CN104004759A - Molecular marker of maize photoperiod sensibility candidate gene zmCCA1 and application thereof - Google Patents
Molecular marker of maize photoperiod sensibility candidate gene zmCCA1 and application thereof Download PDFInfo
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- CN104004759A CN104004759A CN201410254639.2A CN201410254639A CN104004759A CN 104004759 A CN104004759 A CN 104004759A CN 201410254639 A CN201410254639 A CN 201410254639A CN 104004759 A CN104004759 A CN 104004759A
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Abstract
The invention relates to a molecular marker of a maize photoperiod sensibility candidate gene zmCCA1 and an application thereof, and provides a molecular marker SSRA489 in coseparation with the maize photoperiod sensibility candidate gene zmCCA1 and a primer pair capable of amplifying the molecular marker SSRA489. A method for regulating and controlling the molecular marker SSRA489 in the coseparation with the maize photoperiod sensibility candidate gene zmCCA1 comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification by using the primer by taking maize total DNA as a template; detecting an amplified product through denatured 6% polyacrylamide gel electrophoresis; and comparing the size of an objective strip with the size of the molecular marker SSRA489 so as to judge maize photoperiod sensibility according to whether the molecular marker exists or not. The molecular marker SSRA489 in the coseparation with the maize photoperiod sensibility candidate gene zmCCA1 can be used for carrying out genetic structure analysis, genetic map construction, gene positioning, near-isogenic line construction and molecular marker auxiliary selective breeding on the maize photoperiod sensibility.
Description
Technical field
The present invention relates to a kind of molecule marker, specifically a kind of corn photoperiod sensitivity candidate gene zmCCA1 molecule marker and application thereof.
Background technology
Bloom be higher plant shoot apical meristem from nourishing and growing to the transition process of reproductive growth, be the key link of plant in life cycle, in the suitable time, blooming is an important adaptability proterties for plant.The flowering time of plant is subject to the joint effect of environmental factor (as the duration of day, light quality and temperature) and internal factor (as development condition and age).The Photoperiod pathway of Arabidopis thaliana (photoperiod pathway), the approach most important in approach, that research is the most detailed that is that Arabidopis thaliana is bloomed.
Blooming in Photoperiod pathway of Arabidopis thaliana, optical signals Photoreceptors (cryptochrome and phytochrome) perception, and transmitted to biological clock by genes such as ELF3, ELF4, PIF3 and ZTL, FKF and LKP2, make Central oscillator produce rhythm and pace of moving things vibration, by the vibration going down diel rhythm variable signal of Central oscillator, further activate or suppress the expression of floral meristem gene and floral organ gene, thereby control the process of blooming (Sangho etc., 2005 of Arabidopis thaliana; Putterill etc., 2001).
Central oscillator is the core of biological clock; little group composition of CCA1 (CIRCADIAN CLOCK ASSOCIATED1), LHY (the pseudo-response regulator APRRs(Arabidopsis of (LATE ELONGATED HYPOCOTYL) and class Arabidopis thaliana pseudo-response regulators) by two codings transcription factor relevant to Myb; be responsible for the whole regulation and control of biorhythm (Schaffer et al., 1998; Makino et al., 2002; Mizoguchi et al., 2002; Mas et al., 2003).
Research shows, the physiological clock of Arabidopis thaliana is comprised of three interactional biofeedback rings, influences each other and jointly maintained the stable of biological clock rhythm between them.
First biofeedback ring is replied basis member TIMIN G OF CAB EXPRESSION 1 (TOC1) of regulatory factor family by puppet and is formed with CCA1 and L H Y, is called as core feedback loop, is also to study the most thorough feedback loop.TOC1 belongs to APRRs gene family, contain a receiver-1ike structure, and C-end has CCT structural domain at N end, and its major function is be responsible for the interaction between different albumen and appraise and decide position, and TOC1 mainly expresses at night; CCA1 and the LHY transcription factor that contains MYB functional domain of encoding respectively, the aminoacid sequence of the two coding has higher similarity, and gene is similar with protein expression level, all shows the rhythm and pace of moving things of concussion round the clock, mainly in morning, expresses.The diel rhythm of LHY/CCA1 and TOC1 is just in time completely contrary, and TOC1 albumen can activate the expression of CCA1 and LHY at dead of night, and the expression level of CCA1 and LHY is peaked at cockcrow.Due to assembly at night (the evening element, the AAATATCT) combination of LHY and CCA1 albumen energy and TOC1 gene promoter region, thus the expression of inhibition TOC1 gene, so the expression amount of TOC1 reduces by day gradually; Because TOC1 albumen is the necessary promotion factor of LHY and CCA1 genetic expression, the expression amount of LHY and CCA1 gene is lowered along with the minimizing of TOC1 albumen, restraining effect to TOC1 gene reduces gradually, night, LHY and CCA1 protein content were reduced to a minimum, thereby can remove the restraining effect to TOC1 genetic expression.The expression amount of TOC1 rises gradually and peaks, and TOC1 protein content raises gradually, again starts the expression of LHY and CCA1 gene, and the expression amount of CCA1 and LHY restarts to rise, and at dawn, again peaks and starts the circulation of a new round.In this negative feedback loop, three genes have formed the core negative feedback loop of a complicated stable physiological clock jointly, negative regulatory factor suppresses the transcriptional activity of positive regulating factor, and positive regulating factor activates again the transcriptional expression of negative regulatory factor conversely, thereby enters next loop cycle.Based on this feedback regulation mechanism of transcribing with translation skill, the synchronized oscillation that Arabidopis thaliana biological clock is daily cycle property under Photoreceptors regulates changes (Strayer et al, 2000) just.
Second feedback loop is called as " evening loop ", in this feedback loop, GI (GIGAN TEA), ELF3(EARLY FLOWERING 3), EL F4 (EARLY FLOWERING 4) and LUX(LUX ARRHYTHMO) etc. gene, to participate in activating transcribing of CCA1 and LHY with the similar feedback regulation mechanism of TOC1, conversely, a large amount of accumulation along with CCA1 and L H Y albumen, the expression of these genes is suppressed again (Hazen, et al, 2005; Doyle et al, 2002).The myb transcription factor of LUX coding under any illumination and the background of suddenling change all with TOC1 to transcribe the rhythm and pace of moving things identical, its promotor comprises an EE element combining with CCA1/ LHY.CCA1 can directly be attached to the promoter region of ELF4, thereby the expression of ELF4 suppresses by LHY/CCA1 also, equally it to the output rhythm and pace of moving things maintain and the photoinduction of LHY and CCA1 to express be also essential (Putterill et al, 1995; Alabadi et al, 2001).
The 3rd biofeedback ring is called as " morning loop ", and three homologue PRR5, PRR7 and PRR9 by TOC1, become with L H Y shape with CCA1.PRR family protein has the similar pseudo-receiver functional area (PR functional domain) that is called at N end, C end is and the similar CCT functional structure district of CO gene of blooming, downstream, this functional domain is positioned in nucleus, and centre is one section of different intermediate sequence.These genes may play dual role, in physiological clock mechanism, they had had both regulated and controled to physiological clock communicating optical signals, again the CCA1 of Central oscillator and LHY are played to restraining effect, conversely, CCA1 is by their expression level (Wang et al, 1998) of CBS (CCAl mono-binding site) combination regulation and control of direct and PRR7 and PRR9 gene promoter region.PRR7 and PRR9 are activated by CCA1 and LHY in the morning, and contrary PRR5, PRR7 and PRR9 suppress the expression (Mizuno, et al, 2005) of CCA1 and LHY conversely.CCA1 gene is the core of biological clock, in Flowering In Higher Plant control approach, plays an important role.
Summary of the invention
The present invention seeks to the deficiency for solving the problems of the technologies described above, a kind of corn photoperiod sensitivity candidate gene zmCCA1 molecule marker and application thereof are provided, can be used for corn photoperiod sensitivity gene Fine Mapping, detection, can be used for corn molecular breeding.
The invention provides a kind of and corn photoperiod sensitivity candidate gene zmCCA1 be divided into from molecule marker SSRA489; , the nucleotides sequence of this molecule marker is classified SEQ ID NO.1. as.
The corn photoperiod sensitivity candidate gene zmCCA1 that can increase be divided into from the primer pair of molecule marker SSRA489 be:
SSRA489F：5＇- GGGTTGGGTAGGTATGTGA -3＇；
SSRA489R：5＇- CCCCTTTCCTTCTCTCTTC -3＇；
Described regulation and control corn photoperiod sensitivity candidate gene zmCCA1 be divided into from the method for molecule marker be: take total corn DNA as template, with described primer, carry out pcr amplification, amplified production detects through 6% polyacrylamide gel electrophoresis of sex change, the size of object band and molecule marker are contrasted, thereby have or not judgement corn photoperiod sensitivity according to molecule marker.
The present invention and corn photoperiod sensitivity candidate gene zmCCA1 be divided into from the purposes of molecule marker, can be used for corn photoperiod sensitivity to carry out genetic construction analysis, genetic map construction, the assignment of genes gene mapping, structure near isogenic line, molecular marker assisted selection breeding.
Beneficial effect is:
1, the invention discloses a kind of corn photoperiod sensitivity candidate gene zmCCA1 molecule marker; Utilize primer pair of the present invention, the corn gene group DNA of take carries out pcr amplification as template, can obtain and the closely linked molecule marker of corn photoperiod sensitivity gene, and this mark is called after SSRA489 in the present invention.Analysis shows that having or not of this mark be directly connected to the size of corn photoperiod sensitivity, can be used in the prediction of the photoperiod sensitivity of different corn germplasms.This molecule marker structure is simple and clear, easy to use, and utilizes PCR technology for detection rapidly and efficiently, can accurately identify at short notice the parental maize self-mating system of different sources, and effect is fast obvious, has good promotion prospect.It will be understood by those skilled in the art that except obtaining molecule marker of the present invention by above-mentioned pcr amplification method, can also obtain molecule marker of the present invention by chemosynthesis.Corn breeding and resource identify or to the early prediction of different corn material photoperiod sensitivities and screening in, this molecule marker can overcome environmental influence, to the morning of planting material for screening, eliminate to the photoperiod have susceptibility be not suitable with temperate zone environment plant, improve breeding and efficiency of selection.Be applied in the evaluation of corn photoperiod sensitivity, can predict the photoperiodic reaction of different germplasm materials.
Therefore,, according to these features of corn photoperiod candidate gene sequence, these candidate genes undetermined can be developed as to the functional gene molecule marker that depends on PCR.Therefore, when utilizing tropical germplasm to carry out germplasm improvement or inbred line breeding or during the corn hybrid seed commercialization production of hybrid seeds, can utilize these marks to carry out assisted selection and instruct produce actual.This solves the narrow problem in germ plasm resource basis in China's current corn growth for making full use of tropical and subtropical maize germplasm, solve the commercialization production of hybrid seeds region limitation of corn hybrid seed, cost-saving, improve the commodity of cross-fertilize seed, there is important theory and realistic meaning.
Accompanying drawing explanation
Fig. 1 is the electrophoresis banding pattern figure of this corn photoperiod sensitivity candidate gene zmCCA1 molecule marker SSRA48.
Embodiment
The invention provides with corn photoperiod sensitivity candidate gene zmCCA1 be divided into from molecule marker SSRA489, the nucleotides sequence of this molecule marker is classified SEQ ID NO.1. as.
The corn photoperiod sensitivity candidate gene zmCCA1 that can increase be divided into from the primer pair of molecule marker SSRA489 be:
SSRA489F：5＇- GGGTTGGGTAGGTATGTGA -3＇
SSRA489R：5＇- CCCCTTTCCTTCTCTCTTC -3＇；
Described regulation and control corn photoperiod sensitivity candidate gene zmCCA1 be divided into from the method for molecule marker be: take total corn DNA as template, with described primer, carry out pcr amplification, amplified production detects through 6% polyacrylamide gel electrophoresis of sex change, the size of object band and molecule marker are contrasted, thereby have or not judgement corn photoperiod sensitivity according to molecule marker.
Below in conjunction with the concrete example of implementing, further set forth the present invention.Should be understood that these examples are only not used in and limit the scope of the invention for the present invention is described.
embodiment 1,the establishment of corn photoperiod sensitivity major gene CCA1 near isogenic line
Test basic population used and derive from a warm cross combination: yellow morning 4 and CML288, be China Temperate corn inbred line yellow morning 4, insensitive to photoperiodic reaction; CML288 is from the international corn wheat improvement of Mexico center, is selected from the tropical Flint corn self-mating system of Pop24, has showed very strong photoperiod sensitivity in temperate zone.It is donor parents that the present invention be take to the tropical self-mating system CML288 of photoperiod-sensitive, take yellow to the temperate zone self-mating system of photoperiod insensitiveness 4 is early recurrent parent, by back cross breeding program, the method that adopts Phenotypic Selection to combine with molecular marker assisted selection, by hybridizing, backcrossing or selfing, seed selection only contains the near isogenic line of corn photoperiod sensitivity major gene loci.Particularly, first, under the short day environment of Hainan, hybridize and obtain F1, and early 4 backcross with yellow at Second Year F1; Secondly, under day Henan long day environment, backcross population is carried out to Phenotypic Selection, selects the loose powder phase and the phase individual plant in evening extremely of weaving silk, get separately pollen, with yellow early 4 hybridization of pollinate of 4-5 strain, while selfing, and list indicated selfing individual plant, the individual plant that backcrosses originates.Then, under the short day environment of Hainan, with the mark of target site both sides, first carry out foreground selection, with being uniformly distributed in the good SSR mark of 232 polymorphisms on 10 karyomit(e)s of corn, the individual plant that foreground selection is screened carries out genetic background selection.In the ensuing generation, new molecule marker in continual exploitation target area, selects exchange individual plant, finally utilizes mark SSRA489 provided by the present invention, only filters out the near isogenic line individual plant containing corn photoperiod sensitivity major gene loci.
embodiment 2,the application of molecule marker SSR A489 in corn molecular breeding
With the primer of molecule marker SSR A489 of the present invention, to corn individual plant, DNA carries out pcr amplification, and amplified production is carried out to Markers for Detection, molecule marker detected and shows that photoperiod sensitivity is stronger, a little less than not detecting and showing that photoperiod sensitivity.Further, can be by the pcr amplification product of temperate zone parental maize, tropical parental maize and warm heterozygosis F1 generation individual plant DNA in contrast; Wherein tropical parental maize genomic dna is determined and to be contained corn photoperiod sensitivity gene; Temperate zone parental maize genomic dna does not contain corn photoperiod sensitivity gene; If the genotype of the corn individual plant pcr amplification product of surveying is consistent with temperate zone parent's banding pattern, this individual plant adapts to temperate climate condition, can be for further corn research; If the genotype of the corn individual plant pcr amplification product of surveying is consistent with tropical parent's banding pattern, this individual plant is responsive to the weather condition phenotype in temperate zone, show this individual plant can not be under the condition of temperate zone normal growth grow, so this individual plant do not have further utility value, should eliminate; If the genotype of the corn individual plant pcr amplification product of surveying is consistent with the banding pattern of warm heterozygosis F1 generation individual plant, show that this individual plant is heterozygosis individual plant, continue can select insensitive offspring of photoperiod after selfing, this class individual plant also has further utility value, should continue to employ.
Concrete steps are as follows:
(1) extract the genomic dna of corn individual plant to be measured; Extract temperate zone parental maize genomic dna, tropical parental maize genomic dna and warm heterozygosis corn gene group DNA simultaneously;
(2) take respectively genomic dna, temperate zone parental maize genomic dna, tropical parental maize genomic dna and the warm heterozygosis corn gene group DNA of corn individual plant to be measured carries out PCR amplification with the primer of molecule marker SSR A489 as template;
(3) with 6% polyacrylamide gel electrophoresis of sex change, detect PCR amplified production;
(4) statistics banding pattern result; Analyze corn individual plant DNA cloning product genotype to be measured and temperate zone parent, tropical parent or identical with warm heterozygosis corn.
embodiment 3,the identification of polymorphisms of molecule marker SSR A489 between different parents
Extract yellow early 4, CML288, CML310, CML10, B73, Mo17, W22,78599, prosperous seven, Zheng 58, tuck in 478, from 330, the genomic dna of the self-mating system of 86 different sourcess such as red 340 grades, use the above-mentioned self-mating system of primer pair of molecule marker SSR A489 in the present invention to carry out pcr amplification, polyacrylamide gel electrophoresis result shows, molecule marker SSR A489 has polymorphism in the self-mating system of different sources.This result proves, molecule marker SSR A489 can accurately detect the existence of CCA1 gene in any breeding background population.
SEQUENCE LISTING
<110> University Of Science and Technology Of He'nan
<120> corn photoperiod sensitivity candidate gene zmCCA1 molecule mark
Note and application thereof
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 306
<212> DNA
<213> corn
<400> 1
cgcatgtttt tgcctgtcat gttgttggtt gtagttaagt
ccattattgt tactctttgg 60
agtctgtgct gttggggttg ggtaggtatg tgagtctgtt
gttgcctcca cagatctcca 120
ataaatcaca caagatgaga gctgttgctg gtgtgtccta
cctcgtcatt ctttgacggt 180
tgctattcag gtagcccttg caataattaa attaagacag
gggaagagag aaggaaaggg 240
gaacaatgag gaaatgctca tatggctctg gaattcaata
aattagtcat ataatgatat 300
tggtcc
306
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
gggttgggta ggtatgtga
19
<210> 3
<211> 19
<212> DNA
<213> artificial sequence
<400> 3
cccctttcct tctctcttc
19
Claims (4)
1. a corn photoperiod sensitivity candidate gene zmCCA1 molecule marker, is characterized in that: the nucleotides sequence of this molecule marker is classified SEQ ID NO.1. as.
2. corn photoperiod sensitivity candidate gene zmCCA1 molecule marker as claimed in claim 1, is characterized in that: the Nucleotide row of its primer are as follows
SSRA489F：5＇- GGGTTGGGTAGGTATGTGA -3＇；
SSRA489R：5＇- CCCCTTTCCTTCTCTCTTC -3＇。
3. utilize the method that corn photoperiod sensitivity candidate gene zmCCA1 molecule marker carries out the detection of corn photoperiod sensitivity as claimed in claim 2, it is characterized in that: take total corn DNA as template, with described primer, carry out pcr amplification, amplified production detects through 6% polyacrylamide gel electrophoresis of sex change, the size of object band and molecule marker are contrasted, thereby have or not judgement corn photoperiod sensitivity according to molecule marker.
4. corn photoperiod sensitivity candidate gene zmCCA1 molecule marker as claimed in claim 1 is in the early prediction of corn photoperiod sensitivity and the application in screening.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190093A (en) * | 2017-07-24 | 2017-09-22 | 吉林省农业科学院 | For screening the functional molecular marker of corn evening floral formation and its application under long-day conditions |
-
2014
- 2014-06-10 CN CN201410254639.2A patent/CN104004759A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| XINTAO WANG等: "Robust expression and association of ZmCCA1 with circadian rhythms in maize", 《PLANT CELL REP》 * |
| 孙朝辉等: "温带和热带玉米重组自交系群体的株高QTL分析", 《河南农业大学学报》 * |
| 库丽霞等: "玉米光周期敏感相关性状发育动态QTL定位", 《作物学报》 * |
| 张伟强等: "基于温、热带玉米自交系永久F2叶片数QTL分析", 《河南农业大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190093A (en) * | 2017-07-24 | 2017-09-22 | 吉林省农业科学院 | For screening the functional molecular marker of corn evening floral formation and its application under long-day conditions |
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Application publication date: 20140827 |