OsROC1在水稻育种中的应用
文献发布时间:2023-06-19 11:00:24
技术领域
本发明属于农作物育种技术领域,具体涉及水稻最外层细胞特异性基因1(
背景技术
近年来,水稻功能基因组研究的快速发展,为我国水稻遗传育种鉴定了大量的有重要利用价值的基因,水稻育种正迈向设计育种的新时代。水稻育种的创新发展为我国口粮安全提供了重要保障,提高单产仍是未来一段时期内我国水稻育种的主攻方向。因此仍要继续重视发掘、克隆和鉴定新的水稻重要农艺性状调控基因,为设计育种提供元件,服务于新时期水稻育种的精准化、数据化和智能化战略需求。
目前,国内外关于同源异型域-亮氨酸拉链蛋白IV家族中的水稻最外层细胞特异性基因亚族的研究并不多,而该亚族基因在水稻中的作用少之又少,本发明对该亚族成员之一OsROC1(Rice outermost cell-specific gene1)的表达及作用进行了研究,初步明确了
发明内容
本发明的目的是提供了
本发明通过构建敲除
AATAAATGCGATTGCATCCGCTCAACACTCGCCCCCCCTCTCGCAATAACTCCTCCTCCCCATCTCCACATTCCTCCCCCAAATCCCCTCTTCTTCTTCTTCTTCTTCTTCCTCCTCCTCCCATGGCGATCGGCGTCTGCTTCTAGCTGGAGAGATCTCGCTTCGTTTCGAGCTGTCCACCGGTGGTTTTCTCGCATGCGATATCACACGCTTCTTGGAAGCACATCAGGTAGAACAGGGCAGAGATCAAGGCGGATATATACAGCTAGATCTCATCTTGTTGCTGTCGTCTCTAACACAGGTTTTGATGGGATTTTTGTTTTTCTTTGCAGAGTTCTTGGTGCTGACCTTGCCCTAGATCTGCTTCTGACTGTTGCTGTTACGTGGCGTGCAAGCTAGCTATAGCCATTTCTGTGTGTGTAGCTGTACTGGTGGTGAAGAAGAAGATGCAAGCGAATGGGTGGAGGAGCTAGAGGCTTGGCTGGAGGAAGAAGAAGAAGGGGTTGGTGGTGGCTAGCTAGCTAGGGTGCGTGCTTAATTACCTGGCTTCGTTCATGACGCCGGCGAGGCGCATGCCGCCGGTGATCGGCCGGAACGGCGTGGCGTACGAATCGCCGTCGGCGCAGCTGCCCCTCACCCAGGTACGGTAGTTCTTCAGAGATGAACAAGAACCCACCGTATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATTTGCAGCTTTCTTTTTTTGAAGTAAATTTTGAAAAAAAGTTGGTGTACTTTTTGTTTGCAGCTAGTTACTTTATTAATTTGCTTTTCGTTTTCTTTTTTCTTGTTGCGTTTGACTCTTGTACTATAATTTAATTGGCTGTGCATCTGTTTTTCATTTTGTTTTGGAAACGGTTGCTAGCTGTGAATACGTGCATGCATTAATTGGCGTGCCATTCGAGGATACTGAGTTTACTGGCCAATTGATGAGCTCTTTTATTGGAAATCAGAAGTACTGGTTGGAGATATCATACACAGTACAGAGAAAGAGAAGTTGATCTTTTGTTCATACAACTTCATACATGAACATGAGTCAAGCAAGAACTGAAGAAACCATACATGAATACATAAACAAAGTCTCTTCCCTTAGGTCTCTCTAGGTCTTGATCTCTTAATGTTCTAATGATCTATGTTTTTTTAAAAAGAAAAAATTTAAAGGCAGACAGGCACAACAGGTTAAGGGAAGGATTAGTTCATTTTTAGCATCATACTAAAAAGAAAAATAGCTAGCTATATCCATTTTTGACAATCACGTCTCGAAAATACACTATACTGAGCAGAAGCAAGTCAGCAATTTCCAACTTTGCGGCCGAAGTAACTACTCTATCCTATCCCTTTTAGAAAGAAAAAGAAATGGAAGCATGCAAAATTTCCCCTTTTTTATGAGCGTTTCGTATAAGTTTTTCTTTGGTACATCTTAATTGCTTTCTCATTCGATTATTCCAAATTAATCCTGAGCTAGCTATATATGGAACACATATATCTGATGAGTTGGTCTCCTCTCACAAAATACTCTTAATTAATCTCTCCTGAAATTGCAGGCTGATATGCTGGACAGCCATCATCTGCAGCAAGCACTCCAGCAGCAATACTTCGATCAGATCCCGGTGACGACGACGGCGGCGGCGGACAGCGGCGACAACATGCTGCACGGCCGCGCCGACGCCGGCGGGCTGGTTGACGAGTTCGAGAGCAAGTCGTGCAGCGAGAACGTCGACGGCGCCGGCGACGGCCTCTCCGGCGACGACCAGGACCCCAACCAGCGGCCGCGCAAGAAGCGTTACCACCGCCATACCCAGCACCAGATCCAAGAGATGGAAGCGTAAGATTAATTTATCATTTCATCCCTATGTTTTCAATTCTGAGATAATTTTCTTCTCCCTTGCAAATCTTTGTCAGATTGATTAAATGTTATGAATCCTATTCATCAGTTGATCTTTGTAGATTAAGTAAATGTTTGTGAATTTTGATCAGTTTCTTCAAGGAGTGCCCGCACCCAGACGACAAGCAGCGCAAGGAGCTGAGCAGGGAGCTGGGTCTTGAACCTCTGCAGGTTAAATTCTGGTTTCAGAACAAGCGCACACAGATGAAGGCAAGTATAAAAACTCCTTATAGATATATAGAGTGTTCTTTGGTTATTTAATTTGATGGCTTCAGACTAAAATGATTGGGAATTACTGAAAAATAAAATTTTGGTGCACTAAATTCCAAAATCCAAGACTAGGAAAAAAAAACACGTTGGACACTGGGGAAGGACCCAATATCGAATTTATTAAGAAAGAGGGTCAACCAGCTGCACCATGCCATCAGCGCTACACACAGTCACAAAATCCAAGACTAAGAAACGGATTATTGCAATCCATGTTCTTTTCAGTTCATTCACAATATTATTCAAATAGATTATTTATTCCATGCTTTAAAAAATATGTTTTTCTCAAAACAATTTCTCAAAAATTACTGCCTATTTATTTCTTAAAACATTTAATATAAACCTTATAATTAAGTAGATGTTCCGCGACAATAATCTAAATCAAATAGGGGTGAGTTCTAAATCTAAATCAGCTAATAATTTGATTTTTCATTCTTCATACTAGTACAATAATGAGCAGTTTTCTACTTAATTACTGGAGCATGTATAATATTTATATACTTAAGTTACAGTATCTATATGAATATTTAGATTGAACAACGAAGGTATATAATGTTTATATAACTATCTCCATATATCTTTCATGATACTAAATCTTATAGTATCTTTCAGAATTTATATATTCTAATACAAACTATAGTCATCAAAGTTGTGTTTTCTCTAAACCATATTCCAATGCACTTAATTGTTTGTGACCGGAGTTAATTAGTACTGCTATCAATTTTTGGCCAAAAAAGCAAATTAGGTTGTGCACACACGGATGAAACTAGGCGGTACTACTAAGAAAGGACGATGTTTTTTTTTTTTGGGGGGGGGGGGTCTTGAATTAAAATGTTTGGATAACACTGAGGAAAAGTTTTCTCAACTTGAAATCAATGCAAGTGTCTAAACCTCACATAACAATTTGCACTGGCTTGCATTAACCAATTTTCCCGAGGACGCGGATGTTGCTCAGCAGCAAGATTGTTTCCTGCACCACCACAGCAGTTAGTGTTTGAATTGAACCTTGTTGTATCTCTTAATGGAACAGACTATATGAAACAACATTGGGACTCACCATTTAGTTTTAGTTTATGGTTAATTACTATGAAGAAATACTCCATTCGTCTTAAAATATTAGTATTAGTAGAATAATGCTAAGTCAAACATTTTCAGCTTTATCTATTAATAGTCAAAAAAAATAAAACAGATCAATCATTGTAAAATTGATGTTACTAGATTTATCATTAAATAAATTATCATAATATATAACTCTTTTTATTTAAAGCATCTTAACTTTATAGATATTATTGGTTAAATGAGCATCTCGAAAATTGTGTTAAAGTTCAAAAATGCTTATATTTTGGAACGGAGAGAGCATGTTATTTCTTTCTTCACTTCTTTTTTTCATTTCTGATTTATTTTATTTCACTTCTCCCTCTGGCCTCTACTCCTGTTATACATGGTAGATATAGTGAAATATATTCCAGCCCAGACGTGTGTGTGTGTATATATATATGCATCTTTTTGTCTATTGAACAGATGGAAAAATATTGTGTTTTCTGTCAGCTACATATATTGATCGATTGACCCTTACAAAACTATAAATATATCAGTAAAATTAATGTTGTAATAGTATATTGACGATATGTAGTGTTGAATTAATTCAGAACCAGCACGAGAGGCACGAGAACGCGCAGCTGCGGGCGGAGAACGACAAGCTGCGCGCGGAGAACATGCGATACAAGGAGGCCCTGAGCAGCGCGTCGTGCCCCAACTGCGGCGGCCCCGCCGCCCTCGGCGAGATGTCCTTCGACGAGCACCACCTCCGCGTCGAGAACGCCCGCCTCCGCGACGAGATCGACCGCATCTCCGGCATCGCCGCCAAGCACGTCGGCAAGCCCCCCATCGTCTCCTTCCCCGTCCTCTCCTCCCCGCTCGCCGTCGCCGCCGCCCGCTCCCCTCTCGACCTCGCCGGCGCCTACGGCGTCGTCACCCCCGGCCTCGACATGTTCGGCGGCGCCGGCGACCTCCTCCGCGGCGTGCACCCGCTCGACGCCGACAAGCCCATGATCGTGGAGCTCGCCGTCGCCGCCATGGACGAGCTCGTCCAGATGGCCCAGCTCGACGAGCCGCTCTGGTCGTCGTCGTCGGAGCCGGCGGCGGCGTTGCTCGATGAGGAGGAGTACGCGCGCATGTTCCCGCGCGGCCTCGGCCCCAAGCAGTACGGCCTCAAGTCGGAGGCGTCCCGCCATGGCGCCGTCGTCATCATGACGCACAGCAACCTCGTCGAGATCCTCATGGATGTGGTCAGTCTCACAATACTCAAACCTACTACTGCTAGTGCTACCTAAATATGCAGTAATTTCAGTTGAATTTCGCTTGAATAATTTTGCTGCAGAATCAATTCGCGACGGTGTTCTCGAGCATCGTGTCGAGAGCGTCCACGCACGAGGTGTTGTCCACAGGCGTGGCAGGCAACTACAATGGTGCACTGCAAGTGGTACGTACACGTCTTCTTCATCCTCATCGACTACTCGATCAACTCCTTAATTAATCTGTTCATGGCACGCCTCCAATTCCATGCTCCATGGATATATTCTGGAATTAATCCACCAGATATATGATGATCTAAACATTATCGGTTGCAGATGTCAATGGAGTTTCAAGTGCCGTCGCCGCTGGTGCCGACGAGGGAGAGCTATTTCGTCAGGTACTGCAAGAACAACTCCGACGGGACATGGGCCGTCGTCGATGTCTCTCTCGACAGCCTCCGCCCCAGCCCTGTCCAGAAATGCCGGCGCAGGCCGTCTGGCTGCCTCATCCAAGAAATGCCCAATGGCTACTCCAAGGTAATTAATTCAATCTCTATATATATATATCTACAATATTAAAGGATAGTACATATTGTTGTTGTTCATATTTTGTCAGCAAAAGTTGAAATGGTTTGATCGTTCATATTCTGTTGGCAAAGTTAATTTGAAATGTATTGGGTTGTTGTGTGTAGGTGACATGGGTGGAGCATGTGGAGGTGGACGACAGCTCGGTGCACAACATCTACAAGCCATTGGTGAACTCCGGCCTCGCATTCGGCGCGAAACGGTGGGTCGGCACGCTGGACCGGCAATGCGAGCGCCTCGCCAGCGCCATGGCCAGCAACATCCCCAATGGCGACCTTGGAGGTACAAGACGAATCAATCAATCAATTAACCACTCCTACGTCCTACTAATTGCAATTATAAGAAGTACAGATAATTGTGACCAATGAAATGCCACATTACAATCATGATTACTGATGATTAGTATAATTTGTGTATGAAATGTTATGTGTTGTGGTGCAGTGATTACAAGCGTGGAGGGGAGGAAGAGCATGTTGAAGCTGGCGGAGAGGATGGTGGCGAGCTTCTGCGGCGGCGTGACGGCGTCGGTGGCGCACCAGTGGACGACGCTGTCGGGCAGCGGGGCGGAGGACGTGCGCGTCATGACGAGGAAGAGCGTCGACGACCCCGGCAGGCCACCGGGCATCGTGCTCAACGCCGCCACCTCCTTCTGGCTCCCCGTCCCTCCCGCCGCCGTCTTCGACTTCCTCCGCGACGAGACCTCTCGCAGCGAGGTGCCCAATTTGCCCCCCATCCCTAGGGAATTTATCCAATTTGCTGACCAAATTTTATACGTGATCAAATTCATTCCTGACGAGTTTGGATTTCCGCAGTGGGACATTCTGTCCAACGGCGGCGCCGTCCAAGAAATGGCTCACATTGCCAACGGCCGTGACCATGGCAACTCTGTCTCGCTTCTTCGTGTCAATGTAAGTGCCAATTAGTTACTACTCATCAAGGCAATAAATAGCATGTAACGGTCGGTAGCGAATTAGTGTCTGGGTAGCGGATTGCAAATAGCAGGCACTAAGTTCCAATAGCATAATTAAGAAAAATAACCATAAATTTAGATTGTATATCAATATTAACATCAAGTTTCACTTAAAGTTTAAAATTTAGACTGCATGTTTAACTACTACTACATTTCATAACATAAATATATAAGATAAAGAAAACATACAATGCTAGGTTATTCTATCCATATGTAAAGAACACTATCTAAACATTATAGCAATTCAACAAAGTCAATAGCAGGCTGCTATACCTTGTAGCGGCATCGGTAGAGGATCACTACTGCTATATTGCAGCCGCTACGAATGCTATTTATTACCTTCTGCTCATGAATCTGATGATTACACTGTTTACACACGATGAAATCGGGTATCATCATTTCTATAAAATTAAGTAATGATGTGTTTATATATGTATGATTTGTCCATGTGTGTAGAGTGCAAATTCAAACCAGAGCAACATGCTGATCCTGCAAGAGAGCTGCACGGACGCGTCGGGCTCCTACGTGGTGTACGCGCCGGTGGACATCGTGGCGATGAACGTGGTGCTCAACGGCGGCGACCCGGACTACGTGGCGCTGCTGCCGTCGGGGTTCGCCATCCTTCCCGACGGGCCGTCGGGGAACGCGCAGGCCGCCGTCGGGGAGAACGGCTCCGGCTCCGGCGGGGGGTCCCTCCTGACGGTGGCGTTCCAGATCCTCGTCGACTCCGTGCCGACGGCGAAGCTCTCGCTGGGCTCCGTCGCGACGGTGAACAGCCTCATCGCCTGCACGGTGGAGCGCATCAAGGCCGCCGTCTGCAGGGACAGCAACCCTCAGTAGTAGCACCGGCTTCAACCAAATATGATCACGAACAATGTTCCAAGGTACATGCTTGCTACGTTTAGTAAAGCGTTGCTGCACGAATCTCAAAGCACCTACTGAAATGTTTTGAATTTGTGTGTGTGTGTTTTTTTGCAGGAGCAGCAAAATTTTGGAGTTTGATTCAGCAGTAGGGAGTCAAGAACGCACCTCAAACTCTCCTTGCCATGTGGTGCCCTTGCTAGTGAAGAATTTGCGAGGAAACCGACGCGTTTGCGAGAAAATTTTCGCGAAGATACATAGTGTCACCACTATGCAAGGGGGGATGGTTCGGGCATTGACTTCCACCTTTTGATTTTCATCAAATGCATCGAAGAGTGGATGCTGCTACCTTGTTGTTTTTCTGAATCTCTATGTTAGGGCTTCTTTTACCATGTTTAAAAAGGGCTATGAAGCCTCATTTTTATCTGTACTGTTGTGATGTGTTGTCTGTTTGTTCAGACTTCAGGTTCCTGTCCCTTAGCTAGGAGTTGAGAATAGAGAGATTCATGTCCCCCCTTTTGGATTTTATCACATTTCCGATGATTTGTAGACTTGTGGTTGTTGCTTGTTGGTCATTACCATTCTTGTCTCAAA
通过T-DNA插入构建的
附图说明
图1为
图2为野生型植株和
图3为
图4为各节间长度及第二节节间细胞形态大小。A为各节间长度;B为第二节节间细胞形态大小。其中,野生型及
具体实施方式
下面结合附图通过实施例来对本发明进行详细说明,但并不是对本发明的限制,仅仅作示例说明。
实施例1
1.敲除载体的构建和敲除水稻株系的获得
在Oryza sativa cv. Dongjin中构建
植物种植在大田或温室里(14.5小时光照,28℃/9.5小时黑暗,22℃,湿度50%)。种子用含有3%蔗糖的MS培养基(Murashig and Skoog Medium)发芽,种子发芽7天后将幼苗移植到土壤中。
T-DNA插入纯合子突变体植物用以下的引物进行基因型鉴定:
F1:5'-AGGAAGAGCATGTTGAAGCT-3'(SEQ ID NO. 2),
R1:5'-CTACTGAGGGTTGCTGTCCCT-3'(SEQ ID NO. 3)
和5'-AACGCTGATCAATTCCACAG-3' (NGUS1) (SEQ ID NO. 4)。
如图1所示,株系2C-50198中OsROC1被敲除。
2.
提取最上面第二片叶子的中间1.5到2厘米部分的RNA。总RNA提取使用RNAisoPlus(TaKaRa, Shiga, Japan),保证RNA样品260/280的比大于1.8 (NanoDrop 2000;Thermo Scientific, Wilmington, DE, USA)。cDNA合成采用2μg 总RNA、Moloney murineleukemia virus 逆转录酶(Promega, Madison, WI, USA)、RNasin® RibonucleaseInhibitor(Promega)、oligo (dT)18 引物和dNTP。实时定量PCR检测目的基因表达采用SYBR Green I Prime Q-Master mix (GENETBIO, Daejeon, Republic of Korea)和theRotor-Gene Q(QIAGEN, Hilden, Germany)。所有实验至少取样自四棵不同植株,重复三次以上。基因表达变化根据∆∆Ct方法计算。引物的特异性根据熔点曲线确定,OsUbi5作为内参。
OsUbi5引物序列如下:
F:5'-TGAAGACCCTGACTGGGAAG-3'(SEQ ID NO. 5)
R:5'-CACGGTTCAACAACATCCAG -3'(SEQ ID NO. 6)。
OsROC1的qRT-PCR引物如下:
F2:5'-GGTTCAGGGGGTAATTTGTA-3'(SEQ ID NO. 7)
R2:5'-ATGTGAGCCATTTCTTGGAC-3'(SEQ ID NO. 8)。
如图2所示,与野生型植株相比,
3. RNA干扰载体构建和水稻转化
RNAi载体构建引物如下:
RNAi-F:5'-AGATCCCGGTGACGACGACG-3'(SEQ ID NO. 9)
RNAi-R:5'-CAGCGCGTCGTGCCC GGTACC CC-3'(SEQ ID NO. 10)
划线部分为KpnI酶切位点。
使用Pfu高保真酶进行PCR,产物使用乙醇纯化后,用KpnI消化,载体pGA3720使用SmaI消化,载体pGA3426使用KpnI消化,将三种产物连接后,进行大肠杆菌质粒转化。根据冻融方法将RNAi载体转染到农杆菌Agrobacterium tumefaciens LBA4404中。转基因植物通过农杆菌转化方法培育。
如图3所示,通过RNA干扰技术构建了
4. 石蜡组织切片及染色
为进一步揭示
对温室中每棵植株的最长茎秆的节间进行了测量结果显示野生型与
由以上实验可知,通过T-DNA插入构建的
序列表
<110> 南通大学
<120> OsROC1在水稻育种中的应用
<130> 20210106
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7544
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aataaatgcg attgcatccg ctcaacactc gccccccctc tcgcaataac tcctcctccc 60
catctccaca ttcctccccc aaatcccctc ttcttcttct tcttcttctt cctcctcctc 120
ccatggcgat cggcgtctgc ttctagctgg agagatctcg cttcgtttcg agctgtccac 180
cggtggtttt ctcgcatgcg atatcacacg cttcttggaa gcacatcagg tagaacaggg 240
cagagatcaa ggcggatata tacagctaga tctcatcttg ttgctgtcgt ctctaacaca 300
ggttttgatg ggatttttgt ttttctttgc agagttcttg gtgctgacct tgccctagat 360
ctgcttctga ctgttgctgt tacgtggcgt gcaagctagc tatagccatt tctgtgtgtg 420
tagctgtact ggtggtgaag aagaagatgc aagcgaatgg gtggaggagc tagaggcttg 480
gctggaggaa gaagaagaag gggttggtgg tggctagcta gctagggtgc gtgcttaatt 540
acctggcttc gttcatgacg ccggcgaggc gcatgccgcc ggtgatcggc cggaacggcg 600
tggcgtacga atcgccgtcg gcgcagctgc ccctcaccca ggtacggtag ttcttcagag 660
atgaacaaga acccaccgta tatatatata tatatatata tatatatata tatatatata 720
tatatatata tatatatata tatatatata tatatatata tatatatata tatatttgca 780
gctttctttt tttgaagtaa attttgaaaa aaagttggtg tactttttgt ttgcagctag 840
ttactttatt aatttgcttt tcgttttctt ttttcttgtt gcgtttgact cttgtactat 900
aatttaattg gctgtgcatc tgtttttcat tttgttttgg aaacggttgc tagctgtgaa 960
tacgtgcatg cattaattgg cgtgccattc gaggatactg agtttactgg ccaattgatg 1020
agctctttta ttggaaatca gaagtactgg ttggagatat catacacagt acagagaaag 1080
agaagttgat cttttgttca tacaacttca tacatgaaca tgagtcaagc aagaactgaa 1140
gaaaccatac atgaatacat aaacaaagtc tcttccctta ggtctctcta ggtcttgatc 1200
tcttaatgtt ctaatgatct atgttttttt aaaaagaaaa aatttaaagg cagacaggca 1260
caacaggtta agggaaggat tagttcattt ttagcatcat actaaaaaga aaaatagcta 1320
gctatatcca tttttgacaa tcacgtctcg aaaatacact atactgagca gaagcaagtc 1380
agcaatttcc aactttgcgg ccgaagtaac tactctatcc tatccctttt agaaagaaaa 1440
agaaatggaa gcatgcaaaa tttccccttt tttatgagcg tttcgtataa gtttttcttt 1500
ggtacatctt aattgctttc tcattcgatt attccaaatt aatcctgagc tagctatata 1560
tggaacacat atatctgatg agttggtctc ctctcacaaa atactcttaa ttaatctctc 1620
ctgaaattgc aggctgatat gctggacagc catcatctgc agcaagcact ccagcagcaa 1680
tacttcgatc agatcccggt gacgacgacg gcggcggcgg acagcggcga caacatgctg 1740
cacggccgcg ccgacgccgg cgggctggtt gacgagttcg agagcaagtc gtgcagcgag 1800
aacgtcgacg gcgccggcga cggcctctcc ggcgacgacc aggaccccaa ccagcggccg 1860
cgcaagaagc gttaccaccg ccatacccag caccagatcc aagagatgga agcgtaagat 1920
taatttatca tttcatccct atgttttcaa ttctgagata attttcttct cccttgcaaa 1980
tctttgtcag attgattaaa tgttatgaat cctattcatc agttgatctt tgtagattaa 2040
gtaaatgttt gtgaattttg atcagtttct tcaaggagtg cccgcaccca gacgacaagc 2100
agcgcaagga gctgagcagg gagctgggtc ttgaacctct gcaggttaaa ttctggtttc 2160
agaacaagcg cacacagatg aaggcaagta taaaaactcc ttatagatat atagagtgtt 2220
ctttggttat ttaatttgat ggcttcagac taaaatgatt gggaattact gaaaaataaa 2280
attttggtgc actaaattcc aaaatccaag actaggaaaa aaaaacacgt tggacactgg 2340
ggaaggaccc aatatcgaat ttattaagaa agagggtcaa ccagctgcac catgccatca 2400
gcgctacaca cagtcacaaa atccaagact aagaaacgga ttattgcaat ccatgttctt 2460
ttcagttcat tcacaatatt attcaaatag attatttatt ccatgcttta aaaaatatgt 2520
ttttctcaaa acaatttctc aaaaattact gcctatttat ttcttaaaac atttaatata 2580
aaccttataa ttaagtagat gttccgcgac aataatctaa atcaaatagg ggtgagttct 2640
aaatctaaat cagctaataa tttgattttt cattcttcat actagtacaa taatgagcag 2700
ttttctactt aattactgga gcatgtataa tatttatata cttaagttac agtatctata 2760
tgaatattta gattgaacaa cgaaggtata taatgtttat ataactatct ccatatatct 2820
ttcatgatac taaatcttat agtatctttc agaatttata tattctaata caaactatag 2880
tcatcaaagt tgtgttttct ctaaaccata ttccaatgca cttaattgtt tgtgaccgga 2940
gttaattagt actgctatca atttttggcc aaaaaagcaa attaggttgt gcacacacgg 3000
atgaaactag gcggtactac taagaaagga cgatgttttt tttttttggg gggggggggt 3060
cttgaattaa aatgtttgga taacactgag gaaaagtttt ctcaacttga aatcaatgca 3120
agtgtctaaa cctcacataa caatttgcac tggcttgcat taaccaattt tcccgaggac 3180
gcggatgttg ctcagcagca agattgtttc ctgcaccacc acagcagtta gtgtttgaat 3240
tgaaccttgt tgtatctctt aatggaacag actatatgaa acaacattgg gactcaccat 3300
ttagttttag tttatggtta attactatga agaaatactc cattcgtctt aaaatattag 3360
tattagtaga ataatgctaa gtcaaacatt ttcagcttta tctattaata gtcaaaaaaa 3420
ataaaacaga tcaatcattg taaaattgat gttactagat ttatcattaa ataaattatc 3480
ataatatata actcttttta tttaaagcat cttaacttta tagatattat tggttaaatg 3540
agcatctcga aaattgtgtt aaagttcaaa aatgcttata ttttggaacg gagagagcat 3600
gttatttctt tcttcacttc tttttttcat ttctgattta ttttatttca cttctccctc 3660
tggcctctac tcctgttata catggtagat atagtgaaat atattccagc ccagacgtgt 3720
gtgtgtgtat atatatatgc atctttttgt ctattgaaca gatggaaaaa tattgtgttt 3780
tctgtcagct acatatattg atcgattgac ccttacaaaa ctataaatat atcagtaaaa 3840
ttaatgttgt aatagtatat tgacgatatg tagtgttgaa ttaattcaga accagcacga 3900
gaggcacgag aacgcgcagc tgcgggcgga gaacgacaag ctgcgcgcgg agaacatgcg 3960
atacaaggag gccctgagca gcgcgtcgtg ccccaactgc ggcggccccg ccgccctcgg 4020
cgagatgtcc ttcgacgagc accacctccg cgtcgagaac gcccgcctcc gcgacgagat 4080
cgaccgcatc tccggcatcg ccgccaagca cgtcggcaag ccccccatcg tctccttccc 4140
cgtcctctcc tccccgctcg ccgtcgccgc cgcccgctcc cctctcgacc tcgccggcgc 4200
ctacggcgtc gtcacccccg gcctcgacat gttcggcggc gccggcgacc tcctccgcgg 4260
cgtgcacccg ctcgacgccg acaagcccat gatcgtggag ctcgccgtcg ccgccatgga 4320
cgagctcgtc cagatggccc agctcgacga gccgctctgg tcgtcgtcgt cggagccggc 4380
ggcggcgttg ctcgatgagg aggagtacgc gcgcatgttc ccgcgcggcc tcggccccaa 4440
gcagtacggc ctcaagtcgg aggcgtcccg ccatggcgcc gtcgtcatca tgacgcacag 4500
caacctcgtc gagatcctca tggatgtggt cagtctcaca atactcaaac ctactactgc 4560
tagtgctacc taaatatgca gtaatttcag ttgaatttcg cttgaataat tttgctgcag 4620
aatcaattcg cgacggtgtt ctcgagcatc gtgtcgagag cgtccacgca cgaggtgttg 4680
tccacaggcg tggcaggcaa ctacaatggt gcactgcaag tggtacgtac acgtcttctt 4740
catcctcatc gactactcga tcaactcctt aattaatctg ttcatggcac gcctccaatt 4800
ccatgctcca tggatatatt ctggaattaa tccaccagat atatgatgat ctaaacatta 4860
tcggttgcag atgtcaatgg agtttcaagt gccgtcgccg ctggtgccga cgagggagag 4920
ctatttcgtc aggtactgca agaacaactc cgacgggaca tgggccgtcg tcgatgtctc 4980
tctcgacagc ctccgcccca gccctgtcca gaaatgccgg cgcaggccgt ctggctgcct 5040
catccaagaa atgcccaatg gctactccaa ggtaattaat tcaatctcta tatatatata 5100
tctacaatat taaaggatag tacatattgt tgttgttcat attttgtcag caaaagttga 5160
aatggtttga tcgttcatat tctgttggca aagttaattt gaaatgtatt gggttgttgt 5220
gtgtaggtga catgggtgga gcatgtggag gtggacgaca gctcggtgca caacatctac 5280
aagccattgg tgaactccgg cctcgcattc ggcgcgaaac ggtgggtcgg cacgctggac 5340
cggcaatgcg agcgcctcgc cagcgccatg gccagcaaca tccccaatgg cgaccttgga 5400
ggtacaagac gaatcaatca atcaattaac cactcctacg tcctactaat tgcaattata 5460
agaagtacag ataattgtga ccaatgaaat gccacattac aatcatgatt actgatgatt 5520
agtataattt gtgtatgaaa tgttatgtgt tgtggtgcag tgattacaag cgtggagggg 5580
aggaagagca tgttgaagct ggcggagagg atggtggcga gcttctgcgg cggcgtgacg 5640
gcgtcggtgg cgcaccagtg gacgacgctg tcgggcagcg gggcggagga cgtgcgcgtc 5700
atgacgagga agagcgtcga cgaccccggc aggccaccgg gcatcgtgct caacgccgcc 5760
acctccttct ggctccccgt ccctcccgcc gccgtcttcg acttcctccg cgacgagacc 5820
tctcgcagcg aggtgcccaa tttgcccccc atccctaggg aatttatcca atttgctgac 5880
caaattttat acgtgatcaa attcattcct gacgagtttg gatttccgca gtgggacatt 5940
ctgtccaacg gcggcgccgt ccaagaaatg gctcacattg ccaacggccg tgaccatggc 6000
aactctgtct cgcttcttcg tgtcaatgta agtgccaatt agttactact catcaaggca 6060
ataaatagca tgtaacggtc ggtagcgaat tagtgtctgg gtagcggatt gcaaatagca 6120
ggcactaagt tccaatagca taattaagaa aaataaccat aaatttagat tgtatatcaa 6180
tattaacatc aagtttcact taaagtttaa aatttagact gcatgtttaa ctactactac 6240
atttcataac ataaatatat aagataaaga aaacatacaa tgctaggtta ttctatccat 6300
atgtaaagaa cactatctaa acattatagc aattcaacaa agtcaatagc aggctgctat 6360
accttgtagc ggcatcggta gaggatcact actgctatat tgcagccgct acgaatgcta 6420
tttattacct tctgctcatg aatctgatga ttacactgtt tacacacgat gaaatcgggt 6480
atcatcattt ctataaaatt aagtaatgat gtgtttatat atgtatgatt tgtccatgtg 6540
tgtagagtgc aaattcaaac cagagcaaca tgctgatcct gcaagagagc tgcacggacg 6600
cgtcgggctc ctacgtggtg tacgcgccgg tggacatcgt ggcgatgaac gtggtgctca 6660
acggcggcga cccggactac gtggcgctgc tgccgtcggg gttcgccatc cttcccgacg 6720
ggccgtcggg gaacgcgcag gccgccgtcg gggagaacgg ctccggctcc ggcggggggt 6780
ccctcctgac ggtggcgttc cagatcctcg tcgactccgt gccgacggcg aagctctcgc 6840
tgggctccgt cgcgacggtg aacagcctca tcgcctgcac ggtggagcgc atcaaggccg 6900
ccgtctgcag ggacagcaac cctcagtagt agcaccggct tcaaccaaat atgatcacga 6960
acaatgttcc aaggtacatg cttgctacgt ttagtaaagc gttgctgcac gaatctcaaa 7020
gcacctactg aaatgttttg aatttgtgtg tgtgtgtttt tttgcaggag cagcaaaatt 7080
ttggagtttg attcagcagt agggagtcaa gaacgcacct caaactctcc ttgccatgtg 7140
gtgcccttgc tagtgaagaa tttgcgagga aaccgacgcg tttgcgagaa aattttcgcg 7200
aagatacata gtgtcaccac tatgcaaggg gggatggttc gggcattgac ttccaccttt 7260
tgattttcat caaatgcatc gaagagtgga tgctgctacc ttgttgtttt tctgaatctc 7320
tatgttaggg cttcttttac catgtttaaa aagggctatg aagcctcatt tttatctgta 7380
ctgttgtgat gtgttgtctg tttgttcaga cttcaggttc ctgtccctta gctaggagtt 7440
gagaatagag agattcatgt cccccctttt ggattttatc acatttccga tgatttgtag 7500
acttgtggtt gttgcttgtt ggtcattacc attcttgtct caaa 7544
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aggaagagca tgttgaagct 20
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctactgaggg ttgctgtccc t 21
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aacgctgatc aattccacag 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tgaagaccct gactgggaag 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cacggttcaa caacatccag 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggttcagggg gtaatttgta 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgtgagcca tttcttggac 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agatcccggt gacgacgacg 20
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cagcgcgtcg tgcccggtac ccc 23
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