CN114316031B - 一种生产血红素结合蛋白的方法 - Google Patents
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Abstract
本发明公开了一种生产血红素结合蛋白的方法,其特征在于所述方法是将含血红素结合蛋白基因表达框的表达载体转入大豆基因组中并表达,获得富含血红素结合蛋白的大豆种子,分离提取,获得血红素结合蛋白。本发明提供了一种利用转基因大豆种子生产血红蛋白/肌红蛋白的方法,这种方法生产的血红蛋白/肌红蛋白可以作为食品的着色添加剂、风味调节剂;本发明披露了可以在大豆种子中高表达的启动子和调控序列,高表达株系大豆种子中血红蛋白/肌红蛋白含量达到种子重量的0.1%以上;本发明的转基因大豆生产的血红蛋白/肌红蛋白,降低了生产成本,比传统微生物发酵方法节省能源,是一种更加环境友好型的生产方式。
Description
(一)技术领域
本发明涉及血红素结合蛋白的生产,特别涉及利用表达血红素结合蛋白的转基因大豆生产血红素结合蛋白的方法。
(二)背景技术
血红蛋白/肌红蛋白是常见的血红素结合蛋白,包括动物肌红蛋白以及植物血红蛋白。随着植物肉行业的兴起,作为植物肉中一种重要的着色、风味添加剂,血红蛋白/肌红蛋白的需求越来越大。大量生产血红蛋白/肌红蛋白的方法是现实生产中所急需的。
畜禽血可以作为血红蛋白的来源,但是提取过程繁琐,并且有病原菌污染的风险,不适合大规模工业化生产。
微生物发酵也是制备血红蛋白的重要途径,例如,中国专利申请2016800292580.4,中国专利申请202110607028.1分别公开了利用基因工程改造甲基营养型酵母生产大豆血红蛋白,以及利用毕赤酵母重组菌株表达肌红蛋白或血红蛋白的方法。但是微生物发酵比较繁琐,必须加入血红素或者通过基因改造的方法提高微生物中血红素的含量。
转基因植物已成为一种有吸引力的重组蛋白表达系统。由于蛋白含量高、蛋白酶活性低、水分含量低、方便运输和储存等,种子成为表达重组蛋白的理想器官之一。利用种子作为生物反应器表达重组蛋白时,相比较组成型启动子,种子特异性启动子被认为是一个更好的选择(Lau OS,Sun S.Plant seeds as bioreactors for recombinant proteinproduction.Biotechnology Advances,2009,27(6):1015-1022)。
(三)发明内容
本发明目的是针对目前血红蛋白/肌红蛋白的生产方法中存在的缺陷,提供一种利用转基因大豆生产血红素结合蛋白(血红蛋白/肌红蛋白)的方法,所述方法利用植物栽培的可规模化和可持续性生产血红素结合蛋白,且无需考虑血红素供应问题。
本发明采用的技术方案是:
本发明提供一种生产血红素结合蛋白的方法,所述方法是将含血红素结合蛋白基因表达框的表达载体转入大豆基因组中并表达,获得富含血红素结合蛋白的大豆种子,分离提取,获得血红素结合蛋白;所述血红素结合蛋白包括血红蛋白或肌红蛋白。
本发明采用转基因方法将含血红素结合蛋白基因表达框的表达载体转入大豆基因组中,获得含有至少一个拷贝血红素结合蛋白基因的转基因大豆,其种子中高表达红素结合蛋白,质量含量至少为0.1%。所述血红素结合蛋白基因表达框包括启动子、血红素结合蛋白编码基因、终止子。
本发明所述血红素结合蛋白包括但不限于大豆血红蛋白LGH(氨基酸序列SEQ IDNO:1),牛肌红蛋白BMG(氨基酸序列SEQ ID NO:2)。根据大豆密码子偏好性合成相应的核苷酸序列,其中大豆血红蛋白编码基因LGH核苷酸序列如SEQ ID NO:3所示,牛肌红蛋白编码基因BMG的核苷酸序列如SEQ ID NO:4所示。
本发明血红素结合蛋白基因表达框的启动子能够启动血红素结合蛋白在大豆种子中高水平表达,包括组成型启动子和种子特异性启动子,例如拟南芥泛素10启动子pAtUbi10(SEQ ID NO:5中478-1741bp),pGmUbi3启动子(SEQ ID NO:8),pCaMV35S启动子(SEQ ID NO:5中4491-5289bp),pGm2S启动子(SEQ ID NO:7中478-1465bp)等。拟南芥泛素10启动子pAtUbi10调控的血红蛋白/肌红蛋白在大豆种子中的表达量较高,显著高于种子特异性启动子pGm2S。
进一步,本发明的终止子包括但不限于NOS终止子TNOS(SEQ ID NO:9)、拟南芥heat shock protein 18.2基因终止子TAtHSP18.2(SEQ ID NO:5中2180-3075bp)。
进一步,本发明的血红素结合蛋白基因表达框还可在5’端或3’端加入增强基因表达的元件,如烟草RB7核基质结合区调控序列MAR(SEQ ID NO:5中3082-4248bp),增强子,以进一步提高血红蛋白/肌红蛋白在大豆种子中的表达量。所述血红素结合蛋白表达框组成为:启动子-血红素结合蛋白基因-终止子-MAR。
进一步,所述表达载体包含血红素结合蛋白基因表达框和筛选标记基因表达框;筛选标记基因表达框优选由启动子、抗除草剂基因、终止子组成。本发明的筛选标记基因表达框优选耐草甘膦基因表达框(Clemente TE.Progeny analysis of glyphosateselected transgenic soybeans derived from Agrobacterium-mediatedtransformation.Crop Science,2000,40(3):797-803)和耐草铵膦基因表达框(Zhang Z,Xing A,Staswick P,et al.The use of glufosinate as a selective agent inAgrobacterium-mediated transformation of soybean.Plant Cell,Tissue and OrganCulture,1999,56(1):37-46)以及耐啶嘧磺隆基因表达框(专利申请200810164131.8),其中耐草甘膦基因表达框中草甘膦抗性基因g10evo epsps的核苷酸序列如SEQ ID NO:5中5664-6983bp所示,启动子为花椰菜花叶病毒35s启动子pCaMV35S(SEQ ID NO:5中4491-5289bp),终止子为花椰菜花叶病毒35s终止子TCaMV35S(SEQ ID NO:10)。
进一步,本发明所述表达载体的基础载体优选是pCambia1300(NCBI序列编号AF234296)。
进一步,优选本发明所述表达载体的T-DNA元件组成为下列之一:(1)由pAtUbi10启动子、血红蛋白编码基因、终止子、增强α-淀粉酶编码基因表达的元件、花椰菜花叶病毒35s启动子pCaMV35S、抗草甘膦基因g10evo epsps、花椰菜花叶病毒35s终止子TCaMV35S组成,核苷酸序列如SEQ ID NO:5中205-7266bp;(2)由pAtUbi10启动子、肌红蛋白编码基因、终止子、增强α-淀粉酶编码基因表达的元件、花椰菜花叶病毒35s启动子pCaMV35S、抗草甘膦基因g10evo epsps、花椰菜花叶病毒35s终止子TCaMV35S组成,核苷酸序列如SEQ ID NO:6中205-7293bp所示。
本发明所述将含血红素结合蛋白基因的表达载体导入到大豆基因组中的方法如农杆菌介导方法(Hinchee MW,Connor-Ward DV,Newell CA,et al.Production oftransgenic soybean plants using Agrobacterium-mediated DNA transfer.NatureBiotechnology,1988,6(8):915-921),基因枪方法等(Mccabe DE,Swain WF,MartinellBJ,et al.Stable transformation of Soybean(Glycine Max)by particleacceleration.Nature Biotechnology,1988,6(8):923-926)。这些方法都是成熟的方法,本领域的一般技术人员就能够利用这些方法获得转基因大豆。这些不同的方法都可以实现本发明获得表达血红蛋白/肌红蛋白的转基因大豆的目的。比较常用的转化方法是农杆菌介导方法。这种方法的特点是构建一种T-DNA转化体载体,然后通过农杆菌介导将载体中的T-DNA导入到大豆基因组。
本发明还提供了获得具有商业价值的转化体的选择方法:从获得的表达血红蛋白/肌红蛋白的转基因大豆转化体中选择血红蛋白/肌红蛋白表达量至少占种子总重量0.1%的转化体,并进一步筛选在不同代次大豆中表达稳定并且遗传稳定的转化体。
本发明还涉及一种表达载体构建的转基因植物细胞及转基因大豆。
与现有技术相比,本发明有益效果主要体现在:(1)本发明提供了一种利用转基因大豆种子生产血红蛋白/肌红蛋白的方法,这种方法生产的血红蛋白/肌红蛋白可以作为食品的着色添加剂、风味调节剂;(2)本发明披露了可以在大豆种子中高表达的启动子和调控序列,高表达株系大豆种子中血红蛋白/肌红蛋白含量达到种子重量的0.1%以上;(3)本发明的转基因大豆生产的血红蛋白/肌红蛋白,降低了生产成本,比传统微生物发酵方法节省能源,是一种更加环境友好型的生产方式。
(四)附图说明
图1、用于大豆遗传转化的载体GLLGH示意图。
图2、用于大豆遗传转化的载体SLGH示意图。
图3、高表达大豆血红蛋白的转基因大豆种子;CK为非转基因对照皖豆28,1、2、3、4为4个不同的GLLGH转基因株系。
图4、western blot方法检测转基因大豆株系中的大豆血红蛋白;M为蛋白Marker,+为阳性对照,-为阴性对照皖豆28,2、10、15为GLLGH的3个株系,2、5、7为SLGH的3个株系。
图5、吡啶血色原光吸收测定法测定转基因株系中的大豆血红蛋白含量,横坐标代表不同株系。
图6、高表达肌红蛋白的转基因大豆种子;CK为非转基因对照皖豆28,1、2、3、4为4个不同的GLBMG转基因株系。
图7、吡啶血色原光吸收测定法测定转基因株系中的肌红蛋白含量,横坐标代表GLBMG不同株系。
图8、转大豆血红蛋白基因的水稻种子;CK为非转基因对照秀水134,2、3、4、5、6分别为转基因株系OSLGH-2、OSLGH-3、OSLGH-4、OSLGH-5、OSLGH-6。
图9、western blot方法检测转基因水稻中的大豆血红蛋白;M为蛋白Marker,-为阴性对照秀水134,+为阳性对照,1、2、3、4、5、6分别为转基因株系OSLGH-1、OSLGH-2、OSLGH-3、OSLGH-4、OSLGH-5、OSLGH-6。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1、血红蛋白/肌红蛋白基因的合成
通过NCBI基因数据库,直接搜索不同来源的血红蛋白/肌红蛋白基因序列。例如输入Soybean leghemoglobin可以搜寻到若干个大豆血红蛋白基因,随机选取基因LBC2(GeneID:100527379);根据植物密码子偏好性人工合成大豆(Glycine max)血红蛋白基因LGH,氨基酸为SEQ ID NO:1所示,核苷酸序列为SEQ ID NO:3所示。
输入bovine myoglobin可以搜寻到一个牛肌红蛋白基因MB(Gene ID:280695)。根据植物密码子偏好性人工合成牛(Bostaurus)肌红蛋白基因BMG,氨基酸序列为SEQ ID NO:2所示,核苷酸序列为SEQ ID NO:4所示。
实施例2、构建大豆遗传转化载体
理论上,本发明实施例1的2种血红蛋白/肌红蛋白的基因,在5’端添加可在大豆种子中启动基因高表达的启动子,在3’端添加终止子,可以得到在大豆种子中表达血红蛋白/肌红蛋白的表达框,进一步的,血红蛋白/肌红蛋白的表达框可以添加其他增强表达的元件。然后构建入含有选择性标记的植物遗传转化载体。在大豆种子中表达不同血红蛋白/肌红蛋白的植物遗传转化载体的主要区别仅在于血红蛋白/肌红蛋白编码基因的不同。
1、双元T-DNA转化载体GLLGH
下面以实施例1合成的核苷酸序列如SEQ ID NO:3所示的大豆血红蛋白LGH基因为例,分别选择拟南芥pAtUbi10启动子(SEQ ID NO:5中478-1741bp),以及种子特异性的大豆2S球蛋白基因启动子pGM2S(SEQ ID NO:7中478-1465bp)作为5’端控制元件;以拟南芥TAtHsp18.2终止子(SEQ ID NO:5中2180-3075bp)串联烟草RB7核基质结合区调控序列MAR(SEQ ID NO:5中3082-4248bp)作为3’端控制元件。按如下步骤构建:
血红蛋白基因表达框:SEQ ID NO:3所示的大豆血红蛋白LGH编码区5’端接启动子pAtUbi10(SEQ ID NO:5中478-1741bp),3’端接TAtHSP18.2终止子(SEQ ID NO:5中2180-3075bp)和烟草RB7核基质结合区调控序列MAR(SEQ ID NO:5中3082-4248bp),得大豆血红蛋白基因表达框pAtUbi10-LGH-TAtHsp18.2-MAR(SEQ ID NO:5中478-4248bp)。人工合成含有血红蛋白表达框pAtUbi10-LGH-TAtHsp18.2-MAR的片段ALS(SEQ ID NO:5中457-4248bp)。
抗草甘膦基因表达框:草甘膦抗性基因g10evo epsps(SEQ ID NO:5中5664-6983bp)编码区5’端接花椰菜花叶病毒35s启动子pCaMV35S(SEQ ID NO:5中4491-5289bp),TEV5’UTR-AtEPSPS SP序列(SEQ ID NO:5中5296-5657bp),3’端接花椰菜花叶病毒35s终止子TCaMV35S。人工合成含抗草甘膦基因的片段pCaMV35S-TEV5'UTR-AtEPSPS SP-g10evoepsps,记为片段G10evo(核苷酸序列如SEQ ID NO:5中4229-7004bp所示)。
将载体pCAMBIA1300用Hind III/Xho I双酶切,回收6783bp大小的片段FV。以人工合成的片段ALS为模板,使用引物组合(F1,R1)扩增,获得LGH片段。以人工合成的片段G10evo为模板,使用引物组合(F2,R2)扩增得到片段G10evo。将酶切片段FV,与片段LGH和片段G10evo通过无缝克隆的方法重组,转化至大肠杆菌TG1菌株,测序确认,得到双元T-DNA转化载体GLLGH(图1),其核苷酸序列如SEQ ID NO:5所示,组成原件和位置如表3所示。
表1扩增血红蛋白表达框和片段G10evo所使用的引物及目标条带大小
表2扩增血红蛋白表达框和G10evo表达框所使用的引物序列
*黑体部分为同源臂部分,下同。
表3转化载体GLLGH和SLGH的组成元件和位置
2、双元T-DNA转化载体SLGH
血红蛋白基因表达框:SEQ ID NO:3所示的大豆血红蛋白LGH基因编码区5’端接启动子pGM2S(SEQ ID NO:7中478-1465bp),TEV 5’UTR-At2S2SP序列(SEQ ID NO:7中1466-1659bp),3’端接TAtHSP18.2终止子(SEQ ID NO:5中2180-3075bp)和烟草RB7核基质结合区调控序列MAR(SEQ ID NO:5中3082-4248bp),得血红蛋白基因表达框pGM2S-TEV 5’UTR-At2S2 SP-LGH-TAtHsp18.2-MAR(SEQ ID NO:7中478-4163bp)。人工合成含有血红蛋白基因表达框pGM2S-TEV 5’UTR-At2S2SP-LGH-TAtHsp18.2-MAR的片段GLS(457-4163bp)。
抗草甘膦基因表达框:草甘膦抗性基因g10evo epsps(SEQ ID NO:5中5664-6983bp)编码区5’端接花椰菜花叶病毒35s启动子pCaMV35S,TEV 5’UTR-AtEPSPS SP序列(SEQ ID NO:5中5296-5657bp),3’端接花椰菜花叶病毒35s终止子TCaMV35S,人工合成抗草甘膦基因的片段pCaMV35S-TEV5'UTR-AtEPSPS SP-g10evo epsps,记为片段G10evo(SEQ IDNO:5中4229-7004bp)。
将载体pCAMBIA1300用Hind III/Xho I双酶切,回收6783bp大小的片段FV。分别以人工合成的片段GLS为模板,使用表2引物组合(F3,R1)扩增,获得SLGH片段。以人工合成的片段G10evo为模板,使用引物组合(F2,R2)扩增得到片段G10evo。将酶切片段FV,与片段SLGH和片段G10evo通过无缝克隆的方法重组,转化至大肠杆菌TG1菌株,测序确认,得到双元T-DNA转化载体SLGH(图2),其核苷酸序列如SEQ ID NO:7所示,组成原件和位置如表3所示。
3、转化
分别将质粒GLLGH和SLGH电击转入农杆菌EHA105感受态细胞,酶切鉴定正确后以甘油菌保存,获得重组农杆菌,用于大豆转化。
实施例3、转基因大豆的获得
大豆转化流程主要参照Paz的农杆菌介导的half-seed转化法(Paz MM,MartinezJC,Kalvig AB,et al.(2006),Improved cotyledonary node method using analternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation.Plant Cell Reports,25(3):206-213)。具体如下:
①大豆种子灭菌:挑选品质优良的大豆种子(品种:皖豆28),放入干燥器里,用氯气处理16小时进行灭菌。氯气是通过向干燥器中装有100mL 30%(v/v)次氯酸钠水溶液的烧杯中缓慢加入8mL浓盐酸产生。
②种子萌发:将步骤①灭过菌的大豆种子在无菌蒸馏水中室温浸泡16小时,获得萌发的大豆种子。
③农杆菌菌液的制备:将实施例2制备的重组农杆菌在含50mg/L Kan(卡那霉素)和25mg/L利福平的YEP固体培养基上划线,28℃培养2天。刮取YEP培养板上的菌落,均匀悬浮在侵染培养基中,将其浓度调整为OD650=0.5-0.6,即为农杆菌菌液。YEP固体培养基组成:蛋白胨(Typtone)10g,酵母提取物(Yeast extract)10g,NaCl 5g,加水定容至1L。
④外植体的准备和侵染:将步骤②萌发的大豆种子沿着种脐纵向切割均匀切成两瓣,将带有胚轴和胚芽的那一瓣去掉种皮后迅速放入步骤③制备好的农杆菌菌液中,室温下侵染30min,取出放到衬有滤纸无菌培养皿中,封口后于22℃黑暗条件下共培养3天。
⑤芽诱导培养:步骤④共培养后的外植体转入含有30mg/L草甘膦的芽诱导培养基中,于26℃、16h光照/8h黑暗条件下培养3周。芽诱导培养基组成:B5盐3.21g,B5维生素(vitamins)1mL,蔗糖30g,4-吗啉乙磺酸(MES)3mM,琼脂(Agar)8g,加水定容至1L并将pH调至5.6,灭菌后添加200mg特美汀(Timentin)、1.67mg 6-苄基腺嘌呤(6-BA)。
⑥芽伸长和筛选:将步骤⑤诱导后的外植体切掉子叶,带丛生芽的部分转到加入10mg/L草甘膦的芽伸长培养基中,于26℃、16h光照/8h黑暗条件下培养21天。芽伸长培养基组成:MS盐4.33g,B5维生素1mL,蔗糖30g,MES 3mM,天冬酰胺50mg,谷氨酰胺50mg,Agar 8g,加水定容至1L并将pH调至5.6,灭菌后添加0.5mg赤霉素(GA3)、0.1mg吲哚-3-乙酸(IAA)、lmg玉米素核苷(zeatin riboside)、200mg Timentin。
⑦生根和移苗:将步骤⑥伸长后的丛生芽转移到生根培养基生根。当根长出后,洗去根上附着的培养基,将T0代幼苗移栽到土壤中培养,直至收获种子。分别获得GLLGH转基因株系和SLGH转基因株系。生根培养基成:MS盐4.33g,B5vitamins 1mL,蔗糖20g,MES 3mM,Agar 8g,加水定容至1L并将pH调至5.6,灭菌后添加200mg Timentin。
实施例4、转基因大豆种子中大豆血红蛋白的测定
1、颜色观察
转基因大豆种子中大豆血红蛋白是否高表达可通过观察的方法进行初筛,表达量较高的转基因株系种子呈现红色(图3)。CK为非转基因对照皖豆28,1、2、3、4为4个不同的GLLGH转基因株系。4个GLLGH转基因株系均呈红色,而非转基因对照没有观察到红色。
2、Western blot检测
Western blot检测转基因大豆种子中的血红蛋白。检测方法如下:每个GLLGH转基因株系各取一粒呈现红色的种子,每个SLGH转基因株系各取一粒阳性种子(经G10EPSPS试纸条检测,购自上海佑隆生物科技有限公司),另取一粒非转基因种子。将种子充分研磨,各称取30mg,加入300μL PBS缓冲液,振荡混匀3min,于4℃12000rpm离心10min,取上清,适当稀释后取40μL蛋白样品,加入10μL 5×蛋白上样缓冲液混匀,沸水中煮10min,12000rpm离心5min,取上清进行电泳。采用金斯瑞SurePAGE预制胶(4-20%),上样后以200V电泳30min。电泳结束后,采用金斯瑞eBlotTM L1快速湿转仪进行转膜。转膜结束后,将膜浸入含5%脱脂奶粉的TBST溶液中,置于水平摇床上,室温下封闭1h。封闭结束后,去掉封闭液,加入含1%脱脂奶粉的TBST溶液,同时按照1:1000(v/v)比例加入一抗Anti-LegHb,置于水平摇床上,室温下孵育1h;倒掉溶液,用TBST溶液洗膜3次,每次5min;加入含1%脱脂奶粉的TBST溶液,并按照1:5000(v/v)的比例加入辣根过氧化物酶交联的羊抗兔二抗,置于水平摇床上,室温下孵育1h;倒掉溶液,用TBST溶液洗膜3次,每次5min;加入ECL化学发光显色液,室温下孵育2min后进行化学曝光,得到曝光图,结果见图4所示,M为蛋白Marker,+为阳性对照,-为阴性对照皖豆28,2、10、15为GLLGH的3个株系,2、5、7为SLGH的3个株系。3个GLLGH转基因株系均可检测到约为15kDa的目标条带,且信号较强,而3个SLGH转基因株系则检测不到目标条带或是信号比较弱,非转基因对照未检测到目标条带。
阳性对照样品制备:将大豆血红蛋白基因LGH(SEQ ID NO:3)连入载体pET28a(Novagen公司)中,转化大肠杆菌TG1感受态,测序确认,将序列无误的质粒(记为pET28a-LGH)转化表达菌株Transetta,加入终浓度为1mM的异丙基-β-D-硫代半乳糖苷(IPTG)在37℃条件下进行诱导表达。采用Ni-NTA His·Bind树脂(Millipore)对原核表达蛋白进行纯化,具体步骤参照MERCK操作手册:标签蛋白质-亲和层析法03Purification ofhistidine-tagged recombinant proteins。
3、吡啶血色原光吸收测定法
选取转基因株系GLLGH-1、GLLGH-5、GLLGH-6、GLLGH-9、GLLGH-11、GLLGH-14、GLLGH-15种子,通过吡啶血色原光吸收测定法(Senthilkumar M,Amaresan N,Sankaranarayanan A.(2021)Quantitative Estimation of Leghemoglobin Content inLegume Root Nodules.In:Plant-Microbe Interactions.Springer ProtocolsHandbooks.Humana,New York,NY.)进行血红蛋白含量测定。测定方法具体如下:0.3g种子用3mL磷酸盐缓冲液(0.1M Na2HPO4·2H2O/NaH2PO4·H2O,pH 7.4)充分研磨,以10000g(4℃)离心20min,取2mL上清液,与等体积碱性吡啶溶液(0.2M NaOH,4.2M吡啶,溶剂为水)混合,将混合后的溶液分为等体积的两份,一份加入10μL 0.5M连二硫酸钠水溶液,混匀后于2-5min内测定556nm处的吸光值(A556),另一份则加入5μL 0.1M铁氰化钾水溶液,混匀后测定539nm处的吸光值(A539),代入以下公式以计算豆血红蛋白的含量。
D为稀释倍数(计算是基于摩尔消光系数ε=23.4mM-1cm-1)
从图5可以看出,4个转基因株系GLLGH-1、GLLGH-9、GLLGH-11、GLLGH-15种子中的血红蛋白含量超过了0.1%。
实施例5、转基因大豆种子中肌红蛋白的测定
肌红蛋白大豆遗传转化载体的构建过程可以参照实施例2进行,区别仅在于将大豆血红蛋白编码基因(SEQ ID NO:3)替换为肌红蛋白基因(SEQ ID NO:4)。与表达载体GLLGH仅有血红蛋白编码基因差异的表达载体,我们将其命名为GLBMG(SEQ ID NO:6)。由表达载体GLBMG遗传转化所得的转基因株系称之为GLBMG转基因株系。
1、颜色观察
转基因大豆种子中肌红蛋白是否高表达可通过观察的方法进行初筛,表达量较高的转基因株系种子呈现红色(图6)。CK为非转基因对照皖豆28,1、2、3、4为4个不同的GLBMG转基因株系。4个GLBMG转基因株系均呈红色,而非转基因对照没有观察到红色。
2、吡啶血色原光吸收测定法
选取转基因株系GLBMG-1、GLBMG-4、GLBMG-5、GLBMG-8、GLBMG-13、GLBMG-15种子,通过吡啶血色原光吸收测定法进行肌红蛋白含量测定,具体步骤参照实施例4。从图7可以看出,3个转基因株系GLBMG-1、GLBMG-4、GLBMG-5种子中的肌红蛋白含量超过了0.1%。
实施例6、构建水稻遗传转化载体
理论上,本发明实施例1的2种血红蛋白/肌红蛋白的基因,在5’端添加可在水稻种子中启动基因高表达的启动子,在3’端添加终止子,可以得到在水稻种子中表达血红蛋白/肌红蛋白的表达框,进一步的,血红蛋白/肌红蛋白的表达框可以添加其他增强表达的元件。然后构建入含有选择性标记的植物遗传转化载体。下面以转化载体OSLGH的构建过程为例,具体步骤如下:
血红蛋白基因表达框:将大豆血红蛋白LGH基因进行水稻偏爱密码子优化,序列如SEQ ID NO:11中1461-1895所示,血红蛋白编码区5’端接种子特异性启动子pGT1(SEQ IDNO:11中478-1388bp),信号肽GT1SP(SEQ ID NO:11中1389-1460),3’端接TPC终止子(SEQID NO:11中1896-2094bp)和烟草RB7核基质结合区调控序列MAR(SEQ ID NO:5中3082-4248bp),得大豆血红蛋白基因表达框pGT1-GT1SP-LGH-TPC-MAR(SEQ ID NO:11中478-3267bp)。人工合成含有血红蛋白表达框pGT1-GT1SP-LGH-TPC-MAR的片段OLS(SEQ ID NO:11中457-3267bp)。
抗草甘膦基因表达框:将草甘膦抗性基因g10evo epsps进行水稻偏爱密码子优化,序列如SEQ ID NO:11中6533-7849bp所示,编码区5’端接花椰菜花叶病毒35s启动子pCaMV35S(SEQ ID NO:5中4491-5289bp),pZmUbi启动子(SEQ ID NO:11中4304-6328),信号肽AHAS SP(SEQ ID NO:11中6329-6526bp),3’端接花椰菜花叶病毒35s终止子TCaMV35S。人工合成含抗草甘膦基因的片段pCaMV35S-pZmUbi-AHAS SP-g10evo epsps,记为片段OG10evo,核苷酸序列如SEQ ID NO:11中3248-7870bp所示。
将载体pCAMBIA1300用Hind III/Xho I双酶切,回收6783bp大小的片段FV。以人工合成的片段OLS为模板,使用引物组合(F4,R1)扩增,获得OLGH片段。以人工合成的片段OG10evo为模板,使用引物组合(F5,R3)扩增得到片段OG10evo。将酶切片段FV,与片段OLGH和片段OG10evo通过无缝克隆的方法重组,转化至大肠杆菌TG1菌株,测序确认,得到双元T-DNA转化载体OSLGH,其核苷酸序列如SEQ ID NO:11所示,组成原件和位置如表4所示。
将质粒OSLGH电击转入农杆菌LBA4404感受态细胞,酶切鉴定正确后以甘油菌保存,用于水稻转化。
表4转化载体OSLGH的组成元件和位置
实施例7、转基因水稻的获得
采用农杆菌介导的方法(Hiei Y,Komari T.Agrobacterium-mediatedtransformation of rice using immature embryos or calli induced from matureseed.Nat Protoc,2008,3(5):824-34.)进行水稻遗传转化。以水稻品种秀水134作为受体材料。具体步骤如下:
选择生长良好的水稻愈伤组织,用含有转化载体OSLGH的农杆菌菌液侵染水稻愈伤组织,25℃暗培养3天,进一步在含草甘膦400mg L-1的筛选培养基(2NBK培养基+特美汀200mg L-1)上进行筛选培养(28℃暗培养)。每次筛选20d,共筛选2-3次。挑选抗性愈伤组织,将抗性愈伤组织转入分化培养基(NBKRH40培养基,不添加潮霉素)进行分化培养,将分化形成的再生小苗转入生根培养基(NBKFH40培养基,将潮霉素替换为20mg草甘膦)中培养,幼苗生根后炼苗、移栽,得到转基因水稻转化体。
实施例8、转基因水稻种子中大豆血红蛋白的检测
1、颜色观察
将成熟的水稻种子去壳后进行观察,结果显示,在所有转基因株系和非转基因对照的种子中均未观察到红色。图8为部分转基因株系和非转基因水稻的去壳种子对比图,CK为非转基因对照秀水134,2、3、4、5、6分别为转基因株系OSLGH-2、OSLGH-3、OSLGH-4、OSLGH-5、OSLGH-6。
2、Western blot检测
Western blot检测转基因水稻种子中的血红蛋白。每个OSLGH转基因株系各取一粒阳性种子(经G10EPSPS试纸条检测,购自上海佑隆生物科技有限公司),另取一粒非转基因种子作为阴性对照,将种子充分研磨,称量,按10μL/mg样品加入PBS缓冲液,其余操作步骤同实施例4,得到化学曝光图(图9)。M为蛋白Marker,-为阴性对照秀水134,+为阳性对照(同实施例4),1、2、3、4、5、6分别为转基因株系OSLGH-1、OSLGH-2、OSLGH-3、OSLGH-4、OSLGH-5、OSLGH-6。阳性对照可检测到约为15kDa的目标条带,而6个OSLGH转基因株系和非转基因对照均未检测到目标条带。
序列表
<110> 杭州瑞丰生物科技有限公司
<120> 一种生产血红素结合蛋白的方法
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cccgcagttc gtggtggtga aggaggccct cctcaagacc atcaaggagg ccgtgggcga 2100
caagtggtcc gacgagctgt cctccgcctg ggaggtggcc tacgacgagc tggccgccgc 2160
catcaagaag gccttctgac gacatatgaa gatgaagatg aaatatttgg tgtgtcaaat 2220
aaaaagctag tgtgcttaag tttgtgtttt tttcttggct tgttgtgtta tgaatttgtg 2280
gctttttcta atattaaatg aatgtaagat ctcattataa tgaataaaca aatgtttcta 2340
taatccattg tgaatgtttt gttggatctc ttctgcagca tataactact gtatgtgcta 2400
tggtatggac tatggaatat gattaaagat aagatgggct catagagtaa aacgaggcga 2460
gggacctata aacctccctt catcatgcta tttcatgatc tattttataa aataaagatg 2520
tagaaaaaag taagcgtaat aaccgcaaaa caaatgattt aaaacatggc acataatgag 2580
gagattaagt tcggtttacg tttattttag tactaattgt aacgtgagac tacgtatcgg 2640
gaatcgccta attaaagcat taatgcgaac ctgattagat tcaccgaccc tcctatcgtg 2700
tcgacctttc tgtttcttag aattttttgg tagtctatgt actaataatg tcagcttcgt 2760
atttatttca taagcaattt gcatttgcaa tttgtttttt acttttattt ttattgtatt 2820
gtggaatgtg gactcgtacc aacatgaagt tatataccac caaaaaaatt acagttagtc 2880
aaaagattca cgagtgagag ctacttatga ttgtctttta cgtatatgtc taattgtcta 2940
tttgctcaat aatctttgta ctttcttttg tcgttgataa aatcacaaag ttccaaaagt 3000
aatcgaatga tttgctttta agaaaagaac agctcaataa ttcaacatat atctgtacac 3060
acataattga ataagggtac ccgattaaaa atcccaatta tatttggtct aatttagttt 3120
ggtattgagt aaaacaaatt cgaaccaaac caaaatataa atatatagtt tttatatata 3180
tgcctttaag actttttata gaattttctt taaaaaatat ctagaaatat ttgcgactct 3240
tctggcatgt aatatttcgt taaatatgaa gtgctccatt tttattaact ttaaataatt 3300
ggttgtacga tcactttctt atcaagtgtt actaaaatgc gtcaatctct ttgttcttcc 3360
atattcatat gtcaaaatct atcaaaattc ttatatatct ttttcgaatt tgaagtgaaa 3420
tttcgataat ttaaaattaa atagaacata tcattattta ggtatcatat tgatttttat 3480
acttaattac taaatttggt taactttgaa agtgtacatc aacgaaaaat tagtcaaacg 3540
actaaaataa ataaatatca tgtgttatta agaaaattct cctataagaa tattttaata 3600
gatcatatgt ttgtaaaaaa aattaatttt tactaacaca tatatttact tatcaaaaat 3660
ttgacaaagt aagattaaaa taatattcat ctaacaaaaa aaaaaccaga aaatgctgaa 3720
aacccggcaa aaccgaacca atccaaaccg atatagttgg tttggtttga ttttgatata 3780
aaccgaacca actcggtcca tttgcacccc taatcataat agctttaata tttcaagata 3840
ttattaagtt aacgttgtca atatcctgga aattttgcaa aatgaatcaa gcctatatgg 3900
ctgtaatatg aatttaaaag cagctcgatg tggtggtaat atgtaattta cttgattcta 3960
aaaaaatatc ccaagtatta ataatttctg ctaggaagaa ggttagctac gatttacagc 4020
aaagccagaa tacaaagaac cataaagtga ttgaagctcg aaatatacga aggaacaaat 4080
atttttaaaa aaatacgcaa tgacttggaa caaaagaaag tgatatattt tttgttctta 4140
aacaagcatc ccctctaaag aatggcagtt ttcctttgca tgtaactatt atgctccctt 4200
cgttacaaaa attttggact actattggga acttcttctg aaaatagtgg taccgagctc 4260
gaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 4320
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 4380
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 4440
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg ctagagcagc 4500
ttgccaacat ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct 4560
cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg 4620
gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag gaaggtggca 4680
cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctgccgaca 4740
gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 4800
ccacgtcttc aaagcaagtg gattgatgtg ataacatggt ggagcacgac actctcgtct 4860
actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac 4920
aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca 4980
aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg 5040
ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga 5100
gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata 5160
tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac cttcctctat 5220
ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctacaaa 5280
tctatctctc tcgagtcaac acaacatata caaaacaaac gaatctcaag caatcaagca 5340
ttctacttct attgcagcaa tttaaatcat ttcttttaaa gcaaaagcaa ttttctgaaa 5400
attttcacca tttacgaacg atagccatgg ctcaagttag cagaatctgc aatggtgtgc 5460
agaacccatc tcttatctcc aatctctcta aatccagtca aaggaaatct cccttatcgg 5520
tttctctgaa gactcagcag catccacgag cttatccaat ttcttcatct tggggattga 5580
agaagagtgg gatgacttta attggctctg agcttcgtcc tcttaaggtc atgtcttctg 5640
tttccacggc ggagaaggga tccgacgctc ttccagctac cttcgacgtt atcgtgcatc 5700
cagctagaga actcagaggt gaacttagag cacagccatc caagaactac accactagat 5760
acctcctcgc cgctgctctc gctgagggtg aaaccagagt tgttggtgtg gctacctctg 5820
aggatgccga agctatgctc agatgcctca gagattgggg tgctggtgtt gagcttgttg 5880
gtgatgacgc cgtgatcaga ggtttcggtg ctagaccaca ggctggtgtt acccttaacc 5940
caggtaacgc tgctgcggtg gccagactcc ttatgggtgt tgctgctctc acctctggta 6000
caactttcgt taccgattac cctgattccc ttggtaagag acctcagggt gaccttcttg 6060
aagccctcga aagacttggt gcttgggtgt cctccaacga tggtagactc cctatctccg 6120
tttccggtcc agttagaggt ggtacagtgg aggtttccgc cgaaagatcc tcccagtacg 6180
cttccgccct tatgttcctc ggtcctcttc ttcctgacgg actcgaactt agactcaccg 6240
gtgatatcaa gtcccacgct cctcttagac agacacttga caccctctct gatttcggtg 6300
ttagagctac tgcctccgat gaccttagaa gaatctccat ccctggtggt cagaagtaca 6360
gaccaggtag agtgctcgtt cctggtgatt accctggttc cgctgctatc cttaccgccg 6420
ctgctcttct cccaggtgag gttagacttt ctaaccttag agaacacgac ctccagggtg 6480
agaaggaagc tgtgaacgtt cttagagaga tgggtgctga tatcgttaga gaaggtgata 6540
cccttaccgt gagaggtggt agacctctcc acgctgttac tagagatggt gattccttca 6600
ccgacgccgt gcaagctctt accgctgctg ctgccttcgc tgagggtgat accacctggg 6660
aaaacgttgc tactcttaga ctcaaggaat gcgatagaat ctctgacacc agagctgagc 6720
ttgaaagact tggtcttaga gcaagagaga ccgccgattc tctctccgtt actggttctg 6780
ctcaccttgc tggtggtatc accgctgatg gtcacggtga ccacagaatg atcatgcttc 6840
tcacccttct tggtctcaga gcagatgctc cacttagaat caccggtgca caccacatca 6900
gaaagtccta ccctcagttc ttcgctcacc ttgaagctct tggtgctaga ttcgaatacg 6960
ctgaggctac cgcctaatag gagctcgagt ttctccataa taatgtgtga gtagttccca 7020
gataagggaa ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct 7080
tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca 7140
aaatccagta ctaaaatcca gatcccccga attaattcgg cgttaattca gtacattaaa 7200
aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc 7260
ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat 7320
acaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact 7380
ttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac 7440
ggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg 7500
atcaccgcgg tttcaaaatc ggctccgtcg atactatgtt atacgccaac tttgaaaaca 7560
actttgaaaa agctgttttc tggtatttaa ggttttagaa tgcaaggaac agtgaattgg 7620
agttcgtctt gttataatta gcttcttggg gtatctttaa atactgtaga aaagaggaag 7680
gaaataataa atggctaaaa tgagaatatc accggaattg aaaaaactga tcgaaaaata 7740
ccgctgcgta aaagatacgg aaggaatgtc tcctgctaag gtatataagc tggtgggaga 7800
aaatgaaaac ctatatttaa aaatgacgga cagccggtat aaagggacca cctatgatgt 7860
ggaacgggaa aaggacatga tgctatggct ggaaggaaag ctgcctgttc caaaggtcct 7920
gcactttgaa cggcatgatg gctggagcaa tctgctcatg agtgaggccg atggcgtcct 7980
ttgctcggaa gagtatgaag atgaacaaag ccctgaaaag attatcgagc tgtatgcgga 8040
gtgcatcagg ctctttcact ccatcgacat atcggattgt ccctatacga atagcttaga 8100
cagccgctta gccgaattgg attacttact gaataacgat ctggccgatg tggattgcga 8160
aaactgggaa gaagacactc catttaaaga tccgcgcgag ctgtatgatt ttttaaagac 8220
ggaaaagccc gaagaggaac ttgtcttttc ccacggcgac ctgggagaca gcaacatctt 8280
tgtgaaagat ggcaaagtaa gtggctttat tgatcttggg agaagcggca gggcggacaa 8340
gtggtatgac attgccttct gcgtccggtc gatcagggag gatatcgggg aagaacagta 8400
tgtcgagcta ttttttgact tactggggat caagcctgat tgggagaaaa taaaatatta 8460
tattttactg gatgaattgt tttagtacct agaatgcatg accaaaatcc cttaacgtga 8520
gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 8580
tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 8640
ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 8700
gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 8760
tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 8820
cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 8880
gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 8940
actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 9000
ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 9060
gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 9120
atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 9180
tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 9240
tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 9300
aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt 9360
tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg 9420
ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg 9480
gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 9540
gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 9600
ccgtcatcac cgaaacgcgc gaggcagggt gccttgatgt gggcgccggc ggtcgagtgg 9660
cgacggcgcg gcttgtccgc gccctggtag attgcctggc cgtaggccag ccatttttga 9720
gcggccagcg gccgcgatag gccgacgcga agcggcgggg cgtagggagc gcagcgaccg 9780
aagggtaggc gctttttgca gctcttcggc tgtgcgctgg ccagacagtt atgcacaggc 9840
caggcgggtt ttaagagttt taataagttt taaagagttt taggcggaaa aatcgccttt 9900
tttctctttt atatcagtca cttacatgtg tgaccggttc ccaatgtacg gctttgggtt 9960
cccaatgtac gggttccggt tcccaatgta cggctttggg ttcccaatgt acgtgctatc 10020
cacaggaaag agaccttttc gacctttttc ccctgctagg gcaatttgcc ctagcatctg 10080
ctccgtacat taggaaccgg cggatgcttc gccctcgatc aggttgcggt agcgcatgac 10140
taggatcggg ccagcctgcc ccgcctcctc cttcaaatcg tactccggca ggtcatttga 10200
cccgatcagc ttgcgcacgg tgaaacagaa cttcttgaac tctccggcgc tgccactgcg 10260
ttcgtagatc gtcttgaaca accatctggc ttctgccttg cctgcggcgc ggcgtgccag 10320
gcggtagaga aaacggccga tgccgggatc gatcaaaaag taatcggggt gaaccgtcag 10380
cacgtccggg ttcttgcctt ctgtgatctc gcggtacatc caatcagcta gctcgatctc 10440
gatgtactcc ggccgcccgg tttcgctctt tacgatcttg tagcggctaa tcaaggcttc 10500
accctcggat accgtcacca ggcggccgtt cttggccttc ttcgtacgct gcatggcaac 10560
gtgcgtggtg tttaaccgaa tgcaggtttc taccaggtcg tctttctgct ttccgccatc 10620
ggctcgccgg cagaacttga gtacgtccgc aacgtgtgga cggaacacgc ggccgggctt 10680
gtctcccttc ccttcccggt atcggttcat ggattcggtt agatgggaaa ccgccatcag 10740
taccaggtcg taatcccaca cactggccat gccggccggc cctgcggaaa cctctacgtg 10800
cccgtctgga agctcgtagc ggatcacctc gccagctcgt cggtcacgct tcgacagacg 10860
gaaaacggcc acgtccatga tgctgcgact atcgcgggtg cccacgtcat agagcatcgg 10920
aacgaaaaaa tctggttgct cgtcgccctt gggcggcttc ctaatcgacg gcgcaccggc 10980
tgccggcggt tgccgggatt ctttgcggat tcgatcagcg gccgcttgcc acgattcacc 11040
ggggcgtgct tctgcctcga tgcgttgccg ctgggcggcc tgcgcggcct tcaacttctc 11100
caccaggtca tcacccagcg ccgcgccgat ttgtaccggg ccggatggtt tgcgaccgtc 11160
acgccgattc ctcgggcttg ggggttccag tgccattgca gggccggcag acaacccagc 11220
cgcttacgcc tggccaaccg cccgttcctc cacacatggg gcattccacg gcgtcggtgc 11280
ctggttgttc ttgattttcc atgccgcctc ctttagccgc taaaattcat ctactcattt 11340
attcatttgc tcatttactc tggtagctgc gcgatgtatt cagatagcag ctcggtaatg 11400
gtcttgcctt ggcgtaccgc gtacatcttc agcttggtgt gatcctccgc cggcaactga 11460
aagttgaccc gcttcatggc tggcgtgtct gccaggctgg ccaacgttgc agccttgctg 11520
ctgcgtgcgc tcggacggcc ggcacttagc gtgtttgtgc ttttgctcat tttctcttta 11580
cctcattaac tcaaatgagt tttgatttaa tttcagcggc cagcgcctgg acctcgcggg 11640
cagcgtcgcc ctcgggttct gattcaagaa cggttgtgcc ggcggcggca gtgcctgggt 11700
agctcacgcg ctgcgtgata cgggactcaa gaatgggcag ctcgtacccg gccagcgcct 11760
cggcaacctc accgccgatg cgcgtgcctt tgatcgcccg cgacacgaca aaggccgctt 11820
gtagccttcc atccgtgacc tcaatgcgct gcttaaccag ctccaccagg tcggcggtgg 11880
cccatatgtc gtaagggctt ggctgcaccg gaatcagcac gaagtcggct gccttgatcg 11940
cggacacagc caagtccgcc gcctggggcg ctccgtcgat cactacgaag tcgcgccggc 12000
cgatggcctt cacgtcgcgg tcaatcgtcg ggcggtcgat gccgacaacg gttagcggtt 12060
gatcttcccg cacggccgcc caatcgcggg cactgccctg gggatcggaa tcgactaaca 12120
gaacatcggc cccggcgagt tgcagggcgc gggctagatg ggttgcgatg gtcgtcttgc 12180
ctgacccgcc tttctggtta agtacagcga taaccttcat gcgttcccct tgcgtatttg 12240
tttatttact catcgcatca tatacgcagc gaccgcatga cgcaagctgt tttactcaaa 12300
tacacatcac ctttttagac ggcggcgctc ggtttcttca gcggccaagc tggccggcca 12360
ggccgccagc ttggcatcag acaaaccggc caggatttca tgcagccgca cggttgagac 12420
gtgcgcgggc ggctcgaaca cgtacccggc cgcgatcatc tccgcctcga tctcttcggt 12480
aatgaaaaac ggttcgtcct ggccgtcctg gtgcggtttc atgcttgttc ctcttggcgt 12540
tcattctcgg cggccgccag ggcgtcggcc tcggtcaatg cgtcctcacg gaaggcaccg 12600
cgccgcctgg cctcggtggg cgtcacttcc tcgctgcgct caagtgcgcg gtacagggtc 12660
gagcgatgca cgccaagcag tgcagccgcc tctttcacgg tgcggccttc ctggtcgatc 12720
agctcgcggg cgtgcgcgat ctgtgccggg gtgagggtag ggcgggggcc aaacttcacg 12780
cctcgggcct tggcggcctc gcgcccgctc cgggtgcggt cgatgattag ggaacgctcg 12840
aactcggcaa tgccggcgaa cacggtcaac accatgcggc cggccggcgt ggtggtgtcg 12900
gcccacggct ctgccaggct acgcaggccc gcgccggcct cctggatgcg ctcggcaatg 12960
tccagtaggt cgcgggtgct gcgggccagg cggtctagcc tggtcactgt cacaacgtcg 13020
ccagggcgta ggtggtcaag catcctggcc agctccgggc ggtcgcgcct ggtgccggtg 13080
atcttctcgg aaaacagctt ggtgcagccg gccgcgtgca gttcggcccg ttggttggtc 13140
aagtcctggt cgtcggtgct gacgcgggca tagcccagca ggccagcggc ggcgctcttg 13200
ttcatggcgt aatgtctccg gttctagtcg caagtattct actttatgcg actaaaacac 13260
gcgacaagaa aacgccagga aaagggcagg gcggc 13295
<210> 6
<211> 13322
<212> DNA
<213> 未知(Unknown)
<400> 6
agcctgtcgc gtaacttagg acttgtgcga catgtcgttt tcagaagacg gctgcactga 60
acgtcagaag ccgactgcac tatagcagcg gaggggttgg atcaaagtac tttgatcccg 120
aggggaaccc tgtggttggc atgcacatac aaatggacga acggataaac cttttcacgc 180
ccttttaaat atccgttatt ctaataaacg ctcttttctc ttaggtttac ccgccaatat 240
atcctgtcaa acactgatag tttaaactga aggcgggaaa cgacaatctg atccaagctc 300
aagctgctct agcattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 360
gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 420
gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagctttgt 480
atgaactgat gatctaggac cggataagtt cccttcttca tagcgaactt attcaaagaa 540
tgttttgtgt atcattcttg ttacattgtt attaatgaaa aaatattatt ggtcattgga 600
ctgaacacga gtgttaaata tggaccaggc cccaaataag atccattgat atatgaatta 660
aataacaaga ataaatcgag tcaccaaacc acttgccttt tttaacgaga cttgttcacc 720
aacttgatac aaaagtcatt atcctatgca aatcaataat catacaaaaa tatccaataa 780
cactaaaaaa ttaaaagaaa tggataattt cacaatatgt tatacgataa agaagttact 840
tttccaagaa attcactgat tttataagcc cacttgcatt agataaatgg caaaaaaaaa 900
caaaaaggaa aagaaataaa gcacgaagaa ttctagaaaa tacgaaatac gcttcaatgc 960
agtgggaccc acggttcaat tattgccaat tttcagctcc accgtatatt taaaaaataa 1020
aacgataatg ctaaaaaaat ataaatcgta acgatcgtta aatctcaacg gctggatctt 1080
atgacgaccg ttagaaattg tggttgtcga cgagtcagta ataaacggcg tcaaagtggt 1140
tgcagccggc acacacgagt cgtgtttatc aactcaaagc acaaatactt ttcctcaacc 1200
taaaaataag gcaattagcc aaaaacaact ttgcgtgtaa acaacgctca atacacgtgt 1260
cattttatta ttagctattg cttcaccgcc ttagctttct cgtgacctag tcgtcctcgt 1320
cttttcttct tcttcttcta taaaacaata cccaaagagc tcttcttctt cacaattcag 1380
atttcaattt ctcaaaatct taaaaacttt ctctcaattc tctctaccgt gatcaaggta 1440
aatttctgtg ttccttattc tctcaaaatc ttcgattttg ttttcgttcg atcccaattt 1500
cgtatatgtt ctttggttta gattctgtta atcttagatc gaagacgatt ttctgggttt 1560
gatcgttaga tatcatctta attctcgatt agggtttcat agatatcatc cgatttgttc 1620
aaataatttg agttttgtcg aataattact cttcgatttg tgatttctat ctagatctgg 1680
tgttagtttc tagtttgtgc gatcgaattt gtcgattaat ctgagttttt ctgattaaca 1740
gatgggcctc tccgacggcg agtggcagct cgtgctcaac gcctggggca aggtggaggc 1800
cgacgtggcc ggccacggcc aggaggtgct catccgcctc ttcaccggcc acccggagac 1860
cctggagaag ttcgacaagt tcaagcacct caagaccgag gccgagatga aggcctccga 1920
ggacctcaag aagcacggca acaccgtgct caccgccctc ggcggcatcc tcaagaagaa 1980
gggccaccac gaggccgagg tgaagcacct cgccgagtcc cacgccaaca agcacaagat 2040
cccggtgaag tacctggagt tcatctccga cgccatcatc cacgtgctcc acgccaagca 2100
cccgtccgac ttcggcgccg acgcccaggc cgccatgtcc aaggccctgg agctgttccg 2160
caacgacatg gccgcccagt acaaggtgct cggcttccac ggctgacgac atatgaagat 2220
gaagatgaaa tatttggtgt gtcaaataaa aagctagtgt gcttaagttt gtgttttttt 2280
cttggcttgt tgtgttatga atttgtggct ttttctaata ttaaatgaat gtaagatctc 2340
attataatga ataaacaaat gtttctataa tccattgtga atgttttgtt ggatctcttc 2400
tgcagcatat aactactgta tgtgctatgg tatggactat ggaatatgat taaagataag 2460
atgggctcat agagtaaaac gaggcgaggg acctataaac ctcccttcat catgctattt 2520
catgatctat tttataaaat aaagatgtag aaaaaagtaa gcgtaataac cgcaaaacaa 2580
atgatttaaa acatggcaca taatgaggag attaagttcg gtttacgttt attttagtac 2640
taattgtaac gtgagactac gtatcgggaa tcgcctaatt aaagcattaa tgcgaacctg 2700
attagattca ccgaccctcc tatcgtgtcg acctttctgt ttcttagaat tttttggtag 2760
tctatgtact aataatgtca gcttcgtatt tatttcataa gcaatttgca tttgcaattt 2820
gttttttact tttattttta ttgtattgtg gaatgtggac tcgtaccaac atgaagttat 2880
ataccaccaa aaaaattaca gttagtcaaa agattcacga gtgagagcta cttatgattg 2940
tcttttacgt atatgtctaa ttgtctattt gctcaataat ctttgtactt tcttttgtcg 3000
ttgataaaat cacaaagttc caaaagtaat cgaatgattt gcttttaaga aaagaacagc 3060
tcaataattc aacatatatc tgtacacaca taattgaata agggtacccg attaaaaatc 3120
ccaattatat ttggtctaat ttagtttggt attgagtaaa acaaattcga accaaaccaa 3180
aatataaata tatagttttt atatatatgc ctttaagact ttttatagaa ttttctttaa 3240
aaaatatcta gaaatatttg cgactcttct ggcatgtaat atttcgttaa atatgaagtg 3300
ctccattttt attaacttta aataattggt tgtacgatca ctttcttatc aagtgttact 3360
aaaatgcgtc aatctctttg ttcttccata ttcatatgtc aaaatctatc aaaattctta 3420
tatatctttt tcgaatttga agtgaaattt cgataattta aaattaaata gaacatatca 3480
ttatttaggt atcatattga tttttatact taattactaa atttggttaa ctttgaaagt 3540
gtacatcaac gaaaaattag tcaaacgact aaaataaata aatatcatgt gttattaaga 3600
aaattctcct ataagaatat tttaatagat catatgtttg taaaaaaaat taatttttac 3660
taacacatat atttacttat caaaaatttg acaaagtaag attaaaataa tattcatcta 3720
acaaaaaaaa aaccagaaaa tgctgaaaac ccggcaaaac cgaaccaatc caaaccgata 3780
tagttggttt ggtttgattt tgatataaac cgaaccaact cggtccattt gcacccctaa 3840
tcataatagc tttaatattt caagatatta ttaagttaac gttgtcaata tcctggaaat 3900
tttgcaaaat gaatcaagcc tatatggctg taatatgaat ttaaaagcag ctcgatgtgg 3960
tggtaatatg taatttactt gattctaaaa aaatatccca agtattaata atttctgcta 4020
ggaagaaggt tagctacgat ttacagcaaa gccagaatac aaagaaccat aaagtgattg 4080
aagctcgaaa tatacgaagg aacaaatatt tttaaaaaaa tacgcaatga cttggaacaa 4140
aagaaagtga tatatttttt gttcttaaac aagcatcccc tctaaagaat ggcagttttc 4200
ctttgcatgt aactattatg ctcccttcgt tacaaaaatt ttggactact attgggaact 4260
tcttctgaaa atagtggtac cgagctcgaa ttcgtaatca tgtcatagct gtttcctgtg 4320
tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 4380
gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 4440
ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 4500
ggcggtttgc gtattggcta gagcagcttg ccaacatggt ggagcacgac actctcgtct 4560
actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac 4620
aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca 4680
aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg 4740
ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga 4800
gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata 4860
acatggtgga gcacgacact ctcgtctact ccaagaatat caaagataca gtctcagaag 4920
accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc 4980
attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca 5040
aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc 5100
ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 5160
cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc 5220
actatccttc gcaagacctt cctctatata aggaagttca tttcatttgg agaggacacg 5280
ctgaaatcac cagtctctct ctacaaatct atctctctcg agtcaacaca acatatacaa 5340
aacaaacgaa tctcaagcaa tcaagcattc tacttctatt gcagcaattt aaatcatttc 5400
ttttaaagca aaagcaattt tctgaaaatt ttcaccattt acgaacgata gccatggctc 5460
aagttagcag aatctgcaat ggtgtgcaga acccatctct tatctccaat ctctctaaat 5520
ccagtcaaag gaaatctccc ttatcggttt ctctgaagac tcagcagcat ccacgagctt 5580
atccaatttc ttcatcttgg ggattgaaga agagtgggat gactttaatt ggctctgagc 5640
ttcgtcctct taaggtcatg tcttctgttt ccacggcgga gaagggatcc gacgctcttc 5700
cagctacctt cgacgttatc gtgcatccag ctagagaact cagaggtgaa cttagagcac 5760
agccatccaa gaactacacc actagatacc tcctcgccgc tgctctcgct gagggtgaaa 5820
ccagagttgt tggtgtggct acctctgagg atgccgaagc tatgctcaga tgcctcagag 5880
attggggtgc tggtgttgag cttgttggtg atgacgccgt gatcagaggt ttcggtgcta 5940
gaccacaggc tggtgttacc cttaacccag gtaacgctgc tgcggtggcc agactcctta 6000
tgggtgttgc tgctctcacc tctggtacaa ctttcgttac cgattaccct gattcccttg 6060
gtaagagacc tcagggtgac cttcttgaag ccctcgaaag acttggtgct tgggtgtcct 6120
ccaacgatgg tagactccct atctccgttt ccggtccagt tagaggtggt acagtggagg 6180
tttccgccga aagatcctcc cagtacgctt ccgcccttat gttcctcggt cctcttcttc 6240
ctgacggact cgaacttaga ctcaccggtg atatcaagtc ccacgctcct cttagacaga 6300
cacttgacac cctctctgat ttcggtgtta gagctactgc ctccgatgac cttagaagaa 6360
tctccatccc tggtggtcag aagtacagac caggtagagt gctcgttcct ggtgattacc 6420
ctggttccgc tgctatcctt accgccgctg ctcttctccc aggtgaggtt agactttcta 6480
accttagaga acacgacctc cagggtgaga aggaagctgt gaacgttctt agagagatgg 6540
gtgctgatat cgttagagaa ggtgataccc ttaccgtgag aggtggtaga cctctccacg 6600
ctgttactag agatggtgat tccttcaccg acgccgtgca agctcttacc gctgctgctg 6660
ccttcgctga gggtgatacc acctgggaaa acgttgctac tcttagactc aaggaatgcg 6720
atagaatctc tgacaccaga gctgagcttg aaagacttgg tcttagagca agagagaccg 6780
ccgattctct ctccgttact ggttctgctc accttgctgg tggtatcacc gctgatggtc 6840
acggtgacca cagaatgatc atgcttctca cccttcttgg tctcagagca gatgctccac 6900
ttagaatcac cggtgcacac cacatcagaa agtcctaccc tcagttcttc gctcaccttg 6960
aagctcttgg tgctagattc gaatacgctg aggctaccgc ctaataggag ctcgagtttc 7020
tccataataa tgtgtgagta gttcccagat aagggaatta gggttcctat agggtttcgc 7080
tcatgtgttg agcatataag aaacccttag tatgtatttg tatttgtaaa atacttctat 7140
caataaaatt tctaattcct aaaaccaaaa tccagtacta aaatccagat cccccgaatt 7200
aattcggcgt taattcagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc 7260
gtcaatttgt ttacaccaca atatatcctg ccaccagcca gccaacagct ccccgaccgg 7320
cagctcggca caaaatcacc actcgataca ggcagcccat cagtccggga cggcgtcagc 7380
gggagagccg ttgtaaggcg gcagactttg ctcatgttac cgatgctatt cggaagaacg 7440
gcaactaagc tgccgggttt gaaacacgga tgatctcgcg gagggtagca tgttgattgt 7500
aacgatgaca gagcgttgct gcctgtgatc accgcggttt caaaatcggc tccgtcgata 7560
ctatgttata cgccaacttt gaaaacaact ttgaaaaagc tgttttctgg tatttaaggt 7620
tttagaatgc aaggaacagt gaattggagt tcgtcttgtt ataattagct tcttggggta 7680
tctttaaata ctgtagaaaa gaggaaggaa ataataaatg gctaaaatga gaatatcacc 7740
ggaattgaaa aaactgatcg aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc 7800
tgctaaggta tataagctgg tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag 7860
ccggtataaa gggaccacct atgatgtgga acgggaaaag gacatgatgc tatggctgga 7920
aggaaagctg cctgttccaa aggtcctgca ctttgaacgg catgatggct ggagcaatct 7980
gctcatgagt gaggccgatg gcgtcctttg ctcggaagag tatgaagatg aacaaagccc 8040
tgaaaagatt atcgagctgt atgcggagtg catcaggctc tttcactcca tcgacatatc 8100
ggattgtccc tatacgaata gcttagacag ccgcttagcc gaattggatt acttactgaa 8160
taacgatctg gccgatgtgg attgcgaaaa ctgggaagaa gacactccat ttaaagatcc 8220
gcgcgagctg tatgattttt taaagacgga aaagcccgaa gaggaacttg tcttttccca 8280
cggcgacctg ggagacagca acatctttgt gaaagatggc aaagtaagtg gctttattga 8340
tcttgggaga agcggcaggg cggacaagtg gtatgacatt gccttctgcg tccggtcgat 8400
cagggaggat atcggggaag aacagtatgt cgagctattt tttgacttac tggggatcaa 8460
gcctgattgg gagaaaataa aatattatat tttactggat gaattgtttt agtacctaga 8520
atgcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 8580
aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 8640
aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 8700
tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 8760
gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 8820
cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 8880
acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 8940
cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 9000
cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 9060
aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 9120
gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 9180
atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 9240
tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 9300
gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 9360
agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 9420
catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact 9480
ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac 9540
gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 9600
gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagggtgcc 9660
ttgatgtggg cgccggcggt cgagtggcga cggcgcggct tgtccgcgcc ctggtagatt 9720
gcctggccgt aggccagcca tttttgagcg gccagcggcc gcgataggcc gacgcgaagc 9780
ggcggggcgt agggagcgca gcgaccgaag ggtaggcgct ttttgcagct cttcggctgt 9840
gcgctggcca gacagttatg cacaggccag gcgggtttta agagttttaa taagttttaa 9900
agagttttag gcggaaaaat cgcctttttt ctcttttata tcagtcactt acatgtgtga 9960
ccggttccca atgtacggct ttgggttccc aatgtacggg ttccggttcc caatgtacgg 10020
ctttgggttc ccaatgtacg tgctatccac aggaaagaga ccttttcgac ctttttcccc 10080
tgctagggca atttgcccta gcatctgctc cgtacattag gaaccggcgg atgcttcgcc 10140
ctcgatcagg ttgcggtagc gcatgactag gatcgggcca gcctgccccg cctcctcctt 10200
caaatcgtac tccggcaggt catttgaccc gatcagcttg cgcacggtga aacagaactt 10260
cttgaactct ccggcgctgc cactgcgttc gtagatcgtc ttgaacaacc atctggcttc 10320
tgccttgcct gcggcgcggc gtgccaggcg gtagagaaaa cggccgatgc cgggatcgat 10380
caaaaagtaa tcggggtgaa ccgtcagcac gtccgggttc ttgccttctg tgatctcgcg 10440
gtacatccaa tcagctagct cgatctcgat gtactccggc cgcccggttt cgctctttac 10500
gatcttgtag cggctaatca aggcttcacc ctcggatacc gtcaccaggc ggccgttctt 10560
ggccttcttc gtacgctgca tggcaacgtg cgtggtgttt aaccgaatgc aggtttctac 10620
caggtcgtct ttctgctttc cgccatcggc tcgccggcag aacttgagta cgtccgcaac 10680
gtgtggacgg aacacgcggc cgggcttgtc tcccttccct tcccggtatc ggttcatgga 10740
ttcggttaga tgggaaaccg ccatcagtac caggtcgtaa tcccacacac tggccatgcc 10800
ggccggccct gcggaaacct ctacgtgccc gtctggaagc tcgtagcgga tcacctcgcc 10860
agctcgtcgg tcacgcttcg acagacggaa aacggccacg tccatgatgc tgcgactatc 10920
gcgggtgccc acgtcataga gcatcggaac gaaaaaatct ggttgctcgt cgcccttggg 10980
cggcttccta atcgacggcg caccggctgc cggcggttgc cgggattctt tgcggattcg 11040
atcagcggcc gcttgccacg attcaccggg gcgtgcttct gcctcgatgc gttgccgctg 11100
ggcggcctgc gcggccttca acttctccac caggtcatca cccagcgccg cgccgatttg 11160
taccgggccg gatggtttgc gaccgtcacg ccgattcctc gggcttgggg gttccagtgc 11220
cattgcaggg ccggcagaca acccagccgc ttacgcctgg ccaaccgccc gttcctccac 11280
acatggggca ttccacggcg tcggtgcctg gttgttcttg attttccatg ccgcctcctt 11340
tagccgctaa aattcatcta ctcatttatt catttgctca tttactctgg tagctgcgcg 11400
atgtattcag atagcagctc ggtaatggtc ttgccttggc gtaccgcgta catcttcagc 11460
ttggtgtgat cctccgccgg caactgaaag ttgacccgct tcatggctgg cgtgtctgcc 11520
aggctggcca acgttgcagc cttgctgctg cgtgcgctcg gacggccggc acttagcgtg 11580
tttgtgcttt tgctcatttt ctctttacct cattaactca aatgagtttt gatttaattt 11640
cagcggccag cgcctggacc tcgcgggcag cgtcgccctc gggttctgat tcaagaacgg 11700
ttgtgccggc ggcggcagtg cctgggtagc tcacgcgctg cgtgatacgg gactcaagaa 11760
tgggcagctc gtacccggcc agcgcctcgg caacctcacc gccgatgcgc gtgcctttga 11820
tcgcccgcga cacgacaaag gccgcttgta gccttccatc cgtgacctca atgcgctgct 11880
taaccagctc caccaggtcg gcggtggccc atatgtcgta agggcttggc tgcaccggaa 11940
tcagcacgaa gtcggctgcc ttgatcgcgg acacagccaa gtccgccgcc tggggcgctc 12000
cgtcgatcac tacgaagtcg cgccggccga tggccttcac gtcgcggtca atcgtcgggc 12060
ggtcgatgcc gacaacggtt agcggttgat cttcccgcac ggccgcccaa tcgcgggcac 12120
tgccctgggg atcggaatcg actaacagaa catcggcccc ggcgagttgc agggcgcggg 12180
ctagatgggt tgcgatggtc gtcttgcctg acccgccttt ctggttaagt acagcgataa 12240
ccttcatgcg ttccccttgc gtatttgttt atttactcat cgcatcatat acgcagcgac 12300
cgcatgacgc aagctgtttt actcaaatac acatcacctt tttagacggc ggcgctcggt 12360
ttcttcagcg gccaagctgg ccggccaggc cgccagcttg gcatcagaca aaccggccag 12420
gatttcatgc agccgcacgg ttgagacgtg cgcgggcggc tcgaacacgt acccggccgc 12480
gatcatctcc gcctcgatct cttcggtaat gaaaaacggt tcgtcctggc cgtcctggtg 12540
cggtttcatg cttgttcctc ttggcgttca ttctcggcgg ccgccagggc gtcggcctcg 12600
gtcaatgcgt cctcacggaa ggcaccgcgc cgcctggcct cggtgggcgt cacttcctcg 12660
ctgcgctcaa gtgcgcggta cagggtcgag cgatgcacgc caagcagtgc agccgcctct 12720
ttcacggtgc ggccttcctg gtcgatcagc tcgcgggcgt gcgcgatctg tgccggggtg 12780
agggtagggc gggggccaaa cttcacgcct cgggccttgg cggcctcgcg cccgctccgg 12840
gtgcggtcga tgattaggga acgctcgaac tcggcaatgc cggcgaacac ggtcaacacc 12900
atgcggccgg ccggcgtggt ggtgtcggcc cacggctctg ccaggctacg caggcccgcg 12960
ccggcctcct ggatgcgctc ggcaatgtcc agtaggtcgc gggtgctgcg ggccaggcgg 13020
tctagcctgg tcactgtcac aacgtcgcca gggcgtaggt ggtcaagcat cctggccagc 13080
tccgggcggt cgcgcctggt gccggtgatc ttctcggaaa acagcttggt gcagccggcc 13140
gcgtgcagtt cggcccgttg gttggtcaag tcctggtcgt cggtgctgac gcgggcatag 13200
cccagcaggc cagcggcggc gctcttgttc atggcgtaat gtctccggtt ctagtcgcaa 13260
gtattctact ttatgcgact aaaacacgcg acaagaaaac gccaggaaaa gggcagggcg 13320
gc 13322
<210> 7
<211> 13210
<212> DNA
<213> 未知(Unknown)
<400> 7
agcctgtcgc gtaacttagg acttgtgcga catgtcgttt tcagaagacg gctgcactga 60
acgtcagaag ccgactgcac tatagcagcg gaggggttgg atcaaagtac tttgatcccg 120
aggggaaccc tgtggttggc atgcacatac aaatggacga acggataaac cttttcacgc 180
ccttttaaat atccgttatt ctaataaacg ctcttttctc ttaggtttac ccgccaatat 240
atcctgtcaa acactgatag tttaaactga aggcgggaaa cgacaatctg atccaagctc 300
aagctgctct agcattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 360
gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 420
gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagcttcag 480
attaaacgac gccgtttcgt tcctttcgct tcacggctta acgatgtcgt ttctgtctgt 540
gcccaaaaaa taaaggcatt tgttatttgc accagatatt tactaagtgc accctagttt 600
gacaagtagg cgataattac aaatagatgc ggtgcaaata ataaattttg aaggaaataa 660
ttacaaaaga acagaactta tatttacttt attttaaaaa actaaaatga aagaacaaaa 720
aaaagtaaaa aatacaaaaa atgtgcttta accactttca ttatttgtta cagaaagtat 780
gattctactc aaattgatct gttgtatctg gtgctgcctt gtcacactgg cgatttcaat 840
cccctaaaga tatggtgcaa actgcgaagt gatcaatatc tgctcggtta atttagatta 900
attaataata ttcaacgtga tgtaccaaaa aaagacaatt ttttgctcca ttgacaaatt 960
aaacctcatc aaggtaattt ccaaacctat aagcaaaaaa atttcacatt aattggcccg 1020
caatcctatt agtcttatta tactagagta ggaaaaaaaa caattacaca acttgtctta 1080
ttattctcta tgctaatgaa tatttttccc ttttgttaga aatcagtgtt tcctaattta 1140
ttgagtatta attccactca ccgcatatat ttaccgttga ataagaaaat tttacacata 1200
attcttttta aaataaataa tttttttata ctagatctta tatgattacg tgaagccaag 1260
tgggttatac taatgatata taatgtttga tagtaatcag tttataaacc aaatgcatgg 1320
aaatgttacg tggaagcacg taaattaaca agcattgaag caaatgcagc caccgcacca 1380
aaaccacccc acttcacttc cacgtaccat attccatgca actacaacac cctaaaactt 1440
caataaatgc ccccaccttc acttctcaac acaacatata caaaacaaac gaatctcaag 1500
caatcaagca ttctacttct attgcagcaa tttaaatcat ttcttttaaa gcaaaagcaa 1560
ttttctgaaa attttcacca tttacgaacg atagccatgg ctaacaagct cttccttgtt 1620
tgcgctactt tcgctctttg cttccttctt actaacgctg gcgccttcac cgagaagcag 1680
gaggccctcg tgtcctcctc cttcgaggcc ttcaaggcca acatcccgca gtactccgtg 1740
gtgttctaca cctccatcct ggagaaggcc ccggccgcca aggacctctt ctccttcctc 1800
tccaacggcg tggacccgtc caacccgaag ctcaccggcc acgccgagaa gctcttcggc 1860
ctcgtgcgcg actccgccgg ccagctcaag gccaacggca ccgtggtggc cgacgccgcc 1920
ctcggctcca tccacgccca gaaggccatc accgacccgc agttcgtggt ggtgaaggag 1980
gccctcctca agaccatcaa ggaggccgtg ggcgacaagt ggtccgacga gctgtcctcc 2040
gcctgggagg tggcctacga cgagctggcc gccgccatca agaaggcctt ctgacgacat 2100
atgaagatga agatgaaata tttggtgtgt caaataaaaa gctagtgtgc ttaagtttgt 2160
gtttttttct tggcttgttg tgttatgaat ttgtggcttt ttctaatatt aaatgaatgt 2220
aagatctcat tataatgaat aaacaaatgt ttctataatc cattgtgaat gttttgttgg 2280
atctcttctg cagcatataa ctactgtatg tgctatggta tggactatgg aatatgatta 2340
aagataagat gggctcatag agtaaaacga ggcgagggac ctataaacct cccttcatca 2400
tgctatttca tgatctattt tataaaataa agatgtagaa aaaagtaagc gtaataaccg 2460
caaaacaaat gatttaaaac atggcacata atgaggagat taagttcggt ttacgtttat 2520
tttagtacta attgtaacgt gagactacgt atcgggaatc gcctaattaa agcattaatg 2580
cgaacctgat tagattcacc gaccctccta tcgtgtcgac ctttctgttt cttagaattt 2640
tttggtagtc tatgtactaa taatgtcagc ttcgtattta tttcataagc aatttgcatt 2700
tgcaatttgt tttttacttt tatttttatt gtattgtgga atgtggactc gtaccaacat 2760
gaagttatat accaccaaaa aaattacagt tagtcaaaag attcacgagt gagagctact 2820
tatgattgtc ttttacgtat atgtctaatt gtctatttgc tcaataatct ttgtactttc 2880
ttttgtcgtt gataaaatca caaagttcca aaagtaatcg aatgatttgc ttttaagaaa 2940
agaacagctc aataattcaa catatatctg tacacacata attgaataag ggtacccgat 3000
taaaaatccc aattatattt ggtctaattt agtttggtat tgagtaaaac aaattcgaac 3060
caaaccaaaa tataaatata tagtttttat atatatgcct ttaagacttt ttatagaatt 3120
ttctttaaaa aatatctaga aatatttgcg actcttctgg catgtaatat ttcgttaaat 3180
atgaagtgct ccatttttat taactttaaa taattggttg tacgatcact ttcttatcaa 3240
gtgttactaa aatgcgtcaa tctctttgtt cttccatatt catatgtcaa aatctatcaa 3300
aattcttata tatctttttc gaatttgaag tgaaatttcg ataatttaaa attaaataga 3360
acatatcatt atttaggtat catattgatt tttatactta attactaaat ttggttaact 3420
ttgaaagtgt acatcaacga aaaattagtc aaacgactaa aataaataaa tatcatgtgt 3480
tattaagaaa attctcctat aagaatattt taatagatca tatgtttgta aaaaaaatta 3540
atttttacta acacatatat ttacttatca aaaatttgac aaagtaagat taaaataata 3600
ttcatctaac aaaaaaaaaa ccagaaaatg ctgaaaaccc ggcaaaaccg aaccaatcca 3660
aaccgatata gttggtttgg tttgattttg atataaaccg aaccaactcg gtccatttgc 3720
acccctaatc ataatagctt taatatttca agatattatt aagttaacgt tgtcaatatc 3780
ctggaaattt tgcaaaatga atcaagccta tatggctgta atatgaattt aaaagcagct 3840
cgatgtggtg gtaatatgta atttacttga ttctaaaaaa atatcccaag tattaataat 3900
ttctgctagg aagaaggtta gctacgattt acagcaaagc cagaatacaa agaaccataa 3960
agtgattgaa gctcgaaata tacgaaggaa caaatatttt taaaaaaata cgcaatgact 4020
tggaacaaaa gaaagtgata tattttttgt tcttaaacaa gcatcccctc taaagaatgg 4080
cagttttcct ttgcatgtaa ctattatgct cccttcgtta caaaaatttt ggactactat 4140
tgggaacttc ttctgaaaat agtggtaccg agctcgaatt cgtaatcatg tcatagctgt 4200
ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 4260
agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 4320
tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4380
cggggagagg cggtttgcgt attggctaga gcagcttgcc aacatggtgg agcacgacac 4440
tctcgtctac tccaagaata tcaaagatac agtctcagaa gaccaaaggg ctattgagac 4500
ttttcaacaa agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca 4560
cttcatcaaa aggacagtag aaaaggaagg tggcacctac aaatgccatc attgcgataa 4620
aggaaaggct atcgttcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 4680
cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 4740
atgtgataac atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt 4800
ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct 4860
cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg 4920
cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga 4980
cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 5040
aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc 5100
acaatcccac tatccttcgc aagaccttcc tctatataag gaagttcatt tcatttggag 5160
aggacacgct gaaatcacca gtctctctct acaaatctat ctctctcgag tcaacacaac 5220
atatacaaaa caaacgaatc tcaagcaatc aagcattcta cttctattgc agcaatttaa 5280
atcatttctt ttaaagcaaa agcaattttc tgaaaatttt caccatttac gaacgatagc 5340
catggctcaa gttagcagaa tctgcaatgg tgtgcagaac ccatctctta tctccaatct 5400
ctctaaatcc agtcaaagga aatctccctt atcggtttct ctgaagactc agcagcatcc 5460
acgagcttat ccaatttctt catcttgggg attgaagaag agtgggatga ctttaattgg 5520
ctctgagctt cgtcctctta aggtcatgtc ttctgtttcc acggcggaga agggatccga 5580
cgctcttcca gctaccttcg acgttatcgt gcatccagct agagaactca gaggtgaact 5640
tagagcacag ccatccaaga actacaccac tagatacctc ctcgccgctg ctctcgctga 5700
gggtgaaacc agagttgttg gtgtggctac ctctgaggat gccgaagcta tgctcagatg 5760
cctcagagat tggggtgctg gtgttgagct tgttggtgat gacgccgtga tcagaggttt 5820
cggtgctaga ccacaggctg gtgttaccct taacccaggt aacgctgctg cggtggccag 5880
actccttatg ggtgttgctg ctctcacctc tggtacaact ttcgttaccg attaccctga 5940
ttcccttggt aagagacctc agggtgacct tcttgaagcc ctcgaaagac ttggtgcttg 6000
ggtgtcctcc aacgatggta gactccctat ctccgtttcc ggtccagtta gaggtggtac 6060
agtggaggtt tccgccgaaa gatcctccca gtacgcttcc gcccttatgt tcctcggtcc 6120
tcttcttcct gacggactcg aacttagact caccggtgat atcaagtccc acgctcctct 6180
tagacagaca cttgacaccc tctctgattt cggtgttaga gctactgcct ccgatgacct 6240
tagaagaatc tccatccctg gtggtcagaa gtacagacca ggtagagtgc tcgttcctgg 6300
tgattaccct ggttccgctg ctatccttac cgccgctgct cttctcccag gtgaggttag 6360
actttctaac cttagagaac acgacctcca gggtgagaag gaagctgtga acgttcttag 6420
agagatgggt gctgatatcg ttagagaagg tgataccctt accgtgagag gtggtagacc 6480
tctccacgct gttactagag atggtgattc cttcaccgac gccgtgcaag ctcttaccgc 6540
tgctgctgcc ttcgctgagg gtgataccac ctgggaaaac gttgctactc ttagactcaa 6600
ggaatgcgat agaatctctg acaccagagc tgagcttgaa agacttggtc ttagagcaag 6660
agagaccgcc gattctctct ccgttactgg ttctgctcac cttgctggtg gtatcaccgc 6720
tgatggtcac ggtgaccaca gaatgatcat gcttctcacc cttcttggtc tcagagcaga 6780
tgctccactt agaatcaccg gtgcacacca catcagaaag tcctaccctc agttcttcgc 6840
tcaccttgaa gctcttggtg ctagattcga atacgctgag gctaccgcct aataggagct 6900
cgagtttctc cataataatg tgtgagtagt tcccagataa gggaattagg gttcctatag 6960
ggtttcgctc atgtgttgag catataagaa acccttagta tgtatttgta tttgtaaaat 7020
acttctatca ataaaatttc taattcctaa aaccaaaatc cagtactaaa atccagatcc 7080
cccgaattaa ttcggcgtta attcagtaca ttaaaaacgt ccgcaatgtg ttattaagtt 7140
gtctaagcgt caatttgttt acaccacaat atatcctgcc accagccagc caacagctcc 7200
ccgaccggca gctcggcaca aaatcaccac tcgatacagg cagcccatca gtccgggacg 7260
gcgtcagcgg gagagccgtt gtaaggcggc agactttgct catgttaccg atgctattcg 7320
gaagaacggc aactaagctg ccgggtttga aacacggatg atctcgcgga gggtagcatg 7380
ttgattgtaa cgatgacaga gcgttgctgc ctgtgatcac cgcggtttca aaatcggctc 7440
cgtcgatact atgttatacg ccaactttga aaacaacttt gaaaaagctg ttttctggta 7500
tttaaggttt tagaatgcaa ggaacagtga attggagttc gtcttgttat aattagcttc 7560
ttggggtatc tttaaatact gtagaaaaga ggaaggaaat aataaatggc taaaatgaga 7620
atatcaccgg aattgaaaaa actgatcgaa aaataccgct gcgtaaaaga tacggaagga 7680
atgtctcctg ctaaggtata taagctggtg ggagaaaatg aaaacctata tttaaaaatg 7740
acggacagcc ggtataaagg gaccacctat gatgtggaac gggaaaagga catgatgcta 7800
tggctggaag gaaagctgcc tgttccaaag gtcctgcact ttgaacggca tgatggctgg 7860
agcaatctgc tcatgagtga ggccgatggc gtcctttgct cggaagagta tgaagatgaa 7920
caaagccctg aaaagattat cgagctgtat gcggagtgca tcaggctctt tcactccatc 7980
gacatatcgg attgtcccta tacgaatagc ttagacagcc gcttagccga attggattac 8040
ttactgaata acgatctggc cgatgtggat tgcgaaaact gggaagaaga cactccattt 8100
aaagatccgc gcgagctgta tgatttttta aagacggaaa agcccgaaga ggaacttgtc 8160
ttttcccacg gcgacctggg agacagcaac atctttgtga aagatggcaa agtaagtggc 8220
tttattgatc ttgggagaag cggcagggcg gacaagtggt atgacattgc cttctgcgtc 8280
cggtcgatca gggaggatat cggggaagaa cagtatgtcg agctattttt tgacttactg 8340
gggatcaagc ctgattggga gaaaataaaa tattatattt tactggatga attgttttag 8400
tacctagaat gcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 8460
cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 8520
ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 8580
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 8640
gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 8700
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 8760
gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 8820
acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 8880
tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 8940
gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 9000
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 9060
cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 9120
ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 9180
gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 9240
agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 9300
tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag 9360
tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac 9420
ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 9480
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc 9540
agggtgcctt gatgtgggcg ccggcggtcg agtggcgacg gcgcggcttg tccgcgccct 9600
ggtagattgc ctggccgtag gccagccatt tttgagcggc cagcggccgc gataggccga 9660
cgcgaagcgg cggggcgtag ggagcgcagc gaccgaaggg taggcgcttt ttgcagctct 9720
tcggctgtgc gctggccaga cagttatgca caggccaggc gggttttaag agttttaata 9780
agttttaaag agttttaggc ggaaaaatcg ccttttttct cttttatatc agtcacttac 9840
atgtgtgacc ggttcccaat gtacggcttt gggttcccaa tgtacgggtt ccggttccca 9900
atgtacggct ttgggttccc aatgtacgtg ctatccacag gaaagagacc ttttcgacct 9960
ttttcccctg ctagggcaat ttgccctagc atctgctccg tacattagga accggcggat 10020
gcttcgccct cgatcaggtt gcggtagcgc atgactagga tcgggccagc ctgccccgcc 10080
tcctccttca aatcgtactc cggcaggtca tttgacccga tcagcttgcg cacggtgaaa 10140
cagaacttct tgaactctcc ggcgctgcca ctgcgttcgt agatcgtctt gaacaaccat 10200
ctggcttctg ccttgcctgc ggcgcggcgt gccaggcggt agagaaaacg gccgatgccg 10260
ggatcgatca aaaagtaatc ggggtgaacc gtcagcacgt ccgggttctt gccttctgtg 10320
atctcgcggt acatccaatc agctagctcg atctcgatgt actccggccg cccggtttcg 10380
ctctttacga tcttgtagcg gctaatcaag gcttcaccct cggataccgt caccaggcgg 10440
ccgttcttgg ccttcttcgt acgctgcatg gcaacgtgcg tggtgtttaa ccgaatgcag 10500
gtttctacca ggtcgtcttt ctgctttccg ccatcggctc gccggcagaa cttgagtacg 10560
tccgcaacgt gtggacggaa cacgcggccg ggcttgtctc ccttcccttc ccggtatcgg 10620
ttcatggatt cggttagatg ggaaaccgcc atcagtacca ggtcgtaatc ccacacactg 10680
gccatgccgg ccggccctgc ggaaacctct acgtgcccgt ctggaagctc gtagcggatc 10740
acctcgccag ctcgtcggtc acgcttcgac agacggaaaa cggccacgtc catgatgctg 10800
cgactatcgc gggtgcccac gtcatagagc atcggaacga aaaaatctgg ttgctcgtcg 10860
cccttgggcg gcttcctaat cgacggcgca ccggctgccg gcggttgccg ggattctttg 10920
cggattcgat cagcggccgc ttgccacgat tcaccggggc gtgcttctgc ctcgatgcgt 10980
tgccgctggg cggcctgcgc ggccttcaac ttctccacca ggtcatcacc cagcgccgcg 11040
ccgatttgta ccgggccgga tggtttgcga ccgtcacgcc gattcctcgg gcttgggggt 11100
tccagtgcca ttgcagggcc ggcagacaac ccagccgctt acgcctggcc aaccgcccgt 11160
tcctccacac atggggcatt ccacggcgtc ggtgcctggt tgttcttgat tttccatgcc 11220
gcctccttta gccgctaaaa ttcatctact catttattca tttgctcatt tactctggta 11280
gctgcgcgat gtattcagat agcagctcgg taatggtctt gccttggcgt accgcgtaca 11340
tcttcagctt ggtgtgatcc tccgccggca actgaaagtt gacccgcttc atggctggcg 11400
tgtctgccag gctggccaac gttgcagcct tgctgctgcg tgcgctcgga cggccggcac 11460
ttagcgtgtt tgtgcttttg ctcattttct ctttacctca ttaactcaaa tgagttttga 11520
tttaatttca gcggccagcg cctggacctc gcgggcagcg tcgccctcgg gttctgattc 11580
aagaacggtt gtgccggcgg cggcagtgcc tgggtagctc acgcgctgcg tgatacggga 11640
ctcaagaatg ggcagctcgt acccggccag cgcctcggca acctcaccgc cgatgcgcgt 11700
gcctttgatc gcccgcgaca cgacaaaggc cgcttgtagc cttccatccg tgacctcaat 11760
gcgctgctta accagctcca ccaggtcggc ggtggcccat atgtcgtaag ggcttggctg 11820
caccggaatc agcacgaagt cggctgcctt gatcgcggac acagccaagt ccgccgcctg 11880
gggcgctccg tcgatcacta cgaagtcgcg ccggccgatg gccttcacgt cgcggtcaat 11940
cgtcgggcgg tcgatgccga caacggttag cggttgatct tcccgcacgg ccgcccaatc 12000
gcgggcactg ccctggggat cggaatcgac taacagaaca tcggccccgg cgagttgcag 12060
ggcgcgggct agatgggttg cgatggtcgt cttgcctgac ccgcctttct ggttaagtac 12120
agcgataacc ttcatgcgtt ccccttgcgt atttgtttat ttactcatcg catcatatac 12180
gcagcgaccg catgacgcaa gctgttttac tcaaatacac atcacctttt tagacggcgg 12240
cgctcggttt cttcagcggc caagctggcc ggccaggccg ccagcttggc atcagacaaa 12300
ccggccagga tttcatgcag ccgcacggtt gagacgtgcg cgggcggctc gaacacgtac 12360
ccggccgcga tcatctccgc ctcgatctct tcggtaatga aaaacggttc gtcctggccg 12420
tcctggtgcg gtttcatgct tgttcctctt ggcgttcatt ctcggcggcc gccagggcgt 12480
cggcctcggt caatgcgtcc tcacggaagg caccgcgccg cctggcctcg gtgggcgtca 12540
cttcctcgct gcgctcaagt gcgcggtaca gggtcgagcg atgcacgcca agcagtgcag 12600
ccgcctcttt cacggtgcgg ccttcctggt cgatcagctc gcgggcgtgc gcgatctgtg 12660
ccggggtgag ggtagggcgg gggccaaact tcacgcctcg ggccttggcg gcctcgcgcc 12720
cgctccgggt gcggtcgatg attagggaac gctcgaactc ggcaatgccg gcgaacacgg 12780
tcaacaccat gcggccggcc ggcgtggtgg tgtcggccca cggctctgcc aggctacgca 12840
ggcccgcgcc ggcctcctgg atgcgctcgg caatgtccag taggtcgcgg gtgctgcggg 12900
ccaggcggtc tagcctggtc actgtcacaa cgtcgccagg gcgtaggtgg tcaagcatcc 12960
tggccagctc cgggcggtcg cgcctggtgc cggtgatctt ctcggaaaac agcttggtgc 13020
agccggccgc gtgcagttcg gcccgttggt tggtcaagtc ctggtcgtcg gtgctgacgc 13080
gggcatagcc cagcaggcca gcggcggcgc tcttgttcat ggcgtaatgt ctccggttct 13140
agtcgcaagt attctacttt atgcgactaa aacacgcgac aagaaaacgc caggaaaagg 13200
gcagggcggc 13210
<210> 8
<211> 2019
<212> DNA
<213> 未知(Unknown)
<400> 8
tgaatacggg ctaacatggt tatgggctta ggctgcctca ttacccatta catgctctgt 60
tcctaaaaaa aagaagtttg ggcctttatt ctccagtaag ctttttaatt gggctcaaga 120
actttaggta tttaggtgag attcaatgaa aatttataca ttaggcttga tttttttttt 180
caatttacgc aaaaatgaaa gaaaaagtca cactctataa actttatttt tttcaatatt 240
aatacattgt tattgtggat atatcattag cggaaaagta attctaatac cagatttaat 300
tgactttgac taattttttt atgtgatcaa ataatttttt tagcgaaaaa cttatatata 360
tatatatata tatatatata taagttttgt tacttttccg cgtgattttt aatcaaactt 420
tggggataat ttcccctcca attcagccaa aaaaaaaaaa aaactgacac catatattat 480
tagtaggcaa cttgttcgta aatggtgtgg cttatgcgaa tggaaattgg attcgttttc 540
tttaacatca ttattgtttt tgtcaatgag ctatctttta gtcttatgtt attggtgaat 600
ctgtccttaa gttgcagcat ttaacacatc tcctcattag agaaaaaaat tcttccctaa 660
acgatagtaa aaacatctaa taagaaataa gaaagaaaaa ttaggaaaaa gaaaagttca 720
ttaaaaaaat cttttggatt atttttaaaa aaatatctaa atatttttta aatgaataat 780
tttatataaa ctgtaactaa aagtatacaa gtaatgtatg ttaacaaaat acttgaaaaa 840
tctactgaaa atatatctta caaagtgaaa ttaaataaga aagaatttag tggaataatt 900
atgattttat ttaaaaaata attattaaag atttttttgc tccataataa gaaaactttt 960
caattattct tttctggtcc ataataaaaa aaatctagca tgacagcttt tccatagatt 1020
tttaataatg taaaagcagc cgacttcagg caatggatag tggggcccgt atcaacttcg 1080
gacgctccac ttgcaacggg gtgggcccaa tataacaacg acgtcgtaac agataaagcg 1140
aagcttgaag gtgcatgtga ctccgtcaag attacgaaac cgccaactac cacgcaaatt 1200
gcaattctca atttcctaga aggactctcc gaaaatgcat ccaataccaa atattacccg 1260
tgtcataggc accaagtgac accatacatg aacacgcgtc acaatatgac tggagaaggg 1320
ttccacacct tatgctataa aacgccccac acccctcctc cttccttcgc agttcaattc 1380
caatatattc cattctctct gtgtatttcc ctacctctcc cttcaaggtt agtcgatttc 1440
ttctgttttt cttcttcgtt ctttccatga attgtgtatg ttctttgatc aatacgatgt 1500
tgatttgatt gtgttttgtt tggtttcatc gatcttcaat tttcataatc agattcagct 1560
tttattatct ttacaacaac gtccttaatt tgatgattct ttaatcgtag atttgctcta 1620
attagagctt tttcatgtca gatcccttta caacaagcct taattgttga ttcattaatc 1680
gtagattagg gcttttttca ttgattactt cagatccgtt aaacgtaacc atagatcagg 1740
gctttttcat gaattacttc agatccgtta aacaacagcc ttatttttta tacttctgtg 1800
gtttttcaag aaattgttca gatccgttga caaaaagcct tattcgttga ttctatatcg 1860
tttttcgaga gatattgctc agatctgtta gcaactgcct tgtttgttga ttctattgcc 1920
gtggattagg gttttttttc acgagattgc ttcagatccg tacttaagat tacgtaatgg 1980
attttgattc tgatttatct gtgattgttg actcgacag 2019
<210> 9
<211> 248
<212> DNA
<213> 未知(Unknown)
<400> 9
ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat 60
tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac 120
gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat 180
agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt 240
actagatc 248
<210> 10
<211> 191
<212> DNA
<213> 未知(Unknown)
<400> 10
ctcgagtttc tccataataa tgtgtgagta gttcccagat aagggaatta gggttcctat 60
agggtttcgc tcatgtgttg agcatataag aaacccttag tatgtatttg tatttgtaaa 120
atacttctat caataaaatt tctaattcct aaaaccaaaa tccagtacta aaatccagat 180
cccccgaatt a 191
<210> 11
<211> 14161
<212> DNA
<213> 未知(Unknown)
<400> 11
agcctgtcgc gtaacttagg acttgtgcga catgtcgttt tcagaagacg gctgcactga 60
acgtcagaag ccgactgcac tatagcagcg gaggggttgg atcaaagtac tttgatcccg 120
aggggaaccc tgtggttggc atgcacatac aaatggacga acggataaac cttttcacgc 180
ccttttaaat atccgttatt ctaataaacg ctcttttctc ttaggtttac ccgccaatat 240
atcctgtcaa acactgatag tttaaactga aggcgggaaa cgacaatctg atccaagctc 300
aagctgctct agcattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 360
gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 420
gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagcttttg 480
gaaaggtgcc gtgcagttca aacaattagt tagcagtagg gtgttggttt ttgctcacag 540
caataagaag ttaatcatgg tgtaggcaac ccaaataaaa caccaaaata tgcacaaggc 600
agtttgttgt attctgtagt acagacaaaa ctaaaagtaa tgaaagaaga tgtggtgtta 660
gaaaaggaaa caatatcatg agtaatgtgt gagcattatg ggaccacgaa ataaaaagaa 720
cattttgatg agtcgtgtat cctcgatgag cctcaaaagt tctctcaccc cggataagaa 780
acccttaagc aatgtgcaaa gtttgcattc tccactgaca taatgcaaaa taagatatca 840
tcgatgacat agcaactcat gcatcatatc atgcctctct caacctattc attcctactc 900
atctacataa gtatcttcag ctaaatgtta gaacataaac ccataagtca cgtttgatga 960
gtattaggcg tgacacatga caaatcacag actcaagcaa gataaagcaa aatgatgtgt 1020
acataaaact ccagagctat atgtcatatt gcaaaaagag gagagcttat aagacaaggc 1080
atgactcaca aaaattcatt tgcctttcgt gtcaaaaaga ggagggcttt acattatcca 1140
tgtcatattg caaaagaaag agagaaagaa caacacaatg ctgcgtcaat tatacatatc 1200
tgtatgtcca tcattattca tccacctttc gtgtaccaca cttcatatat catgagtcac 1260
ttcatgtctg gacattaaca aactctatct taacatttag atgcaagagc ctttatccca 1320
ctataaatgc acgatgattt ctcattgttt ctcacaaaaa gcattcagtt cattagtcct 1380
acaacaacat ggcatccata aatcgcccca tagttttctt cacagtttgc ttgttcctct 1440
tgtgcgatgg ctccctagcc ggcgccttca ccgagaagca ggaggccctc gtgtcctcct 1500
ccttcgaggc cttcaaggcc aacatcccgc agtactccgt ggtgttctac acctccatcc 1560
tggagaaggc cccggccgcc aaggacctct tctccttcct ctccaacggc gtggacccgt 1620
ccaacccgaa gctcaccggc cacgccgaga agctcttcgg cctcgtgcgc gactccgccg 1680
gccagctcaa ggccaacggc accgtggtgg ccgacgccgc cctcggctcc atccacgccc 1740
agaaggccat caccgacccg cagttcgtgg tggtgaagga ggccctcctc aagaccatca 1800
aggaggccgt gggcgacaag tggtccgacg agctgtcctc cgcctgggag gtggcctacg 1860
acgagctggc cgccgccatc aagaaggcct tctaatagag ctctagatct gttctgcaca 1920
aagtggagta gtcagtcatc gatcaggaac cagacaccag acttttattc atacagtgaa 1980
gtgaagtgaa gtgcagtgca gtgagttgct ggtttttgta caacttagta tgtatttgta 2040
tttgtaaaat acttctatca ataaaatttc taattcctaa aaccaaaatc caggggtacc 2100
cgattaaaaa tcccaattat atttggtcta atttagtttg gtattgagta aaacaaattc 2160
gaaccaaacc aaaatataaa tatatagttt ttatatatat gcctttaaga ctttttatag 2220
aattttcttt aaaaaatatc tagaaatatt tgcgactctt ctggcatgta atatttcgtt 2280
aaatatgaag tgctccattt ttattaactt taaataattg gttgtacgat cactttctta 2340
tcaagtgtta ctaaaatgcg tcaatctctt tgttcttcca tattcatatg tcaaaatcta 2400
tcaaaattct tatatatctt tttcgaattt gaagtgaaat ttcgataatt taaaattaaa 2460
tagaacatat cattatttag gtatcatatt gatttttata cttaattact aaatttggtt 2520
aactttgaaa gtgtacatca acgaaaaatt agtcaaacga ctaaaataaa taaatatcat 2580
gtgttattaa gaaaattctc ctataagaat attttaatag atcatatgtt tgtaaaaaaa 2640
attaattttt actaacacat atatttactt atcaaaaatt tgacaaagta agattaaaat 2700
aatattcatc taacaaaaaa aaaaccagaa aatgctgaaa acccggcaaa accgaaccaa 2760
tccaaaccga tatagttggt ttggtttgat tttgatataa accgaaccaa ctcggtccat 2820
ttgcacccct aatcataata gctttaatat ttcaagatat tattaagtta acgttgtcaa 2880
tatcctggaa attttgcaaa atgaatcaag cctatatggc tgtaatatga atttaaaagc 2940
agctcgatgt ggtggtaata tgtaatttac ttgattctaa aaaaatatcc caagtattaa 3000
taatttctgc taggaagaag gttagctacg atttacagca aagccagaat acaaagaacc 3060
ataaagtgat tgaagctcga aatatacgaa ggaacaaata tttttaaaaa aatacgcaat 3120
gacttggaac aaaagaaagt gatatatttt ttgttcttaa acaagcatcc cctctaaaga 3180
atggcagttt tcctttgcat gtaactatta tgctcccttc gttacaaaaa ttttggacta 3240
ctattgggaa cttcttctga aaatagtgga gctcgaattc gtaatcatgt catagctgtt 3300
tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 3360
gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 3420
gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 3480
ggggagaggc ggtttgcgta ttggctagag cagcttgcca acatggtgga gcacgacact 3540
ctcgtctact ccaagaatat caaagataca gtctcagaag accaaagggc tattgagact 3600
tttcaacaaa gggtaatatc gggaaacctc ctcggattcc attgcccagc tatctgtcac 3660
ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca aatgccatca ttgcgataaa 3720
ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc 3780
acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga 3840
tgtgataaca tggtggagca cgacactctc gtctactcca agaatatcaa agatacagtc 3900
tcagaagacc aaagggctat tgagactttt caacaaaggg taatatcggg aaacctcctc 3960
ggattccatt gcccagctat ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc 4020
acctacaaat gccatcattg cgataaagga aaggctatcg ttcaagatgc ctctgccgac 4080
agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga agacgttcca 4140
accacgtctt caaagcaagt ggattgatgt gatatctcca ctgacgtaag ggatgacgca 4200
caatcccact atccttcgca agaccttcct ctatataagg aagttcattt catttggaga 4260
ggacacgctg aaatcaccag tctctctcta caaatctatc tctctcgacg catgcctaca 4320
gtgcagcgtg acccggtcgt gcccctctct agagataatg agcattgcat gtctaagtta 4380
taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt 4440
atacatatat ttaaacttta ctctacgaat aatataatct atagtactac aataatatca 4500
gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt 4560
ttgacaacag gactctacag ttttatcttt ttagtgtgca tgtgttctcc tttttttttg 4620
caaatagctt cacctatata atacttcatc cattttatta gtacatccat ttagggttta 4680
gggttaatgg tttttataga ctaatttttt tagtacatct attttattct attttagcct 4740
ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaataattt agatataaaa 4800
tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta 4860
aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt 4920
ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca 4980
cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg 5040
ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag 5100
gcggcctcct cctcctctca cggcacggca gctacggggg attcctttcc caccgctcct 5160
tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttcccc 5220
aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa tccacccgtc 5280
ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc taccttctct 5340
agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc atgtttgtgt 5400
tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg cgacctgtac 5460
gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc ctgggatggc 5520
tctagccgtt ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt gcatagggtt 5580
tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt 5640
tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc gttctagatc 5700
ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc tgtatgtgtg 5760
tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg atctaggata 5820
ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt tgttcgcttg 5880
gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg agtagaatac 5940
tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg tcatacatct 6000
tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat acatgttgat 6060
gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 6120
cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 6180
acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 6240
ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 6300
tctgcaggtc gactctagag gatctaccat ggccaccgcc gccgccgcgt ctaccgcgct 6360
cactggcgcc actaccgctg cgcccaaggc gaggcgccgg gcgcacctcc tggccacccg 6420
ccgcgccctc gccgcgccca tcaggtgctc agcggcgtca cccgccatgc cgatggctcc 6480
cccggccacc ccgctccggc cgtggggccc caccgatccc cgcaagggat ccgacgccct 6540
gcccgccacc ttcgacgtga tcgtgcatcc agctcgcgaa ctccgcggcg agcttcgcgc 6600
tcagccatcc aagaactaca ccactcgcta cctcctcgcc gctgccctcg ctgagggcga 6660
gacccgcgtg gtgggcgtgg ctacctctga ggacgccgag gccatgctcc gctgcctccg 6720
cgactggggc gctggcgtgg agcttgtggg cgatgacgcc gtgatccgcg gtttcggcgc 6780
tcgcccacag gccggtgtga ccctcaaccc aggcaacgct ggcgcagtgg cccgcttcct 6840
catgggcgtg gccgctctca cctctggcac cactttcgtg accgactacc cggactccct 6900
cggcaagcgc cctcagggcg acctccttga ggccctcgaa cgcctcggtg cctgggtgtc 6960
ctccaacgac ggtcgcctcc cgatctccgt gtccggccca gtgcgcggtg gcaccgtgga 7020
ggtgtccgcc gagcgctcct cccagtacgc ctccgccctc atgttcctcg gccctctcct 7080
cccggacgga ctcgaactcc gcctcaccgg cgacatcaag tcccacgctc cgctccgcca 7140
gacactcgac accctctctg acttcggcgt gcgcgccact gcctccgacg acctccgccg 7200
catctccatc ccgggtggcc agaagtaccg cccaggccgc gtgctcgtgc cgggcgacta 7260
cccgggctcc gctgccatcc tcaccgccgc tgccctcctc ccaggcgagg tgcgcctctc 7320
taacctccgc gagcacgacc tccagggcga gaaggaggcc gtgaacgtgc tccgcgagat 7380
gggcgctgac atcgtgcgcg agggcgatac cctcaccgtg cgcggtggcc gccctctcca 7440
cgccgtgact cgcgacggcg attccttcac cgacgccgtg caagccctca ccgccgctgc 7500
tgccttcgcc gagggcgaca ccacctggga gaacgtggcc actctccgcc tcaaggagtg 7560
cgaccgcatc tctgacaccc gcgctgagct tgagcgcctc ggcctccgcg cacgcgagac 7620
cgccgactct ctctccgtga ctggctctgc tcacctcgct ggtggcatca ccgccgacgg 7680
ccacggcgac caccgcatga tcatgctcct caccctcctc ggcctccgcg cagacgctcc 7740
actccgcatc accggcgcac accacatccg caagtcctac cctcagttct tcgctcacct 7800
cgaagccctc ggcgctcgct tcgagtacgc tgaggccacc gcctaatagg tcgagtttct 7860
ccataataat gtgtgagtag ttcccagata agggaattag ggttcctata gggtttcgct 7920
catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc 7980
aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc ccccgaatta 8040
attcggcgtt aattcagtac attaaaaacg tccgcaatgt gttattaagt tgtctaagcg 8100
tcaatttgtt tacaccacaa tatatcctgc caccagccag ccaacagctc cccgaccggc 8160
agctcggcac aaaatcacca ctcgatacag gcagcccatc agtccgggac ggcgtcagcg 8220
ggagagccgt tgtaaggcgg cagactttgc tcatgttacc gatgctattc ggaagaacgg 8280
caactaagct gccgggtttg aaacacggat gatctcgcgg agggtagcat gttgattgta 8340
acgatgacag agcgttgctg cctgtgatca ccgcggtttc aaaatcggct ccgtcgatac 8400
tatgttatac gccaactttg aaaacaactt tgaaaaagct gttttctggt atttaaggtt 8460
ttagaatgca aggaacagtg aattggagtt cgtcttgtta taattagctt cttggggtat 8520
ctttaaatac tgtagaaaag aggaaggaaa taataaatgg ctaaaatgag aatatcaccg 8580
gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag atacggaagg aatgtctcct 8640
gctaaggtat ataagctggt gggagaaaat gaaaacctat atttaaaaat gacggacagc 8700
cggtataaag ggaccaccta tgatgtggaa cgggaaaagg acatgatgct atggctggaa 8760
ggaaagctgc ctgttccaaa ggtcctgcac tttgaacggc atgatggctg gagcaatctg 8820
ctcatgagtg aggccgatgg cgtcctttgc tcggaagagt atgaagatga acaaagccct 8880
gaaaagatta tcgagctgta tgcggagtgc atcaggctct ttcactccat cgacatatcg 8940
gattgtccct atacgaatag cttagacagc cgcttagccg aattggatta cttactgaat 9000
aacgatctgg ccgatgtgga ttgcgaaaac tgggaagaag acactccatt taaagatccg 9060
cgcgagctgt atgatttttt aaagacggaa aagcccgaag aggaacttgt cttttcccac 9120
ggcgacctgg gagacagcaa catctttgtg aaagatggca aagtaagtgg ctttattgat 9180
cttgggagaa gcggcagggc ggacaagtgg tatgacattg ccttctgcgt ccggtcgatc 9240
agggaggata tcggggaaga acagtatgtc gagctatttt ttgacttact ggggatcaag 9300
cctgattggg agaaaataaa atattatatt ttactggatg aattgtttta gtacctagaa 9360
tgcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 9420
aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 9480
aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 9540
ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 9600
tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 9660
ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 9720
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 9780
agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 9840
gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 9900
ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 9960
tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 10020
tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 10080
cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 10140
tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 10200
gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 10260
atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc 10320
cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg 10380
cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 10440
ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagggtgcct 10500
tgatgtgggc gccggcggtc gagtggcgac ggcgcggctt gtccgcgccc tggtagattg 10560
cctggccgta ggccagccat ttttgagcgg ccagcggccg cgataggccg acgcgaagcg 10620
gcggggcgta gggagcgcag cgaccgaagg gtaggcgctt tttgcagctc ttcggctgtg 10680
cgctggccag acagttatgc acaggccagg cgggttttaa gagttttaat aagttttaaa 10740
gagttttagg cggaaaaatc gccttttttc tcttttatat cagtcactta catgtgtgac 10800
cggttcccaa tgtacggctt tgggttccca atgtacgggt tccggttccc aatgtacggc 10860
tttgggttcc caatgtacgt gctatccaca ggaaagagac cttttcgacc tttttcccct 10920
gctagggcaa tttgccctag catctgctcc gtacattagg aaccggcgga tgcttcgccc 10980
tcgatcaggt tgcggtagcg catgactagg atcgggccag cctgccccgc ctcctccttc 11040
aaatcgtact ccggcaggtc atttgacccg atcagcttgc gcacggtgaa acagaacttc 11100
ttgaactctc cggcgctgcc actgcgttcg tagatcgtct tgaacaacca tctggcttct 11160
gccttgcctg cggcgcggcg tgccaggcgg tagagaaaac ggccgatgcc gggatcgatc 11220
aaaaagtaat cggggtgaac cgtcagcacg tccgggttct tgccttctgt gatctcgcgg 11280
tacatccaat cagctagctc gatctcgatg tactccggcc gcccggtttc gctctttacg 11340
atcttgtagc ggctaatcaa ggcttcaccc tcggataccg tcaccaggcg gccgttcttg 11400
gccttcttcg tacgctgcat ggcaacgtgc gtggtgttta accgaatgca ggtttctacc 11460
aggtcgtctt tctgctttcc gccatcggct cgccggcaga acttgagtac gtccgcaacg 11520
tgtggacgga acacgcggcc gggcttgtct cccttccctt cccggtatcg gttcatggat 11580
tcggttagat gggaaaccgc catcagtacc aggtcgtaat cccacacact ggccatgccg 11640
gccggccctg cggaaacctc tacgtgcccg tctggaagct cgtagcggat cacctcgcca 11700
gctcgtcggt cacgcttcga cagacggaaa acggccacgt ccatgatgct gcgactatcg 11760
cgggtgccca cgtcatagag catcggaacg aaaaaatctg gttgctcgtc gcccttgggc 11820
ggcttcctaa tcgacggcgc accggctgcc ggcggttgcc gggattcttt gcggattcga 11880
tcagcggccg cttgccacga ttcaccgggg cgtgcttctg cctcgatgcg ttgccgctgg 11940
gcggcctgcg cggccttcaa cttctccacc aggtcatcac ccagcgccgc gccgatttgt 12000
accgggccgg atggtttgcg accgtcacgc cgattcctcg ggcttggggg ttccagtgcc 12060
attgcagggc cggcagacaa cccagccgct tacgcctggc caaccgcccg ttcctccaca 12120
catggggcat tccacggcgt cggtgcctgg ttgttcttga ttttccatgc cgcctccttt 12180
agccgctaaa attcatctac tcatttattc atttgctcat ttactctggt agctgcgcga 12240
tgtattcaga tagcagctcg gtaatggtct tgccttggcg taccgcgtac atcttcagct 12300
tggtgtgatc ctccgccggc aactgaaagt tgacccgctt catggctggc gtgtctgcca 12360
ggctggccaa cgttgcagcc ttgctgctgc gtgcgctcgg acggccggca cttagcgtgt 12420
ttgtgctttt gctcattttc tctttacctc attaactcaa atgagttttg atttaatttc 12480
agcggccagc gcctggacct cgcgggcagc gtcgccctcg ggttctgatt caagaacggt 12540
tgtgccggcg gcggcagtgc ctgggtagct cacgcgctgc gtgatacggg actcaagaat 12600
gggcagctcg tacccggcca gcgcctcggc aacctcaccg ccgatgcgcg tgcctttgat 12660
cgcccgcgac acgacaaagg ccgcttgtag ccttccatcc gtgacctcaa tgcgctgctt 12720
aaccagctcc accaggtcgg cggtggccca tatgtcgtaa gggcttggct gcaccggaat 12780
cagcacgaag tcggctgcct tgatcgcgga cacagccaag tccgccgcct ggggcgctcc 12840
gtcgatcact acgaagtcgc gccggccgat ggccttcacg tcgcggtcaa tcgtcgggcg 12900
gtcgatgccg acaacggtta gcggttgatc ttcccgcacg gccgcccaat cgcgggcact 12960
gccctgggga tcggaatcga ctaacagaac atcggccccg gcgagttgca gggcgcgggc 13020
tagatgggtt gcgatggtcg tcttgcctga cccgcctttc tggttaagta cagcgataac 13080
cttcatgcgt tccccttgcg tatttgttta tttactcatc gcatcatata cgcagcgacc 13140
gcatgacgca agctgtttta ctcaaataca catcaccttt ttagacggcg gcgctcggtt 13200
tcttcagcgg ccaagctggc cggccaggcc gccagcttgg catcagacaa accggccagg 13260
atttcatgca gccgcacggt tgagacgtgc gcgggcggct cgaacacgta cccggccgcg 13320
atcatctccg cctcgatctc ttcggtaatg aaaaacggtt cgtcctggcc gtcctggtgc 13380
ggtttcatgc ttgttcctct tggcgttcat tctcggcggc cgccagggcg tcggcctcgg 13440
tcaatgcgtc ctcacggaag gcaccgcgcc gcctggcctc ggtgggcgtc acttcctcgc 13500
tgcgctcaag tgcgcggtac agggtcgagc gatgcacgcc aagcagtgca gccgcctctt 13560
tcacggtgcg gccttcctgg tcgatcagct cgcgggcgtg cgcgatctgt gccggggtga 13620
gggtagggcg ggggccaaac ttcacgcctc gggccttggc ggcctcgcgc ccgctccggg 13680
tgcggtcgat gattagggaa cgctcgaact cggcaatgcc ggcgaacacg gtcaacacca 13740
tgcggccggc cggcgtggtg gtgtcggccc acggctctgc caggctacgc aggcccgcgc 13800
cggcctcctg gatgcgctcg gcaatgtcca gtaggtcgcg ggtgctgcgg gccaggcggt 13860
ctagcctggt cactgtcaca acgtcgccag ggcgtaggtg gtcaagcatc ctggccagct 13920
ccgggcggtc gcgcctggtg ccggtgatct tctcggaaaa cagcttggtg cagccggccg 13980
cgtgcagttc ggcccgttgg ttggtcaagt cctggtcgtc ggtgctgacg cgggcatagc 14040
ccagcaggcc agcggcggcg ctcttgttca tggcgtaatg tctccggttc tagtcgcaag 14100
tattctactt tatgcgacta aaacacgcga caagaaaacg ccaggaaaag ggcagggcgg 14160
c 14161
Claims (4)
1.一种生产血红素结合蛋白的方法,其特征在于所述方法是将含血红素结合蛋白基因表达框的表达载体转入大豆基因组中并表达,获得富含血红素结合蛋白的大豆种子,分离提取,获得血红素结合蛋白;所述血红素结合蛋白包括血红蛋白或肌红蛋白;
所述血红素结合蛋白基因表达框组成为下列之一:(1)由pAtUbi10启动子、血红蛋白编码基因、终止子、烟草RB7核基质结合区调控序列MAR组成,核苷酸序列如SEQ ID NO:5中478-4248 bp;(2)由pAtUbi10启动子、肌红蛋白编码基因、终止子、烟草RB7核基质结合区调控序列MAR组成,核苷酸序列如SEQ ID NO:6中478-4275 bp所示。
2.如权利要求1所述的方法,其特征在于所述表达载体包含血红素结合蛋白基因表达框和筛选标记基因表达框;筛选标记基因表达框包括耐草甘膦基因表达框、耐啶嘧磺隆基因表达框或耐草铵膦基因表达框。
3.如权利要求2所述的方法,其特征在于所述耐草甘膦基因表达框中草甘膦抗性基因g10evo epsps的核苷酸序列如SEQ ID NO: 5中5661-6977 bp所示,启动子为花椰菜花叶病毒35s的启动子,终止子为花椰菜花叶病毒35s终止子。
4.如权利要求1所述的方法,其特征在于所述表达载体的T-DNA元件组成为下列之一:(1)由pAtUbi10启动子、血红蛋白编码基因、终止子、烟草RB7核基质结合区调控序列MAR、花椰菜花叶病毒35s启动子、抗草甘膦基因g10evo epsps、花椰菜花叶病毒35s终止子组成,核苷酸序列如SEQ ID NO:5中205-7266 bp;(2)由pAtUbi10启动子、肌红蛋白编码基因、终止子、烟草RB7核基质结合区调控序列MAR、花椰菜花叶病毒35s启动子、抗草甘膦基因g10evo epsps、花椰菜花叶病毒35s终止子组成,核苷酸序列如SEQ ID NO:6中205-7293 bp所示。
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