本发明涉及生物
技术领域:
,具体涉及荔枝蒂蛀虫繁殖力关键因子卵黄原蛋白基因csvg及其编码蛋白与应用。
背景技术:
卵黄原蛋白(vitellogenin,vg)基因是特异性存在于非哺乳类雌性动物体内的一种蛋白,该蛋白对于昆虫受精卵的成熟发育有决定性作用。至今,已经有多种昆虫的vg基因被成功克隆和鉴定,但未见有关细蛾科昆虫vg的任何报道。本发明首次在荔枝龙眼产业重大细蛾科害虫-荔枝蒂蛀虫中发现了卵黄原蛋白基因csvg,并对其编码的氨基酸序列进行了分析,补充了细蛾科昆虫卵黄原蛋白基因分子特征的信息。荔枝和龙眼是我国的优势名贵水果,二者的栽培面积及果实产量均居世界第一位。荔枝蒂蛀虫(conopomorphasinensisbradley)是荔枝龙眼栽培中频发、暴发性蛀虫,该虫以幼虫为害,其蛀果率严重时可高达到60%以上,是荔枝龙眼产业“零容忍”的害虫防控对象。但是,在该虫幼虫期和蛹期无法进行防控,在其成虫期唯有反复大量施药才能保证防控效果。高效氯氰菊酯是目前应用于荔枝蒂蛀虫成虫防控最为常见的化学药剂。但是,在实际生产中未见可用于该药剂对荔枝蒂蛀虫繁殖力影响的检测方法,亟待解决该问题。技术实现要素:本发明的目的是提供荔枝龙眼产业重大害虫-荔枝蒂蛀虫卵黄原蛋白基因csvg及其编码蛋白与该基因在评价药物对荔枝蒂蛀虫繁殖力影响中的应用。本发明采取的技术方案为如下:一种荔枝蒂蛀虫卵黄原蛋白基因csvg,其核苷酸序列如seqidno.1所示;该基因的开放阅读框长度为5391bp;由csvg基因编码的卵黄原蛋白的氨基酸序列如seqidno.2所示,该编码蛋白质长度为1796aas,估算等电点为7.99,分子大小为205.8kda,被预测为非稳定蛋白。本发明还提供了一种药物对荔枝蒂蛀虫繁殖力影响的检测方法,其特征在于,通过检测施用药物和未施用药物的荔枝蒂蛀虫卵黄原蛋白基因csvg的表达量来判断该药物施用后荔枝蒂蛀虫繁殖力是否受到影响。优选,具体步骤是分别提取施用药物和未施用药物的荔枝蒂蛀虫的rna,再反转录为cdna,再以real-csvg-f:5’-atgcaaccatagagtcgc-3’和real-csvg-r:5’-atcatcttccgtagataccag-3’作为检测引物检测卵黄原蛋白基因csvg的表达量,如果施用药物的荔枝蒂蛀虫的卵黄原蛋白基因csvg的表达量小于未施用药物的荔枝蒂蛀虫的卵黄原蛋白基因csvg的表达量,则该药物处理后的荔枝蒂蛀虫繁殖力下降;反之则药物处理后的荔枝蒂蛀虫繁殖力上升;如果两者表达量相当,则药物不影响荔枝蒂蛀虫繁殖力。优选,所述的检测卵黄原蛋白基因csvg的表达量是通过荧光定量pcr的方法来检测,所述的荧光定量pcr反应条件为:95℃预变性10s;95℃变性5s,58℃退火15s,72℃延伸20s。本发明还提供了一种检测药物对荔枝蒂蛀虫繁殖力影响的试剂盒,包括pcr反应试剂和pcr反应引物,其特征在于,所述的pcr反应引物为:real-csvg-f:5’-atgcaaccatagagtcgc-3’和real-csvg-r:5’-atcatcttccgtagataccag-3’。优选,还包括real-actin-f:5’-agatctggcaccacacct-3’和real-actin-r:5’-cgataccggtggtacgac-3’。本发明还提供了一种上述的荔枝蒂蛀虫卵黄原蛋白基因csvg基因的克隆方法,其特征在于,包括以下步骤:(1)提取荔枝蒂蛀虫总rna,反转录合成荔枝蒂蛀虫cdna模板;(2)以csvg-f:5’-cmymtgadgtnmgrgtkgcngc-3’,csvg-r:5’-gcaswrtanggccanacrc-3’作为引物,以荔枝蒂蛀虫cdna模板作为模板进行pcr扩增,得到目的片段;(3)根据smarttmracecdnaamplificationkit(clontech)操作指南合成race所需cdna模板,以此cdna模板为模板,以5-csvg-r:5’-atcgaatgcatcatagctg-3’,3-csvg-f:5’-agcatgaaactggtatctacgg-3’作为引物进行pcr扩增,获得目的片段;(4)将步骤(2)和(3)的目的片段进行拼接,得到权利要求1所述的荔枝蒂蛀虫卵黄原蛋白基因csvg基因。与现有的技术相比,本发明的具有以下优点:(1)针对未见细蛾科昆虫vg的任何报道这一问题,本发明提出的荔枝蒂蛀虫卵黄原蛋白基因(vitellogenin,vg)dna序列及其编码的蛋白质序列,补充细蛾科昆虫卵黄原蛋白基因分子特征的信息。(2)针对现有化学农药对荔枝蒂蛀虫繁殖力影响检测技术的局限,提供了一种基于csvg基因研发的高效氯氰菊酯对荔枝蒂蛀虫繁殖力影响的分子检测方法,利用该分子检测方法,可用于高通量检测高效氯氰菊酯对荔枝蒂蛀虫繁殖力的影响,检测的灵敏度和准确度大大提高,检测周期短,稳定性好,对荔枝龙眼实际生产中该药剂的合理使用提供分子参考依据。(3)利用本发明为荔枝蒂蛀虫对高效氯氰菊酯的应答机制研究提供新基因,为实际生产中利用vg检测高效氯氰菊酯对荔枝蒂蛀虫繁殖力影响提供基础。附图说明图1是本发明实施例2实时荧光定量pcr标准曲线结果图。图2是本发明实施例2基于csvg基因研发的高效氯氰菊酯对荔枝蒂蛀虫繁殖力影响的分子检测结果图。具体实施方式为了使本发明的目的、技术方案和有益技术效果更加清晰,以下结合实施例,对本发明进行进一步详细说明。应当理解的是,本说明书中描述的实施例仅仅是为了解释本发明,并非为了限定本发明,实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。实施例1荔枝蒂蛀虫卵黄原蛋白基因csvg的cdna克隆方法,包括以下步骤:(1)以荔枝蒂蛀虫雌性成虫为材料提取总rna,并以所提取的总rna为模板,反转录合成cdna第一链;再以pcr合成荔枝蒂蛀虫cdna模板。(2)根据现有鳞翅目昆虫vg保守区域设计简并引物csvg-f:5’-cmymtgadgtnmgrgtkgcngc-3’和csvg-r:5’-gcaswrtanggccanacrc-3’,见seqidno.3和seqidno.4,(其中r=a/g,y=c/t,m=a/c,k=g/t,s=c/g,w=a/t,h=a/c/t,b=c/g/t,v=a/c/g,d=a/g/t,n=a/c/g/t)。(3)以步骤(1)所述cdna模板与步骤(2)所述简并引物进行pcr扩增,并纯化所得pcr产物。(4)将步骤(3)纯化后的pcr产物进行琼脂糖凝胶电泳,回收目的片段后取纯化产物克隆到pmd18-t载体上,然后转化入感受态细胞dh5α,37℃平板培养过夜,以m13通用引物进行阳性克隆子鉴定。(5)分析阳性克隆子测序结果,针对正确碱基序列,结合smarttmracecdnaamplificationkit(clontech)操作指南设计特异性引物3-csvg-f:5’-agcatgaaactggtatctacgg-3’和5-csvg-r:5’-atcgaatgcatcatagctg-3’,见seqidno.5和seqidno.6。(6)根据smarttmracecdnaamplificationkit(clontech)操作指南,合成race所需cdna模板,结合步骤(5)的特异性引物3-csvg-f和5-csvg-r进行5’-末端和3’-末端序列扩增,纯化此步骤所得pcr产物,琼脂糖凝胶回收目的片段,取纯化后目的片段克隆到pmd18-t载体上,然后转化入感受态细胞dh5α,37℃平板培养过夜,以m13通用引物进行阳性克隆子鉴定。(7)根据步骤(4)和步骤(6)所得的阳性克隆子测序结果,将所测得基因序列拼接后得到荔枝蒂蛀虫卵黄原蛋白基因csvg的全长序列,所述csvg基因的碱基序列如seqidno.1所示,所述csvg基因编码蛋白的氨基酸序列如seqidno.2所示。本发明提供的荔枝蒂蛀虫卵黄原蛋白基因csvg的序列完整,该基因的开放阅读框长度为5391bp,由该基因编码的蛋白质长度为1796aas,估算等电点为7.99,分子大小为205.8kda,被预测为非稳定蛋白,经blast分析,结果表明,与其最接近的蛋白同源性只有47%。本发明提供的荔枝蒂蛀虫卵黄原蛋白基因csvg经blast分析,结果表明:所得基因是新的vg基因,而且是目前仅有细蛾科昆虫vg基因,补充了昆虫卵黄原蛋白基因分子特征的信息,可应用于昆虫繁殖力相关研究。实施例2利用本发明提供的荔枝蒂蛀虫卵黄原蛋白基因csvg全长,借助实时荧光定量pcr技术建立了一种高效氯氰菊酯对荔枝蒂蛀虫繁殖力影响的分子检测方法,该分子检测方法包括以下步骤:(1)以荔枝蒂蛀虫卵黄原蛋白基因csvg为靶标序列,根据实时荧光定量pcr引物设计原则,设计real-csvg-f:5’-atgcaaccatagagtcgc-3’和real-csvg-r:5’-atcatcttccgtagataccag-3’两对特异性检测引物,见seqidno.7和seqidno.8。(2)对处理组(30ppm浓度高效氯氰菊酯喷洒处理)中幸存的荔枝蒂蛀虫雌性成虫和对照组(正常饲养)的荔枝蒂蛀虫雌性成虫分别提取总rna样品,并将两组总rna反转录合成cdna第一链;再以pcr合成对照组和处理组的荔枝蒂蛀虫cdna模板。(3)以步骤(1)所述检测引物real-csvg-f:5’-atgcaaccatagagtcgc-3’和real-csvg-r:5’-atcatcttccgtagataccag-3’与步骤(2)所述cdna模板进行实时荧光定量pcr反应,具体为:首先,分别对内参基因actin(检测引物对为real-actin-f:5’-agatctggcaccacacct-3’和real-actin-r:5’-acgataccggtggtacgac-3’(seqidno.9和seqidno.10)及csvg进行荧光定量pcr标准曲线的制作:①以对照组cdna模板样品,分别作cdna样品原液的10倍、1倍、1/10、1/100、1/1000和1/10000稀释;②在10μl反应体系之中,加入1μl倍比稀释浓度的cdna样品,加入5μlsybrpremixextaq,分别加入待测基因上、下游引物各0.2μl,最后加入ddh2o补足至10μl;③荧光定量pcr反应条件为:95℃预变性10s;95℃变性5s,58℃退火15s,72℃延伸20s的条件下40个循环,收集结果用以制作熔点曲线,以确定扩增产物的特异性,确定引物是否可用于下一步待测样品的检测,所述标准曲线结果如图1所示。接着,实时荧光定量pcr检测对照组与处理组中靶标基因(荔枝蒂蛀虫卵黄原蛋白基因csvg)表达量。①实时荧光定量pcr10μl反应体系:sybrpremixextaq5μlreal-csvg-f0.2μlreal-csvg-r0.2μlcdna模板1μlddh2o补足10μl②实时荧光定量pcr反应条件为:95℃预变性10s;95℃变性5s,58℃退火15s,72℃延伸20s,实验仪器为伯乐cfx96实时荧光定量pcr仪;③结果分析:采用2-△△ct方法比较csvg基因在对照组及高效氯氰菊酯处理组中的表达量差异。结果如表1和图2所示,2-△△ct(正常饲养对照组)/2-△△ct(高效氯氰菊酯处理组)=0.98/0.04=24.5>1,表明高效氯氰菊酯处理后的荔枝蒂蛀虫体csvg基因表达量低于正常饲养组,这一结果与实际产卵量检测结果相符,该药剂处理后荔枝蒂蛀虫繁殖力下降。由此证明本发明的检测方法是可行的。表1csvg基因在对照组及高效氯氰菊酯处理组中的实时荧光定量pcr数据本发明提供的分子检测方法不同于成虫产卵量统计检测方法,不需人工饲养试虫,大大节省劳动力资源,可用于野外高通量检测高效氯氰菊酯对荔枝蒂蛀虫繁殖力的影响,检测的灵敏度和准确度大大提高,稳定性好,对荔枝龙眼实际生产中该药剂的合理使用提供分子参考依据。以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本
技术领域:
的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。序列表<110>广东省农业科学院植物保护研究所<120>荔枝蒂蛀虫卵黄原蛋白基因csvg及其编码蛋白与应用<160>10<170>siposequencelisting1.0<210>1<211>5391<212>dna<213>荔枝蒂蛀虫(conopomorphasinensisbradley)<400>1atgaaggtcctcgtgttagcggcacttttggctgccgcttcttgcgcgaatcttaaaata60aagaaagaagataaaaaacaggggaaaaactcggagacctatgtaacccacagtttgtgg120caagatggcaaagtctacacctatgatgtggagtcgttcaacttggccagcctggaggag180gggagcagcagtggcgccaacactaaggccactctacagctgcgagtaagatctcaaaaa240ctgcttcatgccaaattggtggatgtgagacacgcggcgatgcaccagaaatttaatggt300gagcgccaagcgccagattctctaaaatatgaacctatgaaaagcatggagcatccattt360gagatagtgatgaacatgaatcgcatgtcctctatgaatttgccatcaagcctcaatttg420gaacatgagaattttctcaaaggtatcctaagcactctccaagttgatctcactccttat480cgtaaggctcttgggtctcatgactattacgatcaagaacgcagccaaggacagttcaga540aagatggagaccgacgtcacgggcgactgtgaaacgctgtacagcgtgtcgccgcttact600gggcataacaaaacacagtattacaatgatcaggaaccaattgaaatcactaaaatgaag660gactacggtaaatgcaaccatagagtcgcttatcacttcggtgtaccggccggagccgaa720tggactggaacagctcactcaaataaggaaaatcaattcatggaaagggcgacaaaaacc780cgaattgtcacagggcagcaaggtccgatctacgatgctgaaacaatctcaactgtttac840gtaaacccactaatgtacggacggcagaaagctgaaatttacagtcgagtaagcatgaaa900ctggtatctacggaagatgattctcaaccacaatttgaaatgcaacttaatatgcgacaa960gtaaaaaatttgctttactcattgggttctaagtccgcaaatatcgattatacttcgacg1020tcagcatcgtcagagtctactgaaataccagaagagattgtactgaatgaggaactcgat1080gagcagccggcgacccgagtccgccgctcaccaaaacaaagaggattccgggctgtttca1140gtaaaaaaacttgttatagaggaaggaaatcgtaaatacaaggatgactccgaatctgac1200agttctagtgactcctctacagcttaccagaacgacgaagctcctaagcagaatgaaccc1260gcatatgcttctatttacatggttccccaaagccgaaccccaaacaaacgttcgcctatg1320gttgtccaaaaactagttcaggagtttgcgcaccaacttcaaaatcctaatacaatggcc1380aaatcggataccatttccaaattcaacgtcattgttcatctaattgcaactatgaacagc1440gaagagcttactcaaacaacccgaaatatcgaatctgcaaaatcacctaacgatgatatt1500aaatctgacatggttatggcataccgcgacgctatgacacaagcaggtaccatgcctgca1560ttccaacaaatcaagacttggattaccactaaaaaaatacaaggtgaagaagcagcccaa1620atcatgggatccctcgctcacacgttgcgttaccccacaaaagatctgatgagccagttc1680ttcaatttagcattcagtgatgaagtcagagaacaaagatatttaaacacaaccgcctta1740attagcgctgtgaagttcattaacatgggatatgttaaccaaaaaatgtctagagctcgt1800tacccgacactgatgtacggtcaactgacttcgaataaggataaatttgtcgaaatagaa1860attttgcctcgtctggtaaaagagttggacgaagcaattgctcgcgacgacaaacacaag1920gcacaagtaatgatcaaagcgatgggaactcttggtcataatgccattcttcgcgctttt1980gctccatacttggaaggagaaatagaaacttcaacttatcttcgccgatacatggttgag2040aacctggacgttctggcttatcaaaaggatcacttcgcccgctcagttttgtacaacatt2100atcagaaacacggcagagacccctgaggtaagggtggccgcaatctttaacatcttcgct2160ggaagacccacctcagctatgatgcattcgatggcgtacatgactaaggaagaccctagc2220cagcaagtacgctccgctctcagatcagctattgaaactgccgcggaattgaagaatcct2280cgctattatgaactatctcgaactgcccaagaagtcaaaccattgctgaccaaagaaccc2340gtcaacgtcctgtcttcaagactgcaatacggcgactcttataataaagattacgagttg2400ggggctttcaatgtattaagctacattggaagcgaaaatagtctcatgccaaagtatctg2460aaatattcctggaccaaccaagcaaagggttacaacaataaagacactatggcggtatct2520ctctctgatgcctggtggttcacggaatacttaaaagatcctttacaaataagaagatcg2580tatgaaccgaaaaaactaaatcacaaatataccgcaaaggatgtggctgacctcttaaag2640atcagacacgaccttactgattctcaagaagctgcattctactttagttggatggaacaa2700gagagatattttgctttcagtaaatacgacttcgatattcttagcagcgacatgataaag2760gctttggataaattatcgaagggcgtggactaccactattctaaaactatcaataccaac2820caagtatcagtgatgttcccactagcctctggtatgcctttcatctacagatacaaagag2880cctacattgatgcatgtgaaaggaaaagcaagtgctatgattgctcataaagacaaagct2940acgcgttctaccttcggaatggaaatgcacatgacttacgcgaaaaacttagatggcagc3000gttggcttcttcgacatattgacaaaccactactccagcgttggagttcagaacaaacta3060caattcaatgttccaatcaagattgaactagaaagagaagacaacgaactgaagattgag3120gttgaacctctgcatccggaacaggatatagcgctggcacattacagtgtgtggccctac3180agtgctaggcagaagaaggatgcccacgtcaccgtcgctgtcgaccccaccacgaaagtc3240atagagagagtaaataaagtacataatatcgatatgaggttcggacaagccgcgatagga3300tcgcaattccagatgcaaggatattcatattcaacagactaccgcaacattgctacactg3360ctgaaaaccgacgatctgcttgggaatatggcttacatattcaaccagaaagatatcgct3420ctgacacactacaacttgcgttatctatcaagacaatcgcctaataaacgcatgtctatc3480agtttagtttacgatgagagggaaaagaagaacgacgctaagaaacctttatatccagtg3540cacgagatggctgatgttaagccgaatagcgaaactcgtcgtcaggaaatggctgaccgt3600gtgacaactgaagtgaaagcttccaaggctaaaatcatcgacatgagtgctgtgttccat3660ggagcgcagagttatgaatacgtactaactgctgcaataagcagcagcgacattgaaact3720aaaacaaagtacgccattttctacgcaagaaaccaccccaagcaaggaaacgagcaaagc3780aactttgcaggaagtttcacaactcctgtgatcacatcgaaaaacttcgaagaagctctc3840aaacaggaatccaaatctgtgttcgacggcgaactggtgttaggacagaaggggaacatt3900agagtcaacggatacatggaaagaaccaggcaatacgctgagaagcttcaaagatatcca3960aaggcgcctttgtgcgctgcagataaagctcagttcaaccaacgcttatgctatgcatct4020attctttatgctctttctcccgaccacatcaaggcaaacattgaatacaaggatgtaagc4080ccgtggatcaagaatctgagccacaacatgtataacatggtcagacatgctttcttctgg4140tacgaggaggaaaatatactgaagactatgcccgacggacaagctgaaattgatgtcacc4200atgtcctacaaagaccacatgttcaacatgagcatggcatcccgtgagggggatatgagg4260atttacaacgccccgcttcctaaacagatgatggcagtggttggtgtatcgcctgttgat4320gcgaccgacgaaatggcaaactattttacacaacatcaataccttaaatactgcagcgct4380gagcgtgaaaatatcaagacgttcagcgacaaggcttacaactatacgatgagtggatct4440tggcatgtgctactgctggacgaatggtgggctgctggcaagaggcccgctgacgagctt4500gtgatccttgctcgcaaaccaagcccggatgaacaggagatttacatttcataccaaaat4560tctgaaggagaagaaatggaaatcgaactgaaaccggacaatgttcctggcacggttgca4620atcaatgtcaaatcctccctaactaaattatctcgcgatggttccactcagtacaaacgg4680gatggaaacgataaggtggccctggagtacttctacttgccagacgacagactcatgctc4740aatattcgcgaaggtcgtctcagggcactttacagcggaaggaacattgttatcttggca4800tacggcaaccagaaccatactagaggactctgcggccacatgagcggcgagcctaacgac4860gacttcgtcactcccgatggcaacctcgttgacgaagctgatcagttcgccgcttcctat4920gccctggacagagaatacagtgacccgaaaaccaaaacattgcaagagctggccagaaaa4980aacgcgtaccaaccccgtcgcgtatatccatccgttctgaggtcggatgagtcttggacg5040aagtacaacaaagacaagatgcaaagcatgaagcaacagaaacagcagtcgcagtccatg5100tacagcgccaggagctacgggcagcaggacgtgccctgccgtgtggagaaccaagtgcag5160tactacgagacatatgacgaaatatgtatcagcactcagagactaccttcatgcagagct5220cagtgccgtggggaaggttacattaaaaaatccgcccaggtagtatgcaaatcaaagatg5280gatgaagagttcaaaatgtataaggaagaaatcaaacaaggccagaaccctgaggtgtct5340gggcctccaaagaaacaagagttcagagtacccagctcctgtatggagtaa5391<210>2<211>1796<212>prt<213>荔枝蒂蛀虫(conopomorphasinensisbradley)<400>2metlysvalleuvalleualaalaleuleualaalaalasercysala151015asnleulysilelyslysgluasplyslysglnglylysasnserglu202530thrtyrvalthrhisserleutrpglnaspglylysvaltyrthrtyr354045aspvalgluserpheasnleualaserleuglugluglyserserser505560glyalaasnthrlysalathrleuglnleuargvalargserglnlys65707580leuleuhisalalysleuvalaspvalarghisalaalamethisgln859095lyspheasnglygluargglnalaproaspserleulystyrglupro100105110metlyssermetgluhisprophegluilevalmetasnmetasnarg115120125metsersermetasnleuproserserleuasnleugluhisgluasn130135140pheleulysglyileleuserthrleuglnvalaspleuthrprotyr145150155160arglysalaleuglyserhisasptyrtyraspglngluargsergln165170175glyglnphearglysmetgluthraspvalthrglyaspcysgluthr180185190leutyrservalserproleuthrglyhisasnlysthrglntyrtyr195200205asnaspglngluproilegluilethrlysmetlysasptyrglylys210215220cysasnhisargvalalatyrhispheglyvalproalaglyalaglu225230235240trpthrglythralahisserasnlysgluasnglnphemetgluarg245250255alathrlysthrargilevalthrglyglnglnglyproiletyrasp260265270alagluthrileserthrvaltyrvalasnproleumettyrglyarg275280285glnlysalagluiletyrserargvalsermetlysleuvalserthr290295300gluaspaspserglnproglnpheglumetglnleuasnmetarggln305310315320vallysasnleuleutyrserleuglyserlysseralaasnileasp325330335tyrthrserthrseralasersergluserthrgluileprogluglu340345350ilevalleuasnglugluleuaspgluglnproalathrargvalarg355360365argserprolysglnargglypheargalavalservallyslysleu370375380valileglugluglyasnarglystyrlysaspaspsergluserasp385390395400serserseraspserserthralatyrglnasnaspglualaprolys405410415glnasngluproalatyralaseriletyrmetvalproglnserarg420425430thrproasnlysargserprometvalvalglnlysleuvalglnglu435440445phealahisglnleuglnasnproasnthrmetalalysseraspthr450455460ileserlyspheasnvalilevalhisleuilealathrmetasnser465470475480glugluleuthrglnthrthrargasnilegluseralalysserpro485490495asnaspaspilelysseraspmetvalmetalatyrargaspalamet500505510thrglnalaglythrmetproalapheglnglnilelysthrtrpile515520525thrthrlyslysileglnglygluglualaalaglnilemetglyser530535540leualahisthrleuargtyrprothrlysaspleumetserglnphe545550555560pheasnleualapheseraspgluvalarggluglnargtyrleuasn565570575thrthralaleuileseralavallyspheileasnmetglytyrval580585590asnglnlysmetserargalaargtyrprothrleumettyrglygln595600605leuthrserasnlysasplysphevalgluilegluileleuproarg610615620leuvallysgluleuaspglualailealaargaspasplyshislys625630635640alaglnvalmetilelysalametglythrleuglyhisasnalaile645650655leuargalaphealaprotyrleugluglygluilegluthrserthr660665670tyrleuargargtyrmetvalgluasnleuaspvalleualatyrgln675680685lysasphisphealaargservalleutyrasnileileargasnthr690695700alagluthrprogluvalargvalalaalailepheasnilepheala705710715720glyargprothrseralametmethissermetalatyrmetthrlys725730735gluaspproserglnglnvalargseralaleuargseralaileglu740745750thralaalagluleulysasnproargtyrtyrgluleuserargthr755760765alaglngluvallysproleuleuthrlysgluprovalasnvalleu770775780serserargleuglntyrglyaspsertyrasnlysasptyrgluleu785790795800glyalapheasnvalleusertyrileglysergluasnserleumet805810815prolystyrleulystyrsertrpthrasnglnalalysglytyrasn820825830asnlysaspthrmetalavalserleuseraspalatrptrpphethr835840845glutyrleulysaspproleuglnileargargsertyrgluprolys850855860lysleuasnhislystyrthralalysaspvalalaaspleuleulys865870875880ilearghisaspleuthraspserglnglualaalaphetyrpheser885890895trpmetgluglngluargtyrphealapheserlystyrasppheasp900905910ileleuserseraspmetilelysalaleuasplysleuserlysgly915920925valasptyrhistyrserlysthrileasnthrasnglnvalserval930935940metpheproleualaserglymetpropheiletyrargtyrlysglu945950955960prothrleumethisvallysglylysalaseralametilealahis965970975lysasplysalathrargserthrpheglymetglumethismetthr980985990tyralalysasnleuaspglyservalglyphepheaspileleuthr99510001005asnhistyrserservalglyvalglnasnlysleuglnpheasnval101010151020proilelysilegluleugluarggluaspasngluleulysileglu1025103010351040valgluproleuhisprogluglnaspilealaleualahistyrser104510501055valtrpprotyrseralaargglnlyslysaspalahisvalthrval106010651070alavalaspprothrthrlysvalilegluargvalasnlysvalhis107510801085asnileaspmetargpheglyglnalaalaileglyserglnphegln109010951100metglnglytyrsertyrserthrasptyrargasnilealathrleu1105111011151120leulysthraspaspleuleuglyasnmetalatyrilepheasngln112511301135lysaspilealaleuthrhistyrasnleuargtyrleuserarggln114011451150serproasnlysargmetserileserleuvaltyraspgluargglu115511601165lyslysasnaspalalyslysproleutyrprovalhisglumetala117011751180aspvallysproasnsergluthrargargglnglumetalaasparg1185119011951200valthrthrgluvallysalaserlysalalysileileaspmetser120512101215alavalphehisglyalaglnsertyrglutyrvalleuthralaala122012251230ileserserseraspilegluthrlysthrlystyralailephetyr123512401245alaargasnhisprolysglnglyasngluglnserasnphealagly125012551260serphethrthrprovalilethrserlysasnphegluglualaleu1265127012751280lysglngluserlysservalpheaspglygluleuvalleuglygln128512901295lysglyasnileargvalasnglytyrmetgluargthrargglntyr130013051310alaglulysleuglnargtyrprolysalaproleucysalaalaasp131513201325lysalaglnpheasnglnargleucystyralaserileleutyrala133013351340leuserproasphisilelysalaasnileglutyrlysaspvalser1345135013551360protrpilelysasnleuserhisasnmettyrasnmetvalarghis136513701375alaphephetrptyrgluglugluasnileleulysthrmetproasp138013851390glyglnalagluileaspvalthrmetsertyrlysasphismetphe139514001405asnmetsermetalaserarggluglyaspmetargiletyrasnala141014151420proleuprolysglnmetmetalavalvalglyvalserprovalasp1425143014351440alathraspglumetalaasntyrphethrglnhisglntyrleulys144514501455tyrcysseralagluarggluasnilelysthrpheserasplysala146014651470tyrasntyrthrmetserglysertrphisvalleuleuleuaspglu147514801485trptrpalaalaglylysargproalaaspgluleuvalileleuala149014951500arglysproserproaspgluglngluiletyrilesertyrglnasn1505151015151520sergluglygluglumetgluilegluleulysproaspasnvalpro152515301535glythrvalalaileasnvallysserserleuthrlysleuserarg154015451550aspglyserthrglntyrlysargaspglyasnasplysvalalaleu155515601565glutyrphetyrleuproaspaspargleumetleuasnileargglu157015751580glyargleuargalaleutyrserglyargasnilevalileleuala1585159015951600tyrglyasnglnasnhisthrargglyleucysglyhismetsergly160516101615gluproasnaspaspphevalthrproaspglyasnleuvalaspglu162016251630alaaspglnphealaalasertyralaleuaspargglutyrserasp163516401645prolysthrlysthrleuglngluleualaarglysasnalatyrgln165016551660proargargvaltyrproservalleuargseraspglusertrpthr1665167016751680lystyrasnlysasplysmetglnsermetlysglnglnlysglngln168516901695serglnsermettyrseralaargsertyrglyglnglnaspvalpro170017051710cysargvalgluasnglnvalglntyrtyrgluthrtyraspgluile171517201725cysileserthrglnargleuprosercysargalaglncysarggly173017351740gluglytyrilelyslysseralaglnvalvalcyslysserlysmet1745175017551760aspglugluphelysmettyrlysglugluilelysglnglyglnasn176517701775progluvalserglyproprolyslysglnglupheargvalproser178017851790sercysmetglu1795<210>3<211>22<212>dna<213>人工序列csvg-f(artificialsequencecsvg-f)<400>3cmymtgadgtnmgrgtkgcngc22<210>4<211>19<212>dna<213>人工序列csvg-r(artificialsequencecsvg-r)<400>4gcaswrtanggccanacrc19<210>5<211>22<212>dna<213>人工序列3-csvg-f(artificialsequence3-csvg-f)<400>5agcatgaaactggtatctacgg22<210>6<211>19<212>dna<213>人工序列5-csvg-r(artificialsequence5-csvg-r)<400>6atcgaatgcatcatagctg19<210>7<211>18<212>dna<213>人工序列real-csvg-f(artificialsequencereal-csvg-f)<400>7atgcaaccatagagtcgc18<210>8<211>21<212>dna<213>人工序列real-csvg-r(artificialsequencereal-csvg-r)<400>8atcatcttccgtagataccag21<210>9<211>18<212>dna<213>人工序列real-actin-f(artificialsequencereal-actin-f)<400>9agatctggcaccacacct18<210>10<211>19<212>dna<213>人工序列real-actin-r(artificialsequencereal-actin-r)<400>10acgataccggtggtacgac19当前第1页12