CN110616202A - 柑橘黄化脉明病毒弱毒分离株及其应用 - Google Patents
柑橘黄化脉明病毒弱毒分离株及其应用 Download PDFInfo
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Abstract
本发明公开了一种柑橘黄化脉明病毒弱毒分离株，该弱毒株相对于强毒株产生的基因突变点是：TGB区域的T5306→C，A5482→G，T5792→C，A6058→G，C6096→T，以及位于CP基因上的T6817→C。还公开了该弱毒分离株在防治柑橘黄化脉明病毒强毒株系侵染方面的应用。本发明获得了柑橘黄化脉明病毒弱毒分离株AY001，AY001可有效保护植株免受CYVCV强毒株系的侵染。
Description
技术领域
本发属于病毒技术领域，具体涉及一种柑橘黄化脉明病毒弱毒分离株及其应用。
背景技术
交叉保护(Mild strain cross protection，MSCP)是指植物受某一病毒株系(通常是弱毒株系)侵染后，可以免受同种病毒的不同株系或亲缘关系较近的另一种病毒(即强毒株或挑战毒株)的侵染。植物病毒所表现的交叉保护作用程度不同，若直接接种挑战毒株本应该表现局部坏死症状，当受先接种的病毒干扰后，挑战毒株无症状表现就称作完全保护作用；挑战毒株略有增殖和显出轻症，就属于不完全保护作用；如果与挑战毒株单独侵染时的症状完全相同，就属于完全无保护作用。目前，交叉保护作用主要表现在抑制挑战病毒的复制、限制挑战病毒的移动、延迟植株发病的时间和症状减轻或者不发病四个方面。在生产实践中，交叉保护作用的前提是获得弱毒株系。
获得弱毒株系的传统方法主要有三种：从自然发病的植株中选择，在发病群体中生长状况较好的植株就最有可能是感染了弱毒株系而受到了保护作用，防治柑橘衰退病毒病的弱毒株系就是如此发现的；通过高温诱变获得；化学诱变获得。随着植物病毒侵染性克隆构建技术的发展，研究者可以在明确病毒致病力和基因组功能的基础上，通过定点突变获得弱毒株系。用此方法构建的ZYMV、PRSV、三叶草黄脉病毒(Clover yellow vein virus，CIYVV)和菜豆黄花叶病毒(Bean yellow mosaic virus，BYMV)等弱毒株系已广泛应用于实际生产中。此外，利用植物病毒侵染性克隆表达病毒的基因也能介导对病毒的交叉保护作用。例如，Culver等将TMV的CP基因序列插入PVX侵染性cDNA克隆中，重组质粒接种烟草后能对随后TMV挑战毒株接种产生保护作用。
柑橘黄化脉明病毒(Citrus yellow clearing vein virus，CYVCV)作为2009年我国发现侵染柑橘的一种新病毒，会引起柠檬和酸橙嫩叶脉明、黄化、叶片反卷和皱缩等症状，严重时可导致嫩叶脱落、叶脉坏死，造成树势衰弱、果实产量下降，有时甚至绝收；近几年，该病毒在我国柑橘产区的发生呈现逐年上升趋势。CYVCV不仅可以侵染柠檬、酸橙、香橼，产生严重为害，而且可以侵染甜橙、宽皮柑橘、葡萄柚等柑橘种类。同时，CYVCV在自然界具有高效的传播虫媒，在我国的绝大多数柑橘产区均已检测到CYVCV病毒的存在。因此，CYVCV具有暴发流行的巨大风险，已经成为影响我国柑橘产业尤其是柠檬产业的一个重要问题。
CYVCV为正义单链RNA病毒，病毒粒子呈弯曲线状，属于甲型线性病毒科病毒科(Alphaflexiviridae)印度柑橘病毒属(Mandarivirus)。CYVCV可通过柑橘粉虱和绣线菊蚜(Aphis spiraecola传播，也可通过嫁接以及田间农事操作工具传播。CYVCV基因组包含有6个ORFs，其中ORF2、ORF3和ORF4组成了三基因连锁结构(TGB)。目前，该病的防治措施主要是应用无毒苗木，严格防控传播虫媒并在农事操作中对农事工具进行严格消毒等。利用弱毒株交叉保护是一种具有前景的可行性防控措施。目前，弱毒株交叉保护已经成功地应用于烟草花叶病毒(Rast et al.,1972)、黄瓜花叶病毒(Tien et al.,1991)、可可枝肿病毒(Posnette et al.,1955)、番木瓜环斑病毒(Yeh et al.,1984)等引起的病毒病防治。柑桔衰退病毒弱毒系也成功运用于保护巴西佩拉甜橙(Muller et al.,1998)、日本八朔柑(Sasaki et al,1979)以及美国(Cost and Muller,1980)、澳大利亚(Broadbent et al.,1991)和南非(Van et al.,2000)的葡萄柚等。但针对CYVCV的弱毒系筛选及其在交叉保护中的应用尚未见报道。
发明内容
本发明所要解决的技术问题在于针对上述问题，提供一种柑橘黄化脉明病毒弱毒分离株及其应用。
本发明的技术方案如下：
一种柑橘黄化脉明病毒弱毒分离株，该弱毒株相对于强毒株产生的基因突变点是：TGB区域的T5306→C，A5482→G，T5792→C，A6058→G，C6096→T，以及位于CP基因上的T6817→C。
上述的柑橘黄化脉明病毒弱毒分离株在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏登记号为CGMCC No.18177。
本发明还提供了柑橘黄化脉明病毒弱毒分离株在防治柑橘黄化脉明病毒强毒株系侵染方面的应用。
本发明的有益效果是：获得了柑橘黄化脉明病毒弱毒分离株AY001，AY001可有效保护植株免受CYVCV强毒株系的侵染。
附图说明
图1为尤力克柠檬接种CYVCV弱毒株系AY001后的叶片表现图，其中，A为接种负对照，B为接种正对照，C为接种AY001。
图2为弱毒株与强度株分段多重序列比对与MFE二级结构预测图。
图3为弱毒株与强度株分段多重序列比对与MFE二级结构预测图。
图4为弱毒株与强度株分段多重序列比对与MFE二级结构预测图。
具体实施方式
实施例1构建CYVCV侵染性克隆
按照中国专利申请CN201810367775.0中实施例4中公开的方法构建CYVCV侵染性克隆，抽提酵母质粒并转化大肠杆菌，抽提质粒进行酶切鉴定，结果表明获得了10个CYVCV全长cDNA克隆，其中7个是四川安岳来源的分离株构建的：CYVCV-AY222、CYVCV-AY221、CYVCV-AY212、CYVCV-AY112、CYVCV-AY142、CYVCV-AY141和CYVCV-AY001，另外3个是来源于重庆的分离株构建的：CYVCV-CQ451、CYVCV-CQ331和CYVCV-CQ101。将这10个CYVCV全长cDNA克隆质粒命名为pCY-CYVCV-AY/CQ，具体见表1。
实施例2 CYVCV弱毒系筛选
将实施例1获得的10个CYVCV全长cDNA克隆，提取质粒，转化农杆菌C58C1并通过农杆菌真空浸润法接种指示植物尤力克柠檬，每克隆侵染10株，并以pCY空载体(即为中国专利申请201810367775.0中的载体pCY)为负对照，以强毒株系为正对照。接种植株在光照16h，23℃；黑暗8h，20℃的循环条件下培养。接种后15-120天连续观察症状表现，并于接种后40天进行CYVCV的RT-PCR以及Northern Blot杂交检测，二者均为阳性，且正负对照表现符合预期(即正对照表现出强烈的叶片畸形，黄化，叶脉透明，负对照不表现症状)，根据植株表现CYVCV侵染症状的程度筛选弱毒株系，即不表现CYVCV侵染症状或进表现轻微的CYVCV侵染症状的分离株为弱毒株系。
一、CYVCV的RT-PCR鉴定
抽提尤力克柠檬叶片总RNA，反转录为cDNA，以2×Taq Master Mix(Novoprotein)试剂盒进行PCR扩增检测，反应体系如下：
| 试剂 | 使用量 |
| 2×Taq Master Mix | 10μL |
| DN/RNase-free ddH<sub>2</sub>O | 7μL |
| CYVCV-614F(10μM) | 1μL |
| CYVCV-614R(10μM) | 1μL |
| cDNA | 1μL |
引物CYVCV-614F序列为：5’-TACCGCAGCTATCCATTTCC-3’(SEQ ID NO:1)，引物CYVCV-614R序列为：5’-GCAGAAATCCCGAACCACTA-3’(SEQ ID NO:2)。
PCR扩增条件：94℃3min；(94℃30s，55℃30s，72℃45s)×36；72℃5min。
二、Northern Blot杂交检测
采用植物RNA快速提取试剂盒提取植物组织样品RNA并反转录为cDNA，详细步骤见说明书。
探针制备：
1)PCR反应体系如下
引物序列为：
5982-F:5'-TCACCAAAGGAAGGACTA-3'(SEQ ID NO:3)；
5982-R:5'-AAAATGGAAACTGAAAGC-3'(SEQ ID NO:4)。
2)PCR运行程序如下：94℃预变性5min；(94℃30s，51℃30s，72℃30s)×35；72℃7min。
接着进行后续的转膜、杂交、洗膜、信号检测步骤。
三、结果
10个CYVCV克隆的鉴定结果如表1所示，获得1个CYVCV弱毒株系AY001，获得了6个具有致病力的侵染性克隆，其中，AY221的侵染性和致病力最强。植株症状表现如图1所示。
表1利用尤力克柠檬筛选CYVCV弱毒株系
实施例3CYVCV弱毒株的序列分析
对实施例2得到的弱毒株AY001进行了序列测定，其核酸序列如SEQ ID NO:5所示。
以致病性强弱和侵染性有无将四川安岳来源的7个CYVCV分离物分为A、B两组组，A组为有侵染和致病性的CYVCV-AY212、CYVCV-AY221、CYVCV-AY222，B组为弱毒或无侵染性的CYVCV-AY001、CYVCV-AY141、CYVCV-AY142、CYVCV-AY112。以Clustal W法将这这两组分离物的全基因组cDNA与CYVCV云南分离物(CYVCV-YN)cDNA进行多重序列比对分析，寻找组间统一差异位点。结果共筛选到了6个统一差异碱基位点，这些位点分别位于CYVCV基因组的三基因连锁模块(Triple gene block，TGB)和外壳蛋白(Coatprotein,CP)基因上。对差异位点附近的cDNA序列使用RNAfold web server进行mRNA二级结构预测，并绘制MFE二级结构图(Minimum free energy secondary structures)。
与强毒株CYVCV-AY221(其核酸序列如SEQ ID NO:6所示)的多重序列比对分析，AY001有6个差异碱基位点，这些位点分别位于CYVCV基因组的三基因连锁模块(Triplegene block，TGB)和外壳蛋白(Coatprotein,CP)基因上。分别为位于TGB区域的T5306→C，A5482→G，T5792→C，A6058→G，C6096→T，以及位于CP基因上的T6817→C；对强弱毒分离物序列的mRNA二级结构分析结果，显示A5482→G，A6058→G，C6096→T和T6817→C的突变对其二级结构有明显改变(如图2～4所示)。推测这些位点的突变或包含突变位点的TGB和CP基因可能影响了CYVCV的致病力。
实施例4 CYVCV弱毒株AY001的交叉保护效果
1)将CYVCV弱毒株AY001通过农杆菌介导的真空浸润法接种CYVCV敏感品种尤里克柠檬60株，同时设尤里克柠檬健康对照60株，培养在防虫网室，自然条件生长；
2)3个月后，将携带CYVCV强毒株AY221的毒源植株60株，按随机分布放置至步骤1)的网室中；
3)放入无毒柑橘粉虱2000头至上述网室，每5天对所有植株进行1次晃动，20天后喷杀虫剂杀灭所有粉虱；
4)定期查看健康对照及弱毒株接种植株发病情况，12个月后，进行症状统计及分子检测，评估交叉保护效果。
结果显示，健康对照60株中RT-PCR阳性植株32株，感染率53％，表现典型CYVCV侵染症状的有28株，占47％；弱毒株AY001接种植株只有3株表现出CYVCV侵染症状，占5％，并且与对照相比叶片畸形较轻。表明CYVCV弱毒株AY001对相应强度株具有良好的保护效果。
将CYVCV弱毒株AY001，由中国微生物保藏管理委员会普通微生物中心(简称CGMCC)进行保藏，地址为北京市朝阳区北辰西路1号院3号，保藏日期为2019年7月15号，保藏编号为CGMCC NO.18177，分类命名为柑橘黄化脉明病毒弱毒株。
序列表
<110> 西南大学
<120> 柑橘黄化脉明病毒弱毒分离株及其应用
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<400> 1
taccgcagct atccatttcc 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<400> 2
gcagaaatcc cgaaccacta 20
<210> 3
<211> 18
<212> DNA
<213> 人工序列
<400> 3
tcaccaaagg aaggacta 18
<210> 4
<211> 18
<212> DNA
<213> 人工序列
<400> 4
aaaatggaaa ctgaaagc 18
<210> 5
<211> 7530
<212> DNA
<213> 人工序列
<400> 5
gaaaagcaaa cataaccaac acacacccaa aattgtgatc atcgtctctg catcaacagc 60
tcagctaaga accggagaaa tggccactat taggggtgcc atcgaacgca ttacagacac 120
caccgtacgc actacactcc aagaggaggc atgccgccaa atacgcgctg aacttaaaac 180
cgtcgaacac atcaaccgtt acgctataca gcctgacgcc gccgacgcac ccgaacacct 240
aggcattggt tctaacccct tctctgtggc actgcacacg cacggtgcct gcaaagcgat 300
tgagaaccaa ttactctatg tagttggtac cttactccca aaagaacgtg tcaccatgct 360
cttccttaag aaaagcaagc tcaacatcat aaaacgctgc cctaaattcc aagacatctt 420
cctgaaccaa catattgagc cacgcgacgt ttctaggtac tgcgacttta acgtccaatc 480
cacctccacc agcataccca cacgaacggc ttacatcagt gacacactcc actttatgga 540
ccgtagagac ctagtccgac tattcctaaa cagcccaaat ctagagactc tctacgccac 600
cattgtcctc ccagtcgagg ctgcctacaa acaaccgagc cgctacccag agatatacca 660
gattaactat gacttcgacg gtttccagta catacctggg ggccatggtg gcggcgccta 720
ccaccacgag tttgaacagc tgcaatggtt agacactggc catatccact ggcgtgggcc 780
cgactgtaaa gagattcgca tgacaattac cgcccaaatg ctcgaaagct taggtgcgaa 840
tcatcttttc tgcttcaaac gtgggaaact actcacccca cgtgtacgca ctttcggccg 900
agatacccaa gtgctcttgc ccaaaatctt ccgcccggcc gacaaaaact ttaacagggc 960
aataccacta accctagcta acaaactact cctgtacgct aaatccatta acacagtgac 1020
cttccgcgac gtcgttgcca agacacgcca acttatgaag gacaaggagc ttgaaacata 1080
caccggcaac gacctgttgc atatggccaa ctatctcttc gctgttggag cactgtcagg 1140
agttaactcc tatgaccaat tgcttggcct atccgcctgg gaagcctgca ccgtccgtat 1200
caagaacacc ataacaaatc tttgggaaaa gatcgccgga aagaaagagt tcggcaagct 1260
attggaggcc cttgagtggg aaacattcac atactcacgt caagtgaccg aattcaccgt 1320
cacgggacta cccgccctgc tccccctccc agacatctca gaccaagaag agattttcgc 1380
ccagcaggac gagttagaca aaatcactgc cggcgctacc aagatacgga ccattgacat 1440
tatgcgggga caagctaacc gcgataaccc aaaaactccg accaactcaa caccggccca 1500
gggaccccaa ctgcccgata cccgcgaccc aatcgaccaa gctgcctcca aagagctcgt 1560
caccaagctt caaaaaaaca aacgcattta catccaagac gacgggccgg aataccttat 1620
gggacacacg gctgaagtcc ccgcctggta tctcgaacaa gatgatacca ccactagact 1680
caaaaaccgc tgcgcttggt tcttcggccc acccacctac agatatgggc acaacgacat 1740
tgagtacacc accatagaat actacccttg ggtcgagcgt attggagcca ttttcggcaa 1800
gtacaacacc tgtctcgccc aaacttacga cgccggtgcc cgtattgggt accacgctga 1860
cgacgaagac tgctacgacc ccgacgtcac cgtggtcacc gtcaacctca ctgggaacgc 1920
caccttcctg ctcaagaccc caaccggcac taggacctgg aagcttaaac ctggtgactt 1980
cattatcatg aaacccggtg cccaaaggtg cacaaaacat gccattagag attgcacaac 2040
taaccgcacg tccttaacct tcagatggca ggcccgcact tgccccacta acctcagaaa 2100
gatcactaac ctacccaaag ccaccaacca accccaaacc actgaatggc gtcctgtaac 2160
taaaccacgc cctagcacca ccgcttccag cgacacccag accccacttg tcatagacca 2220
ggaacgaggg tataccacca cctccgacgt gacaccaacg attaggctgc ccgccgaaaa 2280
tggtaataac gccggtgctg gaccctcctc cgcattaacc ctcgccgacc tgaatgataa 2340
ccaagcgccg accactaaca aaaggaaaga gaaacttgag gaaatggtcg aaactggccc 2400
aactatcatg gaacgcttcc tcaacaccgt tcaggaacca acacaagatt tatgggactc 2460
cacctcggaa tccgccgcga gctacctcgc tgaaggcctc ggcatctctg ccatccaagc 2520
actcccttgg gcacctcact tggaactcat caatgcatta ggcttccagg gcactgaaag 2580
gcaatgcggc ccaaacaact gtctcatatg gcccatcacg cattacagag aattgcccag 2640
aagctacaat gtcgaagccc ctccggaagt gcttgagctg ctcgactgcg tcaatagata 2700
ccccactgac gtcccaatgc tcaaaactcg agccgccgcc ttcggttcag acgtcaagaa 2760
tctacgcata agggcacttg ttaagaacca ggacaaacag tggcgcgcat cactcgcact 2820
actttgtgag gaaaatgaac acctcctccc taccactgtt atacacggcg ctggtggctc 2880
cgggaaatcc catttactcc aacagtgggt ggcgtctact gagcgcggaa atgtagtcac 2940
catcttgccc actatcgaac tccttcgaga ctggcaaaac aaatgtccac acgcaccgaa 3000
agagactttc aaaaccttcg agaaagcgct catccagaac agcgcccccg tggttatcat 3060
ggacgactac tctaaacttc ccccagggta tattgaagcc tacgtcagcc tcaagggcca 3120
gtgcaaactg ctcatactca ctggagaccc tagacagagc cactaccacg aggagaaccc 3180
tgaggcctta atctccactc ttgaccccgc cacggactac tttggcaaat tttgcgcata 3240
caacatcaac gccactcata gaaatgccaa aaccttcgcc aatgcgcttg gggtgtactc 3300
tgaaaaagaa atccccacca ctatcacctg ctcatcctac cagaaaagcg gttggccaac 3360
acttgtccca tccatcctta aaagaactgc gctcaacgac atggggcagc gctcactaac 3420
ctacgcaggc tgtcagggac tcaccacccc aaaagagcag atagttcttg acaatgccac 3480
accactatgc tctgacaagg tcatgtacac cgcactatcc agagcggtgg accaaatcca 3540
cttcttcaac actggcccaa atcacactga ttactgggaa aagatgaacg ccacaccttt 3600
cctcaaaact ttcatcgacc acacacgtaa ggagaccttt gctgagcacc aacctgccga 3660
acccacagtg cgtgagtacg caccagcaac gcatttccca ccggctaacg agaacctagc 3720
actagagccg tgggttgaac cgctgactga taaacactcg cgggaactct tccactctgc 3780
cttaggccac agcaactgcg tccagactga aaacaccgta gtccaacttt ttccccacca 3840
acaggccaaa gacgaaacac tcttctggaa aacaatagat gccagaatca aggtaaccac 3900
cccggaagag aacatcaggg cttgcagcat gaccactgat attggcgaca tcttgttcct 3960
aaattacaaa gaagccatgg gcttaccaca agaccccata cccttcgaac aggcactctg 4020
ggactcttgc caagccgagg tgcaactaac ctacctcagc aagcctctgg cagcactcgc 4080
aaacgccgcc caaagacaag accccgactt tgactccaac aaaatccaac tcttcctgaa 4140
atcacaatgg gtcaagaaag tcgagaagat gggctgcctt aagatcaaac ccggccaaac 4200
tattgcgtcc ttcatgcaac agactgtcat gctatacgga actatggctc ggtacatgcg 4260
ccgcatccgc acctcacttt gcccacccga aattatgatc aactgcgaga ccaacccaaa 4320
ccaaatcgga acatgggtgc gtgagtattg gaacttcaac acccagagcc atgagaatga 4380
ctttgaagcg ttcgaccagt cccaagacgc caacatgtta caattcgagc tcattaaggc 4440
caaataccac tcaatccccg aggagatcat cgccgggtat cgacacctga aatgcaacgc 4500
ccacatcttc cttgggacta tatcgatcat gaggttgtct ggtgaagggc cgaccttcgg 4560
cgccaacaca gagtgttcca ttgcatacaa ccacacaagg tattttgtgc ctaaaggcac 4620
cgcccagctc tacgccggag acgactctgc atgcgcgtcg cccctctctg aaaaacccag 4680
cttccaacac atctcaccag aactcagcct caagtctaag gccaagatta gaacccaaca 4740
gaaaggagac tatgcaacct tctgtggctg gctcatcact ccaaaaggcc tgatcaaaaa 4800
ccccacccag ctttacgcct cctggttatt ggctaagcac aacaaagatc tcgcggacgt 4860
ggccagaaac tacgccttgg acttacgcat tgcctatcaa cttaaagacg agctctatga 4920
gttattatca cctgaagaac ttgaccacca ccaactgctc gtccgagaga tgatcaagca 4980
aaaaatgggc catctcctca acctccctga ggggtttaaa acaaactaac tcgaaatgga 5040
cttacctgag ctcctgctat ccaaaaactt cattcgcacc cgattacccc tcgccaaacc 5100
cattgtcata cacgctgttg ctggcgccgg caagacacaa cttctcgaag aattcgcgcg 5160
gtcctcaccc tcaaccaaaa tctactcacc tgtgaaacat cactctaact cactgctgct 5220
ctcgcccttc cacaaagccc tctctgaagc ttccattgtc gacgagtacc ctctaagcca 5280
gatacacgag aacgtcgagt acatccttgc tgaccccatc caatacctgg gtaacccaaa 5340
ccttagaaaa ccccactaca tctgcgcgtc ttcccatcga tttggccatt ccaccgccgc 5400
tctcctaaca aaacttggca ttgaaactta cgcacataaa gaagataccg tccgggtaga 5460
caacatcttc caggctgaac cggacggcca aatcatcgcc tgcgaccgac ccactcaaga 5520
gctcgctgcc agacacacac tagactacct gaggccctgc gaaagcatag gactaacttt 5580
cccacgaacc acgatcctga tctctcacga acttactgca gataccctca ccaaagagat 5640
ttacatcggt ttgacccgcc actctaacca ccttctcata ctcactccgg atgcctctac 5700
aacctcctcc tgaccacact tgggctttcc gactactagc tcttggcgcc gccttagcac 5760
tgctcacttt cacgctcaac cgagacacaa gccgtcacgt cggagatcct tctcactcac 5820
ttccttttgg agggtactac cgagacggca gcaaagtagt ccattataac tccccgagag 5880
ccacaaaacc tagcacccct tccttcctgt acctgacccc aatcttactg atcctactaa 5940
ttcatgcagt caatagactt actaatcctc gccattcttg ttcttgcact cattgccagc 6000
ctattccccg cacctgaacc ttgcacaatt gtagtctctg gtgcctcagc ctccgtagct 6060
aattgcccaa accccgaaca acttgcagag cttgttagag cgcttaaacc ggctaaaccg 6120
gtttaaaact taacacaaac tcgaactcat gagcttcgac tacactcacc ctctctaccg 6180
cagctatcca tttccacact actgcgagtt cgaccggcac caactctgcg accatcatcc 6240
agtactcaaa cctccaacgc acaaacccag cgccccgaac tctctcatgt ctaccgacga 6300
caacaagggc aaacaaccac ttcacccgac accttcgggc cctaacgaca cgaccccgaa 6360
acctatccct gtacccactc cctcaattac gcccacagct gcaggtaagg aaaaccaaga 6420
gcccatcgaa aagcgtatca cacacgcttt ccacgctgaa gcaaaaaccc acaacaatgg 6480
ggtctctcca cctgccttca acccgaacaa catgaatgct gtgccgctga acctgctcaa 6540
cctcaaccta agatactcac cggtcactaa ctccatagct aaccctaaac agaccgaggc 6600
tatcgggaaa gcttgggtcc gcatcttgaa catcgatcct gccaacgtgt tcttatacgc 6660
catcgacctc gccagagctt gcgccgacgc gggctcctcc cctgaagctg atattattgg 6720
agcgaacgaa gatctcaacc ccgttgttga acgaaacgca ttggccctag tggttaggga 6780
tttctgcccg ctgcgcgctt tttgcgctta ctactcccgg gtggtatgga acctcatgat 6840
caaggcggac cagcctccgg ccaactggat gaaatccggg gtagacgaga acgcgaaatt 6900
cgcggcattc gacttcttcc atggtatcct ctcgcccgct tccctgtatg tgcccctaga 6960
gagacaccct acttccgcgg agaggatcgc aaatcaggcc atgttcgctg tgaaaattgc 7020
caacgctcca ggaaatggca cggacctcac gatggaccac gttgccttca ccaaaggaag 7080
gactacccag cactccggcc ttcgcccgac ccctttcaac atctaaccac ctttgaacca 7140
atactcctag catgcctaca agcccttaaa ccactacccc ccgaaatcca aacagctatc 7200
atttcgtgtg tgtgtgactt ctttaattcc ttacgctgtg ccagtagcaa gtaccagggc 7260
accagccgct cagctgtcaa acgtagagcg gctcgcctga actactgcta caagtgtggg 7320
caccccttat atctaaataa acctcacccc tgccgaccag gtcgattgtg ctctgcatct 7380
atctccgagc gcctctgttt gctccacaca ggaccgatta ggcttctaac cgaaaatcct 7440
gttagtgcca gagcagctca cttcctagca cacgagctcc ttgaccccag atgaaacata 7500
aatattcagg ttttcagttt ccattttctg 7530
<210> 6
<211> 7530
<212> DNA
<213> 人工序列
<400> 6
gaaaagcaaa cataaccaac acacacccaa aattgtgatc atcgtctctg catcaacagc 60
tcagctaaga accagagaaa tggccactat taggggtgcc atcgaacgca ttacagacac 120
caccgtacgc actacacttc aagaggaggc atgccgccaa atacgcgctg aacttaaaaa 180
cgtcgaacac atcaaccgtt acgctataca gcctgacgcc gccgacgcac tcgaacacct 240
aggcattggt tctaacccct tctctgtggc actgcacacg cacggtgcct gcaaagctat 300
tgagaaccaa ttactctatg tagttggtac cttactccca aaagagcgtg tcaccatgct 360
cttccttaag aaaagcaagc tcaacatcat gaaacgctgc cctaaattcc aagacatctt 420
cctgaaccaa catattgaac cacgcgacgt ttctaggtac tgcgacttta acgtccaatc 480
cacctccacc agcataccca cacgaacggc ttacatcagt gacacactcc actttatgga 540
ccgtagagac ctagtccgac tattcctaaa cagcccaaat ctagagactc tctacgccac 600
cattgtcctc ccagtcgagg ctgcttacaa acaaccgagc cgctacccag agatatacca 660
gattaactat gatttcgacg gtttccagta catacctgga ggccatgggg gcggcgccta 720
ccaccacgag ttcgaacagc tgcaatggtt agacactggc catatccact ggcgtgggcc 780
cgactgtaaa gagattcgta tgacaattac cgcccaaatg ctcgaaagct taggtgcgaa 840
tcatcttttc tgcttcaaac gtgggaaact acacacccca cgtgtacgca ctttcggccg 900
agatacccaa gtgctcttgc ccaaaatctt ccgcccggcc gacaaaaact ttaacagggc 960
aataccacta accctagcta acaaactact cctgtacgct aaatccatta acacagtgac 1020
cttccgcgac gtcgttgcca agacacgcca acttatgaag gacaaggagc ttgaaacata 1080
caccggcaac gacctgctgc atatggccaa ctatttcttc gctgttggag cactgtcagg 1140
agttaactcc tatgaccaat tgcttggcct atccgcctgg gaagcctgca ccgtccgtat 1200
caagaacacc ataacaaatc tctgggagaa gatcgccgga aagaaagagt tcggcaagct 1260
attggaggcc cttgagtggg aaacattcac atactcacgt caagtgacag gattcaccgt 1320
tacgggactg cccgccctgc tccccctccc agacatctca gaccaagaag agattttcgc 1380
ccagcaggac gagttagaca aaattactgc cggcgctacc aagatacgga ccattgacat 1440
tatgcgggga caagctaacc gcgccaaccc aaaaactccg atcaacccaa caccggccca 1500
gggaccccaa ctgcccgaca cccgcgaccc aatcgaccaa gctgcctcca aagagctcgt 1560
caccaagctt cagaaaaaca aacgcattta catccaagac gacgggccgg aataccttat 1620
gggacacatg gctgaagtcc ccgcctggta tcttgaacaa gatgacacca ccactagact 1680
caaaaaccgc tgcgcttggt tcttcggccc acccacctac agatatgggc acaacgacat 1740
tgagtacacc accatagaat actacccttg ggtcgagcgt attggagcca ttttcggcaa 1800
gtacaacacc tgtctcgctc aaacttacga cgccggagcc cgtattggat accatgctga 1860
cgacgaagac tgctacgacc ccgacgtcac cgtggtcacc gtcaacctca ctgggaacgc 1920
caccttcctg ctcaagaccc taaccggcac taggacctgg aaactcaaac ctggtgactt 1980
cattatcatg aaacccggtg cccaaaggtg tacaaaacat gccattagag actgcacaac 2040
taaccgcaca tccttaacct tcagatggca ggcccgcact tgccccacta acctcagaaa 2100
gatcactaac ctacccaaag ccaccaacca accccaaacc actgaatggc gtcctgtaac 2160
taaaccacgc cctagcacca ccgcttccag cgacacccag accccacctg tcatagacca 2220
ggaacgaggg tataccacca cctccgacgt gacaccaacg attaggctgc ccgccgaaaa 2280
tggtaataac gccggtgctg gaccctcctc cgcattaacc ctcgccgact tgaatgataa 2340
ccaagcgccg accactaaca aaggaaaaga gaaacttgag gaaatggtcg aaactggccc 2400
aactatcatg gaacgcttcc tcaacaccgt tcaggaacca acacaagatt tatgggactc 2460
cgcctctgaa tccgccgcga gctacctcgc tgaaggcctc ggcatctctg ccatccaagc 2520
actcccttgg gcacctcact tggaactcat caatgcatta ggcttccagg gcactgaaag 2580
gcaatacggc ccagacaact gtctcatatg gcccatcacg cattacagag aattgcccag 2640
aagcaacaat gtcgaagccc ctccggaagt gcttgagctg ctcgactgca tcaatagata 2700
ccccactgac gtcccaatgc tcaaaactcg agccgccgcc ttcggttcag acgtcaagaa 2760
tctacgcata ggggcacttg ttaagaacca ggacaaacag tggcgcgcat cactcgcact 2820
actttgtgag gaaaatgaac acctcctccc taccactgta atacacggcg ctggtggctc 2880
cgggaaatcc catttactcc aacagtgggt ggcgtctact gagcgcggaa atgtagtcac 2940
catcttgcca actatcgaac tccttcgaga ctggcaaaac aaatgtccac acgcaccgaa 3000
agagactttc aaaaccttcg agaaagcgct catccagaac agcgcccccg tggttatcat 3060
ggacgactac tctaaacttc ccccagggta tattgaagcc tacgtcagcc tcaagggcca 3120
gtgcaaactt ctcatactca ctggagaccc tagacagagc cactatcatg aggagaaccc 3180
tgaggcctta atctctactc ttgaccccgc cacggactac tttggcaaat tttgtgcata 3240
caacatcaac gccactcata gaaatgccaa aaccttcgcc aatgcgcttg gggtgtactc 3300
tgaaaaagaa atccccacca ctatcacctg ctcatcctac cagaaaagcg gttggccaac 3360
acttgtccca tccatcctta aaagaactgc gctcaacgac atggggcagc gctcactaac 3420
ctacgcaggc tgtcagggac tcaccacccc aaaagtgcag atagttcttg acaacgccac 3480
tccactatgc tctgacaagg tcatgtacac cgcactatct agagcggtgg accaaatcca 3540
cttcttcaac actggcccaa atcacactga ttactgggaa aagatgaacg ccacaccttt 3600
cctcaaaact ttcatcgacc acacacgtga ggagaccttt gctgagcacc aacctgccga 3660
acccacagtg cgtgagtacg caccagcaac gcatttccca ccggctaacg agaacctagc 3720
actagagccg tgggttgaac cgctgactga taaacactcg cgggaactct tccactctgc 3780
cttaggccac agcaactgcg tccagactga aaacaccgta gtccaacttt ttccccacca 3840
acaggccaaa gacgaaacac tcttctggaa aacaatagat gccagaatca agataaccac 3900
cccggaagag aacatcaggg cttgcagcat ggccactgat attggcgaca tcttgttcct 3960
aaattacaaa gaagccatgg gcttaccaca agaccccata cccttcgaac aggcactctg 4020
ggactcttgt caagccgagg tgcaactaac ctacctcagc aagcctctgg cagcactcgc 4080
aaacgccgcc caaagacaag accccgactt tgactccaac aaaatccaac tcttcctgaa 4140
atcacaatgg gtcaagaaag tcgagaagat gggctgcctt aagatcaaac ccggccaaac 4200
tattgcgtct ttcatgcaac agactgtcat gctatacgga actatggctc ggtacatgcg 4260
ccgcatccgc acctcacttt gcccacccga aattatgatc aactgcgaga ccaacccaaa 4320
ccaaatcgga acatgggtgc gtgagtattg gaacttcaac acccagagcc atgagaatga 4380
ctttgaagcg ttcgaccagt cccaagacgc caacatgtta caattcgagc tcattaaggc 4440
caaataccac tcaatccccg aggagatcat cgccgggtat cgacacctga aatgcaacgc 4500
ccacatcttc cttgggacta tatcgatcat gaggttgtct ggtgaagggc cgaccttcga 4560
cgccaacaca gagtgttcca ttgcatacaa ccacacaagg tattttgtgc ctaaaggcac 4620
cgcccagctc tacgccggag acgactctgc atgcgcgtcg cccctctctg aaaaacccag 4680
cttccaacac atctcaccag aactcagcct caagtctaag gccaagatta gaacccaaca 4740
gaaaggagac tttgcaacct tctgtggctg gctcatcact ccaaaaggcc tgatcaaaaa 4800
ccccacccag ctttacgcct cctggttatt ggctaagcac aacaaagatc tcgcggacgt 4860
ggccagaaac tatgccttgg acttacgcat tgcctatcaa cttaaagacg agctctatga 4920
gttattatca cctgaagaac ttgaccacca ccaactgctc gtccgagaga tgatcaagca 4980
caaaatgggc catctcctca acctccctga ggggtttaaa acaaactaac tcgaaatgga 5040
cttacctgag ctcctgctat ccaaaaactt cattcgcacc cgattacccc tcgccaaacc 5100
cattgtcata cacgctgttg ctggcgccgg caagacacaa cttctcgaag aattcgcgcg 5160
gtcctcaccc tcaaccaaaa tctactcacc tgtgaaacat cactctaact cactgctgct 5220
ctcgcccttc cacaaagccc tctctgaagc ttccattgtc gacgagtacc ctctaagcca 5280
gatacacgag aacgtcgagt acatctttgc tgaccccatc caatacctgg gtaacccaaa 5340
ccttagaaaa ccccactaca tctgcgcgtc ttcccatcga tttggccatt ccaccgccgc 5400
tctcctaaca aaacttggca ttgaaactta cgcacataaa gaagataccg tccgggtaga 5460
caacatcttc caggctgaac cagacggcca aatcatcgcc tgcgaccgac ccactcaaga 5520
gctcgctgcc agacacacac tagactacct gaggccctgc gaaagcatag gactaacttt 5580
cccacgaacc acgatcctga tctctcacga acttactgca gataccctca ccaaagagat 5640
ttacatcggt ttgacccgcc actccaacca ccttctcata ctcactccgg atgcctctac 5700
aacctcctcc tgaccacact tgggctttcc gactactagc tcttggcgcc gccttagcac 5760
tgctcacttt cacgctcaac cgagacacaa gtcgtcacgt cggagatcct tctcactcac 5820
ttccttttgg agggtactac cgagacggca gcaaagtagt ccattataac tccccgagag 5880
ccacaaaacc tagcacccct tccttcctgt acctgacccc aatcttactg atcctactaa 5940
ttcatgcagt caatagattt actaatcctc gccattcttg ttcttgcact cattgccagc 6000
ctattccccg cacctgaacc ttgcacaatt gtagtctctg gtgcctcagc ctccgtaact 6060
aattgcccaa accccgaaca acttgcagag cttgtcagag cgcttaaacc ggctaaaccg 6120
gtttaaaact taacacaaac tcgaactcat gagcttcgac tacactcacc ctctctaccg 6180
cagctatcca tttccacact actgcgagtt cgaccggcac caactctgcg accatcatcc 6240
agtactcaaa cctccaacgc acaaacccag cgccccgaac tctctcatgt ctaccgacga 6300
caacaagggc aaacaaccac ttcacccgac accttcgggc cctaacgaca cgaccccgaa 6360
acctatccct gtacccactc cctcaattac gcccacagct gcaggtaagg aaaaccaaga 6420
gcccatcgaa aagcgtatca cacacgcttt ccacgctgaa gcaaaaaccc acaacaatgg 6480
ggtctctcca cctgccttca acccgaacaa catgaatgct gtgccgctga acctgctcaa 6540
cctcaaccta agatactcac cggtcactaa ctccatagct aaccctaaac agaccgaggc 6600
tatcgggaaa gcttgggtcc gcatcttgaa catcgatcct gccaacgtgt tcttatacgc 6660
catcgacctc gccagagctt gcgccgacgc gggctcctcc cctgaagctg atattattgg 6720
agcgaacgaa gatctcaacc ccgttgttga acgaaacgca ttggccctag tggttaggga 6780
tttctgcccg ctgcgcgctt tttgcgctta ctactctcgg gtggtatgga acctcatgat 6840
caaggcggac cagcctccgg ccaactggat gaaatccggg gtagacgaga acgcgaaatt 6900
cgcggcattc gacttcttcc atggtatcct ctcgcccgct tccctgtatg tgcccctaga 6960
gagacaccct acttccgcgg agaggatcgc aaatcaggcc atgttcgctg tgaaaattgc 7020
caacgctcca ggaaatggca cggacctcac gatggaccac gttgccttca ccaaaggaag 7080
gactacccag cactccggcc ttcgcccgac ccctttcaac atctaaccac ctttgaacca 7140
atactcctag catgcctaca agcccttaaa ccactacccc ccgaaatcca aacagctatc 7200
atttcgtgtg tgtgtgactt ctttaattcc ttacgctgtg ccagtagcaa gtaccagggc 7260
accagccgct cagctgtcaa acgtagagcg gctcgcctga actactgcta caagtgtggg 7320
caccccttat atttaaataa acctcacccc tgccgaccag gtcaattgtg ctctgcatct 7380
atctccgagc gcctctgttt gctccacaca ggaccgatta ggtttctaac cgaaaatcct 7440
gttagtgcca gagcagctca cttcctagcg cacgagctcc ttgaccccag atgaaacata 7500
aatattcagg ctttcagttt ccattttctg 7530
Claims (3)
1.一种柑橘黄化脉明病毒弱毒分离株，其特征在于，该弱毒株相对于强毒株产生的基因突变点是：TGB区域的T5306→C，A5482→G，T5792→C，A6058→G，C6096→T，以及位于CP基因上的T6817→C。
2.如权利要求1所述的柑橘黄化脉明病毒弱毒分离株，其特征在于，在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏登记号为CGMCC No.18177。
3.如权利要求1或2所述的柑橘黄化脉明病毒弱毒分离株在防治柑橘黄化脉明病毒强毒株系侵染方面的应用。
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| CN108559759A (zh) * | 2018-04-23 | 2018-09-21 | 西南大学 | 三元穿梭载体及利用其构建clbv侵染性克隆的方法 |
| CN108588113A (zh) * | 2018-04-23 | 2018-09-28 | 西南大学 | 三元穿梭载体及利用其构建cyvcv侵染性克隆的方法 |
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| CN108559759A (zh) * | 2018-04-23 | 2018-09-21 | 西南大学 | 三元穿梭载体及利用其构建clbv侵染性克隆的方法 |
| CN108588113A (zh) * | 2018-04-23 | 2018-09-28 | 西南大学 | 三元穿梭载体及利用其构建cyvcv侵染性克隆的方法 |
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| TIANTIAN CUI等: "Development of Infectious cDNA Clones of Citrus Yellow Vein Clearing Virus Using a Novel and Rapid Strategy", 《PHYTOPATHOLOGY》 * |
| 崔甜甜: "柑橘黄化脉明病毒和柑橘叶斑驳病毒的侵染性克隆构建", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
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