CN113881670B - 抗大豆花叶病毒的转基因植物构建方法 - Google Patents
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Abstract
本发明提供一种抗大豆花叶病毒的转基因植物构建方法。本发明采用表达与大豆花叶病毒基因组RNA同源的短片段amiRNA来抗SMV，具有如下优点：首先microRNA特异性强，产生脱靶的可能性更小，由此microRNA很短而且唯一。而发卡结构产生的siRNA很多，较容易脱靶，影响内源基因的表达。此外microRNA在植物体内更稳定，且可在低温下发挥作用。本发明为抗大豆花叶病毒的植物育种提供有效手段。
Description
技术领域
本发明涉及植物基因工程技术领域，具体地说，涉及一种抗大豆花叶病毒的转基因植物构建方法。
背景技术
转基因技术能够极大地缩短抗病植物品种的选育进程，目前将转基因技术和基因沉默技术相结合已成为培育抗病毒转基因大豆的热门方法。目前已有报道称在大豆内转入产生含有特异片段的发卡结构(hairpin structure)的基因，通过发卡结构被植物内源RNA酶dicer切割产生可以靶向大豆花叶病毒(soybean mosaic virus,SMV)HCPro或CP的编码区的小干扰RNA(siRNA)，或者是靶向与SMV蛋白互作的植物內源基因的mRNA的siRNA，实现对SMV的抗性。
理论上，使用人工microRNA(artificial microRNA,amiRNA)比发卡结构更有优势。首先microRNA更特异，产生脱靶的可能性更小，由于microRNA很短而且唯一。而发卡结构产生的siRNA很多，较容易脱靶，影响内源基因的表达。此外microRNA在植物体内更稳定，且适应在低温下发挥作用。
2006年，美国洛克斐勒大学Niu等以拟南芥(Arabidopsis thaliana,At)pre-miR159a基因为骨架，通过转基因的方法，在拟南芥中表达了靶向芜菁花叶病毒(Turnipmosaic virus,TuMV)基因组RNA的amiRNA，使转基因拟南芥可以抵抗TuMV的侵染。
2008年，中国科学院微生物研究所段成国等发现在拟南芥中转基因表达靶向黄瓜花叶病毒(Cucumber mosaic virus,CMV)基因组RNA的3’非翻译区(untranslatedregions,UTR)的非tRNA结构区的amiRNA能够抗黄瓜花叶病毒侵染。这说明靶向病毒基因组RNA不同部位的amiRNA可以具有不同的抗病毒活性。因此需要对靶向病毒基因组RNA的amiRNA的抗病毒活性进行筛选。
2016年澳大利亚昆士兰大学Mitter等设计靶向TSWV的N基因和NSs基因的amiRNA，通过在烟草中转基因表达，发现靶向N基因的amiRNA介导了对TSWV的抗性。
目前未见利用转基因表达可以靶向SMV基因组RNA的amiRNA进行抗SMV侵染的相关报道。
发明内容
本发明的目的是提供一种抗大豆花叶病毒的转基因植物构建方法。
本发明构思如下：通过转基因技术表达可以靶向SMV基因组的microRNA，从而达到抗SMV的目的。
为了实现本发明目的，第一方面，本发明提供一种miRNA，其是靶向SMV的P1编码区的amiRNA，序列为5’-UGGUUAAAGAUGAUCGCUCAU-3’(SEQ ID NO:4)。
第二方面，本发明提供编码所述miRNA核酸分子。
第三方面，本发明提供含有所述核酸分子的生物材料，所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
第四方面，本发明提供一种抗大豆花叶病毒的制剂，其有效成分为所述miRNA或编码所述miRNA核酸分子或含有所述核酸分子的生物材料。
第五方面，本发明提供所述miRNA或编码所述miRNA核酸分子或含有所述核酸分子的生物材料的以下任一应用：
1)用于制备转基因植物；
2)用于提高植物抗大豆花叶病毒的能力；
3)用于制备抗大豆花叶病毒的制剂或试剂盒。
第六方面，本发明提供一种抗大豆花叶病毒的转基因植物构建方法，所述方法包括：构建含有编码所述miRNA的DNA序列(SEQ ID NO:1)的植物表达载体，转化植物，筛选阳性转基因植株。
所述表达载体可以是植物双元表达载体，如pCambia1301、pCambia3301。
优选地，采用农杆菌介导法转化植物。
所述植物包括但不限于本氏烟、大豆。
本发明采用表达与大豆花叶病毒同源的短片段amiRNA来抗SMV，具有如下优点：首先microRNA特异性强，产生脱靶的可能性更小，由此microRNA很短而且唯一。而发卡结构产生的siRNA很多，较容易脱靶，影响内源基因的表达。此外microRNA在植物体内更稳定，且可在低温下发挥作用。
附图说明
图1为本发明较佳实施例中靶向大豆花叶病毒P1编码区的amiRNA序列设计位点。
图2为本发明较佳实施例中将靶向P1编码区的amiRNA与靶向CP编码区的amiRNA在本氏烟中进行瞬时表达并染毒后SMV的CP蛋白的积累情况。
图3为本发明较佳实施例中DAS-ELISA检测接种SMV后本氏烟转基因家系1和家系2中的SMV含量(**p<0.01,n＝5,单侧t检验)。
图4为本发明较佳实施例中DAS-ELISA检测接种SMV后本氏烟转基因家系A和家系B中的SMV含量(**p<0.01,n＝5,单侧t检验)。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。实施例1靶向SMV的amiRNA的优化设计
本发明利用拟南芥MIR319A的基因序列为骨架(Plant Cell 2006May；18(5):1121-33)，选取大豆花叶病毒4278-1分离物(Genbank Accession#:KT285170.1)、4469-4分离物(Genbank Accession#:HM590055.1)、BYX006分离物(Genbank Accession#:KP710861.1)、GXQZ001分离物(Genbank Accession#:KX834322.1)、SC001分离物(GenbankAccession#:KX834320.1)、NE-N1分离物(Genbank Accession#:KP710869.1)、6202-2分离物(Genbank Accession#:JF833014.1)、SC3分离物(Genbank Accession#:JF833013.1)、SC6分离物(Genbank Accession#:HM590054.1)、SC15分离物(Genbank Accession#:MH919386.1)、SX-Z分离物(Genbank Accession#:KP710870.1)、XFQ020分离物(GenbankAccession#:KP710878.1)、XFQ008分离物(Genbank Accession#:KP710873.1)的P1序列的同源区段，设计了靶向SMV的P1编码区的amiRNA，该amiRNA可以靶向多种SMV流行毒株的基因组RNA序列(图1)。amiRNA的序列为5’-UGGUUAAAGAUGAUCGCUCAU-3’。编码该amiRNA的基因序列如SEQ ID NO:1所示。
进一步地，SMV编码的蛋白还包括外壳蛋白CP。将靶向P1编码区的amiRNA与靶向CP编码区的amiRNA(序列为5’-UAUCUCUCAAAUUCCUCAGUA-3’)在本氏烟(Nicotianabenthamiana)叶片中通过农杆菌注射的方式进行瞬时表达，同时接种大豆花叶病毒4278-1分离物的病叶的汁液，6天后对接种叶片中病毒含量进行Western blot分析，其中用抗SMV病毒CP的多克隆抗体来检测病毒含量。SDS-PAGE电泳及考马斯亮蓝染色的植物总蛋白为蛋白上样量对照。图2结果显示，在本氏烟叶片中瞬时表达靶向P1的amiRNA可以有效抑制SMV的复制，而瞬时表达靶向CP的amiRNA则不能抑制SMV的复制。由此表明P1是一个有效的可以抑制SMV复制的靶点。
实施例2抗大豆花叶病毒的转基因植物的构建
将编码所述amiRNA的DNA序列(SEQ ID NO:1)克隆到用于转基因的植物双元表达载体中，得到用于本氏烟转化的pCambia1301-SMV-P1-amiRNA和用于大豆转化的pCambia3301-SMV-P1-amiRNA，序列分别如SEQ ID NO:2和3所示。
1、抗大豆花叶病毒的转基因本氏烟的制备
以pRS300载体(https://www.addgene.org/22846/)(Plant Cell 2006May；18(5):1121-33.)为模板，利用引物对MiR/A和MiR/P1/IV扩增片段1，引物对MiR/P1/II和MiR/P1/III扩增片段2，引物对MiR/P1/I和MiR/B扩增片段3，将片段1、2、3的PCR片段回收后等比例混合，以引物对MiR/A/NcoI/F和MiR/B/BstEII/R进行重叠PCR(overlapping PCR)，获得拼接了片段1、2、3的片段，此片段含有序列P1-amiRNA。利用NcoI和BstEII酶切载体pCambia1301(https://www.snapgene.com/resources/plasmid-files/？set＝plant_vectors&plasmid＝pCA MBIA1301)得到用于本氏烟转化的pCambia1301-SMV-P1-amiRNA，序列见附件。
利用农杆菌介导的遗传转化，获得稳定表达P1-amiRNA的本氏烟植株，筛选稳定遗传的后代家系。
利用摩擦接种法，对本氏烟植株接种感染了大豆花叶病毒4278-1分离物的病叶的汁液，2周后采上位叶片，经DAS-ELISA法检测，转基因本氏烟植株(本氏烟转基因家系1和家系2)的上位叶片中的SMV含量显著低于非转基因本氏烟植株(图3)。
2、抗大豆花叶病毒的转基因大豆的制备
以pRS300载体(https://www.addgene.org/22846/)(Plant Cell 2006May；18(5):1121-33.)为模板，利用引物对MiR/A和MiR/P1/IV扩增片段1，引物对MiR/P1/II和MiR/P1/III扩增片段2，引物对MiR/P1/I和MiR/B扩增片段3，将片段1、2、3的PCR片段回收后等比例混合，以引物对MiR/A/NcoI/F和MiR/B/BstEII/R进行重叠PCR(overlapping PCR)，获得拼接了片段1、2、3的片段，此片段含有序列P1-amiRNA。利用NcoI和BstEII酶切载体pCambia3301(https://www.snapgene.com/resources/plasmid-files/？set＝plant_vectors&plasmid＝pCA MBIA3301)，得到用于大豆转化的pCambia3301-SMV-P1-amiRNA，序列见附件。
利用农杆菌介导的遗传转化，获得稳定表达P1-amiRNA的大豆植株，筛选稳定遗传的后代家系。
利用摩擦接种法，对大豆植株接种感染了大豆花叶病毒4278-1分离物的病叶的汁液，2周后采上位叶片，经DAS-ELISA法检测，转基因大豆植株的上位叶片中的SMV含量显著低于非转基因大豆植株(图4)。
本实施例中使用的引物序列如下(5′-3′)：
MiR/A：CTGCAAGGCGATTAAGTTGGGTAAC
MiR/B：GCGGATAACAATTTCACACAGGAAACAG
MiR/P1/I：GAtggttaaagatgatcgctcatTCTCTCTTTTGTATTCC
MiR/P1/II：GAatgagcgatcatctttaaccaTCAAAGAGAATCAATGA
MiR/P1/III：GAatcagcgatcatcattaacctTCACAGGTCGTGATATG
MiR/P1/IV：GAaggttaatgatgatcgctgatTCTACATATATATTCCT
MiR/A/NcoI/F：CTGCAAGGCGATTAAGTTGGGTAAC
MiR/B/BstEII/R：GCGGATAACAATTTCACACAGGAAACAG
此前关于amiRNA的研究证明靶向植物病毒基因组的不同位点的amiRNA可能会起到抗病毒的效果，但也有证据显示某些位点可能不会抗病毒。本发明优选了靶向SMV的P1编码区的amiRNA，在转化了此载体的烟草和大豆中可以达到抗病毒的效果。与以往表达发卡结构沉默病毒基因或寄主基因的策略不同，本发明采取了表达amiRNA的策略进行转基因抗病毒。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 南京师范大学
<120> 抗大豆花叶病毒的转基因植物构建方法
<130> KHP211120328.0
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 410
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ctgcagcccc aaacacacgc tcggacgcat attacacatg ttcatacact taatactcgc 60
tgttttgaat tgatgtttta ggaatatata tgtagaatca gcgatcatca ttaaccttca 120
caggtcgtga tatgattcaa ttagcttccg actcattcat ccaaataccg agtcgccaaa 180
attcaaacta gactcgttaa atgaatgaat gatgcggtag acaaattgga tcattgattc 240
tctttgatgg ttaaagatga tcgctcattc tctcttttgt attccaattt tcttgattaa 300
tctttcctgc acaaaaacat gcttgatcca ctaagtgaca tatatgctgc cttcgtatat 360
atagttctgg taaaattaac attttgggtt tatctttatt taaggcatcg 410
<210> 2
<211> 10205
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 60
tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 120
ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgaattc 180
gagctcggta cccggggatc ctctagagtc gacctgcagg catgcaagct tggcactggc 240
cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 300
agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 360
ccaacagttg cgcagcctga atggcgaatg ctagagcagc ttgagcttgg atcagattgt 420
cgtttcccgc cttcagttta gcttcatgga gtcaaagatt caaatagagg acctaacaga 480
actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 540
gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 600
tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 660
cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 720
aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 780
tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 840
cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 900
tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 960
tttggagaga acacggggga ctcttgacca tggctgcagc cccaaacaca cgctcggacg 1020
catattacac atgttcatac acttaatact cgctgttttg aattgatgtt ttaggaatat 1080
atatgtagaa tcagcgatca tcattaacct tcacaggtcg tgatatgatt caattagctt 1140
ccgactcatt catccaaata ccgagtcgcc aaaattcaaa ctagactcgt taaatgaatg 1200
aatgatgcgg tagacaaatt ggatcattga ttctctttga tggttaaaga tgatcgctca 1260
ttctctcttt tgtattccaa ttttcttgat taatctttcc tgcacaaaaa catgcttgat 1320
ccactaagtg acatatatgc tgccttcgta tatatagttc tggtaaaatt aacattttgg 1380
gtttatcttt atttaaggca tcgggtgacc agctcgaatt tccccgatcg ttcaaacatt 1440
tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 1500
tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 1560
agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 1620
atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg 1680
gaattaaact atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg 1740
tttattagaa taacggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt 1800
atgtgcatgc caaccacagg gttcccctcg ggatcaaagt actttgatcc aacccctccg 1860
ctgctatagt gcagtcggct tctgacgttc agtgcagccg tcttctgaaa acgacatgtc 1920
gcacaagtcc taagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt 1980
cgcgtgtttt agtcgcataa agtagaatac ttgcgactag aaccggagac attacgccat 2040
gaacaagagc gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg acgaccagga 2100
cttgaccaac caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa 2160
gatcaccggc accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc 2220
tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc gcgacctact 2280
ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg 2340
ggccgacacc accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga 2400
gttcgagcgt tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg 2460
aggcgtgaag tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga 2520
gctgatcgac caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg 2580
ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg 2640
gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa 2700
tgaacgccaa gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc 2760
attaccgaag agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg 2820
cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg 2880
gcctggccgg ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt 2940
gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat 3000
aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt 3060
caggcaagac gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg 3120
ttctgttagt cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag 3180
atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca 3240
tcggccggcg cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt 3300
ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat 3360
gggccaccgc cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc 3420
tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg 3480
ccgaggcgct ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga 3540
gctacccagg cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg 3600
ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg 3660
aggtaaagag aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg 3720
cagcagcaag gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa 3780
ctttcagttg ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa 3840
gaccattacc gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg 3900
aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac 3960
caggcaccga cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa 4020
gcggctgggt tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg 4080
cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct 4140
ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg 4200
ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc 4260
ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt 4320
cgttccgatg ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt 4380
tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga 4440
cgggcacgta gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct 4500
ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg 4560
agacaagccc ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg 4620
agccgatggc ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac 4680
gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga 4740
gggtgaagcc ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta 4800
catcgagatc gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga 4860
cgtgctgacg gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta 4920
ccgcctggca cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta 4980
cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat 5040
cgggtcaaat gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat 5100
cctagtcatg cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac 5160
ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc 5220
tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat 5280
tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga 5340
gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg 5400
cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac 5460
ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg 5520
ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc 5580
gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg 5640
acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg 5700
atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg 5760
cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc 5820
agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 5880
gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 5940
cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 6000
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 6060
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 6120
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 6180
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 6240
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 6300
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 6360
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 6420
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 6480
tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 6540
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 6600
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 6660
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 6720
aaactcacgt taagggattt tggtcatgca ttctaggtac taaaacaatt catccagtaa 6780
aatataatat tttattttct cccaatcagg cttgatcccc agtaagtcaa aaaatagctc 6840
gacatactgt tcttccccga tatcctccct gatcgaccgg acgcagaagg caatgtcata 6900
ccacttgtcc gccctgccgc ttctcccaag atcaataaag ccacttactt tgccatcttt 6960
cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt 7020
ttccgtcttt aaaaaatcat acagctcgcg cggatcttta aatggagtgt cttcttccca 7080
gttttcgcaa tccacatcgg ccagatcgtt attcagtaag taatccaatt cggctaagcg 7140
gctgtctaag ctattcgtat agggacaatc cgatatgtcg atggagtgaa agagcctgat 7200
gcactccgca tacagctcga taatcttttc agggctttgt tcatcttcat actcttccga 7260
gcaaaggacg ccatcggcct cactcatgag cagattgctc cagccatcat gccgttcaaa 7320
gtgcaggacc tttggaacag gcagctttcc ttccagccat agcatcatgt ccttttcccg 7380
ttccacatca taggtggtcc ctttataccg gctgtccgtc atttttaaat ataggttttc 7440
attttctccc accagcttat ataccttagc aggagacatt ccttccgtat cttttacgca 7500
gcggtatttt tcgatcagtt ttttcaattc cggtgatatt ctcattttag ccatttatta 7560
tttccttcct cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg 7620
aactccaatt cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca 7680
aagttgtttt caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg 7740
tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc 7800
atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga 7860
gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc 7920
ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg 7980
gcaggatata ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga 8040
cgtttttaat gtactgaatt aacgccgaat taattcgggg gatctggatt ttagtactgg 8100
attttggttt taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat 8160
actaagggtt tcttatatgc tcaacacatg agcgaaaccc tataggaacc ctaattccct 8220
tatctgggaa ctactcacac attattatgg agaaactcga gcttgtcgat cgacagatcc 8280
ggtcggcatc tactctattt ctttgccctc ggacgagtgc tggggcgtcg gtttccacta 8340
tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt 8400
gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa 8460
gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg 8520
agcatatacg cccggagtcg tggcgatcct gcaagctccg gatgcctccg ctcgaagtag 8580
cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg 8640
tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg 8700
tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag 8760
tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg 8820
tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc 8880
catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta 8940
agatcggccg cagcgatcgc atccatagcc tccgcgaccg gttgtagaac agcgggcagt 9000
tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc 9060
aggctctcgc taaactcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca 9120
aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg 9180
acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc 9240
tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg 9300
gtgagttcag gctttttcat atctcattgc cccccgggat ctgcgaaagc tcgagagaga 9360
tagatttgta gagagagact ggtgatttca gcgtgtcctc tccaaatgaa atgaacttcc 9420
ttatatagag gaaggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg 9480
gagatatcac atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg 9540
atgctcctcg tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg 9600
atagcctttc ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc 9660
cttttgatga agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc 9720
ctttgttgaa aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg 9780
gagtagacga gagtgtcgtg ctccaccatg ttatcacatc aatccacttg ctttgaagac 9840
gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga 9900
ccactgtcgg cagaggcatc ttgaacgata gcctttcctt tatcgcaatg atggcatttg 9960
taggtgccac cttccttttc tactgtcctt ttgatgaagt gacagatagc tgggcaatgg 10020
aatccgagga ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctttggtct 10080
tctgagactg tatctttgat attcttggag tagacgagag tgtcgtgctc caccatgttg 10140
gcaagctgct ctagccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 10200
gcagc 10205
<210> 3
<211> 9674
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 60
cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 120
aatttcacac aggaaacagc tatgaccatg attacgaatt cgagctcggt acccggggat 180
cctctagagt cgacctgcag gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt 240
gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 300
agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 360
aatggcgaat gctagagcag cttgagcttg gatcagattg tcgtttcccg ccttcagttt 420
agcttcatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt aaagactggc 480
gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt cgtcaacatg 540
gtggagcacg acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa 600
agggcaattg agacttttca acaaagggta atatccggaa acctcctcgg attccattgc 660
ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc 720
catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa 780
gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca 840
aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat 900
ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag aacacggggg 960
actcttgacc atggctgcag ccccaaacac acgctcggac gcatattaca catgttcata 1020
cacttaatac tcgctgtttt gaattgatgt tttaggaata tatatgtaga atcagcgatc 1080
atcattaacc ttcacaggtc gtgatatgat tcaattagct tccgactcat tcatccaaat 1140
accgagtcgc caaaattcaa actagactcg ttaaatgaat gaatgatgcg gtagacaaat 1200
tggatcattg attctctttg atggttaaag atgatcgctc attctctctt ttgtattcca 1260
attttcttga ttaatctttc ctgcacaaaa acatgcttga tccactaagt gacatatatg 1320
ctgccttcgt atatatagtt ctggtaaaat taacattttg ggtttatctt tatttaaggc 1380
atcgggtgac cagctcgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa 1440
gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta 1500
agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta 1560
gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg 1620
ataaattatc gcgcgcggtg tcatctatgt tactagatcg ggaattaaac tatcagtgtt 1680
tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga ataacggata 1740
tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg ccaaccacag 1800
ggttcccctc gggatcaaag tactttgatc caacccctcc gctgctatag tgcagtcggc 1860
ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt cgcacaagtc ctaagttacg 1920
cgacaggctg ccgccctgcc cttttcctgg cgttttcttg tcgcgtgttt tagtcgcata 1980
aagtagaata cttgcgacta gaaccggaga cattacgcca tgaacaagag cgccgccgct 2040
ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg acttgaccaa ccaacgggcc 2100
gaactgcacg cggccggctg caccaagctg ttttccgaga agatcaccgg caccaggcgc 2160
gaccgcccgg agctggccag gatgcttgac cacctacgcc ctggcgacgt tgtgacagtg 2220
accaggctag accgcctggc ccgcagcacc cgcgacctac tggacattgc cgagcgcatc 2280
caggaggccg gcgcgggcct gcgtagcctg gcagagccgt gggccgacac caccacgccg 2340
gccggccgca tggtgttgac cgtgttcgcc ggcattgccg agttcgagcg ttccctaatc 2400
atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc gaggcgtgaa gtttggcccc 2460
cgccctaccc tcaccccggc acagatcgcg cacgcccgcg agctgatcga ccaggaaggc 2520
cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc gctcgaccct gtaccgcgca 2580
cttgagcgca gcgaggaagt gacgcccacc gaggccaggc ggcgcggtgc cttccgtgag 2640
gacgcattga ccgaggccga cgccctggcg gccgccgaga atgaacgcca agaggaacaa 2700
gcatgaaacc gcaccaggac ggccaggacg aaccgttttt cattaccgaa gagatcgagg 2760
cggagatgat cgcggccggg tacgtgttcg agccgcccgc gcacgtctca accgtgcggc 2820
tgcatgaaat cctggccggt ttgtctgatg ccaagctggc ggcctggccg gccagcttgg 2880
ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag taaaacagct 2940
tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata cgcaagggga 3000
acgcatgaag gttatcgctg tacttaacca gaaaggcggg tcaggcaaga cgaccatcgc 3060
aacccatcta gcccgcgccc tgcaactcgc cggggccgat gttctgttag tcgattccga 3120
tccccagggc agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc taaccgttgt 3180
cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc gcgacttcgt 3240
agtgatcgac ggagcgcccc aggcggcgga cttggctgtg tccgcgatca aggcagccga 3300
cttcgtgctg attccggtgc agccaagccc ttacgacata tgggccaccg ccgacctggt 3360
ggagctggtt aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg cctttgtcgt 3420
gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc tggccgggta 3480
cgagctgccc attcttgagt cccgtatcac gcagcgcgtg agctacccag gcactgccgc 3540
cgccggcaca accgttcttg aatcagaacc cgagggcgac gctgcccgcg aggtccaggc 3600
gctggccgct gaaattaaat caaaactcat ttgagttaat gaggtaaaga gaaaatgagc 3660
aaaagcacaa acacgctaag tgccggccgt ccgagcgcac gcagcagcaa ggctgcaacg 3720
ttggccagcc tggcagacac gccagccatg aagcgggtca actttcagtt gccggcggag 3780
gatcacacca agctgaagat gtacgcggta cgccaaggca agaccattac cgagctgcta 3840
tctgaataca tcgcgcagct accagagtaa atgagcaaat gaataaatga gtagatgaat 3900
tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg acgccgtgga 3960
atgccccatg tgtggaggaa cgggcggttg gccaggcgta agcggctggg ttgtctgccg 4020
gccctgcaat ggcactggaa cccccaagcc cgaggaatcg gcgtgacggt cgcaaaccat 4080
ccggcccggt acaaatcggc gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg 4140
cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag 4200
cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga 4260
ttaggaagcc gcccaagggc gacgagcaac cagatttttt cgttccgatg ctctatgacg 4320
tgggcacccg cgatagtcgc agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg 4380
accgacgagc tggcgaggtg atccgctacg agcttccaga cgggcacgta gaggtttccg 4440
cagggccggc cggcatggcc agtgtgtggg attacgacct ggtactgatg gcggtttccc 4500
atctaaccga atccatgaac cgataccggg aagggaaggg agacaagccc ggccgcgtgt 4560
tccgtccaca cgttgcggac gtactcaagt tctgccggcg agccgatggc ggaaagcaga 4620
aagacgacct ggtagaaacc tgcattcggt taaacaccac gcacgttgcc atgcagcgta 4680
cgaagaaggc caagaacggc cgcctggtga cggtatccga gggtgaagcc ttgattagcc 4740
gctacaagat cgtaaagagc gaaaccgggc ggccggagta catcgagatc gagctagctg 4800
attggatgta ccgcgagatc acagaaggca agaacccgga cgtgctgacg gttcaccccg 4860
attacttttt gatcgatccc ggcatcggcc gttttctcta ccgcctggca cgccgcgccg 4920
caggcaaggc agaagccaga tggttgttca agacgatcta cgaacgcagt ggcagcgccg 4980
gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg 5040
agtacgattt gaaggaggag gcggggcagg ctggcccgat cctagtcatg cgctaccgca 5100
acctgatcga gggcgaagca tccgccggtt cctaatgtac ggagcagatg ctagggcaaa 5160
ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc tgtggatagc acgtacattg 5220
ggaacccaaa gccgtacatt gggaaccgga acccgtacat tgggaaccca aagccgtaca 5280
ttgggaaccg gtcacacatg taagtgactg atataaaaga gaaaaaaggc gatttttccg 5340
cctaaaactc tttaaaactt attaaaactc ttaaaacccg cctggcctgt gcataactgt 5400
ctggccagcg cacagccgaa gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc 5460
tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg ccgctcaaaa atggctggcc 5520
tacggccagg caatctacca gggcgcggac aagccgcgcc gtcgccactc gaccgccggc 5580
gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 5640
atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 5700
cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt 5760
agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag 5820
tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc 5880
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5940
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 6000
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 6060
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 6120
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 6180
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 6240
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 6300
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 6360
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 6420
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 6480
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 6540
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 6600
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 6660
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6720
tggtcatgca ttctaggtac taaaacaatt catccagtaa aatataatat tttattttct 6780
cccaatcagg cttgatcccc agtaagtcaa aaaatagctc gacatactgt tcttccccga 6840
tatcctccct gatcgaccgg acgcagaagg caatgtcata ccacttgtcc gccctgccgc 6900
ttctcccaag atcaataaag ccacttactt tgccatcttt cacaaagatg ttgctgtctc 6960
ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt ttccgtcttt aaaaaatcat 7020
acagctcgcg cggatcttta aatggagtgt cttcttccca gttttcgcaa tccacatcgg 7080
ccagatcgtt attcagtaag taatccaatt cggctaagcg gctgtctaag ctattcgtat 7140
agggacaatc cgatatgtcg atggagtgaa agagcctgat gcactccgca tacagctcga 7200
taatcttttc agggctttgt tcatcttcat actcttccga gcaaaggacg ccatcggcct 7260
cactcatgag cagattgctc cagccatcat gccgttcaaa gtgcaggacc tttggaacag 7320
gcagctttcc ttccagccat agcatcatgt ccttttcccg ttccacatca taggtggtcc 7380
ctttataccg gctgtccgtc atttttaaat ataggttttc attttctccc accagcttat 7440
ataccttagc aggagacatt ccttccgtat cttttacgca gcggtatttt tcgatcagtt 7500
ttttcaattc cggtgatatt ctcattttag ccatttatta tttccttcct cttttctaca 7560
gtatttaaag ataccccaag aagctaatta taacaagacg aactccaatt cactgttcct 7620
tgcattctaa aaccttaaat accagaaaac agctttttca aagttgtttt caaagttggc 7680
gtataacata gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg cagcaacgct 7740
ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc atccgtgttt caaacccggc 7800
agcttagttg ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg ccgccttaca 7860
acggctctcc cgctgacgcc gtcccggact gatgggctgc ctgtatcgag tggtgatttt 7920
gtgccgagct gccggtcggg gagctgttgg ctggctggtg gcaggatata ttgtggtgta 7980
aacaaattga cgcttagaca acttaataac acattgcgga cgtttttaat gtactgaatt 8040
aacgccgaat taattcgggg gatctggatt ttagtactgg attttggttt taggaattag 8100
aaattttatt gatagaagta ttttacaaat acaaatacat actaagggtt tcttatatgc 8160
tcaacacatg agcgaaaccc tataggaacc ctaattccct tatctgggaa ctactcacac 8220
attattatgg agaaactcga gtcaaatctc ggtgacgggc aggaccggac ggggcggtac 8280
cggcaggctg aagtccagct gccagaaacc cacgtcatgc cagttcccgt gcttgaagcc 8340
ggccgcccgc agcatgccgc ggggggcata tccgagcgcc tcgtgcatgc gcacgctcgg 8400
gtcgttgggc agcccgatga cagcgaccac gctcttgaag ccctgtgcct ccagggactt 8460
cagcaggtgg gtgtagagcg tggagcccag tcccgtccgc tggtggcggg gggagacgta 8520
cacggtcgac tcggccgtcc agtcgtaggc gttgcgtgcc ttccaggggc ccgcgtaggc 8580
gatgccggcg acctcgccgt ccacctcggc gacgagccag ggatagcgct cccgcagacg 8640
gacgaggtcg tccgtccact cctgcggttc ctgcggctcg gtacggaagt tgaccgtgct 8700
tgtctcgatg tagtggttga cgatggtgca gaccgccggc atgtccgcct cggtggcacg 8760
gcggatgtcg gccgggcgtc gttctgggct catggtagac tcgagagaga tagatttgta 8820
gagagagact ggtgatttca gcgtgtcctc tccaaatgaa atgaacttcc ttatatagag 8880
gaaggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg gagatatcac 8940
atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg atgctcctcg 9000
tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg atagcctttc 9060
ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc cttttgatga 9120
agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc ctttgttgaa 9180
aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg gagtagacga 9240
gagtgtcgtg ctccaccatg ttatcacatc aatccacttg ctttgaagac gtggttggaa 9300
cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg 9360
cagaggcatc ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac 9420
cttccttttc tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga 9480
ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg 9540
tatctttgat attcttggag tagacgagag tgtcgtgctc caccatgttg gcaagctgct 9600
ctagccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 9660
cgacaggttt cccg 9674
<210> 4
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 4
ugguuaaaga ugaucgcuca u 21
Claims (9)
1.一种miRNA，其特征在于，其靶向大豆花叶病毒基因组RNA的P1蛋白编码区，miRNA的序列为5’- UGGUUAAAGAUGAUCGCUCAU-3’。
2.编码权利要求1所述miRNA的核酸分子。
3.含有权利要求2所述核酸分子的生物材料，其特征在于，所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体或工程菌。
4.抗大豆花叶病毒的制剂，其特征在于，有效成分为权利要求1所述的miRNA或权利要求2所述的核酸分子或权利要求3所述的生物材料。
5.权利要求1所述的miRNA或权利要求2所述的核酸分子或权利要求3所述的生物材料的以下任一应用：
1）用于制备转基因植物；
2）用于提高植物抗大豆花叶病毒的能力；
3）用于制备抗大豆花叶病毒的制剂或试剂盒；
所述植物为本氏烟或大豆。
6.抗大豆花叶病毒的转基因植物构建方法，其特征在于，所述方法包括：构建含有编码权利要求1所述miRNA的DNA序列的植物表达载体，转化植物，筛选阳性转基因植株；
所述植物为本氏烟或大豆。
7.根据权利要求6所述的方法，其特征在于，编码所述miRNA的DNA序列如SEQ ID NO:1所示。
8.根据权利要求6所述的方法，其特征在于，所述植物表达载体为pCambia1301或pCambia3301。
9.根据权利要求6所述的方法，其特征在于，采用农杆菌介导法转化植物。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567696A (zh) * | 2016-03-14 | 2016-05-11 | 吉林省农业科学院 | 一种培育抗大豆花叶病毒转基因植物的方法 |
| CN112538488A (zh) * | 2020-12-25 | 2021-03-23 | 中国农业科学院植物保护研究所 | 本氏烟NbSMG7基因在调控植物抗病毒中的应用及转基因植物培育方法 |
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| CN105567696A (zh) * | 2016-03-14 | 2016-05-11 | 吉林省农业科学院 | 一种培育抗大豆花叶病毒转基因植物的方法 |
| CN112538488A (zh) * | 2020-12-25 | 2021-03-23 | 中国农业科学院植物保护研究所 | 本氏烟NbSMG7基因在调控植物抗病毒中的应用及转基因植物培育方法 |
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| Detection and evolutionary analysis of soybean miRNAs responsive to soybean mosaic virus;Xianchao Yin等;《Planta 》;20130118;第237卷;第1213-1225页 * |
| Soybean mosaic virus: a successful potyvirus with a wide distribution but restricted natural host range;M. R. HAJIMORAD等;《MOLECULAR PLANT PATHOLOGY》;20181231;第19卷(第7期);第1563-1579页 * |
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