CN105695459A - Molecular marker of maize transcription factor ZmPIF3 transgenic rice and application of molecular marker - Google Patents
Molecular marker of maize transcription factor ZmPIF3 transgenic rice and application of molecular marker Download PDFInfo
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Abstract
The invention discloses a molecular marker of maize transcription factor ZmPIF3 transgenic rice and application of the molecular marker and belongs to the field of crop genetic breeding. A maize transcription factor ZmPIF3 transgenic rice material which is obtained already has obvious water-saving drought-enduring phenotype. According to a nucleotide sequence of maize transcription factor ZmPIF3 gene, a primer of the molecular marker is designed in CDS, PCR technology is applied to acquire DNA segments of ZmPIF3 and sequence the DNA segments, and the primer of the molecular marker is obtained according to specific segment screening of ZmPIF3. The molecular marker is high in repeatability, stable in amplification, simple and convenient to operate and low in cost and can be used for detecting maize ZmPIF3 genes in rice background, and a solid foundation is laid for molecular marker assisted selection in the future by utilizing ZmPIF3 gene transgenic rice.
Description
Technical field
The present invention relates to the molecular marker of a kind of Maize Transcription Factor ZmPIF3 transgenic paddy rice and application thereof, belong to field of crop genetic breeding。
Background technology
1, Semen Maydis ZmPIF3 transcription factor brief introduction
Whole world arid, semiarid zone account for the half of cultivated area, and these area water supplies are not enough, forest cover dredges rare, the deterioration of the ecological environment, severe water and soil erosion, frequent natural calamity。Even if when soil moisture is sufficient, water deficit also usually can occur, thus having influence on the physiological process such as photosynthesis, matter transportation, protein synthesis and cell elongation。Arid can cause plant dehydration in various degree, causes a series of physiological metabolism change in plant, so the abiotic stress such as arid, high salt is called again water stress or osmotic stress。
The process of plant response osmotic stress is a complex process relating to polygenes, multi signal approach, polygenes product, generally these genes and expression product thereof can be divided into two classes: i.e. functional protein and adjustment albumen。Functional protein refers to the protein directly worked in opposing osmotic stress；And regulating albumen is participate in various signal transduction or controlling gene expression in adverse circumstance, the albumen indirectly shielded, it specifically includes that the transcription factor etc. that transmission signal and controlling gene are expressed。
Plant can induce synthesis compound degeneration-resistant in a large number and protein, these compounds relevant to degeneration-resistant border and protein again by transcription factor regulation and control on transcriptional level under environment stress。Transcription factor by being combined with cis acting element, can start transcribing of degeneration-resistant correlation function gene, makes plant make the metabolism adjustment adapting to environment stress by the expression of degeneration-resistant functional gene。In recent years, in the molecular breeding improving crop anti-adversity, research emphasis forwards improvement to from improvement Individual genes or strengthens the transcription factor of one or more performance pivotal role gradually, multiple functional gene so can be promoted to play a role, to obtaining the plant of comprehensive degeneration-resistant character improvement。
Phytochrome acting factor (phytochrome-interactingfactors, PIFs), also referred to as PILs (phytochromeinteractingfactor-like), belongs to the 15th subtribe of arabidopsis bHLH transcription factor family。Can there is specific effect with CANNTG sequence (also known as E-box) in plant PIFs family transcription factor, regulate in promoter the expression of the functional gene containing E-box element or controlling gene, thus the various resistance reaction of involved in plant。Maize Transcription Factor ZmPIF3 transgenic paddy rice is obtained, by the research to its offspring's population physiology biochemical function, it has been found that the Transgenic Rice Plants of ZmPIF3 gene has obvious drought resisting phenotype currently with agriculture bacillus mediated transgenic technology；But there is presently no the molecular marker following the trail of Semen Maydis ZmPIF3 gene, and it is very necessary to the later use of ZmPIF3 transgenic line to develop the effective specific molecular marker of ZmPIF3 gene。
2, the development of molecular marking technique
2.1 based on the molecular marker of molecular hybridization
Restriction fragment length polymorphism (RestrictionFragmentLengthPolymorphism, RFLP) Grodzicker in 1974 etc. have founded RFLP technology, genetic marker (Grodzickeretal as adenovirus temperature sensitive mutation, 1974), it is a kind of first generation genetic marker based on DNA-DNA is hybridized。Utilize the genomic DNA that the cutting of specific restricted enzyme is different, obtain the DNA fragmentation differed in size, isolate different band by gel electrophoresis, then carry out Southern hybridization and radiography with cloned DNA probe, namely obtain the RFLP collection of illustrative plates of reflection individual specificity。
The molecular marker of 2.2 PCR-based technology
2.2.1 randomly amplified polymorphic DNA (RandomAmplifiedPolymorphismDNA, RAPD)
Nineteen ninety is utilized the method (Williamsetal, 1990) of detection DNA polymorphism that round pcr develops by Williams et al.。It utilizes random primer (being generally 8～10bp) to react non-fixed point amplification of DNA fragments by PCR, then by the polymorphism of gel electrophoresis analysis amplified production DNA fragmentation。Amplified fragments polymorphism just reflects the DNA polymorphism of genome respective regions。But RAPD is affected relatively big by reaction condition, thus the repeatability of its detection is poor。
2.2.2 sequence tagged site (SequenceTaggedSites, STS)
Olson in 1989 etc. propose, and are to the general designation with the specific molecule labelling that primer sequence carries out PCR specific amplified。By designing specific primer so that it is particular combination site is combined in genomic dna sequence, thus can be used to specific region in amplification gene group, analyze its polymorphism。
2.2.3 simple repeated sequence (SimpleSequenceRepeat, SSR)
Also referred to as microsatellite DNA (Tautzetal, 1989；Loveetal, 1990), be a class by motif (motif) tandem repetitive sequence of 1～6 base composition, wherein the most common is that dinucleotide repeats and Trinucleotide repeats, such as (CA)n、(AT)n、(TG)n、(GGC)n、(GAT)nDeng。The core sequence structure of each microsatellite DNA is identical, and recurring unit's number is generally 10～60, and they are distributed widely in most eukaryotic gene group, and its height polymorphism is mainly derived from the difference of tandem sequence repeats number。Microsatellite sequence both sides are usually relatively conservative single-copy sequence, primer is designed according to conserved sequence, micro-satellite fragment is expanded by PCR reaction, owing to core sequence tandem sequence repeats number is different, it is thus able to amplify the PCR primer of different length, amplified production is carried out gel electrophoresis, determines polymorphism according to the size of isolated fragment。What SSR marker generally detected is single multiple alleles site, and in codominant inheritance, therefore heterozygote and homozygote can be differentiated。But, traditional SSR molecular marker development approach workload is big, need to spend a large amount of human and material resources, and inefficient。
2.2.4 district (inter-simplesequencerepeat, ISSR) between simple repeated sequence
A kind of molecular marker based on micro-satellite that Zietkeiwitcz proposed equal to 1994, its detection is the polymorphism in one section of short dna sequence between two SSR。Owing to SSR sequence is distributed widely in the genome of higher organism, so ISSR has very strong polymorphism。At present, ISSR labelling is widely used in the researchs such as various plants cultivar identification, genetic mapping, gene mapping, genetic diversity, but does not study in E. elongata.
2.2.5 amplification polymorphism (inter-retrotransposonamplifiedpolymorphism, IRAP) between retrotransposon site
Its principle is according to the conserved sequence design primer of LTR in retrotransposon, these primers in PCR process can with LTR sequence anneals, thus the fragment amplified between the retrotransposon member of adjacent same family。Primer can also be designed according to the relative conserved sequence of reverse transcriptase gene to carry out expanding (Kalendaretal.1999)。At present, IRAP molecular marking technique be applied to Fructus Hordei Vulgaris (Kalendaretal, 1999；Kalendaretal, 2000；Leighetal, 2003), in the genetic research of the plant such as Citrus (Bret ó etal, 2001), net thatch (Yannicetal, 2004), Rhizoma Solani tuber osi。2.2.6 the micro-satellite amplification polymorphism (retrotransposonmicrosatelliteamplifiedpolymorphism, REMAP) of retrotransposon
A kind of labelling technique (Kalendaretal based on retrotransposon, 1999), REMAP know-why is the LTR conserved sequence according to retrotransposon and microsatellite sequence design primer, then PCR is carried out, amplify retrotransposon and contiguous micro-intersatellite fragment, thus the polymorphism detected between retrotransposon and contiguous simple repeated sequence。
2.3 based on the DNA marker of restriction enzyme digestion and round pcr
Amplified fragment length polymorphism (AmplifiedFragmentLengthPolymorphism, AFLP) AFLP is Dutch scientist Zbaeau and Vos in 1993 a kind of new method (Zbaeauetal, 1993) detecting DNA polymorphism grown up。AFLP is RFLP and the PCR product combined, first with restricted enzyme, genomic DNA cut into different size of DNA fragmentation, double-strand manual splice is made to connect with endonuclease bamhi phase limit again, template DNA as amplified reaction, then expand in advance with the complementary strand of manual splice for primer, finally on the basis of joint complementary strand, 1～3 selective kernel thuja acid of interpolation is made primer pair template DNA gene and is carried out selective amplification again, by the DNA cloning fragment that polyacrylamide gel electrophoresis separation detection obtains, detect polymorphism according to the difference of expanding fragment length。Along with constantly improving and development of technology, AFLP technology is widely used to plant germplasm and identifies (Thomasetal, 1995), genetic map construction (Marguesetal, 1998), the aspect such as genes of interest location and genetic polymorphism detection (Pakniyatetal, 1997)。
2.4 based on the molecular marker of DNA chip technology
2.4.1 single nucleotide polymorphism (SingleNucleotidePolymorphism, SNP)
The third generation DNA genetic marker that Lander proposed in 1996, refers to the DNA sequence polymorphism caused by single nucleotide diversity in genomic level, and its frequency of polymorphism is more than 1%。From molecular level, the difference of single core thuja acid is detected。
2.4.2 multiformity microarray technology (Diversityarraystechnology, DArT)
Jaccoud develops a kind of new molecular marking technique multiformity microarray technology (Diversityarraystechnology, DArT) equal to calendar year 2001, and is successfully applied in rice research。DArT technology is that the method relying on chip hybridization is to distinguish the difference of seat polymorphism in genome。By relevant for warp after the genomic DNA mixed in equal amounts of different samples to be detected restriction enzyme ferment treatment, select to reclaim different size DNA fragmentation and a series of DNA operation according to electrophoresis result and reach genome complexity and reduce, this part DNA is genomic representation (Genomicrepresentation), and is fixed on slide by correlated process by this part DNA to be formed the chip of lattice array。Individually the genomic representation obtained is processed for probe through same restriction endonuclease with different samples, and form corresponding probe combinations chip is hybridized, owing to the genomic dna sequence of different samples is variant, thus it is inconsistent with the efficiency of same point sequence hybridization on chip, point only complementary with DNA probe on chip just has hybridization signal, can distinguish the power of different colours hybridization signal or with or without the genetic difference determining sample to be detected by scanner。
Summary of the invention
For the shortcoming existed in prior art with not enough, the invention provides a kind of Maize Transcription Factor ZmPIF3 transgenic paddy rice molecular marker and application thereof；The transgenic paddy rice material of acquired Maize Transcription Factor ZmPIF3 gene has drought-enduring phenotype of significantly economizing on water；Nucleotide sequence according to Maize Transcription Factor ZmPIF3 gene, the primer of molecular marker of the present invention is designed in the CDS of protein coding region, round pcr is used to obtain the DNA fragmentation of Maize Transcription Factor ZmPIF3 and check order, obtain the primer of molecular marker of the present invention according to the specific fragment screening of Semen Maydis ZmPIF3, and obtain Maize Transcription Factor ZmPIF3 transgenic paddy rice molecular marker；This molecular marker is reproducible, it is stable, easy and simple to handle, with low cost to expand。
Technical scheme is as follows:
The molecular marker of Maize Transcription Factor ZmPIF3 transgenic paddy rice of the present invention, length is 691bp, and its extension increasing sequence is such as shown in SEQIDNo.1。
The primer of the molecular marker of Maize Transcription Factor ZmPIF3 transgenic paddy rice of the present invention, its sequence is such as shown in SEQIDNo.2 and SEQIDNo.3:
SEQIDNo.2:ZmPIF3-F5'CCGACGGCTGCTAACCT3'；
SEQIDNo.3:ZmPIF3-R5'TTCCGATTCCGAGACCCT3'。
The invention also discloses the application in detection transgenic line or molecular marker assisted selection breeding of the molecular marker of Maize Transcription Factor ZmPIF3 transgenic paddy rice。
The preparation method of the molecular marker of Maize Transcription Factor ZmPIF3 transgenic paddy rice, it is characterized in that, described method is as follows: the nucleotide sequence according to Maize Transcription Factor ZmPIF3, the primer of described molecular marker is designed in protein-coding region, round pcr is used to obtain the DNA fragmentation of Maize Transcription Factor ZmPIF3 and this DNA fragmentation is checked order, the primer of described molecular marker is obtained, thus obtaining the molecular marker of described Maize Transcription Factor ZmPIF3 transgenic paddy rice according to the specific fragment screening of Maize Transcription Factor ZmPIF3。
This research is with Semen Maydis ZmPIF3 transcription factor transgenic paddy rice DNA for experimental group, wild rice (military fortune round-grained rice 7) DNA is matched group, the differential fragment of sequence between experimental group and matched group can be distinguished by regular-PCR amplification, these fragments can be considered the specific fragment of Semen Maydis ZmPIF3 transcription factor transgenic paddy rice DNA, in conjunction with the enrichment of regular-PCR, the DNA fragmentation of Semen Maydis ZmPIF3 can be amplified efficiently。Recovered, order-checking, finally changes into Semen Maydis ZmPIF3 transcription factor transgenic paddy rice specific molecular marker。
The present invention has following beneficial effect:
1, the present invention utilizes round pcr, it is thus achieved that the molecular marker 1 that Maize Transcription Factor ZmPIF3 transgenic paddy rice is special, and success efficiency is up to 100%。
2, the molecular marker of the present invention is reproducible, amplification is stable, easy and simple to handle, with low cost, can be used to the Semen Maydis ZmPIF3 gene detecting in Oryza sativa L. background, for utilizing from now on, ZmPIF3 gene transgenic rice breeding carries out molecular marker assisted selection offer solid foundation。
Accompanying drawing explanation
Fig. 1 is Maize Transcription Factor ZmPIF3 transgenic paddy rice T2Detect for fluorescent quantitation in homozygous lines；Wherein, WT-wild type, VC-empty carrier, OE1-OE13 is ZmPIF3 transgenic paddy rice；
Fig. 2 (a) and (b) are the drought tolerance research in soil of the Maize Transcription Factor ZmPIF3 transgenic paddy rice；Wherein, WT-wild type, VC-empty carrier, OE3, OE5, OE11 are ZmPIF3 transgenic paddy rice；
Fig. 3 is the amplification in different Maize Transcription Factor ZmPIF3 transgenic paddy rice materials of the genome specific molecular marker；Wherein, swimming lane 1-9 is ZmPIF3 transgenic paddy rice T0 generation, and swimming lane 10 is negative control；
Fig. 4 is the Detection of Stability of Maize Transcription Factor ZmPIF3 transgenic paddy rice genome specific molecular marker；Wherein, M is MarkerDL2000, and swimming lane 1 is plasmid, and swimming lane 2,3 is WT, VC, and swimming lane 4-15 is 12 ZmPIF3 transgenic paddy rice T2Generation pure lines。
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is described further。
Embodiment 1: the acquisition of experiment material
Primers according to ZmPIF3 gene, utilizes RT-PCR technology amplification from the total cDNA of corn variety Zheng Dan 958 (being purchased from Yangzhou Tian Yuan Seed company) to obtain ZmPIF3 gene；Utilize Agrobacterium-mediated transformation to wild rice plant (military fortune round-grained rice 7), obtain obtaining Maize Transcription Factor ZmPIF3 transgenic paddy rice。
Embodiment 2:PCR primer sequence
Nucleotide sequence according to the transcription factor ZmPIF3 gene that Maize is correlated with, designs primer in protein-coding region, and the sequence of primer is as follows:
ZmPIF3-F:5'-CCGACGGCTGCTAACCT-3'(SEQIDNo.2)；
ZmPIF3-R:5'-TTCCGATTCCGAGACCCT-3'(SEQIDNo.3)；
Its amplified fragments is 691bp, is the sequence (SEQIDNo.1) of the molecular marker of Maize Transcription Factor ZmPIF3 transgenic paddy rice。
Embodiment 3: the qualification of Maize Transcription Factor ZmPIF3 transgenic paddy rice positive plant
Adopting SDS phenol-chloroform method Trace bio-element oryza sativa genomic dna, its step is as follows:
1. take two panels young leaflet tablet (about 0.2g), shred in the centrifuge tube loading 2ml, be placed in liquid nitrogen and cool down, smash to pieces to Powdered with chopsticks；
2. add the extraction buffer A of 700 μ l, gently after mixing, in 65 DEG C of water-bath 30min (every 5min turns upside down mixing once)；
3. taking out and be slightly cooled to room temperature, add isopyknic phenol/chloroform (each 350 μ l), turn upside down fully mixing, extracting 10min；
4.12000rpm, centrifugal 5min, draw supernatant in a new centrifuge tube；
5. adding the isopropanol of 0.7 times of volume, mix gently, room temperature places 10min, it is seen that flocculent deposit；
6.12000rpm, centrifugal 10min, supernatant discarded；
7. the ethanol purge precipitation of the 70% of addition 700 μ l；
8.12000rpm, centrifugal 5min, supernatant discarded；
9. dry under room temperature；
10. the TER adding 30 μ l dissolves, after 37 DEG C of temperature bath 60min, and-20 DEG C of preservations。
Taking 1 μ lDNA as template, carry out pcr amplification with the primer in embodiment 2, amplification condition is: 94 DEG C of preheating 5min；94 DEG C, 40s, 58 DEG C, 40s, 72 DEG C, 2min, totally 35 circulations；72 DEG C, 10min。With transgenic paddy rice DNA for template, the purpose fragment that length is 691bp can be amplified, it was demonstrated that genes of interest ZmPIF3 has been integrated in rice genome。
Embodiment 4: the degeneration-resistant analysis after transcription factor ZmPIF3 rice transformation
Obtain 13 transformation plants that isozygoty, carry out ZmPIF3 transgenic paddy rice T2Detecting for fluorescent quantitation in homozygous lines, result is shown in these 13 transgenic homozygous materials ZmPIF3 and all expresses, not producer reticent (as shown in Figure 1)。Choose three transgenic lines of OE3, OE5 and OE11 to be further analyzed, mainly have herein below:
The siccocolous research of plant。When carrying out Osmotic treatment, than the plant of more consistent wild type and ZmPIF3 transgenic line, growth about 40 days and growing way in soil being carried out natural drought Stress treatment, they, after 7 days, are replaced in In water and carry out renewal cultivation by drought stress。As in figure 2 it is shown, after drought stress 7 days, the blade table of WT lines reveals serious etiolation, plant part dead, though and transfer-gen plant to have partial blade to occur sallow, but the major part blade of plant and stem still keep green。After recovering 10 days, WT lines cannot recover and dead, and transfer-gen plant but can turn green by restoration ecosystem。Above-mentioned result of the test shows, when being subjected to drought stress, transgenic paddy rice shows higher drought stress tolerance than wild rice, and after drought stress processes termination, transgenic paddy rice shows resume speed faster than wild rice。
Embodiment 5: the amplification of Semen Maydis ZmPIF3 transcription factor transgenic paddy rice specific molecular marker and stability
Whether the genome specific molecular marker obtained stably is applied to extremely important, utilize same primers that ZmPIF3 transgenic paddy rice positive material is carried out PCR, result shows that the genome specific molecular marker developed is highly stable, it is possible to follow the tracks of Semen Maydis ZmPIF3 transcription factor。Transport, with force, the Semen Maydis ZmPIF3 transcription factor transgenic paddy rice that round-grained rice 7 is background, select above-mentioned primer pair T0, T2 generation to expand。From Fig. 3 result it can be seen that gained amplification label is consistent with expection stripe size, it was shown that T0 has the existence of Semen Maydis ZmPIF3 gene in generation。Same molecular is marked in the different generations of same transgenic line and can stablize heredity, and Fig. 4 shows that all there is Semen Maydis ZmPIF3 gene stable existence in different individual plants in this strain T2 generation。
Claims (4)
1. the molecular marker of Maize Transcription Factor ZmPIF3 transgenic paddy rice, it is characterised in that the extension increasing sequence of described molecular marker is such as shown in SEQIDNo.1。
2. the primer of the molecular marker of the Maize Transcription Factor ZmPIF3 transgenic paddy rice described in a claim 1, it is characterised in that the sequence of the primer of described molecular marker is such as shown in SEQIDNo.2 and SEQIDNo.3:
SEQIDNo.2:ZmPIF3-F5'CCGACGGCTGCTAACCT3'；
SEQIDNo.3:ZmPIF3-R5'TTCCGATTCCGAGACCCT3'。
3. the molecular marker of the Maize Transcription Factor ZmPIF3 transgenic paddy rice described in a claim 1 application in detection transgenic line or molecular marker assisted selection breeding。
4. the preparation method of the molecular marker of a Maize Transcription Factor ZmPIF3 transgenic paddy rice, it is characterized in that, described method is as follows: the nucleotide sequence according to Maize Transcription Factor ZmPIF3, the primer as shown in SEQIDNo.2 and SEQIDNo.3 is designed in protein-coding region, round pcr is used to obtain the DNA fragmentation of Maize Transcription Factor ZmPIF3 and this DNA fragmentation is checked order, the primer as shown in SEQIDNo.2 and SEQIDNo.3 is obtained according to the specific fragment screening of Maize Transcription Factor ZmPIF3, thus obtaining the molecular marker of described Maize Transcription Factor ZmPIF3 transgenic paddy rice。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079535A (en) * | 2019-04-05 | 2019-08-02 | 华南农业大学 | Corn ZmPIF3s mutein, its encoding gene and its application in breeding |
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| CN104513823A (en) * | 2014-11-24 | 2015-04-15 | 扬州大学 | Drought and salt tolerant transgenic plant preparation method |
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- 2016-04-20 CN CN201610248547.2A patent/CN105695459A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103305507A (en) * | 2013-05-29 | 2013-09-18 | 四川农业大学 | Corn photoperiod gene specific molecular marker and application thereof |
| CN104513823A (en) * | 2014-11-24 | 2015-04-15 | 扬州大学 | Drought and salt tolerant transgenic plant preparation method |
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| Title |
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| YONG GAO ET AL.: "A maize phytochrome‑interacting factor 3 improves drought and salt stress tolerance in rice", 《PLANT MOL BIOL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079535A (en) * | 2019-04-05 | 2019-08-02 | 华南农业大学 | Corn ZmPIF3s mutein, its encoding gene and its application in breeding |
| CN110079535B (en) * | 2019-04-05 | 2022-01-28 | 华南农业大学 | Maize ZmPIF3s mutant protein, coding gene thereof and application thereof in breeding |
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