一种基因组核苷酸定点替换的方法与流程

本发明涉及一种基因组核苷酸定点替换的方法。
背景技术：
：随着crispr-cas9(theclusteredregularlyinterspacedshortpalindromicrepeats-crispr-associatedprotein9)技术的出现和发展，目前已经成为强有力的基因组编辑手段，被广泛应用到很多组织和细胞中。crispr/cas9protein–rna复合物通过向导rna(guiderna)定位于靶点上，切割产生dna双链断裂(dsb，dsdnabreak)，而后，生物体会本能的启动dna修复机制修复dsb。一般有两种修复机制，一种是非同源末端连接(nhej，non-homologousendjoining)，一种是同源重组(hdr，homology-directedrepair)，通常情况下，nhej修复占大多数，因此，修复产生的随机的indels(insertionsordeletions)比精确修复高很多。对于碱基精确替换，因为hdr效率低以及需要dna模板，所以使用hdr实现碱基精确替换的应用受到很大的限制。2016年，davidliu和akihikokondo两个实验室分别独立报道了两个不同类型的胞嘧啶碱基编辑器(cbe，cytosinebaseeditor)，原理都是通过使用胞苷脱氨酶实现直接对单个的c(胞嘧啶，cytosine)碱基进行编辑，而不再通过产生dsb和启动hdr修复，大大提高了c替换为t(胸腺嘧啶，thymine)的碱基编辑效率。pmcda1(activation-inducedcytidinedeaminase(aid)orthologfromsealamprey)就是其中使用的一种胞苷脱氨酶。在所测试的pmcda1编辑器中，spcas9n(d10a)&pmcda1&ugi(尿嘧啶dna糖化酶抑制剂，uracildnaglycosylaseinhibitor)碱基编辑系统的平均突变率较高，一是因为ugi可以抑制udg(尿嘧啶dna糖化酶，uracildnaglycosylase)催化清除dna中u(尿嘧啶，uracil)，二是因为spcas9n(d10a)在非编辑链上产生切口，诱导真核错配修复机制或long-patchber(base-excisionrepair)修复机制，促使u:g错配更多的偏好性修复成u:a。spcas9n(d10a)连带着pmcda1通过sgrna定位到靶点，pmcda1催化非配对的单链dna上的c发生胞嘧啶脱氨反应变成u，通过dna的修复使得u与a(腺嘌呤，adenine)配对，又通过dna复制，最终使得t与a配对，从而实现了c到t的转换。植物中使用spcas9进行基因组编辑时具有一定的脱靶效应，spcas9n(d10a)与pmcda融合使用进行碱基编辑，即使用spcas9n(d10a)&pmcda1&ugi碱基编辑系统时可能存在潜在的脱靶风险。虽然植物不同于动物，在后代可以通过遗传分离去除脱靶位点，但是由于一些潜在的脱靶位点可能是未知的，所以，后代中就很难有目的性的去除。因此，降低脱靶效应也是植物中一直以来的技术优化方向。技术实现要素：本发明的目的是提供一种基因组核苷酸定点替换的方法。本发明提供了一种碱基编辑系统，包括融合蛋白和sgrna；所述融合蛋白由切刻酶、胞嘧啶核苷脱氨酶pmcda1和尿嘧啶dna糖化酶抑制剂ugi组成；所述切刻酶如序列表的序列15自n端第1-1423位氨基酸所示；所述sgrna的核苷酸序列如序列表的序列3自5’端第441-526位所示。所述胞嘧啶核苷脱氨酶pmcda1如序列表的序列15自n端第1521-1728位氨基酸所示。所述尿嘧啶dna糖化酶抑制剂ugi如序列表的序列15自n端第1736-1833位氨基酸所示。本发明还保护表达所述的碱基编辑系统的重组表达载体、表达盒、重组细胞或重组菌。表达所述融合蛋白的表达盒具体可为表达盒甲。当靶序列如表1所示时，表达所述sgrna的表达盒具体可为表达盒乙。本发明还保护一种用于基因组碱基替换的重组表达载体，包括表达盒甲和表达盒乙；所述表达盒甲表达所述融合蛋白；所述表达盒乙包括n个元件乙；所述元件乙包括所述sgrna和靶序列；所述重组表达载体可靶向n个不同的靶序列进行碱基替换。所述元件乙还包括pre-trna的核苷酸序列；所述pre-trna的核苷酸序列如序列表的序列3自5’端第344-420所示。所述元件乙自5’端依次为pre-trna的核苷酸序列、靶点序列和sgrna的核苷酸序列。以上任一所述表达盒甲中由启动子甲启动所述切刻酶的编码基因、胞嘧啶核苷脱氨酶pmcda1的编码基因和尿嘧啶dna糖化酶抑制剂ugi的编码基因表达。所述表达盒甲自5’端依次包括启动子甲、切刻酶的编码基因、胞嘧啶核苷脱氨酶pmcda1的编码基因、尿嘧啶dna糖化酶抑制剂ugi的编码基因和终止子甲。所述启动子甲具体可为osubq3启动子。所述osubq3启动子的核苷酸序列如序列表的序列1自5’端第1290-3003位所示。所述终止子甲具体可为camv35s终止子。所述camv35s终止子的核苷酸序列如序列表的序列1自5’端第8518-8712位。所述切刻酶的编码基因如序列表的序列1自5’端第3010-7278位所示。所述胞嘧啶核苷脱氨酶pmcda1的编码基因如序列表的序列1自5’端第7570-8193位所示。所述尿嘧啶dna糖化酶抑制剂ugi编码基因如序列表的序列1自5’端第8215-8511位所示。所述表达盒甲具体可如序列表的序列1自5’端第1290-8712位所示。以上任一所述表达盒乙中由启动子乙启动所述元件乙表达。所述表达盒乙自5’端依次为启动子乙、元件乙和终止子乙。所述启动子乙具体可为osu3启动子。所述osu3启动子的核苷酸序列如序列表的序列3自5’端第1-337位所示。所述终止子乙具体可为osu3终止子。所述osu3终止子的核苷酸序列如序列表的序列3自5’端第893-1183位所示。当靶序列如表1所示时，所述表达盒乙具体可如序列表的序列3所示。当靶序列如表1所示时，以上任一所述重组表达载体具体可为将序列表的序列1自5’端第131位-1283位替换为序列表的序列3得到的环状质粒。本发明还保护所述融合蛋白。本发明还保护所述融合蛋白的编码基因。所述融合蛋白的编码基因具体可如序列表的序列1自5’端第3010-8511位所示(其中，第3010-7278位为切刻酶的编码基因，第7570-8193位为胞嘧啶核苷脱氨酶pmcda1的编码基因，第8215-8511位为尿嘧啶dna糖化酶抑制剂ugi的编码基因)。本发明还保护一种植物基因组碱基替换的方法，包括如下步骤：利用以上任一所述碱基编辑系统完成植物基因组碱基替换。本发明还保护一种植物基因组碱基替换的方法，包括如下步骤：将以上任一所述的重组表达载体导入目的植物，实现植物基因组碱基替换。本发明还保护所述碱基编辑系统，或，以上任一所述的重组表达载体、表达盒、重组细胞或重组菌，或，所述的融合蛋白，在植物基因组碱基替换中的应用。以上任一所述碱基替换为将碱基c替换为t。以上任一所述所述植物具体可为水稻，更具体可为日本晴水稻。本发明首次将高保真酶espcas9(1.1)n和优化的sgrna运用到pmcda1介导的碱基编辑系统中，大部分靶点提高了c·t碱基替换的替换效率；sgrna(wt)+espcas9(1.1)n&pmcda1&ugi相对于sgrna(wt)+spcas9n&pmcda1&ugi,大大降低了c·t碱基替换的替换效率；而sgrna(modified)+espcas9(1.1)n&pmcda1&ugi则能够增加c·t碱基替换的替换效率，使得c·t碱基替换的替换效率与sgrna(wt)+spcas9n&pmcda1&ugi相当甚至更高；sgrna(modified)+espcas9(1.1)n&pmcda1&ugi脱靶效率几乎为零，相对于sgrna(wt)+spcas9n&pmcda1&ugi，大幅度降低了脱靶效应。附图说明图1为sgrna(wt)+spcas9n&pmcda1&ugi和sgrna(wt)+espcas9(1.1)n&pmcda1&ugi的c·t碱基替换效率。图2为sgrna(wt)+espcas9(1.1)n&pmcda1&ugi和sgrna(modified)+espcas9(1.1)n&pmcda1&ugi的c·t碱基替换效率。图3为脱靶效率检测结果。具体实施方式以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。日本晴水稻：参考文献：梁卫红,王高华,杜京尧,等.硝普钠及其光解产物对日本晴水稻幼苗生长和5种激素标记基因表达的影响[j].河南师范大学学报(自然版),2017(2):48-52.；公众可以从北京市农林科学院获得。下述实施例中靶标基因、靶点名称和序列如表1所示。表1靶标基因靶点名称靶点序列osalscs650cgcgtccatggagatccaccoscdc48cs651gaccagccagcgtctggcgcosnrt1.1bcs652cggcgacggcgagcaagtgg实施例1、sgrna(modified)+espcas9(1.1)n&pmcda1&ugi系统c·t碱基替换效率一、基因组编辑载体的构建1、sgrna(wt)+espcas9(1.1)n&pmcda1&ugi：人工合成序列表的序列1所示的环状质粒。序列表的序列1包括如下三个表达盒：序列1自5’端第131位-1283位为表达盒i，其中，第131-467位为osu3启动子的核苷酸序列，第474-550位为pre-trna的核苷酸序列，第551-570位为cs650靶点的核苷酸序列，第571-646位为sgrna(wt)的核苷酸序列，第647-723位为pre-trna的核苷酸序列，第724-743位为cs651靶点的核苷酸序列，第744-819位为sgrna(wt)的核苷酸序列，第820-896位为pre-trna的核苷酸序列，第897-916位为cs652靶点的核苷酸序列，第917-992位为sgrna(wt)的核苷酸序列，第993-1283位为osu3终止子的核苷酸序列。序列1自5’端第1290-8712位为表达盒ⅱ，其中，第1290-3003位为osubq3启动子的核苷酸序列，第3010-7278位为espcas9(1.1)n的核苷酸序列(不含有终止密码子)，第7570-8193位为pmcda1的核苷酸序列，第8215-8511位为ugi的核苷酸序列，第8518-8712位为camv35s终止子的核苷酸序列。序列1自5’端第8787-12064位为表达盒ⅲ，其中，第8787-10779为zmubi1启动子的核苷酸序列，第10786-11811位为潮霉素的核苷酸序列，第11812-12064位为nos终止子的核苷酸序列。2、sgrna(wt)+spcas9n&pmcda1&ugi：将序列表的序列1自5’端第3010-7278位替换为序列表的序列2得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi与sgrna(wt)+espcas9(1.1)n&pmcda1&ugi的区别仅在于采用spcas9n替换了espcas9(1.1)n。3、sgrna(modified)+espcas9(1.1)n&pmcda1&ugi：将序列表的序列1自5’端第131位-1283位(表达盒i)替换为序列表的序列3得到的环状质粒。序列表的序列3中，自5’端第1-337位为osu3启动子的核苷酸序列，第344-420位为pre-trna的核苷酸序列，第421-440位为cs650靶点的核苷酸序列，第441-526位为sgrna(modified)的核苷酸序列，第527-603位为pre-trna的核苷酸序列，第604-623位为cs651靶点的核苷酸序列，第624-709位为sgrna(modified)的核苷酸序列，第710-786位为pre-trna的核苷酸序列，第787-806位为cs652靶点的核苷酸序列，第807-892位为sgrna(modified)的核苷酸序列，第893-1183位为osu3终止子的核苷酸序列。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi和sgrna(wt)+espcas9(1.1)n&pmcda1&ugi的区别仅在于采用sgrna(modified)替换了sgrna(wt)。二、在水稻愈伤组织中进行基因编辑将步骤一构建的三个载体分别按照如下步骤1-5进行操作：1、将载体导入农杆菌lba4404(唯地生物，上海，cat#:ac1030)，得到重组菌，采用yep培养基培养重组菌，得到菌液od600nm为0.2的侵染液。2、选取日本晴水稻种子，剥去种皮，灭菌洗涤后，均匀的点入在n6培养基中，28℃暗培养4-6周以诱导愈伤组织的产生。3、将步骤2得到的水稻愈伤组织浸泡在步骤1得到的侵染液中浸泡10min，取愈伤组织接种于含有两层滤纸的培养皿上，25℃黑暗下培养3天(培养基为含有100mg/l的特美汀的n6培养基)，然后愈伤组织在筛选培养基中(含有50mg/l潮霉素的n6培养基，ph5.7)28℃黑暗下筛选培养2周，转入新配置的筛选培养基中再次进行筛选培养2周，获得抗性愈伤。4、提取步骤3得到的抗性愈伤的基因组dna，采用引物f(5’-attatgtagcttgtgcgtttcg-3’)和引物r(5’-gatgaagagcttatcgacgt-3’)组成的引物对进行pcr扩增；将获得的扩增产物进行琼脂糖凝胶电泳，如电泳图显示有1150bp大小的条带，说明对应的愈伤为阳性抗性愈伤。5、对步骤4得到的阳性抗性愈伤dna(每个载体选择15块)，分别采用cs650靶点引物(cs650-f和cs650-r)、cs651靶点引物(cs651-f和cs651-r)和cs652靶点引物(cs652-f和cs652-r)进行pcr扩增，然后将扩增产物测序。cs650-f:5’-taagaaccaccagcgacacc-3’；cs650-r:5’-ggtaattgtgcttggtgatggag-3’；cs651-f:5’-acatcgagatggagaagcgg-3’；cs651-r:5’-ccatgctccaatcgatgaatac-3’；cs652-f:5’-ttacgaactttataactttgtcgg-3’；cs652-r:5’-atggaggcgatgaggaagac-3’。对于cs650靶点，发生c·t碱基替换的位点对应为：cgtgtccatggagatccacc；对于cs651靶点，发生c·t碱基替换的位点对应为：gattagccagcgtctggcgc；对于cs652靶点，发生c·t碱基替换的位点对应为：cggtgacggcgagcaagtgg。三个靶点分别统计c·t碱基替换的阳性抗性愈伤数，碱基替换效率为15块阳性抗性愈伤中发生c·t碱基替换的阳性抗性愈伤所占的比例。结果如图1和图2所示。实验结果表明，相对于sgrna(wt)+spcas9n&pmcda1&ugi，sgrna(wt)+espcas9(1.1)n&pmcda1&ugi对靶点的c·t碱基替换的替换效率显著下降(图1)；而sgrna(modified)+espcas9(1.1)n&pmcda1&ugi对靶点的c·t碱基替换效率相对于sgrna(wt)+espcas9(1.1)n&pmcda1&ugi显著增加(图2)，增至与sgrna(wt)+spcas9n&pmcda1&ugi相当甚至更高(图1和图2)。实施例2、sgrna(modified)+espcas9(1.1)n&pmcda1&ugi系统脱靶效应一、基因编辑载体的构建sgrna(wt)+spcas9n&pmcda1&ugi-t1：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列4得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t2：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列5得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t3：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列6得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t4：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列7得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t5：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列8得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t6：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列9得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t7：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列10得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t8：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列11得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t9：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列12得到的环状质粒。sgrna(wt)+spcas9n&pmcda1&ugi-t10：将sgrna(wt)+spcas9n&pmcda1&ugi载体中自5’端第551-916位替换为序列表的序列13得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t1：将序列表的序列1自5’端第551-916位替换为序列表的序列4得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t2：将序列表的序列1自5’端第551-916位替换为序列表的序列5得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t3：将序列表的序列1自5’端第551-916位替换为序列表的序列6得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t4：将序列表的序列1自5’端第551-916位替换为序列表的序列7得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t5：将序列表的序列1自5’端第551-916位替换为序列表的序列8得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t6：将序列表的序列1自5’端第551-916位替换为序列表的序列9得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t7：将序列表的序列1自5’端第551-916位替换为序列表的序列10得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t8：将序列表的序列1自5’端第551-916位替换为序列表的序列11得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t9：将序列表的序列1自5’端第551-916位替换为序列表的序列12得到的环状质粒。sgrna(wt)+espcas9(1.1)n&pmcda1&ugi-t10：将序列表的序列1自5’端第551-916位替换为序列表的序列13得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi载体的表达盒i替换为序列表的序列14得到的环状质粒。序列表的序列14中，自5’端第421-440位为靶点序列。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t2：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列5得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t3：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列6得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t4：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列7得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t5：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列8得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t6：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列9得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t7：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列10得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t8：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列11得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t9：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列12得到的环状质粒。sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t10：将sgrna(modified)+espcas9(1.1)n&pmcda1&ugi-t1载体的靶点序列替换为序列表的序列13得到的环状质粒。二、在水稻愈伤组织中进行基因编辑1、将步骤一得到的载体分别按照实施例1中步骤二的1-4进行操作，得到阳性抗性愈伤。2、每个载体从步骤1得到的阳性抗性愈伤中随机选取8块，将其dna进行混合，分别采用cs652靶点引物(cs652-f和cs652-r)进行pcr扩增，以第一轮pcr产物为模板，将正向和反向条码加入pcr产物末端进行文库构建，使用illuminahiseq2500高通量测序平台测序，测序深度为10000x(石家庄博瑞迪生物技术有限公司)，目标靶点区检测c·t碱基替换和indels，靶点区任何c·t碱基替换和indels均计入脱靶突变，脱靶效率为检测到的有突变的reads数占总reads数的比例。结果如图3所示。实验结果表明，sgrna(modified)+espcas9(1.1)n&pmcda1&ugi脱靶效率几乎为零。序列表<110>北京市农林科学院<120>一种基因组核苷酸定点替换的方法<160>15<170>siposequencelisting1.0<210>1<211>18476<212>dna<213>人工序列(artificialsequence)<400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agtccgttatcaacttgaaaaagtggcaccgagtcggtgcaacaaagcaccagt540ggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggt600gcagaccagccagcgtctggcgcgtttcagagctatgctggaaacagcatagcaagttga660aataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcaacaaagcacc720agtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggct780ggtgcacggcgacggcgagcaagtgggtttcagagctatgctggaaacagcatagcaagt840tgaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttt900ttcgttttgcattgagttttctccgtcgcatgtttgcagttttattttccgttttgcatt960gaaatttctccgtctcatgtttgcagcgtgttcaaaaagtacgcagctgtatttcactta1020tttacggcgccacattttcatgccgtttgtgccaactatcccgagctagtgaatacagct1080tggcttcacacaacactggtgacccgctgacctgctcgtacctcgtaccgtcgtacggca1140cagcatttggaattaaagggtgtgatcgatactgcttgctgct1183<210>4<211>20<212>dna<213>人工序列(artificialsequence)<400>4tagcgacggcgagcaagtgg20<210>5<211>20<212>dna<213>人工序列(artificialsequence)<400>5cgatgacggcgagcaagtgg20<210>6<211>20<212>dna<213>人工序列(artificialsequence)<400>6cggcagcggcgagcaagtgg20<210>7<211>20<212>dna<213>人工序列(artificialsequence)<400>7cggcgatagcgagcaagtgg20<210>8<211>20<212>dna<213>人工序列(artificialsequence)<400>8cggcgacgatgagcaagtgg20<210>9<211>20<212>dna<213>人工序列(artificialsequence)<400>9cggcgacggcaggcaagtgg20<210>10<211>20<212>dna<213>人工序列(artificialsequence)<400>10cggcgacggcgaataagtgg20<210>11<211>20<212>dna<213>人工序列(artificialsequence)<400>11cggcgacggcgagcgggtgg20<210>12<211>20<212>dna<213>人工序列(artificialsequence)<400>12cggcgacggcgagcaaacgg20<210>13<211>20<212>dna<213>人工序列(artificialsequence)<400>13cggcgacggcgagcaagtaa20<210>14<211>817<212>dna<213>人工序列(artificialsequence)<400>14gaagcaacttaaagttatcaggcatgcatggatcttggaggaatcagatgtgcagtcagg60gaccatagcacaagacaggcgtcttctactggtgctaccagcaaatgctggaagccggga120acactgggtacgttggaaaccacgtgatgtgaagaagtaagataaactgtaggagaaaag180catttcgtagtgggccatgaagcctttcaggacatgtattgcagtatgggccggcccatt240acgcaattggacgacaacaaagactagtattagtaccacctcggctatccacatagatca300aagctgatttaaaagagttgtgcagatgatccgtggcggatccaacaaagcaccagtggt360ctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgca420tagcgacggcgagcaagtgggtttcagagctatgctggaaacagcatagcaagttgaaat480aaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttttttcgtt540ttgcattgagttttctccgtcgcatgtttgcagttttattttccgttttgcattgaaatt600tctccgtctcatgtttgcagcgtgttcaaaaagtacgcagctgtatttcacttatttacg660gcgccacattttcatgccgtttgtgccaactatcccgagctagtgaatacagcttggctt720cacacaacactggtgacccgctgacctgctcgtacctcgtaccgtcgtacggcacagcat780ttggaattaaagggtgtgatcgatactgcttgctgct817<210>15<211>1833<212>prt<213>人工序列(artificialsequence)<400>15metasptyrlysasphisaspglyasptyrlysasphisaspileasp151015tyrlysaspaspaspasplysmetalaprolyslyslysarglysval202530glyilehisglyvalproalaalaasplyslystyrserileglyleu354045alaileglythrasnservalglytrpalavalilethraspglutyr505560lysvalproserlyslysphelysvalleuglyasnthrasparghis65707580serilelyslysasnleuileglyalaleuleupheaspserglyglu859095thralaglualathrargleulysargthralaargargargtyrthr100105110argarglysasnargilecystyrleuglngluilepheserasnglu115120125metalalysvalaspaspserphephehisargleuglugluserphe130135140leuvalglugluasplyslyshisgluarghisproilepheglyasn145150155160ilevalaspgluvalalatyrhisglulystyrprothriletyrhis165170175leuarglyslysleuvalaspserthrasplysalaaspleuargleu180185190iletyrleualaleualahismetilelyspheargglyhispheleu195200205ilegluglyaspleuasnproaspasnseraspvalasplysleuphe210215220ileglnleuvalglnthrtyrasnglnleupheglugluasnproile225230235240asnalaserglyvalaspalalysalaileleuseralaargleuser245250255lysserargargleugluasnleuilealaglnleuproglyglulys260265270lysasnglyleupheglyasnleuilealaleuserleuglyleuthr275280285proasnphelysserasnpheaspleualagluaspalalysleugln290295300leuserlysaspthrtyraspaspaspleuaspasnleuleualagln305310315320ileglyaspglntyralaaspleupheleualaalalysasnleuser325330335aspalaileleuleuseraspileleuargvalasnthrgluilethr340345350lysalaproleuseralasermetilelysargtyraspgluhishis355360365glnaspleuthrleuleulysalaleuvalargglnglnleuproglu370375380lystyrlysgluilephepheaspglnserlysasnglytyralagly385390395400tyrileaspglyglyalaserglnglugluphetyrlyspheilelys405410415proileleuglulysmetaspglythrglugluleuleuvallysleu420425430asnarggluaspleuleuarglysglnargthrpheaspasnglyser435440445ileprohisglnilehisleuglygluleuhisalaileleuargarg450455460glngluaspphetyrpropheleulysaspasnargglulysileglu465470475480lysileleuthrpheargileprotyrtyrvalglyproleualaarg485490495glyasnserargphealatrpmetthrarglysserglugluthrile500505510thrprotrpasnpheglugluvalvalasplysglyalaseralagln515520525serpheilegluargmetthrasnpheasplysasnleuproasnglu530535540lysvalleuprolyshisserleuleutyrglutyrphethrvaltyr545550555560asngluleuthrlysvallystyrvalthrgluglymetarglyspro565570575alapheleuserglygluglnlyslysalailevalaspleuleuphe580585590lysthrasnarglysvalthrvallysglnleulysgluasptyrphe595600605lyslysileglucyspheaspservalgluileserglyvalgluasp610615620argpheasnalaserleuglythrtyrhisaspleuleulysileile625630635640lysasplysasppheleuaspasnglugluasngluaspileleuglu645650655aspilevalleuthrleuthrleuphegluaspargglumetileglu660665670gluargleulysthrtyralahisleupheaspasplysvalmetlys675680685glnleulysargargargtyrthrglytrpglyargleuser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