Summary of the invention
The invention provides a kind of TALEN rite-directed mutagenesis system preferring to plant genome DNA, the nucleotide sequence of four identification modules that described TALEN rite-directed mutagenesis system comprises is as follows respectively: identification in conjunction with the nucleotide sequence of the NI module of base A as shown in SEQ ID NO:13; Identify in conjunction with the nucleotide sequence of the HD module of base C as shown in SEQ ID NO:14; Identify in conjunction with the nucleotide sequence of the NG module of base T as shown in SEQ ID NO:15; Identify in conjunction with the nucleotide sequence of the NN module of bases G as shown in SEQ ID NO:16.
Present invention also offers a kind of method of acquisition bentazone sensitive plant newly, be to provide a kind of in monocotyledons more specifically, in paddy rice, preferably formulate the method to the responsive plant of bentazone.Present method is using the nucleotide sequence of first exon initiator codon back of paddy rice Bel gene as target sequence, the nucleotide sequence of described target sequence is as shown in SEQ ID NO:10, wherein the sequence of the 17th to the 34th of SEQ ID NO:10 is intervening sequence, and both sides are the Module recognition sequence (respectively called after L and R) of TALENs.In the present invention, the L for this target sequence holds the TALEN-L designed to comprise TALE gene and Fok1 fusion rotein, and its nucleotide sequence is as shown in SEQ ID NO:6; R for this target sequence holds the TALEN-R designed to comprise TALE gene and Fok1 fusion rotein, and its nucleotide sequence is as shown in SEQ ID NO:8.The nucleotide sequence of this TALEN-L and TALEN-R is building up to same expression vector, under the driving of composition type expression promoter, is transferred to after in paddy rice, effectively can obtain Bel gene by the responsive plant of bentazone suddenlyd change.Achieve the actual demand that paddy rice Bel gene is practiced shooting accurately and efficiently.
Present invention also offers a kind of plant expression constructs, described construct contains SEQ ID NO:6 and/or the nucleotide sequence shown in SEQ ID NO:8.More specifically, the TALEN-L of described construct starts expression by Ubiquitin promotor, and TALEN-R is expressed by 35s promoters driven.
The mutant material screening means that current TALEN method obtains mainly contain: Phenotypic Observation, gene sequencing checking etc.Material for the bad observation of phenotype can only carry out gene sequencing, because progeny material is more, causes workload huge, cost up.Specific to bentazone sensitive mutant, phenotype is easily observed, but has phenotype plant can be dead gradually and can not receive seed, so can not carry out the screening of bentazone susceptibility in T0 generation, and order-checking exists mosaic and is not easy order-checking, and program is loaded down with trivial details, high in cost of production problem.
For solving the problem, present invention also offers a kind of method of screening this type of mutant, particularly for transforming the transfer-gen plant obtained, screening mutant plants by the method for high resolving power solubility curve (HRM).HRM (HighResolution Melt), Chinese is translated into " high resolving power melting " is the up-to-date SNP and mutation research instrument that rose in the world in recent years.This detection method is not subject to the limitation of mutating alkali yl site and type, without the need to sequence-specific probes, directly runs high resolving power and melts, can complete the analysis to sample genotype after PCR terminates.The invention provides a species specific HRM primer Ben-F for detecting paddy rice Bel transgenation situation and Ben-R, its nucleotide sequence is as shown in SEQ ID NO:11 and SEQ ID NO:12.
The present invention realizes successively through the following steps:
(1) Preference of monocotyledons codon and the nucleotide sequence of paddy rice Bel gene order to TALEN are transformed, this sequence, as shown in SEQ ID NO:6 in sequence table and SEQ ID NO:8, hands over precious biotechnology (Dalian) company limited to synthesize by improved nucleotide sequence;
(2) the driving expression of Pubi and P35S promotor downstream is connected to by synthesizing a pair TALEN nucleotide fragments obtained; Pubi and P35S promotor nucleotide sequence is respectively as shown in SEQ ID NO:5 in sequence table and SEQ ID NO:2.
(3) conversion of plant, particularly, preferred rice transformation;
(4) with the T0 obtained for transgenic paddy rice genome for template, carry out HRM analysis.Design one couple of PCR primers particularly, product is made to comprise Bel gene target sequence fragment, PCR primer length is about 120bp, solubility curve is obtained with Lightscanner (USIdaho) scanning, by comparing transfer-gen plant and WT lines PCR primer melting temperature difference screens possible mutant plant, utilize this method can a large amount of screening transgenic plant of fast and easy;
(5) by plant Bel transgenation situation that sequence verification screens.Screen for checking the mutant plants Bel gene obtained whether to suddenly change and how to suddenly change, by the DNA fragmentation of pcr amplification with target sequence, its real sequence of sequence verification.
(6) Bentazon herbicide sensitivity experiments is carried out to the mutant screened.Screening the Bel transgenation strain that obtains and WT lines is planted in same flowerpot simultaneously, it is new that plant to be planted grows to 3 leaves one, sprays with the bentazone of 1200mg/L, and one week " Invest, Then Investigate " plant leaves dries up situation, and take pictures (Fig. 4).
" promotor " of the present invention is one section of DNA sequence dna being positioned at that structure gene 5' holds upstream, can activate RNA polymerase, make it combine exactly with template DNA and have the specificity of transcription initiation.
" nucleotide sequence " of the present invention is putting in order of nucleic acid (DNA and RNA) nucleotide.In many situations, nucleotide sequence determines higher structure and the biological function of nucleic acid, and namely different sequence has different higher structures and different biological functions.
" plant expression vector " of the present invention refer to well known in the prior art, can in vegetable cell any one carrier of constant expression alien gene, as pCAMBIA1300, pBI121 etc.
" conversion " of the present invention refer to well known in the prior art, can by any one methods for plant transformation of exogenous gene transfered plant cell or plant tissue, as agrobacterium-mediated transformation and particle gun etc.
" transgenic plant " of the present invention refer to the plant individual being integrated with foreign gene obtained by gene transfer technique.In usual conversion of plant or transgenic plant genome, the stable nucleotide sequence with foreign gene, stably can entail the next generation by this exogenous nucleotide sequence.
The present invention is compared with the technology of existing establishment bentazone sensitive plant, it is advantageous that easily and fast, scientific research personnel the Mutants homozygous plant in multiple different mutational site can be obtained within the time of a year and a half by this technology, greatly save human and material resources, and the mutant obtained is not with transgene component, eliminate people to genetically modified misgivings, new germ plasm resource formulated to plant genetic engineering significant.
Embodiment 5, the application of bentazone sensitive paddy plant in hybrid seeding
Bentazone sensitive rice transformation restorer, hybridisation rice producing method for seed can be reformed: do male parent by the restorer with bentazone sensitive gene, after free pollination, just male parent restorer can be killed by spraying bentazone, therefore can realize Parent mixed seeding miscegenation in breeding of hybrid rice, be conducive to improving outcrossing rate, reduce labor intensity, realize mechanized operation.Gone if forward in two-line sterile line, what can solve that the two-line hybrid rice production of hybrid seeds worries causes purity inadequate because of temperature variation, give and produce damnous great difficult problem.
The present invention with the responsive plant of bentazone (military fortune No. 7, round-grained rice) for male parent, the male sterile plant of Huang Huazhan sudden change is maternal, adjustment date of seeding, makes its flower synchronization, two row are maternal, a line male parent, and 20 meters of row are long, plant 30 row altogether, August 28 sprayed the bentazone of 1200mg/L, and after the week, male parent is all wilted death, September 29 mechanical harvesting.Get the seed seedling-raising that 1kg is dried, 3 leaf phases sprayed bentazone, did not find dead plant of wilting after one week.The seed not mixing male parent selfing and produce is described in gathered in the crops seed.
SEQUENCE LISTING
<110> Unnamed Xingwang System Crop Design Front Laboratory (Beijing) Co., Ltd.
Shenzhen Crop Molecular Design Breeding Institute
Shenzhen Xingwang Biological Seed Industry Co., Ltd.
Xingwang Investment Pty Ltd.
The fixed point of a <120> paddy rice Bel gene knocks out system and application thereof
<130>
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 2308
<212> DNA
<213> paddy rice (Oryza Sativa)
<400> 1
tcacagaaac acatcacaca ttcgtgagct cagcttagcc atggataacg cctacattat 60
tgccattctc tctgtagcta tcctcttctt gctccactac tacctcctcg gccgcggcaa 120
tggcggggcg gcgcggctgc cgccgggtcc accggccgtc ccgatcctgg gacacctcca 180
cctcgtcaag aagccgatgc acgccaccat gtcccgcctc gccgagcggt acgggccggt 240
gttctcgctg cgcctcgggt cgcggcgcgc cgtggtggtg tcgtcgccgg ggtgcgccag 300
ggagtgcttc accgagcacg acgtgacctt cgcgaaccgg cccaggttcg agtcgcagct 360
gctggtctcg ttcaacggcg ccgcgctcgc cacggcgagc tacggcgcgc actggcgcaa 420
cctccgccgg atcgtcgccg tgcagctgct ctccgcgcac cgcgtcggcc tcatgtcggg 480
gctcatcgcc ggcgaggtcc gcgccatggt gcggaggatg taccgcgccg cggccgcgtc 540
ccccgccggc gccgcgcgca tccagctgaa gcggaggctg ttcgaggtct ccctcagcgt 600
gctcatggag accatcgccc acaccaaggc gacccgcccc gagacggacc cggacaccga 660
catgtccgtg gaagcccagg agtttaagca ggtcgtcgac gagatcatcc cgcacatcgg 720
cgcggccaac ctgtgggact acttgccggc gctccggtgg ttcgacgtgt tcggcgtcag 780
gaggaagatc ctcgccgctg taagccggag ggacgcgttc cttcgccgcc tgatcgacgc 840
ggagcggcgg aggctggacg acggcgacga gggcgagaag aagagcatga tcgccgtgct 900
gctcactctg cagaagacag agccggaggt gtacaccgat aacatgatca cagctctaac 960
ggcggtgagt tcatcttctg ctgttttacc tttctgatat ctgaattctc tcattggtgc 1020
gtaatttttt ttttttggct gtcatcggta tagctttctt aagcactcag tagccttgca 1080
attataaaaa gaaaaacaat cagtagcttt ttacatgctt tgagtcagtc agtagcagtg 1140
tggcactatc agcattcagc agtattcatg ttgtttgcta atcactatca tggtttgagt 1200
cagcacaatc agtagctttt gacatggttt gagtcagcag tatcttttct aggaactgaa 1260
ttagttattc atttagtaca acttgtttgt ctgtctattg attgctttaa attatttctt 1320
ctatgcaacc ctctaatcct agtatagtac tagcctttta tatgaagaat catcaataat 1380
tttcttctca ctttcagtgt agctttactt aatgaatatt ttgaaagatc gcctagttgc 1440
cttattataa ttgtataaaa ggaaaacaat cagtagcctt ttacatggtt gagtcagcga 1500
gttatcagga gtactattgt ttatcatggt agtagcacga tagactattc aacccgggcg 1560
attaaaatcc ttctcccaaa tttgtattct ttgttttttc ctcaaaaatt caccaaactc 1620
ttggaactat tatagttcag ttttagacaa aaaaaaaatg aaatcttagt tccaccctct 1680
tgccgtgcag aacttgttcg gagcaggaac agagacaacc tcgacgacat cagaatgggc 1740
gatgtcgcta ctgctgaacc accccgacac actcaagaaa gcgcaagccg agatcgacgc 1800
atccgtcggc aactctcgcc tgatcaccgc cgacgacgtg actcgcctcg gctacctcca 1860
gtgcatcgtc agggagacgc tccgcctgta ccccgccgcg ccgatgctcc tcccgcacga 1920
gtcctccgcc gactgcaagg tcggcggcta caacatcccg cgcgggtcga tgttgctcat 1980
caacgcgtac gccatccacc gtgacccggc ggtgtgggag gagccggaga agttcatgcc 2040
ggagaggttc gaggacggcg ggtgcgacgg caatctcttg atgccgttcg ggatggggag 2100
gcggaggtgc cccggcgaga cgctggcgct gcgcacagtg gggttggtgc tgggcacgct 2160
gatccagtgc ttcgactggg agagggtcga cggcgtggag gtcgacatga ctgaaggtgg 2220
cgggctcacc atccccaagg tcgtgccgtt ggaggccatg tgcaggccgc gcgacgccat 2280
gggtggtgtt cttcgcgagc tcgtctga 2308
<210> 2
<211> 538
<212> DNA
<213> synthetic
<400> 2
catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca 60
gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga 120
gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 180
aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 240
tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 300
ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg 360
acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 420
agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 480
gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggact 538
<210> 3
<211> 1023
<212> DNA
<213> synthetic
<400> 3
gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 60
cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 120
aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 180
ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 240
ggagtttagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 300
agacctgcct gaaaccgaac tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc 360
gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 420
cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 480
ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 540
gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 600
caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 660
gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 720
tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccacg 780
actccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 840
caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 900
cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 960
tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagaa 1020
ata 1023
<210> 4
<211> 174
<212> DNA
<213> cauliflower mosaic virus (cauliflower mosaic virus)
<400> 4
gccccagcac tcgtccgagg gcaaagaaat agagtagatg ccgaccggat ctgtcgatcg 60
acaagctcga gtttctccat aataatgtgt gagtagttcc cagataaggg aattagggtt 120
cctatagggt ttcgctcatg tgttgagcat ataagaaacc cttagtatgt attt 174
<210> 5
<211> 1988
<212> DNA
<213> corn (Zea mays)
<400> 5
ccagtgcagc gtgacccggt cgtgcccctc tctagagata atgagcattg catgtctaag 60
ttataaaaaa ttaccacata ttttttttgt cacacttgtt tgaagtgcag tttatctatc 120
tttatacata tatttaaact ttactctacg aataatataa tctatagtac tacaataata 180
tcagtgtttt agagaatcat ataaatgaac agttagacat ggtctaaagg acaattgagt 240
attttgacaa caggactcta cagttttatc tttttagtgt gcatgtgttc tccttttttt 300
ttgcaaatag cttcacctat ataatacttc atccatttta ttagtacatc catttagggt 360
ttagggttaa tggtttttat agactaattt ttttagtaca tctattttat tctattttag 420
cctctaaatt aagaaaacta aaactctatt ttagtttttt tatttaataa tttagatata 480
aaatagaata aaataaagtg actaaaaatt aaacaaatac cctttaagaa attaaaaaaa 540
ctaaggaaac atttttcttg tttcgagtag ataatgccag cctgttaaac gccgtcgacg 600
agtctaacgg acaccaacca gcgaaccagc agcgtcgcgt cgggccaagc gaagcagacg 660
gcacggcatc tctgtcgctg cctctggacc cctctcgaga gttccgctcc accgttggac 720
ttgctccgct gtcggcatcc agaaatgcgt ggcggagcgg cagacgtgag ccggcacggc 780
aggcggcctc ctcctcctct cacggcacgg cagctacggg ggattccttt cccaccgctc 840
cttcgctttc ccttcctcgc ccgccgtaat aaatagacac cccctccaca ccctctttcc 900
ccaacctcgt gttgttcgga gcgcacacac acacaaccag atctccccca aatccacccg 960
tcggcacctc cgcttcaagg tacgccgctc gtcctccccc cccccccctc tctaccttct 1020
ctagatcggc gttccggtcc atggttaggg cccggtagtt ctacttctgt tcatgtttgt 1080
gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga tgcgacctgt 1140
acgtcagaca cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg 1200
gctctagccg ttccgcagac gggatcgatt tcatgatttt ttttgtttcg ttgcataggg 1260
tttggtttgc ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct 1320
tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga 1380
tcggagtaga attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg 1440
tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga 1500
taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct 1560
tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat 1620
actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat 1680
cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt atacatgttg 1740
atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta 1800
accttgagta cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat 1860
atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc tgccttcata 1920
cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta 1980
cttggtac 1988
<210> 6
<211> 2778
<212> DNA
<213> synthetic
<400> 6
atggctccaa agaagaagcg taaggtagac tacaaagacc atgacggtga ttataaagat 60
catgacatcg attacaagga tgacgatgac aagggatctg tggatctacg cacgctcggc 120
tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 180
cacgaggcac tggtcggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 240
ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 300
gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 360
gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 420
cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 480
aatgcactga cgggtgcccc cctgaacctg accccggagc aggtggtggc catcgctagt 540
aatattggtg gcaaacaggc tcttgagacg gttcagcgcc tccttccagt tctctgtcaa 600
gcccacggac tcaccccaga tcaagttgta gcgattgcta gtaacaatgg tggcaaacag 660
gctcttgaaa ccgtacagcg cctactgcca gttctctgtc aagcccacgg tctgactccg 720
gagcaagttg tagcgattgc tagtcatgac ggtggcaaac aggctcttga aacagtccaa 780
cgactacttc cagttctctg tcaagcccac ggccttactc ctgagcaagt tgtagcgatt 840
gctagtaatg ggggtggcaa acaggctctt gagactgttc agcgccttct accagttctc 900
tgtcaagccc acggcctgac gcccgagcaa gttgtagcga ttgctagtaa tattggtggc 960
aaacaggctc ttgagacggt tcagcgcctc cttccagttc tctgtcaagc ccacggactc 1020
accccagatc aagttgtagc gattgctagt catgacggtg gcaaacaggc tcttgaaaca 1080
gtccaacgac tacttccagt tctctgtcaa gcccacggcc ttactcctga gcaagttgta 1140
gcgattgcta gtaatattgg tggcaaacag gctcttgaga cggttcagcg cctccttcca 1200
gttctctgtc aagcccacgg actcacccca gatcaagttg tagcgattgc tagtaacaat 1260
ggtggcaaac aggctcttga aaccgtacag cgcctactgc cagttctctg tcaagcccac 1320
ggtctgactc cggagcaagt tgtagcgatt gctagtaata ttggtggcaa acaggctctt 1380
gagacggttc agcgcctcct tccagttctc tgtcaagccc acggactcac cccagatcaa 1440
gttgtagcga ttgctagtaa caatggtggc aaacaggctc ttgaaaccgt acagcgccta 1500
ctgccagttc tctgtcaagc ccacggtctg actccggagc aagttgtagc gattgctagt 1560
aatattggtg gcaaacaggc tcttgagacg gttcagcgcc tccttccagt tctctgtcaa 1620
gcccacggac tcaccccaga tcaagttgta gcgattgcta gtaacaatgg tggcaaacag 1680
gctcttgaaa ccgtacagcg cctactgcca gttctctgtc aagcccacgg tctgactccg 1740
gagcaagttg tagcgattgc tagtaatatt ggtggcaaac aggctcttga gacggttcag 1800
cgcctccttc cagttctctg tcaagcccac ggactcaccc cagatcaagt tgtagcgatt 1860
gctagtaata ttggtggcaa acaggctctt gagacggttc agcgcctcct tccagttctc 1920
tgtcaagccc acggactcac cccagatcaa gttgtagcga ttgctagcaa tggcggcggc 1980
aggccggcgc tggagagcat tgttgcccag ttatctcgcc ctgatccggc gttggccgcg 2040
ttgaccaacg accacctcgt cgccttggcc tgcctcggcg gacgtcctgc gctggatgca 2100
gtgaaaaagg gattgccgca cgcgccggcc ttgatcaaaa gaaccaatcg ccgtattccc 2160
gaacgcacat cccatcgcgt tgccggatcc caactagtca aaagtgaact ggaggagaag 2220
aaatctgaac ttcgtcataa attgaaatat gtgcctcatg aatatattga attaattgaa 2280
attgccagaa atcccactca ggatagaatt cttgaaatga aggtaatgga attttttatg 2340
aaagtttatg gatatagagg tgagcatttg ggtggatcaa ggaaaccgga cggagcaatt 2400
tatactgtcg gatctcctat tgattacggt gtgatcgtgg atactaaagc ttatagcgga 2460
ggttataatc tgccaattgg ccaagcagat gccatgcaaa gctatgtcga agaaaatcaa 2520
acacgaaaca aacatatcaa ccctaatgaa tggtggaaag tctatccatc ttctgtaacg 2580
gaatttaagt ttttatttgt gagtggtcac tttaaaggaa actacaaagc tcagcttaca 2640
cgattaaatc atatcactaa ttgtaatgga gctgttctta gtgtagaaga gcttttaatt 2700
ggtggagaaa tgattaaagc cggcacatta accttagagg aagtgagacg gaaatttaat 2760
aacggcgaga taaacttt 2778
<210> 7
<211> 482
<212> DNA
<213> Arabidopis thaliana (Arabidopsis thaliana)
<400> 7
aggcctccca gctttcgtcc gtatcatcgg tttcgacaac gttcgtcaag ttcaatgcat 60
cagtttcatt gcccacacac cagaatccta ctaagtttga gtattatggc attggaaaag 120
ctgttttctt ctatcatttg ttctgcttgt aatttactgt gttctttcag tttttgtttt 180
cggacatcaa aatgcaaatg gatggataag agttaataaa tgatatggtc cttttgttca 240
ttctcaaatt attattatct gttgttttta ctttaatggg ttgaatttaa gtaagaaagg 300
aactaacagt gtgatattaa ggtgcaatgt tagacatata aaacagtctt tcacctctct 360
ttggttatgt cttgaattgg tttgtttctt cacttatctg tgtaatcaag tttactatga 420
gtctatgatc aagtaattat gcaatcaagt taagtacagt ataggctttt tgtgtcgagg 480
gg 482
<210> 8
<211> 2880
<212> DNA
<213> synthetic
<400> 8
atggctccaa agaagaagcg taaggtagac tacaaagacc atgacggtga ttataaagat 60
catgacatcg attacaagga tgacgatgac aagggatctg tggatctacg cacgctcggc 120
tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 180
cacgaggcac tggtcggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 240
ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 300
gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 360
gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 420
cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 480
aatgcactga cgggtgcccc cctgaacctg accccggagc aggtggtggc catcgctagt 540
catgacggtg gcaaacaggc tcttgagacc gtccaacgcc ttctaccagt tctctgtcaa 600
gcccacggac taaccccagc gcaagttgta gcgattgcta gtaatattgg tggcaaacag 660
gctcttgaga cggttcagcg cctccttcca gttctctgtc aagcccacgg actcacccca 720
gatcaagttg tagcgattgc tagtaacaat ggtggcaaac aggctcttga aaccgtacag 780
cgcctactgc cagttctctg tcaagcccac ggtctgactc cggagcaagt tgtagcgatt 840
gctagtcatg acggtggcaa acaggctctt gaaacagtcc aacgactact tccagttctc 900
tgtcaagccc acggccttac tcctgagcaa gttgtagcga ttgctagtaa tgggggtggc 960
aaacaggctc ttgagactgt tcagcgcctt ctaccagttc tctgtcaagc ccacggcctg 1020
acgcccgagc aagttgtagc gattgctagt aatgggggtg gcaaacaggc tcttgagact 1080
gttcagcgcc ttctaccagt tctctgtcaa gcccacggcc tgacgcccga gcaagttgta 1140
gcgattgcta gtaatattgg tggcaaacag gctcttgaga cggttcagcg cctccttcca 1200
gttctctgtc aagcccacgg actcacccca gatcaagttg tagcgattgc tagtaacaat 1260
ggtggcaaac aggctcttga aaccgtacag cgcctactgc cagttctctg tcaagcccac 1320
ggtctgactc cggagcaagt tgtagcgatt gctagtcatg acggtggcaa acaggctctt 1380
gagaccgtcc aacgccttct accagttctc tgtcaagccc acggactaac cccagcgcaa 1440
gttgtagcga ttgctagtca tgacggtggc aaacaggctc ttgagaccgt ccaacgcctt 1500
ctaccagttc tctgtcaagc ccacggacta accccagcgc aagttgtagc gattgctagt 1560
aatattggtg gcaaacaggc tcttgagacg gttcagcgcc tccttccagt tctctgtcaa 1620
gcccacggac tcaccccaga tcaagttgta gcgattgcta gtaatggggg tggcaaacag 1680
gctcttgaga ctgttcagcg ccttctacca gttctctgtc aagcccacgg cctgacgccc 1740
gagcaagttg tagcgattgc tagtaacaat ggtggcaaac aggctcttga aaccgtacag 1800
cgcctactgc cagttctctg tcaagcccac ggtctgactc cggagcaagt tgtagcgatt 1860
gctagtaaca atggtggcaa acaggctctt gaaaccgtac agcgcctact gccagttctc 1920
tgtcaagccc acggtctgac tccggagcaa gttgtagcga ttgctagtaa caatggtggc 1980
aaacaggctc ttgaaaccgt acagcgccta ctgccagttc tctgtcaagc ccacggtctg 2040
actccggagc aagttgtagc gattgctagc aatggcggcg gcaggccggc gctggagagc 2100
attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc 2160
gtcgccttgg cctgcctcgg cggacgtcct gcgctggatg cagtgaaaaa gggattgccg 2220
cacgcgccgg ccttgatcaa aagaaccaat cgccgtattc ccgaacgcac atcccatcgc 2280
gttgccggat cccaactagt caaaagtgaa ctggaggaga agaaatctga acttcgtcat 2340
aaattgaaat atgtgcctca tgaatatatt gaattaattg aaattgccag aaatcccact 2400
caggatagaa ttcttgaaat gaaggtaatg gaatttttta tgaaagttta tggatataga 2460
ggtgagcatt tgggtggatc aaggaaaccg gacggagcaa tttatactgt cggatctcct 2520
attgattacg gtgtgatcgt ggatactaaa gcttatagcg gaggttataa tctgccaatt 2580
ggccaagcac gagaaatgca acgatatgtc gaagaaaatc aaacacgaaa caaacatatc 2640
aaccctaatg aatggtggaa agtctatcca tcttctgtaa cggaatttaa gtttttattt 2700
gtgagtggtc actttaaagg aaactacaaa gctcagctta cacgattaaa tcatatcact 2760
aattgtaatg gagctgttct tagtgtagaa gagcttttaa ttggtggaga aatgattaaa 2820
gccggcacat taaccttaga ggaagtgaga cggaaattta ataacggcga gataaacttt 2880
<210> 9
<211> 231
<212> DNA
<213> synthetic
<400> 9
agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 60
aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 120
tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 180
gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg g 231
<210> 10
<211> 49
<212> DNA
<213> paddy rice (Oryza Sativa)
<400> 10
cagcttagcc atggataacg cctacattat tgccattctc tctgtagct 49
<210> 11
<211> 22
<212> DNA
<213> synthetic
<400> 11
acagaaacac atcacacatt cg 22
<210> 12
<211> 25
<212> DNA
<213> synthetic
<400> 12
tagtagtgga gcaagaagag gatag 25
<210> 13
<211> 102
<212> DNA
<213> synthetic
<400> 13
cttactcctg agcaagttgt agcgattgct agtaatattg gtggcaaaca ggctcttgaa 60
acagtccaac gactacttcc agttctctgt caagcccacg gc 102
<210> 14
<211> 102
<212> DNA
<213> synthetic
<400> 14
cttactcctg agcaagttgt agcgattgct agtcatgacg gtggcaaaca ggctcttgaa 60
acagtccaac gactacttcc agttctctgt caagcccacg gc 102
<210> 15
<211> 102
<212> DNA
<213> synthetic
<400> 15
cttactcctg agcaagttgt agcgattgct agtaatgggg gtggcaaaca ggctcttgaa 60
acagtccaac gactacttcc agttctctgt caagcccacg gc 102
<210> 16
<211> 102
<212> DNA
<213> synthetic
<400> 16
cttactcctg agcaagttgt agcgattgct agtaacaatg gtggcaaaca ggctcttgaa 60
acagtccaac gactacttcc agttctctgt caagcccacg gc 102