本发明涉及发酵领域,具体涉及一种过表达aro9或aro10的酿酒酵母基因工程菌株及其构建方法和应用
背景技术:
生物膜,又称生物被膜,是自然界当中微生物之间相互合作自发形成的多细胞群体,微生物细胞分泌出来的胞外聚合物(eps)组成的聚合体称为生物膜,生物膜在微生物的生命活动中起着非常重要的作用,为其提供一个稳定的内环境,具有抵抗外界恶劣环境的作用,它可以提高反应底物以及产物的耐受性,具有提高发酵产量等工业化优点,对于工业化应用有一定的发展前景。同时,它还承担着细胞间以及细胞与介质间的运输、信息传递等功能,因此提高生物膜稳定性对于微生物的生长具有重要的意义。
从以前的相关研究可以看出群体感应对于酿酒酵母细胞的成膜有一定的影响,但是相关报道少之又少。因此,本发明提供了一种酿酒酵母基因工程菌株及其构建方法和应用。
技术实现要素:
发明目的:本发明所要解决的技术问题是针对现有技术的不足,提供一种酿酒酵母基因工程菌株。
本发明还要解决的技术问题是提供上述酿酒酵母基因工程菌株的构建方法。
本发明进一步要解决的技术问题是提供上述酿酒酵母基因工程菌株的应用。
发明思路:本发明以原始酿酒酵母为出发菌株,研究群体感应的合成关键基因aro9、aro10对于酿酒酵母生物膜形成的影响。首先,分别构建过表达aro9基因和过表达aro10基因的酿酒酵母基因工程菌株,将酿酒酵母基因工程菌株进行结晶紫染色、细胞吸附等实验观察生物膜形成差异,并进行连续化固定发酵观察糖耗和乙醇产量的影响。
为了解决上述第一个技术问题,本发明公开了一种酿酒酵母基因工程菌株,其在原始酿酒酵母中过表达aro9基因或过表达aro10基因。
其中,所述原始酿酒酵母为saccharomycescerevisiaes288c。
其中,所述重组质粒pyx212-aro9(过表达aro9基因后)的核苷酸序列如seqidno:1所示,过表达前aro9基因的核苷酸序列如seqidno:3所示。
其中,所述重组质粒pyx212-aro10(过表达aro10基因后)的核苷酸序列如seqidno:2所示,过表达前aro10基因的核苷酸序列如seqidno:4所示。
为了解决上述第二个技术问题,本发明公开了上述酿酒酵母基因工程菌株的构建方法,在原始酿酒酵母导入携带aro9的重组质粒,或携带aro10的重组质粒。
优选地,所述酿酒酵母基因工程菌株的构建方法为在原始酿酒酵母导入携带aro9的pyx212重组质粒,或携带aro10的pyx212重组质粒。
具体地,所述酿酒酵母基因工程菌株的构建方法包括如下步骤:
(1)以原始酿酒酵母的基因组为模板,扩增aro9基因或aro10基因;
(2)将步骤(1)得到的基因插入pyx212质粒,得到携带aro9基因的pyx212重组质粒或携带aro10基因的pyx212重组质粒;
(3)将步骤(2)所得重组质粒转化到e.colidh5α的感受态细胞中,从e.colidh5α中提取重组质粒;
(4)将步骤(3)提取所得重组质粒转化到原始酿酒酵母的感受态细胞中,即得到酿酒酵母工程菌。
步骤(1)中,所述扩增为采用pcr扩增。
步骤(1)中,所述扩增aro9基因的引物如下:
ttccttattcagttagctagctgagctcatgactgctggttctgcccccc(seqidno:5)aro9-f;
gcctgactacgcatgatatctcgagctctcaacttttatagttgtcaaaa(seqidno:6)aro9-r。
步骤(1)中,所述扩增aro10基因的引物如下:
cagttagctagctgagctcatggcacctgttacaattgaa(seqidno:7)aro10-f;
acgcatgatatctcgagctcctattttttatttcttttaa(seqidno:8)aro10-r。
步骤(2)中,用nhe1(aro10)和saci(aro9)限制性内切酶酶切质粒,将步骤(1)得到的基因插入pyx212质粒的酶切位点之间。
为了解决上述第三个技术问题,本发明公开了上述酿酒酵母基因工程菌株在发酵产乙醇中的应用。
其中,上述酿酒酵母基因工程菌株在发酵产乙醇的过程中增加生物被膜产量。
其中,发酵培养基为:60g/l葡萄糖、4g/l蛋白胨、3g/l酵母膏、3g/l磷酸二氢钾、0.5g/l硫酸镁、4g/l硫酸铵、0.05g/l七水合硫酸锌、0.05g/l七水合硫酸亚铁。
其中,所述发酵为固定化发酵。
其中,所述发酵为固定化连续发酵。
其中,固定化载体为含棉纤维的材料。
其中,固定化载体的用量为20-60g/l发酵液;优选地,固定化载体的用量为40g/l发酵液。
有益效果:与现有技术相比,本发明具有如下优势:
(1)本发明通过过表达aro9或aro10基因,所得酿酒酵母基因工程菌株能提高生物膜的形成量,过表达aro9的酿酒酵母基因工程菌株和过表达aro10的酿酒酵母基因工程菌株的生物膜形成分别提高了18%和10%。
(2)本发明通过过表达aro9或aro10基因,所得酿酒酵母基因工程菌株,通过固定化连续发酵,以棉纤维为载体,经过6批发酵周期,乙醇产量均提高了将近15%,且发酵周期缩短了6h。
附图说明
下面结合附图和具体实施方式对本发明做更进一步的具体说明,本发明的上述和/或其他方面的优点将会变得更加清楚。
图1为aro9和aro10pcr扩增片段。
图2为pyx212-aro9/10酶切验证。
图3为aro9、aro10过表达菌株菌落pcr验证。
图4为原始菌和重组菌的生物膜结晶紫染色实验。
图5为细胞吸附实验。
图6为原始菌和重组菌葡萄糖消耗速率。
图7为原始菌和重组菌的乙醇产量。
具体实施方式
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例1:过表达aro9、aro10基因,获得重组菌
一、提取原始酿酒酵母菌株saccharomycescerevisiaes288c的基因组
使用takara公司的真菌基因组提取试剂盒(dr.gentletm(fromyeast)highrecovery)
(1)取od1.2-1.8的原始酿酒酵母saccharomycescerevisiaes288c过夜培养液至2ml微型管中,12000rpm,离心1min,弃上清液;
(2)向步骤(1)所得沉淀中加入500μl的gentleyeastsolutiona轻松震荡并充分悬浮沉淀于37℃,温浴1h,期间轻轻震荡离心管数次;
(2)加入100μl的gentleyeastsolutionb,轻微震荡,混匀后于70℃水浴10min;
(3)加入200μl的gentleyeastsolutionc,轻微震荡,混匀后于冰上静置5min;
(4)将微型管于4℃下,转速12000rpm离心5min;
(5)将上清液移入新的微型管中,加入400μl的异丙醇,充分颠倒混匀;
(6)将微型管于4℃下,转速12000rpm离心5min,弃上清液;
(7)向沉淀中加入500μl预冷的70%乙醇,上下颠倒洗涤沉淀,于4℃下,转速12000rpm离心5min;
(8)弃上清液,室温下自然干燥dna,沉淀至无乙醇气味;
(9)加入50μl的tebuffer/ddh2o,溶解基因组dna。
二、运用pcr技术分别扩增目的基因aro9、aro10
(1)以步骤一提取的酿酒酵母基因组为模板,采用pcr技术扩增aro9和aro10基因。
反应体系如下表:
表1pcr反应体系
扩增aro9基因的引物如下:
ttccttattcagttagctagctgagctcatgactgctggttctgcccccc(seqidno:5)aro9-f;
gcctgactacgcatgatatctcgagctctcaacttttatagttgtcaaaa(seqidno:6)aro9-r。
扩增aro10基因的引物如下:
cagttagctagctgagctcatggcacctgttacaattgaa(seqidno:7)aro10-f;
acgcatgatatctcgagctcctattttttatttcttttaa(seqidno:8)aro10-r。
使用1.2%(1.2g/100ml)琼脂糖凝胶对上述pcr产物(aro9和aro10基因片段)进行电泳,结果见图1。
(2)胶回收纯化片段aro9和aro10
使用takara试剂盒(takaraminibestagarosegeldnaextractionkitver.4.0)
2.1使用tbe缓冲液制作琼脂糖凝胶,然后对目的片段aro9和aro10进行琼脂糖凝胶电泳;
2.2在紫外灯下切出含有目的片段aro9和aro10的琼脂糖凝胶,用纸巾吸尽表面液体;
2.3切碎胶块,称量胶块重。计算体积时,以1mg=1μl进行计算;
2.4向胶块中加入溶解液buffergm,buffergm加量没过胶块即可,均匀混合后室温15-25℃溶解胶块,此时应间断振荡混合,使胶块充分溶解;
2.5将试剂盒中的过滤柱安置于2ml收集管上;
2.6将步骤2.4得到的溶液转移至过滤柱中,12000rpm离心1min,弃滤液;
2.7将700μl的bufferwb加入过滤柱中,室温12000rpm离心1min,弃滤液;
2.8重复操作步骤2.7;
2.9将过滤柱安置于2ml收集管上,室温12000rpm离心1min,弃滤液;
2.10将过滤柱安置于新的1.5ml的离心管上,在过滤柱膜的中央处加入60μl灭菌水,室温静置1min;
2.11室温12000rpm离心1min洗脱aro9和aro10片段;
2.12对纯化后的aro9和aro10目的片段进行琼脂糖凝胶电泳验证,计算浓度;其中aro9和aro10基因的核苷酸序列分别如seqidno:3和seqidno:4所示。
三、提取质粒pyx212
利用axygen的提质粒试剂盒(axypreptmplasmidminiprepkit)
(1)从平板培养基上挑选e.colidh5α单菌落(内含pyx212,其核苷酸序列见seqidno:9),其中e.colidh5α购自上海生工生物工程(上海)股份有限公司,no.b528413,pet-28a的保存方法参考按照《分子克隆实验指南》(黄培堂等译,中国,科学出版社,2002,第三版)中方法进行;然后接种至5ml的含有抗生素的lb液体培养基中(抗性为卡那霉素50μg/ml),37℃过夜培养12-16h;
(2)取2ml的过夜培养菌液,12000rpm离心1min,弃上清;
(3)用250μl的4℃预冷的buffers1(含rnasea)充分悬浮细菌沉淀;
(4)加入250μl的buffers2轻轻上下翻转混合4-6次,使菌体充分裂解,形成透明溶液;
(5)加入350μl的buffers3轻轻上下翻转混合6-8次,直至形成紧实凝集块;
(6)室温12000rpm离心10min,取上清液;
(7)将试剂盒中的过滤柱安置于2ml收集管上;
(8)将步骤(6)得到的上清液转移至过滤柱中,12000rpm离心1min,弃滤液;
(9)将500μl的bufferw1加入过滤柱中,12000rpm离心1min,弃滤液;
(10)将700μl的bufferw2加入过滤柱中,12000rpm离心1min,弃滤液;
(11)重复操作步骤(10);
(12)重新将过滤柱安置于2ml收集管上,12000rpm离心1min,除尽残留洗液;
(13)将过滤柱安置于新的1.5ml的离心管上,在过滤柱膜的中央处加入30-50μl的65℃灭菌水,室温静置1min;
(14)12000rpm离心1min洗脱dna;
(15)对提取后的质粒pyx212进行琼脂糖凝胶电泳验证,计算浓度。
四、利用限制性内切酶nhei(aro10)或者saci(aro9)酶切质粒pyx212
表2aro9酶切反应体系
表3aro10酶切反应体系
37℃酶切30min,跑胶验证(如图2),确认片段正确后进行胶回收(步骤如二)
五、分别连接目的基因aro9和aro10和酶切后质粒pyx212得到重组质粒pyx212-aro9、pyx212-aro10。
表4aro9一步克隆体系
表5aro10一步克隆体系
37℃水浴30min后,立即冰浴五min,-20℃保存备用。
六、将重组质粒pyx212-aro9、pyx212-aro10转化到e.colidh5α
(1)从-80℃冰箱中取出一管200μle.colidh5α感受态细胞(上海生工生物工程(上海)股份有限公司no.b528413)悬液,室温下解冻,解冻后立即置于冰上;
(2)加入步骤五得到的重组质粒pyx212-aro9、pyx212-aro10溶液于e.colidh5α感受态细胞悬液中,轻轻摇匀,冰上放置30min;
(3)42℃水浴热激45-90s,立即置于冰上冷却5min;
(4)加入1ml的lb液体培养基混匀后,37℃振荡培养40min,使菌体恢复正常生长状态;
(5)将上述菌液摇匀后取100μl涂布于lb抗性板,倒置培养皿,37℃恒温培养箱培养12h;其中lb抗性板的配方如下:氯化钠10g/l、胰蛋白胨10g/l、酵母粉5g/l、琼脂粉20g/l,121℃灭菌20min,冷却后加卡那霉素至终浓度为50μg/ml;
(6)从抗性板上挑取单菌落,保菌后,按步骤三提取质粒pyx212-aro9、pyx212-aro10,对提取后的质粒pyx212-aro9、pyx212-aro10进行琼脂糖凝胶电泳验证和测序验证,其核苷酸序列分别如seqidno:1和seqidno:2所示。
七、将重组质粒pyx212-aro9、pyx212-aro10电转化到原始酿酒酵母中
(1)将10μl的pyx212-aro9、pyx212-aro10重组质粒与酿酒酵母感受态混匀,于冰上预冷30min;
(2)将混合液转移至2mm电转杯中,冰浴5min;
(3)擦干电转杯外部水分,放入电转仪,进行电转(电压1500v,电容25μf,电阻200ω);
(4)向电转杯中加入1mlypd培养基,混匀后转移到1.5ml离心管中,30℃,200rpm,复苏1h;
(5)离心。弃600上清液,用剩下的400重悬沉淀,取200菌液涂布在g418抗性的ypd平板上,30℃培养至长出菌落;
(6)菌落pcr验证是否为pyx212-aro9、pyx212-aro10重组酿酒酵母菌株。结果正确,见图3。
根据凝胶电泳的结果表明:得到了3株aro9过表达菌株、1株aro10过表达菌株。保藏菌株后用于后续实验。
实施例2:重组菌96孔板结晶紫染色实验
(1)各取100μl甘油管里的原始菌、两种重组菌分别加入到已经灭过菌的5mlypd培养基中进行24h活化培养;
(2)将od600=1的原始菌、两种重组菌以10%的体积比接入新鲜ypd液体培养基中,30℃摇床慢摇5h,od值到达0.8-1.0左右停止培养;
(3)取200μl的步骤(2)所得菌液加入96孔聚苯乙烯微孔板中,于30℃培养箱培养12-36h;
(4)将步骤(3)所得培养液全部吸出,每孔加入200μl的无菌pbs缓冲液清洗板孔3次,轻轻拍干;
(5)每孔加入200μl1%的结晶紫溶液,室温下染色5min,之后用自来水缓缓冲洗,酿干;
(6)酿干后,每孔加入200μl33%的冰乙酸溶液,在37℃培养箱作用30min溶解结晶紫;
(7)用酶标仪测定培养孔中的溶液od值,取平均值。
实验结果如图4所示,在生物膜结晶紫染色实验中,aro9对生物膜形成能力有最明显的影响,过表达菌株生物膜形成提高18%;aro10基因过表达菌株生物膜提高10%。
实施例3:细胞吸附实验
(1)分别挑取原始菌株saccharomycescerevisiaes288c和重组菌株pyx212-aro9、pyx212-aro10单菌落至5ml/50mlypd中,在30℃、200rpm摇床中培养12h,得到培养菌液;
(2)将步骤(1)所得培养菌液用无菌水分别稀释至od600=1,得到细胞;
(3)在6孔板中加入步骤(2)所得细胞,爬片,随后先后加入200μl步骤(1)所得菌液和3mlypd发酵液,密封后至30℃培养箱培养72h;
其中ypd发酵培养基为:60g/l葡萄糖、4g/l蛋白胨、3g/l酵母膏、3g/l磷酸二氢钾、0.5g/l硫酸镁、4g/l硫酸铵、0.05g/l七水合硫酸锌、0.05g/l七水合硫酸亚铁;
(4)培养结束后用移液枪吸取发酵液,然后加入3mlpbs缓冲液清洗细胞爬片两次;
(5)然后加入1ml在-20℃预冷的细胞固定液2%戊二醛水溶液,固定十分钟后,舍弃固定液;
(6)再次用3mlpbs缓冲液清洗细胞爬片两次,然后将细胞爬片放置于显微镜下观察。
如图5所示,原始菌株在细胞爬片上分布较散且稀疏,而aro9、aro10过表达菌株在细胞爬片上分布更加密集,可以清楚的观察到细胞间更加黏附在一起,形成密集的细胞聚集体。因此可以证明aro9、aro10对于生物膜的生长有重要作用。
实施例4:生物膜固定化连续发酵比较糖耗和乙醇产量
(1)将原始菌株与重组菌pyx212-aro9、pyx212-aro10分别加入到已经灭过菌的5mlypd培养基中进行24h活化培养;
(2)各取大概2.5ml培养液加入含有棉纤维固定化材料(毛巾)(40g/l发酵液)的ypd发酵培养基中,置于35℃、200rpm的摇床中培养至耗糖为0g/l时停止发酵;
每隔3h取样,使用测糖仪计算出葡萄糖浓度;同时,乙醇产量的测定采用气相色谱仪,使用30m毛细管柱和火焰离子检测器(fid),使用前柱箱要进行升温;
其中ypd发酵培养基为:60g/l葡萄糖、4g/l蛋白胨、3g/l酵母膏、3g/l磷酸二氢钾、0.5g/l硫酸镁、4g/l硫酸铵、0.05g/l七水合硫酸锌、0.05g/l七水合硫酸亚铁;
(3)本实验一共分为6批连续发酵,一共耗时66h,每隔3h取一次样,糖耗为0g/l的时候一批次的发酵结束。每一批次发酵结束后,便以20%的接种量将培养液重新接种到新的培养基中培养。以此类推,直到第6批发酵结束。
固定化发酵实验结果如下图所示:由图6可以得到第一批固定化发酵过程中,在第6h的时候,过表达aro9、aro10菌株的耗糖相比原始菌株变快,在后面几批的固定化发酵实验当中,原始菌和重组菌的耗糖速率都明显提高,并且从第三批发酵开始,原始菌和重组菌的耗糖速率都趋于稳定。这说明固定化发酵能够明显提高发酵周期,第一批发酵周期是15h,到第二批的发酵周期是12h,之后的发酵周期缩短到9h。另外,由图7可以得到,过表达aro9、aro10菌株的乙醇产量都略高于原始菌株,且从第四批发酵开始,重组菌株pyx212-aro9的乙醇产量高于原始菌株3.5g/l,重组菌株pyx212-aro10的乙醇产量高于原始菌株3.8g/l,两者的乙醇产量均提高了大约15%。
本发明提供了一种酿酒酵母基因工程菌株及其构建方法和应用的思路及方法,具体实现该技术方案的方法和途径很多,以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
序列表
<110>南京工业大学
<120>一种酿酒酵母基因工程菌株及其构建方法和应用
<160>9
<170>siposequencelisting1.0
<210>1
<211>11538
<212>dna
<213>过表达aro9基因(pyx212-aro9)
<400>1
ctgtatgtgttttttgtagttatagatttaagcaagaaaagaatacaaacaaaaaattga60
aaaagattgatttagaattaaaaagaaaaatatttacgtaagaagggaaaatagtaaatg120
ttgcaagttcactaaactcctaaattatgctgccctttatattccctgttacagcagccg180
agccaaaggtatataggctcctttgcattagcatgcgtaacaaaccacctgtcagtttca240
accgaggtggtatccgagagaattgtgtgattgctttaattaatttcggagaatctcaca300
tgccactgaagattaaaaactggatgccagaaaaggggtgtccgagtgtaacatcaatag360
aggaagctgaaaagtcttagaacgggtaatcttccaccaacctgatgggttcctagatat420
aatctcgaagggaataagtagggtgataccgtcagaagtgtctgaatgtattgaggtcct480
cacagtttaaatcccgctcacactaacgtaggattattataactcaaaaaaatggcatta540
ttctaagtaagttaaatatccgtaatctttaaacactatgtagttaggtctccctcacaa600
tcagtccatttgggtagcacggtcctcaggacgtatctattgatggattcgtccagttcc660
atcaccattacgctcccgttaggaacattggtaaacgattcaaactcttcgtatgtccat720
ctaaaccatttcatcaggaatactctggaataaataccatgtgtaactaggacaacaaca780
tctctgggtcttctctcttgcctatcatggaagtgcctgaataaagtctcttggaaactg840
gcgactctgtcatatacatctgccgcacttctccatgagggaatctgaagaagaatgacc900
atacgtagatccccaattcttgaagacgaaagggcctcgtgatacgcctatttttatagg960
ttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgc1020
gcggaacccctatttgtttatttttctaaatacattcaaatatgtatcgctcatgagaca1080
ataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacattt1140
ccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccaga1200
aacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcga1260
actggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaat1320
gatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggca1380
agagcaactcggtcgccgcatacactattctcagaatgacttggttgagtacaccagtca1440
cagaaaagcatcttacggatggcatgacagtaagagaattatgcagttcctgccataacc1500
atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaagagctaa1560
ccgctttttttcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagc1620
tgaatgaagccataccaaacgacgagcgtgacaccacgatgccatcaagctcgacggccg1680
gcccggtaccataacttcgtatagcatacattatacgaagttatttgaagtcggacagtg1740
agtgtagtcttgagaaattctgaagccgtatttttattatcagtgagtcagtcatcagga1800
gatcctctacgccggacgcatcgtggccgacctgcagggggggggggggcgctgaggtct1860
gcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagcca1920
gaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgatttt1980
gaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatcctt2040
caactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatg2100
ctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaa2160
tgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttc2220
tgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcgg2280
tctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaata2340
aggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagc2400
ttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatca2460
ctcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcga2520
tcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgcc2580
agcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgtt2640
ttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttg2700
atggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaaca2760
tcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttccca2820
tacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttataccca2880
tataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttga2940
atatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcat3000
gatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctt3060
tcccccccccccctgcaggtcggcatcaccggcgccacaggtgcggttgctggcgcctat3120
atcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgt3180
ttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttg3240
catgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggcataact3300
tcgtatagcatacattatacgaagttatggtacccaattcgccctatagtgagtcgtatt3360
tcgagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcaca3420
attccacacaacataggagccggaagcataaagtgtaaagcctggggtgcctaatgagtg3480
aggtaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcg3540
tgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgc3600
tcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgaccggta3660
tcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaag3720
aacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcg3780
tttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagagg3840
tggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg3900
cgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggga3960
agcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgc4020
tccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggt4080
aactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccact4140
ggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg4200
cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtt4260
accttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggt4320
ggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcct4380
ttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttg4440
gtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtttt4500
aaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagt4560
gaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtc4620
gtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccg4680
cgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggcc4740
gagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgg4800
gaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctaca4860
ggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacga4920
tcaaggcgagttacatgatcccccatgttgtccaaaaaagcggttagctccttcggtcct4980
ccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactg5040
cataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactca5100
accaagtcattctgagaatagtgtatgcggcgagcgagttgctcttgcccggcgtcaata5160
cgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttct5220
tcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccact5280
cgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaa5340
acaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactc5400
atactcttcctttttcaatattattgaagcatttatcacggttattgtctcatgagcgga5460
tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccga5520
aaagtgccacctgacgtactagtgagagtgcggccgcagctttgaagaaaaatgcgcctt5580
attcaatctttgctataaaaaatggcccaaaatctcacattggaagacatttgatgacct5640
catttctttcaatgaagggcctaacggagttgactaatgttgtgggaaattggagcgata5700
agggtgcttctgccgtggccaggacaacgtatactcatcagataacagcaatacctgatc5760
actacttcgcactagtttctcggtactatgcatatgatccaatatcaaaggaaatgatag5820
cattgaaggatgagactaatccaattgaggagtggcagcatatagaacagctaaagggta5880
gtgctgaaggaagcatacgataccccgcatggaatgggataatatcacaggaggtactag5940
actacctttcatcctacataaatagacgcatataagtacgcatttaagcataaacacgca6000
ctatgccgttcttctcatgtatatatatatacaggcaacacgcagatataggtgcgacgt6060
gaacagtgagctgtatgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaa6120
acgctttgaagttcctattccgaagttcctattctctagaaagtataggaacttcagagc6180
gcttttgaaaaccaaaagcgctctgaagacgcactttcaaaaaaccaaaaacgcaccgga6240
ctgtaacgagctactaaaatattgcgaataccgcttccacaaacattgctgaaaagtatc6300
tctttgctatatatctctgtgctatatccctatataacctacccatccacctttcgctcc6360
ttgaacttgcatctaaactcgacctctacattttttatgtttatctctagtattactctt6420
tagacaaaaaaattgtagtaagaactattcatagagtgaatcgaaaacaatacgaaaatg6480
taaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttct6540
gaccaatgaagaatcatcaacgctatcactttctgttcacaaagtatgcggaatccacat6600
cggtatagaatataatcggggatgcctttatcttgaaaaaatgcacccgcagcttcgcta6660
gtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagagaaaatagacacc6720
aaagtagccttcttctaaccttaacggacctacagtgcaaaaagttatcaagagactgca6780
ttatagagcgcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgc6840
tctcgggatgcatttttgtagaacaaaaaagaagtatagattctttgttggtaaaatagc6900
gctctcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaatt6960
agcgctctcgcgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaa7020
atagcgctttcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaa7080
aaattagcgctctcgcgttgcatttttgttctacaaaatgaagcacagatgcttcgttaa7140
caaagatatgctattgaagtgcaagatggaaacgcagaaaatgaaccggggatgcgacgt7200
gcaagattacctatgcaatagatgcaatagtttctccaggaaccgaaatacatacattgt7260
cttccgtaaagcgctagactatatattattatacaggttcaaatatactatctgtttcag7320
ggaaaactcccaggttcggatgttcaaaattcaatgatgggtaacaagtacgatcgtaaa7380
tctgtaaaacagtttgtcggatattaggctgtatctcctcaaagcgtattcgaatatcat7440
tgagaagctgcagcgtcacatcggataataatgatggcagccattgtagaagtgcctttt7500
gcatttctagtctctttctcggtctagctagttttactacatcgcgaagatagaatctta7560
gatcacactgcctttgctgagctggatcaatagagtaacaaaagagtggtaaggcctcgt7620
taaaggacaaggacctgagcggaagtgtatcgtacagtagacggagtatactagtatagt7680
ctatagtccgtggaattgatccactagttctagagcggccgcacgtctaagaaaccatta7740
ttatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtt7800
tcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtc7860
tgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggt7920
gtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatacca7980
cagcttttcaattcaattcatcattttttttttattcttttttttgatttcggtttcttt8040
gaaatttttttgattcggtaatctccgaacagaaggaagaacgaaggaaggagcacagac8100
ttagattggtatatatacgcatatgtagtgttgaagaaacatgaaattgcccagtattct8160
taacccaactgcacagaacaaaaacctgcaggaaacgaagataaatcatgtcgaaagcta8220
catataaggaacgtgctgctactcatcctagtcctgttgctgccaagctatttaatatca8280
tgcacgaaaagcaaacaaacttgtgtgcttcattggatgttcgtaccaccaaggaattac8340
tggagttagttgaagcattaggtcccaaaatttgtttactaaaaacacatgtggatatat8400
tgactgatttttcgatggagggcacagttaagccgctaaaggcattatccgccaagtaca8460
attttttactcttcgaagacagaaaatttgctgacattggtaatacagtcaaattgcagt8520
actctgcgggtgtatacagaatagcagaatgggcagacattacgaatgcacacggtgtgc8580
tggggccaggtattgttagcggtttgaagcaggcggcagaagaagtaacaaaggaaccta8640
gaggccttttgatgttagcagaattgtcatgcaagggctccctatctactggagaatata8700
ctaagggtactgttgacattgcgaagagcgacaaagattttgttatcggctttattgctc8760
aaagagacatgggtggaagagatgaaggttacgattggttgattatgacacccggtgtgg8820
gtttagatgacaagggagacgcattgggtcaacagtatagaaccgtggatgatgtggtct8880
ctacaggatctgacattattattgttggaagaggactatttgcaaagggaagggatgcta8940
aggtacagggtgaacgttacagaaaagcaggctgggaagcatatttgagaagatgcggcc9000
agcaaaactaaaaaactgtattataagtaaatgcatgtatactaaactcacaaattagag9060
cttcaatttaattatatcagttattaccctatgcggtgtgaaataccgcacagatgcgta9120
aggagaaaataccgcatcaggaaattgtaaacgttaatattttgttaaaattcgcgttaa9180
atttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttata9240
aatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccac9300
tattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcc9360
cactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaaccactaa9420
atcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtgg9480
cgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcgg9540
tcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcgc9600
gccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgc9660
tattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccag9720
ggttttcccagtcacgacgttgtaaaacgacggccagtgaattgaattaaccctcactaa9780
agggaacaaaagctggagctagacaaagacaaaaaaaaaaagggatgtatcggtcagtca9840
ttaatactaggaacaagagaaaggaaaaacgttcattgttccttattcagttagctagct9900
gagctcatgactgctggttctgccccccctgttgattacacttccttaaagaagaacttc9960
caaccgtttctctccagaagagtagaaaatagatctctgaaaagcttttgggatgcttct10020
gatatctcagatgacgtcattgagctagctggtggaatgccaaacgagagattttttcct10080
atcgaatctatggatttgaaaatatcaaaagttccttttaatgataacccaaaatggcat10140
aattcgtttaccacggcgcatttggacttgggatcccccagtgagctacccattgcacgt10200
tctttccaatatgcagaaaccaagggtttaccccctctcttacattttgttaaagatttt10260
gtgtccagaattaatcgcccagccttttccgatgagacggagtctaactgggatgtcatc10320
ctttctggcgggtccaacgattcaatgtttaaggtttttgaaacaatttgcgacgaatcg10380
accactgtgatgattgaagagtttactttcaccccggctatgtccaatgtggaggctaca10440
ggagcaaaagtcatccccatcaagatgaacctgaccttcgacagagagtcccagggtatt10500
gatgtcgaatatctaacgcagttgctcgataattggtcaactggaccatacaaagactta10560
aacaagccaagggtcctatataccattgcaacgggccaaaatcctaccgggatgtctgtc10620
ccccagtggaaaagagagaaaatttaccagttggcccaaagacacgatttcctcattgtt10680
gaagatgatccctacggttatctgtactttccttcctataatccgcaagagccattagaa10740
aacccttaccattctagcgacctgactactgaacggtatttgaatgattttttaatgaaa10800
tcattcttgactttggatacagatgcccgtgtcatccgtttggagactttttctaaaatt10860
tttgctcctggattaaggttatccttcatcgttgctaataaattccttttgcaaaaaatc10920
ttggatttggccgacattactacaagggcccccagtggtacctcacaagctattgtttat10980
tctacaataaaggcaatggctgagtccaacttatcgtcctctctttctatgaaagaagca11040
atgtttgagggttggataagatggataatgcagattgcttctaaatacaatcataggaaa11100
aatcttactttgaaagccttatacgaaacagaatcttaccaagctggtcagtttaccgtt11160
atggaaccctccgcgggtatgttcatcattattaaaatcaattgggggaatttcgataga11220
cctgacgatttgccgcaacagatggatattttagataagttcttgctgaagaatggtgtt11280
aaagtagtgcttggttataaaatggctgtttgcccaaattattcaaagcagaattcagat11340
tttctaagactcaccatcgcctatgcaagggatgatgatcagttgattgaagcttccaaa11400
agaatcggtagtggcataaaagaattttttgacaactataaaagttgagagctcgagata11460
tcatgcgtagtcaggcacatcatacggatacccgggtcgacgcgtaagcttgtgggccct11520
aggatccatggtgaattc11538
<210>2
<211>11904
<212>dna
<213>过表达aro10基因(pyx212-aro10)
<400>2
ctgtatgtgttttttgtagttatagatttaagcaagaaaagaatacaaacaaaaaattga60
aaaagattgatttagaattaaaaagaaaaatatttacgtaagaagggaaaatagtaaatg120
ttgcaagttcactaaactcctaaattatgctgccctttatattccctgttacagcagccg180
agccaaaggtatataggctcctttgcattagcatgcgtaacaaaccacctgtcagtttca240
accgaggtggtatccgagagaattgtgtgattgctttaattaatttcggagaatctcaca300
tgccactgaagattaaaaactggatgccagaaaaggggtgtccgagtgtaacatcaatag360
aggaagctgaaaagtcttagaacgggtaatcttccaccaacctgatgggttcctagatat420
aatctcgaagggaataagtagggtgataccgtcagaagtgtctgaatgtattgaggtcct480
cacagtttaaatcccgctcacactaacgtaggattattataactcaaaaaaatggcatta540
ttctaagtaagttaaatatccgtaatctttaaacactatgtagttaggtctccctcacaa600
tcagtccatttgggtagcacggtcctcaggacgtatctattgatggattcgtccagttcc660
atcaccattacgctcccgttaggaacattggtaaacgattcaaactcttcgtatgtccat720
ctaaaccatttcatcaggaatactctggaataaataccatgtgtaactaggacaacaaca780
tctctgggtcttctctcttgcctatcatggaagtgcctgaataaagtctcttggaaactg840
gcgactctgtcatatacatctgccgcacttctccatgagggaatctgaagaagaatgacc900
atacgtagatccccaattcttgaagacgaaagggcctcgtgatacgcctatttttatagg960
ttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgc1020
gcggaacccctatttgtttatttttctaaatacattcaaatatgtatcgctcatgagaca1080
ataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacattt1140
ccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccaga1200
aacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcga1260
actggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaat1320
gatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggca1380
agagcaactcggtcgccgcatacactattctcagaatgacttggttgagtacaccagtca1440
cagaaaagcatcttacggatggcatgacagtaagagaattatgcagttcctgccataacc1500
atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaagagctaa1560
ccgctttttttcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagc1620
tgaatgaagccataccaaacgacgagcgtgacaccacgatgccatcaagctcgacggccg1680
gcccggtaccataacttcgtatagcatacattatacgaagttatttgaagtcggacagtg1740
agtgtagtcttgagaaattctgaagccgtatttttattatcagtgagtcagtcatcagga1800
gatcctctacgccggacgcatcgtggccgacctgcagggggggggggggcgctgaggtct1860
gcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagcca1920
gaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgatttt1980
gaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatcctt2040
caactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatg2100
ctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaa2160
tgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttc2220
tgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcgg2280
tctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaata2340
aggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagc2400
ttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatca2460
ctcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcga2520
tcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgcc2580
agcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgtt2640
ttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttg2700
atggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaaca2760
tcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttccca2820
tacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttataccca2880
tataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttga2940
atatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcat3000
gatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctt3060
tcccccccccccctgcaggtcggcatcaccggcgccacaggtgcggttgctggcgcctat3120
atcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgt3180
ttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttg3240
catgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggcataact3300
tcgtatagcatacattatacgaagttatggtacccaattcgccctatagtgagtcgtatt3360
tcgagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcaca3420
attccacacaacataggagccggaagcataaagtgtaaagcctggggtgcctaatgagtg3480
aggtaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcg3540
tgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgc3600
tcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgaccggta3660
tcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaag3720
aacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcg3780
tttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagagg3840
tggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg3900
cgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggga3960
agcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgc4020
tccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggt4080
aactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccact4140
ggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg4200
cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtt4260
accttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggt4320
ggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcct4380
ttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttg4440
gtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtttt4500
aaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagt4560
gaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtc4620
gtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccg4680
cgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggcc4740
gagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgg4800
gaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctaca4860
ggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacga4920
tcaaggcgagttacatgatcccccatgttgtccaaaaaagcggttagctccttcggtcct4980
ccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactg5040
cataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactca5100
accaagtcattctgagaatagtgtatgcggcgagcgagttgctcttgcccggcgtcaata5160
cgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttct5220
tcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccact5280
cgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaa5340
acaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactc5400
atactcttcctttttcaatattattgaagcatttatcacggttattgtctcatgagcgga5460
tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccga5520
aaagtgccacctgacgtactagtgagagtgcggccgcagctttgaagaaaaatgcgcctt5580
attcaatctttgctataaaaaatggcccaaaatctcacattggaagacatttgatgacct5640
catttctttcaatgaagggcctaacggagttgactaatgttgtgggaaattggagcgata5700
agggtgcttctgccgtggccaggacaacgtatactcatcagataacagcaatacctgatc5760
actacttcgcactagtttctcggtactatgcatatgatccaatatcaaaggaaatgatag5820
cattgaaggatgagactaatccaattgaggagtggcagcatatagaacagctaaagggta5880
gtgctgaaggaagcatacgataccccgcatggaatgggataatatcacaggaggtactag5940
actacctttcatcctacataaatagacgcatataagtacgcatttaagcataaacacgca6000
ctatgccgttcttctcatgtatatatatatacaggcaacacgcagatataggtgcgacgt6060
gaacagtgagctgtatgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaa6120
acgctttgaagttcctattccgaagttcctattctctagaaagtataggaacttcagagc6180
gcttttgaaaaccaaaagcgctctgaagacgcactttcaaaaaaccaaaaacgcaccgga6240
ctgtaacgagctactaaaatattgcgaataccgcttccacaaacattgctgaaaagtatc6300
tctttgctatatatctctgtgctatatccctatataacctacccatccacctttcgctcc6360
ttgaacttgcatctaaactcgacctctacattttttatgtttatctctagtattactctt6420
tagacaaaaaaattgtagtaagaactattcatagagtgaatcgaaaacaatacgaaaatg6480
taaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttct6540
gaccaatgaagaatcatcaacgctatcactttctgttcacaaagtatgcggaatccacat6600
cggtatagaatataatcggggatgcctttatcttgaaaaaatgcacccgcagcttcgcta6660
gtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagagaaaatagacacc6720
aaagtagccttcttctaaccttaacggacctacagtgcaaaaagttatcaagagactgca6780
ttatagagcgcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgc6840
tctcgggatgcatttttgtagaacaaaaaagaagtatagattctttgttggtaaaatagc6900
gctctcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaatt6960
agcgctctcgcgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaa7020
atagcgctttcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaa7080
aaattagcgctctcgcgttgcatttttgttctacaaaatgaagcacagatgcttcgttaa7140
caaagatatgctattgaagtgcaagatggaaacgcagaaaatgaaccggggatgcgacgt7200
gcaagattacctatgcaatagatgcaatagtttctccaggaaccgaaatacatacattgt7260
cttccgtaaagcgctagactatatattattatacaggttcaaatatactatctgtttcag7320
ggaaaactcccaggttcggatgttcaaaattcaatgatgggtaacaagtacgatcgtaaa7380
tctgtaaaacagtttgtcggatattaggctgtatctcctcaaagcgtattcgaatatcat7440
tgagaagctgcagcgtcacatcggataataatgatggcagccattgtagaagtgcctttt7500
gcatttctagtctctttctcggtctagctagttttactacatcgcgaagatagaatctta7560
gatcacactgcctttgctgagctggatcaatagagtaacaaaagagtggtaaggcctcgt7620
taaaggacaaggacctgagcggaagtgtatcgtacagtagacggagtatactagtatagt7680
ctatagtccgtggaattgatccactagttctagagcggccgcacgtctaagaaaccatta7740
ttatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtt7800
tcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtc7860
tgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggt7920
gtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatacca7980
cagcttttcaattcaattcatcattttttttttattcttttttttgatttcggtttcttt8040
gaaatttttttgattcggtaatctccgaacagaaggaagaacgaaggaaggagcacagac8100
ttagattggtatatatacgcatatgtagtgttgaagaaacatgaaattgcccagtattct8160
taacccaactgcacagaacaaaaacctgcaggaaacgaagataaatcatgtcgaaagcta8220
catataaggaacgtgctgctactcatcctagtcctgttgctgccaagctatttaatatca8280
tgcacgaaaagcaaacaaacttgtgtgcttcattggatgttcgtaccaccaaggaattac8340
tggagttagttgaagcattaggtcccaaaatttgtttactaaaaacacatgtggatatat8400
tgactgatttttcgatggagggcacagttaagccgctaaaggcattatccgccaagtaca8460
attttttactcttcgaagacagaaaatttgctgacattggtaatacagtcaaattgcagt8520
actctgcgggtgtatacagaatagcagaatgggcagacattacgaatgcacacggtgtgc8580
tggggccaggtattgttagcggtttgaagcaggcggcagaagaagtaacaaaggaaccta8640
gaggccttttgatgttagcagaattgtcatgcaagggctccctatctactggagaatata8700
ctaagggtactgttgacattgcgaagagcgacaaagattttgttatcggctttattgctc8760
aaagagacatgggtggaagagatgaaggttacgattggttgattatgacacccggtgtgg8820
gtttagatgacaagggagacgcattgggtcaacagtatagaaccgtggatgatgtggtct8880
ctacaggatctgacattattattgttggaagaggactatttgcaaagggaagggatgcta8940
aggtacagggtgaacgttacagaaaagcaggctgggaagcatatttgagaagatgcggcc9000
agcaaaactaaaaaactgtattataagtaaatgcatgtatactaaactcacaaattagag9060
cttcaatttaattatatcagttattaccctatgcggtgtgaaataccgcacagatgcgta9120
aggagaaaataccgcatcaggaaattgtaaacgttaatattttgttaaaattcgcgttaa9180
atttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttata9240
aatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccac9300
tattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcc9360
cactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaaccactaa9420
atcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtgg9480
cgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcgg9540
tcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcgc9600
gccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgc9660
tattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccag9720
ggttttcccagtcacgacgttgtaaaacgacggccagtgaattgaattaaccctcactaa9780
agggaacaaaagctggagctagacaaagacaaaaaaaaaaagggatgtatcggtcagtca9840
ttaatactaggaacaagagaaaggaaaaacgttcattgttccttattcagttagctagca9900
tggcacctgttacaattgaaaagttcgtaaatcaagaagaacgacaccttgtttccaacc9960
gatcagcaacaattccgtttggtgaatacatatttaaaagattgttgtccatcgatacga10020
aatcagttttcggtgttcctggtgacttcaacttatctctattagaatatctctattcac10080
ctagtgttgaatcagctggcctaagatgggtcggcacgtgtaatgaactgaacgccgctt10140
atgcggccgacggatattcccgttactctaataagattggctgtttaataaccacgtatg10200
gcgttggtgaattaagcgccttgaacggtatagccggttcgttcgctgaaaatgtcaaag10260
ttttgcacattgttggtgtggccaagtccatagattcgcgttcaagtaactttagtgatc10320
ggaacctacatcatttggtcccacagctacatgattcaaattttaaagggccaaatcata10380
aagtatatcatgatatggtaaaagatagagtcgcttgctcggtagcctacttggaggata10440
ttgaaactgcatgtgaccaagtcgataatgttatccgcgatatttacaagtattctaaac10500
ctggttatatttttgttcctgcagattttgcggatatgtctgttacatgtgataatttgg10560
ttaatgttccacgtatatctcaacaagattgtatagtatacccttctgaaaaccaattgt10620
ctgacataatcaacaagattactagttggatatattccagtaaaacacctgcgatccttg10680
gagacgtactgactgataggtatggtgtgagtaactttttgaacaagcttatctgcaaaa10740
ctgggatttggaatttttccactgttatgggaaaatctgtaattgatgagtcaaacccaa10800
cttatatgggtcaatataatggtaaagaaggtttaaaacaagtctatgaacattttgaac10860
tgtgcgacttggtcttgcattttggagtcgacatcaatgaaattaataatgggcattata10920
cttttacttataaaccaaatgctaaaatcattcaatttcatccgaattatattcgccttg10980
tggacactaggcagggcaatgagcaaatgttcaaaggaatcaattttgcccctattttaa11040
aagaactatacaagcgcattgacgtttctaaactttctttgcaatatgattcaaatgtaa11100
ctcaatatacgaacgaaacaatgcggttagaagatcctaccaatggacaatcaagcatta11160
ttacacaagttcacttacaaaagacgatgcctaaatttttgaaccctggtgatgttgtcg11220
tttgtgaaacaggctcttttcaattctctgttcgtgatttcgcgtttccttcgcaattaa11280
aatatatatcgcaaggatttttcctttccattggcatggcccttcctgccgccctaggtg11340
ttggaattgccatgcaagaccactcaaacgctcacatcaatggtggcaacgtaaaagagg11400
actataagccaagattaattttgtttgaaggtgacggtgcagcacagatgacaatccaag11460
aactgagcaccattctgaagtgcaatattccactagaagttatcatttggaacaataacg11520
gctacactattgaaagagccatcatgggccctaccaggtcgtataacgacgttatgtctt11580
ggaaatggaccaaactatttgaagcattcggagacttcgacggaaagtatactaatagca11640
ctctcattcaatgtccctctaaattagcactgaaattggaggagcttaagaattcaaaca11700
aaagaagcgggatagaacttttagaagtcaaattaggcgaattggatttccccgaacagc11760
taaagtgcatggttgaagcagcggcacttaaaagaaataaaaaataggctagctgagctc11820
gagatatcatgcgtagtcaggcacatcatacggatacccgggtcgacgcgtaagcttgtg11880
ggccctaggatccatggtgaattc11904
<210>3
<211>1542
<212>dna
<213>过表达前aro9基因(aro9)
<400>3
atgactgctggttctgccccccctgttgattacacttccttaaagaagaacttccaaccg60
tttctctccagaagagtagaaaatagatctctgaaaagcttttgggatgcttctgatatc120
tcagatgacgtcattgagctagctggtggaatgccaaacgagagattttttcctatcgaa180
tctatggatttgaaaatatcaaaagttccttttaatgataacccaaaatggcataattcg240
tttaccacggcgcatttggacttgggatcccccagtgagctacccattgcacgttctttc300
caatatgcagaaaccaagggtttaccccctctcttacattttgttaaagattttgtgtcc360
agaattaatcgcccagccttttccgatgagacggagtctaactgggatgtcatcctttct420
ggcgggtccaacgattcaatgtttaaggtttttgaaacaatttgcgacgaatcgaccact480
gtgatgattgaagagtttactttcaccccggctatgtccaatgtggaggctacaggagca540
aaagtcatccccatcaagatgaacctgaccttcgacagagagtcccagggtattgatgtc600
gaatatctaacgcagttgctcgataattggtcaactggaccatacaaagacttaaacaag660
ccaagggtcctatataccattgcaacgggccaaaatcctaccgggatgtctgtcccccag720
tggaaaagagagaaaatttaccagttggcccaaagacacgatttcctcattgttgaagat780
gatccctacggttatctgtactttccttcctataatccgcaagagccattagaaaaccct840
taccattctagcgacctgactactgaacggtatttgaatgattttttaatgaaatcattc900
ttgactttggatacagatgcccgtgtcatccgtttggagactttttctaaaatttttgct960
cctggattaaggttatccttcatcgttgctaataaattccttttgcaaaaaatcttggat1020
ttggccgacattactacaagggcccccagtggtacctcacaagctattgtttattctaca1080
ataaaggcaatggctgagtccaacttatcgtcctctctttctatgaaagaagcaatgttt1140
gagggttggataagatggataatgcagattgcttctaaatacaatcataggaaaaatctt1200
actttgaaagccttatacgaaacagaatcttaccaagctggtcagtttaccgttatggaa1260
ccctccgcgggtatgttcatcattattaaaatcaattgggggaatttcgatagacctgac1320
gatttgccgcaacagatggatattttagataagttcttgctgaagaatggtgttaaagta1380
gtgcttggttataaaatggctgtttgcccaaattattcaaagcagaattcagattttcta1440
agactcaccatcgcctatgcaagggatgatgatcagttgattgaagcttccaaaagaatc1500
ggtagtggcataaaagaattttttgacaactataaaagttga1542
<210>4
<211>1908
<212>dna
<213>过表达前aro10基因(aro10)
<400>4
atggcacctgttacaattgaaaagttcgtaaatcaagaagaacgacaccttgtttccaac60
cgatcagcaacaattccgtttggtgaatacatatttaaaagattgttgtccatcgatacg120
aaatcagttttcggtgttcctggtgacttcaacttatctctattagaatatctctattca180
cctagtgttgaatcagctggcctaagatgggtcggcacgtgtaatgaactgaacgccgct240
tatgcggccgacggatattcccgttactctaataagattggctgtttaataaccacgtat300
ggcgttggtgaattaagcgccttgaacggtatagccggttcgttcgctgaaaatgtcaaa360
gttttgcacattgttggtgtggccaagtccatagattcgcgttcaagtaactttagtgat420
cggaacctacatcatttggtcccacagctacatgattcaaattttaaagggccaaatcat480
aaagtatatcatgatatggtaaaagatagagtcgcttgctcggtagcctacttggaggat540
attgaaactgcatgtgaccaagtcgataatgttatccgcgatatttacaagtattctaaa600
cctggttatatttttgttcctgcagattttgcggatatgtctgttacatgtgataatttg660
gttaatgttccacgtatatctcaacaagattgtatagtatacccttctgaaaaccaattg720
tctgacataatcaacaagattactagttggatatattccagtaaaacacctgcgatcctt780
ggagacgtactgactgataggtatggtgtgagtaactttttgaacaagcttatctgcaaa840
actgggatttggaatttttccactgttatgggaaaatctgtaattgatgagtcaaaccca900
acttatatgggtcaatataatggtaaagaaggtttaaaacaagtctatgaacattttgaa960
ctgtgcgacttggtcttgcattttggagtcgacatcaatgaaattaataatgggcattat1020
acttttacttataaaccaaatgctaaaatcattcaatttcatccgaattatattcgcctt1080
gtggacactaggcagggcaatgagcaaatgttcaaaggaatcaattttgcccctatttta1140
aaagaactatacaagcgcattgacgtttctaaactttctttgcaatatgattcaaatgta1200
actcaatatacgaacgaaacaatgcggttagaagatcctaccaatggacaatcaagcatt1260
attacacaagttcacttacaaaagacgatgcctaaatttttgaaccctggtgatgttgtc1320
gtttgtgaaacaggctcttttcaattctctgttcgtgatttcgcgtttccttcgcaatta1380
aaatatatatcgcaaggatttttcctttccattggcatggcccttcctgccgccctaggt1440
gttggaattgccatgcaagaccactcaaacgctcacatcaatggtggcaacgtaaaagag1500
gactataagccaagattaattttgtttgaaggtgacggtgcagcacagatgacaatccaa1560
gaactgagcaccattctgaagtgcaatattccactagaagttatcatttggaacaataac1620
ggctacactattgaaagagccatcatgggccctaccaggtcgtataacgacgttatgtct1680
tggaaatggaccaaactatttgaagcattcggagacttcgacggaaagtatactaatagc1740
actctcattcaatgtccctctaaattagcactgaaattggaggagcttaagaattcaaac1800
aaaagaagcgggatagaacttttagaagtcaaattaggcgaattggatttccccgaacag1860
ctaaagtgcatggttgaagcagcggcacttaaaagaaataaaaaatag1908
<210>5
<211>50
<212>dna
<213>aro9-f
<400>5
ttccttattcagttagctagctgagctcatgactgctggttctgcccccc50
<210>6
<211>50
<212>dna
<213>aro9-r
<400>6
gcctgactacgcatgatatctcgagctctcaacttttatagttgtcaaaa50
<210>7
<211>40
<212>dna
<213>aro10-f
<400>7
cagttagctagctgagctcatggcacctgttacaattgaa40
<210>8
<211>40
<212>dna
<213>aro10-r
<400>8
acgcatgatatctcgagctcctattttttatttcttttaa40
<210>9
<211>9990
<212>dna
<213>质粒(pyx212)
<400>9
ctgtatgtgttttttgtagttatagatttaagcaagaaaagaatacaaacaaaaaattga60
aaaagattgatttagaattaaaaagaaaaatatttacgtaagaagggaaaatagtaaatg120
ttgcaagttcactaaactcctaaattatgctgccctttatattccctgttacagcagccg180
agccaaaggtatataggctcctttgcattagcatgcgtaacaaaccacctgtcagtttca240
accgaggtggtatccgagagaattgtgtgattgctttaattaatttcggagaatctcaca300
tgccactgaagattaaaaactggatgccagaaaaggggtgtccgagtgtaacatcaatag360
aggaagctgaaaagtcttagaacgggtaatcttccaccaacctgatgggttcctagatat420
aatctcgaagggaataagtagggtgataccgtcagaagtgtctgaatgtattgaggtcct480
cacagtttaaatcccgctcacactaacgtaggattattataactcaaaaaaatggcatta540
ttctaagtaagttaaatatccgtaatctttaaacactatgtagttaggtctccctcacaa600
tcagtccatttgggtagcacggtcctcaggacgtatctattgatggattcgtccagttcc660
atcaccattacgctcccgttaggaacattggtaaacgattcaaactcttcgtatgtccat720
ctaaaccatttcatcaggaatactctggaataaataccatgtgtaactaggacaacaaca780
tctctgggtcttctctcttgcctatcatggaagtgcctgaataaagtctcttggaaactg840
gcgactctgtcatatacatctgccgcacttctccatgagggaatctgaagaagaatgacc900
atacgtagatccccaattcttgaagacgaaagggcctcgtgatacgcctatttttatagg960
ttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgc1020
gcggaacccctatttgtttatttttctaaatacattcaaatatgtatcgctcatgagaca1080
ataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacattt1140
ccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccaga1200
aacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcga1260
actggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaat1320
gatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggca1380
agagcaactcggtcgccgcatacactattctcagaatgacttggttgagtacaccagtca1440
cagaaaagcatcttacggatggcatgacagtaagagaattatgcagttcctgccataacc1500
atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaagagctaa1560
ccgctttttttcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagc1620
tgaatgaagccataccaaacgacgagcgtgacaccacgatgccatcaagctcgacggccg1680
gcccggtaccataacttcgtatagcatacattatacgaagttatttgaagtcggacagtg1740
agtgtagtcttgagaaattctgaagccgtatttttattatcagtgagtcagtcatcagga1800
gatcctctacgccggacgcatcgtggccgacctgcagggggggggggggcgctgaggtct1860
gcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagcca1920
gaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgatttt1980
gaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatcctt2040
caactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatg2100
ctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaa2160
tgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttc2220
tgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcgg2280
tctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaata2340
aggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagc2400
ttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatca2460
ctcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcga2520
tcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgcc2580
agcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgtt2640
ttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttg2700
atggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaaca2760
tcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttccca2820
tacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttataccca2880
tataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttga2940
atatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcat3000
gatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctt3060
tcccccccccccctgcaggtcggcatcaccggcgccacaggtgcggttgctggcgcctat3120
atcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgt3180
ttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttg3240
catgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggcataact3300
tcgtatagcatacattatacgaagttatggtacccaattcgccctatagtgagtcgtatt3360
tcgagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcaca3420
attccacacaacataggagccggaagcataaagtgtaaagcctggggtgcctaatgagtg3480
aggtaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcg3540
tgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgc3600
tcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgaccggta3660
tcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaag3720
aacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcg3780
tttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagagg3840
tggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg3900
cgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggga3960
agcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgc4020
tccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggt4080
aactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccact4140
ggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg4200
cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtt4260
accttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggt4320
ggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcct4380
ttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttg4440
gtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtttt4500
aaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagt4560
gaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtc4620
gtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccg4680
cgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggcc4740
gagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgg4800
gaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctaca4860
ggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacga4920
tcaaggcgagttacatgatcccccatgttgtccaaaaaagcggttagctccttcggtcct4980
ccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactg5040
cataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactca5100
accaagtcattctgagaatagtgtatgcggcgagcgagttgctcttgcccggcgtcaata5160
cgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttct5220
tcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccact5280
cgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaa5340
acaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactc5400
atactcttcctttttcaatattattgaagcatttatcacggttattgtctcatgagcgga5460
tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccga5520
aaagtgccacctgacgtactagtgagagtgcggccgcagctttgaagaaaaatgcgcctt5580
attcaatctttgctataaaaaatggcccaaaatctcacattggaagacatttgatgacct5640
catttctttcaatgaagggcctaacggagttgactaatgttgtgggaaattggagcgata5700
agggtgcttctgccgtggccaggacaacgtatactcatcagataacagcaatacctgatc5760
actacttcgcactagtttctcggtactatgcatatgatccaatatcaaaggaaatgatag5820
cattgaaggatgagactaatccaattgaggagtggcagcatatagaacagctaaagggta5880
gtgctgaaggaagcatacgataccccgcatggaatgggataatatcacaggaggtactag5940
actacctttcatcctacataaatagacgcatataagtacgcatttaagcataaacacgca6000
ctatgccgttcttctcatgtatatatatatacaggcaacacgcagatataggtgcgacgt6060
gaacagtgagctgtatgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaa6120
acgctttgaagttcctattccgaagttcctattctctagaaagtataggaacttcagagc6180
gcttttgaaaaccaaaagcgctctgaagacgcactttcaaaaaaccaaaaacgcaccgga6240
ctgtaacgagctactaaaatattgcgaataccgcttccacaaacattgctgaaaagtatc6300
tctttgctatatatctctgtgctatatccctatataacctacccatccacctttcgctcc6360
ttgaacttgcatctaaactcgacctctacattttttatgtttatctctagtattactctt6420
tagacaaaaaaattgtagtaagaactattcatagagtgaatcgaaaacaatacgaaaatg6480
taaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttct6540
gaccaatgaagaatcatcaacgctatcactttctgttcacaaagtatgcggaatccacat6600
cggtatagaatataatcggggatgcctttatcttgaaaaaatgcacccgcagcttcgcta6660
gtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagagaaaatagacacc6720
aaagtagccttcttctaaccttaacggacctacagtgcaaaaagttatcaagagactgca6780
ttatagagcgcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgc6840
tctcgggatgcatttttgtagaacaaaaaagaagtatagattctttgttggtaaaatagc6900
gctctcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaatt6960
agcgctctcgcgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaa7020
atagcgctttcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaa7080
aaattagcgctctcgcgttgcatttttgttctacaaaatgaagcacagatgcttcgttaa7140
caaagatatgctattgaagtgcaagatggaaacgcagaaaatgaaccggggatgcgacgt7200
gcaagattacctatgcaatagatgcaatagtttctccaggaaccgaaatacatacattgt7260
cttccgtaaagcgctagactatatattattatacaggttcaaatatactatctgtttcag7320
ggaaaactcccaggttcggatgttcaaaattcaatgatgggtaacaagtacgatcgtaaa7380
tctgtaaaacagtttgtcggatattaggctgtatctcctcaaagcgtattcgaatatcat7440
tgagaagctgcagcgtcacatcggataataatgatggcagccattgtagaagtgcctttt7500
gcatttctagtctctttctcggtctagctagttttactacatcgcgaagatagaatctta7560
gatcacactgcctttgctgagctggatcaatagagtaacaaaagagtggtaaggcctcgt7620
taaaggacaaggacctgagcggaagtgtatcgtacagtagacggagtatactagtatagt7680
ctatagtccgtggaattgatccactagttctagagcggccgcacgtctaagaaaccatta7740
ttatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtt7800
tcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtc7860
tgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggt7920
gtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatacca7980
cagcttttcaattcaattcatcattttttttttattcttttttttgatttcggtttcttt8040
gaaatttttttgattcggtaatctccgaacagaaggaagaacgaaggaaggagcacagac8100
ttagattggtatatatacgcatatgtagtgttgaagaaacatgaaattgcccagtattct8160
taacccaactgcacagaacaaaaacctgcaggaaacgaagataaatcatgtcgaaagcta8220
catataaggaacgtgctgctactcatcctagtcctgttgctgccaagctatttaatatca8280
tgcacgaaaagcaaacaaacttgtgtgcttcattggatgttcgtaccaccaaggaattac8340
tggagttagttgaagcattaggtcccaaaatttgtttactaaaaacacatgtggatatat8400
tgactgatttttcgatggagggcacagttaagccgctaaaggcattatccgccaagtaca8460
attttttactcttcgaagacagaaaatttgctgacattggtaatacagtcaaattgcagt8520
actctgcgggtgtatacagaatagcagaatgggcagacattacgaatgcacacggtgtgc8580
tggggccaggtattgttagcggtttgaagcaggcggcagaagaagtaacaaaggaaccta8640
gaggccttttgatgttagcagaattgtcatgcaagggctccctatctactggagaatata8700
ctaagggtactgttgacattgcgaagagcgacaaagattttgttatcggctttattgctc8760
aaagagacatgggtggaagagatgaaggttacgattggttgattatgacacccggtgtgg8820
gtttagatgacaagggagacgcattgggtcaacagtatagaaccgtggatgatgtggtct8880
ctacaggatctgacattattattgttggaagaggactatttgcaaagggaagggatgcta8940
aggtacagggtgaacgttacagaaaagcaggctgggaagcatatttgagaagatgcggcc9000
agcaaaactaaaaaactgtattataagtaaatgcatgtatactaaactcacaaattagag9060
cttcaatttaattatatcagttattaccctatgcggtgtgaaataccgcacagatgcgta9120
aggagaaaataccgcatcaggaaattgtaaacgttaatattttgttaaaattcgcgttaa9180
atttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttata9240
aatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccac9300
tattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcc9360
cactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaaccactaa9420
atcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtgg9480
cgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcgg9540
tcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcgc9600
gccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgc9660
tattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccag9720
ggttttcccagtcacgacgttgtaaaacgacggccagtgaattgaattaaccctcactaa9780
agggaacaaaagctggagctagacaaagacaaaaaaaaaaagggatgtatcggtcagtca9840
ttaatactaggaacaagagaaaggaaaaacgttcattgttccttattcagttagctagct9900
gagctcgagatatcatgcgtagtcaggcacatcatacggatacccgggtcgacgcgtaag9960
cttgtgggccctaggatccatggtgaattc9990