CN105483136A - wheat pre-harvest sprouting tolerance gene TaZFP18 and application thereof - Google Patents
wheat pre-harvest sprouting tolerance gene TaZFP18 and application thereof Download PDFInfo
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Abstract
The invention discloses a wheat pre-harvest sprouting tolerance gene TaZFP18. The gene includes the nucleotide sequence shown as SEQ ID NO.1, or includes the nucleotide sequence shown as complementary with the SEQ ID NO.1 sequence, and the gene encodes a CCCH type zinc finger protein 18 and can serve as the molecular marker gene of a wheat pre-harvest sprouting tolerance variety. The wheat pre-harvest sprouting tolerance gene TaZFP18 is a novel major wheat pre-harvest sprouting tolerance gene, can be utilized to develop a molecular marker so as to rapidly identify whether wheat belongs to the pre-harvest sprouting tolerance variety or not and provides a new gene resources for breeding of wheat pre-harvest sprouting tolerance varieties.
Description
Technical field
What the present invention relates to is the technical field of Crop Genetic Breeding, in particular grow wheat anti growing out gene TaZFP18 and an application thereof.
Background technology
Pre-harvest sprouting (Pre-harvestsprouting, PHS) refers to that meeting with rainy weather before crop closes on results causes kernel direct to be connected on the phenomenon of fringe portion germination.Wheat generation Pre-harvest sprouting can the serious stability reducing Yield and qualities.If it is longer to meet with the continuous rainfall weather time before results, then Spike sprouting harm is more serious.Therefore, the wheat breed cultivating ear germinating resistance persistence strong contributes to reducing this risk.
The factor affecting Spike sprouting mainly contains outside environmental elements and interior factor.Light and temperature is essential environmental factors; Interior factor mainly contains Grain Dormancy, kernel seed coat colour, fringe portion form, seed water absorption character, alpha-amylase activity and endogenous hormones etc., and wherein Grain Dormancy is the central genetic mechanism affecting ear germinating resistance.
Forefathers utilize the method for linkage analysis and association analysis to located the relevant QTL of a large amount of wheat seed dormancy/ear germinating resistance, nearly cover wheat 21 karyomit(e)s.But the QTL identified not is functional indicia, it only represents a certain section on karyomit(e), have impact on high efficiency and the accuracy of molecular mark selection.At present, wheat ear germinating resistance correlation function gene is mostly out identified by homologous clone.But cultivating the higher stronger kind of persistence that has concurrently of ear germinating resistance level also needs to continue to excavate anti growing out major gene, to meet the demand of the polygene molecule aggregation breeding of wheat ear germinating resistance.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide grow wheat anti growing out gene TaZFP18 and an application thereof, to provide a kind of new wheat anti growing out major gene, meet the demand of the polygene molecule aggregation breeding of wheat ear germinating resistance.
The present invention is achieved by the following technical solutions:
The invention provides a grow wheat anti growing out gene TaZFP18, described gene comprises the nucleotide sequence as shown in SEQIDNO.1, or comprise the nucleotide sequence with SEQIDNO.1 complementary, this genes encoding CCCH type zinc finger protein 18, can be used as the molecular marker gene of the anti-sense kind of wheat ear germinating.
Present invention also offers the application of a kind of above-mentioned wheat anti growing out gene TaZFP18 in the anti-sense kind of qualification wheat ear germinating.
Present invention also offers a kind of method utilizing above-mentioned wheat anti growing out gene TaZFP18 to identify the anti-sense kind of wheat ear germinating, comprise the following steps:
(1) with Wheat volatiles DNA for template, with C8DBS6BL-F and C8DBS6BL-R for Auele Specific Primer, carry out pcr amplification, the sequence of described C8DBS6BL-F and C8DBS6BL-R is:
SEQIDNO.2：C8DBS6BL-F：CGCGACAGGAAGTTTGC
SEQIDNO.3：C8DBS6BL-R：ATTGAAGAGGATTGGAGGG
(2) if step (1) pcr amplification obtains nucleotide sequence as shown in SEQIDNO.1 or its complementary sequence, then this wheat is ear germinating resistance kind, if step (1) does not increase obtain nucleotide sequence as shown in SEQIDNO.1 or its complementary sequence, then this wheat is Spike sprouting perception kind.
Preferably, in described step (1), with the genomic dna of wheat seed for template carries out pcr amplification.
The present invention has the following advantages compared to existing technology: the invention provides grow wheat anti growing out gene TaZFP18 and an application thereof, this wheat anti growing out gene TaZFP18 is a kind of new wheat anti growing out major gene, utilize its exploitation molecule marker, can identify rapidly whether wheat is ear germinating resistance kind, also for wheat ear germinating resistance breeding provides new genetic resources.
Accompanying drawing explanation
Fig. 1 is the qualification result electrophorogram of the anti-sense kind of wheat ear germinating, wherein, and M:Marker, 1: the red awns spring 21,2: capital 411,3: Suining sticks together wheat, 4: Jimai 22,5: Fuling must wheat, 6: tobacco grower 19,7: Yongchuan white shell shell wheat, 8: Zhongyou9507.
Embodiment
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
1, material
The ordinary method that the present embodiment method therefor is all known to those skilled in the art dawn if no special instructions, the materials such as reagent used, if no special instructions, are commercially available purchase product, germ plasm resource used, all can obtain from national germplasm resource bank.
2, method
2.1SDS-Tris saturated phenol method extracting wheat genomic dna
(1) each 2-3 grain of seed of 8 grow wheat kinds is got respectively, be placed in 2ml sterile centrifugation tube, utilize high-throughput tissue grinder grind into powder, described wheat breed is the red awns spring 21, capital 411, Suining sticks together wheat, Jimai 22, Fuling palpus palpus wheat, tobacco grower 19, Yongchuan white shell shell wheat, Zhongyou9507, its Zhong jing 411, Jimai 22, tobacco grower 19, Zhongyou9507 is by commercially available acquisition, the red awns spring 21, Suining sticks together wheat, Fuling palpus palpus wheat, Yongchuan white shell shell wheat (C.Changet.al.IdentifyingallelesofViviparous-1Bassociated withpre-harvestsproutinginmicro-corecollectionsofChinese wheatgermplasm, MolecularBreedingMarch2010, Volume25, Issue3, pp481-490) be local variety, directly acquisition is bought by national germplasm resource bank or locality.
(2) 1.2mlDNA Extraction buffer (200mMTris-Cl, 250mMNaCl, 25mMEDTA, 0.5%SDS, 2% β-ME) is added.
(3) 60 DEG C of water-bath 45min, period, interrupted oscillation was fully to extract.
(4) the centrifugal 10min of 12000rpm under room temperature.
(5) shift in supernatant liquor to new 2ml sterile centrifugation tube, the saturated phenol/chloroform of equal-volume Tris adding precooling is different/amylalcohol (volume ratio is 25:24:1), in putting upside down mixing 15min on ice, period interrupted oscillation.
(6) the centrifugal 10min of 12000rpm under room temperature.
(7) shift supernatant liquor in new 2ml sterile centrifugation tube, repeating step (5), (6), fully to remove albumen.
(8) supernatant liquor is shifted in new 1.5ml sterile centrifugation tube, add 0.6 times of Virahol (300 μ l) and 1/10 times of volume (50 μ l) NaAc (pH5.2), leave standstill 17min on ice after abundant mixing mixing, DNA white precipitate is fully separated out.
(9) 4 DEG C of centrifugal 10min of 10000rpm.
(10) abandon supernatant, add 70% ethanol rinse 2 times of precooling, then use dehydrated alcohol rinsing 1 time, room temperature is dried, and adds 100 μ l and wherein to spend the night dissolving containing 1 × TE damping fluid (or distilled water) of 2 μ l10mg/mlRNase enzymes.
(11) on NanoVuePlus micro-spectrophotometer, detect DNA concentration, and unification is diluted to 50ng/ul working fluid, for subsequent use.
2.2PCR amplification and detection
PCR amplification system is 20 μ L, comprises 10 × buffer (containing 2.0mmolL
-1mg
2+) 2.0 μ L, 2.5mmolL
-1dNTPs1.6 μ L, 5U μ L
-1taqDNApolymerase0.2 μ L, 10 μm of olL
-1the each 0.8 μ L of primer, 2.0 μ L template DNA (50-60ng μ L
-1), ddH
2o13.8 μ L.
Described primer sequence is:
SEQIDNO.2：C8DBS6BL-F：CGCGACAGGAAGTTTGC
SEQIDNO.3：C8DBS6BL-R：ATTGAAGAGGATTGGAGGG
Response procedures is: 94 DEG C of denaturation 5min; 35 circulations (72 DEG C extend 1min for 95 DEG C of sex change 30s, 60 DEG C of annealing 30s); 72 DEG C extend 10min; 4 DEG C of preservations.
PCR primer utilize mass ratio be 1.2% agarose gel electrophoresis carry out analyzing and testing, GelStain fluorescent dyeing, BIO-RAD gel imaging system scanning takes pictures.
Result as shown in Figure 1, found that the wheat that to stick together in red awns spring 21, Suining, Fuling must wheat, all amplify 790bp object fragment in these 4 ear germinating resistance kinds of Yongchuan white shell shell wheat, and in Beijing 411, Jimai 22, tobacco grower 19, in these 4 Spike sprouting perception kinds of Zhongyou9507 all without amplified production.
The checking of 2.3 goal gene
Carry out glue recovery to ear germinating resistance kind amplified production, be connected in pGEM-T carrier and carry out cloning and sequencing, sequencing result verifies that this amplified production is really TaZFP18 Gene Partial gene order, namely consistent with nucleotide sequence shown in SEQIDNO.1.Find that amplified production comprises 452bp coding region sequence and 338bp3' non-translational region sequence by compare of analysis, and this amplified production is the G-patch domain sequence of TaZFP18 zinc finger protein, main relevant with mRNA specific combination, therefore, the disappearance of this position sequence may cause this zinc finger transcription factor can not normally be combined with Downstream regulatory gene, thus affects the expression of downstream genes involved.
3, the checking of functional label gene in different groups
3.1 seed germination indexes (GI) measure
Gather in the crops wheat stem fringe material respectively in wheat dough stage, room-dry 2 days, deposit in-20 DEG C of refrigerators immediately to maintain the dormant trait of seed, treat that total Test material results are complete, carry out germination test in the lump.Choose 10 ripening stages consistent stem fringe, manual threshing, each material is got 50 and is carried out seed germination experiment, repeats for 2 times.Seed ventral groove is placed in culture dish down, 9ml sterilized water is added in culture dish, be placed in artificial climate incubator (20 DEG C, illumination 14h, dark 10h, humidity 80%), 24 as a child started to add up each culture dish germinating seed number afterwards, add up rudiment number in the every day same time and reject chitting piece (showing money or valuables one carries unintentionally for rudiment standard of perfection with embryo portion), within 3 days, calculating seed germination index (GI) afterwards.
Seed germination index (GI) calculation formula: GI=[(3 × n1+2 × n2+1 × n3)/(3 × N)] × 100%
Wherein n1, n2, n3 are the kernal numbers of seed the 1st day, institute's rudiment every day in the 2nd day, the 3rd day, and N refers to total grain number.
The 3.2 whole Spike sprouting rates (FS) in field measure
Run into natural precipitation weather during harvesting wheat, retain 10 wheat stem fringes in field and carry out the whole Spike sprouting rate mensuration of natural precipitation.Results are placed in Constant Temp. Oven, 150 DEG C of flash bakings, manual threshing, statistics chitting piece number and total grain number, calculate the whole Spike sprouting rate (fieldsprouting, FS) in field
Field whole Spike sprouting rate (FS) calculation formula: FS=(10 Spike sprouting number ÷ 10 total grain panicle number) × 100%
The checking of 3.3 functional label genes in wheat Mini core collection sources group
Get 235 parts of wheat Mini core collection resources, the method of above-mentioned steps 2 is utilized to carry out pcr amplification, measure and add up the whole Spike sprouting rate (FS) of seed germination index (GI) and field of each germ plasm resource, do significance of difference analysis and correlation analysis to statistics, result is as shown in following table 1,2.
3.5 the checking in the RILs colony family in functional label gene 411/ red awns spring 21 in Beijing
Get the RILs colony family in capital 411/ red awns spring 21, the method of above-mentioned steps 2 is utilized to carry out pcr amplification, measure and add up the whole Spike sprouting rate (FS) of seed germination index (GI) and field of each germ plasm resource, significance of difference analysis and correlation analysis are done to statistics, result is as shown in following table 1,2, in table, the seed germination index that GI3, GI7, GI15 measure after representing results respectively for the 3rd day, the 7th day, the 15th day.
Spike sprouting phenotypic difference significance analysis between table 1 different genotype
Note:
*for P < 0.01, reach pole conspicuous level;
*for P < 0.05, reach conspicuous level;
afor standard deviation
Table 2 carries the allelic variation of 790bp and the correlation analysis of different Spike sprouting phenotype
Note:
*for P < 0.01, reach pole conspicuous level;
*for P < 0.05, reach conspicuous level
Found that, in 235 parts of wheat Mini core collection resources, carry the genotypic material of 790bp and there is higher ear germinating resistance, containing this genotypic material, then not there is the whole Spike sprouting rate of higher germination index and field, significance of difference analytical results shows, both are difference extremely remarkable (P < 0.01) on GI3, GI7 in 2014 and GI3, FS phenotype in 2015, significant difference (P < 0.05) in GI15 phenotype in 2015.Further from different Spike sprouting phenotype, correlation analysis is carried out to different genotype, result shows to carry the allelic variation of 790bp and GI3, GI7 in 2014 and GI3, FS phenotype in 2015 in extremely remarkable negative correlation (P < 0.01), is remarkable negative correlation (P < 0.05) with GI15 phenotype in 2015.
In addition to significance of difference analysis and the correlation analysis of capital 411/ Hong Mangchun 21RILs colony family, result also shows, between the Spike sprouting phenotype that different allelic variation is corresponding, difference extremely significantly (P < 0.01); Allelic variation and all Spike sprouting phenotypes of carrying 790bp are all extremely remarkable negative correlation (P < 0.01).
Claims (4)
1. a grow wheat anti growing out gene TaZFP18, it is characterized in that, described gene comprises the nucleotide sequence as shown in SEQIDNO.1, or comprises the nucleotide sequence with SEQIDNO.1 complementary.
2. the application of wheat anti growing out gene TaZFP18 as claimed in claim 1 in the anti-sense kind of qualification wheat ear germinating.
3. utilize wheat anti growing out gene TaZFP18 as claimed in claim 1 to identify a method for the anti-sense kind of wheat ear germinating, it is characterized in that, comprise the following steps:
(1) with Wheat volatiles DNA for template, with C8DBS6BL-F and C8DBS6BL-R for Auele Specific Primer, carry out pcr amplification, the sequence of described C8DBS6BL-F and C8DBS6BL-R is:
SEQIDNO.2：C8DBS6BL-F：CGCGACAGGAAGTTTGC
SEQIDNO.3：C8DBS6BL-R：ATTGAAGAGGATTGGAGGG
(2) if step (1) pcr amplification obtains nucleotide sequence as shown in SEQIDNO.1 or its complementary sequence, then this wheat is ear germinating resistance kind, if step (1) does not increase obtain nucleotide sequence as shown in SEQIDNO.1 or its complementary sequence, then this wheat is Spike sprouting perception kind.
4. a kind of method utilizing wheat anti growing out gene TaZFP18 to identify the anti-sense kind of wheat ear germinating according to claim 3, is characterized in that, in described step (1), with the genomic dna of wheat seed for template carries out pcr amplification.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671060A (en) * | 2016-04-26 | 2016-06-15 | 杨燕 | Wheat pre-harvest sprouting resistance related gene and application thereof |
| CN106636372A (en) * | 2016-12-01 | 2017-05-10 | 安徽农业大学 | Molecular marker for identifying wheatear sprouting resistance based on TAMFT-3A gene and method thereof |
| CN108642064A (en) * | 2018-05-21 | 2018-10-12 | 安徽农业大学 | Wheat seed suspend mode duration gene TaCNGC-2A and its functional label |
| CN113528700A (en) * | 2021-07-22 | 2021-10-22 | 湖北省农业科学院粮食作物研究所 | KASP molecular marker kit for detecting wheat ear germination resistance, detection method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1904049A (en) * | 2006-08-08 | 2007-01-31 | 中国农业科学院作物科学研究所 | Wheat ear germinating resistance related gene and its application |
| CN1904050A (en) * | 2006-08-08 | 2007-01-31 | 中国农业科学院作物科学研究所 | Wheat ear germinating resistance related gene and its application |
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| CN1904050A (en) * | 2006-08-08 | 2007-01-31 | 中国农业科学院作物科学研究所 | Wheat ear germinating resistance related gene and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105671060A (en) * | 2016-04-26 | 2016-06-15 | 杨燕 | Wheat pre-harvest sprouting resistance related gene and application thereof |
| CN105671060B (en) * | 2016-04-26 | 2019-07-23 | 杨燕 | Wheat ear germinating resistance related gene and its application |
| CN106636372A (en) * | 2016-12-01 | 2017-05-10 | 安徽农业大学 | Molecular marker for identifying wheatear sprouting resistance based on TAMFT-3A gene and method thereof |
| CN106636372B (en) * | 2016-12-01 | 2020-06-09 | 安徽农业大学 | Molecular marker and method for identifying wheat ear germination resistance |
| CN108642064A (en) * | 2018-05-21 | 2018-10-12 | 安徽农业大学 | Wheat seed suspend mode duration gene TaCNGC-2A and its functional label |
| CN108642064B (en) * | 2018-05-21 | 2021-11-26 | 安徽农业大学 | Wheat seed dormancy duration gene TaCNGC-2A and functional marker thereof |
| CN113528700A (en) * | 2021-07-22 | 2021-10-22 | 湖北省农业科学院粮食作物研究所 | KASP molecular marker kit for detecting wheat ear germination resistance, detection method and application |
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