CN106148558B - A kind of anti-sense molecule label of corn nicosulfuron and its application - Google Patents
A kind of anti-sense molecule label of corn nicosulfuron and its application Download PDFInfo
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- CN106148558B CN106148558B CN201610829112.7A CN201610829112A CN106148558B CN 106148558 B CN106148558 B CN 106148558B CN 201610829112 A CN201610829112 A CN 201610829112A CN 106148558 B CN106148558 B CN 106148558B
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Abstract
The present invention relates to field of plant genetic project technology, provide a kind of anti-sense molecule label of corn nicosulfuron, which is located on the nsfy gene of No. 5 chromosomes of corn；Pass through the application of the molecular labeling, it whether can detecte in corn variety or self-mating system containing corn nicosulfuron sensitivity, resistant gene, to accelerate the breeding process of corn nicosulfuron resistant variety, due to using dedicated nicosulfuron sensitivity, resistance molecule label, fast accurate is not only screened, not affected by environment, selection target is clear, and production cost has been saved, substantially increase the efficiency of selection and quality of corn nicosulfuron resistant variety or strain.
Description
Technical field
The present invention relates to field of plant genetic project technology, provide a kind of anti-sense molecule label of corn nicosulfuron and its
Using.
Background technique
Corn is important one of the cereal crops in China.Currently, China per capita cultivated land only has 0.093 hectare, with production
The reduction in the soil and freshwater resources of grain, staple food supply safety problem are increasingly prominent.Life of the herbicide nicosulfuron in corn
In production, have use most wide, safe highest, dosage is minimum, it is cheap the advantages that, by liking for peasant and breeder.Closely
It is found over year, more and more corn inbred lines and cenospecies are sensitive to nicosulfuron, have seriously affected normal corn breeding
And production.
The sensibility of corn nicosulfuron is controlled by Recessive genes nsfy, and resistance is controlled by dominant single-gene nsfy.
Corn nicosulfuron resistant material is screened in traditional breeding method needs large-scale field selection, heavy workload, low efficiency, it is difficult to
Meet present needs.
Summary of the invention
The present inventor is directed to the case where above-mentioned prior art, in conjunction with studying for a long period of time and exploring, provides a kind of jade
Two molecular labelings of rice nicosulfuron sensitivity, resistance, the length of two labels are 277bp and 261bp, are all located at jade
In No. 5 chromosome nsfy genes of rice；Whether by the application of molecular labeling, can detecte in corn variety or self-mating system has cigarette
Sulfometuron Methyl resistant gene, to accelerate the breeding process of corn nicosulfuron resistant variety.It is phonetic due to using dedicated corn cigarette
Grand sensitive and resistance the molecular labeling of sulphur, not only screens fast accurate, not affected by environment, selection target is clear, and saves
Production cost, substantially increases the efficiency of selection and quality of corn nicosulfuron resistant variety or self-mating system, develops actual effect
Property molecular labeling, develop corn nicosulfuron perception marker assisted selection system, can with sequencing be applied to breeding practice.
The specific technical solution that inventor provides is as follows:
Present invention corn nicosulfuron sensitive molecule label obtained is located in corn nicosulfuron sensitive gene nsfy；
Its nucleotide sequence is as described in SEQ ID NO:1；
Corn nicosulfuron resistance molecule label is located in corn nicosulfuron resistant gene nsfy；Its nucleotide sequence is such as
Described in SEQ ID NO:5；
Above-mentioned two molecular labeling according to the SNP site in gene nsfy in the prior art by the present inventor for the first time
What exploitation obtained；
After obtaining above-mentioned two molecular labeling, using in the Markers for Detection corn nicosulfuron kind or self-mating system
Whether the gene nsfy containing nicosulfuron resistance, the specific steps are that:
(1) it detects in corn nicosulfuron kind or self-mating system and whether contains nicosulfuron sensitive gene nsfy:
Using the leaf DNA of primer amplified target plant, the segment of 295bp, nucleotide sequence such as SEQ are obtained
Shown in ID NO:2, later using being detected whether containing nucleotide sequence as described in SEQ ID NO:1 after the single-minded enzyme digestion of AvaII
Segment；
Criterion are as follows: occur as described in SEQ ID NO:1 in kind or self-mating system with corn nicosulfuron sensitivity
277bp electrophoresis band；The segment will not be cut off in kind or self-mating system without corn nicosulfuron sensitivity, so will not
Occur；
In order to cooperate the molecular labeling, inventor devises its specific primer,
Forward primer sequence: 5 '-GCGTGCTTCACGGAGCAGGAC-3 ' (as shown in SEQ ID NO:3)；
Reverse primer sequences: 5 '-TGAGCACGCCGAGGGAGA-3 ' (as shown in SEQ ID NO:4).
(2) detect in corn nicosulfuron kind or self-mating system whether the gene nsfy containing nicosulfuron resistance:
Using the leaf DNA of primer amplified target plant, the segment of 294bp, nucleotide sequence such as SEQ are obtained
Shown in ID NO:6, later using being detected whether containing nucleotide sequence as described in SEQ ID NO:5 after the single-minded enzyme digestion of BclI
Segment；
Criterion are as follows: occur as described in SEQ ID NO:5 in kind or self-mating system with corn nicosulfuron resistance
261bp electrophoresis band；The segment will not be cut off in kind or self-mating system without corn nicosulfuron resistance, so will not
Occur；
In order to cooperate the molecular labeling, inventor devises its specific primer,
Forward primer sequence: 5 '-CGTCGCCTTCCTCTTCTTGG-3 ' (as shown in SEQ ID NO:7)；
Reverse primer sequences: 5 '-ATGGACTCGAAGCGCGGGCGGTTGGCGATGATC-3 ' are (such as SEQ ID NO:8 institute
Show).
Determine whether can determine that the site by the amplification of specific primer, and to the digestion result of product
The sensitive, resistant gene containing corn nicosulfuron, so you can learn that whether corn variety to be measured or strain have nicosulfuron
Sensitive, resistant phenotype so as to better breeding and is applied to breed improvement.
In conclusion the present inventor provides a kind of anti-sense molecule label of corn nicosulfuron for the first time, which is located at jade
On the nsfy gene of No. 5 chromosome of rice；By the application of the molecular labeling, it can detecte in corn variety or self-mating system and whether contain
There are corn nicosulfuron sensitivity, resistant gene, it is special due to using to accelerate the breeding process of corn nicosulfuron resistant variety
Nicosulfuron is sensitive, resistance molecule marks, and not only screens fast accurate, and not affected by environment, selection target is clear, and
Production cost has been saved, the efficiency of selection and quality of corn nicosulfuron resistant variety or strain are substantially increased.
Detailed description of the invention
Fig. 1 is using molecular labeling of the present invention and determination method to the sensitive self-mating system of corn nicosulfuron and corn
The digestion verification result electrophoresis grayscale image of nicosulfuron resistance self-mating system,
It is found that the electrophoresis band of 277bp occurs after digestion in corn nicosulfuron sensitivity self-mating system in figure, and corn cigarette
Sulfometuron Methyl resistance self-mating system does not occur then；
Fig. 2 is using molecular labeling of the present invention and determination method to the sensitive self-mating system of corn nicosulfuron and corn
The digestion verification result electrophoresis grayscale image of nicosulfuron resistance self-mating system,
It is found that the electrophoresis band of 261bp occurs after digestion in corn nicosulfuron resistance self-mating system in figure, and corn cigarette
Sulfometuron Methyl sensitivity self-mating system does not occur then.
Specific embodiment
Further definition is of the invention in following embodiment, according to above description and these embodiments, those skilled in the art
Member can determine essential characteristic of the invention, and without departing from the spirit and scope of the invention, can be to the present invention
It makes various changes and modifications, so that it is applicable in various uses and condition.In addition to special indicate, of the present invention is this
The field prior art；
The application of 1 corn nicosulfuron resistance related molecular marker of embodiment
1. the leaf DNA of corn is extracted
Maize leaf DNA is extracted using routine techniques；
2. target product expands
Forward primer sequence: 5 '-CGTCGCCTTCCTCTTCTTGG-3 ' (as shown in SEQ ID NO:7)；Reverse primer sequence
Column: 5 '-ATGGACTCGAAGCGCGGGCGGTTGGCGATGATC-3 ' (as shown in SEQ ID NO:8).
PCR amplification: its PCR amplification system is 20 μ L
Amplification condition:
It can be obtained the segment of 294bp by above-mentioned amplification, nucleotide sequence is as shown in SEQ ID NO:6.
The single-minded digestion of PCR product:
10 μ L of digestion system:
Single-minded enzyme BclI:0.3 μ L
PCR product: 3 μ L
10×NE buffer:0.7μL
DdH2O:6μL
Endonuclease reaction condition: the 0.3 single-minded enzyme of μ L BclI (market is bought), 50 DEG C of water-bath 2h are added in pcr amplification product.
Then digestion system is placed into 65 DEG C of 5min；
After above-mentioned amplified production is separated by electrophoresis on 8% polyacrylamide gel respectively, amplified production molecular size range
For 294bp, after amplified production digestion, occurs such as SEQ ID in kind or self-mating system with nicosulfuron resistant gene nsfy
The electrophoresis band of 261bp described in NO:5；In kind or self-mating system without nicosulfuron resistant gene nsfy the segment not by
It cuts away, without the segment；As shown in Figure 2.
The application of 2 corn nicosulfuron sensitivity related molecular marker of embodiment
1. the leaf DNA of corn is extracted,
Maize leaf DNA is extracted using routine techniques；Blade is respectively derived from corn nicosulfuron resistance self-mating system and jade
Rice nicosulfuron sensitivity self-mating system；
2. target product expands
Forward primer sequence: 5 '-GCGTGCTTCACGGAGCAGGAC-3 ' (as shown in SEQ ID NO:3)；Reverse primer
Sequence: 5 '-TGAGCACGCCGAGGGAGA-3 ' (as shown in SEQ ID NO:4).
PCR amplification: its PCR amplification system is 20 μ L
Amplification condition:
It can be obtained the segment of 295bp by above-mentioned amplification, nucleotide sequence is as shown in SEQ ID NO:2.
The single-minded digestion of PCR product:
10 μ L of digestion system:
Single-minded enzyme AvaII:0.3 μ L
PCR product: 3 μ L
10×NE buffer:0.7μL
DdH2O:6μL
Endonuclease reaction condition: the 0.3 single-minded enzyme of μ L AvaII (market is bought), 37 DEG C of water-bath 2h are added in pcr amplification product.
Then digestion system is placed into 65 DEG C of 5min.
After above-mentioned amplified production is separated by electrophoresis on 8% polyacrylamide gel respectively, amplified production molecular size range
For 295bp, after amplified production digestion, occur such as SEQ ID NO in kind or strain with nicosulfuron sensitive gene nsfy:
The electrophoresis band of 277bp described in 1；The segment is not cut off in kind or strain without nicosulfuron sensitive gene nsfy,
Without the segment；As shown in Figure 1.
<110>Shandong Agricultural University
<120>a kind of anti-sense molecule label of corn nicosulfuron and its application
<160>8
<210>1
<211>277
<212>DNA
<213>corn
<400>1
gaccagagct tcgccaaccg cccgcgcttc gagtccatgc gcctcgtgtc cttcgacggc 60
gcccagctct cggtgtccag ctacgggccc tactggcgca ccctccgccg cgtcgccgcc 120
gtgcagctcc tctccgcgca ccgcgtcgcc tgcatgtccc ccgtcatctg cgccgaggtg 180
cgcgccatgg tgcgccggat ggctcgccta gccgcgggcg gcgccgcgcg cgtccagctc 240
aggcggcgcc tgttcgagct ctccctcggc gtgctca 277
<210>2
<211>295
<212>DNA
<213>corn
<400>2
cgtgcttca cggagcagga ccagagcttc gccaaccgcc cgcgcttcga gtccatgcgc 60
ctcgtgtcct tcgacggcgc ccagctctcg gtgtccagct acgggcccta ctggcgcacc 120
ctccgccgcg tcgccgccgt gcagctcctc tccgcgcacc gcgtcgcctg catgtccccc 180
gtcatctgcg ccgaggtgcg cgccatggtg cgccggatgg ctcgcctagc cgcgggcggc 240
gccgcgcgcg tccagctcag gcggcgcctg ttcgagctct ccctcggcgt gctca 295
<210>3
<211>21
<212>DNA
<213>artificial sequence
<400>3
gcgtgcttca cggagcagga c 21
<210>4
<211>18
<212>DNA
<213>artificial sequence
<400>4
tgagcacgcc gagggaga 18
<210>5
<211>261
<212>DNA
<213>corn
<400>5
cgtcgccttc ctcttcttgg tccactacct cgtgggccgc gccgccgccg gcggcggcaa 60
ggggaggaag cggctgccac cgagccctct ggccatcccg ttcctcggcc acctccacct 120
cgtcaagacg ccgttccact cggcgctggg ccgcctcgcg gagcgccacg gcccggtgtt 180
ctccctgcgc atggggtgcc gccgcgcggt ggtcgtgtcc tcgccggagt gcgccagggc 240
gtgcttcacg gagcacgaca t 261
<210>6
<211>294
<212>DNA
<213>corn
<400>6
cgtcgccttc ctcttcttgg tccactacct cgtgggccgc gccgccgccg gcggcggcaa 60
ggggaggaag cggctgccac cgagccctct ggccatcccg ttcctcggcc acctccacct 120
cgtcaagacg ccgttccact cggcgctggg ccgcctcgcg gagcgccacg gcccggtgtt 180
ctccctgcgc atggggtgcc gccgcgcggt ggtcgtgtcc tcgccggagt gcgccagggc 240
gtgcttcacg gagcacgaca tgatcatcgc caaccgcccg cgcttcgagt ccat 294
<210>7
<211>20
<212>DNA
<213>artificial sequence
<400>7
cgtcgccttc ctcttcttgg 20
<210>8
<211>33
<212>DNA
<213>artificial sequence
<400>8
atggactcga agcgcgggcg gttggcgatg atc 33
Claims (2)
1. a kind of anti-sense molecule label of corn nicosulfuron, by corn nicosulfuron perception molecular labeling and corn nicosulfuron
Resistance molecule label composition, it is characterised in that: the corn nicosulfuron perception molecular labeling is located at corn nicosulfuron perception
In gene nsfy, nucleotide sequence is as shown in SEQ ID NO:1；
The corn nicosulfuron resistance molecule label is located in corn nicosulfuron resistant gene nsfy, and nucleotide sequence is such as
Shown in SEQ ID NO:5.
2. the anti-sense molecule of corn nicosulfuron described in claim 1 marks answering in corn nicosulfuron resistant variety breeding
With.
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