本发明公开了一种爪哇根结线虫效应基因
1.一种爪哇根结线虫效应基因Mj-nulg,其特征在于核苷酸序列如SEQxa0IDxa0NO:1~3任意一条所示。
2.一种爪哇根结线虫效应蛋白MJ-NULG,其特征在于氨基酸序列如SEQxa0IDxa0NO:4所示。
3.在严格条件下与权利要求1所述爪哇根结线虫效应基因Mj-nulg杂交且编码与爪哇根结线虫寄生相关蛋白的DNA分子。
4.与权利要求1所述爪哇根结线虫效应基因Mj-nulg具有90%以上同源性且编码爪哇根结线虫寄生相关蛋白的DNA分子。
5.一种爪哇根结线虫效应蛋白MJ-NULG,其特征在于在SEQxa0IDxa0NO:4所示氨基酸序列的氨基末端或羧基末端连接有特定标签,所述特定标签为5~6个精氨酸,或2~12个组氨酸,或如SEQxa0IDxa0NO:5所示序列,或如SEQxa0IDxa0NO:6所示序列,或如SEQxa0IDxa0NO:7所示序列。
6.一种重组表达载体,其特征在于由出发载体的多克隆位点插入权利要求1所述爪哇根结线虫效应基因Mj-nulg,或该基因的部分外显子片段如SEQxa0IDxa0NO:8~10任一所示序列构建而成。
7.一种重组基因表达盒,其特征在于含有权利要求1所述爪哇根结线虫效应基因Mj-nulg。
8.转基因细胞系,其特征在于含有权利要求1所述爪哇根结线虫效应基因Mj-nulg。
9.一种重组菌,其特征在于含有权利要求1所述爪哇根结线虫效应基因Mj-nulg。
10.权利要求1所述爪哇根结线虫效应基因Mj-nulg在制备转基因植物中的应用。技术领域
本发明属生物基因工程技术领域,具体涉及一种爪哇根结线虫效应基因Mj-nulg,相关蛋白及应用。
背景技术
根结线虫是一类非常重要的植物寄生线虫,广泛分布于世界各地,能侵染超过3000种不同植物。根结线虫每年造成大概770亿美元的经济损失,随着作物复种指数的提高和设施农业的发展,根结线虫的发生和危害将会不断的加剧。由于根结线虫的根内寄生、寄主范围广泛的特性以及抗根结线虫种质资源的缺乏限制了化学防治、轮作和抗病育种等防治方法在控制根结线虫危害中的应用。因此,寻找其他有效的防治根结线虫的方法是植物生产所长期面临的艰巨任务。RNA干扰技术(RNAi)的出现使得开发更为有效的防治根结线虫的方法成为可能。RNAi能通过靶向抑制根结线虫特定基因功能而限制根结线虫侵染、寄生和繁殖因而能高效地减少根结线虫的危害而且对其他非靶标生物较为安全。RNAi现象最早在秀丽小杆线虫中发现,随后在植物、无脊椎动物和脊椎动物中都相继发现了这种现象。RNAi技术通过dsRNA特异性的降解靶mRNA,影响或抑制该目的基因功能,从而影响该种生物的生长、发育、繁殖甚至能杀死该种生物。该技术相比其他防治方法的最大优点是能定向作用于特定的某种(某类)害虫而不会影响在该生境中的其他生物。应用农杆菌介导的病毒诱导的基因沉默(VIGS)技术相对于传统的使用组织培养方法获得RNAi转基因植株更加简单快速,使得我们并不需要通过复杂的组培方法就可快速获得抗线虫植株。
发明内容
本发明的目的在于提供一种新的爪哇根结线虫效应基因Mj-nulg。本发明的另一目的是提供上述爪哇根结线虫效应基因Mj-nulg的相关蛋白MJ-NULG。本发明的又一目的是提供上述爪哇根结线虫效应基因Mj-nulg的应用。本发明通过以下技术方案实现上述目的:一种爪哇根结线虫(Meloidogynexa0javanica)效应基因Mj-nulg,其核苷酸序列如SEQxa0IDxa0NO:1~3任意一条所示。其中SEQxa0IDxa0NO:1为全基因序列(包括5’端非编码区、3’端非编码区、内含子和外显子),SEQxa0IDxa0NO:2为编码区序列(包括内含子和外显子),SEQxa0IDxa0NO:3为编码序列(即cDNA序列,仅为外显子)。在严格条件下与上述爪哇根结线虫效应基因Mj-nulg杂交且编码与爪哇根结线虫寄生相关蛋白的DNA分子,或者与上述爪哇根结线虫效应基因Mj-nulg有90%以上同源性且编码根结线虫寄生相关蛋白的DNA分子,也在本发明的保护范围之内。所述严格条件为:可在0.1×SSC、0.1%xa0SDS的溶液中,65°C条件下杂交并洗膜。一种爪哇根结线虫效应蛋白MJ-NULG,其氨基酸序列如SEQxa0IDxa0NO:4所示,或该氨基酸序列经过一个或一个以上氨基酸残基的取代和/或缺失和/或添加并且与爪哇根结线虫寄生相关的衍生蛋白。为了使爪哇根结线虫效应蛋白MJ-NULG便于纯化,可以在该序列(SEQxa0IDxa0NO:4)的氨基末端或羧基末端连接上特定的标签,所述特定标签为5~6个精氨酸,或2~12个组氨酸,或如SEQxa0IDxa0NO:5所示序列,或如SEQxa0IDxa0NO:6所示序列,或如SEQxa0IDxa0NO:7所示序列。如表1,但不限于表1:表1xa0标签序列 标签 残基数量 序列 Poly-Arg 5-6(常为5个) RRRRR Poly-His 2-12(常为6个) HHHHHH FLAG 8 DYKDDDK(SEQ ID NO:5) Strep-TagII 8 WSHPQFEK(SEQ ID NO:6) c-myc 10 EQKLISEEDL(SEQ ID NO:7) 上述蛋白可以人工合成,也可以先合成其编码基因,再进行生物表达得到。一种重组表达载体,是由出发载体的多克隆位点插入上述爪哇根结线虫效应基因Mj-nulg,或该基因的部分外显子片段如SEQxa0IDxa0NO:8~10任一所示序列构建而成。其中SEQxa0IDxa0NO:8为Mj-nulg全基因序列(SEQxa0IDxa0NO:1)的第369至691核苷酸序列,SEQxa0IDxa0NO:9为第951至1305核苷酸序列,SEQxa0IDxa0NO:10为第1712至1852核苷酸序列。上述表达载体的出发载体可以是一般表达载体或RNAi载体,可以根据需要选择。对于制备转基因抗病植物,载体pTRV2较优,该载体是一种植物RNAi载体,将含有爪哇根结线虫效应基因Mj-nulg的该RNAi载体导入植物,可以在植物中产生所述基因的dsRNA,当根结线虫侵染该植株后,dsRNA进入根结线虫的体内,使所述基因的mRNA降解,引发所述基因的失活,严重的减弱了线虫的寄生能力,从而抑制线虫的侵染、寄生、繁殖和传播。对于含有上述爪哇根结线虫效应基因Mj-nulg的重组基因表达盒、转基因细胞系或重组菌,也属于本发明的保护范围。上述爪哇根结线虫效应基因Mj-nulg在制备转基因植物中的应用,所述转基因植物的目的植物优选为双子叶植物或单子叶植物,尤其是番茄,如番茄夏红1号。作为上述应用的一个方面,可以将含有爪哇根结线虫效应基因Mj-nulg的重组表达载体利用农杆菌导入至目的植物中,或者通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导入或基因枪等常规生物学方法转化植物细胞或组织,并将转化的植物组织培育成植株,得到抗根结线虫的转基因植物。与现有技术相比,本发明具有以下有益效果:本发明提供了一种新的有利于提高植物抗根结线虫活性的基因序列及其编码蛋白,为进一步培育有广谱抗虫性的植物新品种奠定基础。
附图说明
图1为3’RACExa0扩增获得的片段的电泳结果;3为3’RACE的电泳结果;M为分子量标记。图2为5’RACExa0扩增获得的片段的电泳结果;xa03为5’RACE的电泳结果;M为分子量标记。图3为扩增Mj-nulg基因的电泳结果;xa03为扩增Mj-nulg基因的电泳结果;M为分子量标记。图4为爪哇根结线虫不同发育阶段Mj-nulg基因的相对表达量。图5xa0为MJ-NULG蛋白在爪哇根结线虫中组织定位。A:为侵染前的二龄幼虫,B:为侵染阶段三龄幼虫,C:为对照,没有加入抗血清。DG:表示背食道腺细胞。图6xa0为使用免疫定位技术研究MJ-NULG蛋白的分泌情况。A:根结线虫侵染5天后的根结切片,B:为根结线虫侵染10天后的根结切片,C:为根结线虫侵染10天后的根结切片,D为为根结线虫侵染21天后的根结切片,E:为根结线虫侵染10天后的根结切片,加入的是对照的未免疫的抗血清,F:为根结线虫侵染10天后的根结切片,未作任何处理。图7xa0为pBI121-nulgxa0载体示意图。图8为pTRV2-nulgxa0载体示意图。图9为表达pTRV2-nulgxa0载体的植株,与对照组植株抗根结线虫能力差异的实验。
具体实施方式
下面通过实施例进一步解释本发明,但对本发明不做任何形式的限定。实施例中无特殊说明,均为本领域常规实验方法;所用实验材料如无特殊说明,均购自常规生化试剂商店。实施例中定量实验均设置三次重复,结果取平均值。生物材料来源如下:番茄夏红一号:购于华南农业大学科技发展有限公司;爪哇根结线虫:自行采集,参考文献:Huxa0M,xa0X.,xa0Zhuo,xa0K.,xa0Liao,xa0J,xa0L.xa0Multiplexxa0PCRxa0forxa0thexa0simultaneousxa0identificationxa0andxa0detectionxa0ofxa0Meloidogynexa0incognita,xa0M.xa0enterolobiixa0andxa0M.xa0javanicaxa0usingxa0DNAxa0extractedxa0directlyxa0fromxa0individualxa0galls.xa0Phytopathology,xa02011,xa0101(11):xa01270-1277。以下生物材料为发明人获赠得到:拟南芥克伦比亚生态型:参考文献:叶青,侯智慧,刘菁,等.xa0H₂O₂介导H₂S诱导的拟南芥气孔关闭.xa0植物生理学报,2011,47xa0(12):1195-1200。载体pTRV1和载体pTRV2:参考文献:Dubreuil,xa0G.,xa0M.xa0Magliano,xa0M.xa0P.xa0Dubrana,xa0etxa0al.xa0Tobaccoxa0rattlexa0virusxa0mediatesxa0genexa0silencingxa0inxa0axa0plantxa0parasiticxa0root-knotxa0nematode.xa0Journalxa0ofxa0Experimentalxa0Botany,xa02009,xa060(14):xa04041-4050。载体pBI121:参考文献:王华新,曹家树,向珣,等.xa0pBI121xa0表达载体及其转化植株的快速鉴定.xa0浙江大学学报(xa0农业与生命科学版),2008,34(2):137-142。根癌农杆菌EHA105:参考文献:Ryu,xa0C.xa0M.,xa0A.xa0Anand,xa0L.xa0Kang,xa0etxa0al.xa0Agrodrench:xa0axa0novelxa0andxa0effectivexa0agroinoculationxa0methodxa0forxa0virus-inducedxa0genexa0silencingxa0inxa0rootsxa0andxa0diversexa0Solanaceousxa0species.xa0Plantxa0Journal,xa02004,xa040(2):xa0322-331。以上生物材料公众可以从申请人处获得。 实施例1爪哇根结线虫MJ-NULG蛋白及其编码基因的克隆 (1)本实验室使用抑制差减杂交(SSH)的方法,从爪哇根结线虫中获得了一批在二龄幼虫中表达量比卵的表达量明显增高的EST序列。发明人通过NCBI上的BLASTNxa0程序比较上述EST序列与其它物种中发现的基因的同源性,选择了一条仅出现在根结线虫属中的EST序列进行克隆。(2)使用TRIZOLxa0法提取爪哇根结线虫二龄幼虫RNA,然后使用Clontechxa0RACE试剂盒将RNA反转录为cDNA。(3)3’RACE片段的获得:使用引物对,D153R2:GTTGAGGAAGAGACCCGTGAGAA,(SEQxa0IDxa0NO:11)和Clontechxa0RACE试剂盒的锚定引物NUP,以步骤(2)获得的cDNA为模板进行扩增。PCR扩增体系:cDNAxa02μL,两条引物各3μL,10×KODxa0plusxa0Bufferxa05μL,MgSO₄xa02μL,dNTPxa05μL,KODxa0plusxa0DNAxa0polymerasexa01μL,xa0ddH₂Oxa034μL。PCR扩增程序:94°Cxa03min,30×(94°Cxa030s,66°Cxa030s,68°Cxa02min),68°Cxa05min,20°C保存。琼脂糖电泳检测扩增结果,见图1。(4)5’RACE片段的获得:使用引物对,D155R2:CTTTTCACGTTCATCAGCACCAG,(SEQxa0IDxa0NO:12)和Clontechxa0RACE试剂盒的锚定引物NUP,以步骤(2)获得的cDNA为模板进行扩增。PCR扩增体系:cDNAxa02μL,两条引物各3μL,10×xa0KODxa0plusxa0Bufferxa05μL,MgSO₄xa02μL,dNTPxa05μL,KODxa0plusxa0DNAxa0polymerasexa01μL,ddH₂Oxa034μL。PCR扩增程序同(3),20°C保存。琼脂糖电泳检测扩增结果,见图2。(5)对3’RACE片段、5’RACE片段和SSH获得的EST片段进行拼接后获得Mj-nulg的基因全长,再使用引物对D15cdsF:ATGAAAACCTCAGTGTTGTGCCTAT(SEQxa0IDxa0NO:13)和D15cdsR:TTAATGGCCCTTTCCTTTAGATTCTA(SEQxa0IDxa0NO:14)xa0进行扩增,验证序列正确性。PCR扩增程序同(3)。结果见图3,测序后获得的序列如序列表中SEQxa0IDxa0NO:1xa0所示,并命名为Mj-nulg,该基因编码的蛋白命名为MJ-NULG,如序列表序列中SEQxa0IDxa0NO:4所示。 实施例2xa0Mj-nulg基因在爪哇根结线虫不同发育阶段的表达分析 (1)使用TRIZOLxa0法提取不同发育阶段的爪哇根结线虫的RNA,并将RNA反转录为cDNA。(2)使用引物qD15AF:AAAGAAAAACGTACAGAAAAGACCA(SEQxa0IDxa0NO:15)和qD15AR:AACTTCCTCAACAGGTTTCTTCTCTxa0(SEQxa0IDxa0NO:16)对Mj-nulg进行荧光定量PCR分析,以爪哇根结线虫β-actin基因为内参,引物为qactinF:GACGGTCAAGTTATTACTGTGGAAA(SEQxa0IDxa0NO:17)和qactinR:GTAAAGGTCTTTACGGATGTCTATG(SEQxa0IDxa0NO:18)。反应条件:94°Cxa030s,40×(94°Cxa030s,xa060°Cxa030s,72°Cxa01min)。结果见图4。图4结果表明,Mj-nulgxa0基因在爪哇根结线虫侵染后二龄幼虫(侵染番茄5天后)时表达量最高,这与编码根结线虫效应蛋白基因的表达模式一致。证明该基因在爪哇根结线虫的寄生中发挥重要作用。 实施例3xa0MJ-NULG蛋白在爪哇根结线虫中的组织定位 常规方法制备MJ-NULG蛋白,免疫兔制备抗血清,检测该蛋白在组织中的定位。具体步骤如下:(1)使用纯化的MJ-NULG蛋白作为抗原免疫新西兰兔,40天后采集抗血清。(2)收集新鲜孵化的爪哇根结线虫二龄幼虫和寄生在根内的三龄幼虫,用灭菌水清洗数次后,加入4%甲醛固定24小时。(3)用灭菌水清洗数次后,加入5%xa0BSAxa037°Cxa0处理30分钟。(4)加入MJ-NULGxa0抗血清,对照组则加入未免疫的抗血清,4°C处理16小时。(5)用灭菌水清洗数次后,加入HRP标记的抗兔IgG抗体,室温处理1小时。(6)用灭菌水清洗数次后,加入HRP显色液,黑暗静置10分钟后观察,结果如图5。图5结果表明,MJ-NULG蛋白在爪哇根结线虫的背食道腺中表达,这与其他根结线虫效应蛋白基因的组织表达模式一致。进一步说明该基因在爪哇根结线虫的寄生中发挥重要作用。 实施例xa04xa0MJ-NULG蛋白的分泌及定位情况 (1)xa0使用纯化的MJ-NULG蛋白作为抗原免疫新西兰兔,40天后采集抗血清。(2)xa0收集新鲜孵化的爪哇根结线虫二龄幼虫并接种到番茄幼苗根部,待线虫发育到相应阶段收集根结,用灭菌水清洗后,加入4%甲醛固定。(3)xa0使用酒精进行梯度脱水,并用石蜡在65°C条件下包埋。然后制作成5微米厚的切片。(4)xa0显微镜下观察找出合适的切片后,进行脱蜡处理。(5)xa0然后加入5%xa0BSAxa037°Cxa0处理30分钟。(6)xa0加入MJ-NULGxa0抗血清,对照组则加入未免疫的抗血清,4°Cxa0处理16小时。(7)xa0用灭菌水清洗数次后,加入HRP标记的抗兔IgG抗体,室温处理1小时。(8)xa0用灭菌水清洗数次后,加入HRP显色液,黑暗静置10分钟后观察,结果如图5。图6结果表明,MJ-NULG蛋白能被爪哇根结线虫分泌到植物细胞中,并定位于植物细胞的细胞核。说明该蛋白作用于植物细胞核并对根结线虫的成功寄生有重要作用。 实施例xa05xa0Mj-nulg基因在爪哇根结线虫寄生中的作用 (1)使用TRIZOLxa0法提取爪哇根结线虫二龄幼虫RNA,并将RNA反转录为cDNA。(2)使用引物对D15nsFbam:cgcggatccxa0ATGGATGATAAAGAAGTTGAGAAGCATAA(SEQxa0IDxa0NO:19,斜体字为酶切位点BamHxa0I)/D15cdsRsac:cgagctcxa0TTAATGGATGATAAAGAAGTTGAGAAGCATAA(SEQxa0IDxa0NO:20,斜体字为酶切位点Sacxa0I)进行PCR扩增,PCR扩增程序:94°Cxa03min,30×(94°Cxa030s,60°Cxa030s,68°Cxa02min),68°Cxa05min。将得到的PCR产物使用限制性内切酶Salxa0I和Sphxa0I进行酶切,并纯化回收。(3)同时使用限制性内切酶BamHxa0I和Sacxa0Ixa0酶切载体pBI121,并纯化回收。(4)将(2)和(3)得到的产物进行连接,获得重组表达载体pBI121-nulg。根据测序结果,对重组表达载体pBI121-nulg进行结构描述如下:骨架为pBI121,在骨架载体BamHxa0I和Sacxa0Ixa0酶切位点之间插入了双链DNA片段A(来自序列SEQxa0IDxa0NO:3的5’端第58至825核苷酸所示的DNA分子),如图7。并将载体pBI121-nulg转化根癌农杆菌EHA105得到重组农杆菌。(5)将哥伦比亚生态型拟南芥播种到灭菌土上。45天后,待拟南芥长出花序后,将重组农杆菌使用xa00.01Mxa0KH₂PO₄重悬浮并混合后接种到拟南芥花序。正常培养30天后,收取种子。(6)将步骤(5)所述的种子进行表面消毒后,播种到0.5×MS培养基中进行筛选(培养基中加入了500mg/L羧苄青霉素和25mg/L潮霉素B)。(7)将能在步骤(6)中所述的MS培养基中生长的拟南芥植株移栽到灭菌土中培养,然后收获种子。(8)将步骤(7)所述的种子进行继代培养至T3代,取纯合的转化系进行实验。(9)步骤(8)所述植株,每棵植株接种约500条爪哇根结线虫二龄幼虫。接种后,保持温度在23°C-26°C之间,42天后计算成熟雌虫、根结的数量。实验进行三次重复,结果取平均值。结果见表2。表2xa0各组寄生指数的统计结果 xa0 野生型拟南芥 转化系1拟南芥 转化系2拟南芥 调查株数 45 47 42 成熟雌虫数(条/株) 27±0.6a 45±2.6b 52±3b 根结数(个/株) 20±1.7a 42±3.4b 40±2.1b xa0注:表中数据为平均值±标准误,表中同一指标同行中小写字母不同者示差异显著(p<0.05,Duncan’s法)表2xa0结果表明,表达爪哇根结线虫Mj-nulg基因的拟南芥株系,每株植株上成熟雌虫、根结和数量与野生型拟南芥比较显著增加,说明该基因能增强爪哇根结线虫的感病性,进一步说明该基因在根结线虫寄生中有重要作用。 实施例6xa0xa0Mj-nulg基因在培育抗根结线虫植物中的应用 (1)使用TRIZOLxa0法提取爪哇根结线虫RNA,并将RNA反转录为cDNA。(2)使用引物对D15RNAiF3:cgagctcgGAAAACCTCAGTGTTGTGCCTAT(SEQxa0IDxa0NO:21,斜体字为酶切位点Sacxa0I)/D15RNAiR322:ctagtctagaTTTCATCAGAAACTTCCTCAACAGG(,SEQxa0IDxa0NO:22,斜体字为酶切位点Xbaxa0I)进行PCR扩增,PCR扩增程序:94°Cxa03min,30×(94°Cxa030s,60°Cxa030s,68°Cxa01min),68°Cxa05min。将得到的PCR产物使用限制性内切酶Sacxa0I和Xbaxa0I进行酶切,并纯化回收。(3)同时使用限制性内切酶Sacxa0I和Xbaxa0I酶切载体pTRV2,并纯化回收。(4)将(2)和(3)得到的产物进行连接,获得重组表达载体pTRV2-nulg。根据测序结果,对重组表达载体pTRV2-nulg进行结构描述如下:骨架为pTRV2,在骨架载体Xbaxa0I和Sacxa0I酶切位点之间插入了双链DNA片段B(来自序列SEQxa0IDxa0NO:3的5’端第3至322核苷酸所示的DNA分子),如图8。(5)将载体pTRV1(编码TRV病毒的RNA聚合酶1,为病毒侵染植株所必须)转化根癌农杆菌EHA105到重组农杆菌A。(6)将载体pTRV2-nulg转化根癌农杆菌EHA105得到重组农杆菌B,将载体pTRV2转化根癌农杆菌EHA105得到对照农杆菌。(7)将番茄夏红一号种子进行表面消毒后播种到灭菌的沙壤土中。14天后,幼苗长出第4或第5片真叶;将重组农杆菌A、B使用xa00.01Mxa0KH₂PO₄重悬浮并混合后接种到幼苗的根部。生长21天后,取新长出的叶片进行检测。(8)对照组A的处理方法同(7)所述,但把对照农杆菌代替重组农杆菌B。对照组B则不接种农杆菌菌液。(9)步骤(6)、(7)和(8)所述的植株,每棵植株接种约200条爪哇根结线虫二龄幼虫。接种后,保持温度在23°C-26°C之间,42天后计算成熟雌虫、根结和卵的数量。实验进行三次重复,结果取平均值。结果见表3。表3xa0各组寄生指数的统计结果 xa0 实验组番茄 对照A组番茄 对照B组番茄 调查株数 37 32 38 成熟雌虫数(条/株) 24.7±5.4a 158.3±32b 154.6±17b 根结数(个/株) 12.7±8a 90±14b 94±27b 卵数(个/株) 70±3a 1140±205b 820±174b 注:表中数据为平均值±标准误,表中同一指标同行中小写字母不同者示差异显著(p<0.05,Duncan’s法)表3xa0和图9所示表明,表达沉默爪哇根结线虫的Mj-nulgxa0基因后,能显著地减少每株植株上成熟雌虫、根结和卵的数量,即沉默Mj-nulgxa0基因后,能显著地抑制根结线虫的寄生能力能力。xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0SEQUENCExa0LISTINGxa0<110>xa0xa0华南农业大学xa0<120>xa0xa0一种爪哇根结线虫效应基因Mj-nulg,相关蛋白及应用xa0<130>xa0xa0<160>xa0xa022xa0xa0xa0xa0<170>xa0xa0PatentInxa0versionxa03.3xa0<210>xa0xa01<211>xa0xa01974<212>xa0xa0DNA<213>xa0xa0人工序列xa0<400>xa0xa01gagaacggttxa0aaaaataaagxa0ggagagaagaxa0aagagaaaaaxa0aataaaaagtxa0tggagaaaagxa0xa0xa0xa0xa060xa0aaacgcgcagxa0tcaaaataatxa0aaaagaaagaxa0gaaaaaatgtxa0tattgtttaaxa0tatatattttxa0xa0xa0xa0120xa0ttcaattttgxa0ttttattaaaxa0tgattgatatxa0tacttaacaaxa0aaatatcaagxa0agagtaagaaxa0xa0xa0xa0180xa0aacgcgtggcxa0tactttaaacxa0agatttttttxa0tatcaaacaaxa0ccgttttcatxa0aaacaatttgxa0xa0xa0xa0240xa0gcaaaaaggaxa0ggcgtcgtctxa0caataaacacxa0aaattttaagxa0atgtataaaaxa0gtgttcgagtxa0xa0xa0xa0300xa0caaaacaagtxa0aaatcattccxa0cagttttaatxa0ttaatttttaxa0ataccacattxa0aaaaatataaxa0xa0xa0xa0360xa0acaaaatgaaxa0aacctcagtgxa0ttgtgcctatxa0ttttgattatxa0ttctattattxa0tctatttatgxa0xa0xa0xa0420xa0ccgatgataaxa0agaagttgagxa0aagcataaagxa0ttgaggaagaxa0gacaaaagaaxa0aaacgtgatgxa0xa0xa0xa0480xa0ataaagaactxa0tactcataaaxa0gttgaggatgxa0agacaaaagaxa0gaaacgtgatxa0gataaagaaaxa0xa0xa0xa0540xa0aacgtacagaxa0aaagaccataxa0acaggaaagaxa0agcatgtcgaxa0agcagatgagxa0ccaaaatccgxa0xa0xa0xa0600xa0aaacttctggxa0tgctgatgaaxa0cgtgaaaagcxa0gtattacagcxa0aaagaaaccaxa0acagagaagaxa0xa0xa0xa0660xa0aacctgttgaxa0ggaagtttctxa0gatgaaacaaxa0ggtgagaaaaxa0tatttttaaaxa0aatttatattxa0xa0xa0xa0720xa0gagaaaacaaxa0tattgatattxa0tgtggagataxa0cttatgtataxa0ttacccatatxa0ttaaaaattcxa0xa0xa0xa0780xa0tataaatgttxa0ctatagacatxa0gatctagattxa0ttggaagataxa0ttctgtgattxa0atgcacctgcxa0xa0xa0xa0840xa0gatcatgtagxa0gcgcataactxa0tctctataacxa0ccggaaataaxa0ttttgtagttxa0tatctacttcxa0xa0xa0xa0900xa0tttaaaaacaxa0tcacaaaattxa0tttaaatgtaxa0aataaatcaaxa0attttaacagxa0agaaaaacgcxa0xa0xa0xa0960xa0actactaaagxa0aaaagaagccxa0tacaaaacccxa0gctgacgctgxa0aagagtccgcxa0agctgaagagxa0xa0xa01020xa0agccgtgaaaxa0agcgtactatxa0cccaaaaaagxa0gaaaagccaaxa0aaaccgttgaxa0ggaagaatctxa0xa0xa01080xa0aaagttgaagxa0acgaaactcgxa0cgagaagcgcxa0ttgactggaaxa0ccccaaagaaxa0gacaaaacctxa0xa0xa01140xa0gctgatgctgxa0aggaagagacxa0tcctaaggttxa0gaggaagagaxa0cccgtgagaaxa0gcgattggcaxa0xa0xa01200xa0acaacaaaaaxa0aagaaaagatxa0caagccagctxa0gatgacgaatxa0ccaaagtcgaxa0ggaagagactxa0xa0xa01260xa0aaggaaaagaxa0gaacaataaaxa0gaaggaaaagxa0acaaaaacagxa0ctgatgtaagxa0tatacaggggxa0xa0xa01320xa0aggaatgttgxa0taaaatggttxa0tttgagatcaxa0ggataggactxa0agttttttttxa0tataaatatgxa0xa0xa01380xa0agtggttggcxa0attctaagttxa0tatagctttaxa0ggttacttttxa0tggtcatctaxa0aactataaatxa0xa0xa01440xa0ttatctacctxa0cgatccataaxa0tacttactagxa0ggttatataaxa0gaaagttatgxa0tgagtatgttxa0xa0xa01500xa0aaagtaaggaxa0tcattgagttxa0gtttcaatcgxa0tgaaaatcgcxa0cagggaacgaxa0ggccaggatgxa0xa0xa01560xa0cttactatttxa0tcatattaagxa0taaaataaccxa0aatttattttxa0tcgggtcatcxa0atattaaaatxa0xa0xa01620xa0agagttcaatxa0aatataaagtxa0gagagtgtgaxa0ttttaaagttxa0gtaattctttxa0taagcaatttxa0xa0xa01680xa0ttaaaattcaxa0aaactttcaaxa0atattttttaxa0gattgaagcaxa0gaagcagacgxa0aacccagagaxa0xa0xa01740xa0ggagcgtatgxa0acaaagaaaaxa0ctagcacagcxa0agagaaaaagxa0ccaattcataxa0cccgcgcagcxa0xa0xa01800xa0tcgtgcaacaxa0aagactggaaxa0ccaaaacaatxa0agaatctaaaxa0ggaaagggccxa0attaaatatcxa0xa0xa01860xa0tgataaatttxa0gtctattataxa0tttagtatgtxa0agatttaattxa0gaaagagaaaxa0gagatgattcxa0xa0xa01920xa0aatcttaaatxa0attttttaatxa0tctttcatttxa0tttgacgatgxa0aaaataaaagxa0aaggxa0xa0xa0xa0xa0xa0xa0xa0xa01974xa0xa0<210>xa0xa02<211>xa0xa01490<212>xa0xa0DNA<213>xa0xa0人工序列xa0<400>xa0xa02atgaaaacctxa0cagtgttgtgxa0cctatttttgxa0attatttctaxa0ttatttctatxa0ttatgccgatxa0xa0xa0xa0xa060xa0gataaagaagxa0ttgagaagcaxa0taaagttgagxa0gaagagacaaxa0aagaaaaacgxa0tgatgataaaxa0xa0xa0xa0120xa0gaacttactcxa0ataaagttgaxa0ggatgagacaxa0aaagagaaacxa0gtgatgataaxa0agaaaaacgtxa0xa0xa0xa0180xa0acagaaaagaxa0ccataacaggxa0aaagaagcatxa0gtcgaagcagxa0atgagccaaaxa0atccgaaactxa0xa0xa0xa0240xa0tctggtgctgxa0atgaacgtgaxa0aaagcgtattxa0acagcaaagaxa0aaccaacagaxa0gaagaaacctxa0xa0xa0xa0300xa0gttgaggaagxa0tttctgatgaxa0aacaaggtgaxa0gaaaatatttxa0ttaaaaatttxa0atattgagaaxa0xa0xa0xa0360xa0aacaatattgxa0atatttgtggxa0agatacttatxa0gtatattaccxa0catatttaaaxa0aattctataaxa0xa0xa0xa0420xa0atgttctataxa0gacatgatctxa0agattttggaxa0agatattctgxa0tgattatgcaxa0cctgcgatcaxa0xa0xa0xa0480xa0tgtaggcgcaxa0taacttctctxa0ataacccggaxa0aataattttgxa0tagtttatctxa0acttctttaaxa0xa0xa0xa0540xa0aaacatcacaxa0aaatttttaaxa0atgtaaataaxa0atcaaattttxa0aacagagaaaxa0aacgcactacxa0xa0xa0xa0600xa0taaagaaaagxa0aagcctacaaxa0aacccgctgaxa0cgctgaagagxa0tccgcagctgxa0aagagagccgxa0xa0xa0xa0660xa0tgaaaagcgtxa0actatcccaaxa0aaaaggaaaaxa0gccaaaaaccxa0gttgaggaagxa0aatctaaagtxa0xa0xa0xa0720xa0tgaagacgaaxa0actcgcgagaxa0agcgcttgacxa0tggaaccccaxa0aagaagacaaxa0aacctgctgaxa0xa0xa0xa0780xa0tgctgaggaaxa0gagactcctaxa0aggttgaggaxa0agagacccgtxa0gagaagcgatxa0tggcaacaacxa0xa0xa0xa0840xa0aaaaaaagaaxa0aagatcaagcxa0cagctgatgaxa0cgaatccaaaxa0gtcgaggaagxa0agactaaggaxa0xa0xa0xa0900xa0aaagagaacaxa0ataaagaaggxa0a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