1
Or recombinant bacterium.
4. haynaldia villosa NAM-V1 gene described in claim 1, or express described in claim 2
The application in improving crop protein content of box, recombinant vector or recombinant bacterium, the most described crop
For Semen Tritici aestivi, Fructus Hordei Vulgaris, rye (Secale cereale L.), Herba bromi japonici, haynaldia villosa.
Application the most according to claim 4, it is characterised in that described in claim 3
Expression cassette, recombinant vector or recombinant bacterium, imported or gene by agriculture bacillus mediated, pollen tube
The method of rifle proceeds in crop, cultivates the genetically modified crops of high grain protein content.
6. the molecular marker CauNAM-V1 of haynaldia villosa NAM-V1 gene described in claim 1,
It is characterized in that, the primer expanding this molecular marker for specific PCR is: forward primer
F5'-TCCCCGGTATGCCATGTC-3' and reverse primer
R5'-AAGATACCGCTAGACCGTGA-3'。
7. NAM-V1 gene and/or the Pm21 in detection crop of molecular marker described in claim 6
Application in gene, the most described crop is Semen Tritici aestivi, Fructus Hordei Vulgaris, rye (Secale cereale L.), Herba bromi japonici, haynaldia villosa.
8. molecular marker described in claim 6 is at Semen Tritici aestivi high protein content and/or mildew-resistance
Application in molecular mark.
9. molecular marker described in claim 6 in screening or is identified high protein content and/or resists white
Application in powder disease wheat breed.
10. for detecting NAM-V1 gene and/or the primer of Pm21 gene, its feature in crop
It is, including forward primer F5'-TCCCCGGTATGCCATGTC-3' and reverse primer
R5'-AAGATACCGCTAGACCGTGA-3'。
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| CN107699630A (en) * | 2017-10-27 | 2018-02-16 | 南京农业大学 | Chain molecular labeling and its application in breeding with wheat resistance genes Pm21 |
| CN107699630B (en) * | 2017-10-27 | 2021-06-01 | 南京农业大学 | Molecular marker linked with wheat disease-resistant gene Pm21 and application thereof in breeding |
| CN116640768A (en) * | 2023-04-11 | 2023-08-25 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Wheat powdery mildew resistance gene PmTR1 and application thereof |
| CN116640768B (en) * | 2023-04-11 | 2024-02-13 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Wheat powdery mildew resistance gene PmTR1 and application thereof |
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