CN115216464A - 获得α-法尼烯以及β-法尼烯的重组微生物及其构建方法 - Google Patents

获得α-法尼烯以及β-法尼烯的重组微生物及其构建方法 Download PDF

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CN115216464A
CN115216464A CN202110417823.4A CN202110417823A CN115216464A CN 115216464 A CN115216464 A CN 115216464A CN 202110417823 A CN202110417823 A CN 202110417823A CN 115216464 A CN115216464 A CN 115216464A
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刘天罡
叶紫玲
石彬
黄阳磊
邝照琳
林晓莹
黄曼
马田
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Abstract

本发明公开了获得α‑法尼烯以及β‑法尼烯的重组微生物及其构建方法，属于合成生物学领域。本发明获得了性能优异的α‑法尼烯合酶和β‑法尼烯合酶，包括序列如SEQ ID NO.1‑6所示的α‑法尼烯合酶或β‑法尼烯合酶。本发明的重组微生物在CEN.PK2‑1D的基础上，含有5个法尼烯合酶基因，含有额外的MVA途径基因ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1，以及额外一个ERG20和一个tHMG1基因。

Description

获得α-法尼烯以及β-法尼烯的重组微生物及其构建方法
技术领域
本发明属于合成生物学领域，具体涉及一种获得α-法尼烯以及β-法尼烯的重组微生物及其构建方法。
背景技术
法尼烯是一种倍半萜化合物，其在农业、工业生产中具有广泛应用。目前法尼烯可作为现有基于石油的二烯烃单体替代物用于增粘树脂的生产之中，使其软化点、稳定性和热稳定性都有所提高。同时，法尼烯是众多植物挥发性物质的的主要成分，根据植物-害虫之间的信号联系机理以及植物挥发性物质在害虫诱杀方面的天然优势，法尼烯常用作害虫引诱剂，结合昆虫诱杀器用于农业生产。聚合法尼烯也作为胶基成分代替源自于石油原料的人造聚合物，由于其为食品可接受聚合物，故也用于口香胶基等食品之中。目前通过植物提取、化学合成和生物合成方法得到法尼烯。微生物发酵可不受植物生长、杂质众多等因素限制，成本较低且比起化学合成法，其对环境污染较小，是一种经济、绿色的合成方式。
目前针对于法尼烯的微生物合成主要采用大肠杆菌、酿酒酵母、解脂耶氏酵母，均获得了一定的进展。而目前所使用的生产α-法尼烯的酶基本均是苹果来源，而β-法尼烯合酶均是青蒿来源。
发明内容
本发明的目的之一在于提供一种性能优异的法尼烯合酶，以及所述法尼烯合酶在构建产法尼烯的重组微生物中的应用。
一种性能优异的法尼烯合酶，为α-法尼烯合酶或β-法尼烯合酶；其中，α-法尼烯合酶为氨基酸序列如SEQ ID NO.1所示的茶树来源的α-法尼烯合酶，或以氨基酸序列如SEQID NO.1所示的茶树来源的α-法尼烯合酶为基础，含有下述突变中的一种或两种的α-法尼烯合酶突变体：W281C、C455N；或α-法尼烯合酶为氨基酸序列如氨基酸序列如SEQ ID NO.3所示的梨来源的α-法尼烯合酶，或以氨基酸序列如SEQ ID NO.3所示的梨来源的α-法尼烯合酶为基础，含有下述突变中的一种或多种的α-法尼烯合酶突变体：G252E、D10G、A78T；β-法尼烯合酶为氨基酸序列如SEQ ID NO.2所示的洋甘菊来源的β-法尼烯合酶，或以氨基酸序列如SEQ ID NO.2所示的洋甘菊来源的β-法尼烯合酶为基础，含有下述突变中的一种或多种的β-法尼烯合酶突变体：F11S、M35T、T319S、I434T、I460V、K59R、S204Y。
上述法尼烯合酶合成法尼烯的性能更为优异，其可用于生产法尼烯或构建产法尼烯的重组微生物，提高法尼烯的产量。
本发明的目的之二在于提供一种产法尼烯的重组微生物，以及所述重组微生物的构建方法和在生产法尼烯中的应用。
一种产法尼烯的重组微生物，为产α-法尼烯的重组微生物或产β-法尼烯的重组微生物。其中，产α-法尼烯的重组微生物中基因的拷贝数为ERG10：ERG13：tHMG1：ERG12：ERG8：MVD1：IDI1：ERG20：aFS＝2：2：X：2：2：2：2：2：X，X为大于等于1的整数；产β-法尼烯的重组微生物中基因的拷贝数为ERG10：ERG13：tHMG1：ERG12：ERG8：MVD1：IDI1：ERG20：bFS＝2：2：X：2：2：2：2：2：X，X为大于等于1的整数。其中，ERG10为编码乙酰乙酰辅酶A硫解酶的基因，ERG13为编码HMG-CoA合酶的基因，tHMG1为编码HMG-CoA还原酶的基因，ERG12为编码甲羟戊酸激酶的基因，ERG8为编码甲羟戊酸-5-磷酸激酶的基因，MVD1为编码甲羟戊酸焦磷酸脱羧酶的基因，IDI1为编码异戊二烯焦磷酸异构酶的基因，ERG20为编码法尼烯焦磷酸合酶的基因，aFS为编码α-法尼烯合酶的基因，bFS为编码β-法尼烯合酶的基因。优选的，aFS编码的α-法尼烯合酶为氨基酸序列如SEQ ID NO.1所示的茶树来源的α-法尼烯合酶或氨基酸序列如SEQ ID NO.3所示的梨来源的α-法尼烯合酶或上述α-法尼烯合酶突变体；bFS编码的β-法尼烯合酶为氨基酸序列如SEQ ID NO.2所示的洋甘菊来源的β-法尼烯合酶或上述β-法尼烯合酶突变体，更为优选的，aFS编码的α-法尼烯合酶的氨基酸序列如SEQ ID NO.4所示(在梨来源的α-法尼烯合酶的基础上，含有G252E突变)；bFS编码的β-法尼烯合酶的氨基酸序列如SEQ ID NO.5所示(在洋甘菊来源的β-法尼烯合酶的基础上，同时含有F11S、M35T、T319S、I434T、I460V突变)，或bFS编码的β-法尼烯合酶的氨基酸序列如SEQ ID NO.6所示(在洋甘菊来源的β-法尼烯合酶的基础上，同时含有F11S、M35T、T319S、I434T、I460V、K59R、S204Y突变)。
更为优选的，ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20在NCBI中的Accession/GENE id见下表。
Figure BDA0003026658980000021
Figure BDA0003026658980000031
优选的，所述的产法尼烯的重组微生物以酿酒酵母为宿主；更为优选的，所述的产法尼烯的重组微生物以酿酒酵母CEN.PK2-1D菌株为宿主。
优选的，所述的产α-法尼烯的重组微生物在酿酒酵母CEN.PK2-1D的基础上，含有5个SEQ ID NO.3所示或SEQ ID NO.4所示α-法尼烯合酶的编码基因，含有额外的MVA途径基因(ERG10、ERG13、THMG1、ERG12、ERG8、MVD1、IDI1)以及额外一个ERG20基因，额外一个tHMG1基因。更为优选的，所述的产α-法尼烯的重组微生物为将下表所示基因通过质粒转到酿酒酵母CEN.PK2-1D中，使相关基因整合到酿酒酵母CEN.PK2-1D染色体特定位置后得到。
Figure BDA0003026658980000032
优选的，所述的产β-法尼烯的重组微生物在酿酒酵母CEN.PK2-1D的基础上，含有5个SEQ ID NO.5所示或SEQ ID NO.6所示β-法尼烯合酶的编码基因，含有额外的MVA途径基因(ERG10、ERG13、THMG1、ERG12、ERG8、MVD1、IDI1)以及额外一个ERG20基因，额外一个tHMG1基因。更为优选的，所述的产β-法尼烯的重组微生物为将下表所示基因通过质粒转到酿酒酵母CEN.PK2-1D中，使相关基因整合到酿酒酵母CEN.PK2-1D染色体特定位置后得到。
Figure BDA0003026658980000041
上表中，相关启动子、基因或终止子的序列见下：
>pTEF1：AGCTCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTCTAGAACTAGTGGATCCCCCGGGaaaa；
>cas9：ATGGACAAGAAGTACTCCATTGGGCTCGATATCGGCACAAACAGCGTCGGtTGGGCCGTCATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAGTTCTGGGCAATACCGATCGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGGAGACGGCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAATCGGATCTGCTACCTGCAGGAGATCTTTAGTAATGAGATGGCTAAGGTGGATGACTCTTTCTTCCATAGGCTGGAGGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCCACCCAATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTACCCAACCATATATCATCTGAGGAAGAAGCTTGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACCTGAACCCAGACAACAGCGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAATCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCTGAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCCTGGGGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAACTTTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGACACCTACGATGATGATCTCGACAATCTGCTGGCCCAGATCGGCGACCAGTACGCAGACCTTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATTCTGCGAGTGAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAGCACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTACGCCGGATACATTGACGGCGGAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGGCACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCACTTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGGCGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTTTTTGAAAGATAACAGGGAAAAGATTGAGAAAATCCTCACATTTCGGATACCCTACTATGTAGGCCCCCTCGCCCGGGGAAATTCCAGATTCGCGTGGATGACTCGCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAAAATCTGCCTAACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTTATAACGAGCTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCATTCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCAAGACGAACCGGAAAGTTACCGTGAAACAGCTCAAAGAAGACTATTTCAAAAAGATTGAATGTTTCGACTCTGTTGAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGATCTCCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGCGCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACAAGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATGGATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAAGGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAACCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGATCATATCGTGCCCCAGTCTTTTCTCAAAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAAATAGAGGGAAGAGTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAGGCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAACAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTCTTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGATCAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTGTGA；
>tCYC1：TCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGC；
>tGAL10pGAL7：Tttgccagcttactatccttcttgaaaatatgcactctatatcttttagttcttaattgcaacacatagatttgctgtataacgaattttatgctattttttaaatttggagttcagtgataaaagtgtcacagcgaatttcctcacatgtagggaccgaattgtttacaagttctctgtaccaccatggagacatcaaagattgaaaatctatggaaagatatggacggtagcaacaagaatatagcacgagccgcgaagttcatttcgttacttttgatatcgctcacaactattgcgaagcgcttcagtgaaaaaatcataaggaaaagttgtaaatattattggtagtattcgtttggtaaagtagagggggtaatttttcccctttattttgttcatacattcttaaattgctttgcctctccttttggaaagctatacttcggagcactgttgagcgaaggctcattagatatattttctgtcattttccttaacccaaaaataagggaaagggtccaaaaagcgctcggacaactgttgaccgtgatccgaaggactggctatacagtgttcacaaaatagccaagctgaaaataatgtgtagctatgttcagttagtttggctagcaaagatataaaagcaggtcggaaatatttatgggcattattatgcagagcatcaacatgataaaaaaaaacagttgaatattccctcaaaa；
>tADH1：GCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGG；
>pGAL1pGAL10：Tatagttttttctccttgacgttaaagtatagaggtatattaacaattttttgttgatacttttatgacatttgaataagaagtaatacaaaccgaaaatgttgaaagtattagttaaagtggttatgcagcttttgcatttatatatctgttaatagatcaaaaatcatcgcttcgctgattaattaccccagaaataaggctaaaaaactaatcgcattattatcctatggttgttaatttgattcgttgatttgaaggtttgtggggccaggttactgccaatttttcctcttcataaccataaaagctagtattgtagaatctttattgttcggagcagtgcggcgcgaggcacatctgcgtttcaggaacgcgaccggtgaagaccaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaatatagcaatgagcagttaagcgtattactgaaagttccaaagagaaggtttttttaggctaagataatggggctctttacatttccacaacatataagtaagattagatatggatatgtatatggtggtattgccatgtaatatgattattaaacttctttgcgtccatccaaaaaaaaagtaagaatttttgaaaattcaatataa；
>tHMG1：ACTTAGTCATACGTCATTGGTATTCTCTTGAAAAAGAAGCACAACAGCACCATGTGTTACGTAAAATATTTACTTTATAGTTTGTACGTCATAATTTCTTCCATATTACAAGTTCGTGCATATATAGAAAGAATTCTGTTGTTGTAATTGTCATAACTATTGAGCTTTACCTGAAAATTCAACGAAAAAAACTCAAAAACCACATGCTTCTCTTGAGTCATGCGGTTCCTTTCCCTTATGAGTGAAAATCTTCCTTTTTTAGCTATGTGCGCCATCCGATAAATGTAGGAGCAATGAAGC；
>tERG20：AACTAACGCTAATCGATAAAACATTAGATTTCAAACTAGATAAGGACCATGTATAAGAACTATATACTTCCAATATAATATAGTATAAGCTTTAAGATAGTATCTCTCGATCTACCGTTCCACGTGACTAGTCCAAGGATTTTTTTTAA；
>tPGK1：ATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAATTACGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTCCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTAATAACTCGAAAATTCTGCGTTCGTT；
>pURA3：TTCAATTCATCATTTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCTTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATC；
>tURA3：aaaactgtattataagtaaatgcatgtatactaaactcacaaattagagcttcaatttaattatatcagttattaccc。
进一步地，所述的产法尼烯的重组微生物还可以敲除其GAL80基因，敲除Gal80后，菌株可以实现在不添加半乳糖诱导的情况下合成法尼烯，可以减少实验流程，减小发酵成本。
上述产法尼烯的重组微生物的构建方法，为将基因ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20、Pyc-aFS/Mac-bFS克隆到多个质粒上，再将质粒转入到宿主中，经过筛选，获得表达各基因的重组微生物。优选的，所述的产法尼烯的重组微生物的构建方法，包括如下步骤：构建上表中所示的质粒，将质粒转到酿酒酵母CEN.PK2-1D中，使相关基因整合到酿酒酵母CEN.PK2-1D的染色体上，得到产法尼烯的重组微生物。
上述产法尼烯的重组微生物可用于生产法尼烯。
本发明的目的之三在于提供一种构建突变株文库的方法，其包括如下步骤：使用低保真DNA聚合酶taq聚合酶，并调整体系中镁离子浓度，进行PCR扩增关键基因，获得含有突变的DNA片段，克隆到表达载体。将上述构建获得的表达载体转入表达宿主，如酿酒酵母等，涂布筛选平板获得突变株文库。
本发明的目的之四在于提供一种高效的法尼烯合成相关酶的正向突变体筛选的方法，包括如下步骤：从上述筛选平板接种至高通量种子培养板(包括但不限于96孔板、384孔板)，菌株生长到对数生长期时，转接至发酵培养板(包括但不限于96孔板、384孔板)，待菌株产量达到最高，加入萃取剂进行高通量产物萃取，将萃取获得的产物转移至新的平板(包括但不限于96孔板、384孔板)，加入广谱显色剂(包括但不限于碘、香草醛浓硫酸显色剂、钼酸铈、茴香醛、高锰酸钾)，反应(常温或高温或低温，静置或摇晃)足够时间后，酶标仪检测最大吸收值。测序确定正突变位点。本发明通过随机突变筛选获得的性能提升的含一个或多个点突变的α-法尼烯合酶或β-法尼烯合酶突变体。其中，所述的萃取剂为棕榈酸异丙酯、白油(液体石蜡)、油酸甲酯或菜籽油。使用棕榈酸异丙酯、白油(液体石蜡)油酸甲酯或菜籽油作为萃取剂，不会对法尼烯的产量产生影响，基于此，本发明还提供一种萃取剂在萃取法尼烯中的应用。
本发明的目的之五在于提供一种法尼烯高产菌株的构建方法，其包括如下步骤：将含有至少一个正突变的法尼烯合酶导入可用于法尼烯生产的底盘菌株，通过调整MVA途径关键基因tHMG1和法尼烯合酶的比例，使法尼烯的产量进一步提升，获得法尼烯高产菌株。从而可以利用该重组微生物培养获得高产量的法尼烯。
本发明的目的之六在于提供一种以某一种高产菌株为基础，一步法构建获得另一种产物高产菌株的方法，包括如下步骤：以产物A高产的菌株为底盘，通过crispr基因编辑方法，gRNA靶向产物合成基因，donor DNA包含产物B合成基因，通过crispr基因编辑技术实现关键基因一次性替换，从而获得产物B高产菌株。
本发明中验证并比较了几种不同来源的法尼烯合酶，结果发现了性能优于目前常用的法尼烯合酶。并且经过工程改造，获得了性能提升的β-法尼烯合酶，最终还获得了高产量的法尼烯合成酵母菌株。
附图说明
图1是质粒pZY600的构建示意图。
图2是质粒pZY413的构建示意图。
图3是质粒pZY412L的构建示意图。
图4是质粒pZY414的构建示意图。
图5是质粒pZY900的构建示意图。
图6是质粒AFS系列、BFS系列的构建示意图。
图7是不同菌株发酵的法尼烯产量结果图。
图8是质粒pAFS1-P2、pAFS1-P3、pAFS1-P4、pAFS1-P5、pAFS1-P6、pAFS1-P7的构建示意图。
图9是β-法尼烯高产菌株的产量结果图。
具体实施方式
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1底盘酵母菌株所需载体构建
(1)质粒pZY600
质粒pZY600的作用是整合Cas9蛋白，以便后续使用Crispr-cas9基因编辑技术进行菌株改造。质粒pZY600的相关特征：△ChrXII-2：Hygr_pTEF1_Cas9_tCYC1(图1)，其中，pTEF1_Cas9_tCYC1指的是表达TEF1启动子控制表达的Cas9，终止子为cyc1，筛选标记为Hygr即潮霉素；ΔchrXII-2指的是该表达框插入到的染色体的位置。
质粒pZY600的具体构建过程：以质粒pZY403为模板，用引物G6001-F和G6001-R通过PCR扩增得到片段G6001(用于整合Cas9基因的ChrXII-2位点同源左、右臂，引入NotI酶切位点，含有氨苄抗性与ura标签的质粒骨架)；以pZY403为模板，用引物G6002-F和G6002-R通过PCR扩增得到片段G6002(潮霉素抗性基因及其启动子TEF终止子TEF)；以p43802(addgene)为模板，用引物G6003-F和G6003-R通过PCR扩增得到片段G6003(基因Cas9及其启动子TEF1终止子CYC1)。通过DNA assemble(酵母组装)的方法将以上片段在酿酒酵母体内重组构建pZY600，然后在大肠杆菌内扩增，酶切验证以及测序正确后，得到pZY600。NotI酶切线性化后得到带有目的基因的片段，其序列如SEQ ID NO.7所示。
其中，质粒pZY403的构建为：以酵母30000B基因组为模板，用引物Z4031-F和Z4031-R通过PCR扩增得到片段Z4031(用于整合ERG13基因的ChrXII-2位点同源左臂)；以酵母30000B基因组为模板，用引物Z4032-F和Z4032-R通过PCR扩增得到片段Z4032(启动子)；以酵母30000B基因组为模板，用引物Z4033-F和Z4033-R通过PCR扩增得到片段Z4033(ERG13基因)；以酵母30000B基因组为模板，用引物Z4034-F和Z4034-R通过PCR扩增得到片段Z4034(终止子)；以质粒pZY402(详见CN 111019850 A)为模板，用引物Z4035-F和Z4035-R通过PCR扩增得到片段Z4035(筛选标记)；以酵母30000B基因组为模板，用引物Z4036-F和Z4036-R通过PCR扩增得到片段Z4036(用于整合ERG13基因的ChrXII-2位点同源右臂)；以pZY402(详见CN 111019850 A)为模板，用引物Z4037-F和Z4037-R通过PCR扩增得到片段Z4037(引入NotI酶切位点，含有氨苄抗性与ura标签的质粒骨架)；。通过DNA assemble(酵母组装)的方法将以上片段在酿酒酵母体内重组构建pZY403，然后在大肠杆菌内扩增，酶切验证以及测序正确后，得到pZY403。
构建上述质粒所用各引物序列见下表：
Figure BDA0003026658980000111
Figure BDA0003026658980000121
(2)质粒pZY413和gRNA质粒pZY607
质粒pZY413的作用是过表达MVA途径中的相关基因，为后续进行基因筛选提供一个高效的FPP前体合成底盘菌株。质粒pZY413的相关特征：△ChrXI-3：ERG8_pGAL1pGAL10_tHMG1_pGAL7_ERG12(图2)，用启动子GAL1、GAL7、GAL10分别控制表达基因ERG8、ERG12、THMG1，插入的染色体位点为ChrXI-3。
质粒pZY413的具体构建过程：以质粒pZY402(详见CN 111019850 A)为模板，用引物4131-F和4121-R通过PCR扩增得到片段4131(用于整合ERG8、tHMG1、ERG12基因的同源右臂，引入NotI酶切位点以及含有氨苄抗性与ura标签的质粒骨架的一部分)；以pZY402为模板，用引物4122-F和4132-R通过PCR扩增得到片段4132(含有氨苄抗性与ura标签的质粒骨架的剩余部分，用于整合ERG8、tHMG1、ERG12基因的ChrXI-3位点同源右臂的一部分，引入NotI酶切位点)；以pZY402为模板，用引物4133-F和4133-R通过PCR扩增得到片段4133(用于整合ERG8、tHMG1、ERG12基因的ChrXI-3位点同源右臂的剩余部分，目的基因ERG8、tHMG1、ERG12及其启动子终止子的一部分)；以pZY402为模板，用引物4134-F和4134-R通过PCR扩增得到片段4134(目的基因ERG8、tHMG1、ERG12及其启动子终止子的剩余部分)。通过DNAassemble的方法将以上片段在酿酒酵母体内重组构建pZY413，然后在大肠杆菌内扩增，酶切验证以及测序正确后，得到pZY413。NotI酶切线性化后得到带有目的基因的片段，其序列如SEQ ID NO.8所示。
pZY607是靶向ChrXI-3位点的gRNA质粒，用于Crispr-Cas9基因编辑技术中靶点识别切割酿酒酵母基因组DNA的工具质粒，其在质粒p43803(http://www.addgene.org/43803/)的基础上用atatgtctctaattttggaa替换了guide-RNA序列。以质粒p43803为模板，用引物6071-F和G6031-R通过PCR扩增得到片段6071(用于靶向识别的20bp靶点序列、gRNAscafold、终止子SUP4以及含有氨苄抗性与ura标签的质粒骨架的一部分)；以p43803为模板，用引物G6032-F和6072-R通过PCR扩增得到片段6072(含有氨苄抗性与ura标签的质粒骨架的剩余部分，用于靶向识别的20bp靶点序列以及启动子SNR52)。通过DNA assemble的方法将以上片段在酿酒酵母体内重组构建pZY607。
构建上述质粒所用各引物序列见下表：
Figure BDA0003026658980000122
Figure BDA0003026658980000131
(3)质粒pZY412L和gRNA质粒pZY606
质粒pZY412L的作用是过表达MVA途径中的相关基因，为后续进行基因筛选提供一个高效的FPP前体合成底盘菌株。质粒pZY412L的相关特征：△ChrXII-4：IDI1_pGAL1pGAL10_ERG10_pGAL7_MVD1(图3)，用启动子GAL1、GAL7、GAL10分别控制表达基因IDI1、MVD1、ERG10，插入的染色体位点为ChrXII-4。
质粒pZY412L的具体构建过程：用引物412-F和412-R通过PCR无模板变性退火得到片段pZY412 SPD(一段无意义的DNA序列，用于替换pZY410(详见CN 111019850 A)中的g418抗性基因，含有SalI和SpeI酶切位点的粘性末端)，此无模板变性退火PCR的反应体系为10μL，其中引物分别为4.5μL，10×T4 DNA ligase buffer为1μL，设置以下PCR程序：95℃，5min；降落PCR，95℃，1min，-1℃/cycle，70cycle；25℃，5min；12℃，∞(该片段经无模板变性退火PCR后取出可于-40℃暂存)。用SalI和SpeI酶切pZY401(详见CN 111019850 A)后胶回收获得12835bp的大片段pZY412 VPD(含有目的基因MVD1、ERG10、IDI1以及氨苄抗性与ura筛选标记的载体片段)。将片段pZY412 SPD与片段pZY412 VPD按照摩尔比3:1的比例用T4 DNA连接酶在16℃条件下过夜酶连(约20h)，酶连产物转入大肠杆菌DH10B感受态细胞扩增，酶切验证以及测序正确后，得到pZY412L的质粒。NotI酶切线性化后得到带有目的基因的片段，其序列如SEQ ID NO.9所示。
pZY606是靶向Chr XII-4位点的gRNA质粒，用于Crispr-Cas9基因编辑技术中靶点识别切割酿酒酵母基因组DNA的工具质粒，其在质粒p43803的基础上用gcttcaagaattgagtaaac替换了guide-RNA序列。以质粒p43803为模板，用引物6061-F和G6031-R通过PCR扩增得到片段6061(用于靶向识别的20bp靶点序列、gRNA scafold、终止子SUP4以及含有氨苄抗性与ura标签的质粒骨架的一部分)；以p43803为模板，用引物G6032-F和6062-R通过PCR扩增得到片段6062(含有氨苄抗性与ura标签的质粒骨架的剩余部分，用于靶向识别的20bp靶点序列以及启动子SNR52)。通过DNA assemble的方法将以上片段在酿酒酵母体内重组构建pZY606。
构建上述质粒所用各引物序列见下表：
Figure BDA0003026658980000141
(4)质粒pZY414和gRNA质粒pZY608
质粒pZY414的作用是过表达MVA途径中的相关基因，为后续进行基因筛选提供一个高效的FPP前体合成底盘菌株。质粒pZY414的相关特征：△ChrX-3：pGAL1_ERG13_pGAL10_tHMG1，用启动子GAL1、GAL10分别控制表达基因ERG13、THMG1，插入的染色体位点为ChrX-3。
质粒pZY414的具体构建过程：以酿酒酵母菌株CEN.PK2-1D(30000B)基因组为模板，用引物4141-F和4141-R通过PCR扩增得到片段4141(用于整合ERG13、tHMG1基因的ChrX-3位点同源右臂，引入NotI酶切位点)；以质粒pRS426为模板，用引物4142-F和4142-R通过PCR扩增得到片段4142(含有氨苄抗性与ura标签的质粒骨架)；以30000B基因组为模板，用引物4143-F和4143-R通过PCR扩增得到片段4143(用于整合ERG13、tHMG1基因的ChrX-3位点同源右臂，引入NotI酶切位点)；以pZY410(详见CN 111019850 A)为模板，用引物4144-F和4144-R通过PCR扩增得到片段4144(目的基因ERG13、tHMG1及其启动子终止子)。通过DNAassemble的方法将以上片段在酿酒酵母体内重组构建pZY414，然后在大肠杆菌内扩增，酶切验证以及测序正确后，得到pZY414的质粒。NotI酶切线性化后得到带有目的基因的片段，其序列如SEQ ID NO.10所示。
pZY608是靶向ChrX-3位点的gRNA质粒，用于Crispr-Cas9基因编辑技术中靶点识别切割酿酒酵母基因组DNA的工具质粒，其在质粒p43803的基础上用ctaatgtgtccgcgtttcta替换了guide-RNA序列。以质粒p43803为模板，用引物6081-F和G6031-R通过PCR扩增得到片段6081(用于靶向识别的20bp靶点序列、gRNA scafold、终止子SUP4以及含有氨苄抗性与ura标签的质粒骨架的一部分)；以p43803为模板，用引物G6032-F和6082-R通过PCR扩增得到片段6082(含有氨苄抗性与ura标签的质粒骨架的剩余部分，用于靶向识别的20bp靶点序列以及启动子SNR52)。通过DNA assemble的方法将以上片段在酿酒酵母体内重组构建pZY608，然后在大肠杆菌内扩增，酶切验证以及测序正确后，得到pZY608。
构建上述质粒所用各引物序列见下表：
Figure BDA0003026658980000151
实施例2底盘酵母菌株构建
将质粒pZY600线性化后片段转入酿酒酵母菌株CEN.PK2-1D，根据同源重组来整合到染色体上，其整合位点为ChrXII-2，筛选标记为潮霉素(Hygr)，至此成功构建背景菌株JCR1。
将质粒pZY413线性化后片段，与质粒pZY607一同转化至酿酒酵母JCR1，筛选标记为尿嘧啶(URA3)，5-FOA反筛去除质粒后获得菌株JCR23。
将质粒pZY414线性化后片段，与质粒pZY608一同转化至酿酒酵母JCR23，筛选标记为尿嘧啶(URA3)，5-FOA反筛去除质粒后获得菌株JCR25。
将质粒pZY412L线性化后片段，与质粒pZY606一同转化至酿酒酵母JCR25，筛选标记为尿嘧啶(URA3)，5-FOA反筛去除质粒后获得菌株JCR27。
Figure BDA0003026658980000152
Figure BDA0003026658980000161
实施例3含有不同来源的法尼烯合酶的表达载体的构建
(1)质粒pZY900
质粒pZY900的相关特征：△LEU2：LEU2(URA3)_TCYC1_LacZ_pGAL10pGAL1_ERG20_tERG20(图5)，用启动子GAL1、GAL10分别控制表达基因ERG20、LacZ，筛选标记为Leu2，插入的染色体位点为Leu2。
质粒pZY900具体构建过程：以酵母S288c基因组为模板，900-1F/1R、900-2F/2R、900-6F/6R、900-7F/7R分别扩增获得片段9001(Leu2的左同源臂)、9002(终止子tTDH2)、9006(基因ERG20与终止子tERG20)、9007(Leu2右臂)；以酵母30000B的基因组为模板，用引物900-3F/3R、900-5F/5R分别扩增获得片段9003(终止子tCYC1)和9005(启动子pGAL1和Pgal10)；以引物900-8F/8R扩增获得质粒骨架(引入MssI酶切位点，筛选标记)。通过DNAassemble(酵母组装)的方法将以上片段在酿酒酵母体内重组构建pZY900，然后在大肠杆菌内扩增，酶切验证以及测序正确后，得到pZY900，序列如SEQ ID NO.11所示。
构建上述质粒所用各引物序列见下表：
Figure BDA0003026658980000162
Figure BDA0003026658980000171
(2)质粒AFS系列，BFS系列
△LEU2：LEU2(URA3)_TCYC1_TS_pGAL10pGAL1_ERG20_tERG20
质粒AFS、BFS的具体构建过程：不同来源的α-法尼烯合酶、β-法尼烯合酶的氨基酸序列可在GeneBank查询(Accession number见下表)，编码各酶的核苷酸均根据酿酒酵母密码子偏爱性做了优化，优化后的序列由金斯瑞公司合成。其中，编码梨来源α-法尼烯合酶的核苷酸序列见SEQ ID NO.12，编码茶树来源α-法尼烯合酶的核苷酸序列见SEQ ID NO.13，编码洋甘菊来源β-法尼烯合酶的核苷酸序列见SEQ ID NO.14。通过PCR扩增基因使其带有BsaI酶切位点，随后与通用质粒pZY900通过Goden gate assembly方式组装获得表达载体。质粒pAFS1-6中的法尼烯合酶的来源依次为梨、樱桃、毛果杨、蓖麻、茶树、苹果；质粒pBFS1-8中法尼烯合酶的来源依次为洋甘菊、向日葵、野菊花、除虫菊、刺菜蓟、莴苣、青蒿、禾谷镰刀菌。
Figure BDA0003026658980000172
Figure BDA0003026658980000181
构建上述质粒所用各引物序列见下表：
Figure BDA0003026658980000182
Figure BDA0003026658980000191
实施例4法尼烯产生菌株的构建
分别将实施例3中构建的质粒转入菌株JCR27，筛选平板为Sc-ura(合成酵母氮源YNB6.7g/L，葡萄糖20g/L，缺尿嘧啶的混合氨基酸粉末1.3g/L，2％琼脂粉)。
这些菌株通过摇瓶发酵培养，具体方法如下：从转化平板挑取单克隆到含有5mL种子培养基的PA瓶中，种子培养基配方为：YNB(6.7g/L)，酵母粉(10g/L)，葡萄糖(20g/L)，缺尿嘧啶的混合氨基酸粉末(1.3g/L)。种子30℃摇床培养过夜后进行转接，按照初始OD600＝0.1转接，转接后覆盖20％的有机相(正十二烷或肉豆蔻酸异丙酯、棕榈酸异丙酯、白油(液体石蜡)、油酸甲酯或菜籽油)置于30℃摇床开始摇瓶发酵，发酵培养基配方为：蛋白胨(20g/L)，酵母粉(10g/L)，葡萄糖(10g/L)，半乳糖(10g/L)。发酵结束收样，有机相GCMS检测产物组成。能检测到产物的，α-法尼烯合酶包括苹果(Md-aFS)、樱桃(Pyr-aFS)、梨(Pyc-aFS)、茶树(Cas-aFS)、毛果杨(Pot-aFS)来源，β-法尼烯合酶包括青蒿(Aa-bFS)、洋甘菊(Mac-bFS)、野菊花(Chi-aFS)、禾谷镰刀菌(FgJ03939)来源。产量结果如图7所示。
据我们所知，这是第一次在微生物中表达梨、茶树来源的α-法尼烯合酶并成功检测到了产物。也是第一次在微生物中表达野菊花来源的β-法尼烯合酶并成功检测到了产物。梨来源的α-法尼烯合酶以及洋甘菊来源的β-法尼烯合酶活性更高，其中此前研究中未进行异源表达的茶树来源的α-法尼烯合酶也具有较好的性能。
实施例5法尼烯合酶突变文库的构建
采用的方式为随机突变，即设计引物，使用低保真DNA聚合酶对法尼烯合成基因进行扩增，调整镁离子浓度(2mM-12mM)，PCR扩增关键基因，获得含有突变的DNA片段，与表达载体pZY900(BsaI切后)一同通过醋酸锂转化法转入菌株JCR27通过酵母组装获得含有突变体的菌株，涂布Sc-ura筛选平板。
对于梨来源的α-法尼烯合酶的随机突变，引物为pAFS1-mutation-F和pAFS1-mutation-R；对于洋甘菊来源的β-法尼烯合酶的随机突变，引物为pBFS1-mutation-F和pBFS1-mutation-R；对茶树来源的α-法尼烯合酶的随机突变，引物为pAFS5-mutation-F和pAFS5-mutation-R。
各引物序列见下表：
Figure BDA0003026658980000201
实施例6含有法尼烯合酶突变体的菌株的高通量筛选与表征
在实施例5中的平板上挑选阳性克隆至含有Sc-ura缺陷型培养基的高通量种子培养板，挑选含有野生型法尼烯合酶的菌株作为对照，999rpm、30℃培养，菌株生长到对数生长期时，转接至含有发酵培养基的发酵培养板，加入覆盖剂减少产物的挥发，待菌株产量达到最高，加入萃取剂(正十二烷、肉豆蔻酸异丙酯、液体石蜡、白油、棕榈酸异丙酯)进行高通量产物萃取，将萃取获得的产物转移至新的平板，加入广谱显色剂(香草醛浓硫酸显色剂)，反应(65℃，20min)，酶标仪检测最大吸收值。选择吸光度高于野生型的菌株，提酵母质粒后转大肠杆菌扩增后提质粒，一代测序确定正突变位点。结果显示对于洋甘菊来源的β-法尼烯合酶，具有提高酶活性的氨基酸突变包括以下F11S、M35T、T319S、I434T、I460V、K59R、S204Y产量提升倍数见下表。
Figure BDA0003026658980000202
将所获得的有益突变进行各种组合，将所获得的质粒转入菌株JCR27，并进一步通过96孔板发酵评估，最终获得了提升倍数最大的组合，其中pBFS45(F11S、M35T、T319S、I434T、I460V)以及pBFS46(F11S、M35T、T319S、I434T、I460V、K59R、S204Y)，具有最大的产量提升，β-法尼烯合酶的最优突变体产量相较于野生型提升了近2倍，产量分别达到了430mg/L、470mg/L。两者产量相近，选择这两个突变体进行后续构建。
构建质粒pBFS45的过程如下。用引物pBFS1-1F/pBFS45-2R从pBFS20质粒(即以pBFS20质粒为模板，下同)扩增获得Mac-bFS-I460V，用引物pBFS45-3F/R从pBFS18质粒扩增获得Mac-bFS-I434T，用引物pBFS45-4F/R从pBFS15质粒扩增获得Mac-bFS-T319S，用引物pBFS45-5F/R从pBFS12质粒扩增获得Mac-bFS-M35T，用引物pBFS45-6F/R从pBFS9质粒扩增获得Mac-bFS-F11S。随后通过重叠延伸PCR将这些片段连接起来，再通过Golden Gateassembly的方法克隆到pZY900获得质粒pBFS45。
构建质粒pBFS45所用引物序列见下表：
Figure BDA0003026658980000211
构建质粒pBFS46的过程如下。用引物pBFS1-1F/pBFS46-1R从pBFS45质粒(即以pBFS45质粒为模板，下同)扩增获得Mac-bFS-A1341G，用引物pBFS46-2F/R从pBFS45质粒扩增获得Mac-bFS-S204Y，用引物pBFS46-3F/R从pBFS45质粒扩增获得Mac-bFS-K59R，用引物pBFS46-4F/pBFS45-6R从pBFS45质粒扩增获得Mac-bFS-K59R。随后通过重叠延伸PCR将这些片段连接起来，再通过Golden Gate assembly的方法克隆到pZY900获得质粒pBFS46。
构建质粒pBFS46所用引物序列见下表：
Figure BDA0003026658980000212
Figure BDA0003026658980000221
同样的突变方法对梨来源与茶树来源的α-法尼烯合酶进行突变筛选，结果梨来源获得提升的突变体结果如下。
Figure BDA0003026658980000222
茶树来源的突变体产量为野生型的1.52倍，摇瓶产量达到了257.85mg/L。
Figure BDA0003026658980000223
实施例7法尼烯高产菌株所需质粒的构建
(1)实现α-法尼烯的高产，构建质粒pAFS1-P2、pAFS1-P3、pAFS1-P4、pAFS1-P5、pAFS1-P6、pAFS1-P7，这些质粒的构建示意图见图8。
梨来源的α-法尼烯合酶基因由金斯瑞公司，按照酿酒酵母密码子优化(SEQ IDNO.12)并合成。所获得的质粒用于Pyc-afs基因扩增所需的模板。
用引物pAFS1-P2-1F/R、pAFS1-P2-3F/R、pAFS1-P2-5F/R、pAFS1-P2-7F/R、pAFS1-P2-8F/R从CEN.PK2-1D(CEN.PK2-1D酵母基因组DNA，下同)扩增获得Ura3 left arm、tCYC1、pGAL10-pGAL1、tPGK1、Ura3 right arm。用引物pAFS1-P2-2F/R从pRS423质粒扩增获得His3marker，用引物pAFS1-P2-4F/R从S288C(S288C酵母基因组DNA，下同)扩增获得tHMG1，用引物pAFS1-P2-6F/R扩增获得Pyc-aFS，用引物pAFS1-P2-9F/R从pRS426质粒扩增获得质粒骨架，随后这些片段通过DNA assemble的方法组装获得pAFS1-P2。
用引物pAFS1-P2-1F/R、pAFS1-P2-3F/pAFS1-P3-3R、pAFS1-P3-5F/pAFS1-P2-5R、pAFS1-P2-7F/R、pAFS1-P2-8F/R从CEN.PK2-1D扩增获得Ura3 left arm、tCYC1、pGAL10-pGAL1、tPGK1、Ura3 right arm，用引物pAFS1-P2-2F/R从pRS423扩增获得His3 marker，用引物pAFS1-P2-6F/R扩增获得Pyc-aFS，用引物pAFS1-P2-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pAFS1-P3。
用引物pAFS1-P2-1F/R、pAFS1-P2-3F/pAFS1-P4-3R、pAFS1-P4-5F/pAFS1-P2-5R、pAFS1-P2-7F/R、pAFS1-P2-8F/R从CEN.PK2-1D扩增获得Ura3 left arm、tCYC1、pGAL10-pGAL1、tPGK1、Ura3 right arm。用引物pAFS1-P2-2F/R从pRS423扩增获得His3 marker，用引物pAFS1-P4-4F/R、pAFS1-P2-6F/R扩增获得Pyc-aFS，用引物pAFS1-P2-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pAFS1-P4。
用引物pAFS1-P5-1F/R、pAFS1-P5-3F/R、pAFS1-P5-5F/R、pAFS1-P5-7F/R、pAFS1-P5-8F/R从CEN.PK2-1D扩增获得YPRCdelta15 left arm、tCYC1、pGAL10-pGAL1、tPGK1、YPRCdelta15 right arm。用引物pAFS1-P5-2F/R从pRS424扩增获得Trp1 marker，用引物pAFS1-P5-4F/R、pAFS1-P5-6F/R扩增获得Pyc-aFS，用引物pAFS1-P5-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pAFS1-P5。
用引物pAFS1-P5-1F/R、pAFS1-P6-5F/pAFS1-P5-5R、pAFS1-P5-7F/R、pAFS1-P5-8F/R从CEN.PK2-1D扩增获得YPRCdelta15 left arm、pGAL10-pGAL1、tPGK1、YPRCdelta15right arm。用引物pAFS1-P5-2F/pAFS1-P6-2R从pRS424扩增获得Trp1 marker，用引物pAFS1-P6-3F/R从S288C扩增获得tGPM1，用引物pAFS1-P5-6F/R扩增获得Pyc-aFS，用引物pAFS1-P5-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pAFS1-P6。
用引物pAFS1-P5-1F/R、pAFS1-P5-3F/pAFS1-P7-3R、pAFS1-P7-5F/pAFS1-P5-5R、pAFS1-P5-7F/R、pAFS1-P5-8F/R从CEN.PK2-1D扩增获得YPRCdelta15 left arm、tCYC1、pGAL10-pGAL1、tPGK1、YPRCdelta15 right arm。用引物pAFS1-P5-2F/R从pRS424扩增获得Trp1 marker，用引物pAFS1-P7-4F/R从S288C扩增获得tHMG1，用引物pAFS1-P5-6F/R扩增获得Pyc-aFS，用引物pAFS1-P5-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pAFS1-P7。
构建上述质粒所用引物序列见下表：
Figure BDA0003026658980000231
Figure BDA0003026658980000241
Figure BDA0003026658980000251
Figure BDA0003026658980000261
Figure BDA0003026658980000271
Figure BDA0003026658980000281
pAFS38-P2、pAFS38-P3、pAFS38-P4、pAFS38-P5、pAFS38-P6、pAFS38-P7的构建过程与pAFS1-P2、pAFS1-P3、pAFS1-P4、pAFS1-P5、pAFS1-P6、pAFS1-P7的构建过程一样，只是将构建pAFS1-P2、pAFS1-P3、pAFS1-P4、pAFS1-P5、pAFS1-P6、pAFS1-P7时的模板ppAFS1替换为pAFS38。
(2)实现β-法尼烯的高产，构建质粒pBFS45、pBFS45-P2、pBFS45-P3、pBFS45-P4、pBFS45-P5、pBFS45-P6、pBFS45-P7。构建质粒pBFS46、pBFS46-P2、pBFS46-P3、pBFS46-P4、pBFS46-P5、pBFS46-P6、pBFS46-P7。
质粒pBFS45的构建见实施例6。
用引物pAFS1-P2-1F/R、pAFS1-P2-3F/R、pAFS1-P2-5F/pBFS45-P2-5R、pBFS45-P2-7F/pAFS1-P2-7R、pAFS1-P2-8F/R从CEN.PK2-1D扩增获得Ura3 left arm、tCYC1、pGAL10-pGAL1、tPGK1、Ura3 right arm。用引物pAFS1-P2-2F/R从pRS423扩增获得His3 marker，用引物pAFS1-P2-4F/R从S288C扩增获得tHMG1，用引物pBFS45-P2-6F/pBFS45-P2-6R从pBFS45扩增获得Mac-bFS(F11S，M35T，T319S，I434T，I460V)，用引物pAFS1-P2-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pBFS45-P2。
用引物pAFS1-P2-1F/R、pAFS1-P2-3F/pAFS1-P3-3R、pAFS1-P3-5F/pBFS45-P3-5R、pBFS45-P3-7F/pAFS1-P2-7R、pAFS1-P2-8F/R从CEN.PK2-1D扩增获得Ura3 left arm、tCYC1、pGAL10-pGAL1、tPGK1、Ura3 right arm，用引物pAFS1-P2-2F/R从pRS423扩增获得His3 marker，用引物pBFS45-P3-6F/R扩增获得Mac-bFS(F11S，M35T，T319S，I434T，I460V)，用引物pAFS1-P2-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pBFS45-P3。
用引物pAFS1-P2-1F/R、pAFS1-P2-3F/pBFS45-P4-3R、pBFS45-P4-5F/pBFS45-P4-5R、pBFS45-P4-7F/pAFS1-P2-7R、pAFS1-P2-8F/R从CEN.PK2-1D扩增获得Ura3 left arm、tCYC1、pGAL10-pGAL1、tPGK1、Ura3 right arm。用引物pAFS1-P2-2F/R从pRS423扩增获得His3 marker，用引物pBFS45-P4-4F/R、pBFS45-P4-6F/R从pBFS45扩增获得Mac-bFS(F11S，M35T，T319S，I434T，I460V)，用引物pAFS1-P2-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pBFS45-P4。
用引物pAFS1-P5-1F/R、pAFS1-P5-3F/pAFS1-P7-3R、pAFS1-P7-5F/pBFS45-P5-5R、pBFS45-P5-7F/pAFS1-P5-7R、pAFS1-P5-8F/R从CEN.PK2-1D扩增获得YPRCdelta15 leftarm、tCYC1、pGAL10-pGAL1、tPGK1、YPRCdelta15 right arm。用引物pAFS1-P5-2F/R从pRS424扩增获得Trp1 marker，用引物pAFS1-P7-4F/pAFS1-P7-4R从S288C扩增获得tHMG1，用引物pBFS45-P5-6F/R从pBFS45扩增获得Mac-bFS(F11S，M35T，T319S，I434T，I460V)，用引物pAFS1-P5-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pBFS45-P5。
用引物pAFS1-P5-1F/R、pAFS1-P6-5F/pBFS45-P6-5R、pBFS45-P6-7F/pAFS1-P5-7R、pAFS1-P5-8F/R从CEN.PK2-1D扩增获得YPRCdelta15 left arm、pGAL10-pGAL1、tPGK1、YPRCdelta15 right arm。用引物pAFS1-P5-2F/pAFS1-P6-2R从pRS424扩增获得Trp1marker、用引物pAFS1-P6-3F/R从S288C扩增获得tGPM1，用引物pBFS45-P6-6F/R从pBFS45扩增获得Mac-bFS(F11S，M35T，T319S，I434T，I460V)，用引物pAFS1-P5-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pBFS45-P6。
用引物pAFS1-P5-1F/R、pAFS1-P5-3F/pBFS45-P7-3R、pBFS45-P7-5F/R、pBFS45-P7-7F/pAFS1-P5-7R、pAFS1-P5-8F/R从CEN.PK2-1D扩增获得YPRCdelta15 left arm、tCYC1、pGAL10-pGAL1、tPGK1、YPRCdelta15 right arm。用引物pAFS1-P5-2F/R从pRS424扩增获得Trp1 marker，用引物pBFS45-P7-4F/R、pBFS45-P7-6F/R从pBFS45扩增获得Mac-bFS(F11S，M35T，T319S，I434T，I460V)，用引物pAFS1-P5-9F/R从pRS426扩增获得质粒骨架，随后这些片段组装获得pBFS45-P7。
质粒pBFS46的构建见实施例6。
pBFS46-P2、pBFS46-P3、pBFS46-P4、pBFS46-P5、pBFS46-P6、pBFS46-P7的构建过程与pBFS45-P2、pBFS45-P3、pBFS45-P4、pBFS45-P5、pBFS45-P6、pBFS45-P7的构建过程一样，只是将构建pBFS45-P2、pBFS45-P3、pBFS45-P4、pBFS45-P5、pBFS45-P6、pBFS45-P7时的模板pBFS45替换为pBFS46。
其中，按照酿酒酵母密码子优化的含F11S、M35T、T319S、I434T、I460V突变的Mac-bFS的核苷酸序列如SEQ ID NO.16所示；按照酿酒酵母密码子优化的含F11S、M35T、T319S、I434T、I460V、K59R、S204Y突变的Mac-bFS的核苷酸序列如SEQ ID NO.17所示。
构建上述质粒所用引物序列见下表：
Figure BDA0003026658980000301
Figure BDA0003026658980000311
Figure BDA0003026658980000321
Figure BDA0003026658980000331
Figure BDA0003026658980000341
Figure BDA0003026658980000351
实施例8β-法尼烯高产菌株的构建
用Mssi线性化质粒pBFS45，回收带有目的基因的片段，转化至酿酒酵母JCR27，根据同源重组来整合到染色体上，其整合位点为LEU2，筛选标记为亮氨酸，构建菌株JVA122。线性化质粒pBFS45-P2、pBFS45-P3、pBFS45-P4，回收带有目的基因的片段，给分别转化至酿酒酵母JVA122，根据同源重组来整合到染色体上，其整合位点为URA3，筛选标记为组氨酸，构建菌株JVA124、JVA125、JVA129。线性化pBFS45-P5，回收带有目的基因的片段，分别转化至酿酒酵母JVA124、JVA125、JVA129，根据同源重组来整合到染色体上，其整合位点为YPRCdelta15，筛选标记为色氨酸，构建菌株JVA127、JVA130、JVA134。线性化pBFS45-P6，回收带有目的基因的片段，分别转化至酿酒酵母JVA124、JVA125、JVA129，根据同源重组来整合到染色体上，其整合位点为YPRCdelta15，筛选标记为色氨酸，构建菌株JVA128、JVA131、JVA135。线性化pBFS45-P7，回收带有目的基因的片段，分别转化至酿酒酵母JVA124、JVA125、JVA129，根据同源重组来整合到染色体上，其整合位点为YPRCdelta15，筛选标记为色氨酸，构建菌株JVA127、JVA138、JVA139。经过摇瓶发酵，这些菌株都能展现出良好得性能，β-法尼烯得产量均能达到较高水平，产量范围为200mg/L-800mg/L(图9)。选择其中产量最高的菌株JVA139进行GAL80基因的敲除(敲除盒pZY521的构建：以酵母30000B的基因组为模板，使用引物5201-F/5211-R扩增获得GAL80的左同源臂；以pZY900为模板，使用引物5212-F/5212-R扩增获得筛选标记URA3；使用引物5213-F/5203-R扩增获得GAL80的右同源臂；)以消除发酵过程中半乳糖的使用，构建获得菌株JVA140。
上述引物序列见下表：
Figure BDA0003026658980000352
Figure BDA0003026658980000361
由图9可见，含有不同基因个数(Thmg1与法尼烯合酶)的菌株之间产量存在明显差异，在不敲除gal80的菌株中，在野生型菌株CEN.PK2-1D菌株的基础上，含有5个β-法尼烯合酶，含有额外的MVA途径基因(ERG10、ERG13、THMG1、ERG12、ERG8、MVD1、IDI1)以及额外一个ERG20，额外一个Thmg1的菌株具有最高的β-法尼烯产量。因此调整Thmg1与法尼烯合酶的比例对于高产具有很大意义。
同样的，线性化质粒pBFS46、pBFS46-P4、pBFS46-P7，依次转入菌株JCR27，获得菌株JVA139-pBFS46，进行GAL80基因的敲除构建获得菌株JVA140-pBFS46。
实施例9α-法尼烯高产菌株的构建
可以通过传统的同源重组，类似于实施例8中构建β-法尼烯高产菌株的方式进行α-法尼烯高产菌株的构建，构建获得相应的菌株JSA126-JSA138。还可以以获得的高产β-法尼烯的菌株为底盘，采用crispr-cas9基因编辑技术，使用α-法尼烯合酶一次性替换β-法尼烯合酶，获得高产α-法尼烯合酶菌株。质粒pAFS-gRNA的构建过程：以质粒pKlURA3为模板，用引物pAFS-gRNA-F和pAFS-gRNA-R通过PCR扩增得到PD pAFS-gRNA，以pCAS9为载体骨架，用Golden Gate的方法，构建质粒pAFS-gRNA，酶切验证以及测序正确后，得到pAFS-gRNA。
上述引物序列见下表：
引物 序列(5’-3’)
pAFS-gRNA-F aaaggtctcaGATCGCTGGCATCAACAATGGGAAGTTTTAGAGCTAGAAATAGCA
pAFS-gRNA-R AAAGGTCTCAAAACTCTAGACTTTTTCGATGATGTAGTTTCT
线性化质粒pAFS1、pAFS1-P4、pAFS1-P5，回收带有目的基因的片段，与β-法尼烯高产菌株构建质粒相对应(pAFS1 vs pBFS45、pAFS1-P4 vs pBFS45-P4、pAFS1-P5 vspBFS45-P7)、与pAFS-gRNA一同转入菌株JVA139，筛选标记为尿嘧啶，长出单克隆后，划线5-FOA平板，构建得到菌株JSA132Crispr。
菌株JSA132Crispr经过摇瓶发酵，α-法尼烯的产量均能达到较高水平，产量为473±35mg/L。进行GAL80基因的敲除(敲除盒pZY521的构建：以酵母30000B的基因组为模板，使用引物5201-F/5211-R扩增获得GAL80的左同源臂；以pZY900为模板，使用引物5212-F/5212-R扩增获得筛选标记URA3；使用引物5213-F/5203-R扩增获得GAL80的右同源臂；)以消除发酵过程中半乳糖的使用，构建获得菌株JSA145，摇瓶产量为520mg/L。菌株JSA145在野生型菌株CEN.PK2-1D的基础上，含有5个α-法尼烯合酶(Pyc-aFS)，含有额外的MVA途径基因(ERG10、ERG13、THMG1、ERG12、ERG8、MVD1、IDI1)以及额外一个ERG20，额外一个Thmg1，且敲除了GAL80基因。
同样的，线性化质粒pAFS38、pAFS38-P4、pAFS38-P5，回收带有目的基因的片段，与β-法尼烯高产菌株构建质粒相对应(pAFS38 vs pBFS45、pAFS38-P4 vs pBFS45-P4、pAFS38-P5 vs pBFS45-P7)、与pAFS-gRNA一同转入菌株JVA139，筛选标记为尿嘧啶，长出单克隆后，划线5-FOA平板，构建得到菌株JSA132-pAFS38-Crispr，摇瓶产量为580mg/L。进行GAL80基因的敲除构建获得菌株JSA145-pAFS38-Crispr，摇瓶产量达到598mg/L。菌株JSA145-pAFS38-Crispr在野生型菌株CEN.PK2-1D的基础上，含有5个α-法尼烯合酶(Pyc-aFS(G252E))基因(SEQ ID NO.18)，含有额外的MVA途径基因(ERG10、ERG13、THMG1、ERG12、ERG8、MVD1、IDI1)以及额外一个ERG20，额外一个Thmg1，且敲除了GAL80基因。
实施例10法尼烯高产菌株的发酵罐发酵
参照文献(van Hoek,P.；de Hulster,E.；van Di jken,J.P.；Pronk,J.T.Fermentative capacity in high-cell-density fed-batch cultures of baker’syeast.Biotechnol.Bioeng.2000,68,517-523.)中所记载的发酵培养基，对所构建的菌株，α-法尼烯合成菌株、β-法尼烯菌株(菌株JSA145、JSA145-pAFS38-Crispr、JVA140、JVA140-pBFS46)进行分批补料发酵，在发酵过程中添加覆盖剂以实现原位萃取，覆盖剂可为正十二烷、肉豆蔻酸异丙酯、棕榈酸异丙酯、液体石蜡、白油。发酵过程控制溶氧在20％以上，pH为5，葡萄糖浓度为1-2g/L，乙醇浓度为5-10g/L。最终在发酵罐上，菌株JSA145的α-法尼烯产量达到了28g/L，菌株JSA145-pAFS38-Crispr的α-法尼烯产量达到了36g/L。菌株JVA140的β-法尼烯产量达到了50g/L，菌株JVA140-pBFS46的β-法尼烯产量达到了55g/L。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
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Asn Ala Ser Glu Glu Ile Ala Arg Lys Asn Ile Lys Gly Met Ile Asp
500 505 510
Asn Ala Trp Lys Lys Val Asn Gly Lys Cys Phe Thr Thr Asn Gln Val
515 520 525
Pro Phe Leu Ser Ser Phe Met Asn Asn Ala Thr Asn Met Ala Arg Val
530 535 540
Ala His Ser Leu Tyr Lys Asp Gly Asp Gly Phe Gly Asp Gln Glu Lys
545 550 555 560
Gly Pro Arg Thr His Ile Leu Ser Leu Leu Phe Gln Pro Leu Val Asn
565 570 575
<210> 4
<211> 576
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Glu Phe Arg Val His Leu His Ala Asp His Glu Gln Lys Ile Leu
1 5 10 15
Gln Asn Gln Met Lys Pro Glu His Glu Ala Ser Tyr Leu Ile Asn Gln
20 25 30
Arg Arg Ser Ala Asn Tyr Lys Pro Asn Ile Trp Lys Asn Asp Phe Leu
35 40 45
Asp Gln Ser Leu Ile Ser Lys Tyr Asp Gly Asp Glu Tyr Arg Lys Leu
50 55 60
Ser Glu Lys Leu Ile Glu Glu Val Lys Ile Tyr Ile Ser Ala Glu Thr
65 70 75 80
Lys Asp Leu Val Ala Lys Leu Glu Leu Ile Asp Ser Val Arg Lys Leu
85 90 95
Gly Leu Ala Asn His Phe Glu Lys Glu Ile Lys Glu Ala Leu Asp Gly
100 105 110
Ile Ala Ala Ile Glu Ser Asp Asn Leu Gly Thr Arg Asp Asp Leu Tyr
115 120 125
Gly Thr Ala Leu His Phe Lys Ile Leu Arg Gln His Gly Tyr Lys Val
130 135 140
Ser Gln Asp Ile Phe Gly Arg Phe Met Asp Glu Lys Asp Thr Leu Glu
145 150 155 160
Asn His His Phe Ala His Leu Lys Gly Met Leu Glu Leu Phe Glu Ala
165 170 175
Ser Asn Leu Gly Phe Glu Gly Glu Asp Ile Leu Asp Glu Ala Lys Ala
180 185 190
Ser Leu Thr Leu Ala Leu Arg Asp Ser Gly His Ile Cys Tyr Pro Asp
195 200 205
Ser Asn Leu Ser Arg Asp Val Val His Ser Leu Glu Leu Pro Ser His
210 215 220
Arg Arg Val Gln Trp Phe Asp Val Lys Trp Gln Ile Asn Ala Tyr Glu
225 230 235 240
Lys Asp Ile Cys Arg Val Asn Ala Thr Leu Leu Glu Leu Ala Lys Leu
245 250 255
Asn Phe Asn Met Val Gln Ala Gln Leu Gln Lys Asp Leu Arg Glu Ala
260 265 270
Ser Lys Trp Trp Ala Asn Leu Gly Ile Ala Asp Asn Leu Lys Phe Ala
275 280 285
Arg Asp Arg Leu Val Glu Cys Phe Ala Cys Ala Val Gly Val Ala Phe
290 295 300
Glu Pro Glu Tyr Ser Ser Phe Arg Ile Cys Leu Thr Lys Val Ile Asn
305 310 315 320
Leu Val Leu Ile Ile Asp Asp Val Tyr Asp Ile Tyr Gly Ser Glu Glu
325 330 335
Glu Leu Lys His Phe Thr Asn Ala Val Asp Arg Trp Asp Ser Arg Glu
340 345 350
Thr Glu Gln Leu Pro Glu Cys Met Lys Met Cys Phe Gln Val Leu Tyr
355 360 365
Asn Thr Thr Cys Glu Ile Ala His Glu Ile Glu Lys Asp Asn Gly Trp
370 375 380
Asn Gln Val Leu Pro Gln Leu Thr Lys Val Trp Ala Asp Phe Cys Lys
385 390 395 400
Ala Leu Leu Val Glu Ala Glu Trp Tyr Asn Lys Ser His Ile Pro Thr
405 410 415
Leu Glu Glu Tyr Leu Arg Asn Gly Cys Asp Ser Ser Ser Val Ser Ile
420 425 430
Leu Leu Val His Ser Phe Phe Ser Ile Thr His Glu Gly Thr Lys Glu
435 440 445
Met Ala Asp Phe Leu His Lys Asn Glu Asp Leu Leu Tyr Asn Leu Ser
450 455 460
Leu Ile Val Arg Leu Asn Asn Asp Leu Gly Thr Ser Ala Ala Glu Gln
465 470 475 480
Glu Arg Gly Asp Ser Pro Ser Ser Ile Val Cys Tyr Met Arg Glu Val
485 490 495
Asn Ala Ser Glu Glu Ile Ala Arg Lys Asn Ile Lys Gly Met Ile Asp
500 505 510
Asn Ala Trp Lys Lys Val Asn Gly Lys Cys Phe Thr Thr Asn Gln Val
515 520 525
Pro Phe Leu Ser Ser Phe Met Asn Asn Ala Thr Asn Met Ala Arg Val
530 535 540
Ala His Ser Leu Tyr Lys Asp Gly Asp Gly Phe Gly Asp Gln Glu Lys
545 550 555 560
Gly Pro Arg Thr His Ile Leu Ser Leu Leu Phe Gln Pro Leu Val Asn
565 570 575
<210> 5
<211> 574
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ser Thr Ile Pro Val Ser Ser Val Ser Ser Ser Ser Ser Ala Ser
1 5 10 15
Pro Leu Val Leu Asp Asp Lys Leu Ser Thr Lys Gln Asp Val Val Arg
20 25 30
His Thr Thr Asn Phe Ser Ala Ser Ile Trp Gly Asp Gln Phe Leu Thr
35 40 45
Tyr His Glu Pro Glu Asp Leu Val Ile Lys Lys Gln Gln Val Glu Gln
50 55 60
Leu Lys Glu Glu Val Lys Lys Glu Leu Met Ala Ile Lys Gly Ser Asn
65 70 75 80
Asp Pro Gln Gln His Ile Lys Leu Met Glu Leu Ile Asp Ser Val Gln
85 90 95
Arg Leu Gly Ile Ala Tyr His Phe Glu Glu Glu Ile Glu Glu Ala Leu
100 105 110
Gln His Ile His Val Thr Tyr Gly Glu His Trp Val Asp Lys Glu Asn
115 120 125
Leu Gln Ser Val Ser Leu Trp Phe Arg Leu Leu Arg Gln Gln Gly Phe
130 135 140
Asn Val Ser Ser Gly Val Phe Lys Asp Tyr Met Asp Glu Lys Gly Asn
145 150 155 160
Phe Lys Glu Ser Leu Cys Asn Asp Ala Gln Gly Ile Leu Ala Leu Tyr
165 170 175
Glu Ala Ala Tyr Met Arg Val Glu Gly Glu Thr Arg Leu Asp Lys Ala
180 185 190
Leu Glu Phe Thr Lys Val His Leu Asp Ile Ile Ser Lys Asp Pro Ser
195 200 205
Cys Asp Ser Tyr Leu Arg Thr Arg Val His Gln Ala Leu Lys Gln Pro
210 215 220
Leu Arg Arg Arg Leu Ala Arg Ile Glu Ala Leu His Tyr Met Pro Val
225 230 235 240
Tyr Gln Gln Asp Ser Ser His Asn Glu Val Leu Leu Lys Leu Ala Lys
245 250 255
Val Asp Phe Ser Val Leu Gln Ser Met His Lys Lys Glu Leu Ser His
260 265 270
Ile Cys Lys Trp Trp Lys Asp Leu Asp Leu Gln Asn Lys Leu Pro Tyr
275 280 285
Val Arg Asp Arg Val Val Glu Gly Tyr Phe Trp Ile Leu Ser Ile Tyr
290 295 300
Tyr Glu Pro Gln His Ala Arg Thr Arg Met Phe Leu Met Lys Ser Cys
305 310 315 320
Met Trp Leu Val Val Leu Asp Asp Thr Phe Asp Asn Tyr Gly Thr Tyr
325 330 335
Glu Glu Leu Glu Ile Phe Ala Gln Ala Val Glu Arg Trp Ser Ile Ser
340 345 350
Cys Met Asp Met Leu Pro Glu Tyr Met Lys Leu Ile Tyr Gln Glu Leu
355 360 365
Val Asn Leu His Val Asp Met Glu Glu Ser Leu Glu Lys Glu Gly Lys
370 375 380
Thr Tyr Gln Ile His Tyr Val Lys Glu Met Ala Lys Glu Leu Val Arg
385 390 395 400
Asn Tyr Leu Val Glu Ala Arg Trp Leu Lys Glu Gly Tyr Met Pro Thr
405 410 415
Leu Glu Glu Tyr Met Ser Val Ser Met Val Thr Gly Thr Tyr Gly Leu
420 425 430
Met Thr Ala Arg Ser Tyr Val Gly Arg Asp Asp Ile Val Thr Glu Asp
435 440 445
Thr Phe Lys Trp Val Ser Ser Tyr Pro Pro Ile Val Lys Ala Ser Cys
450 455 460
Val Ile Val Arg Leu Met Asp Asp Ile Val Ser His Lys Glu Glu Gln
465 470 475 480
Glu Arg Gly His Val Ala Ser Ser Ile Glu Cys Tyr Ser Lys Glu Ser
485 490 495
Gly Ala Thr Glu Glu Glu Ala Cys Glu Tyr Ile Ser Ser Lys Val Glu
500 505 510
Asp Ala Trp Lys Val Ile Asn Arg Glu Ser Leu Arg Pro Thr Ala Val
515 520 525
Pro Phe Pro Leu Leu Met Pro Ala Ile Asn Leu Ala Arg Met Cys Glu
530 535 540
Val Leu Tyr Ser Val Asn Asp Gly Phe Thr His Ala Glu Gly Asp Met
545 550 555 560
Lys Ser Tyr Met Lys Ser Tyr Phe Val His Pro Met Val Ile
565 570
<210> 6
<211> 574
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ser Thr Ile Pro Val Ser Ser Val Ser Ser Ser Ser Ser Ala Ser
1 5 10 15
Pro Leu Val Leu Asp Asp Lys Leu Ser Thr Lys Gln Asp Val Val Arg
20 25 30
His Thr Thr Asn Phe Ser Ala Ser Ile Trp Gly Asp Gln Phe Leu Thr
35 40 45
Tyr His Glu Pro Glu Asp Leu Val Ile Lys Arg Gln Gln Val Glu Gln
50 55 60
Leu Lys Glu Glu Val Lys Lys Glu Leu Met Ala Ile Lys Gly Ser Asn
65 70 75 80
Asp Pro Gln Gln His Ile Lys Leu Met Glu Leu Ile Asp Ser Val Gln
85 90 95
Arg Leu Gly Ile Ala Tyr His Phe Glu Glu Glu Ile Glu Glu Ala Leu
100 105 110
Gln His Ile His Val Thr Tyr Gly Glu His Trp Val Asp Lys Glu Asn
115 120 125
Leu Gln Ser Val Ser Leu Trp Phe Arg Leu Leu Arg Gln Gln Gly Phe
130 135 140
Asn Val Ser Ser Gly Val Phe Lys Asp Tyr Met Asp Glu Lys Gly Asn
145 150 155 160
Phe Lys Glu Ser Leu Cys Asn Asp Ala Gln Gly Ile Leu Ala Leu Tyr
165 170 175
Glu Ala Ala Tyr Met Arg Val Glu Gly Glu Thr Arg Leu Asp Lys Ala
180 185 190
Leu Glu Phe Thr Lys Val His Leu Asp Ile Ile Tyr Lys Asp Pro Ser
195 200 205
Cys Asp Ser Tyr Leu Arg Thr Arg Val His Gln Ala Leu Lys Gln Pro
210 215 220
Leu Arg Arg Arg Leu Ala Arg Ile Glu Ala Leu His Tyr Met Pro Val
225 230 235 240
Tyr Gln Gln Asp Ser Ser His Asn Glu Val Leu Leu Lys Leu Ala Lys
245 250 255
Val Asp Phe Ser Val Leu Gln Ser Met His Lys Lys Glu Leu Ser His
260 265 270
Ile Cys Lys Trp Trp Lys Asp Leu Asp Leu Gln Asn Lys Leu Pro Tyr
275 280 285
Val Arg Asp Arg Val Val Glu Gly Tyr Phe Trp Ile Leu Ser Ile Tyr
290 295 300
Tyr Glu Pro Gln His Ala Arg Thr Arg Met Phe Leu Met Lys Ser Cys
305 310 315 320
Met Trp Leu Val Val Leu Asp Asp Thr Phe Asp Asn Tyr Gly Thr Tyr
325 330 335
Glu Glu Leu Glu Ile Phe Ala Gln Ala Val Glu Arg Trp Ser Ile Ser
340 345 350
Cys Met Asp Met Leu Pro Glu Tyr Met Lys Leu Ile Tyr Gln Glu Leu
355 360 365
Val Asn Leu His Val Asp Met Glu Glu Ser Leu Glu Lys Glu Gly Lys
370 375 380
Thr Tyr Gln Ile His Tyr Val Lys Glu Met Ala Lys Glu Leu Val Arg
385 390 395 400
Asn Tyr Leu Val Glu Ala Arg Trp Leu Lys Glu Gly Tyr Met Pro Thr
405 410 415
Leu Glu Glu Tyr Met Ser Val Ser Met Val Thr Gly Thr Tyr Gly Leu
420 425 430
Met Thr Ala Arg Ser Tyr Val Gly Arg Asp Asp Ile Val Thr Glu Asp
435 440 445
Thr Phe Lys Trp Val Ser Ser Tyr Pro Pro Ile Val Lys Ala Ser Cys
450 455 460
Val Ile Val Arg Leu Met Asp Asp Ile Val Ser His Lys Glu Glu Gln
465 470 475 480
Glu Arg Gly His Val Ala Ser Ser Ile Glu Cys Tyr Ser Lys Glu Ser
485 490 495
Gly Ala Thr Glu Glu Glu Ala Cys Glu Tyr Ile Ser Ser Lys Val Glu
500 505 510
Asp Ala Trp Lys Val Ile Asn Arg Glu Ser Leu Arg Pro Thr Ala Val
515 520 525
Pro Phe Pro Leu Leu Met Pro Ala Ile Asn Leu Ala Arg Met Cys Glu
530 535 540
Val Leu Tyr Ser Val Asn Asp Gly Phe Thr His Ala Glu Gly Asp Met
545 550 555 560
Lys Ser Tyr Met Lys Ser Tyr Phe Val His Pro Met Val Ile
565 570
<210> 7
<211> 9255
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gacattgaac tcttggccga ctcttctgat gaggctttcg aaaaatctac tttcattaac 60
acaattgagt tcattgtgca cgatatttgg gtcgaaacgt tgataaagaa cataaactta 120
ttacaaatgt ttgatgcaga tttgaaatgc tacaatgaca agtacataat ttgcaagcta 180
aagggtcagt atccctccac aaatatagtg gacataatgt gccgtttgaa gcatttttcc 240
aattctattt tggaaacttt caaattcgga atcgagctaa aagaacaaga tcaatgccac 300
aatagaaaca ctattatcaa ctacgttctt ttcagtagag tattttcaac gattgtttta 360
gagattcaaa aatgctttat cttgatcgtt aagttcatgt attctgtcaa ttttttagaa 420
aaattttccg atgaagtatt tttgtcattt attgaaattc tcataaaaat tgtctttgag 480
caccagattc cgcagttatt tttgggaatt gatgaaatta ttcaactctg gttaaagaac 540
aatgaaggaa aacggcaaca actcttgagc gcttggtgca acggaactgt tcaagatatg 600
aagcaaagcc aacaaagaga gtcctcaaac gcagaatctg aatcaatcac ttcaagtaca 660
gaagaggacg aagaaggcct gcaattcaat aaatgggatg tgattgaacc atttattgat 720
aatatcaagg ctttgaatca atcaaaatcc catatgtgac gcagcgataa aaccgcggca 780
tgcaaacatc tacacaatta gcaagggcaa tccatatttt gtcttttcgc gccctggaaa 840
ggcctaagta atgtcgtaaa cgcattctat ctgtacttca actctcctct gtgcattggt 900
ttgtgcaaat cacattttac gatactgcca gatatatgca aaaagagaaa accaagggac 960
cagaacaaag caaaattacg atattcttcg aattccttcg tgcttgacta agacaaaggg 1020
atggacgtag cgatttttag cgggccaaga actggttccg aaaaagcaca ggtacaccga 1080
accctcagct aaggagggac agcaccgatg cggaaggaca aactttcttt ttgcctatca 1140
cagtatctta tcgagctaac tattttcgac acacatgaaa aagcagaaat attaacgaaa 1200
aagaaaagaa agaccatgtc atgtacgggc aatcagaatc tgtaacaagc gccatttttt 1260
tttctgtatc gggccctcct tactgctctc cttccgtgta acgcgttatg gctgcaggtc 1320
gacaaccctt aatataactt cgtataatgt atgctatacg aagttattag gtctagagat 1380
ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag gcccagaata 1440
ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca 1500
tttagcccat acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg 1560
cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac gctcccctca 1620
cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt 1680
tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc 1740
acatcacatc cgaacataaa caaccatggg taaaaagcct gaactcaccg cgacgtctgt 1800
cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg 1860
cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa 1920
tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc 1980
gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat 2040
ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt 2100
tctgcagccg gtcgcggagg caatggatgc gatcgctgcg gccgatctta gccagacgag 2160
cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat 2220
atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag 2280
tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt 2340
ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat 2400
aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa 2460
catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg 2520
gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct 2580
tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg 2640
tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg 2700
cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg 2760
acgccccagc actcgtccga gggcaaagga ataatcagta ctgacaataa aaagattctt 2820
gttttcaaga acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca 2880
aatgttagcg tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta 2940
agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg 3000
tcgattcgat actaacgagc tcatagcttc aaaatgtttc tactcctttt ttactcttcc 3060
agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc acagcatact 3120
aaatttcccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa ggtttggaaa 3180
agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa aaatttttat 3240
cacgtttctt tttcttgaaa attttttttt tgattttttt ctctttcgat gacctcccat 3300
tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt 3360
ctattacaac tttttttact tcttgctcat tagaaagaaa gcatagcaat ctaatctaag 3420
ttttctagaa ctagtggatc ccccgggaaa aatggacaag aagtactcca ttgggctcga 3480
tatcggcaca aacagcgtcg gttgggccgt cattacggac gagtacaagg tgccgagcaa 3540
aaaattcaaa gttctgggca ataccgatcg ccacagcata aagaagaacc tcattggcgc 3600
cctcctgttc gactccgggg agacggccga agccacgcgg ctcaaaagaa cagcacggcg 3660
cagatatacc cgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat 3720
ggctaaggtg gatgactctt tcttccatag gctggaggag tcctttttgg tggaggagga 3780
taaaaagcac gagcgccacc caatctttgg caatatcgtg gacgaggtgg cgtaccatga 3840
aaagtaccca accatatatc atctgaggaa gaagcttgta gacagtactg ataaggctga 3900
cttgcggttg atctatctcg cgctggcgca tatgatcaaa tttcggggac acttcctcat 3960
cgagggggac ctgaacccag acaacagcga tgtcgacaaa ctctttatcc aactggttca 4020
gacttacaat cagcttttcg aagagaaccc gatcaacgca tccggagttg acgccaaagc 4080
aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg cacagctccc 4140
tggggagaag aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc 4200
caactttaaa tctaacttcg acctggccga agatgccaag cttcaactga gcaaagacac 4260
ctacgatgat gatctcgaca atctgctggc ccagatcggc gaccagtacg cagacctttt 4320
tttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgc gagtgaacac 4380
ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccacca 4440
agacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat 4500
tttcttcgat cagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga 4560
ggaattttac aaatttatta agcccatctt ggaaaaaatg gacggcaccg aggagctgct 4620
ggtaaagctt aacagagaag atctgttgcg caaacagcgc actttcgaca atggaagcat 4680
cccccaccag attcacctgg gcgaactgca cgctatcctc aggcggcaag aggatttcta 4740
cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttc ggatacccta 4800
ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc gcaaatcaga 4860
agagaccatc actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc 4920
cttcatcgaa aggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa 4980
acactctctg ctgtacgagt acttcacagt ttataacgag ctcaccaagg tcaaatacgt 5040
cacagaaggg atgagaaagc cagcattcct gtctggagag cagaagaaag ctatcgtgga 5100
cctcctcttc aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag actatttcaa 5160
aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc 5220
cctgggaacg tatcacgatc tcctgaaaat cattaaagac aaggacttcc tggacaatga 5280
ggagaacgag gacattcttg aggacattgt cctcaccctt acgttgtttg aagataggga 5340
gatgattgaa gaacgcttga aaacttacgc tcatctcttc gacgacaaag tcatgaaaca 5400
gctcaagagg cgccgatata caggatgggg gcggctgtca agaaaactga tcaatgggat 5460
ccgagacaag cagagtggaa agacaatcct ggattttctt aagtccgatg gatttgccaa 5520
ccggaacttc atgcagttga tccatgatga ctctctcacc tttaaggagg acatccagaa 5580
agcacaagtt tctggccagg gggacagtct tcacgagcac atcgctaatc ttgcaggtag 5640
cccagctatc aaaaagggaa tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt 5700
aatgggaagg cataagcccg agaatatcgt tatcgagatg gcccgagaga accaaactac 5760
ccagaaggga cagaagaaca gtagggaaag gatgaagagg attgaagagg gtataaaaga 5820
actggggtcc caaatcctta aggaacaccc agttgaaaac acccagcttc agaatgagaa 5880
gctctacctg tactacctgc agaacggcag ggacatgtac gtggatcagg aactggacat 5940
caatcggctc tccgactacg acgtggatca tatcgtgccc cagtcttttc tcaaagatga 6000
ttctattgat aataaagtgt tgacaagatc cgataaaaat agagggaaga gtgataacgt 6060
cccctcagaa gaagttgtca agaaaatgaa aaattattgg cggcagctgc tgaacgccaa 6120
actgatcaca caacggaagt tcgataatct gactaaggct gaacgaggtg gcctgtctga 6180
gttggataaa gccggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca 6240
cgtggcccaa attctcgatt cacgcatgaa caccaagtac gatgaaaatg acaaactgat 6300
tcgagaggtg aaagttatta ctctgaagtc taagctggtc tcagatttca gaaaggactt 6360
tcagttttat aaggtgagag agatcaacaa ttaccaccat gcgcatgatg cctacctgaa 6420
tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctg aatttgttta 6480
cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaatagg 6540
caaggccacc gctaagtact tcttttacag caatattatg aattttttca agaccgagat 6600
tacactggcc aatggagaga ttcggaagcg accacttatc gaaacaaacg gagaaacagg 6660
agaaatcgtg tgggacaagg gtagggattt cgcgacagtc cggaaggtcc tgtccatgcc 6720
gcaggtgaac atcgttaaaa agaccgaagt acagaccgga ggcttctcca aggaaagtat 6780
cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa aaagattggg accccaagaa 6840
atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga 6900
gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatgga 6960
gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat ataaagaggt 7020
caaaaaagac ctcatcatta agcttcccaa gtactctctc tttgagcttg aaaacggccg 7080
gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg cactgccctc 7140
taaatacgtt aatttcttgt atctggccag ccactatgaa aagctcaaag ggtctcccga 7200
agataatgag cagaagcagc tgttcgtgga acaacacaaa cactaccttg atgagatcat 7260
cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt 7320
gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattat 7380
ccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac 7440
catagacaga aagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca 7500
gtcaattacg gggctctatg aaacaagaat cgacctctct cagctcggtg gagacagcag 7560
ggctgacccc aagaagaaga ggaaggtgtg atctcttctc gagtcatgta attagttatg 7620
tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga 7680
caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta 7740
tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata 7800
ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg cccgccatcc 7860
agtgtcgaaa acgagctctc gagaaccctt aatataactt cgtataatgt atgctatacg 7920
aagttattag gtgatatcag atccactagt ggctactatc ggcgactctc tcgaaatttt 7980
tcttaacgcg tccttgtact gcgtctaacg cttttgccac ttggatttct attataggaa 8040
atagtctcac ttactgggcg acgaattttc gcgttttgat gaagcacagg aagaatttct 8100
tttttttttg gcttcttctg gttccgtttt ttacgcgcac aaatctaaaa aaagaaataa 8160
ttataaccta gtctcgaaaa ttttcatcga tccattcgtt cctttttttc gattttttca 8220
gatcaaaatt cttgtttctt tctttgtctt agtttatatt aaaagatatt ttgattttac 8280
tcctgaacta tttattcttt ctaagaaggc cagaacacta cagctgtttt aaccgactac 8340
gaagttctcc attctcgaac actagccttc atttaccaaa caggaactag cgtatatcat 8400
tagtccttat tcgaaaagag attggtagat atttattgta gtttgtgaga aggagaaaat 8460
actgtcattg gactgatagt tagaggacat taacctctct tacgttcgct caaaaaaatt 8520
aaaataagca agtagctgaa atcaagtctt tcatacaacg gtcagaccat gaacactgat 8580
caacaacctt atcagggcca aacggactat acccagggac caggtaacgg gcaaagtcag 8640
gaacaagact atgaccaata tggccagcct ttgtatcctt cacaagctga tggttactac 8700
gatccaaatg tcgctgctgg tactgaagct gatatgtatg gtcaacaacc accaaacgag 8760
tcttacgacc aagactacac aaacggtgaa tactatggtc aaccgccaaa tatggctgct 8820
caagacggtg aaaacttctc ggattttagc agttacggcc ctcctggaac acctggatat 8880
gatagctatg gtggtcagta taccgcttct caaatgagtt atggagaacc aaattcgtcg 8940
ggtacctcga ctccaattta cggtaattat gacccaaatg ctatcgctat ggctttgcca 9000
aatgaacctt atcccgcttg gactgctgac tctcaatctc ccgtttcgat cgagcaaatc 9060
gaagatatct ttattgattt gaccaacaga ctcgggttcc aaagagactc catgagaaat 9120
atgtttgatc attttatggt tctcttggac tctaggtcct cgagaatgtc tcctgatcaa 9180
gctttactat ctttacatgc cgactacatt ggtggcgata ctgctaacta taaaaaatgg 9240
tattttgctg ctcag 9255
<210> 8
<211> 8416
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gcagagaatc ctcgggtttg taaaccatgt tatagttaaa ccccgatatt acaccattaa 60
aatcatttct aaatatgcgg tcgtcgatga tcagctgcag ggaaatgcca aaatatggac 120
cccatagagg tcttggcttg ttcatatcgt tgggtcgatt tctgttccca gtgttatttg 180
gttgaatggg aacagtcgcg tcttgcccca tcttgtctct gccgctttcg tcgaatctga 240
ctttgcaatt gacgttgcat atcaacgtgc cggatcccgg aatagcgtga tagtctactc 300
ctgttagggc atgttgtgtt tgtacgacct ggttttgcca catttgcaga aaaactgttg 360
cttgcgcgaa gggggtagca ttaaatatta ttctgcagtt gtttggatta aaaagctgta 420
cgaattgggc caatttgttg gagtccggat catccaaatg cgctaatatc ttcttcgtaa 480
atgtggccgt tatttgtgct tgatttgcgt cattcattac catagtatta ctattggtgt 540
tcatatttct gttattactg ttattagttt ggcgttttgc ctgacgatca gcctggtggg 600
gccaagtata tactataaaa acaggttagt tacttgctct atgcgtttgc gcatcctctt 660
tttacttttt ttttttcagt aaagcctaag cataaatcgt tttatacgta cgacacgttc 720
aacttttctt ggttagtagt ggcaatctct gcaatacata cagggagtca tggtctatca 780
tcttgtccaa tcaaagaagc atcggttcag atcgagcaaa ctgtagggag aaaggaaagt 840
agaaatgcag agtgtgctat atgtccaatc tcggttttgt agtttggatg tcattagaga 900
tctaccaccc aaccggctgc tttcatgtgg aacagaaaag aaatcggggc gcttcctctt 960
ctgtattcct ttaattaacg tttttattca gccatctaac catcataccc ccatacggta 1020
acaaaacctc ttctaagaaa agaagtctct gctcctccgc catcttattt ttattcgctg 1080
cgcgcgttta ttgtcgcatc gctagccagc aaaaagttgg ttgccttttt ttacctaaaa 1140
aagacacatc taactgatta gttttccgtt ttaggatatt gacgccaagc gtgcgtctga 1200
ttgcaaatta aagccttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg 1260
ttacatgcgt acacgcgttt gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt 1320
cttaatacta acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact 1380
ccttcctttt cggttagagc ggatgtgggg ggagggcgtg aatgtaagcg tgacataact 1440
aattacatga tatcgacaaa ggaaaagggg cctgtttatt tatcaagata agtttccgga 1500
tctttttctt tcctaacacc ccagtcagcc tgagttacat ccagccattg aaccttagaa 1560
aatcttttgt catcagcggt ttgagcccta agatcaacat cttgcttagc aatcactgca 1620
atggcgtcat aaccaccagc accaggtatt aagcaagtaa gaactccttt taaggtctgg 1680
caatcatcca ataagctagt ttgtacggga ggttcgatat cggcaccaga ttctttagtt 1740
atttttctaa aggaacgtct aattgtggca actgcatctc taacttctgt gatctcagga 1800
tacttttgac aggtacagtc attcctctca agagactcaa atatctgatc gctgtaatcg 1860
tcatgagtct cgtgtaagcg atctagttta gatagtccat ccataaatct agaatttgca 1920
tgatcgagtt ctgtatatat tttcaagctt tccggcatat gcgaatcata ccaatttttt 1980
accttctgga ccagttttac tgtttctgaa ccattcttaa tatcgcccat ccataaagtt 2040
aatcccgaag gtaaatggtt acttttaatc gttatattcc agtcttcttc attaaccaaa 2100
tgcgccagtt tactgccgta agtagcactt ccaatatctg gcaaattaga gattaatgcg 2160
ggtgggaatc ttctatatct gatagatcca tatgctgccg ccgctacatc aaacccgctt 2220
ccaattttac cctgagcttg acaatgagca acttgtgata aattatgaat aacttctcta 2280
tatttgtcta cattattttc caggtccgat acaaaaaagg aggccaaagc tgtagttaaa 2340
actgtgacta aacctgccga ggagcccagc cctgttttgg gaacttcttc aattctgtgc 2400
gaatgaaaac tcaatcttct gttgccacga tgttcggtaa cgctgtcctc ctgagaatgg 2460
taggcatcat cagagaaaat atcaataacg aacaagtttc tattgcagta gtcgtccatg 2520
ttaggcttaa agtagctaaa tacgttagcg ataacttttt caatgaaagg gttcttagat 2580
ccgcctatcg aaacaggaat gaagccagtt ttaggactta tatggtacag ccactcccca 2640
tctttaaatt gtttactttt cacacgcact tcaaacttat cagactcttg caatgaaccg 2700
taaggatggg ctacagcatg cattcttgcc gataatccga ctacaaatgc ttcatatttc 2760
ggatctaaaa ctaaatatcc accagctagt aacgctttcc ctggggcact gaaggctctc 2820
aactctgaca ttatagtttt ttctccttga cgttaaagta tagaggtata ttaacaattt 2880
tttgttgata cttttatgac atttgaataa gaagtaatac aaaccgaaaa tgttgaaagt 2940
attagttaaa gtggttatgc agcttttgca tttatatatc tgttaataga tcaaaaatca 3000
tcgcttcgct gattaattac cccagaaata aggctaaaaa actaatcgca ttattatcct 3060
atggttgtta atttgattcg ttgatttgaa ggtttgtggg gccaggttac tgccaatttt 3120
tcctcttcat aaccataaaa gctagtattg tagaatcttt attgttcgga gcagtgcggc 3180
gcgaggcaca tctgcgtttc aggaacgcga ccggtgaaga ccaggacgca cggaggagag 3240
tcttccgtcg gagggctgtc gcccgctcgg cggcttctaa tccgtacttc aatatagcaa 3300
tgagcagtta agcgtattac tgaaagttcc aaagagaagg tttttttagg ctaagataat 3360
ggggctcttt acatttccac aacatataag taagattaga tatggatatg tatatggtgg 3420
tattgccatg taatatgatt attaaacttc tttgcgtcca tccaaaaaaa aagtaagaat 3480
ttttgaaaat tcaatataaa tggttttaac caataaaaca gtcatttctg gatcgaaagt 3540
caaaagttta tcatctgcgc aatcgagctc atcaggacct tcatcatcta gtgaggaaga 3600
tgattcccgc gatattgaaa gcttggataa gaaaatacgt cctttagaag aattagaagc 3660
attattaagt agtggaaata caaaacaatt gaagaacaaa gaggtcgctg ccttggttat 3720
tcacggtaag ttacctttgt acgctttgga gaaaaaatta ggtgatacta cgagagcggt 3780
tgcggtacgt aggaaggctc tttcaatttt ggcagaagct cctgtattag catctgatcg 3840
tttaccatat aaaaattatg actacgaccg cgtatttggc gcttgttgtg aaaatgttat 3900
aggttacatg cctttgcccg ttggtgttat aggccccttg gttatcgatg gtacatctta 3960
tcatatacca atggcaacta cagagggttg tttggtagct tctgccatgc gtggctgtaa 4020
ggcaatcaat gctggcggtg gtgcaacaac tgttttaact aaggatggta tgacaagagg 4080
cccagtagtc cgtttcccaa ctttgaaaag atctggtgcc tgtaagatat ggttagactc 4140
agaagaggga caaaacgcaa ttaaaaaagc ttttaactct acatcaagat ttgcacgtct 4200
gcaacatatt caaacttgtc tagcaggaga tttactcttc atgagattta gaacaactac 4260
tggtgacgca atgggtatga atatgatttc taaaggtgtc gaatactcat taaagcaaat 4320
ggtagaagag tatggctggg aagatatgga ggttgtctcc gtttctggta actactgtac 4380
cgacaaaaaa ccagctgcca tcaactggat cgaaggtcgt ggtaagagtg tcgtcgcaga 4440
agctactatt cctggtgatg ttgtcagaaa agtgttaaaa agtgatgttt ccgcattggt 4500
tgagttgaac attgctaaga atttggttgg atctgcaatg gctgggtctg ttggtggatt 4560
taacgcacat gcagctaatt tagtgacagc tgttttcttg gcattaggac aagatcctgc 4620
acaaaatgtt gaaagttcca actgtataac attgatgaaa gaagtggacg gtgatttgag 4680
aatttccgta tccatgccat ccatcgaagt aggtaccatc ggtggtggta ctgttctaga 4740
accacaaggt gccatgttgg acttattagg tgtaagaggc ccgcatgcta ccgctcctgg 4800
taccaacgca cgtcaattag caagaatagt tgcctgtgcc gtcttggcag gtgaattatc 4860
cttatgtgct gccctagcag ccggccattt ggttcaaagt catatgaccc acaacaggaa 4920
acctgctgaa ccaacaaaac ctaacaattt ggacgccact gatataaatc gtttgaaaga 4980
tgggtccgtc acctgcatta aatcctaatt tgccagctta ctatccttct tgaaaatatg 5040
cactctatat cttttagttc ttaattgcaa cacatagatt tgctgtataa cgaattttat 5100
gctatttttt aaatttggag ttcagtgata aaagtgtcac agcgaatttc ctcacatgta 5160
gggaccgaat tgtttacaag ttctctgtac caccatggag acatcaaaga ttgaaaatct 5220
atggaaagat atggacggta gcaacaagaa tatagcacga gccgcgaagt tcatttcgtt 5280
acttttgata tcgctcacaa ctattgcgaa gcgcttcagt gaaaaaatca taaggaaaag 5340
ttgtaaatat tattggtagt attcgtttgg taaagtagag ggggtaattt ttccccttta 5400
ttttgttcat acattcttaa attgctttgc ctctcctttt ggaaagctat acttcggagc 5460
actgttgagc gaaggctcat tagatatatt ttctgtcatt ttccttaacc caaaaataag 5520
ggaaagggtc caaaaagcgc tcggacaact gttgaccgtg atccgaagga ctggctatac 5580
agtgttcaca aaatagccaa gctgaaaata atgtgtagct atgttcagtt agtttggcta 5640
gcaaagatat aaaagcaggt cggaaatatt tatgggcatt attatgcaga gcatcaacat 5700
gataaaaaaa aacagttgaa tattccctca aaaatgtcat taccgttctt aacttctgca 5760
ccgggaaagg ttattatttt tggtgaacac tctgctgtgt acaacaagcc tgccgtcgct 5820
gctagtgtgt ctgcgttgag aacctacctg ctaataagcg agtcatctgc accagatact 5880
attgaattgg acttcccgga cattagcttt aatcataagt ggtccatcaa tgatttcaat 5940
gccatcaccg aggatcaagt aaactcccaa aaattggcca aggctcaaca agccaccgat 6000
ggcttgtctc aggaactcgt tagtcttttg gatccgttgt tagctcaact atccgaatcc 6060
ttccactacc atgcagcgtt ttgtttcctg tatatgtttg tttgcctatg cccccatgcc 6120
aagaatatta agttttcttt aaagtctact ttacccatcg gtgctgggtt gggctcaagc 6180
gcctctattt ctgtatcact ggccttagct atggcctact tgggggggtt aataggatct 6240
aatgacttgg aaaagctgtc agaaaacgat aagcatatag tgaatcaatg ggccttcata 6300
ggtgaaaagt gtattcacgg taccccttca ggaatagata acgctgtggc cacttatggt 6360
aatgccctgc tatttgaaaa agactcacat aatggaacaa taaacacaaa caattttaag 6420
ttcttagatg atttcccagc cattccaatg atcctaacct atactagaat tccaaggtct 6480
acaaaagatc ttgttgctcg cgttcgtgtg ttggtcaccg agaaatttcc tgaagttatg 6540
aagccaattc tagatgccat gggtgaatgt gccctacaag gcttagagat catgactaag 6600
ttaagtaaat gtaaaggcac cgatgacgag gctgtagaaa ctaataatga actgtatgaa 6660
caactattgg aattgataag aataaatcat ggactgcttg tctcaatcgg tgtttctcat 6720
cctggattag aacttattaa aaatctgagc gatgatttga gaattggctc cacaaaactt 6780
accggtgctg gtggcggcgg ttgctctttg actttgttac gaagagacat tactcaagag 6840
caaattgaca gcttcaaaaa gaaattgcaa gatgatttta gttacgagac atttgaaaca 6900
gacttgggtg ggactggctg ctgtttgtta agcgcaaaaa atttgaataa agatcttaaa 6960
atcaaatccc tagtattcca attatttgaa aataaaacta ccacaaagca acaaattgac 7020
gatctattat tgccaggaaa cacgaattta ccatggactt cataagcgaa tttcttatga 7080
tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt 7140
gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg 7200
ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cggttacgtg 7260
gattgagcca gcaatacaga tcattattaa actgttttgt acatgatgtt agtatataat 7320
cgtaaagctt ttctaatatg tataccttat acatggaact ccacagaact tgcaaacata 7380
ccaaaaatcc tttattcttg ttcactcatt ttacatcaaa aaataatatt tcagttatta 7440
aggaaaataa aaaaatagat tagagaagca ttttgaagaa atagtatatt cttttattga 7500
acctaagagc gtgatatttt tactcgaaat aaaatacgaa aaatctatac actcatcttt 7560
ccgactacta ttggctcctg ctcaaaaaaa gagggaaaaa aagctccaaa attctatctt 7620
ttcctatcgc tcctgtccta tccttattac gttcattact attttaatac tatccattct 7680
tttattttca gtctaaaaaa aacatttctc ataacgggaa aagcaaaaaa atgtcaagct 7740
tatacatcaa aacaccactg catgcattat ctgctggtcc ggattctcat gcaaatagct 7800
cctattatga caatctttta ttgccttcat tttccaattt atccagtaat atcagcagga 7860
acaatatcac aaccgataac aatataaatt cagcaagtcc taggaagtat tcgtttcatt 7920
cgctcaatgt gtcgccaatt ctttccccaa tatctttggc taacgaaata cttggaaaaa 7980
aatcaaatac tgcaccagca tcgccacatc atatggatta caatccaatt tcctcattaa 8040
caccaggcaa ttcaccagaa ttcaacaagg caagcttatc tcagattagt tttacaaatc 8100
cattgaacta tgggtctggc ttaggttttt cctctaattc acaacctcga ctaccattgt 8160
tagacaggct atcgtccgtc tctttatcta agagaccgga gcgcccacaa caaagcctac 8220
catcactaag gcatctgcaa ttattaccca gtcctctttt acaagaaaac gcagcacgct 8280
tccctgatac atcaaaacgt acatctaatt ggaaaactga tcttacccat tggtgtaagg 8340
atacaaacta tcaagactat gttaaaatac gcgaagaagt ggctcatttc aagccattaa 8400
gtatacccaa cttaac 8416
<210> 9
<211> 7388
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gactcaatga cttcgggatc taattcagca taaataatct cttctccctc gccagcttca 60
gctacaatct tgcctctggg gtccaccact atagagtggc catatgcatg gtaagaactc 120
tgtagattcc ttgcgggaga acataacatc acgtacactt ggttgtcaac tgccctgctt 180
cttgccagta aatgccaatg caagggccct gtgacagtgt tgaatgcact agggtagatc 240
atggcaaacg cacccttgcg tgcacttagc attgcgagtt ccggaaacct catgtcatag 300
catataccta cgccaaactt accatatttc gtgtcaatgg tagtagactt ctctccaggg 360
ctcaaggttt cactttcatg gaacgatata ccgttaggaa tgtctacatc gaaaagatgg 420
accttccgat gcttgtcaat caacttgcca tcctcgttga aaattataga ggtgttgtag 480
attttgtctg tctttggatc caattccgga attgttcctc ctaccaagat gattttaaac 540
ttattggcta aattagacag aaattgaact gacgtcgaag gctctttcgg attgataact 600
tctgaatact ttctaaactg atcagtggag tacggagaat tgaagcattc tggcaatacg 660
accaacttgg tatccggctg ttccttcata gccctttcaa tgaacgttgc agccctttga 720
agattggcca ttttgtcagg actcgagcct gacagttgga ccaacgcaac tttaattttt 780
tgtgaaagaa tcttcgaagc actcatactg gcgatcttca cgccctcctg ctattacaaa 840
agctgtgttt ttacaagaat caaattaagt tagcaagata tcatacaaca ttattgataa 900
tttcaatatc gtgttcgtac ctgatgacgt atctgtgcat tgataaggcc cgcatggttt 960
cagaaagcag agcggaacga ttccaaatta gtggccttgt gctttgcatg tcaattgtgt 1020
taccttcagc tcgtggattt gttttatcaa tacacagtct acagtcaagn nnnnnnnnnt 1080
atcaaatttt gcgttcgagc gtataaaata gccgctgtag ctacttaagt tcctgttcag 1140
ccggtagagg tgtggtcaat aagagcgacc tcatgctata cctgagaaag caacctgacc 1200
tacaggaaag agttactcaa gaataagaat tttcgtttta aaacctaaga gtcactttaa 1260
aatttgtata cacttatttt ttttataact tatttaataa taaaaatcat aaatcataag 1320
aaattcgctt attcctttgg tagaccagtc tttgcgtcaa tcaaagattc gtttgtttct 1380
tgtgggcctg aaccgacttg agttaaaatc actctggcaa catccttttg caactcaaga 1440
tccaattcac gtgcagtaaa gttagatgat tcaaattgat ggttgaaagc ctcaagctgc 1500
tcagtagtaa atttcttgtc ccatccagga acagagccaa acaatttata gataaatgca 1560
aagagtttcg actcattttc agctaagtag tacaacacag catttggacc tgcatcaaac 1620
gtgtatgcaa cgattgtttc tccgtaaaac tgattaatgg tgtggcacca actgatgata 1680
cgcttggaag tgtcattcat gtagaatatt ggagggaaag agtccaaaca tgtggcatgg 1740
aaagagttgg aatccatcat tgtttccttt gcaaaggtgg cgaaatcttt ttcaacaatg 1800
gctttacgca tgacttcaaa tctctttggt acgacatgtt caattctttc tttaaatagt 1860
tcggaggttg ccacggtcaa ttgcataccc tgagtggaac tcacatcctt tttaatatcg 1920
ctgacaacta ggacacaagc tttcatctga ggccagtcag agctgtctgc gatttgtact 1980
gccatggaat catgaccatc ttcagctttt cccatttccc aggccacgta tccgccaaac 2040
aacgatctac aagctgaacc agaccccttt cttgctattc tagatatttc tgaagttgac 2100
tgtggtaatt ggtataactt agcaattgca gagaccaatg cagcaaagcc agcagcggag 2160
gaagctaaac cagctgctgt aggaaagtta ttttcggaga caatgtggag tttccattga 2220
gataatgtgg gcaatgaggc gtccttcgat tccatttcct ttcttaattg gcgtaggtcg 2280
cgcagacaat tttgagttct ttcattgtcg atgctgtgtg gttctccatt taaccacaaa 2340
gtgtcgcgtt caaactcagg tgcagtagcc gcagaggtca acgttctgag gtcatcttgc 2400
gataaagtca ctgatatgga cgaattggtg ggcagattca acttcgtgtc ccttttcccc 2460
caatacttaa gggttgcgat gttgacgggt gcggtaacgg atgctgtgta aacggtcatt 2520
tttgagggaa tattcaactg ttttttttta tcatgttgat gctctgcata ataatgccca 2580
taaatatttc cgacctgctt ttatatcttt gctagccaaa ctaactgaac atagctacac 2640
attattttca gcttggctat tttgtgaaca ctgtatagcc agtccttcgg atcacggtca 2700
acagttgtcc gagcgctttt tggacccttt cccttatttt tgggttaagg aaaatgacag 2760
aaaatatatc taatgagcct tcgctcaaca gtgctccgaa gtatagcttt ccaaaaggag 2820
aggcaaagca atttaagaat gtatgaacaa aataaagggg aaaaattacc ccctctactt 2880
taccaaacga atactaccaa taatatttac aacttttcct tatgattttt tcactgaagc 2940
gcttcgcaat agttgtgagc gatatcaaaa gtaacgaaat gaacttcgcg gctcgtgcta 3000
tattcttgtt gctaccgtcc atatctttcc atagattttc aatctttgat gtctccatgg 3060
tggtacagag aacttgtaaa caattcggtc cctacatgtg aggaaattcg ctgtgacact 3120
tttatcactg aactccaaat ttaaaaaata gcataaaatt cgttatacag caaatctatg 3180
tgttgcaatt aagaactaaa agatatagag tgcatatttt caagaaggat agtaagctgg 3240
caaatcatat cttttcaatg acaatagagg aagcaccacc accaccatta caaatggcgg 3300
caacaccgat cttacctcct tcttgctgta agatggatag cagtgtaaca accactctag 3360
caccagaaca acccaatggg tgacctagag caacagcacc accatataca ttaaccttag 3420
atgggtctag cttcaaaatc ttagtgttca ccaaaccgac aaccgaaaag gcttcattga 3480
attcaaagta atcaacagaa ttgatgtctt cgatgccagc atgtttcaaa gcctttggaa 3540
ctgcaagaga tggagcccat gtaaaatcag ctggttgatg agcggcctca ccccaacctt 3600
tgataatagc caaaggcttc aaattctttt ccttcaaaac tttttcggaa accaagatga 3660
cggctgcagc accatcgttg attggagaag cgttagcggc agtaacagta ccgttttctt 3720
tttggaaaac agtccttgca gatctcaatt tttcaacgtg taatctagca ggttcctcgt 3780
ccttcgtgac ttgagtatca ggcttacctc taaatccctt aatggtaaca ggtacaattt 3840
cattgtcgaa tttaccttcc ttttgagatt tttgagattt ttggtaggat tcgatggcaa 3900
aattgtcttg ttgttctcta gtaatatccc aatcacgggc acacttttct gcgtgtacac 3960
ccatggctag accatcgtac gcatcgttca acccatctct ttcgacacca tcaacaagaa 4020
cagtttggcc aaatttggca cccgcacggg ctgctggcat gtagtatggt gcgttagtca 4080
tagattcaca accaccagct acgacaacat cagcattacc acatttgatg gattgagcac 4140
ccaaaatgat tgccttcata gcggatgcac agaccttgtt aactgtgctt gcaacgatat 4200
gattactcaa accggcagcc aaagcaactt gtctggccgg agcttggccc aaattggcag 4260
aaagaacgtt accaaaaata atttcgtcaa aatccttgga tgcatccaat tctggaacct 4320
tagccaaggc gccttttaaa gcaacagcac ccaattccac tgctgtcttg gaggatagag 4380
aaccctggaa tgaaccaatt ggggttctgg cagtcgatac aatgtaaacg ttctgagaca 4440
tttatattga attttcaaaa attcttactt tttttttgga tggacgcaaa gaagtttaat 4500
aatcatatta catggcaata ccaccatata catatccata tctaatctta cttatatgtt 4560
gtggaaatgt aaagagcccc attatcttag cctaaaaaaa ccttctcttt ggaactttca 4620
gtaatacgct taactgctca ttgctatatt gaagtacgga ttagaagccg ccgagcgggc 4680
gacagccctc cgacggaaga ctctcctccg tgcgtcctgg tcttcaccgg tcgcgttcct 4740
gaaacgcaga tgtgcctcgc gccgcactgc tccgaacaat aaagattcta caatactagc 4800
ttttatggtt atgaagagga aaaattggca gtaacctggc cccacaaacc ttcaaatcaa 4860
cgaatcaaat taacaaccat aggataataa tgcgattagt tttttagcct tatttctggg 4920
gtaattaatc agcgaagcga tgatttttga tctattaaca gatatataaa tgcaaaagct 4980
gcataaccac tttaactaat actttcaaca ttttcggttt gtattacttc ttattcaaat 5040
gtcataaaag tatcaacaaa aaattgttaa tatacctcta tactttaacg tcaaggagaa 5100
aaaactataa tgactgccga caacaatagt atgccccatg gtgcagtatc tagttacgcc 5160
aaattagtgc aaaaccaaac acctgaagac attttggaag agtttcctga aattattcca 5220
ttacaacaaa gacctaatac ccgatctagt gagacgtcaa atgacgaaag cggagaaaca 5280
tgtttttctg gtcatgatga ggagcaaatt aagttaatga atgaaaattg tattgttttg 5340
gattgggacg ataatgctat tggtgccggt accaagaaag tttgtcattt aatggaaaat 5400
attgaaaagg gtttactaca tcgtgcattc tccgtcttta ttttcaatga acaaggtgaa 5460
ttacttttac aacaaagagc cactgaaaaa ataactttcc ctgatctttg gactaacaca 5520
tgctgctctc atccactatg tattgatgac gaattaggtt tgaagggtaa gctagacgat 5580
aagattaagg gcgctattac tgcggcggtg agaaaactag atcatgaatt aggtattcca 5640
gaagatgaaa ctaagacaag gggtaagttt cactttttaa acagaatcca ttacatggca 5700
ccaagcaatg aaccatgggg tgaacatgaa attgattaca tcctatttta taagatcaac 5760
gctaaagaaa acttgactgt caacccaaac gtcaatgaag ttagagactt caaatgggtt 5820
tcaccaaatg atttgaaaac tatgtttgct gacccaagtt acaagtttac gccttggttt 5880
aagattattt gcgagaatta cttattcaac tggtgggagc aattagatga cctttctgaa 5940
gtggaaaatg acaggcaaat tcatagaatg ctataaacag gccccttttc ctttgtcgat 6000
atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 6060
gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 6120
ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta caaacgcgtg 6180
tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 6240
ttaatttgca cgctgcaggt cgacaaccct taatataggt gatatcagat ccactagtgg 6300
caattcccca ttagagtcaa ataaaaggat gcctcacgga ggtatgttac ccgcgctatt 6360
tcacatggct cattgaatta gaggtggaat ttggtgtacc ctcccctcct catctgatga 6420
agtagtgatc cgacaattct taaaagttgt agacattact tttaccacca actaagttgt 6480
atttatattg ctacccttat ccttttatat ctaactagcg ctcataaggt tggggcaata 6540
ctaaaactgt gttcttattc aactcattaa atacgtggca gtacgtaccc tattagaaac 6600
aataggaaac agcagagtcg gaagaagcca aatgccagat ttgaagtcca aaaccttgtc 6660
aagccaatct ttgggagcgg ctattcctcc agaaattgtg taccaaatac ttacatacca 6720
gtttagggat ttgttaagaa atgaccatcc aggtacagca gaaaagttta acgaaaactt 6780
aactacattt gtcaaaagta atttaactgt taataagaca ttctcccata tttgtcaagt 6840
tctcatttat agatattgta atttaactac cgcaaagaga tttcacggtc ttttacagac 6900
tttaaaggga aatagatgcc tttgcaataa gattgaagta gccgatttcc aagaattaac 6960
gtctattgga ctggggaggt ccagtgagat gaataaaatg attaaaaatc taactaacga 7020
aactcttctt gagtttctaa tgctcacaaa agcaaactta agggaattct tagcatgtga 7080
aaatattcaa gacgatttag acgataatat cataaagtat attctatctc cagaaaaagt 7140
cttgagtgta gtggatttct gtggctgttc aggtaccact ttcacagaaa gttttattaa 7200
ggccctggat aaatactacc ctaataaatc gattgatcag taccggctgg aacctattca 7260
acaaaattat cagattacct gcttaggcct aaatgattgt attgatctac catcacacgt 7320
tctttggaag atcttaaaaa tgttaccaga actacaaaaa cttgatttat ctcatacctc 7380
cattgatg 7388
<210> 10
<211> 6357
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctcaaattac acacatacag ataggtcaca tcatagaggt gataatgttt tgggtgatgc 60
ctctttaggc gattcgttta attcaatgcc agattttact ccaagaaatt tcttgatacc 120
tacattaaaa aaatcacggg aaaagttcga gcctcatcta tccaatccaa accaacattt 180
tggaaatgta accttctaat aatttgtcat ttgactgacg aatcgttagg cacagttata 240
tagttatata cacatagaca tacatatgtt cgcatatata ttatattact atgcattcac 300
ctaaatagct actgctctat taatacagtg cttttttgat ggctttaatt taacttatta 360
gacattactt tgcgtttatt cccctccaat atatttttat gtttgttttt cttgaacatt 420
ttatgatttc ttgctcttgg cggctactga ttttgttaag caactcatca agaggtcaaa 480
aaaagtagaa cgagatcttt gtgttcggtt acccggctca gatcctaact tggtatgttt 540
attcgtataa gttactgttg tccacaggca atactctgca gaaaattaaa acggcattaa 600
tgctaggaca accagaattg ttactactgt atgtgcgata gttgataact gcaacattat 660
gcccggtata ttctcaaaaa accctattac tgcatacgaa gaaatcgcaa gagaaatctt 720
tcggtttgga aaagctcact gtgaggttcc ttggagccaa tagtaataca gcacaatcca 780
aggaaaaatc tggcctatat gcaaggaagg agagatagtc aaaagcattc tttcccctag 840
aagttggtgc atatatggca tcgttaaaac atattacccc caaaatttct tctctaaacg 900
atgtgcttgg cctttgtttt ggtttttgat gtcggtcgtt tgaggcccct tgcggaaaat 960
cgagatcgcc gaatggcacg cgagggaagg gaaataaggt ttaaaggcac tgaaacaata 1020
ggcaagaagt aggcgagagc cgacatacga gagcttcatt gctcctacat ttatcggatg 1080
gcgcacatag ctaaaaaagg aagattttca ctcataaggg aaaggaaccg catgactcaa 1140
gagaagcatg tggtttttga gtttttttcg ttgaattttc aggtaaagct caatagttat 1200
gacaattaca acaacagaat tctttctata tatgcacgaa cttgtaatat ggaagaaatt 1260
atgacgtaca aactataaag taaatatttt acgtaacaca tggtgctgtt gtgcttcttt 1320
ttcaagagaa taccaatgac gtatgactaa gtttaggatt taatgcaggt gacggaccca 1380
tctttcaaac gatttatatc agtggcgtcc aaattgttag gttttgttgg ttcagcaggt 1440
ttcctgttgt gggtcatatg actttgaacc aaatggccgg ctgctagggc agcacataag 1500
gataattcac ctgccaagac ggcacaggca actattcttg ctaattgacg tgcgttggta 1560
ccaggagcgg tagcatgcgg gcctcttaca cctaataagt ccaacatggc accttgtggt 1620
tctagaacag taccaccacc gatggtacct acttcgatgg atggcatgga tacggaaatt 1680
ctcaaatcac cgtccacttc tttcatcaat gttatacagt tggaactttc aacattttgt 1740
gcaggatctt gtcctaatgc caagaaaaca gctgtcacta aattagctgc atgtgcgtta 1800
aatccaccaa cagacccagc cattgcagat ccaaccaaat tcttagcaat gttcaactca 1860
accaatgcgg aaacatcact ttttaacact tttctgacaa catcaccagg aatagtagct 1920
tctgcgacga cactcttacc acgaccttcg atccagttga tggcagctgg ttttttgtcg 1980
gtacagtagt taccagaaac ggagacaacc tccatatctt cccagccata ctcttctacc 2040
atttgcttta atgagtattc gacaccttta gaaatcatat tcatacccat tgcgtcacca 2100
gtagttgttc taaatctcat gaagagtaaa tctcctgcta gacaagtttg aatatgttgc 2160
agacgtgcaa atcttgatgt agagttaaaa gcttttttaa ttgcgttttg tccctcttct 2220
gagtctaacc atatcttaca ggcaccagat cttttcaaag ttgggaaacg gactactggg 2280
cctcttgtca taccatcctt agttaaaaca gttgttgcac caccgccagc attgattgcc 2340
ttacagccac gcatggcaga agctaccaaa caaccctctg tagttgccat tggtatatga 2400
taagatgtac catcgataac caaggggcct ataacaccaa cgggcaaagg catgtaacct 2460
ataacatttt cacaacaagc gccaaatacg cggtcgtagt cataattttt atatggtaaa 2520
cgatcagatg ctaatacagg agcttctgcc aaaattgaaa gagccttcct acgtaccgca 2580
accgctctcg tagtatcacc taattttttc tccaaagcgt acaaaggtaa cttaccgtga 2640
ataaccaagg cagcgacctc tttgttcttc aattgttttg tatttccact acttaataat 2700
gcttctaatt cttctaaagg acgtattttc ttatccaagc tttcaatatc gcgggaatca 2760
tcttcctcac tagatgatga aggtcctgat gagctcgatt gcgcagatga taaacttttg 2820
actttcgatc cagaaatgac tgttttattg gttaaaacca tttatattga attttcaaaa 2880
attcttactt tttttttgga tggacgcaaa gaagtttaat aatcatatta catggcaata 2940
ccaccatata catatccata tctaatctta cttatatgtt gtggaaatgt aaagagcccc 3000
attatcttag cctaaaaaaa ccttctcttt ggaactttca gtaatacgct taactgctca 3060
ttgctatatt gaagtacgga ttagaagccg ccgagcgggc gacagccctc cgacggaaga 3120
ctctcctccg tgcgtcctgg tcttcaccgg tcgcgttcct gaaacgcaga tgtgcctcgc 3180
gccgcactgc tccgaacaat aaagattcta caatactagc ttttatggtt atgaagagga 3240
aaaattggca gtaacctggc cccacaaacc ttcaaatcaa cgaatcaaat taacaaccat 3300
aggataataa tgcgattagt tttttagcct tatttctggg gtaattaatc agcgaagcga 3360
tgatttttga tctattaaca gatatataaa tgcaaaagct gcataaccac tttaactaat 3420
actttcaaca ttttcggttt gtattacttc ttattcaaat gtcataaaag tatcaacaaa 3480
aaattgttaa tatacctcta tactttaacg tcaaggagaa aaaactataa tgaaactctc 3540
aactaaactt tgttggtgtg gtattaaagg aagacttagg ccgcaaaagc aacaacaatt 3600
acacaataca aacttgcaaa tgactgaact aaaaaaacaa aagaccgctg aacaaaaaac 3660
cagacctcaa aatgtcggta ttaaaggtat ccaaatttac atcccaactc aatgtgtcaa 3720
ccaatctgag ctagagaaat ttgatggcgt ttctcaaggt aaatacacaa ttggtctggg 3780
ccaaaccaac atgtcttttg tcaatgacag agaagatatc tactcgatgt ccctaactgt 3840
tttgtctaag ttgatcaaga gttacaacat cgacaccaac aaaattggta gattagaagt 3900
cggtactgaa actctgattg acaagtccaa gtctgtcaag tctgtcttga tgcaattgtt 3960
tggtgaaaac actgacgtcg aaggtattga cacgcttaat gcctgttacg gtggtaccaa 4020
cgcgttgttc aactctttga actggattga atctaacgca tgggatggta gagacgccat 4080
tgtagtttgc ggtgatattg ccatctacga taagggtgcc gcaagaccaa ccggtggtgc 4140
cggtactgtt gctatgtgga tcggtcctga tgctccaatt gtatttgact ctgtaagagc 4200
ttcttacatg gaacacgcct acgattttta caagccagat ttcaccagcg aatatcctta 4260
cgtcgatggt catttttcat taacttgtta cgtcaaggct cttgatcaag tttacaagag 4320
ttattccaag aaggctattt ctaaagggtt ggttagcgat cccgctggtt cggatgcttt 4380
gaacgttttg aaatatttcg actacaacgt tttccatgtt ccaacctgta aattggtcac 4440
aaaatcatac ggtagattac tatataacga tttcagagcc aatcctcaat tgttcccaga 4500
agttgacgcc gaattagcta ctcgcgatta tgacgaatct ttaaccgata agaacattga 4560
aaaaactttt gttaatgttg ctaagccatt ccacaaagag agagttgccc aatctttgat 4620
tgttccaaca aacacaggta acatgtacac cgcatctgtt tatgccgcct ttgcatctct 4680
attaaactat gttggatctg acgacttaca aggcaagcgt gttggtttat tttcttacgg 4740
ttccggttta gctgcatctc tatattcttg caaaattgtt ggtgacgtcc aacatattat 4800
caaggaatta gatattacta acaaattagc caagagaatc accgaaactc caaaggatta 4860
cgaagctgcc atcgaattga gagaaaatgc ccatttgaag aagaacttca aacctcaagg 4920
ttccattgag catttgcaaa gtggtgttta ctacttgacc aacatcgatg acaaatttag 4980
aagatcttac gatgttaaaa aataatcatg taattagtta tgtcacgctt acattcacgc 5040
cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc 5100
cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt 5160
cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag 5220
aaggttttgg gacgctcgaa ggctttaatt tgcccacttt tcaatgaaac ggatattgat 5280
atgctagtaa aaggacgagc tcaagagcga aaatataagt aaagaattcg agtgcacttg 5340
tctccatgca gcaagatttc atatgagtct tttttatctt tttacttttt acattacacg 5400
atatgcactt tatgaaaatt taacgaggtt ggaagccgga taatcaacca aaatcaggca 5460
cgaaggcaca ctcgtatatg catgttgttg aaactctgtt acgctgaact aacaatcaca 5520
catgtagagg tcaccgggaa aagttgcgac cccatggaag gtcgatctct tcgtttggct 5580
ttgcttggct ggcggcattg cgcttcttcg cttatacccg tctcttgacg ctcgagctcg 5640
ttcattgaga tacctttatt cttgcacatt ttctggcttt tttcgctact cgggtacatg 5700
taatcatgca cacagaaggt gctgtagggt gaaagttcct ttgtgctgtc gtttgttttt 5760
aatgccaaac tttccggtga tcaataacca cctctttttc cttcaggaaa ccttattatt 5820
gttcttggat agtactagga agtatataag gaacctcgat tttggtattg cacggctata 5880
cacatctaag aaactttgta taaaaggtgg ctaccctatt catagcttga tatcaatagg 5940
ccatctcatc actttttatt gaaaaggaaa ggagggaaat atatctgatt caaattactt 6000
gtttgcttct ctttaagaca aaagcataga taatttcagc gtggaacgcc ggaataagat 6060
tggtaccctc gtcagaaagt tacaaatacc gcttcatctt caaaatgact tcaccggaat 6120
cactatcttc tcgtcatatc aggcaaggaa ggacatacac aaccacagac aaggtcatat 6180
cgcggtcgtc gtcgtactca tctaatagtt caatatctaa agattacggc gatcacacac 6240
ccttgtccgt cagcagtgca gcttcagaga cattaccctc acctcagtat atgccgataa 6300
ggacattcaa tacaatgcct acagctggcc caacgccttt acatttattt caaaatg 6357
<210> 11
<211> 10554
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aacagttgcg cagcctgaat ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg 60
cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 120
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 180
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 240
aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 300
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 360
tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 420
ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 480
ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 540
agggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat 600
ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc 660
ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc 720
ttccaacaat aataatgtca gatcctgtag aaaccacatc atccacggtt ctatactgtt 780
gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt 840
aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt 900
cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc 960
atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct 1020
gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc 1080
attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag 1140
caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct 1200
taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa 1260
ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg 1320
aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac 1380
taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc 1440
tgcaggtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa 1500
cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct 1560
gttcggagat taccgaatca aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa 1620
aaaaaaatga tgaattgaat tgaaaagctg tggtatggtg cactctcagt acaatctgct 1680
ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 1740
gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 1800
tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 1860
gcctattttt ataggttaat gtcatgataa taatggtttc ttagtatgat ccaatatcaa 1920
aggaaatgat agcattgaag gatgagacta atccaattga ggagtggcag catatagaac 1980
agctaaaggg tagtgctgaa ggaagcatac gataccccgc atggaatggg ataatatcac 2040
aggaggtact agactacctt tcatcctaca taaatagacg catataagta cgcatttaag 2100
cataaacacg cactatgccg ttcttctcat gtatatatat atacaggcaa cacgcagata 2160
taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt tcggaagcgc 2220
tcgttttcgg aaacgctttg aagttcctat tccgaagttc ctattctcta gaaagtatag 2280
gaacttcaga gcgcttttga aaaccaaaag cgctctgaag acgcactttc aaaaaaccaa 2340
aaacgcaccg gactgtaacg agctactaaa atattgcgaa taccgcttcc acaaacattg 2400
ctcaaaagta tctctttgct atatatctct gtgctatatc cctatataac ctacccatcc 2460
acctttcgct ccttgaactt gcatctaaac tcgacctcta cattttttat gtttatctct 2520
agtattactc tttagacaaa aaaattgtag taagaactat tcatagagtg aatcgaaaac 2580
aatacgaaaa tgtaaacatt tcctatacgt agtatataga gacaaaatag aagaaaccgt 2640
tcataatttt ctgaccaatg aagaatcatc aacgctatca ctttctgttc acaaagtatg 2700
cgcaatccac atcggtatag aatataatcg gggatgcctt tatcttgaaa aaatgcaccc 2760
gcagcttcgc tagtaatcag taaacgcggg aagtggagtc aggctttttt tatggaagag 2820
aaaatagaca ccaaagtagc cttcttctaa ccttaacgga cctacagtgc aaaaagttat 2880
caagagactg cattatagag cgcacaaagg agaaaaaaag taatctaaga tgctttgtta 2940
gaaaaatagc gctctcggga tgcatttttg tagaacaaaa aagaagtata gattctttgt 3000
tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa aaatgcagct cagattcttt 3060
gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta caaaaatgaa gcacagattc 3120
ttcgttggta aaatagcgct ttcgcgttgc atttctgttc tgtaaaaatg cagctcagat 3180
tctttgtttg aaaaattagc gctctcgcgt tgcatttttg ttctacaaaa tgaagcacag 3240
atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 3300
ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 3360
taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 3420
tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 3480
gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 3540
atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 3600
ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 3660
cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 3720
ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 3780
aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 3840
ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 3900
gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 3960
ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 4020
gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 4080
ggagccggtg agcgtgggtc gcgcggtatc attgcagcac tggggccaga tggtaagccc 4140
tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 4200
cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 4260
tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 4320
atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 4380
tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 4440
tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 4500
ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 4560
cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 4620
ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 4680
gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 4740
tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 4800
gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 4860
ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 4920
tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 4980
ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 5040
tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 5100
attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 5160
tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 5220
ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc 5280
aacgcaatta atgtgagtta cctcactcat taggcacccc aggctttaca ctttatgctt 5340
ccggctccta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat 5400
gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc 5460
tagtagttta aacataacga gaacacacag gggcgctatc gcacagaatc aaattcgatg 5520
actggaaatt ttttgttaat ttcagaggtc gcctgacgca tatacctttt tcaactgaaa 5580
aattgggaga aaaaggaaag gtgagagcgc cggaaccggc ttttcatata gaatagagaa 5640
gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta tttaaggacc 5700
tattgttttt tccaataggt ggttagcaat cgtcttactt tctaactttt cttacctttt 5760
acatttcagc aatatatata tatatatttc aaggatatac cattctaatg tctgccccta 5820
agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga aatcacagcc gaagccatta 5880
aggttcttaa agctatttct gatgttcgtt ccaatgtcaa gttcgatttc gaaaatcatt 5940
taattggtgg tgctgctatc gatgctacag gtgttccact tccagatgag gcgctggaag 6000
cctccaagaa ggctgatgcc gttttgttag gtgctgtggg tggtcctaaa tggggtaccg 6060
gtagtgttag acctgaacaa ggtttactaa aaatccgtaa agaacttcaa ttgtacgcca 6120
acttaagacc atgtaacttt gcatccgact ctcttttaga cttatctcca atcaagccac 6180
aatttgctaa aggtactgac ttcgttgttg tcagagaatt agtgggaggt atttactttg 6240
gtaagagaaa ggaagacgat ggtgatggtg tcgcttggga tagtgaacaa tacaccgttc 6300
cagaagtgca aagaatcaca agaatggccg ctttcatggc cctacaacat gagccaccat 6360
tgcctatttg gtccttggat aaagctaatg ttttggcctc ttcaagatta tggagaaaaa 6420
ctgtggagga aaccatcaag aacgaattcc ctacattgaa ggttcaacat caattgattg 6480
attctgccgc catgatccta gttaagaacc caacccacct aaatggtatt ataatcacca 6540
gcaacatgtt tggtgatatc atctccgatg aagcctccgt tatcccaggt tccttgggtt 6600
tgttgccatc tgcgtccttg gcctctttgc cagacaagaa caccgcattt ggtttgtacg 6660
aaccatgcca cggttctgct ccagatttgc caaagaataa ggtcaaccct atcgccacta 6720
tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt gcctgaagaa ggtaaggcca 6780
ttgaagatgc agttaaaaag gttttggatg caggtatcag aactggtgat ttaggtggtt 6840
ccaacagtac caccgaagtc ggtgatgctg tcgccgaaga agttaagaaa atccttgctt 6900
aaatttaact ccttaagtta ctttaatgat ttagttttta ttattaataa ttcatgctca 6960
tgacatctca tatacacgtt tataaaactt aaatagattg aaaatgtatt aaagattcct 7020
cagggattcg atttttttgg aagtttttgt ttttttttcc ttgagatgct gtagtatttg 7080
ggaacaatta tacaatcgaa agatatatgc ttacattcga ccgttttagc cgtgatcatt 7140
atcctatagt aacataacct gaagcataac tgacactact atcatcaata cttgtcacat 7200
gagaactctg tgaataatta ggccactgaa atttgatgcc tgaaggaccg gcatcacgga 7260
ttttcgataa agcacttagt atcacactaa ttggcttttc gcgcaaatta aagccttcga 7320
gcgtcccaaa accttctcaa gcaaggtttt cagtataatg ttacatgcgt acacgcgttt 7380
gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt cttaatacta acataactat 7440
aaaaaaataa atagggacct agacttcagg ttgtctaact ccttcctttt cggttagagc 7500
ggatgtgggg ggagggcgtg aatgtaagcg tgacataact aattacatga tatcgacaaa 7560
ggaaaagggg cctgttgaga cctcagccgc tacagggcgc gtcccattcg ccattcaggc 7620
tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 7680
aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 7740
gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 7800
caggaccacc acggctcgtg cgccagcttt tgttcccttt agtgagggtt aattgcgcgc 7860
ttggcgtaat catggtggtc tcacatttat attgaatttt caaaaattct tacttttttt 7920
ttggatggac gcaaagaagt ttaataatca tattacatgg caataccacc atatacatat 7980
ccatatctaa tcttacttat atgttgtgga aatgtaaaga gccccattat cttagcctaa 8040
aaaaaccttc tctttggaac tttcagtaat acgcttaact gctcattgct atattgaagt 8100
acggattaga agccgccgag cgggcgacag ccctccgacg gaagactctc ctccgtgcgt 8160
cctggtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 8220
acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac 8280
ctggccccac aaaccttcaa atcaacgaat caaattaaca accataggat aataatgcga 8340
ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat 8400
taacagatat ataaatgcaa aagctgcata accactttaa ctaatacttt caacattttc 8460
ggtttgtatt acttcttatt caaatgtcat aaaagtatca acaaaaaatt gttaatatac 8520
ctctatactt taacgtcaag gagaaaaaac tataatggct tcagaaaaag aaattaggag 8580
agagagattc ttgaacgttt tccctaaatt agtagaggaa ttgaacgcat cgcttttggc 8640
ttacggtatg cctaaggaag catgtgactg gtatgcccac tcattgaact acaacactcc 8700
aggcggtaag ctaaatagag gtttgtccgt tgtggacacg tatgctattc tctccaacaa 8760
gaccgttgaa caattggggc aagaagaata cgaaaaggtt gccattctag gttggtgcat 8820
tgagttgttg caggcttact tcttggtcgc cgatgatatg atggacaagt ccattaccag 8880
aagaggccaa ccatgttggt acaaggttcc tgaagttggg gaaattgcca tcaatgacgc 8940
attcatgtta gaggctgcta tctacaagct tttgaaatct cacttcagaa acgaaaaata 9000
ctacatagat atcaccgaat tgttccatga ggtcaccttc caaaccgaat tgggccaatt 9060
gatggactta atcactgcac ctgaagacaa agtcgacttg agtaagttct ccctaaagaa 9120
gcactccttc atagttactt tcaagactgc ttactattct ttctacttgc ctgtcgcatt 9180
ggccatgtac gttgccggta tcacggatga aaaggatttg aaacaagcca gagatgtctt 9240
gattccattg ggtgaatact tccaaattca agatgactac ttagactgct tcggtacccc 9300
agaacagatc ggtaagatcg gtacagatat ccaagataac aaatgttctt gggtaatcaa 9360
caaggcattg gaacttgctt ccgcagaaca aagaaagact ttagacgaaa attacggtaa 9420
gaaggactca gtcgcagaag ccaaatgcaa aaagattttc aatgacttga aaattgaaca 9480
gctataccac gaatatgaag agtctattgc caaggatttg aaggccaaaa tttctcaggt 9540
cgatgagtct cgtggcttca aagctgatgt cttaactgcg ttcttgaaca aagtttacaa 9600
gagaagcaaa tagaactaac gctaatcgat aaaacattag atttcaaact agataaggac 9660
catgtataag aactatatac ttccaatata atatagtata agctttaaga tagtatctct 9720
cgatctaccg ttccacgtga ctagtccaag gatttttttt aaaaagattc tcttttttta 9780
tgatatttgt acataaactt tataaatgaa attcataata gaaacgacac gaaattacaa 9840
aatggaatat gttcataggg tagacgaaac tatatacgca atctacatac atttatcaag 9900
aaggagaaaa aggaggatgt aaaggaatac aggtaagcaa attgatacta atggctcaac 9960
gtgataagga aaaagaattg cactttaaca ttaatattga caaggaggag ggcaccacac 10020
aaaaagttag gtgtaacaga aaatcatgaa actatgattc ctaatttata tattggagga 10080
ttttctctaa aaaaaaaaaa atacaacaaa taaaaaacac tcaatgacct gaccatttga 10140
tggagtttaa gtcaatacct tcttgaacca tttcccataa tggtgaaagt tccctcaaga 10200
attttactct gtcagaaacg gccttaacga cgtagtcgac ctcctcttca gtactaaatc 10260
taccaatacc aaatctgatg gaagaatggg ctaatgcatc atccttaccc agcgcatgta 10320
aaacataaga aggttctagg gaagcagatg tacaggctga acccgaggat aatgcgatat 10380
cccttagtgc catcaataaa gattctcctt ccacgtaggc gaaagaaacg ttaacacacc 10440
ctggataacg atgatctgga gatccgttca acgtggtatg ttcagcggat aatagacctt 10500
tgactaattt atcggatagt cttttgatgt gagcttggtc gttgtcgttt aaac 10554
<210> 12
<211> 1731
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atggaattcc gtgttcatct acacgctgac cacgaacaaa agatcttgca aaaccaaatg 60
aagccagaac acgaagcttc ttacttgatt aaccaaagaa gatctgctaa ctacaagcca 120
aacatctgga agaacgattt tttggaccaa tccttgatct ccaagtacga tggtgatgaa 180
tacagaaaat tgtctgaaaa attgattgaa gaggttaaga tttacatttc cgctgaaacc 240
aaggacttag tcgctaagtt ggaattgatc gattctgtca gaaagttggg cttggctaac 300
catttcgaaa aggaaatcaa ggaagctttg gatggtattg ctgctatcga atctgacaac 360
ttaggtacca gagatgactt gtacggtacc gcattgcact tcaagatctt aagacaacac 420
ggttacaagg tttctcaaga tatcttcggt agattcatgg acgaaaagga cactttggaa 480
aaccaccact ttgctcattt gaagggtatg ttggaattgt ttgaagcctc taaccttggt 540
ttcgaaggtg aagacatctt ggatgaagct aaggcctctt taactttggc tttgagagac 600
tctggtcaca tctgttaccc agactccaac ttatcaagag atgttgttca ctctttagaa 660
ttgccatccc acagacgtgt tcaatggttc gatgtcaagt ggcaaattaa tgcttacgaa 720
aaggatattt gcagagtcaa cgctacctta ctcggtttgg ccaagttgaa cttcaacatg 780
gttcaagctc aattgcaaaa ggatttaaga gaagcttcca aatggtgggc caacctaggt 840
atcgctgata acttgaagtt cgcccgtgac agattggtcg aatgctttgc ctgtgctgtt 900
ggtgttgctt tcgaaccaga atactcttcc ttcagaatct gtttgaccaa ggtcatcaac 960
ctagtgttga ttattgacga cgtctacgac atctacggtt ccgaagaaga attaaagcac 1020
ttcactaacg cggttgacag atgggattcc agagaaactg aacaacttcc tgaatgtatg 1080
aagatgtgtt tccaggtttt gtacaacact acttgtgaaa tagctcacga aattgaaaag 1140
gacaatggtt ggaaccaagt cttgccacaa ttgacaaaag tctgggctga tttctgtaag 1200
gctttgttag ttgaagctga atggtacaac aagtctcata tcccaacttt ggaagaatat 1260
ctgagaaacg gttgtgattc ttcgtccgtt tccatcctct tggtccactc cttcttctct 1320
atcacccacg aaggaaccaa ggaaatggcc gacttcttgc acaagaacga agatttgcta 1380
tacaacttgt ccttgattgt tagattgaac aacgacttgg gtacttctgc tgctgaacaa 1440
gaaagaggtg actctccatc ttctattgtc tgttacatga gagaggtcaa cgcttctgaa 1500
gaaattgctc gtaagaacat taagggtatg attgacaatg cttggaagaa agtcaacggt 1560
aagtgtttca ccaccaacca agttccattc ttgtcctctt tcatgaacaa tgccactaac 1620
atggctagag ttgcccatag cttgtataag gacggtgatg gtttcggtga ccaagaaaaa 1680
ggtccaagaa ctcacatttt gtctttgttg ttccaaccat tggttaactg a 1731
<210> 13
<211> 1683
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atggactgtt ccaagggtat gttggctgac caacaacatg tcttgcaatg ccaaagaaga 60
tctgctaact acaagccaaa catttggaag tacgatttgt tgcaatcctt aacaacccaa 120
tacaacgaag aagactacaa gagacaagtc gaaaagttga aggaagaagt tatgtgtatg 180
ttcatggaag ctgtcgactt ggtaactaaa ttagagttca ttgataacat cgaaaaattg 240
ggtttagcca acttgtttga aatggaaatc aaggaagcct tggacactat cgttgccttc 300
aagaataatt acccaatcac tgaagaaggt ttgtacgcta acgctttgtt cttccgtttg 360
ttgagacaac acggtttcaa tattagtcaa gatatcttca gaggtttcac ttcaaagatg 420
ggtactttca acaagaccac taactgtgat actaaggcta tgttggaatt attggaagct 480
tcccatttgg ctttggaagg tgaaaatatc ttgattgaag ccaaagcctt ctctactggt 540
atcttgaaga acatcactaa ttccaagaac gttgagaaca aggttaccaa gcaattgtct 600
cacgctttgg aattgccatt gcaatggaga gttcaatggt atgatgtcaa gtctcacatc 660
cacgaattgg aaaacaacga caacactaac tctgtcttgt tgaaactagc taagttgaac 720
tttaacattg tccaagctac ccaccaaaac gatttgaaag aaatctccag atggtggaag 780
aacctttgtt tggtcgaaaa cttgtctttc accagaaaca gattggttga atcctactta 840
tgggccgtcg gtgttgcttt cgaaccacaa cacaagtcct tcagaaagtg gttaaccaag 900
gctatcaact tcgttttgat cattgacgac atttacgaca tttacggttc tttggaagaa 960
ctagaatgtt tcaccaactc tgttgaaaga tgggatgaca aggaaattca aagattgcca 1020
gaatgtatga agatctgttt ccaagccttc tacaacacca ccaacgaagt cgctcacgaa 1080
atccaaaagg aaaagggttg gaagaactcc atcttgccat acttgcacaa agtctgggct 1140
gatttctgta aggctctttt agttgaagcc agatggtaca acaaatctta cactccaacc 1200
ctcgatgaat acttggaaaa cggtttggtt tctagctccg gtccagtctt gtctctgcta 1260
gctttctttt ccgttatgga agaaactact cgtgaagaat tggttgactt gttggccaag 1320
tctgacgttt tggtccattg cacttccatg attatcagat tgtgtaacga tttgggtacc 1380
tctgctgctg aattggaaag aggtgacgct ccatcttcta ttttgtgttt catgcgtgaa 1440
gcgaatgtct ctgaagaaat tgctagaaag aagatccgta ctaccattaa ggacacctgg 1500
aacaagatta accacgaatt cattactcaa tctccattct tgcaaccttt tgtcaaatat 1560
accgttaaca ctgctagagt tgctcatttt atctaccaac acggcgatgg tttcggtaac 1620
caagacagag aaaccagagc tcaagttttg tccatgttga ttgaaccact gaagatcaac 1680
tga 1683
<210> 14
<211> 1725
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgtctacta tccctgtttc cagtgtttct ttctctagtt ctgcctctcc attggtattg 60
gatgataaat tatctaccaa gcaagatgtt gtcagacaca ccatgaactt ttctgcttct 120
atctggggtg accaattcct aacataccac gaaccagaag atttggtcat caagaagcaa 180
caagttgaac aattgaagga agaagttaag aaggaattga tggctatcaa gggttctaac 240
gacccacaac aacacatcaa attaatggaa ttgatcgact ctgttcaaag attgggtatc 300
gcttaccatt tcgaagaaga aattgaagaa gctttgcaac acattcacgt tacttatggt 360
gaacactggg ttgacaagga aaacttgcaa tccgtcagct tgtggttcag attactaaga 420
caacaaggtt tcaacgtttc ttccggtgtt ttcaaggact acatggacga aaaaggtaac 480
ttcaaggaat ctttgtgtaa cgatgctcaa ggtatcttgg ctttgtacga agctgcttac 540
atgagagttg aaggtgaaac cagattggac aaggctttgg aattcaccaa ggttcatttg 600
gatatcattt ctaaggatcc atcttgtgat tcttacttga gaactcgtgt tcaccaagcc 660
ctgaaacaac cattaagaag aagattggcc agaatcgaag cattacacta catgccagtc 720
taccaacaag actcttctca caacgaagtt ttactaaagt tggctaaggt cgatttctct 780
gttttgcaat ctatgcacaa gaaagaattg tcacatatct gtaagtggtg gaaggacttg 840
gatttgcaaa acaagttgcc atacgttcgt gacagagtcg ttgaaggtta cttctggatt 900
ttgtccatct actacgaacc tcaacatgcc agaaccagaa tgttcttgat gaagacttgt 960
atgtggcttg ttgttttgga tgacactttt gacaattacg gtacctacga agagttggaa 1020
attttcgctc aagctgtcga aagatggagt atttcatgca tggatatgtt gccagaatat 1080
atgaaattga tttaccaaga attagttaac ttgcacgtcg acatggaaga atccttggaa 1140
aaggaaggta agacttacca aatccattac gtcaaggaaa tggccaagga attggtccgt 1200
aactacttgg tcgaagcccg ttggttgaag gagggttaca tgccaacctt agaagaatac 1260
atgtccgtat ccatggttac cggtacttac ggtttgatga ttgctagatc ctacgttggt 1320
agagacgaca ttgtcactga agacactttc aaatgggttt cttcttaccc accaatcatc 1380
aaggcttcct gtgttattgt ccgtctgatg gacgatattg tctcccacaa ggaagaacaa 1440
gaaagaggtc acgttgcttc ttccattgaa tgttacagca aggaatctgg tgctaccgaa 1500
gaagaagctt gtgaatacat ctcctccaag gtcgaagacg cttggaaggt catcaacaga 1560
gaatcattaa gaccaactgc tgttccattc ccattgttga tgccagctat taatttggcc 1620
agaatgtgtg aagtcttata ctctgtcaac gatggtttca ctcacgctga aggcgacatg 1680
aagtcctata tgaagtccta ctttgtccac ccaatggtca tttga 1725
<210> 15
<211> 371
<212> PRT
<213> Fusarium graminearum
<400> 15
Met Pro His Lys His Val Pro Leu Arg Pro Val Lys Leu Thr Phe Asp
1 5 10 15
Pro Val Gly Ser Asn Thr Leu Gly Val Pro Thr Leu Asp Phe Glu Ser
20 25 30
Leu Phe Arg Glu Asp Ser Val Ser Glu Asp Ala Pro Leu Val Ile Tyr
35 40 45
Pro Glu Asp Met Gly Val Pro Trp Asn Thr Ser Leu Pro Trp Thr Arg
50 55 60
Gln Ser Lys Phe Trp Ala Tyr Ala Glu Ala Ala Gly Tyr Glu Met Ala
65 70 75 80
Asn Gly Ile Ser Leu Asp Lys Ala Ser Glu Arg Gly Thr Leu Pro Met
85 90 95
Glu Leu Met Asp Glu Arg Arg Lys Trp Lys Ile Asp Glu Leu Val Glu
100 105 110
Asp Ala Ile Ser Cys Cys Ala Tyr Leu Tyr Pro Thr Ser Ser Pro Thr
115 120 125
Arg Leu Ala Leu Leu Thr Gln Ser Val Leu Leu Leu Phe Leu His Asp
130 135 140
Asp Val Ile Glu Arg Gly Ala Thr Gln Asn Glu Thr Thr Val Val Asp
145 150 155 160
Glu Phe Leu Ser Met Ala Pro Lys Asn Arg His Leu Lys Lys Phe Trp
165 170 175
Ser Asp Val Leu Glu Cys Asp Pro Val Leu Gly Pro Asp Leu Leu Tyr
180 185 190
Ala Ile His Ala Phe Val Arg Asp Gly Arg Val Lys Ser Pro Phe Lys
195 200 205
Gln Asp His Tyr Ala Thr Leu Ala Asp Tyr Met Leu Tyr Arg Arg Asn
210 215 220
Asp Val Gly Lys Thr Phe Met Ile Ala Ala Ile Arg Phe Gly Ser Gly
225 230 235 240
Val Gln Gln Thr Arg Glu Glu Leu Ala Pro Phe Asp Glu Leu Ala Asp
245 250 255
Leu Tyr Val Arg His Ser Ile Leu Ile Asn Asp Leu Tyr Ser Tyr Asp
260 265 270
Lys Glu Val His Glu Val Lys Thr Ile Asp Ala Ser Ile Val Asn Ala
275 280 285
Val Ala Val Thr Glu Gln Leu Leu Ser Val Ser Pro Asp Leu Ala Lys
290 295 300
Asn Leu Thr Arg Ala Ile Thr Phe Asp Met Glu Lys Glu Phe Tyr Gly
305 310 315 320
Ile Cys Glu Lys Phe Met His Ser Pro Asp Ile Asn Asp Arg Gln Arg
325 330 335
Val Phe Val Thr Ala Leu Phe Asp Ala Leu Thr Gly Asn Ile Phe His
340 345 350
Ser Ala Thr Leu Ser Arg Tyr Val Arg His Gly Glu Arg Pro Leu Pro
355 360 365
Cys Lys Cys
370
<210> 16
<211> 1725
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgtctacta tccctgtttc cagtgtttct tcctctagtt ctgcctctcc attggtattg 60
gatgataaat tatctaccaa gcaagatgtt gtcagacaca ccaccaactt ttctgcttct 120
atctggggtg accaattcct aacataccac gaaccagaag atttggtcat caagaagcaa 180
caagttgaac aattgaagga agaagttaag aaggaattga tggctatcaa gggttctaac 240
gacccacaac aacacatcaa attaatggaa ttgatcgact ctgttcaaag attgggtatc 300
gcttaccatt tcgaagaaga aattgaagaa gctttgcaac acattcacgt tacttatggt 360
gaacactggg ttgacaagga aaacttgcaa tccgtcagct tgtggttcag attactaaga 420
caacaaggtt tcaacgtttc ttccggtgtt ttcaaggact acatggacga aaaaggtaac 480
ttcaaggaat ctttgtgtaa cgatgctcaa ggtatcttgg ctttgtacga agctgcttac 540
atgagagttg aaggtgaaac cagattggac aaggctttgg aattcaccaa ggttcatttg 600
gatatcattt ctaaggatcc atcttgtgat tcttacttga gaactcgtgt tcaccaagcc 660
ctgaaacaac cattaagaag aagattggcc agaatcgaag cattacacta catgccagtc 720
taccaacaag actcttctca caacgaagtt ttactaaagt tggctaaggt cgatttctct 780
gttttgcaat ctatgcacaa gaaagaattg tcacatatct gtaagtggtg gaaggacttg 840
gatttgcaaa acaagttgcc atacgttcgt gacagagtcg ttgaaggtta cttctggatt 900
ttgtccatct actacgaacc tcaacatgcc agaaccagaa tgttcttgat gaagtcttgt 960
atgtggcttg ttgttttgga tgacactttt gacaattacg gtacctacga agagttggaa 1020
attttcgctc aagctgtcga aagatggagt atttcatgca tggatatgtt gccagaatat 1080
atgaaattga tttaccaaga attagttaac ttgcacgtcg acatggaaga atccttggaa 1140
aaggaaggta agacttacca aatccattac gtcaaggaaa tggccaagga attggtccgt 1200
aactacttgg tcgaagcccg ttggttgaag gagggttaca tgccaacctt agaagaatac 1260
atgtccgtat ccatggttac cggtacttac ggtttgatga ctgctagatc ctacgttggt 1320
agagacgaca ttgtcactga agacactttc aaatgggttt cttcttaccc accaatcgtc 1380
aaggcttcct gtgttattgt ccgtctgatg gacgatattg tctcccacaa ggaagaacaa 1440
gaaagaggtc acgttgcttc ttccattgaa tgttacagca aggaatctgg tgctaccgaa 1500
gaagaagctt gtgaatacat ctcctccaag gtcgaagacg cttggaaggt catcaacaga 1560
gaatcattaa gaccaactgc tgttccattc ccattgttga tgccagctat taatttggcc 1620
agaatgtgtg aagtcttata ctctgtcaac gatggtttca ctcacgctga aggcgacatg 1680
aagtcctata tgaagtccta ctttgtccac ccaatggtca tttga 1725
<210> 17
<211> 1725
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
atgtctacta tccctgtttc cagtgtttct tcctctagtt ctgcctctcc attggtattg 60
gatgataaat tatctaccaa gcaagatgtt gtcagacaca ccaccaactt ttctgcttct 120
atctggggtg accaattcct aacataccac gaaccagaag atttggtcat caagaggcaa 180
caagttgaac aattgaagga agaagttaag aaggaattga tggctatcaa gggttctaac 240
gacccacaac aacacatcaa attaatggaa ttgatcgact ctgttcaaag attgggtatc 300
gcttaccatt tcgaagaaga aattgaagaa gctttgcaac acattcacgt tacttatggt 360
gaacactggg ttgacaagga aaacttgcaa tccgtcagct tgtggttcag attactaaga 420
caacaaggtt tcaacgtttc ttccggtgtt ttcaaggact acatggacga aaaaggtaac 480
ttcaaggaat ctttgtgtaa cgatgctcaa ggtatcttgg ctttgtacga agctgcttac 540
atgagagttg aaggtgaaac cagattggac aaggctttgg aattcaccaa ggttcatttg 600
gatatcattt ataaggatcc atcttgtgat tcttacttga gaactcgtgt tcaccaagcc 660
ctgaaacaac cattaagaag aagattggcc agaatcgaag cattacacta catgccagtc 720
taccaacaag actcttctca caacgaagtt ttactaaagt tggctaaggt cgatttctct 780
gttttgcaat ctatgcacaa gaaagaattg tcacatatct gtaagtggtg gaaggacttg 840
gatttgcaaa acaagttgcc atacgttcgt gacagagtcg ttgaaggtta cttctggatt 900
ttgtccatct actacgaacc tcaacatgcc agaaccagaa tgttcttgat gaagtcttgt 960
atgtggcttg ttgttttgga tgacactttt gacaattacg gtacctacga agagttggaa 1020
attttcgctc aagctgtcga aagatggagt atttcatgca tggatatgtt gccagaatat 1080
atgaaattga tttaccaaga attagttaac ttgcacgtcg acatggaaga atccttggaa 1140
aaggaaggta agacttacca aatccattac gtcaaggaaa tggccaagga attggtccgt 1200
aactacttgg tcgaagcccg ttggttgaag gagggttaca tgccaacctt agaagaatac 1260
atgtccgtat ccatggttac cggtacttac ggtttgatga ctgctagatc ctacgttggt 1320
agagacgaca ttgtcactga ggacactttc aaatgggttt cttcttaccc accaatcgtc 1380
aaggcttcct gtgttattgt ccgtctgatg gacgatattg tctcccacaa ggaagaacaa 1440
gaaagaggtc acgttgcttc ttccattgaa tgttacagca aggaatctgg tgctaccgaa 1500
gaagaagctt gtgaatacat ctcctccaag gtcgaagacg cttggaaggt catcaacaga 1560
gaatcattaa gaccaactgc tgttccattc ccattgttga tgccagctat taatttggcc 1620
agaatgtgtg aagtcttata ctctgtcaac gatggtttca ctcacgctga aggcgacatg 1680
aagtcctata tgaagtccta ctttgtccac ccaatggtca tttga 1725
<210> 18
<211> 1731
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atggaattcc gtgttcatct acacgctgac cacgaacaaa agatcttgca aaaccaaatg 60
aagccagaac acgaagcttc ttacttgatt aaccaaagaa gatctgctaa ctacaagcca 120
aacatctgga agaacgattt tttggaccaa tccttgatct ccaagtacga tggtgatgaa 180
tacagaaaat tgtctgaaaa attgattgaa gaggttaaga tttacatttc cgctgaaacc 240
aaggacttag tcgctaagtt ggaattgatc gattctgtca gaaagttggg cttggctaac 300
catttcgaaa aggaaatcaa ggaagctttg gatggtattg ctgctatcga atctgacaac 360
ttaggtacca gagatgactt gtacggtacc gcattgcact tcaagatctt aagacaacac 420
ggttacaagg tttctcaaga tatcttcggt agattcatgg acgaaaagga cactttggaa 480
aaccaccact ttgctcattt gaagggtatg ttggaattgt ttgaagcctc taaccttggt 540
ttcgaaggtg aagacatctt ggatgaagct aaggcctctt taactttggc tttgagagac 600
tctggtcaca tctgttaccc agactccaac ttatcaagag atgttgttca ctctttagaa 660
ttgccatccc acagacgtgt tcaatggttc gatgtcaagt ggcaaattaa tgcttacgaa 720
aaggatattt gcagagtcaa cgctacctta ctcgaattgg ccaagttgaa cttcaacatg 780
gttcaagctc aattgcaaaa ggatttaaga gaagcttcca aatggtgggc caacctaggt 840
atcgctgata acttgaagtt cgcccgtgac agattggtcg aatgctttgc ctgtgctgtt 900
ggtgttgctt tcgaaccaga atactcttcc ttcagaatct gtttgaccaa ggtcatcaac 960
ctagtgttga ttattgacga cgtctacgac atctacggtt ccgaagaaga attaaagcac 1020
ttcactaacg cggttgacag atgggattcc agagaaactg aacaacttcc tgaatgtatg 1080
aagatgtgtt tccaggtttt gtacaacact acttgtgaaa tagctcacga aattgaaaag 1140
gacaatggtt ggaaccaagt cttgccacaa ttgacaaaag tctgggctga tttctgtaag 1200
gctttgttag ttgaagctga atggtacaac aagtctcata tcccaacttt ggaagaatat 1260
ctgagaaacg gttgtgattc ttcgtccgtt tccatcctct tggtccactc cttcttctct 1320
atcacccacg aaggaaccaa ggaaatggcc gacttcttgc acaagaacga agatttgcta 1380
tacaacttgt ccttgattgt tagattgaac aacgacttgg gtacttctgc tgctgaacaa 1440
gaaagaggtg actctccatc ttctattgtc tgttacatga gagaggtcaa cgcttctgaa 1500
gaaattgctc gtaagaacat taagggtatg attgacaatg cttggaagaa agtcaacggt 1560
aagtgtttca ccaccaacca agttccattc ttgtcctctt tcatgaacaa tgccactaac 1620
atggctagag ttgcccatag cttgtataag gacggtgatg gtttcggtga ccaagaaaaa 1680
ggtccaagaa ctcacatttt gtctttgttg ttccaaccat tggttaactg a 1731

Claims (10)

1.一种法尼烯合酶，其特征在于：所述的法尼烯合酶为α-法尼烯合酶突变体或β-法尼烯合酶突变体；
所述的α-法尼烯合酶为氨基酸序列如SEQ ID NO.1所示的茶树来源的α-法尼烯合酶，或以氨基酸序列如SEQ ID NO.1所示的茶树来源的α-法尼烯合酶为基础，含有下述突变中的一种或两种的α-法尼烯合酶突变体：W281C、C455N；或α-法尼烯合酶为氨基酸序列如氨基酸序列如SEQ ID NO.3所示的梨来源的α-法尼烯合酶，或以氨基酸序列如SEQ ID NO.3所示的梨来源的α-法尼烯合酶为基础，含有下述突变中的一种或两种的α-法尼烯合酶突变体：G252E、D10G、A78T；
所述的β-法尼烯合酶为氨基酸序列如SEQ ID NO.2所示的洋甘菊来源的β-法尼烯合酶，或以氨基酸序列如SEQ ID NO.2所示的洋甘菊来源的β-法尼烯合酶为基础，含有下述突变中的一种或多种的β-法尼烯合酶突变体：F11S、M35T、T319S、I434T、I460V、K59R、S204Y。
2.权利要求1所述的法尼烯合酶的在生产法尼烯或构建产法尼烯的重组微生物中的应用。
3.一种产法尼烯的重组微生物，其特征在于：所述的重组微生物为产α-法尼烯的重组微生物或产β-法尼烯的重组微生物；
所述的产α-法尼烯的重组微生物中基因的拷贝数为ERG10：ERG13：tHMG1：ERG12：ERG8：MVD1：IDI1：ERG20：aFS＝2：2：X：2：2：2：2：2：X，X为大于等于1的整数；
所述的产β-法尼烯的重组微生物中基因的拷贝数为ERG10：ERG13：tHMG1：ERG12：ERG8：MVD1：IDI1：ERG20：bFS＝2：2：X：2：2：2：2：2：X，X为大于等于1的整数；
其中，ERG10为编码乙酰乙酰辅酶A硫解酶的基因，ERG13为编码HMG-CoA合酶的基因，tHMG1为编码HMG-CoA还原酶的基因，ERG12为编码甲羟戊酸激酶的基因，ERG8为编码甲羟戊酸-5-磷酸激酶的基因，MVD1为编码甲羟戊酸焦磷酸脱羧酶的基因，IDI1为编码异戊二烯焦磷酸异构酶的基因，ERG20为编码法尼烯焦磷酸合酶的基因，aFS为编码α-法尼烯合酶的基因，bFS为编码β-法尼烯合酶的基因。
4.根据权利要求3所述的产法尼烯的重组微生物，其特征在于：
aFS编码的α-法尼烯合酶为氨基酸序列如SEQ ID NO.1所示的茶树来源的α-法尼烯合酶或权利要求1中所述的α-法尼烯合酶突变体或氨基酸序列如SEQ ID NO.3所示的梨来源的α-法尼烯合酶或权利要求1中所述的α-法尼烯合酶突变体；
bFS编码的β-法尼烯合酶为氨基酸序列如SEQ ID NO.2所示的洋甘菊来源的β-法尼烯合酶或权利要求1中所述的β-法尼烯合酶突变体。
5.根据权利要求3所述的产法尼烯的重组微生物，其特征在于：aFS编码的α-法尼烯合酶的氨基酸序列如SEQ ID NO.4所示；bFS编码的β-法尼烯合酶的氨基酸序列如SEQ IDNO.5或SEQ ID NO.6所示。
6.根据权利要求3所述的产法尼烯的重组微生物，其特征在于：所述的重组微生物以酿酒酵母为宿主。
7.根据权利要求3所述的产法尼烯的重组微生物，其特征在于：
所述的产α-法尼烯的重组微生物在酿酒酵母CEN.PK2-1D的基础上，含有5个SEQ IDNO.3所示或SEQ ID NO.4所示α-法尼烯合酶的编码基因，含有额外的MVA途径基因ERG10、ERG13、THMG1、ERG12、ERG8、MVD1、IDI1，以及额外一个ERG20基因，额外一个tHMG1基因；
所述的产β-法尼烯的重组微生物在酿酒酵母CEN.PK2-1D的基础上，含有5个SEQ IDNO.5所示或SEQ ID NO.6所示β-法尼烯合酶的编码基因，含有额外的MVA途径基因ERG10、ERG13、THMG1、ERG12、ERG8、MVD1、IDI1，以及额外一个ERG20基因，额外一个tHMG1基因。
8.根据权利要求3-7任一项说书的产法尼烯的重组微生物，其特征在于：所述的重组微生物敲除了GAL80基因。
9.权利要求3-8任一项所述的产法尼烯的重组微生物的构建方法，其特征在于：所述的构建方法为将基因ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20、Pyc-aFS/Mac-bFS克隆到多个质粒上，再将线性化后含有目的基因的片段转入到宿主中，经过筛选，获得表达各基因的重组微生物。
10.权利要求3-8任一项所述的产法尼烯的重组微生物在生产法尼烯中的应用。
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