CN105132440B - With the wheat leaf blade chlorophyll content and relevant gene of grain weight, its Indel labels and application - Google Patents
With the wheat leaf blade chlorophyll content and relevant gene of grain weight, its Indel labels and application Download PDFInfo
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Abstract
The present invention relates to weigh relevant gene with wheat leaf blade chlorophyll content and grainTaBAS1‑2BAnd its Indel labels.The present invention is isolated and cloned into from wheat and wheat leaf blade chlorophyll content and the relevant gene of grain weight for the first timeTaBAS1‑2B, and according to being clonedTaBAS1‑2BGene order develops the labels of the Indel with polymorphism in ' capital 411 ' and ', and analyzes its effect to phenotype in 194 parts of wheat nature kinds between 21 ' two kinds of red awns spring, finally develop and geneTaBAS1‑2BThe functional label for isolating, and being closely related again with wheat leaf blade chlorophyll content and grain.Indel labels can explain that wheat spends the 8.2% and 4.4% of rear boot leaf chlorophyll content and mass of 1000 kernel phenotypic variation respectively, wherein molecular size range 494bp（Seq ID No.3）Band to grain weight increase have synergistic effect, molecular size range 493bp（Seq ID No.4）Band to grain weight reduction have synergistic effect.
Description
Technical field
The present invention relates to genetic engineering and molecular biology fields, specifically, being related to and wheat leaf blade chlorophyll content
With the relevant gene of grain weight, its Indel labels and application.
Background technology
Wheat is one of most important cereal crops in the world, with the growth of population, the reduction of cultivated area and grain
The continuous improvement of production cost is eaten, high yield, Breeding For Super High-yielding become urgent problem to be solved in China's wheat breeding.Wheat growth
Process is the process of substance production constantly to be carried out by photosynthesis, and green blade is the main source of substance production.Suitable
Under conditions of preferably, the about 70-90% of grain yield derives from the photosynthate for spending rear boot leaf manufacture during wheat grain filling
(Austin etc., 1997；Bidinger etc., 1997).Thus, extend wheat flag leaf functional period, promotes the more light of vane manufacturing
Product is closed, to improve the basis that yield is improving yield of wheat.
In higher plant, chlorophyll is photosynthetic pigments important in chloroplaset, absorb and the use of luminous energy in play
Important role.Some researches show that, the decline of pustulation period chlorophyll content in leaf blades significantly affects the photosynthetic rate of plant, to
Directly result in the decline (Araus etc., 1997) of photosynthetic efficiency.Wang etc. (2008) is pointed out in its research, is increased chlorophyll and is contained
Amount is to improve an effective way of plant biological yield and grain weight.
After by biology and abiotic stress, active oxygen in plant (reactive oxygen species,
ROS) content will increase, and cause to poison to cell, chloroplaset is caused to damage, to cause the decline of photosynthetic rate, final shadow
It rings to grain weight and yield.Plant forms more perfect ROS and removes enzyme system, wherein peroxiredoxin during evolution
(peroxiredoxin, Prx) protect chloroplaset and other organelles from oxidative damage in play an important role.Plant mistake
It is a chloroplast protein by nuclear gene encoding that oxide, which restores protein B AS1, belongs to AhpC families.The albumen be sulfydryl according to
Bad peroxidase restores hydrogen peroxide by the Cys residues of catalysis, relies on the chloroplaset thioredoxin reduction of NADPH
Enzyme keeps the reduction-state of BAS1.Ruiz etc. (2006) confirms that NTRC-BAS1 accesses are a kind of efficient for leaf in rice
Oxidation-reduction system of the green body from oxidative damage.
Although the research for restoring the structure and function of albumen for peroxide in plant at present is more, in plant
Internal specific mechanism of action is still imperfectly understood.It is more rare about the research of Prx report in wheat, it not yet finds at present small
The correlative study of wheat BAS1 genes is reported.The content of leaf chlorophyll, Yi Jiyu whether can be influenced about the expression of BAS1 genes
Whether grain weight is related still unapparent.Thus, research peroxiredoxin gene BAS1 is to further appreciate that ROS removes enzyme system
Mechanism of action provide foundation, simultaneously for study its effect for playing in improving wheat yield and grain weight also have it is important
Meaning.
Invention content
The object of the present invention is to provide weigh relevant gene TaBAS1-2B with wheat leaf blade chlorophyll content and grain.
It is a further object of the present invention to provide the Indel isolated with wheat cdna TaBAS1-2B labels and applications.
In order to realize the object of the invention, provided by the invention and wheat leaf blade chlorophyll content and the relevant gene of grain weight
TaBAS1-2B is obtained by electronic cloning combination conventional PCR method.According to the wheat thioredoxin mistake announced on NCBI
CDS sequences (the GenBank of oxide enzyme (Thiol-specific antioxidant protein)：AB000405.1 it) searches for
The est sequence screened (CA484935.1, CK214438.1, CK217637.1) is carried out electronics spelling by wheat est database
It connects, to splice sequence as template, wheat is used for primer B1, B2 and B3 using 5.0 Software for Design three of Primer Premier
The acquisition of BAS1 gene cDNAs, two further pairs primer G1 and G2 are used for the acquisition of wheat BAS1 genes (primer information is shown in Table 1).
Table 1 is for expanding wheat TaBAS1-2B genes and its primer of cDNA sequence
PCR react respectively using the cDNA in wheat breed ' capital 411 ' and genomic DNA as template, then will using primer B1,
B2 is connected by digestion successively with the product that B3 is expanded, and finally obtains the cDNA sequence of wheat BAS1 genes, size is
846bp connects the product expanded using primer G1 and G2 by digestion successively, finally obtains wheat BAS1 gene total orders
Row, size 2856bp.The gene is positioned using four body material of wheat ' China spring ' nullisomic, as a result the gene is positioned
It is TaBAS1-2B by the unnamed gene on wheat 2B chromosomes.
Specifically, provided by the invention and wheat leaf blade chlorophyll content and the relevant gene TaBAS1-2B of grain weight, core
Nucleotide sequence is：
I) nucleotide sequence shown in Seq ID No.1；Or
Ii) nucleotide sequence shown in Seq ID No.1 be substituted, lack and/or increase one or more nucleotide and
Express the nucleotide sequence of identical function protein；Or
Iii) under strict conditions with sequence hybridizes shown in Seq ID No.1 nucleotide sequence；
The stringent condition be in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS,
Hybridize at 65 DEG C, the solution is used in combination to wash film.
The cDNA sequence of the gene TaBAS1-2B is as shown in Seq ID No.2.
The present invention also provides carrier, engineering bacteria and transgenic cell lines containing the gene TaBAS1-2B.
According to the gene TaBAS1-2B sequences that clone obtains, ' capital 411 ' and ' developed between 21 ' two kinds of red awns spring
Indel labels with polymorphism are marked using 5.0 Software for Design of Primer Premier is a pair of for expanding the Indel
The primer of note, and its effect to phenotype is analyzed in 194 parts of wheat nature kinds, it finally develops and the gene
What TaBAS1-2B was isolated, and the functional label being closely related again with wheat leaf blade chlorophyll content and grain.
It is for expanding the primer pair that the Indel is marked：
Sense primer F：5’-CGCAGTGCCTGTCGTTTC-3’(Seq ID No.5)
Downstream primer R：5’-TCACATACTTCTTCCCAAT-3’(Seq ID No.6)
The primer pair amplifies weigh relevant Indel marker characteristics with wheat leaf blade compared with high chlorophyll content and higher grain
Band is 494bp, and nucleotide sequence is as shown in Seq ID No.3；The primer pair amplifies it is green with the relatively low leaf of wheat leaf blade
Cellulose content and the relevant Indel marker characteristics band of relatively low grain weight are 493bp, and nucleotide sequence is as shown in Seq ID No.4.
The gene TaBAS1-2B clones of the present invention and its particular technique route of Indel marker developments are as shown in Figure 1.
The present invention also provides Indel labels to have the wheat compared with high chlorophyll content and higher grain weight in screening blade
Application in germ plasm resource.Include the following steps：
1) genomic DNA of plant to be measured is extracted；
2) it using the genomic DNA of plant to be measured as template, using the primers F and R (Seq ID No.5 and 6), carries out
Pcr amplification reaction；
3) pcr amplification product is detected, if it is possible to amplify the feature item of the nucleotide sequence as shown in Seq ID No.3
Band, then plant to be measured, which is blade, has the wheat resource compared with high chlorophyll content and higher grain weight.
Wherein, the amplification system that PCR reactions use is calculated as with 10 μ l：Template DNA 100ng, 10 μM of primers Fs and each 0.2 μ of R
0.8 μ l, 5U/ μ l Taq archaeal dna polymerases of l, 2.5mM dNTP 0.12 μ l, Mg containing 25mM2+10 × PCR reaction buffers 1.0
μ l, ddH2O complements to 10 μ l.
PCR reaction amplification program be：94 DEG C 5 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 3 minutes, 36 cycle；72
DEG C 10 minutes.
Preferably, pcr amplification product is detected using 6% denaturing polyacrylamide gel electrophoresis in step 3), then silver staining
Colour developing.
The present invention further provides the Indel to mark the application in wheat molecular marker assistant breeding.
The present invention is isolated and cloned into from wheat and wheat leaf blade chlorophyll content and the relevant gene of grain weight for the first time
TaBAS1-2B, and according to the TaBAS1-2B gene orders cloned ' capital 411 ' and ' is being developed between 21 ' two kinds of red awns spring
The Indel labels of polymorphism are provided, and its effect to phenotype is analyzed in 194 parts of wheat nature kinds, are finally developed
It is isolated with gene TaBAS1-2B, and the functional label being closely related again with wheat leaf blade chlorophyll content and grain.It should
Indel labels can explain that wheat spends the 8.2% and 4.4% of rear boot leaf chlorophyll content and mass of 1000 kernel phenotypic variation respectively,
In, the band that molecular size range is 494bp (Seq ID No.3) has synergistic effect to the increase of grain weight, and molecular size range is
The band of 493bp (Seq ID No.4) has synergistic effect to the reduction of grain weight.The exploitation of the molecular labeling is High-yield Wheat point
Sub- assistant breeding provides a feasible approach.
Description of the drawings
Fig. 1 is gene TaBAS1-2B clones and its particular technique route map of Indel marker developments of the present invention.
Fig. 2 is that Indel marks the equipotential type analysis in Wheat Cultivars in the embodiment of the present invention 3；Wherein, swimming lane
1 is river agriculture 972, and swimming lane 2 is all wheats 16, and swimming lane 3 is precious wheat 8, and swimming lane 4 is whirlpool wheat No. 8, and swimming lane 5 is peace agriculture 9267, and swimming lane 6 is
Shandong wheat 23, swimming lane 7 are Shanxi wheat 31, and swimming lane 8 is all wheats 18, and swimming lane 9 is peace agriculture 1014, and swimming lane 10 is short by anti-58, and swimming lane 11 is river
Wheat 42, swimming lane 12 are Jimai 20, and swimming lane 13 is general wheat No. 5, and M is DNA Marker.
Fig. 3 is to be determined gene TaBAS1-2B using four body material of wheat ' China spring ' nullisomic in the embodiment of the present invention 3
The result of position；Wherein, swimming lane 1 is wheat ' China spring ' (CS), and swimming lane 2-7 is respectively CS N2AT2B, CS N2AT2D, CS
N2BT2A、CS N2BT2D、CS N2DT2A、CS N2DT2B。
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular cloning:A laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.
Sequencing is completed by raw work bioengineering Shanghai (share) Co., Ltd in following embodiment.
The acquisition of 1 wheat TaBAS1-2B genes of embodiment
It is that routine is combined by electronic cloning with wheat leaf blade chlorophyll content and the relevant gene TaBAS1-2B of grain weight
What PCR method obtained.According to the wheat thioredoxin peroxidase (Thiol-specific announced on NCBI
Antioxidant protein) CDS sequences (GenBank：AB000405.1 wheat est database) is searched for, by what is screened
Est sequence (CA484935.1, CK214438.1, CK217637.1) carries out electronic splicing, to splice sequence as template, utilizes
5.0 Software for Design three of Primer Premier is used for primer B1, B2 and B3 the acquisition of wheat BAS1 gene cDNAs, and in addition two
The acquisition of wheat BAS1 genes is used for primer G1 and G2, primer information to be shown in Table 1.
Table 1 is for expanding wheat TaBAS1-2B genes and its primer of cDNA sequence
PCR reacts respectively using the cDNA in wheat breed ' capital 411 ' and genomic DNA as template, in BIO-RAD My
It is carried out on Cycler 1.0PCR instrument.After PCR amplification, 1.5% agarose gel electrophoresis is carried out, is then cut in the UV lamp
Glue recycles amplified production, carries out TA cloning and sequencings, then splices the amplified production of primer B1, B2 and B3, finally obtain
The cDNA sequence (Seq ID No.2) of wheat BAS1 genes, size 846bp spell the product of primer G1 and G2 amplification
It connects, finally obtains wheat BAS1 genes complete sequence (Seq ID No.1), size 2856bp.
Wherein, the amplification system that PCR reactions use is calculated as with 10 μ l：Template DNA 100ng, 10 μM of primers Fs and each 0.2 μ of R
0.8 μ l, 5U/ μ l Taq archaeal dna polymerases of l, 2.5mM dNTP 0.12 μ l, Mg containing 25mM2+10 × PCR reaction buffers 1.0
μ l, ddH2O complements to 10 μ l.
PCR reaction amplification program be：94 DEG C 5 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 3 minutes, 36 cycle；72
DEG C 10 minutes.In 4 DEG C of preservations.
SDS-Tris saturated phenol method extracting wheat leaves genomic DNAs are used in the present embodiment, the specific method is as follows：
(1) the fresh young leaflet tablets of 2g, liquid nitrogen grinding is taken to be placed in 2ml sterile centrifugation tubes at fine powder.
(2) be added 1.2ml DNA Extraction buffers (200mM Tris-Cl, pH8.0,250mM NaCl, 25mM EDTA,
PH8.0,0.5%SDS, 2% β-ME are uniformly mixed, β-ME fresh additions before use).
(3) 55 DEG C of water-bath 45min, during which intermittent oscillation is fully to extract.
(4) 12000rpm centrifuges 10min at room temperature.
(5) it shifting in supernatant to new 2ml sterile centrifugation tubes, the isometric Tris saturated phenols/chloroform for being added precooling is different/
Amylalcohol (volume ratio 25:24:1), in overturning mixing 15min on ice, during which intermittent oscillation.
(6) 12000rpm centrifuges 10min at room temperature.
(7) it shifts in supernatant to new 2ml sterile centrifugation tubes, repeats step (5), (6), fully to remove deproteinized.
(8) shift supernatant to new 1.5ml sterile centrifugation tubes in, be added 0.6 times of volume isopropanol (300 μ l) and
The 50 μ l of 5M NaAC solution that the pH of 0.1 times of volume is 5.2,17min is stood after being sufficiently mixed mixing on ice.
(9) there are white flock DNA precipitations, 4 DEG C, 10000rpm centrifuges 10min.
(10) supernatant is abandoned, 70% ethyl alcohol that precooling is added rinses 2 times, is then rinsed 1 time with absolute ethyl alcohol, and room temperature is dried,
1 × TE buffer solutions (or distilled water) of the 100 μ l containing 2 μ l 10mg/ml RNase enzymes is added to dissolve overnight, 10 times of dilutions are spare.
(11) with the quality and concentration of the Wheat volatiles DNA of 1% agarose Detection and Extraction.
The preparation method of wheat cDNA is as follows：
1, the extraction that wheat leaf blade total serum IgE is carried out using TaKaRa companies kit (Code NO.9769), by kit
Operating instruction carries out.
2, the conjunction of the first chains of cDNA is carried out using the reverse transcription reaction kit of TaKaRa companies (Code NO.6210A)
At.Method is as follows：
(1) in Microtube reaction mixture is prepared by table 2.
2 reaction mixture of table
It is rapid cooling on ice after (2) 65 DEG C of heat preservation 5min.
(3) in above-mentioned Microtube pipes inverse transcription reaction liquid is prepared by table 3.
3 inverse transcription reaction liquid of table
(4) slow mixing.
(5) following conditions are pressed and carries out reverse transcription reaction：30 DEG C of 10min, 42 DEG C~50 DEG C 30~60min；95 DEG C of 5min, so
Afterwards in cooled on ice to get wheat cDNA.
* the exploitation of 2 wheat TaBAS1-2B gene functions of embodiment label
According to the gene TaBAS1-2B sequences for cloning acquisition in embodiment 1, in ' capital 411 ' and ' 21 ' two product of red awns spring
Inter-species develops the labels of the Indel with polymorphism, a pair of for expanding using 5.0 Software for Design of Primer Premier
The primer of Indel label, and analyze its effect to phenotype in 194 parts of wheat nature kinds, is finally developed and institute
State what gene TaBAS1-2B was isolated, and the functional label being closely related again with wheat leaf blade chlorophyll content and grain.
It is for expanding the primer pair that the Indel is marked：
Sense primer F：5’-CGCAGTGCCTGTCGTTTC-3’(Seq ID No.5)
Downstream primer R：5’-TCACATACTTCTTCCCAAT-3’(Seq ID No.6)
The primer pair amplifies weigh relevant Indel marker characteristics with wheat leaf blade compared with high chlorophyll content and higher grain
Band is 494bp, and nucleotide sequence is as shown in Seq ID No.3；The primer pair amplifies it is green with the relatively low leaf of wheat leaf blade
Cellulose content and the relevant Indel marker characteristics band of relatively low grain weight are 493bp, and nucleotide sequence is as shown in Seq ID No.4.
3 wheat TaBAS1-2B genes different genotype of embodiment and the correlation for spending rear boot leaf chlorophyll content and grain weight
1, wheat spends the measurement of rear different times chlorophyll content and seed mass of 1000 kernel
Chlorophyll measuring：Are marked to 194 parts of wheat nature kinds, and respectively 10,15,25,28 and 31 after spending florescence
It measures boot leaf chlorophyll content.Each kind chooses 3 more consistent Main Shoot Flag Leavess of flowering period, and each boot leaf uniformly selects
The site of upper, middle and lower 3 is taken, chlorophyll content is measured with SPAD-502 chlorophyll meters, is finally averaged as each family of measurement
The chlorophyll levels of boot leaf.
Mass of 1000 kernel measures：1000 complete wheat seeds are taken at random, repeats, is weighed with one thousandth balance, twice twice
Average value is denoted as final mass of 1000 kernel value.
2, the extraction of wheat leaf blade genomic DNA is the same as embodiment 1.
3, the amplification of target product.The system and program of pcr amplification reaction are the same as embodiment 1.
After PCR amplification, add denaturation Dual-indicator (DNA loading buffer) 95 DEG C of denaturation in PCR instrument
10min is placed on ice immediately after, waits being cooled to room temperature, the electrophoresis on 8% denaturing polyacrylamide gel, and then silver staining is aobvious
Color.Wheat breed with relatively low mass of 1000 kernel amplifies the target stripe of 493bp sizes, and the kind with higher mass of 1000 kernel expands
Increase the target stripe (Fig. 2) for 494bp sizes.
4, the positioning of TaBAS1-2B genes and its Indel labels on chromosome
Gene TaBAS1-2B is positioned using four body material of wheat ' China spring ' nullisomic, as a result the gene and its
Indel labels are located on wheat 2B chromosomes (Fig. 3).
5, the genotyping of different mass of 1000 kernel wheat breeds and to spend the related of rear boot leaf chlorophyll content and mass of 1000 kernel
Property
Variance is carried out to boot leaf chlorophyll content after the spending of two kinds of genotype and mass of 1000 kernel in 194 parts of wheat nature kinds
Analysis, while correlation is carried out between rear boot leaf chlorophyll content and mass of 1000 kernel with spending to the allelic variation of TaBAS1-2B genes
Analysis, the results are shown in Table 4.
In 4 Wheat Cultivars of table rear boot leaf chlorophyll content and the mass of 1000 kernel significance of difference point are spent between two kinds of genotype
Analysis
Note：The horizontal P values 0.05,0.01 and 0.001 of the significance of difference, respectively by *, * * and * * * marks；A genotype is
494bp banding patterns, 1 B gene type are 493bp banding patterns.
Table 4 the result shows that, the mass of 1000 kernel of A genotype (494bp) is significantly higher than the mass of 1000 kernel of 1 B gene type (493bp).The base
Mass of 1000 kernel of the equipotential type of cause in 194 parts of materials has reached the pole level of signifiance.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (5)
1. the Indel for spending rear Flag Leaf Blade chlorophyll content and mass of 1000 kernel related gene TaBAS1-2B to isolate with wheat is marked,
It is characterized in that, the primer pair for expanding the Indel labels is：
Sense primer F：5’-CGCAGTGCCTGTCGTTTC-3’
Downstream primer R：5’-TCACATACTTCTTCCCAAT-3’
The primer pair amplifies spend rear Flag Leaf Blade to be marked compared with high chlorophyll content and the relevant Indel of higher mass of 1000 kernel with wheat
Note characteristic bands are 494bp, and nucleotide sequence is as shown in Seq ID No.3；The primer pair amplifies spend rear flag with wheat
The relatively low chlorophyll content of leaf blade and the relevant Indel marker characteristics band of relatively low mass of 1000 kernel are 493bp, and nucleotide sequence is such as
Shown in Seq ID No.4.
2. the boot leaf after screening is spent of Indel labels described in claim 1 has small compared with high chlorophyll content and higher mass of 1000 kernel
Application in wheat germ plasm resource, which is characterized in that include the following steps：
1）Extract the genomic DNA of plant to be measured；
2）Using the genomic DNA of plant to be measured as template, using primers F described in claim 1 and R, pcr amplification reaction is carried out；
3）Detect pcr amplification product, if it is possible to amplify the characteristic bands of the nucleotide sequence as shown in Seq ID No.3, then
Plant to be measured is the wheat resource for spending rear Flag Leaf Blade to have compared with high chlorophyll content and higher mass of 1000 kernel.
3. application according to claim 2, which is characterized in that step 2）The amplification system that middle PCR reactions use is with 10 μ l
It is calculated as：Template DNA 100ng, 10 μM of primers Fs and each 0.8 μ l, 5U/ μ l Taq archaeal dna polymerases of 0.2 μ l, 2.5mM dNTP of R
0.12 μ l, Mg containing 25mM2+10 × PCR reaction buffers 1.0 μ l, ddH2O complements to 10 μ l.
4. application according to claim 2, which is characterized in that step 2）Middle PCR reacts the amplification program used：94℃
5 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 3 minutes, 36 cycle；72 DEG C 10 minutes.
5. application according to claim 2, which is characterized in that step 3）It is middle to use 6% denaturing polyacrylamide gel electrophoresis
Pcr amplification product is detected, then silver staining develops the color.
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