具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明,本发明所用试剂和原材料均可通过市售获得。
本发明实施例中所涉及的培养基的配方如下:
(1)YPD培养基:蛋白胨20g/L,酵母提取物10g/L,葡萄糖20g/L(固体YPD培养基在配制时添加20g/L的琼脂粉)。
(2)YPG培养基:蛋白胨20g/L,酵母提取物10g/L,葡萄糖2g/L,半乳糖18g/L。
(3)SD-URA培养基:YNB培养基6.7g/L,URA(尿嘧啶)缺陷氨基酸(100X)10mL/L,葡萄糖20g/L(固体培养基配制时添加20g/L的琼脂粉)。
(4)SD-MET培养基:YNB培养基6.7g/L,MET(甲硫氨酸)缺陷氨基酸(100X)10mL/L,葡萄糖20g/L(固体培养基配制时添加20g/L的琼脂粉)。
(5)SD-LEU培养基:YNB培养基6.7g/L,LEU(亮氨酸)缺陷氨基酸(100X)10mL/L,葡萄糖20g/L(固体培养基配制时添加20g/L的琼脂粉)。
(6)SD-HIS培养基:YNB培养基6.7g/L,HIS(组氨酸)缺陷氨基酸(100X)10mL/L,葡萄糖20g/L(固体培养基配制时添加20g/L的琼脂粉)。
缺陷氨基酸母液(100X):腺嘌呤硫酸盐0.25g,精氨酸0.12g,天冬氨酸0.6g,谷氨酸0.6g,组氨酸0.12g,亮氨酸0.36g,赖氨酸0.18g,甲硫氨酸0.12g,苯丙氨酸0.3g,丝氨酸2.25g,苏氨酸1.2g,色氨酸0.24g,酪氨酸0.18g,缬氨酸0.9g,尿嘧啶0.12g,用ddH2O定容到57mL,根据需要可不加任意一种氨基酸配置成缺陷氨基酸母液(100X)。上述原料均购买自Sigma-Aldrich。
(7)发酵培养基:25g/L葡萄糖,(NH4)2SO4 15g/L,KH2PO4 8g/L,MgSO4 3g/L,ZnSO4·7H2O 0.72g/L,维生素溶液12mL/L,以及微量金属盐溶液10mL/L。
(8)补料培养基:葡萄糖585g/L,KH2PO4 9g/L,MgSO4 2.5g/L,K2SO4 3.5g/L,Na2SO40.28g/L,维生素溶液12mL/L,以及微量金属盐溶液10mL/L;其中,
维生素溶液:维生素H 0.05g/L,泛酸钙1g/L,烟酸1g/L,肌醇25g/L,盐酸硫胺素1g/L,盐酸吡哆醇1g/L和对氨基苯甲酸0.2g/L。
微量金属盐溶液:EDTA(乙二胺四乙酸)15g/L,ZnSO4·7H2O 10.2g/L,MnCl2·4H2O0.5g/L,CuSO4 0.5g/L,CoCl2·6H2O 0.86g/L,Na2MoO4·2H2O 0.56g/L,CaCl2·2H2O 3.84g/L和FeSO4·7H2O 5.12g/L。
(9)PDA培养基:马铃薯葡萄糖粉末6g/L(固体培养基配制时添加20g/L的琼脂粉)。
(10)玉米黑粉菌培养基:马铃薯葡萄糖粉末6g/L,KH2PO4 3.0g/L,MgSO4·7H2O1.5g/L(固体培养基配制时添加20g/L的琼脂粉)。
实施例1酵母内源性基因、启动子、终止子和枯草芽孢杆菌GDH基因的克隆
1、酵母基因组的提取
(1)挑取酿酒酵母BY4741(购于ATCC)的单克隆于5mL YPD培养基中,30℃培养20~24h,而后4000rpm离心5min,集菌,并置于研钵中。
(2)液氮速冻,研磨,待液氮挥干后,加入1mL DNAiso Reagent(宝日医生物技术有限公司),混匀。
(3)将裂解液转移至离心管中,于4℃或室温12,000rpm离心10min。
(4)将上清液转移到新的离心管内,加入1/2体积的无水乙醇,混匀,室温4000rpm离心,去上清。
(5)用75%(v/v)的乙醇清洗沉淀2次,并挥干残留乙醇,加入50μL ddH2O溶解,作为PCR克隆模板。
2、酵母内源性基因和表达元件的克隆
(1)以上述获得的酵母基因组为模板,分别克隆以下7个基因、5个启动子和5个终止子(扩增引物见表1):
ERG8(引物ERG8-F、ERG8-R)、ERG10(引物ERG10-F、ERG10-R)、tHMG1(引物tHMG1-F、tHMG1-R)、ERG12(引物ERG12-F、ERG12-R)、ERG13(引物ERG13-F、ERG13-R)、ERG19(引物ERG19-F、ERG19-R),IDI1(引物IDI1-F、IDI1-R)和UPC2(引物UPC2-F、UPC2-R)基因;其中,ERG8、ERG10、tHMG1、ERG12、ERG13、ERG19、IDI1和UPC2的基因序列分别如SEQ ID NO.1~8所示。
PPGK1(引物PPGK1-F、PPGK1-R)、PTEF1(引物PTEF1-F、PTEF1-R)、PTDH3(引物PTDH3-F、PTDH3-R)、PTDH2(引物PTDH2-F、PTDH2-R)和PTPI1(引物PTPI1-F、PTPI1-R)启动子,其核苷酸序列分别如SEQ ID NO.9~13所示。
TADH1(引物TADH1-F、TADH1-R)、TCYC1(引物TCYC1-F、TCYC1-R)、TTDH2(引物TTDH2-F、TTDH2-R)、TPYX212(引物TPYX212-F、TPYX212-R)和TFBA1(引物TFBA1-F、TFBA1-R)终止子,其核苷酸序列分别如SEQ ID NO.14~18所示。
PCR反应体系:phanta max高保真酶(Phanta Max Super-Fidelity DNAPolymerase)0.5μL,dNTP(10mM)0.5μL,2x Phanta Max Buffer 10μL,上下游特异性引物各0.5μL,ddH2O 7μl,模板1μL,总反应体系20μL。
PCR扩增反应条件为:95℃1min;95℃30s,50-60℃30s,72℃1-2min,30循环;72℃7min。
DNA片段纯化:PCR反应结束后,琼脂糖凝胶电泳检测条带,并使用普通琼脂糖凝胶DNA回收试剂盒(天根生化科技有限公司)进行胶回收,具体操作过程见说明书。
(2)DNA片段连接pEASY-Blunt载体
DNA片段连接平末端pEASY-Blunt(全式金生物技术有限公司),转化DH5α菌株,具体操作见产品说明书。
(3)37℃培养14~16h后,挑选单菌落做菌落PCR。
PCR反应体系:Easy Taq聚合酶0.2μL,dNTPs(2.5mM)0.8μL,10x Easy Taq Buffer1μL,通用引物M13F(表1)(10μM)0.3μL,M13R(表1)(10μM)0.3μL,DMSO 1μL,ddH2O 6.4μL。
PCR扩增条件为:95℃5min;95℃30s,55℃30s,72℃1-3min,30个循环;72℃7min。
反应结束后,琼脂糖凝胶电泳检测阳性转化子,送样测序。
3、UPC2的定点突变
UPC2是正向调控酵母MVA途径的重要转录因子,可以促进萜类化合物的底物IPP和DMAPP的合成,将该转录因子的第888位的甘氨酸(Glycine)突变为天门冬氨酸(Aspartate),能进一步提高其调控效率。利用PCR的方法,以pEASY-Blunt-UPC2质粒(pEASY-Blunt-UPC2质粒由UPC2基因构建到pEASY-Blunt载体上制备得到,具体参考实施例1)为模板,进行定点突变(引物序列见表1),具体步骤如下:
(1)PCR反应系:Phanta Max Super-Fidelity DNA Polymerase 0.5μL,dNTP 0.5μL,2x Phanta Max buffer 10μL,正向、反向引物各1μL(引物见表1:上游引物UPC2-1-F和下游引物UPC2-1-R),ddH2O 6μL,模板1μL,总反应体系20μL。
(2)PCR反应条件:95℃3min;95℃30s,62℃30s,72℃2min,18个循环;72℃7min。
(3)向PCR产物中加入0.5μL限制性内切酶DpnΙ,37℃消化1h。
(4)将消化体系全部转化大肠杆菌DH5α,37℃倒置培养14~18h。
(5)菌落PCR挑选阳性转化子进行测序。
UPC2定点突变得到的UPC2-1的核苷酸序列如SEQ ID NO.19所示。
实施例2、植物来源的基因的克隆
1、植物组织RNA的提取
利用快速通用RNA提取试剂盒(北京华越洋生物科技有限公司)提取长春花、丹参、拟南芥(长春花、丹参、拟南芥均可通过常规市售购买得到)叶片中的总RNA,具体操作过程见说明书。
2、mRNA反转录成cDNA
采用Hiscript II Reverse Transcriptase试剂盒(南京诺维赞生物科技有限公司)将RNA反转录成cDNA,具体操作见试剂盒说明书。
3、目的基因的克隆
以长春花cDNA为模板克隆CrTPS18和CrCYP71D349基因,以拟南芥cDNA为模板克隆AtCPR1基因,以丹参cDNA为模板克隆SmFPPS基因,引物见表1,具体实施方法可参考实施例1。克隆得到的CrTPS18、CrCYP71D349、AtCPR1和SmFPPS的基因序列如SEQ ID NO.20~23所示。
表1.本发明涉及的克隆基因和表达元件所用引物序列
实施例3、CRISPR/Cas9技术敲除酿酒酵母中的目的基因
1、目的基因spacer核酸序列的设计和载体的构建
参考文献(Bao Z,Xiao H,Liang J et al.Homology-integrated CRISPR–Cas(HI-CRISPR)system for one-step multigene disruption in Saccharomycescerevisiae.ACS Synthetic Biology,2015,5:585-594))中的CRISPR/Cas9靶点设计原则,设计消耗法尼基焦磷酸(FPP)的LPP1和以及消耗辅酶NADPH的DPP1基因,GDH1三个基因的spacer序列,并委托苏州泓迅生物科技有限公司合成。LPP1、DPP1、GDH1基因的crRNAspacer核酸序列如SEQ ID NO.24所示,crRNA spacer序列通过限制性内切酶BsaΙ酶切连接到pCRCT载体(武汉淼灵生物科技有限公司)上。
2、pCRCT载体转化酿酒酵母敲除目的基因
(1)利用Zymo Research Frozen-EZ Yeast Transformation II KitTM酵母转化试剂盒(上海懋康生物科技有限公司)进行酵母转化,将pCRCT载体转化酿酒酵母BY4741,具体转化方法参照产品说明书,30℃培养4~5d(天)。
(2)利用营养缺陷型培养基SD-URA和菌落PCR筛选阳性转化子。
(3)将阳性转化子接种于4mL SD-URA培养基中,30℃、220rpm条件下培养2d。
(4)取100μL菌液接于新鲜SD-URA培养基中培养2d。
(5)将菌液稀释后涂于SD-URA缺陷平板上,挑选单克隆摇菌2d,离心后集菌,利用DNAiso Reagent基因组提取试剂盒提取基因组。
(6)PCR克隆目的基因,产物直接测序,检测目的基因的敲除情况。
(7)将已经敲除目的基因的菌株用YPD培养基传代培养十天,以去除pCRCT载体,得到菌株BY4741-1。
实施例4、各表达模块的构建
1、利用重叠PCR构建基因表达模块I~X
(1)各模块的构建都通过重叠PCR技术,模块中各连接的片段通过PCR克隆,加上40~50bp的重叠区域,重叠区域碱基退火温度在60~70℃之间。
(2)第一轮反应体系:Phanta Max Super-Fidelity DNA Polymerase 0.5μL,dNTP(10mM)0.5μL,2x Phanta Max Buffer 10μL,DNA片段1~5μL,上下游引物各0.5μL(见表2),加ddH2O至20μL;其中引物序列见表2。
第一轮PCR反应条件:95℃3min;95℃30s,60~70℃30s,72℃1~4min,15个循环;72℃7min。
(3)取第一轮PCR反应液1μL作为第二轮PCR模板,第二轮PCR体系:Phanta MaxSuper-Fidelity DNA Polymerase 0.5μL,dNTP(10mM)0.5μL,2x Phanta Max Buffer 10μL,上下游引物各0.5μL(引物为连接成完整DNA片段上下游引物,见表2),ddH2O 7μL,模板1μL,总反应体系20μL。
第二轮PCR反应条件:95℃3min;95℃30s,50~60℃30s,72℃1~4min,30个循环;72℃5min。
(4)胶回收并连接pEASY-Blunt载体(全式金生物技术有限公司)测序。
按上述步骤共构建以下10个模块:
(a)将ERG8、PTDH2和TPYX212重叠,构建基因表达模块PTDH2-ERG8-TPYX212,命名为模块I;其中,第一轮PCR克隆ERG8引物为I-ERG8-F、I-ERG8-R,克隆PTDH2引物为PTDH2-F、I-PTDH2-R,克隆TPYX212引物为I-TPYX212-F、TPYX212-R;第二轮PCR引物为PTDH2-F、TPYX212-R;
(b)将ERG10、PPGK1和TADH1重叠,构建基因模块PPGK1-ERG10-TADH1,命名为模块II;其中,第一轮PCR克隆ERG10引物为II-ERG10-F、II-ERG10-R,克隆PPGK1引物为PPGK1-F、II-PPGK1-R,克隆TADH1引物为II-TADH1-F、TADH1-R;第二轮PCR引物为PPGK1-F、TADH1-R;
(c)将ERG12、PTDH3和TTDH2重叠,构建基因表达模块PTDH3-ERG12-TTDH2,命名为模块III;其中,第一轮PCR克隆ERG12引物为III-ERG12-F、III-ERG12-R,克隆PTDH3引物为PTDH3-F、III-PTDH3-R,克隆TTDH2引物为III-TTDH2-F、TTDH2-R;第二轮PCR引物为PTDH3-F、TTDH2-R;
(d)将ERG13、PTEF1和TCYC1重叠,构建基因表达模块PTEF1-ERG13-TCYC1,命名为模块IV;其中,第一轮PCR克隆ERG13引物为IV-ERG13-F、IV-ERG13-R,克隆PTEF1引物为PTEF1-F、IV-PTEF1-R,克隆TCYC1引物为IV-TCYC1-F、TCYC1-R;第二轮PCR引物为PTEF1-F、TCYC1-R;
(e)将ERG19、PTPI1和TFBA1重叠,构建基因表达模块PTPI1-ERG19-TFBA1,命名为模块V;其中,第一轮PCR克隆ERG19引物为V-ERG19-F、V-ERG19-R,克隆PTPI1引物为PTPI1-F、V-PTPI1-R,克隆TFBA1物为V-TFBA1-F、TFBA1-R;第二轮PCR引物为PTPI1-F、TFBA1-R;
(f)将CrTPS18、PTPI1和TFBA1重叠,构建基因表达模块PTPI1-CrTPS18-TFBA1,命名为模块VI;其中,第一轮PCR克隆CrTPS18引物为VI-CrTPS18-F、VI-CrTPS18-R,克隆PTPI1引物为PTPI1-F、VI-PTPI1-R,克隆TFBA1物为VI-TFBA1-F、TFBA1-R;第二轮PCR引物为PTPI1-F、TFBA1-R;
(g)将CrCYP71D349、PPGK1和TADH1重叠,构建基因表达模块PPGK1-CrCYP71D349-TADH1,命名为模块VII;其中,第一轮PCR克隆CrCYP71D349引物为VII-CrCYP71D349-F、VII-CrCYP71D349-R,克隆PPGK1引物为PPGK1-F、VII-PPGK1-R,克隆TADH1物为II-TADH1-F、TADH1-R;第二轮PCR引物为PPGK1-F、TADH1-R;
(h)将AtCPR1、PTEF1和TCYC1重叠,构建基因表达模块PTEF1-AtCPR1-TCYC1,命名为模块VIII;其中,第一轮PCR克隆AtCPR1引物为VIII-AtCPR1-F、VIII-AtCPR1-R,克隆PTEF1引物为PTEF1-F、VIII-PTEF1-R,克隆TCYC1引物为VIII-TCYC1-F、TCYC1-R;第二轮PCR引物为PTEF1-F、TCYC1-R;
(i)将tHMG1、PPGK1、PTEF1和UPC2-1重叠,构建基因表达模块tHMG1-PPGK1-PTEF1-UPC2-1,命名为模块IX;其中,第一轮PCR克隆tHMG1引物为IX-tHMG1-F、IX-tHMG1-R,克隆PPGK1引物为IX-PPGK1-F、IX-PPGK1-R,克隆PTEF1引物为IX-PTEF1-F、IX-PTEF1-R,克隆UPC2-1引物为IX-UPC2-1-F、IX-UPC2-1-R,第二轮PCR引物为IX-tHMG1-F、IX-UPC2-1-R;
(j)将IDI1、SmFPPS、PTDH2、PTDH3重叠,构建基因表达模块IDI1-PPGK1-PTEF1-SmFPPS,命名为模块X;其中,第一轮PCR克隆IDI1引物为X-IDI1-F、X-IDI1-R,克隆PTDH2引物为X-PTDH2-F、X-PTDH2-R,克隆PTDH3引物为X-PTDH3-F、X-PTEF1-R,克隆SmFPPS引物为X-SmFPPS-F、X-SmFPPS-R,第二轮PCR引物为X-IDI1-F、IX-SmFPPS-R。
表2构建基因表达模块所用引物序列
2、整合载体pCfB2875-1和pCfB2798-1的构建
(1)利用限制性内切酶SfaAI线性化载体pCfB2875(Addgene),酶切体系:pCfB2875载体1μg,SfaAI 0.5μL,10x Fast Digest Buffer 2μL,加水补齐到20μL体系。37℃酶切1h,胶回收线性化载体。
(2)将模块IX从pEASY-Blunt-tHMG1-PPGK1-PTEF1-UPC2-1(即上述模块IX连接pEASY-Blunt载体获得)上用SfaAI切下来,酶切体系同载体pCfB2875线性化酶切体系。
(3)将模块IX通过T4连接酶连接到载体pCfB875sfaΙ酶切位点,连接体系:线性化的pCfB2875 20~100ng,模块IX 50~500ng,T4DNA Ligase 0.5μL,10x T4DNA LigaseBuffer 1μL,加水补齐到10μL体系。
(4)16℃连接1h,将连接产物转化DH5α感受态细胞,37℃培养14~16h,通过菌落PCR筛选和提取质粒测序,获得连接好的载体pCfB2875-1。
(5)含有模块X的整合表达载体pCfB2798-1的构建方法如pCfB2875-1。
实施例5、构建工程酵母菌
1、BY4741-3菌株的构建
(1)用限制性内切酶NotΙ将包含模块IX的TCYC1-IX-TADH1表达单元和筛选标记KIURA3(KIURA3的核苷酸序列如SEQ ID NO.25所示,由苏州泓迅生物科技有限公司合成)的DNA整合片段从载体pCfB2875-1(实施例4步骤2构建)切下来。酶切体系:载体pCfB2875-1 3~5μg,NotΙ限制性内切酶1~2μL,10x Fast Digest Buffer 5μL,补水至50μL,37℃酶切1~2h。跑电泳检测酶切效果,并纯化。
(2)取5μL上述纯化的DNA片段,转化酿酒酵母BY4741-1(方法同实施例3步骤2),将模块整合到酵母BY4741-1染色体的TY3位点,用尿嘧啶缺陷型培养基(SD-URA培养基)平板进行筛选,得到工程菌株BY4741-2。
(3)用限制性内切酶NotΙ将包含模块X的TCYC1-X-TADH1表达单元和筛选标记KILEU2(KILEU2的核苷酸序列如SEQ ID NO.26所示,由苏州泓迅生物科技有限公司合成)的DNA整合片段从载体pCfB2798-1(实施例4步骤2构建)切下来,并整合到酵母菌株BY4741-2染色体TY4位点,整合方法同上所述,利用LEU营养缺陷型培养基(SD-LEU培养基)平板筛选,得到工程菌株BY4741-3。
2、敲除筛选标记
(1)将pSH65载体(武汉淼灵生物科技有限公司)转化BY4741-3菌株(ZymoResearch Frozen-EZ Yeast Transformation II KitTM酵母转化试剂盒;上海懋康生物科技有限公司),用含有zeocin(100μg/mL)的YPD培养基筛选。
(2)挑选单菌落用含有zeocin(100μg/mL)的YPD培养基培养四天,通过划板稀释挑选单菌落,提取基因组,通过PCR克隆出整合的DNA片段,通过测序筛选得到KILEU2和KIURA3都被敲除的菌株(通过Cre/LoxP体系酶敲除KILEU2和KIURA3筛选标记),再用YPD培养基传代培养十天,以丢掉pSH65质粒,得到工程菌株BY4741-4。
3、酵母菌株BY4741-5的构建
(1)利用酵母体内同源重组的方法,将模块I、II、III、IV和V整合到工程菌株BY4741-4染色体的HIS3位点,通过PCR给相邻模块和筛选标记MET15(核苷酸序列如SEQ IDNO.27所示,由苏州泓迅生物科技有限公司合成)加上50bp的重叠区域,同时在模块1的5'端和MET15的3'端加上50bp同源臂用于整合到HIS3位点,整合顺序为模块I-II-III-IV-V-MET15(即将这5个模块和筛选标记MET15一起转化到酿酒酵母内会发生体内同源重组连接成完整的一条DNA片段,并且由于两端的模块I和MET15带有50bp特异性的同源臂,使得这个DNA片段可以整合到酵母染色体的HIS3位点)。其中,克隆模块I的引物为HIS3-1-F、HIS3-1-R,克隆模块II的引物为PPGK1-F、HIS3-2-R,克隆模块III引物为PTDH3-F、HIS3-3-R,克隆模块IV的引物为PTEF1-F、HIS3-4-R,克隆模块V的引物为PTPI1-F、HIS3-5R,克隆MET15的引物为HIS3-MET15(KILEU2,HIS3,KIURA3)-F、HIS3-MET15-R。
(2)将上述6个片段转化工程菌株BY4741-4,用SD-MET缺陷型培养基筛选和菌落PCR鉴定,得到阳性菌株BY4741-5。
4、5-epi-jinkoh-eremol高产酿酒酵母工程菌株CR-1的构建
将模块VI整合到BY4741-5菌株染色体NDT80位点上,所用筛选标记HIS3(HIS3的核苷酸序列如SEQ ID NO.28所示,由苏州泓迅生物科技有限公司合成),利用HIS缺陷型培养基(SD-HIS培养基)进行筛选,得到菌株CR-1。克隆模块VI的引物为NDT80-8-F、NDT80(GAL80)-8-R,克隆模块IX的引物为PPGK1-F、NDT80(GAL80)-9-R,克隆模块X的引物为PTEF1-F、NDT80(GAL80)-10-R,克隆筛选标记HIS3的引物为HIS3-MET15(KILEU2,HIS3,KIURA3)-F、NDT80-HIS3-R。克隆方法参考实施例5步骤3,构建菌株CR-1。
5、debneyol高产酿酒酵母工程菌株CR-2的构建
利用KIURA3(KIURA3的核苷酸序列如SEQ ID NO.27所示,由苏州泓迅生物科技有限公司合成)筛选标记第二次将模块VII和VIII整合到CR-1菌株染色体Gal80位点上,利用URA缺陷型培养基(SD-URA培养基)进行筛选。克隆模块VII的引物为PPGK1-F、NDT80(GAL80)-9-R,克隆模块VIII的引物为PTEF1-F、NDT80(GAL80)-10-R,克隆KIURA3的引物为HIS3-MET15(KILEU2,HIS3,KIURA3)-F、GAL80-KIURA3-R。方法参考实施例5步骤3(酵母菌株BY4741-7的构建),构建菌株CR-2。
表3.模块整合酵母染色体引物
实施例6、菌株CR-1、CR-2发酵生成雅槛蓝醇型倍半萜
1、菌株CR-1发酵生成5-epi-jinkoh-eremol
(1)将菌株CR-1的单菌落接种到15mL的YPD培养基中,30℃220rpm培养OD值至2~3。
(2)将菌液接种到3瓶100mL的发酵培养基中,每瓶接种5mL,30℃220rpm培养OD值至8~10。
(3)将上述3瓶100mL菌液合并,都接种到3L的发酵培养基中进行发酵。发酵条件为:温度30℃,转速300~1000rpm,pH5.0(用氨水调节),溶氧值30%,通气量3~20L/min;当溶氧值达到60%时,启动补料***进行补料,使培养基中葡萄糖的含量维持在5g/L;发酵时间为168h。
(4)菌株CR-1发酵生成debneyol的方法与菌株CR-1发酵生成5-epi-jinkoh-eremol相同。
2、菌株CR-2发酵生成5-epi-jinkoh-eremol和debneyol:方法同上。
3、发酵产物的萃取、检测及纯化
(1)取上述发酵液加入等体积的正己烷或乙酸乙酯超声萃取20min,静置24h,取200μL有机层于液相小瓶中,用安捷伦气质联用仪7890B-5977B进行GC-MS检测。检测方法:HP-5ms毛细管柱;进样量1μL,不分流;进样口温度250℃,起始温度50℃保持3min,而后以20℃/min的速率升到70℃保持1min,接着15℃/min的速率升到300℃保持3min;ElectronIonization离子源,能量强度70eV;MS溶剂延迟设为12min,开启电压倍增模式,增益因子设为1。离子检测设为离子选择模式(SIM)并检测m/z 189,136,119,105等离子。
(2)将剩余上述萃取的有机层旋转蒸发,浓缩至干燥,复溶于少量正己烷中,然后按1:40的比例过200~300目硅胶柱。柱纯化条件为:6倍柱体积的正己烷,6倍柱体积的正己烷/丙酮(40:1),3倍柱体积的正己烷/丙酮(30:1),3倍柱体积的正己烷/丙酮(20:1),3倍柱体积的正己烷/丙酮(10:1)。洗脱期间,利用薄层层析(TLC)检测目标产物,将含有目标产物的馏分经气相色谱分析后合并浓缩,并用高效液相色谱(HPLC)进一步分离纯化。最后,将含有目标产物的馏分合并,用氮气吹干备用。
(3)结果:酿酒酵母中重建雅槛蓝醇型倍半萜的生物合成途径如图1所示;酵母菌株发酵产物的GC-MS总离子流图和质谱图如图2所示;CR-1和CR-2菌株高密度发酵生产5-epi-jinkoh-eremol和debneyol的产量如图3所示:5-epi-jinkoh-eremol由CrCYP71D349基因进一步氧化产生debneyol,CR1菌株没有CrCYP71D349基因,故只产生5-epi-jinkoh-eremol;CR2菌株引入了CrCYP71D349基因,故5-epi-jinkoh-eremol基本都全被转化为debneyol。
实施例7、雅槛蓝醇型倍半萜抗植物病原真菌的应用
1、植物病原真菌的活化和雅槛蓝醇型倍半萜母液的配置
(1)利用马铃薯葡萄糖固体培养基(PDA)和玉米黑粉病菌培养基活化植物病原真菌立枯丝核菌Rhizoctonia solani和玉米黑粉病菌Ustilago maydis(两种菌株均购自中国普通微生物菌种保藏中心,CGMCC)。
(2)将菌株CR1发酵得到的5-epi-jinkoh-eremol和菌株CR2发酵得到的debneyol分别用DMSO(二甲基亚砜)配成100mg/mL的母液,,同时将抗植物真菌的阳性药Validamycin用DMSO配成50mg/mL的母液。
2、雅槛蓝醇型倍半萜在刺五加上对立枯丝核菌的抗性应用
(1)将5-epi-jinkoh-eremol、debneyol和Validamycin母液用无菌蒸馏水稀释成50μg/mL的工作液,并均匀喷洒在刺五加活体植株叶片表面。
(2)用打孔器从活化立枯丝核菌的PDA培养基上取直径为5mm的菌饼,将菌饼放到喷洒化合物的刺五加叶片上。将接种后的刺五加置于光照培养箱中,培养箱条件为:16h光照/8h黑暗,25℃,湿度70-80%。
(3)48h后,统计并比较接种叶片上的病斑面积,以评估抗病效果。结果如图4A所示,对照组和50μg/mL的阳性药Validamycin处理的刺五加叶片被立枯丝核菌侵染,并产生坏死斑;同浓度的5-epi-jinkoh-eremol和debneyol处理的刺五加叶片没有被侵染,无坏死斑的产生。这表明两种雅槛蓝醇型倍半萜对腐生营养型病原真菌立枯丝核菌能够产生抗病性。
3、雅槛蓝醇型倍半萜在玉米上对玉米黑粉病菌的抗性应用
(1)将5-epi-jinkoh-eremol、debneyol和Validamycin母液用无菌蒸馏水稀释成50μg/mL的工作液,并均匀喷洒在玉米(三叶一心苗期)叶片表面。
(2)从活化玉米黑粉病菌的培养基上挑取部分菌落,接种于液体玉米黑粉病专用培养基中,30℃,200rpm培养至OD值为1。4500rpm离心集菌,用等体积的无菌蒸馏水重悬。
(3)用1mL无菌注射器(带针头)将重悬的玉米黑粉病菌注射玉米第一片叶下的茎部,接种后,置于光照培养箱中,培养箱条件为:16h光照/8h黑暗,25℃,湿度70-80%。
(4)72h后,观察并比较玉米第一片叶上的病斑面积,以评估抗病效果。结果如图4B所示,对照组玉米叶片褪绿面积基本占据了整个叶片,而50μg/mL的5-epi-jinkoh-eremol和debneyol与100μg/mL的阳性药Validamycin处理的玉米叶片褪绿面积明显很小,表明两种雅槛蓝醇型倍半萜对活体营养型病原真菌玉米黑粉病菌具有显著的抗病活性。
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
<120> 高产雅槛蓝醇型倍半萜的酿酒酵母工程菌及其构建方法与应用
<160> 155
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1356
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 1
atgtcagagt tgagagcctt cagtgcccca gggaaagcgt tactagctgg tggatattta 60
gttttagata caaaatatga agcatttgta gtcggattat cggcaagaat gcatgctgta 120
gcccatcctt acggttcatt gcaagggtct gataagtttg aagtgcgtgt gaaaagtaaa 180
caatttaaag atggggagtg gctgtaccat ataagtccta aaagtggctt cattcctgtt 240
tcgataggcg gatctaagaa ccctttcatt gaaaaagtta tcgctaacgt atttagctac 300
tttaaaccta acatggacga ctactgcaat agaaacttgt tcgttattga tattttctct 360
gatgatgcct accattctca ggaggatagc gttaccgaac atcgtggcaa cagaagattg 420
agttttcatt cgcacagaat tgaagaagtt cccaaaacag ggctgggctc ctcggcaggt 480
ttagtcacag ttttaactac agctttggcc tccttttttg tatcggacct ggaaaataat 540
gtagacaaat atagagaagt tattcataat ttagcacaag ttgctcattg tcaagctcag 600
ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat atggatctat cagatataga 660
agattcccac ccgcattaat ctctaatttg ccagatattg gaagtgctac ttacggcagt 720
aaactggcgc atttggttga tgaagaagac tggaatatta cgattaaaag taaccattta 780
ccttcgggat taactttatg gatgggcgat attaagaatg gttcagaaac agtaaaactg 840
gtccagaagg taaaaaattg gtatgattcg catatgccag aaagcttgaa aatatataca 900
gaactcgatc atgcaaattc tagatttatg gatggactat ctaaactaga tcgcttacac 960
gagactcatg acgattacag cgatcagata tttgagtctc ttgagaggaa tgactgtacc 1020
tgtcaaaagt atcctgaaat cacagaagtt agagatgcag ttgccacaat tagacgttcc 1080
tttagaaaaa taactaaaga atctggtgcc gatatcgaac ctcccgtaca aactagctta 1140
ttggatgatt gccagacctt aaaaggagtt cttacttgct taatacctgg tgctggtggt 1200
tatgacgcca ttgcagtgat tactaagcaa gatgttgatc ttagggctca aaccgctaat 1260
gacaaaagat tttctaaggt tcaatggctg gatgtaactc aggctgactg gggtgttagg 1320
aaagaaaaag atccggaaac ttatcttgat aaataa 1356
<210> 2
<211> 1197
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 2
atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt 60
tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct 120
aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt 180
tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat 240
catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg 300
ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct 360
atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact 420
gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg 480
ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat 540
tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat 600
gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag 660
gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa 720
aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc 780
gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt ggctattatc 840
aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca 900
gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa 960
ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca 1020
tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt 1080
gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt 1140
gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatga 1197
<210> 3
<211> 1584
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 3
atggctgcag accaattggt gaaaactgaa gtcaccaaga agtcttttac tgctcctgta 60
caaaaggctt ctacaccagt tttaaccaat aaaacagtca tttctggatc gaaagtcaaa 120
agtttatcat ctgcgcaatc gagctcatca ggaccttcat catctagtga ggaagatgat 180
tcccgcgata ttgaaagctt ggataagaaa atacgtcctt tagaagaatt agaagcatta 240
ttaagtagtg gaaatacaaa acaattgaag aacaaagagg tcgctgcctt ggttattcac 300
ggtaagttac ctttgtacgc tttggagaaa aaattaggtg atactacgag agcggttgcg 360
gtacgtagga aggctctttc aattttggca gaagctcctg tattagcatc tgatcgttta 420
ccatataaaa attatgacta cgaccgcgta tttggcgctt gttgtgaaaa tgttataggt 480
tacatgcctt tgcccgttgg tgttataggc cccttggtta tcgatggtac atcttatcat 540
ataccaatgg caactacaga gggttgtttg gtagcttctg ccatgcgtgg ctgtaaggca 600
atcaatgctg gcggtggtgc aacaactgtt ttaactaagg atggtatgac aagaggccca 660
gtagtccgtt tcccaacttt gaaaagatct ggtgcctgta agatatggtt agactcagaa 720
gagggacaaa acgcaattaa aaaagctttt aactctacat caagatttgc acgtctgcaa 780
catattcaaa cttgtctagc aggagattta ctcttcatga gatttagaac aactactggt 840
gacgcaatgg gtatgaatat gatttctaaa ggtgtcgaat actcattaaa gcaaatggta 900
gaagagtatg gctgggaaga tatggaggtt gtctccgttt ctggtaacta ctgtaccgac 960
aaaaaaccag ctgccatcaa ctggatcgaa ggtcgtggta agagtgtcgt cgcagaagct 1020
actattcctg gtgatgttgt cagaaaagtg ttaaaaagtg atgtttccgc attggttgag 1080
ttgaacattg ctaagaattt ggttggatct gcaatggctg ggtctgttgg tggatttaac 1140
gcacatgcag ctaatttagt gacagctgtt ttcttggcat taggacaaga tcctgcacaa 1200
aatgttgaaa gttccaactg tataacattg atgaaagaag tggacggtga tttgagaatt 1260
tccgtatcca tgccatccat cgaagtaggt accatcggtg gtggtactgt tctagaacca 1320
caaggtgcca tgttggactt attaggtgta agaggcccgc atgctaccgc tcctggtacc 1380
aacgcacgtc aattagcaag aatagttgcc tgtgccgtct tggcaggtga attatcctta 1440
tgtgctgccc tagcagccgg ccatttggtt caaagtcata tgacccacaa caggaaacct 1500
gctgaaccaa caaaacctaa caatttggac gccactgata taaatcgttt gaaagatggg 1560
tccgtcacct gcattaaatc ctaa 1584
<210> 4
<211> 1332
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 4
atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60
gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120
ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat 180
cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa 240
ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tcttttggat 300
ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat 360
atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta 420
cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg 480
gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag 540
catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga 600
atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660
ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc 720
ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780
gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc 840
ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga tgacgaggct 900
gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga 960
ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020
gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080
ttgttacgaa gagacattac tcaagagcaa attgacagct tcaaaaagaa attgcaagat 1140
gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200
gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag tattccaatt atttgaaaat 1260
aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac gaatttacca 1320
tggacttcat aa 1332
<210> 5
<211> 1476
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 5
atgaaactct caactaaact ttgttggtgt ggtattaaag gaagacttag gccgcaaaag 60
caacaacaat tacacaatac aaacttgcaa atgactgaac taaaaaaaca aaagaccgct 120
gaacaaaaaa ccagacctca aaatgtcggt attaaaggta tccaaattta catcccaact 180
caatgtgtca accaatctga gctagagaaa tttgatggcg tttctcaagg taaatacaca 240
attggtctgg gccaaaccaa catgtctttt gtcaatgaca gagaagatat ctactcgatg 300
tccctaactg ttttgtctaa gttgatcaag agttacaaca tcgacaccaa caaaattggt 360
agattagaag tcggtactga aactctgatt gacaagtcca agtctgtcaa gtctgtcttg 420
atgcaattgt ttggtgaaaa cactgacgtc gaaggtattg acacgcttaa tgcctgttac 480
ggtggtacca acgcgttgtt caactctttg aactggattg aatctaacgc atgggatggt 540
agagacgcca ttgtagtttg cggtgatatt gccatctacg ataagggtgc cgcaagacca 600
accggtggtg ccggtactgt tgctatgtgg atcggtcctg atgctccaat tgtatttgac 660
tctgtaagag cttcttacat ggaacacgcc tacgattttt acaagccaga tttcaccagc 720
gaatatcctt acgtcgatgg tcatttttca ttaacttgtt acgtcaaggc tcttgatcaa 780
gtttacaaga gttattccaa gaaggctatt tctaaagggt tggttagcga tcccgctggt 840
tcggatgctt tgaacgtttt gaaatatttc gactacaacg ttttccatgt tccaacctgt 900
aaattggtca caaaatcata cggtagatta ctatataacg atttcagagc caatcctcaa 960
ttgttcccag aagttgacgc cgaattagct actcgcgatt atgacgaatc tttaaccgat 1020
aagaacattg aaaaaacttt tgttaatgtt gctaagccat tccacaaaga gagagttgcc 1080
caatctttga ttgttccaac aaacacaggt aacatgtaca ccgcatctgt ttatgccgcc 1140
tttgcatctc tattaaacta tgttggatct gacgacttac aaggcaagcg tgttggttta 1200
ttttcttacg gttccggttt agctgcatct ctatattctt gcaaaattgt tggtgacgtc 1260
caacatatta tcaaggaatt agatattact aacaaattag ccaagagaat caccgaaact 1320
ccaaaggatt acgaagctgc catcgaattg agagaaaatg cccatttgaa gaagaacttc 1380
aaacctcaag gttccattga gcatttgcaa agtggtgttt actacttgac caacatcgat 1440
gacaaattta gaagatctta cgatgttaaa aaataa 1476
<210> 6
<211> 1191
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 6
atgaccgttt acacagcatc cgttaccgca cccgtcaaca tcgcaaccct taagtattgg 60
gggaaaaggg acacgaagtt gaatctgccc accaattcgt ccatatcagt gactttatcg 120
caagatgacc tcagaacgtt gacctctgcg gctactgcac ctgagtttga acgcgacact 180
ttgtggttaa atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc 240
gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc cacattatct 300
caatggaaac tccacattgt ctccgaaaat aactttccta cagcagctgg tttagcttcc 360
tccgctgctg gctttgctgc attggtctct gcaattgcta agttatacca attaccacag 420
tcaacttcag aaatatctag aatagcaaga aaggggtctg gttcagcttg tagatcgttg 480
tttggcggat acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca 540
gtacaaatcg cagacagctc tgactggcct cagatgaaag cttgtgtcct agttgtcagc 600
gatattaaaa aggatgtgag ttccactcag ggtatgcaat tgaccgtggc aacctccgaa 660
ctatttaaag aaagaattga acatgtcgta ccaaagagat ttgaagtcat gcgtaaagcc 720
attgttgaaa aagatttcgc cacctttgca aaggaaacaa tgatggattc caactctttc 780
catgccacat gtttggactc tttccctcca atattctaca tgaatgacac ttccaagcgt 840
atcatcagtt ggtgccacac cattaatcag ttttacggag aaacaatcgt tgcatacacg 900
tttgatgcag gtccaaatgc tgtgttgtac tacttagctg aaaatgagtc gaaactcttt 960
gcatttatct ataaattgtt tggctctgtt cctggatggg acaagaaatt tactactgag 1020
cagcttgagg ctttcaacca tcaatttgaa tcatctaact ttactgcacg tgaattggat 1080
cttgagttgc aaaaggatgt tgccagagtg attttaactc aagtcggttc aggcccacaa 1140
gaaacaaacg aatctttgat tgacgcaaag actggtctac caaaggaata a 1191
<210> 7
<211> 867
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 7
atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg 60
caaaaccaaa cacctgaaga cattttggaa gagtttcctg aaattattcc attacaacaa 120
agacctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct 180
ggtcatgatg aggagcaaat taagttaatg aatgaaaatt gtattgtttt ggattgggac 240
gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300
ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360
caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420
catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480
ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540
actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600
gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660
aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720
gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780
tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840
gacaggcaaa ttcatagaat gctataa 867
<210> 8
<211> 2742
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 8
atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60
aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120
cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180
tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240
acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300
gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360
cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420
tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480
gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540
gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600
ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660
ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720
caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780
ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840
attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900
caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960
caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020
caacagcagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080
gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140
actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200
atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260
tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320
actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380
gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440
aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500
acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560
tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620
ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680
cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740
gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800
accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860
tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920
gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980
ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040
agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100
agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160
aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220
acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280
gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340
gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400
ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460
cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520
gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580
gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640
gacgaataca gtggaggtgg tggtatgcat atgatgctag atttcctcgg tggcggatta 2700
ccatcgatga caacaacaaa tttctctgat ttttcgttat ga 2742
<210> 9
<211> 984
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 9
ggaagtacct tcaaagaatg gggtcttatc ttgttttgca agtaccactg agcaggataa 60
taatagaaat gataatatac tatagtagag ataacgtcga tgacttccca tactgtaatt 120
gcttttagtt gtgtattttt agtgtgcaag tttctgtaaa tcgattaatt tttttttctt 180
tcctcttttt attaacctta atttttattt tagattcctg acttcaactc aagacgcaca 240
gatattataa catctgcata ataggcattt gcaagaatta ctcgtgagta aggaaagagt 300
gaggaactat cgcatacctg catttaaaga tgccgatttg ggcgcgaatc ctttattttg 360
gcttcaccct catactatta tcagggccag aaaaaggaag tgtttccctc cttcttgaat 420
tgatgttacc ctcataaagc acgtggcctc ttatcgagaa agaaattacc gtcgctcgtg 480
atttgtttgc aaaaagaaca aaactgaaaa aacccagaca cgctcgactt cctgtcttcc 540
tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 600
tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 660
tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 720
aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 780
accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 840
cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 900
caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 960
ctttttacaa caaatataaa acaa 984
<210> 10
<211> 419
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 10
cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 60
tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 120
tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 180
gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 240
ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 300
aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 360
ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaa 419
<210> 11
<211> 676
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 11
tcgagtttat cattatcaat actgccattt caaagaatac gtaaataatt aatagtagtg 60
attttcctaa ctttatttag tcaaaaaatt agccttttaa ttctgctgta acccgtacat 120
gcccaaaata gggggcgggt tacacagaat atataacatc gtaggtgtct gggtgaacag 180
tttattcctg gcatccacta aatataatgg agcccgcttt ttaagctggc atccagaaaa 240
aaaaagaatc ccagcaccaa aatattgttt tcttcaccaa ccatcagttc ataggtccat 300
tctcttagcg caactacaga gaacaggggc acaaacaggc aaaaaacggg cacaacctca 360
atggagtgat gcaacctgcc tggagtaaat gatgacacaa ggcaattgac ccacgcatgt 420
atctatctca ttttcttaca ccttctatta ccttctgctc tctctgattt ggaaaaagct 480
gaaaaaaaag gttgaaacca gttccctgaa attattcccc tacttgacta ataagtatat 540
aaagacggta ggtattgatt gtaattctgt aaatctattt cttaaacttc ttaaattcta 600
cttttatagt tagtcttttt tttagtttta aaacaccaag aacttagttt cgaataaaca 660
cacataaaca aacaaa 676
<210> 12
<211> 874
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 12
cttatcttga cgggtattct gagcatctta ctcagtttca agatctttta atgtccaaaa 60
acatttgagc cgatctaaat acttctgtgt tttcattaat ttataaattg tactctttta 120
agacatggaa agtaccaaca tcggttgaaa cagtttttca tttacttatg gtttattggt 180
ttttccagtg aatgattatt tgtcgttacc ctttcgtaaa agttcaaaca cgtttttaag 240
tattgtttag ttgctctttc gacatatatg attatccctg cgcggctaaa gttaaggatg 300
caaaaaacat aagacaactg aagttaattt acgtcaatta agttttccag ggtaatgatg 360
ttttgggctt ccactaattc aataagtatg tcatgaaata cgttgtgaag agcatccaga 420
aataatgaaa agaaacaacg aaactgggtc ggcctgttgt ttcttttctt taccacgtga 480
tctgcggcat ttacaggaag tcgcgcgttt tgcgcagttg ttgcaacgca gctacggcta 540
acaaagccta gtggaactcg actgatgtgt tagggcctaa aactggtggt gacagctgaa 600
gtgaactatt caatccaatc atgtcatggc tgtcacaaag accttgcgga ccgcacgtac 660
gaacacatac gtatgctaat atgtgttttg atagtaccca gtgatcgcag acctgcaatt 720
tttttgtagg tttggaagaa tatataaagg ttgcactcat tcaagatagt ttttttcttg 780
tgtgtctatt cattttatta ttgtttgttt aaatgttaaa aaaaccaaga acttagtttc 840
aaattaaatt catcacacaa acaaacaaaa caaa 874
<210> 13
<211> 926
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 13
tcttcaagaa ttggggatct acgtatggtc attcttcttc agattccctc atggagaagt 60
gcggcagatg tatatgacag agtcgccagt ttccaagaga ctttattcag gcacttccat 120
gataggcaag agagaagacc cagagatgtt gttgtcctag ttacacatgg tatttattcc 180
agagtattcc tgatgaaatg gtttagatgg acatacgaag agtttgaatc gtttaccaat 240
gttcctaacg ggagcgtaat ggtgatggaa ctggacgaat ccatcaatag atacgtcctg 300
aggaccgtgc tacccaaatg gactgattgt gagggagacc taactacata gtgtttaaag 360
attacggata tttaacttac ttagaataat gccatttttt tgagttataa taatcctacg 420
ttagtgtgag cgggatttaa actgtgagga cctcaataca ttcagacact tctgacggta 480
tcaccctact tattcccttc gagattatat ctaggaaccc atcaggttgg tggaagatta 540
cccgttctaa gacttttcag cttcctctat tgatgttaca ctcggacacc ccttttctgg 600
catccagttt ttaatcttca gtggcatgtg agattctccg aaattaatta aagcaatcac 660
acaattctct cggataccac ctcggttgaa actgacaggt ggtttgttac gcatgctaat 720
gcaaaggagc ctatatacct ttggctcggc tgctgtaaca gggaatataa agggcagcat 780
aatttaggag tttagtgaac ttgcaacatt tactattttc ccttcttacg taaatatttt 840
tctttttaat tctaaatcaa tctttttcaa ttttttgttt gtattctttt cttgcttaaa 900
tctataacta caaaaaacac atacag 926
<210> 14
<211> 165
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 14
cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat 60
acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt 120
tcctgtaggt caggttgctt tctcaggtat agcatgaggt cgctc 165
<210> 15
<211> 190
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 15
atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60
ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120
gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180
acgctcgaag 190
<210> 16
<211> 400
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 16
atttaactcc ttaagttact ttaatgattt agtttttatt attaataatt catgctcatg 60
acatctcata tacacgttta taaaacttaa atagattgaa aatgtattaa agattcctca 120
gggattcgat ttttttggaa gtttttgttt ttttttcctt gagatgctgt agtatttggg 180
aacaattata caatcgaaag atatatgctt acattcgacc gttttagccg tgatcattat 240
cctatagtaa cataacctga agcataactg acactactat catcaatact tgtcacatga 300
gaactctgtg aataattagg ccactgaaat ttgatgcctg aaggaccggc atcacggatt 360
ttcgataaag cacttagtat cacactaatt ggcttttcgc 400
<210> 17
<211> 569
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 17
tagggcccac aagcttacgc gtcgacccgg gtatccgtat gatgtgcctg actacgcatg 60
atatctcgag ctcagctagc taactgaata aggaacaatg aacgtttttc ctttctcttg 120
ttcctagtat taatgactga ccgatacatc cctttttttt tttgtctttg tctagctcca 180
gcttttgttc cctttagtga gggttaattc aattcactgg ccgtcgtttt acaacgtcgt 240
gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 300
agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 360
aatggcgaat ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 420
acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 480
ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 540
ttagggttcc gatttagtgg tttacggca 569
<210> 18
<211> 400
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 18
gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60
gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120
tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180
caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 240
gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 300
ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 360
actcttcgag ttctttgtaa agtctttcat agtagcttac 400
<210> 19
<211> 2742
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 19
atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60
aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120
cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180
tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240
acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300
gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360
cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420
tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480
gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540
gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600
ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660
ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720
caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780
ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840
attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900
caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960
caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020
caacagcagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080
gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140
actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200
atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260
tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320
actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380
gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440
aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500
acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560
tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620
ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680
cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740
gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800
accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860
tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920
gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980
ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040
agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100
agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160
aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220
acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280
gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340
gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400
ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460
cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520
gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580
gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640
gacgaataca gtggaggtgg tgatatgcat atgatgctag atttcctcgg tggcggatta 2700
ccatcgatga caacaacaaa tttctctgat ttttcgttat ga 2742
<210> 20
<211> 1685
<212> DNA
<213> 长春花(Catharanthus roseus)
<400> 20
atgggcaggg aagtagtgat aatgatggct tctttcgtcg acaatggggt tcttcgtcca 60
ataaaaaaat tccctcctag catatggggt gataccttca attctttcga gtgggatcat 120
gaggcatcag aaaaacttgg caaggaaatg aaaatattgg aaaaagatgc gaggaacatg 180
ttacaagctg atacaagaaa tgagacagta aaggacataa ttactttgat caatactttt 240
gagcggctcg ggctttcgta taagtttgag aaagagatag aagatcatct ggaacgactt 300
gtccattctt ttgattatga tggaaatcaa catgatttgc tcacggtttc tctcctgttt 360
agaattctca ggcaacacgg atatgaaatc tcctcaggta tcttcaaaaa attcatggac 420
aagaatgggg aatttaaaga agagatcatt ggcaatgatg tgaaaggtgt attaagcctg 480
tatgaagcat cttacgtgag ggggcatgga gaagatattc ttgaaaaagc attggttttt 540
acaaaaggcc atctcatgag aattcttcct gaaataatag aatctcctct tggaaaacaa 600
gtaatatatg ctcttgaaca accactccat agaggtgttc caagacttga ggctcgtcat 660
tacatctcca tttatgagga agaccaagaa actaaaaatg aagcacttct tagactcgca 720
aaattggatt tcaatctatt gcaaatgtta cacaagaaag agatatgtga gattacaagg 780
tggtggaaga aatcggactt tatgggaaaa cttccttatc taagagatag ggtggtggaa 840
tgctattttt gggcaatagg aatatacttt gaacctaagt attctcttgc tcgtataatg 900
gccagcaaag ttgtggccat gacttcaatc atggatgaca cctatgactc ttatggcata 960
attgaagaac ttgaagtttt cacttctgct gtcgaaaggt ggagcattga agaaattgat 1020
agactcccaa gttacatgaa gatagcctac atggcacttc tcaacctgta cgaagaattt 1080
gatgaaaaac taaaagaaca gggacgatcc tttgcagttc aatattccaa agaaagaatg 1140
aagcagctaa taagaagcta tgacaaagaa gctaagtggt tttatgaaag atcagatgat 1200
gtgcctagtt ttgatgaata catggaaaat gcaatatcaa ctagcactta cctcgtactc 1260
atgccgtcgt tgttgttggg gatggaatct gcaagcaggg aagtgtttga ttgggtcatg 1320
aacaatccta gtatagttgt ggctagtgcc aaagttggtc gatgcacaga cgacgtcgct 1380
acttattcgg ttgagaaagc aaggggtcaa ccagcctgcg gaatcgaatg gtacatgaaa 1440
gaacatggtg tctctaaaga agaaactttc aaaaaatttc atgaaattgt tgaagattct 1500
tggaaagata ttaataagga acttgtccga tcatcctcga ttccaatgga tattctagtg 1560
agggctttaa accaagcaag agtgatcgac gtggtttata agcatgatca agatggttat 1620
actcaccctg aaaaggttct caaaccccat atcaaggcct tacttgttga tcctataagc 1680
attaa 1685
<210> 21
<211> 1516
<212> DNA
<213> 长春花(Catharanthus roseus)
<400> 21
atgggctttc agattccttt aaacttcatt gccttctttg tattcctatt gttgtcttct 60
attttattag tgaaacagag gaatagaaaa tcattaggaa agaaaaaact gcccccagga 120
ccccggaagt taccattgat tggaaaccta cacaatttga taggtggact tcctcatcat 180
attttcagag atttatctcg aaaatatgga cctcttattc acctacaatt gggtcaagta 240
ggtaccattt tgatatcttc accacgttta gcaaaagagg tgatgaaaac tcatgatctt 300
acctttgcaa caaggccgga caatcttgcc ggagatgtca tgttctatgg tagcacagat 360
attgtatttg ccaaatacgg cgagtactgg agacaaatgc gtaaaatcag tgtcttagaa 420
ctcttcagcg cgaaaaatgt ccggtcattt ggttctataa gaatggatga atcattactt 480
atgattgcgt ctatacgaga atcagttggt aaagcggtta atctaagcac aaaacttgca 540
aactatacaa gttctgtggt ttgcagggca gcatttggta ggttatgtcc tgaccaacat 600
gagtttattg agttagttga tgaagcatct gttttagcag caggctttga tattggtgac 660
ctttttccat cattaaagtt tattcagttt ttgactggat taaagcctaa attaatgaag 720
gttcataata aggttgacaa gattttggac catgtaatta atgagcatag aaaaaacatg 780
ggaaggagaa atggtgagtt tggtgaagaa gacttaactg attcacttct aagaattcaa 840
caaagtggtg gtgaccttca atttcccatc tccgacaaca atattaaggc aatcttgttt 900
gatgtgtttg gtgcgggaac agaaacttca tccacaataa cagaatgggc cttgtcagaa 960
ttaattaaaa atccagatat gatgaacaag gcacaaactg aaataaggca agccttcaag 1020
ggaaagaaaa ggccgattga agaggctgat cttcaaggcc taagttatct caagtgtgta 1080
attaaagaaa cactgaggct atatcctgca gcgcctttat tggttcctcg tgaatgcaga 1140
gaggactgtg aattggatgg atattttata ccaaagaaat caagggtaat tgttaatgct 1200
tgggcaattg gaagagatcc tgagtattgg cctaatgcaa acagttttat tcccgaaaga 1260
tttgagaatt cttcaactga tttcaccggt aatcactttg aattaatacc atttgggtca 1320
ggaaggagga gttgtcctgg aatgctgttt ggtatagcta atattgagct tcctttagct 1380
cttcttttat accacttcaa ctggagtctc ccagatggcc ttacttccga aactttggac 1440
atgtctgaga cttggggaat aacaactcca aggaaatatg atcttcacct aatccctaca 1500
tcttattatc ctttaa 1516
<210> 22
<211> 2079
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 22
atgacttctg ctttgtatgc ttccgatttg tttaagcagc tcaagtcaat tatggggaca 60
gattcgttat ccgacgatgt tgtacttgtg attgcaacga cgtctttggc actagtagct 120
ggatttgtgg tgttgttatg gaagaaaacg acggcggatc ggagcgggga gctgaagcct 180
ttgatgatcc ctaagtctct tatggctaag gacgaggatg atgatttgga tttgggatcc 240
gggaagacta gagtctctat cttcttcggt acgcagactg gaacagctga gggatttgct 300
aaggcattat ccgaagaaat caaagcgaga tatgaaaaag cagcagtcaa agtcattgac 360
ttggatgact atgctgccga tgatgaccag tatgaagaga aattgaagaa ggaaactttg 420
gcatttttct gtgttgctac ttatggagat ggagagccta ctgacaatgc tgccagattt 480
tacaaatggt ttacggagga aaatgaacgg gatataaagc ttcaacaact agcatatggt 540
gtgtttgctc ttggtaatcg ccaatatgaa cattttaata agatcgggat agttcttgat 600
gaagagttat gtaagaaagg tgcaaagcgt cttattgaag tcggtctagg agatgatgat 660
cagagcattg aggatgattt taatgcctgg aaagaatcac tatggtctga gctagacaag 720
ctcctcaaag acgaggatga taaaagtgtg gcaactcctt atacagctgt tattcctgaa 780
taccgggtgg tgactcatga tcctcggttt acaactcaaa aatcaatgga atcaaatgtg 840
gccaatggaa atactactat tgacattcat catccctgca gagttgatgt tgctgtgcag 900
aaggagcttc acacacatga atctgatcgg tcttgcattc atctcgagtt cgacatatcc 960
aggacgggta ttacatatga aacaggtgac catgtaggtg tatatgctga aaatcatgtt 1020
gaaatagttg aagaagctgg aaaattgctt ggccactctt tagatttagt attttccata 1080
catgctgaca aggaagatgg ctccccattg gaaagcgcag tgccgcctcc tttccctggt 1140
ccatgcacac ttgggactgg tttggcaaga tacgcagacc ttttgaaccc tcctcgaaag 1200
tctgcgttag ttgccttggc ggcctatgcc actgaaccaa gtgaagccga gaaacttaag 1260
cacctgacat cacctgatgg aaaggatgag tactcacaat ggattgttgc aagtcagaga 1320
agtcttttag aggtgatggc tgcttttcca tctgcaaaac ccccactagg tgtatttttt 1380
gctgcaatag ctcctcgtct acaacctcgt tactactcca tctcatcctc gccaagattg 1440
gcgccaagta gagttcatgt tacatccgca ctagtatatg gtccaactcc tactggtaga 1500
atccacaagg gtgtgtgttc tacgtggatg aagaatgcag ttcctgcgga gaaaagtcat 1560
gaatgtagtg gagccccaat ctttattcga gcatctaatt tcaagttacc atccaaccct 1620
tcaactccaa tcgttatggt gggacctggg actgggctgg caccttttag aggttttctg 1680
caggaaagga tggcactaaa agaagatgga gaagaactag gttcatcttt gctcttcttt 1740
gggtgtagaa atcgacagat ggactttata tacgaggatg agctcaataa ttttgttgat 1800
caaggcgtaa tatctgagct catcatggca ttctcccgtg aaggagctca gaaggagtat 1860
gttcaacata agatgatgga gaaggcagca caagtttggg atctaataaa ggaagaagga 1920
tatctctatg tatgcggtga tgctaagggc atggcgaggg acgtccaccg aactctacac 1980
accattgttc aggagcagga aggtgtgagt tcgtcagagg cagaggctat agttaagaaa 2040
cttcaaaccg aaggaagata cctcagagat gtctggtga 2079
<210> 23
<211> 1050
<212> DNA
<213> 丹参(Salvia miltiorrhiza)
<400> 23
atggcgaatc tgaacggaga gtcggcggat ctgagggcga cgtttctggg ggtttattcg 60
gtgcttaaat ctgagctctt gaacgaccct gctttcgagt ggactgatgg ttctcgtcaa 120
tgggtcgagc gtatgctgga ctataatgta cctggaggga aattaaaccg aggcctgtca 180
gtcattgata gctacaagtt actaaaagga ggaaaagatc taactgatga tgaagtgttt 240
ctagctagtg ctcttggctg gtgtgttgaa tggctccagg catattttct tgtacttgat 300
gatattatgg ataattctca cacacgacgt ggtcagccat gctggtttag agtccccaag 360
gttggtatga ttgccataaa tgatggaatc attctccgga accatatccc cagaattctt 420
aagaagcact tcagaacaaa gccttactat gttgatctgc tggatttgtt caatgaggtg 480
gaatttcaaa ctgcttctgg acagatgata gatttaatta ccactattga aggagaaaaa 540
gatttatcaa aatactcatt gcctcttcat cgccgcattg ttcagtacaa gacggcctac 600
tactcatttt acctcccagt tgcttgtgcg ttgctcatgg cgggtgagga cctggagaaa 660
catccaacag tgaaggatgt gcttattaat atgggaatat actttcaagt acaggatgac 720
tatttagatt gctttggtga gcctgaaaag attgggaaga ttggaacaga tattgaagat 780
ttcaaatgtt cttggctggt tgtaaaggcc ctggagcttt gtaacgaaga acagaagaaa 840
actcttttcg agcactatgg aaaggaagat ccagctgatg ttgcaaaaat caaagtcctc 900
tataatgaga ttaatctaca aggtgtgttt gctgagtttg agagcaagag ctacgagaaa 960
ctaaatagct cgattgaagc tcatcccagc aaatctgtgc aagcagtgct caagtctttc 1020
ttgggcaaga tatacaagag gcagaaataa 1050
<210> 24
<211> 448
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ccaaaacaat tggattggaa gactaagacc agattttcta gatcgttgcc aacctgttgc 60
cattggacac tttatttact gcaaaagatg tgtgtacgac taagaatgat cgttgccaac 120
ctgttgagtt ttagagctat gctgttttga atggtcccaa aactgatata ctggggtcat 180
caagactaaa ttcgatgttt tggcccctag gtaatctccg aatagaggaa taatatcgta 240
catagaccaa ttatcatgta ctggttttgg cccctaggta ccagttttag agctatgctg 300
ttttgaatgg tcccaaaaca gcagtttcaa agacggcaat agcttctgga gtggaaccca 360
tgttggaaca taaacttgac accttgagca accaaggcct tggcttcttc accgctgacg 420
gaacccatgt tggaaccttg ttttagag 448
<210> 25
<211> 1536
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ataacttcgt atagcataca ttatacgaag ttatattaag ggttctcgag agctcgtttt 60
atttaggttc tatcgaggag aaaaagcgac aagaagagat agaccatgga taaactgatt 120
atgttctaaa cactcctcag aagctcatcg aactgtcatc ctgcgtgaag attaaaatcc 180
aacttagaaa tttcgagctt acggagacaa tcatatggga gaagcaattg gaagatagaa 240
aaaaggtact cggtacataa atatatgtga ttctgggtag aagatcggtc tgcattggat 300
ggtggtaacg cattttttta cacacattac ttgcctcgag catcaaatgg tggttattcg 360
tggatctata tcacgtgatt tgcttaagaa ttgtcgttca tggtgacact tttagctttg 420
acatgattaa gctcatctca attgatgtta tctaaagtca tttcaactat ctaagatgtg 480
gttgtgattg ggccattttg tgaaagccag tacgccagcg tcaatacact cccgtcaatt 540
agttgcacca tgtccacaaa atcatatacc agtagagctg agactcatgc aagtccggtt 600
gcatcgaaac ttttacgttt aatggatgaa aagaaaacca atttgtgtgc ttctcttgac 660
gttcgttcga ctgatgagct attgaaactt gttgaaacgt tgggtccata catttgcctt 720
ttgaaaacac acgttgatat cttggatgat ttcagttatg agggtactgt cgttccattg 780
aaagcattgg cagagaaata caagttcttg atatttgagg acagaaaatt cgccgatatc 840
ggtaacacag tcaaattaca atatacatcg ggcgtttacc gtatcgcaga atggtctgat 900
atcaccaacg cccacggggt tactggtgct ggtattgttg ctggcttgaa acaaggtgcg 960
caagaggtca ccaaagaacc aaggggatta ttgatgcttg ctgaattatc ttccaagggt 1020
tctctagcac acggtgaata tactaagggt accgttgata ttgcaaagag tgataaagat 1080
ttcgttattg ggttcattgc tcagaacgat atgggaggaa gagaagaagg gtttgattgg 1140
ctaatcatga ccccaggtgt aggtttagac gacaaaggcg atgcattggg tcagcagtac 1200
agaaccgtcg acgaagttgt aagtggtgga tcagatatca tcattgttgg cagaggactt 1260
ttcgccaagg gtagagatcc taaggttgaa ggtgaaagat acagaaatgc tggatgggaa 1320
gcgtaccaaa agagaatcag cgctccccat taattataca ggaaacttaa tagaacaaat 1380
cacatattta atctaatagc cacctgcatt ggcacggtgc aacactcact tcaacttcat 1440
cttacaaaag atcacgtgat ctgttgtatt gggatctcta gacctaataa cttcgtatag 1500
catacattat acgaagttat attaagggtt gtcgac 1536
<210> 26
<211> 2352
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ataacttcgt atagcataca ttatacgaag ttatattaag ggttctcgag agctcgctgt 60
gaagatccca gcaaaggctt acaaagtgtt atctcttttg agacttgttg agttgaacac 120
tggtgttttc atcaaactta ccaaggacgt gtacccattg ttgaaacttg tatcaccata 180
tattgttatc ggacaacctt cacttgcatc tatccgttct ttaatccaaa agagatctag 240
aataatgtgg caaaggccag aagataaaga accaaaagag ataatcttga atgacaacaa 300
tatcgttgaa gagaaattag gtgatgaagg tgtcatttgt atcgaggata tcatccatga 360
gatttcgacg ttgggcgaaa atttctcgaa atgtactttc ttcctattac cattcaaatt 420
gaacagagaa gtcagtggat tcggtgccat ctcccgtttg aataaactga aaatgcgcga 480
acaaaacaag gagactcgtc aaatttcaaa cgctgccacg gctccagtta tccaagtaga 540
tatcgactca atgatttcca agttgaattg attaactata aaaggaaaat atctgtacaa 600
tagacatcgg gctcccattg gccctaccca catatgtaga aatacattac tctattcact 660
actgcattta gttatgttta acatttgata tagcagacta ccgccaggca caatatattc 720
cccttccctc ttgccattcg ctgtacttgt ggtggattcc aattcagcgc agtcacgtgc 780
tagtaatcac cgcatttttt tcttttcctt tcaggctaaa accggttccg ggcctgatcc 840
ctgcactcat tttctaacgg aaaaccttca gaagcataac tacccattcc agtttagagt 900
catgacaggt tcaacatcag atgcttcata tacttttata tattgaatta tataaatata 960
tctatgtact ctaagtaagt acatctgctt taacgcattc ctacatttgc ttcgatttat 1020
ttttattgtt gatacctatt tgaagaagta aaaagtatcc cacactacac agattatacc 1080
atgtctaaga atatcgttgt cctaccgggt gatcacgtcg gtaaagaagt tactgacgaa 1140
gctattaagg tcttgaatgc cattgctgaa gtccgtccag aaattaagtt caatttccaa 1200
catcacttga tcgggggtgc tgccatcgat gccactggca ctcctttacc agatgaagct 1260
ctagaagcct ctaagaaagc cgatgctgtc ttactaggtg ctgttggtgg tccaaaatgg 1320
ggtacgggcg cagttagacc agaacaaggt ctattgaaga tcagaaagga attgggtcta 1380
tacgccaact taagaccatg taactttgct tctgattctt tactagatct ttctcctttg 1440
aagcctgaat atgcaaaggg taccgatttc gtcgtcgtta gagaattggt tggtggtatc 1500
tactttggtg aaagaaaaga agatgaaggt gacggagttg cttgggactc tgagaaatac 1560
agtgttcctg aagttcaaag aattacaaga atggctgctt tcttggcatt gcaacaaaac 1620
ccaccattac caatctggtc acttgacaag gctaacgtgc ttgcctcttc cagattgtgg 1680
agaaagactg ttgaagaaac catcaagact gagttcccac aattaactgt tcagcaccaa 1740
ttgatcgact ctgctgctat gattttggtt aaatcaccaa ctaagctaaa cggtgttgtt 1800
attaccaaca acatgtttgg tgatattatc tccgatgaag cctctgttat tccaggttct 1860
ttgggtttat taccttctgc atctctagct tccctacctg acactaacaa ggcattcggt 1920
ttgtacgaac catgtcatgg ttctgcccca gatttaccag caaacaaggt taacccaatt 1980
gctaccatct tatctgcagc tatgatgttg aagttatcct tggatttggt tgaagaaggt 2040
agggctcttg aagaagctgt tagaaatgtc ttggatgcag gtgtcagaac cggtgacctt 2100
ggtggttcta actctaccac tgaggttggc gatgctatcg ccaaggctgt caaggaaatc 2160
ttggcttaat tatacaggaa acttaataga acaaatcaca tatttaatct aatagccacc 2220
tgcattggca cggtgcaaca ctcacttcaa cttcatctta caaaagatca cgtgatctgt 2280
tgtattggga tctctagacc taataacttc gtatagcata cattatacga agttatatta 2340
agggttgtcg ac 2352
<210> 27
<211> 2077
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ataacttcgt ataatgtatg ctatacgaag ttattaggtc tagagatccc aatacaacag 60
atcacgtgat cttttgtaag atgaagttga agtgagtgtt gcaccgtgcc aatgcaggtg 120
gctattagat taaatatgtg atttgttcta ttaagtttcc tgtataatta tggtttttgg 180
ccagcgaaaa cagtttcaaa agattgctgg aagtctgcaa taatgtcatc aataaattcg 240
ataccaacag agacacgaat taagtccttg gtaacaccag atgccaactt ttctttgtca 300
tttaattgtt tgtgggtagt gaagtatgga gcaatgacta aggtcttggc atcaccaaca 360
ttggccaagt tagaggcaag ctttaaattg tcaacaactt gagcaccaga aagtttgaat 420
gggtcagttt ccttgtcggc atttggtaag tcttttacac cgaaagataa gacaccaccg 480
aaaccgttag atagatactt cttagcattt tcatgatgag aatgagatgc taaaccaggg 540
tatgaaaccc aagatacgta tggggattgt tctaaccatt tggctaactt caatgcattt 600
tcaccgtgtc tttcagctct caaagataat gtttcaacac cttgtagtag caagaaagag 660
gcaaatgggt tcatcaatgg acccaaatct cttaatagtt cagttctaac atgaacgatg 720
tatgccaagt taccgtaggc ttcattgtag atagtaccgt gatatccttc ggcaggttga 780
gagaattgag ggaacttttc tgggtagtcc ttccatggga acttaccaga gtcaacaata 840
ataccaccga tagtagtacc atgaccacca atccatttgg tagcagaatg tgttacaata 900
tcagcaccgt atttaattgg ctgacagaag taaccaccgg caccaaatgt gttgtcaacg 960
acaactggaa taccgtgttt gtgagcaatt gcaacaattt tttcaaaatc cggaacattg 1020
tactttggat taccaatggt ttccaaataa acagccttgg ttctttcatc aaagaccttt 1080
tcgaattctt ctggattgtc accttcaaca aatctagcct cgataccaaa tcttttgaac 1140
gagattttga actggttata agtaccaccg tataagtaag aagtggaaac gatgttgtca 1200
ccagtgtgtg ccaaaccttg gatggcaagg gtttgagcgg cttgaccgga ggaaacagcc 1260
aaagcagcag caccaccttc taaagcagca attctttctt ccaaaacatt actggttggg 1320
ttttggaaac gggaatagac gtaacctgga acttctagac caaacaattg cgaaccatgc 1380
ttagagtttt cgaaaacata agaagtggtg gcgtaaattg gtacagctct ggatctgtga 1440
gcattgtcac cagggttctc ttggccggcg tgtagttgaa cagtatcgaa atgagatggc 1500
atggtgcaac taattgacgg gagtgtattg acgctggcgt actggctttc acaaaatggc 1560
ccaatcacaa ccacatctta gatagttgaa atgactttag ataacatcaa ttgagatgag 1620
cttaatcatg tcaaagctaa aagtgtcacc atgaacgaca attcttaagc aaatcacgtg 1680
atatagatcc acgaataacc accatttgat gctcgaggca agtaatgtgt gtaaaaaaat 1740
gcgttaccac catccaatgc agaccgatct tctacccaga atcacatata tttatgtacc 1800
gagtaccttt tttctatctt ccaattgctt ctcccatatg attgtctccg taagctcgaa 1860
atttctaagt tggattttaa tcttcacgca ggatgacagt tcgatgagct tctgaggagt 1920
gtttagaaca taatcagttt atccatggtc tatctcttct tgtcgctttt tctcctcgat 1980
agaacctaaa taaaacgagc tctcgagaac ccttaatata acttcgtata atgtatgcta 2040
tacgaagtta ttgcaggtct catctggaat ataattc 2077
<210> 28
<211> 1348
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ataacttcgt ataatgtatg ctatacgaag ttatcttaac tatgcggcat cagagcagat 60
tgtactgaga gtgcaccata aattcccgtt ttaagagctt ggtgagcgct aggagtcact 120
gccaggtatc gtttgaacac ggcattagtc agggaagtca taacacagtc ctttcccgca 180
attttctttt tctattactc ttggcctcct ctagtacact ctatattttt ttatgcctcg 240
gtaatgattt tcattttttt ttttccccta gcggatgact cttttttttt cttagcgatt 300
ggcattatca cataatgaat tatacattat ataaagtaat gtgatttctt cgaagaatat 360
actaaaaaat gagcaggcaa gataaacgaa ggcaaagatg acagagcaga aagccctagt 420
aaagcgtatt acaaatgaaa ccaagattca gattgcgatc tctttaaagg gtggtcccct 480
agcgatagag cactcgatct tcccagaaaa agaggcagaa gcagtagcag aacaggccac 540
acaatcgcaa gtgattaacg tccacacagg tatagggttt ctggaccata tgatacatgc 600
tctggccaag cattccggct ggtcgctaat cgttgagtgc attggtgact tacacataga 660
cgaccatcac accactgaag actgcgggat tgctctcggt caagctttta aagaggccct 720
actggcgcgt ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc 780
cagagcggtg gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag 840
ggagaaagta ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga 900
ggctagcaga attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag 960
tgagagtgcg ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac 1020
caacgatgtt ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc 1080
agcatacgat atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat 1140
acgaacagta tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga 1200
acgaggcgcg ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg 1260
gatctatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaataact 1320
tcgtataatg tatgctatac gaagttat 1348
<210> 29
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tgtcagagtt gagagccttc ag 22
<210> 30
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ttatttatca agataagttt ccgg 24
<210> 31
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
atgtctcaga acgtttacat tgtatc 26
<210> 32
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tcatatcttt tcaatgacaa tagagg 26
<210> 33
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
atggctgcag accaattggt gaaaac 26
<210> 34
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ttaggattta atgcaggtga cgg 23
<210> 35
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
atgtcattac cgttcttaac ttctg 25
<210> 36
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ttatgaagtc catggtaaat tcgtg 25
<210> 37
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
atgaaactct caactaaact ttgttg 26
<210> 38
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ttatttttta acatcgtaag atcttc 26
<210> 39
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
atgaccgttt acacagcatc c 21
<210> 40
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ttattccttt ggtagaccag tctttg 26
<210> 41
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
atgactgccg acaacaatag tatgc 25
<210> 42
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ttatagcatt ctatgaattt gcctgtc 27
<210> 43
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
atgagcgaag tcggtataca g 21
<210> 44
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
tcataacgaa aaatcagaga aatttg 26
<210> 45
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ggaagtacct tcaaagaatg ggg 23
<210> 46
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ttgttttata tttgttgtaa aaagtag 27
<210> 47
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
gcacacacca tagcttcaaa atg 23
<210> 48
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
ttgtaattaa aacttagatt agattgc 27
<210> 49
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
tcgagtttat cattatcaat actgc 25
<210> 50
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
tttgtttgtt tatgtgtgtt tattcg 26
<210> 51
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
cttatcttga cgggtattct gagc 24
<210> 52
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
tttgttttgt ttgtttgtgt gatg 24
<210> 53
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
tcttcaagaa ttggggatct acg 23
<210> 54
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
ctgtatgtgt tttttgtagt tatagatt 28
<210> 55
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
cgaatttctt atgatttatg atttttat 28
<210> 56
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
gagcgacctc atgctatacc tgag 24
<210> 57
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
atccgctcta accgaaaagg aagg 24
<210> 58
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
cttcgagcgt cccaaaacct tctc 24
<210> 59
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
atttaactcc ttaagttact ttaatg 26
<210> 60
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
gcgaaaagcc aattagtgtg atac 24
<210> 61
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
tagggcccac aagcttacgc g 21
<210> 62
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
tgccgtaaac cactaaatcg gaac 24
<210> 63
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
gttaattcaa attaattgat atagttt 27
<210> 64
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
gtaagctact atgaaagact ttacaaag 28
<210> 65
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
atggcgaatc tgaacggaga g 21
<210> 66
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
ttatttctgc ctcttgtata tcttg 25
<210> 67
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
atgggcaggg aagtagtgat 20
<210> 68
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
ttaatgctta taggatcaac 20
<210> 69
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
atgggctttc agattccttt a 21
<210> 70
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
ttaaaggata ataagatgta g 21
<210> 71
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
atgacttctg ctttgtatgc 20
<210> 72
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
tcaccagaca tctctgaggt 20
<210> 73
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
gaatacagtg gaggtggtga tatgcatatg atgctaga 38
<210> 74
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
tctagcatca tatgcatatc accacctcca ctgtattc 38
<210> 75
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
cttatcttga cgggtattct gagc 24
<210> 76
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
gcactgaagg ctctcaactc tgacattttg ttttgtttgt ttgtgtgatg 50
<210> 77
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
catcacacaa acaaacaaaa caaaatgtca gagttgagag ccttcagtgc 50
<210> 78
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
tcgacgcgta agcttgtggg ccctattatt tatcaagata agtttccgga 50
<210> 79
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
tccggaaact tatcttgata aataataggg cccacaagct tacgcgtcga 50
<210> 80
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
tgccgtaaac cactaaatcg gaac 24
<210> 81
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
ggaagtacct tcaaagaatg ggg 23
<210> 82
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
atacaatgta aacgttctga gacatttgtt ttatatttgt tgtaaaaagt 50
<210> 83
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
actttttaca acaaatataa aacaaatgtc tcagaacgtt tacattgtat 50
<210> 84
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
aaaatcataa atcataagaa attcgtcata tcttttcaat gacaatagag 50
<210> 85
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
ctctattgtc attgaaaaga tatgacgaat ttcttatgat ttatgatttt 50
<210> 86
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
tcgagtttat cattatcaat actgcc 26
<210> 87
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
cagaagttaa gaacggtaat gacattttgt ttgtttatgt gtgtttattc 50
<210> 88
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
gaataaacac acataaacaa acaaaatgtc attaccgttc ttaacttctg 50
<210> 89
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
attaaagtaa cttaaggagt taaatttatg aagtccatgg taaattcgtg 50
<210> 90
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
gcgaaaagcc aattagtgtg atac 24
<210> 91
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
gcacacacca tagcttcaaa atg 23
<210> 92
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
caacaaagtt tagttgagag tttcatttgt aattaaaact tagattagat tgc 53
<210> 93
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
gcaatctaat ctaagtttta attacaaatg aaactctcaa ctaaactttg ttg 53
<210> 94
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
ctccttcctt ttcggttaga gcggatttat tttttaacat cgtaagatct tc 52
<210> 95
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
gaagatctta cgatgttaaa aaataaatcc gctctaaccg aaaaggaagg ag 52
<210> 96
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
cttcgagcgt cccaaaacct tctc 24
<210> 97
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 97
tcttcaagaa ttggggatct acg 23
<210> 98
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 98
aacggatgct gtgtaaacgg tcatctgtat gtgttttttg tagttatag 49
<210> 99
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 99
ctataactac aaaaaacaca tacagatgac cgtttacaca gcatccgtt 49
<210> 100
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 100
actatatcaa ttaatttgaa ttaacttatt cctttggtag accagtcttt g 51
<210> 101
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 101
caaagactgg tctaccaaag gaataagtta attcaaatta attgatatag t 51
<210> 102
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 102
gtaagctact atgaaagact ttacaaagaa c 31
<210> 103
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 103
ctcagcatcg tccccaagct ccccatctgt atgtgttttt tgtagttata g 51
<210> 104
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 104
ctataactac aaaaaacaca tacagatggg cagggaagta gtgat 45
<210> 105
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 105
actatatcaa ttaatttgaa ttaacttaat gcttatagga tcaac 45
<210> 106
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 106
ctttgcatat ctcagagcaa actgagttaa ttcaaattaa ttgatatagt 50
<210> 107
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 107
ggaaggttcc tgattacgaa tggtttcatt tgttttatat ttgttgtaaa aagtag 56
<210> 108
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 108
ctacttttta caacaaatat aaaacaaatg ggctttcaga ttccttta 48
<210> 109
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 109
ataaaaatca taaatcataa gaaattcgtt aaaggataat aagatgtag 49
<210> 110
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 110
tgccatatac cgtgctcctc cttaacgaat ttcttatgat ttatgatttt tat 53
<210> 111
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 111
ccctttccta tcttcaactt ccacatttgt aattaaaact tagattagat tgc 53
<210> 112
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 112
gcaatctaat ctaagtttta attacaaatg acttctgctt tgtatgct 48
<210> 113
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 113
ctccttcctt ttcggttaga gcggattcac cagacatctc tgaggta 47
<210> 114
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 114
cgaaaagttg tgtttatcgt taattaaatc cgctctaacc gaaaaggaag gag 53
<210> 115
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 115
gcgatcgctt aggatttaat gcaggtgacg 30
<210> 116
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 116
actttttaca acaaatataa aacaaatgga ccaattggtg aaaactgaag 50
<210> 117
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 117
cttcagtttt caccaattgg tccatttgtt ttatatttgt tgtaaaaagt 50
<210> 118
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 118
tagaaacatt ttgaagctat ggtgtgtggg aagtaccttc aaagaatggg g 51
<210> 119
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 119
ccccattctt tgaaggtact tcccacacac catagcttca aaatgtttct a 51
<210> 120
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 120
cttgtgattc tgtataccga cttcgctcat ttgtaattaa aacttagatt agattgc 57
<210> 121
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 121
gcaatctaat ctaagtttta attacaaatg agcgaagtcg gtatacagaa tcacaag 57
<210> 122
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 122
gcgatcgctc ataacgaaaa atcagagaaa tttg 34
<210> 123
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 123
gcgatcgctt atagcattct atgaatttgc ctg 33
<210> 124
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 124
ctacttttta caacaaatat aaaacaaatg actgccgaca acaatagtat gc 52
<210> 125
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 125
gcatactatt gttgtcggca gtcatttgtt ttatatttgt tgtaaaaagt ag 52
<210> 126
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 126
ccgactctcc gttcagattc gccatttgta attaaaactt agattagatt gc 52
<210> 127
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 127
gcaatctaat ctaagtttta attacaaatg gcgaatctga acggagagtc gg 52
<210> 128
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 128
gcgatcgctt atttctgcct cttgtatatc ttgc 34
<210> 129
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 129
cgcgattttt gttatcaaaa agcattttgt ttgtttatgt gtgtttattc g 51
<210> 130
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 130
tgctcagaat acccgtcaag ataagtcgag tttatcatta tcaatactgc 50
<210> 131
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 131
gcagtattga taatgataaa ctcgacttat cttgacgggt attctgagca 50
<210> 132
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 132
acttttcctt ttaaatccgg atacattttg ttttgtttgt ttgtgtgatg a 51
<210> 133
<211> 78
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 133
cagaacaggc cacacaatcg caagtgatta acgtccacac aggtataggg cttatcttga 60
cgggtattct gagcatct 78
<210> 134
<211> 74
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 134
cagtggtact tgcaaaacaa gataagaccc cattctttga aggtacttcc tgccgtaaac 60
cactaaatcg gaac 74
<210> 135
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 135
ggaagtacct tcaaagaatg ggg 23
<210> 136
<211> 74
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 136
aattatttac gtattctttg aaatggcagt attgataatg ataaactcga gagcgacctc 60
atgctatacc tgag 74
<210> 137
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 137
tcgagtttat cattatcaat actgc 25
<210> 138
<211> 74
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 138
tctggaagag taaaaaagga gtagaaacat tttgaagcta tggtgtgtgc gcgaaaagcc 60
aattagtgtg atac 74
<210> 139
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 139
gcacacacca tagcttcaaa atg 23
<210> 140
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 140
gagggaatct gaagaagaat gaccatacgt agatccccaa ttcttgaaga cttcgagcgt 60
cccaaaacct tc 72
<210> 141
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 141
tcttcaagaa ttggggatct acg 23
<210> 142
<211> 78
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 142
ataacttcgt atagcataca ttatacgaag ttatattaag ggttgtcgac gtaagctact 60
atgaaagact ttacaaag 78
<210> 143
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 143
gtcgacaacc cttaatataa cttcg 25
<210> 144
<211> 77
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 144
ccttgaacgc actctcacta cggtgatgat cattcttgcc tcgcagacaa ataacttcgt 60
atagcataca ttatacg 77
<210> 145
<211> 73
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 145
catatgtttt agccccaatc ataatctaac cattccacaa atgaaacaat tcttcaagaa 60
ttggggatct acg 73
<210> 146
<211> 78
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 146
cagtggtact tgcaaaacaa gataagaccc cattctttga aggtacttcc gtaagctact 60
atgaaagact ttacaaag 78
<210> 147
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 147
ataacttcgt atagcataca ttatacgaag ttatattaag ggttgtcgac gagcgacctc 60
atgctatacc tgaga 75
<210> 148
<211> 77
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 148
attcattctc cactacgact cttgacttct ctcttgattc taatttgacg ataacttcgt 60
atagcataca ttatacg 77
<210> 149
<211> 73
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 149
gttttgatct tctcgttgaa gactcttcag tagaaagcag attaagagtg gcacacacca 60
tagcttcaaa atg 73
<210> 150
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 150
ggaagtacct tcaaagaatg gggtcttatc ttgttttgca agtaccactg cttcgagcgt 60
cccaaaacct tc 72
<210> 151
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 151
tctggaagag taaaaaagga gtagaaacat tttgaagcta tggtgtgtgc gagcgacctc 60
atgctatacc tg 72
<210> 152
<211> 74
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 152
ataacttcgt atagcataca ttatacgaag ttatattaag ggttgtcgac cttcgagcgt 60
cccaaaacct tctc 74
<210> 153
<211> 77
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 153
ctccatattc ttcataatta acgtggtctc tgtgcaaata aaaagtggaa ataacttcgt 60
atagcataca ttatacg 77
<210> 154
<211> 77
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 154
aaaattgagt aatgccactg cttttcccac tttagagtca tttgcatcat gcacacacca 60
tagcttcaaa atgtttc 77
<210> 155
<211> 77
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 155
cattggctag cgggaagtcg tttgctctag ttccactgta gtaaaggacc ataacttcgt 60
atagcataca ttatacg 77