CN103160505A - Corn wrky gene intron length polymorphism molecular marker - Google Patents
Corn wrky gene intron length polymorphism molecular marker Download PDFInfo
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- CN103160505A CN103160505A CN2013100787811A CN201310078781A CN103160505A CN 103160505 A CN103160505 A CN 103160505A CN 2013100787811 A CN2013100787811 A CN 2013100787811A CN 201310078781 A CN201310078781 A CN 201310078781A CN 103160505 A CN103160505 A CN 103160505A
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Abstract
The invention relates to the field of a bio-genetic engineering technology and genetic breeding and especially relates to a corn WRKY gene intron length polymorphism molecular marker. Primers are designed in exon zones adjacent to a WRKY gene; the WRKY gene is subjected to PCR amplification in different corn inbred lines such as a Huangzaosi inbred line, a B73 inbred line, a Mo17 inbred line and a Dan340 inbred line so that DNA fragments having different lengths are obtained; and PCR amplification protocols and reaction systems aiming at the different primers are determined. The corn WRKY gene intron length polymorphism molecular marker can be used as a functional gene marker for construction of a corn genetic linkage map, has fast and obvious effects and a good popularization prospect, and provides a marker-assisted selection foundation for corn gene and QTL locus cloning.
Description
Technical field
The present invention relates to biology gene engineering technology and genetic breeding field, be specifically related to corn WRKY gene intron length polymorphism molecule marker.
Background technology
Corn (Zea maize) is important food crop, plays an important role in national economy.Different genetic backgrounds according to corn germplasm, can be divided into the red bone in Luda (red 340), tangsipingtou (yellow morning four), Lancaster(MO17 to China's corn) and four hereditary monoids of Reid (B73), the corn hybrid seed of promoting the use of in a lot of agriculture productions is all by these four kinds of Different groups germplasms hybridization assembly.Corn self rotary series yellow early four, B73, Mo17, red 340 belong to respectively the red bone monoid in tangsipingtou, Reid, Lancaster and Luda, except possessing the typical hereditary property that represents separately monoid, also have high yield, combining ability high, disease-resistant, to the excellent proterties such as the photoperiod is insensitive, be the representative Inbred Lines that utilizes in maize genetic improvement and breeding process in the Different groups assembling cross-fertilize seed, most widely used general in the corn breeding process.
WRKY is a class transcription factor, has a plurality of family members in plant.A lot of WRKY genes are relevant with multiple environment stresses such as disease-resistant, anti-salt, drought resisting, anti-low temperature, high temperature resistances.Gene intron is one section non-coding sequence of two exons that adjoin, and the selective pressure that is subject to during due to evolution is less, so there is the difference of length or nucleotide sequence under long-term planting environment condition between different self-mating systems.Gene extron nucleotide coding amino acid, large for guaranteeing that the correct functionating of gene is subject to selective pressure during evolution, relatively conservative be subject to process when evolving in, the variation of length of nucleotides and sequence is all very little between different self-mating systems.Intron sequences between two exons that adjoin can be developed as molecule marker according to this specific character.Intron Length Polymorphism mark ILP(Intron length Polymorphism, be called for short ILP) be a kind of molecule marker that the exploitation of functional gene (as the WRKY gene) sequence is formed, although some gene is developed by ILP at present, and be applied to build genetic linkage maps, but the exploitation of WRKY-ILP mark up to now and application there is no report.The present invention is by the sequences Design ILP special primer to two exon regions that adjoin of corn WRKY gene, i.e. WRKY-ILP molecule marker.Through special PCR system and reaction conditions, obtain discrepant degree of length by pcr amplification in the representative Inbred Lines of four corn monoids, running gel is shown as the WRKY-ILP molecule marker of different corn inbred lines.This will and improve the conventional corn Cultivar for the efficient transformation of corn WRKY gene molecule assisted Selection basis will be provided.
Summary of the invention
The purpose of this invention is to provide corn WRKY gene intron length polymorphism molecule marker.
According to corn WRKY gene intron length polymorphism molecule marker of the present invention, comprising:
ZmWRKY1-ILP-F1:5’-GACATCCCTTCGGACGAGTA-3’
ZmWRKY1-ILP-R1:5 '-CCTCGTACGTCACCACCAG-3 '; Or
ZmWRKY2-ILP-F2:5’-GCAAGTACGGCCAGAAGC-3’
ZmWRKY2-ILP-R2:5 '-ACGTGACGATCACCTTGTCC-3; Or
ZmWRKY3-ILP-F3:5’-GTACCGCTGGAGGAAGTACG-3'
ZmWRKY3-ILP-R3:5 '-CCCTCGTAGGTGGTGATGA-3 '; Or
ZmWRKY4-ILP-F4:5’-GACAGCTGACGCTGAGAGACT-3’
ZmWRKY4-ILP-R4:5 '-GTAGGCGATGATCTCGGTGA-3 '; Or
ZmWRKY5-ILP-F5:5’-GCTGAGCCTCACCTGTCG-3’
ZmWRKY5-ILP-R5:5 '-ACTCGTAGCCGTCGTCGTAG-3; Or
ZmWRKY6-ILP-F6:5’-CCTCCACCACCACTTCCAT-3’
ZmWRKY6-ILP-R6:5 '-CTTGTTGGCGATGATCGTG-3 '; Or
ZmWRKY7-ILP-F7:5’-AGAGGCGACCATGGAGGT-3’
ZmWRKY7-ILP-R7:5’-CATCCCTGGTCCTTGTGC-3’。
According to WRKY gene intron length polymorphism molecule marker of the present invention, wherein,
Gene ZmWRKY1 (GRMZM2G071907) is positioned at 11,677,947 to 11,679,542 of corn inbred line B73Chr2, altogether 1596bp;
Gene ZmWRKY2 (GRMZM2G040298) is positioned at corn inbred line B73Chr3199, and 190,076 to 199,191,712, be total to 1637bp;
Gene ZmWRKY3 (GRMZM2G018721) is positioned at 1,923,404 to 1,925,109 of corn inbred line B73Chr7, altogether 1706bp;
Gene ZmWRKY4 (GRMZM2G125653) is positioned at 109,903,288 to 109,905,118 of corn inbred line B73Chr7, altogether 1831bp;
Gene ZmWRKY5 (GRMZM2G099593) is positioned at 148,933,289 to 148,934,702 of corn inbred line B73Chr2, altogether 1414bp;
Gene ZmWRKY6 (GRMZM2G151444) is positioned at 9,028,660 to 9,036,594 of corn inbred line B73Chr3, altogether 7935bp;
Gene ZmWRKY7 (GRMZM2G059562) is positioned at 183,069,351 to 183,070,645 of corn inbred line B73Chr3, altogether 1295bp.
Therefore, according to technical scheme of the present invention, can not only provide polymorphic molecular marker for the genetic map that builds between four genetic groups of corn, can also provide molecular information basic in the cultivated maize background for the transformation of corn WRKY gene efficient.Develop the specific mark primer of specific amplified corn WRKY gene by the present invention, can use the regular-PCR technology and accurately identify fast the Intron Length Polymorphism in different genetic group corns of WRKY gene.
In order to realize purpose of the present invention, the technical solution used in the present invention is as follows:
(1) obtain corn WRKY gene-correlation sequence from the MaizeGDB database, and the sequence that obtains is identified, screen two exons that adjoin (intron sequences is longer) nucleotide sequence and analyse in depth;
(2) the some WRKY-ILP special primers of exon nucleotide sequence design that adjoin according to two of WRKY genes;
(3) increase obtaining represent Inbred Lines pellet 340, yellow morning four, MO17 and the B73 of WRKY-ILP special primer to four red bones in hereditary monoid Luda of Chinese maize, tangsipingtou, Lancaster, Reid in step (2), and continue to optimize the improvement primer, obtain different lengths WRKY gene fragment.Specificity and the stability of checking WRKY-ILP primer in self-mating system pellet 340, yellow morning four, MO17 and B73, and definite pcr amplification program and reaction system for different primers.
Enthusiasm effect of the present invention and use as follows:
1, the PCR system used of the present invention, do not need special PCR instrument and special reaction reagent, and the product that common commercial company produces all can reach requirement.
What 2, in PCR condition of the present invention, expanding effect is had the greatest impact is the design of special primer.The WRKY gene fragment that the WRKY-ILP specific molecular marker of developing in the present invention all obtains from the present invention, and the specific mark primer PCR amplified fragments that is used for amplification differs greatly on length, can not occur mutually obscuring or disturbing, can guarantee that result is accurate.
3, utilize base specific to design 7 pairs of primers, structure is simple and clear, and is easy to use, and utilizes round pcr to detect rapidly and efficiently, can accurately identify at short notice the corn inbred line of different sources, and effect is fast obvious, and good promotion prospect is arranged.
4, the corn WRKY-ILP specific molecular marker that obtains of the present invention is for the efficient transformation of corn WRKY gene and improve the conventional corn Cultivar molecule assisted Selection basis is provided.
Description of drawings
Fig. 1 shows that corn ZmWRKY-ILP specific molecular marker detects collection of illustrative plates, is followed successively by from left to right the 1:ZmWRKY1-ILP specific molecular marker and detects collection of illustrative plates; 2: corn ZmWRKY2-ILP specific molecular marker detects collection of illustrative plates; 3: corn ZmWRKY3-ILP specific molecular marker detects collection of illustrative plates; 4: corn ZmWRKY4-ILP specific molecular marker detects collection of illustrative plates; 5: corn ZmWRKY5-ILP specific molecular marker detects collection of illustrative plates; 6: corn ZmWRKY6-ILP specific molecular marker detects collection of illustrative plates; 7: corn ZmWRKY7-ILP specific molecular marker detects collection of illustrative plates.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but be not limitation of the present invention, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Embodiment 1:
1, extracting genome DNA
Adopt the CTAB method to extract genomic dna (in self-mating system pellet 340, yellow morning four, MO17 and B73).
2, WRKY-ILP special primer design
Obtain ZmWRKY1 gene-correlation sequence GRMZM2G071907 from the MaizeGDB database, according to DNASTAR2.0 software sequences comparison result, two upstream and downstream exon 2s that adjoin at the ZmWRKY1 gene and 3 design respectively primer, obtain upstream ZmWRKY1-ILP-F1 and downstream ZmWRKY1-ILP-R1 special primer by repeatedly optimizing screening, because WRKY gene intron selective pressure in the long-term evolution process is little, there is difference in length in different genetic group corn inbred lines.And exon is relatively conservative during evolution, the coding region is very conservative in the corn inbred line of different genetic groups, substantially there is not the encoding sequence difference in length, so with the primers of two exon regions that adjoin of certain WRKY gene, this special primer has namely represented the special primer of certain specific WRKY gene.Running gel is shown as the WRKY-ILP molecule marker of different corn inbred lines.
This primer is specific as follows:
ZmWRKY1-ILP-F1:5’-GACATCCCTTCGGACGAGTA-3’
ZmWRKY1-ILP-R1:5’-CCTCGTACGTCACCACCAG-3’
The amplification of ZmWRKY1 gene specific molecular marker:
3.1PCR reaction system
3.2 the evaluation of corn ZmWRKY1-ILP specific molecular marker
(1) 6.0% polyacrylate hydrogel carries out pcr amplification product to be separated.
(2) polyacrylate hydrogel after electrophoresis is taken pictures with camera and analyzed.
1, with the 1st step of embodiment 1
2, design of primers
Obtain ZmWRKY2-ILP gene-correlation sequence GRMZM2G099593 from the MaizeGDB database, according to DNASTAR2.0 software sequences comparison result, the upstream and downstream exon region that adjoins two of ZmWRKY2 genes designs respectively primer, obtain the ZmWRKY2-ILP special primer through repeatedly optimizing to improve, this specific primer PCR product gel electrophoresis showed is the WRKY-ILP molecule marker of different corn inbred lines:
ZmWRKY2-ILP-F2:5’-GCAAGTACGGCCAGAAGC-3’
ZmWRKY2-ILP-R2:5’-ACGTGACGATCACCTTGTCC-3’
3, with the 3rd step of embodiment 1
3.1 with the 3.1st step of embodiment 1
3.2 with the 3.2nd step of embodiment 1
1, with embodiment step 1
2, design of primers
Obtain ZmWRKY3-ILP gene-correlation sequence GRMZM2G151444 from the MaizeGDB database, according to DNASTAR2.0 software sequences comparison result, the upstream and downstream exon region that adjoins two of ZmWRKY3 genes designs respectively primer, obtain the ZmWRKY3-ILP special primer through repeatedly optimizing to improve, this specific primer PCR product gel electrophoresis showed is the WRKY-ILP molecule marker of different corn inbred lines:
ZmWRKY3-ILP-F3:5’-GTACCGCTGGAGGAAGTACG-3'
ZmWRKY3-ILP-R3:5’-CCCTCGTAGGTGGTGATGA-3’
3, with the 3rd step of embodiment 1
3.1 with the 3.1st step of embodiment 1
3.2 with the 3.2nd step of embodiment 1
1, with embodiment 1 step 1
2, design of primers
Obtain ZmWRKY4-ILP gene-correlation sequence GRMZM2G040298 from the MaizeGDB database, according to DNASTAR2.0 software sequences comparison result, the upstream and downstream exon region that adjoins two of ZmWRKY4 genes designs respectively primer, obtain the ZmWRKY4-ILP special primer through repeatedly optimizing to improve, this specific primer PCR product gel electrophoresis showed is the WRKY-ILP molecule marker of different corn inbred lines:
ZmWRKY4-ILP-F4:5’-GACAGCTGACGCTGAGAGACT-3’
ZmWRKY4-ILP-R4:5’-GTAGGCGATGATCTCGGTGA-3’
3, with embodiment 1 step 3
3.1 with the 3.1st step of embodiment 1
3.2 with the 3.2nd step of embodiment 1
Embodiment 5
1, with the 1st step of embodiment 1
2, design of primers
Obtain ZmWRKY5-ILP gene-correlation sequence GRMZM2G040298 from the MaizeGDB database, according to DNASTAR2.0 software sequences comparison result, the upstream and downstream exon region that adjoins two of ZmWRKY5 genes designs respectively primer, obtain the ZmWRKY5-ILP special primer through repeatedly optimizing to improve, this specific primer PCR product gel electrophoresis showed is the WRKY-ILP molecule marker of different corn inbred lines:
ZmWRKY5-ILP-F5:5’-GCTGAGCCTCACCTGTCG-3’
ZmWRKY5-ILP-R5:5’-ACTCGTAGCCGTCGTCGTAG-3’
3, with the 3rd step of embodiment 1
3.1 with the 3.1st step of embodiment 1
3.2 with the 3.2nd step of embodiment 1
1, with embodiment 1 step 1
2, design of primers
Obtain ZmWRKY6-ILP gene-correlation sequence GRMZM2G125653 from the MaizeGDB database, according to DNASTAR2.0 software sequences comparison result, the upstream and downstream exon region that adjoins two of ZmWRKY6 genes designs respectively primer, obtain the ZmWRKY6-ILP special primer through repeatedly optimizing to improve, this specific primer PCR product gel electrophoresis showed is the WRKY-ILP molecule marker of different corn inbred lines:
ZmWRKY6-ILP-F6:5’-CCTCCACCACCACTTCCAT-3’
ZmWRKY6-ILP-R6:5’-CTTGTTGGCGATGATCGTG-3’
3, with the 3rd step of embodiment 1
3.1 with the 3.1st step of embodiment 1
3.2 with the 3.2nd step of embodiment 1
1, with embodiment 1 step 1
2, design of primers
Obtain ZmWRKY7 gene-correlation sequence RMZM2G036703 from the MaizeGDB database, according to DNASTAR2.0 software sequences comparison result, the upstream and downstream exon region that adjoins two of ZmWRKY7 genes designs respectively primer, obtain the ZmWRKY7-ILP special primer through repeatedly optimizing to improve, this specific primer PCR product gel electrophoresis showed is the WRKY-ILP molecule marker of different corn inbred lines:
ZmWRKY7-ILP-F7:5’-AGAGGCGACCATGGAGGT-3’
ZmWRKY7-ILP-R7:5’-CATCCCTGGTCCTTGTGC-3’
3, with the 3rd step of embodiment 1
3.1 with the 3.1st step of embodiment 1
3.2 with the 3.2nd step of embodiment 1
Embodiment 8
As shown in Figure 1, by above-mentioned steps, final optimization pass obtains the different self-mating system WRKY-ILP of corn specific molecular marker primer:
1, specific amplified corn ZmWRKY1-ILP molecule marker:
ZmWRKY1-ILP-F1:5’-GACATCCCTTCGGACGAGTA-3’
ZmWRKY1-ILP-R1:5’-CCTCGTACGTCACCACCAG-3’
Carry out pcr amplification in Maize Inbred Line Dan 340, yellow morning four, MO17 and B73, the amplified fragments size is 271bp.The amplified reaction program is as follows:
PCR reaction system (20 μ l): 1 * PCR damping fluid
2mM?MgCl
2
0.2mMdNTP?Mix
0.5UTaq?Enzyme
0.5 μ M ZmWRKY1-ILP primer
The 80ng DNA profiling
The PCR program is:
Step194 ℃ of denaturation 5min
Step294 ℃ of sex change 30sec
Step355 ℃ of annealing 30sec
Step472 ℃ is extended 45sec
Step5Go?to?Step2For39Times
Step672 ℃ is extended 10min
Electrophoresis detection:
6% polyacrylate hydrogel electrophoresis detection.Huang early four, in B73, Mo17, red 340 4 self-mating systems, B73 amplifies the general band of electrophoresis of 271bp, yellow morning four, red 340PCR also amplify the 271bp electrophoretic band, and Mo17 amplifies less than the general band of the electrophoresis of 271bp, shows the length polymorphism of this mark.
2, specific amplified corn ZmWRKY2-ILP molecule marker:
ZmWRKY2-ILP-F2:5’-GCAAGTACGGCCAGAAGC–3’
ZmWRKY2-ILP-R2:5’-ACGTGACGATCACCTTGTCC–3’
Carry out pcr amplification in Maize Inbred Line Dan 340, yellow morning four, MO17 and B73, the amplified fragments size is 264bp.The amplified reaction program is as follows:
PCR reaction system (20 μ l): 1 * PCR damping fluid
2mM?MgCl
2
0.2mMdNTP?Mix
0.5UTaq?Enzyme
0.5 μ M ZmWRKY2-ILP primer
The 80ng DNA profiling
The PCR program is:
Step194 ℃ of denaturation 5min
Step294 ℃ of sex change 30sec
Step358 ℃ of annealing 30sec
Step472 ℃ is extended 45sec
Step5Go?to?Step2For39Times
Step672 ℃ is extended 10min
Electrophoresis detection:
6% polyacrylate hydrogel electrophoresis detection.Huang early four, in B73, Mo17, red 340 4 self-mating systems, B73 amplifies the general band of electrophoresis of 264bp, yellow morning four, red 340PCR also amplify the 264bp electrophoretic band, and Mo17 amplifies less than the general band of the electrophoresis of 317bp, shows the length polymorphism of this mark.
3, specific amplified corn ZmWRKY3-ILP molecule marker:
ZmWRKY3-ILP-F3:5’-GTACCGCTGGAGGAAGTACG-3'
ZmWRKY3-ILP-R3:5’-CCCTCGTAGGTGGTGATGA-3’
Carry out pcr amplification in Maize Inbred Line Dan 340, yellow morning four, MO17 and B73, the amplified fragments size is 263bp.The amplified reaction program is as follows:
PCR reaction system (20 μ l): 1 * PCR damping fluid
2mM?MgCl
2
0.2mMdNTP?Mix
0.5UTaq?Enzyme
0.5 μ M ZmWRKY3-ILP primer
The 80ng DNA profiling
The PCR program is:
Step194 ℃ of denaturation 5min
Step294 ℃ of sex change 30sec
Step358 ℃ of annealing 30sec
Step472 ℃ is extended 45sec
Step5Go?to?Step2For39Times
Step672 ℃ is extended 10min
Electrophoresis detection:
6% polyacrylate hydrogel electrophoresis detection.Huang early four, in B73, Mo17, red 340 4 self-mating systems, B73 amplifies the general band of electrophoresis of 263bp; Yellow morning four, pellet 340, Mo17 amplify the electrophoretic band less than 263bp through PCR, and these three amplified fragments sizes are identical, show the length polymorphism of this mark.
4, specific amplified corn ZmWRKY4-ILP molecule marker:
ZmWRKY4-ILP-F4:5’-GACAGCTGACGCTGAGAGACT-3’
ZmWRKY4-ILP-R4:5’-GTAGGCGATGATCTCGGTGA-3’
Carry out pcr amplification in Maize Inbred Line Dan 340, yellow morning four, MO17 and B73, the amplified fragments size is 276bp.The amplified reaction program is as follows:
PCR reaction system (20 μ l): 1 * PCR damping fluid
2mM?MgCl
2
0.2mMdNTP?Mix
0.5UTaq?Enzyme
0.5 μ M ZmWRKY4-ILP primer
The 80ng DNA profiling
The PCR program is:
Step194 ℃ of denaturation 5min
Step294 ℃ of sex change 30sec
Step357 ℃ of annealing 30sec
Step472 ℃ is extended 45sec
Step5Go?to?Step2For39Times
Step672 ℃ is extended 7min
Electrophoresis detection:
Huang early four, in B73, Mo17, red 340 4 self-mating systems, B73 amplifies the general band of electrophoresis of 264bp, amplify the electrophoretic band less than 276bp yellow morning four, and Mo17, red 340 amplifies greater than the general band of the electrophoresis of 276bp, and red 340 electrophoretic bands show the length polymorphism of this mark greater than Mo17.
5, specific amplified corn ZmWRKY5-ILP molecule marker:
ZmWRKY5-ILP-F5:5’-GCTGAGCCTCACCTGTCG-3’
ZmWRKY5-ILP-R5:5’-ACTCGTAGCCGTCGTCGTAG-3’
Carry out pcr amplification in Maize Inbred Line Dan 340, yellow morning four, MO17 and B73, the amplified fragments size is 317bp.The amplified reaction program is as follows:
PCR reaction system (20 μ l): 1 * PCR damping fluid
2mM?MgCl
2
0.2mMdNTP?Mix
0.5UTaq?Enzyme
0.5 μ M ZmWRKY5-ILP primer
The 80ng DNA profiling
The PCR program is:
Step194 ℃ of denaturation 5min
Step294 ℃ of sex change 30sec
Step356 ℃ of annealing 30sec
Step472 ℃ is extended 45sec
Step5Go?to?Step2For39Times
Step672 ℃ is extended 10min
Electrophoresis detection:
6% polyacrylate hydrogel electrophoresis detection.Huang early four, in B73, Mo17, red 340 4 self-mating systems, B73 amplifies the general band of electrophoresis of 317bp, yellow early four pcr amplifications go out the electrophoretic band greater than 263bp, and Mo17, the red 340 general bands of identical electrophoresis that amplify less than 317bp, show the length polymorphism of this mark.
6, specific amplified corn ZmWRKY6-ILP molecule marker:
ZmWRKY6-ILP-F6:5’-CCTCCACCACCACTTCCAT-3’
ZmWRKY6-ILP-R6:5’-CTTGTTGGCGATGATCGTG-3’
Carry out pcr amplification in Maize Inbred Line Dan 340, yellow morning four, MO17 and B73, the amplified fragments size is 276bp.The amplified reaction program is as follows:
PCR reaction system (20 μ l): 1 * PCR damping fluid
2mM?MgCl
2
0.2mMdNTP?Mix
0.5UTaq?Enzyme
0.5 μ M ZmWRKY6-ILP primer
The 80ng DNA profiling
The PCR program is:
Step194 ℃ of denaturation 5min
Step294 ℃ of sex change 30sec
Step350 ℃ of annealing 30sec
Step472 ℃ is extended 45sec
Step5Go?to?Step2For39Times
Step672 ℃ is extended 8min
Electrophoresis detection:
6% polyacrylate hydrogel electrophoresis detection.Huang early four, in B73, Mo17, red 340 4 self-mating systems, B73 amplifies the general band of electrophoresis of 276bp, yellow early four, Mo17, red 340 amplifies identical electrophoretic band greater than 276bp through PCR, shows the length polymorphism of this mark.
7, specific amplified corn ZmWRKY7-ILP molecule marker:
ZmWRKY7-ILP-F7:5’-AGAGGCGACCATGGAGGT-3’
ZmWRKY7-ILP-R7:5’-CATCCCTGGTCCTTGTGC-3’
Carry out pcr amplification in Maize Inbred Line Dan 340, yellow morning four, MO17 and B73, the amplified fragments size is 243bp.The amplified reaction program is as follows:
PCR reaction system (20 μ l): 1 * PCR damping fluid
2mM?MgCl
2
0.2mMdNTP?Mix
0.5UTaq?Enzyme
0.5 μ M ZmWRKY7-ILP primer
The 80ng DNA profiling
The PCR program is:
Step194 ℃ of denaturation 5min
Step294 ℃ of sex change 30sec
Step356 ℃ of annealing 30sec
Step472 ℃ is extended 45sec
Step5Go?to?Step2For39Times
Step672 ℃ is extended 10min
Electrophoresis detection:
6% polyacrylate hydrogel electrophoresis detection.Huang early four, in B73, Mo17, red 340 4 self-mating systems, Mo17 and B73 amplify the general band of electrophoresis of 243bp, and be yellow early four and the red 340 identical electrophoretic bands that all amplify less than 243bp, shows the length polymorphism of this mark.
Claims (1)
1. corn WRKY gene intron length polymorphism molecule marker, is characterized in that, described corn WRKY gene intron length polymorphism molecule marker comprises:
ZmWRKY1-ILP-F1:5’-GACATCCCTTCGGACGAGTA-3’
ZmWRKY1-ILP-R1:5 '-CCTCGTACGTCACCACCAG-3 '; Or
ZmWRKY2-ILP-F2:5’-GCAAGTACGGCCAGAAGC-3’
ZmWRKY2-ILP-R2:5 '-ACGTGACGATCACCTTGTCC-3; Or
ZmWRKY3-ILP-F3:5’-GTACCGCTGGAGGAAGTACG-3'
ZmWRKY3-ILP-R3:5 '-CCCTCGTAGGTGGTGATGA-3 '; Or
ZmWRKY4-ILP-F4:5’-GACAGCTGACGCTGAGAGACT-3’
ZmWRKY4-ILP-R4:5 '-GTAGGCGATGATCTCGGTGA-3 '; Or
ZmWRKY5-ILP-F5:5’-GCTGAGCCTCACCTGTCG-3’
ZmWRKY5-ILP-R5:5 '-ACTCGTAGCCGTCGTCGTAG-3; Or
ZmWRKY6-ILP-F6:5’-CCTCCACCACCACTTCCAT-3’
ZmWRKY6-ILP-R6:5 '-CTTGTTGGCGATGATCGTG-3 '; Or
ZmWRKY7-ILP-F7:5’-AGAGGCGACCATGGAGGT-3’
ZmWRKY7-ILP-R7:5’-CATCCCTGGTCCTTGTGC-3’。
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| CN103695418A (en) * | 2013-12-25 | 2014-04-02 | 四川农业大学 | Maize phosphate starvation responses intron length polymorphism marker for corn |
| CN104561273A (en) * | 2014-12-17 | 2015-04-29 | 国家烟草质量监督检验中心 | Method for identifying tobacco product by using lycopene epsilon cyclase gene ILP marker |
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| CN1632117A (en) * | 2004-11-22 | 2005-06-29 | 浙江大学 | Molecule mark and its development method |
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| CN104561273A (en) * | 2014-12-17 | 2015-04-29 | 国家烟草质量监督检验中心 | Method for identifying tobacco product by using lycopene epsilon cyclase gene ILP marker |
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