技术领域technical field
本发明属于生物技术领域，涉及一种穿梭质粒，具体涉及一种用于大肠杆菌-鸭疫里默氏杆菌的高效穿梭质粒。The invention belongs to the field of biotechnology and relates to a shuttle plasmid, in particular to a high-efficiency shuttle plasmid for Escherichia coli-Riemerella anatipestifer.
背景技术Background technique
基因敲除技术是研究基因功能的常用手段，细菌缺失某个基因后往往会引起它的表型发生变化，但是表型的改变可能是由于这个基因被敲除后影响下游基因的表达造成的。所以，将被缺基因克隆到穿梭质粒并进行功能回补研究非常必要。Gene knockout technology is a common method for studying gene function. The deletion of a certain gene in bacteria often causes its phenotype to change, but the change in phenotype may be caused by the gene being knocked out and affecting the expression of downstream genes. Therefore, it is necessary to clone the deficient gene into a shuttle plasmid and conduct functional complementation research.
目前，能用于鸭疫里默氏杆菌基因回补的穿梭质粒数量有限，且酶切位点较少，接合转移效率低下，十分不利于基因回补实验的进行。At present, the number of shuttle plasmids that can be used for gene complementation of Riemerella anatipestifer is limited, and there are few restriction sites, and the efficiency of conjugative transfer is low, which is very unfavorable for gene complementation experiments.
发明内容Contents of the invention
有鉴于此，本发明的目的在于提供一种用于大肠杆菌-鸭疫里默氏杆菌的高效穿梭质粒。In view of this, the object of the present invention is to provide a high-efficiency shuttle plasmid for Escherichia coli-Rimerella anatipestifer.
本发明还提供了该穿梭质粒的制备方法及其应用。The invention also provides the preparation method and application of the shuttle plasmid.
为达到上述目的，本发明提供如下技术方案：To achieve the above object, the present invention provides the following technical solutions:
一种用于大肠杆菌-鸭疫里默氏杆菌的高效穿梭质粒，其基因序列如SEQ ID NO.1所示。A high-efficiency shuttle plasmid for Escherichia coli-Rimerella anatipestifer, the gene sequence of which is shown in SEQ ID NO.1.
上述穿梭质粒的制备方法，包括步骤：The preparation method of the above-mentioned shuttle plasmid comprises the steps of:
(1)将转移位点oriT序列克隆至质粒pPM5中，得质粒pPM5+oriT；(1) Cloning the transfer site oriT sequence into plasmid pPM5 to obtain plasmid pPM5+oriT;
(2)将鸭疫里默氏杆菌复制起始基因pRA0726 ori克隆至质粒pPM5+oriT中，所得质粒命名为pFY01；(2) The replication initiation gene pRA0726 ori of Riemerella anatipestifer was cloned into the plasmid pPM5+oriT, and the resulting plasmid was named pFY01;
(3)在pFY01上克隆一段带有多个内切酶位点的高表达启动子序列High EXP，所得质粒命名为pFY02。(3) A high expression promoter sequence High EXP with multiple endonuclease sites was cloned on pFY01, and the resulting plasmid was named pFY02.
优选的，oriT序列、pRA0726 ori和High EXP在NCBI中的序列号分别为AF047518.1、JF268689.1和CP003787.1。Preferably, the sequence numbers of the oriT sequence, pRA0726 ori and High EXP in NCBI are AF047518.1, JF268689.1 and CP003787.1, respectively.
优选的，步骤(1)的具体方法是：以质粒pEX18GM(Gene.1998；212(1):77–86)为模板，以SEQ ID NO.2和SEQ ID NO.3所示序列为引物进行PCR扩增，PCR产物经限制性内切酶SphI和HindIII酶切质粒pPM5(Infect Immun.2015,83(1):300-310)和oriT片段，用DNA连接试剂盒连接载体片段pPM5与基因片段oriT序列，并制备E.coli DH5α感受态细胞，将连接产物转化至E.coli DH5α，培养细菌，分离长出的单菌落，用PCR的方法鉴定出阳性克隆，扩大培养阳性克隆菌株，用质粒小提试剂盒提取得到质粒pPM5+oriT。Preferably, the specific method of step (1) is: use the plasmid pEX18GM (Gene.1998; 212(1):77-86) as a template, and use the sequences shown in SEQ ID NO.2 and SEQ ID NO.3 as primers PCR amplification, PCR products were digested with restriction enzymes SphI and HindIII plasmid pPM5 (Infect Immun.2015,83(1):300-310) and oriT fragments, and DNA ligation kit was used to connect the vector fragment pPM5 and the gene fragment oriT sequence, and prepare E.coli DH5α competent cells, transform the ligation product into E.coli DH5α, culture the bacteria, isolate the single colonies grown, identify positive clones by PCR method, expand and cultivate positive clone strains, and use plasmid The plasmid pPM5+oriT was obtained by extracting with the small extraction kit.
进一步优选的，采用HindIII酶单酶切进行质粒pPM5+oriT的鉴定。Further preferably, the identification of the plasmid pPM5+oriT is carried out by single-digestion with HindIII enzyme.
进一步优选的，细菌的培养方法是：用加有100μg/ml氨苄西林的LB固体培养基培养细菌。Further preferably, the method for culturing the bacteria is: culturing the bacteria with LB solid medium added with 100 μg/ml ampicillin.
进一步优选的，所述PCR扩增条件为98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸30s)，30个循环，72℃延伸7min，22℃，∞。Further preferably, the PCR amplification conditions are pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C for 30s), 30 cycles, extension at 72°C for 7min, 22°C, ∞.
进一步优选的，PCR扩增结束后和酶切结束后，分别用DNA纯化试剂盒回收PCR产物oriT序列和长片段酶切产物。Further preferably, after PCR amplification and enzyme digestion, DNA purification kits are used to recover the PCR product oriT sequence and long fragment enzyme digestion products respectively.
优选的，步骤(2)的具体方法是：以质粒pLMF01(Genbank:KU963002)为模板，以SEQID NO.4和SEQ ID NO.5所示序列为引物进行PCR扩增，PCR产物经限制性内切酶SphI和PstI酶切pPM5+oriT和pRA0726 ori片段，用DNA连接试剂盒连接载体片段pPM5+oriT与基因片段pRA0726 ori，并制备E.coli DH5α感受态细胞，将连接产物转化至E.coli DH5α，用含100μg/ml氨苄抗性的培养板培养细菌，分离长出的单菌落，用PCR的方法鉴定出阳性克隆，扩大培养阳性克隆菌株，用质粒小提试剂盒提取得到质粒pFY01。Preferably, the specific method of step (2) is: using the plasmid pLMF01 (Genbank: KU963002) as a template, using the sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers to carry out PCR amplification, and the PCR product is subjected to restriction internal Dicer SphI and PstI digest pPM5+oriT and pRA0726 ori fragments, use a DNA ligation kit to connect the vector fragment pPM5+oriT and the gene fragment pRA0726 ori, and prepare E.coli DH5α competent cells, and transform the ligation product into E.coli For DH5α, the bacteria were cultured on a culture plate containing 100 μg/ml ampicillin resistance, and the grown single colonies were isolated. The positive clones were identified by PCR, and the positive clones were expanded and cultured, and the plasmid pFY01 was obtained by extraction with a plasmid mini-extraction kit.
进一步优选的，采用SphI酶单酶切进行质粒pFY01的鉴定。Further preferably, the identification of the plasmid pFY01 is carried out by single-digestion with SphI enzyme.
进一步优选的，细菌的培养方法是：用加有100μg/ml氨苄西林的LB固体培养基培养细菌。Further preferably, the method for culturing the bacteria is: culturing the bacteria with LB solid medium added with 100 μg/ml ampicillin.
进一步优选的，所述PCR扩增条件为98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸1min，30个循环，72℃延伸7min，22℃，∞。Further preferably, the PCR amplification conditions are pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C for 1min, 30 cycles, extension at 72°C for 7min, 22°C, ∞.
进一步优选的，PCR扩增结束后和酶切结束后，分别用DNA纯化试剂盒回收PCR产物pRA0726 ori和长片段酶切产物。Further preferably, after the PCR amplification and enzyme digestion, the PCR product pRA0726 ori and the long fragment enzyme digestion product are respectively recovered with a DNA purification kit.
优选的，步骤(3)的具体方法是：以RA CH-1菌株基因组(BMC genomics.2014；15:479)为模板，以SEQ ID NO.6和SEQ ID NO.7所示序列为引物进行PCR扩增，PCR产物经限制性内切酶SalI和XbaI酶切质粒pFY01和High EXP片段，用DNA连接试剂盒连接载体片段pFY01与基因片段High EXP，并制备E.coli DH5α感受态细胞，将连接产物转化至E.coliDH5α，培养细菌，分离长出的单菌落，用PCR的方法鉴定出阳性克隆，扩大培养阳性克隆菌株，用质粒小提试剂盒提取得到质粒pFY02。Preferably, the specific method of step (3) is: using the RA CH-1 strain genome (BMC genomics.2014; 15:479) as a template, and using the sequences shown in SEQ ID NO.6 and SEQ ID NO.7 as primers PCR amplification, the PCR product was digested with restriction endonuclease SalI and XbaI plasmid pFY01 and High EXP fragments, and the DNA ligation kit was used to connect the vector fragment pFY01 and the gene fragment High EXP, and prepare E.coli DH5α competent cells. The ligation product was transformed into E.coliDH5α, the bacteria were cultured, and the grown single colony was isolated. The positive clone was identified by PCR method, and the positive clone strain was expanded and cultured, and the plasmid pFY02 was obtained by extraction with a plasmid mini-extraction kit.
进一步优选的，采用XbaI酶单酶切进行质粒pFY02的鉴定。Further preferably, the identification of plasmid pFY02 is carried out by single digestion with XbaI enzyme.
进一步优选的，细菌的培养方法是：用加有100μg/ml氨苄西林的LB固体培养基培养细菌。Further preferably, the method for culturing the bacteria is: culturing the bacteria with LB solid medium added with 100 μg/ml ampicillin.
进一步优选的，所述PCR扩增条件为98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸20s，30个循环，72℃延伸7min，22℃，∞。Further preferably, the PCR amplification conditions are pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C for 20s, 30 cycles, extension at 72°C for 7min, 22°C, ∞.
进一步优选的，PCR扩增结束后和酶切结束后，分别用DNA纯化试剂盒回收PCR产物High EXP和长片段酶切产物。Further preferably, after the PCR amplification and enzyme digestion, the PCR product High EXP and the long fragment enzyme digestion product are respectively recovered with a DNA purification kit.
上述穿梭质粒在鸭疫里默氏杆菌缺失株的基因回补中的应用。The application of the above-mentioned shuttle plasmid in the gene complementation of the Riemerella anatipestifer deletion strain.
本发明的有益效果在于：The beneficial effects of the present invention are:
本发明的穿梭质粒是在质粒pPM5的基础上进行改造的，由于此质粒中的复制起始位点与鸭疫里默氏杆菌的复制起始位点不同，为了使其能够在鸭疫里默氏杆菌中稳定的存在，将鸭疫里默氏杆菌复制起始基因(pRA0726 ori)克隆到此质粒中；为了提高其结合转移效率，将转移位点(oriT)克隆到此质粒中，使此穿梭质粒能够通过高效的结合转移导入鸭疫里默氏杆菌中，且能稳定的复制。为了方便基因片段插入穿梭质粒上且提高基因的表达量，将在穿梭质粒上克隆一段带有多个内切酶位点的高表达启动子序列(High EXP)。The shuttle plasmid of the present invention is transformed on the basis of plasmid pPM5. Since the replication origin site in this plasmid is different from that of Riemerella anatipestifer, in order to enable it to Stable existence in bacilli, the replication initiation gene (pRA0726 ori) of Riemeria anatipestifer was cloned into this plasmid; in order to improve its combined transfer efficiency, the transfer site (oriT) was cloned into this plasmid, making this The shuttle plasmid can be introduced into Riemerella anatipestifer through efficient conjugation transfer, and can replicate stably. In order to facilitate the insertion of gene fragments into the shuttle plasmid and increase the expression level of the gene, a high expression promoter sequence (High EXP) with multiple endonuclease sites will be cloned on the shuttle plasmid.
本发明所得穿梭质粒可以在鸭疫里默氏杆菌中稳定存在，用于基因敲除株的回补。质粒较小且具有多个多克隆位点，易于操作。结合转移效率非常高。The shuttle plasmid obtained in the present invention can stably exist in Riemerella anatipestifer, and is used for the complementation of gene knockout strains. Plasmids are small and have multiple multiple cloning sites for easy manipulation. The binding transfer efficiency is very high.
附图说明Description of drawings
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention provides the following drawings for illustration:
图1为E.coli DH5α+pPM5+oriT菌落PCR鉴定图，即扩增的oriT片段后的电泳图，其中M是DNA marker，1泳道是以质粒pEX18GM为模板的阳性对照，2～4泳道是以E.coli DH5α+pPM5+oriT单克隆菌为模板；Figure 1 is the PCR identification diagram of E.coli DH5α+pPM5+oriT colony, that is, the electrophoresis of the amplified oriT fragment, where M is a DNA marker, lane 1 is a positive control with plasmid pEX18GM as a template, and lanes 2 to 4 are Use E.coli DH5α+pPM5+oriT monoclonal bacteria as template;
图2为质粒pPM5+oriT酶切(HindⅢ)鉴定图，是用限制性内切酶(HindⅢ)酶切鉴定质粒的电泳图，其中M是DNA marker，1泳道是质粒pPM5+oriT，2泳道是pPM5；Figure 2 is the identification diagram of plasmid pPM5+oriT enzyme digestion (HindⅢ), which is the electrophoresis diagram of restriction endonuclease (HindⅢ) enzyme digestion identification plasmid, wherein M is DNA marker, 1 lane is plasmid pPM5+oriT, and 2 lanes are pPM5;
图3为E.coli DH5α+pFY01菌落PCR鉴定图，是扩增的pRA0726 ori片段后的电泳图，其中M是DNA marker，1泳道是以质粒pLMF01为模板的阳性对照，2-3泳道是以E.coliDH5α+pFY01单克隆菌为模板；Figure 3 is the PCR identification map of E.coli DH5α+pFY01 colony, which is the electrophoresis image of the amplified pRA0726 ori fragment, where M is the DNA marker, the 1st lane is the positive control of the plasmid pLMF01 as the template, and the 2-3 lanes are the positive control of the plasmid pLMF01. E.coliDH5α+pFY01 monoclonal bacteria as a template;
图4为质粒pFY01酶切(SphI)鉴定图，是用限制性内切酶(SphI)酶切鉴定质粒的电泳图，其中M是DNA marker，1泳道是质粒pPM5+oriT，2泳道是pFY01；Fig. 4 is a plasmid pFY01 enzyme digestion (SphI) identification diagram, which is an electrophoresis diagram of a restriction endonuclease (SphI) enzyme digestion identification plasmid, wherein M is a DNA marker, the 1st swimming lane is the plasmid pPM5+oriT, and the 2nd swimming lane is pFY01;
图5为E.coli DH5α+pFY02菌落PCR鉴定图，是扩增的High EXP片段后的电泳图，其中M是DNA marker，1泳道是以RA CH-1菌株为模板的阳性对照，2-6泳道是以E.coli DH5α+pFY02单克隆菌为模板；Figure 5 is the PCR identification map of E.coli DH5α+pFY02 colony, which is the electrophoresis map of the amplified High EXP fragment, where M is a DNA marker, and lane 1 is a positive control with RA CH-1 strain as a template, 2-6 The lane is based on E.coli DH5α+pFY02 monoclonal bacteria as template;
图6为质粒pFY02酶切(XbaI)鉴定图，是用限制性内切酶(XbaI)酶切鉴定质粒的电泳图，其中M是DNA marker，1泳道是质粒pFY01，2泳道是pFY02；Fig. 6 is the identification diagram of plasmid pFY02 digestion (XbaI), which is the electrophoresis diagram of restriction endonuclease (XbaI) digestion identification plasmid, wherein M is a DNA marker, 1 lane is plasmid pFY01, and 2 lanes are pFY02;
图7为E.coli S17.1+pFY02菌落PCR鉴定图，是扩增的oriT+pRA0726 ori片段后的电泳图，其中M是DNA marker，1泳道是以质粒pFY02为模板的阳性对照，2～4泳道是以E.coli S17.1+pFY02单克隆菌为模板；Figure 7 is the PCR identification diagram of E.coli S17.1+pFY02 colony, which is the electrophoresis diagram after the amplified oriT+pRA0726 ori fragment, where M is a DNA marker, lane 1 is a positive control with plasmid pFY02 as a template, and lane 2- Lane 4 is based on E.coli S17.1+pFY02 monoclonal bacteria as a template;
图8为RA ATCC+pFY02菌落PCR鉴定图，是扩增的oriT+pRA0726 ori片段后的电泳图，其中M是DNA marker，1泳道是以质粒pFY02为模板的阳性对照，2～4泳道是以RA ATCC+pFY02单克隆菌为模板；Figure 8 is the RA ATCC+pFY02 colony PCR identification diagram, which is the electrophoresis of the amplified oriT+pRA0726 ori fragment, where M is a DNA marker, the 1st lane is a positive control with the plasmid pFY02 as a template, and the 2nd to 4th lanes are RA ATCC+pFY02 monoclonal bacteria as template;
图9为阳性克隆RA ATCC+pFY02传10代菌落PCR鉴定图，是扩增的oriT+pRA0726ori片段后的电泳图，其中M是DNA marker，1泳道是以质粒pFY02为模板的阳性对照，2～3泳道是以RA ATCC+pFY02单克隆菌传十代后的菌落为模板；Figure 9 is the positive clone RA ATCC+pFY02 passage 10 generation colony PCR identification diagram, which is the electrophoresis diagram after the amplified oriT+pRA0726ori fragment, where M is a DNA marker, lane 1 is a positive control with plasmid pFY02 as a template, lane 2～ Lane 3 is based on the colonies of RA ATCC+pFY02 monoclonal bacteria passed for ten generations as a template;
图10为结合转移后长出的菌落，是结合转移后长出的菌落图，1是使用普通穿梭质粒pLMF01长出的菌落，2是使用穿梭质粒pFY02长出的菌；Figure 10 shows the colonies grown after combination transfer, which is the colony diagram grown after combination transfer, 1 is the colony grown using the common shuttle plasmid pLMF01, and 2 is the bacteria grown using the shuttle plasmid pFY02;
图11为pFY02的质粒图谱；Figure 11 is the plasmid map of pFY02;
图12为pPM5+oriT+pRA0726 ori构建得到pFY01的质粒图谱；Figure 12 is the plasmid map of pFY01 obtained by constructing pPM5+oriT+pRA0726 ori;
图13为pFY01构建得到pFY02的质粒图谱；Figure 13 is the plasmid map of pFY02 obtained by constructing pFY01;
图14为E.coli DH5α+pFY02::tonB2菌落PCR鉴定图，是扩增的tonB2片段后的电泳图，其中M是DNA marker，1泳道是以RA ATCC11845(BMC genomics.2014；15:479)为模板的阳性对照，2-4泳道是以E.coli DH5α+pFY02::tonB2单克隆菌为模板；Figure 14 is the PCR identification map of E.coli DH5α+pFY02::tonB2 colony, which is the electrophoresis map of the amplified tonB2 fragment, where M is a DNA marker, and lane 1 is based on RA ATCC11845 (BMC genomics.2014; 15:479) As the positive control of the template, lanes 2-4 use E.coli DH5α+pFY02::tonB2 monoclonal bacteria as the template;
图15为RA ATCCΔtonB2+pFY02::tonB2菌落PCR鉴定图，是扩增的tonB2片段后的电泳图，其中M是DNA marker，1泳道是以RA ATCC11845为模板的阳性对照，2泳道是以RAATCCΔtonB2+pFY02::tonB2单克隆菌为模板；Figure 15 is the PCR identification map of RA ATCCΔtonB2+pFY02::tonB2 colony, which is the electrophoresis image of the amplified tonB2 fragment, where M is the DNA marker, the 1st lane is the positive control of RA ATCC11845 as the template, and the 2nd lane is the RAATCCΔtonB2+ pFY02::tonB2 monoclonal bacteria as a template;
图16为RA ATCC野生株、RA ATCC tonB2敲除株，RA ATCC tonB2回补株生长曲线。Figure 16 shows the growth curves of RA ATCC wild strain, RA ATCC tonB2 knockout strain, and RA ATCC tonB2 complementation strain.
具体实施方式detailed description
下面将结合附图，对本发明的优选实施例进行详细的描述。The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
实施例1：Example 1:
重组质粒pPM5+oriT的构建Construction of recombinant plasmid pPM5+oriT
本发明的穿梭质粒是在质粒pPM5的基础上进行改造的，为了提高其结合转移效率，申请人将转移位点oriT序列(NCBI中序列号为AF047518.1)克隆到此质粒中。The shuttle plasmid of the present invention is modified on the basis of plasmid pPM5. In order to improve its combined transfer efficiency, the applicant cloned the transfer site oriT sequence (the sequence number in NCBI is AF047518.1) into this plasmid.
根据oriT序列设计两条PCR引物：Design two PCR primers according to the oriT sequence:
上游引物：5'-CCCAAGCTTCGCCTGATGCGGTATTTTCTCC-3'，如SEQ ID NO.2所示；Upstream primer: 5'-CCCAAGCTTCGCCTGATGCGGTATTTTTCTCC-3', as shown in SEQ ID NO.2;
下游引物：5'-ACATGCATGCCTAGAGTCGATCTTCGCCAGC-3'，如SEQ ID NO.3所示。Downstream primer: 5'-ACATGCATGCCTAGAGTCGATCTTCGCCAGC-3', as shown in SEQ ID NO.3.
以质粒pEX18GM为模板，SEQ ID NO.2和SEQ ID NO.3所示的引物用PCR扩增oriT序列，扩增程序为：98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸30s，30个循环，72℃延伸7min，22℃，∞。用DNA纯化回收试剂盒回收PCR产物oriT序列，用限制性内切酶SphI和HindIII酶切质粒pPM5和oriT片段，用DNA纯化回收试剂盒回收长片段酶切产物。用DNA连接试剂盒连接载体片段pPM5与基因片段oriT，并制备E.coli DH5α感受态细胞，将连接产物转化至E.coli DH5α，用加有100μg/ml氨苄西林的LB固体培养基培养细菌，分离长出的单菌落，用PCR的方法鉴定出阳性克隆(鉴定oriT片段，约800bp)，结果如图1。用LB液体培养基扩大培养阳性克隆菌株，用质粒小提试剂盒提取质粒，用酶(HindⅢ)单酶切的方法鉴定，将新构建质粒命名为pPM5+oriT，酶切结果如图2。即质粒pPM5+oriT构建成功。Using the plasmid pEX18GM as a template, the primers shown in SEQ ID NO.2 and SEQ ID NO.3 were used to amplify the oriT sequence by PCR. The amplification program was: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, 72°C ℃ extension 30s, 30 cycles, 72 ℃ extension 7min, 22 ℃, ∞. The PCR product oriT sequence was recovered with a DNA purification and recovery kit, the plasmid pPM5 and oriT fragments were digested with restriction enzymes SphI and HindIII, and the long fragment digestion product was recovered with a DNA purification and recovery kit. Use the DNA ligation kit to connect the vector fragment pPM5 and the gene fragment oriT, and prepare E.coli DH5α competent cells, transform the ligation product into E.coli DH5α, and culture the bacteria in LB solid medium with 100 μg/ml ampicillin, The grown single colonies were isolated, and positive clones were identified by PCR (identification of the oriT fragment, about 800 bp), the results are shown in Figure 1. Use LB liquid medium to amplify and cultivate the positive cloned strain, extract the plasmid with a plasmid mini-extraction kit, and identify it with a single enzyme (HindⅢ) digestion method. The newly constructed plasmid is named pPM5+oriT, and the digestion results are shown in Figure 2. That is, the plasmid pPM5+oriT was constructed successfully.
实施例2：Example 2:
重组质粒pFY01的构建Construction of recombinant plasmid pFY01
由于此质粒中的复制起始位点与鸭疫里默氏杆菌的复制起始位点不同，为了使其能够在鸭疫里默氏杆菌中稳定的存在，申请人将鸭疫里默氏杆菌复制起始基因pRA0726ori(NCBI中序列号为JF268689.1)克隆到此质粒中。Since the replication initiation site in this plasmid is different from that of Riemerella anatipestifer, in order to enable it to exist stably in Riemerella anatipestifer, the applicant introduced Riemerella anatipestifer The replication initiation gene pRA0726ori (sequence number JF268689.1 in NCBI) was cloned into this plasmid.
根据复制起始基因pRA0726 ori设计两条PCR引物：Design two PCR primers based on the replication initiation gene pRA0726 ori:
上游引物：5'-ACATGCATGCCTATTTAGGCATTAGCCCTC-3'，如SEQ ID NO.4所示；Upstream primer: 5'-ACATGCATGCCTATTTAGGCATTAGCCCTC-3', as shown in SEQ ID NO.4;
下游引物：5'-AAAACTGCAGCCAATGCATTGGAACAGATCTCGTATAGAGCTCG-3'，如SEQ IDNO.5所示。Downstream primer: 5'-AAAACTGCAGCCAATGCATTGGAACAGATCTCGTATAGAGCTCG-3', as shown in SEQ ID NO.5.
以pLMF01(Genbank:KU963002)为模板，SEQ ID NO.4和SEQ ID NO.5所示的引物用PCR扩增pRA0726 ori基因序列，扩增程序为：98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸1min，30个循环，72℃延伸7min，22℃，∞。用DNA纯化回收试剂盒回收PCR产物pRA0726 ori，用限制性内切酶SphI和PstI酶切质粒pPM5+oriT和pRA0726 ori片段，用DNA纯化回收试剂盒回收长片段酶切产物。用DNA连接试剂盒连接载体片段pPM5+oriT与基因片段pRA0726 ori，并制备E.coli DH5α感受态细胞，将连接产物转化至E.coli DH5α，用加有100μg/ml氨苄西林的LB固体培养基培养细菌，分离长出的单菌落，用PCR的方法鉴定出阳性克隆(鉴定pRA0726 ori片段，约2000bp)，结果如图3。用LB液体培养基扩大培养阳性克隆菌株，用质粒小提试剂盒提取质粒，用酶(SphI)单酶切的方法鉴定，将新构建质粒命名为pFY01，酶切结果如图4。即pFY01构建成功(图12)。Using pLMF01 (Genbank: KU963002) as a template, the primers shown in SEQ ID NO.4 and SEQ ID NO.5 were used to amplify the pRA0726 ori gene sequence by PCR. The amplification program was: 98°C pre-denaturation for 30s; 98°C denaturation for 10s, Anneal at 55°C for 30s, extend at 72°C for 1min, 30 cycles, extend at 72°C for 7min, 22°C, ∞. The PCR product pRA0726 ori was recovered with a DNA purification and recovery kit, the plasmid pPM5+oriT and pRA0726 ori fragments were digested with restriction enzymes SphI and PstI, and the long fragment digestion product was recovered with a DNA purification and recovery kit. Use the DNA ligation kit to connect the vector fragment pPM5+oriT and the gene fragment pRA0726 ori, and prepare E.coli DH5α competent cells, transform the ligated product into E.coli DH5α, and use LB solid medium with 100 μg/ml ampicillin Bacteria were cultured, and single colonies grown were isolated, and positive clones were identified by PCR (identification of the pRA0726 ori fragment, about 2000 bp), the results are shown in Figure 3. Use LB liquid medium to amplify and cultivate the positive clone strain, extract the plasmid with a plasmid mini-extraction kit, and identify it with a single enzyme (SphI) digestion method. The newly constructed plasmid is named pFY01, and the digestion results are shown in Figure 4. That is, pFY01 was constructed successfully (Fig. 12).
实施例3：Example 3:
重组质粒pFY02的构建Construction of recombinant plasmid pFY02
为了方便基因片段插入穿梭质粒上且提高基因的表达量，申请人在穿梭质粒上克隆一段带有多个内切酶位点的高表达启动子序列High EXP(NCBI中序列号为CP003787.1)。In order to facilitate the insertion of gene fragments into the shuttle plasmid and increase the expression of the gene, the applicant cloned a high expression promoter sequence High EXP with multiple endonuclease sites on the shuttle plasmid (the sequence number in NCBI is CP003787.1) .
根据高表达启动子High EXP序列设计两条PCR引物：Design two PCR primers according to the high expression promoter High EXP sequence:
上游引物：5'-ACGCGTCGACGTCGGCCATATTTCAAAAATTTAACTTAAACC-3'，如SEQ IDNO.6所示；Upstream primer: 5'-ACGCGTCGACGTCGGCCATATTTCAAAAATTTAACTTAAACC-3', as shown in SEQ ID NO.6;
下游引物：Downstream primers:
5'-TGCTCTAGAGCAGCGCGCAGGCCTGCTAGCCCGCGGAATTTTAAATAATTTTTTTAAATTTG-3'，如SEQ ID NO.7所示。5'-TGCTCTAGAGCAGCGCGCAGGCCTGCTAGCCCGCGGAATTTTAAATAATTTTTTTAAATTTG-3', as shown in SEQ ID NO.7.
其中如SEQ ID NO.7所示的下游引物中添加了酶切位点SacII，NheI，StuI，BsshII。Wherein the downstream primer shown in SEQ ID NO.7 has added restriction sites SacII, NheI, StuI, BsshII.
以RA CH-1菌株为模板，如SEQ ID NO.6和SEQ ID NO.7所示的为引物用PCR扩增High EXP基因序列，扩增程序为：98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸20s，30个循环，72℃延伸7min，22℃，∞。用DNA纯化回收试剂盒回收PCR产物High EXP，用限制性内切酶SalI和XbaI酶切质粒pFY01和High EXP片段，用DNA纯化回收试剂盒回收长片段酶切产物。用DNA连接试剂盒连接载体片段pFY01与基因片段High EXP，并制备E.coli DH5α感受态细胞，将连接产物转化至E.coli DH5α，用加有100μg/ml氨苄西林的LB固体培养基培养细菌，分离长出的单菌落，用PCR的方法鉴定出阳性克隆(鉴定High EXP片段，约250bp)，结果如图5。用LB液体培养基扩大培养阳性克隆菌株，用质粒小提试剂盒提取质粒，用酶(XbaI)单酶切的方法鉴定，将新构建质粒命名为pFY02，酶切结果如图6。即pFY02(图11和图13)构建成功，其基因序列如SEQ ID NO.1所示：Use the RA CH-1 strain as a template, and use the primers shown in SEQ ID NO.6 and SEQ ID NO.7 as primers to amplify the High EXP gene sequence by PCR. The amplification program is: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s , annealing at 55°C for 30s, extension at 72°C for 20s, 30 cycles, extension at 72°C for 7min, 22°C, ∞. The PCR product High EXP was recovered with a DNA purification and recovery kit, the plasmid pFY01 and High EXP fragments were digested with restriction enzymes SalI and XbaI, and the long fragment digestion product was recovered with a DNA purification and recovery kit. Use the DNA ligation kit to connect the vector fragment pFY01 and the gene fragment High EXP, and prepare E.coli DH5α competent cells, transform the ligated product into E.coli DH5α, and culture the bacteria in LB solid medium with 100 μg/ml ampicillin , isolate the single colony that grew out, and identify positive clones (identification of High EXP fragment, about 250bp) with the method of PCR, the results are shown in Figure 5. Use LB liquid medium to amplify and cultivate the positive cloned strain, extract the plasmid with a plasmid mini-extraction kit, and identify it with a single enzyme (XbaI) digestion method. The newly constructed plasmid is named pFY02, and the digestion results are shown in Figure 6. That is, pFY02 (Figure 11 and Figure 13) was constructed successfully, and its gene sequence is shown in SEQ ID NO.1:
AAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTCGCCTGATGCGGTATTTTCTCCTTACGCATATCGACATCCGCCCTCACCGCCAGGAACGCAACCGCAGCCTCATCACGCCGGCGCTTCTTGGCCGCGCGGGATTCAACCCACTCGGCCAGCTCGTCGGTGTAGCTCTTTGGCATCGTCTCTCGCCTGTCCCCTCAGTTCAGTAATTTCCTGCATTTGCCTGTTTCCAGTCGGTAGATATTCCACAAAACAGCAGGGAAGCAGCGCTTTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGGGTTCGTGTAGACTTTCCTTGGTGTATCCAACGGCGTCAGCCGGGCAGGATAGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCCGGCTACCGCCGGCGTAACAGATGAGGGCAAGCGGATGGCTGATGAAACCAAGCCAACCAGGAAGGGCAGCCCACCTATCAAGGTGTACTGCCTTCCAGACGAACGAAGAGCGATTGAGGAAAAGGCGGCGGCGGCCGGCATGAGCCTGTCGGCCTACCTGCTGGCCGTCGGCCAGGGCTACAAAATCACGGGCGTCGTGGACTATGAGCACGTCCGCGAGCTGGCCCGCATCAATGGCGACCTGGGCCGCCTGGGCGGCCTGCTGAAACTCTGGCTCACCGACGACCCGCGCACGGCGCGGTTCGGTGATGCCACGATCCTCGCCCTGCTGGCGAAGATCGACTCTAGGCATGCCTATTTAGGCATTAGCCCTCTTTTTAACCTGACTTAAAAAATAATTAAATCATAAGATATTAAATATCAAGTGATTATGATTTACTATGCTATACATAATATCACCCAGTGTTAGAAAATTCTTGCTATGCTTTTTGTTTGTTTTTTTTCATTCGTGAACTTTTAGAATACTTTTAAAATAAACTCAAATTTATAAACTATTGATTTTCAATATTATACAAAACCAAACTTATTAACATTAAACTTATTTCTATTACCTTTAAAATAAACTACTAAAAGACAAATCTACCAATCCTCGGAACTATTTAATAAATAGGAAGTTATATAAAAATAATGAACTTTTAGAATACTTTATTAGTCTTTTTAGCTCCAAAAATGAACTTTTAGAATACTTTTTTTGAACTTTTAGAATACTTTTTTAAACTTGCTTTTTAGTAAGTTCATTTTTTATATATTTGTATTCTAAAAGTGCATTTTATATTCATAACTCTCTATATATGGCAAAAGTAGATAATAAACTAATGTTTCAGTCCTATATTTTTACTACGGCTAAATATGATTTTTCAGTCTATGAAAAGCGTATACTATATAGGCAAATAGAAATAGAACAGGCATTGCTAAATAATGAGGCGTTGAAAGATTGTATCAAAATAGATACTAACCTATGGGGGGATAAAAAATATACAATTCCAATTTCTATGCTGTTAGCTAATGATGAAGATAAAAATTATTATAAAATTAAAAAAGCATTCAAAGACCTAAAATCAAAGAACATTGAGTACGAAGATGAAAAAGAATACGCTTGCTTTGGTGTTATTGATAAGTTTTGGATTGATAAATATGGAAGAAATGTTACTTGGCAATCTGACAAAAGAATTGTTGAAGCGGTAATGGATTTTACAAGAGGATGGAGAAAATATGAATTGAAGATTGCTATGCAGTTTGAGACTATTTATGCAATGCGTTTTTACGAACTGATAGCTAATAAAACCAAACCAATAACATATGAAGTGTCATTTCTTGAAAAAATGTTTCAATTGGAAAATAAGTACAGGAACCCTCAGGGTAATCTTAATATCAGTATGTTTAAACGCCGTGTATTAGACCCCGCTAAAAACGAGTTAGATAAATGTTCTCCATACACTTTTGATTATACTCTTTCAAAGGATAAAAAAATTATTTCTCTAATTCCTATTTTCCAAGAACAATTTGCAGATGAAAGTATGAAGATGATCAAAATAGAAGAGGAAAAAGATATACACTCTGTATTAACTCAAAGAGAAATTGATGTTTTtACAAACGAATTTGGTTTTACACAACAAGGTTTATTAAACAACTATGCCCTATTCAAAGATTGTAAGGCTACACTTCCATCAAACTATTCTTTCTTCCTTTTTCAAGAGATAAGAAAAAGTCGGACTAAGAAAGGAAAAATATCTCCTGCTTATGTGATTGGGATTATAAGAAATATGCTTAAAGATTTCAAAAAAGAACAACAATAGGCTTATGAAAAAATATACAACAAAAGAAATCGCAGAGCTTTTTGGCGTCAGCGAACGGACAATCCAGCGACACATAACGACACTAAGCGACAACATATCAAATACCGACAGAAAGGGCTTTTCAATACCAGAAGACATTGTTTTCTTTCTTGCCGAGCGACACCATTACGACATTTTAGCGACAACGAACGACAACGAACGACAGGATAAAAATGAAGAATTTCCTCATATTGAATACTTCACAGAAGAGGAATACCAAGAGTTTAAAAAAAGGATCACAGAGTACCCTTTTTTGAAAGAGCAAATTAAACTGTCTAATGAACATTTGGATACTCTAAAAACACAAATAGAATATTTCAGACTATCTTACAATAAGCAATTGGATATTCACGAAAAATTAATCGGAGCGTTCCGAGAAAGAAACTTTATTGAAGCTAAAGAAAAGGGGATGGATAAATAGCTCCTAATAATTAAAGAAGTATTAGAAAAAAAATGCTATATAAAAACTTAATCGAGCTCTATACGAGATCTGTTCCAATGCATTGGCTGCAGGTCGACGTCGGCCATATTTCAAAAATTTAACTTAAACCACTGATTTACAAAAATGTTTATTTGCAAAACTATTTTACAAAGATATTACAATAATTTTTCTAGCACAAATTTATTTCTCACCATATCTTTGCCTTGTTCAAGTGAGTGAACAGAGTAACAAACAAAAAAAGAAACAAATTTAAAAAAATTATTTAAAATTCCGCGGGCTAGCAGGCCTGCGCGCTGCTCTAGAACTCGAGCATCATCATCATCATCATTGAACTAGTGGAGTTGCTTCTCTTTTCGGGAGTGGAAAACTGAAAGAACTGGAACGTGCCAACGAAAAACTGCAAGACGAGGTTTCAAAACGGAACACCAATATTGAAAAATTGCAGAGCCAAGTACAGCAGATGCAGAAACAGCATGATACGCAAATCCACAATCTCAGAGAAATGCACAGGCAGGAACTTGACATGAAAGAAAAAGAACTGTCACGGCTCGCCAGAATCATAGACAAGGCTTTCAGGTGGTTTCCGATGTTCAGGGAAATGCTGCGCATGGAAAAGTTTTGTGCCATGCTGGGATTCTCTAAAGAAATGACTGAAAGTCTTATAGTCAAAAAAGAAGCCCTGAAATGTAGCGGTAAAATCTATTCCGAGCAACACAGGCGGAACTTTGATATAAAGGATGATATTTTAAGGGTGGAAAATGACCCTGACGATGAAAGCAGGCTGAACCTGACAATAAACAGGAAGCCGATTGCCGACTGGTTCAGGGAGCAATGGCACAGGCTTAGATATGGAGCAAGAGTGCCGCAACAGGAAGAAAGAAAAAGTAGAGGATTCAAATTATAATAGAAGCAATTTGATTAGTAATCTAAAAGCACTCCGATAACGATTAGAGTGCTTTTAGATTGTTTATCATTAATTATCAAAGCAAGTGCAGTTTAAGATTTTACTGAAGTTTGCATTAATAAAGAATATACTACAGCTGATATATGCGCAACATATTGTGACGCTTGTGATTTATTTCCCTTGAAATCCTTAACAAATACCGCTAAGGTATAACTGATATTATTAGGCAGACATATATAGGCAACATCATTGTGAGCTGCAAGAACACCATTTTCATTAACATAACCTGAACCTGTCTTATGCGCTATAACAACCCCTTCTTTATCAAGAAGTGGAGCTGCTATCCTATCTACACCTGTTTTGCATTCTTTTAACGTATTCTTAATGAAACTTTGTTTCTCATCATCGATAAGACCTTCAGTAAACAAACGATTCATCAACATTGCAGCACCAAGAGGAGATGTATAGTTAGAGTAAGCCTTGTTATGGTCAGCCGACATTTCCTCTTCCGTATAAGCTATCTGAAAACTTGAACGAGGAATGAGTGTGGCTATAAAACTATCTGTTTGAGCGACATTAACCATATCCTTAAACATAAGGTTGCTTGCATTGTTGTCACTCTGAGTAAGAGTATAACGCAGCAAATCTCTCACTGTCAATGATATGACTGGCCCTGAATAATCTTTCAGCATAGGACTCCAAGTCTTTGGGTCAAGTTTATCCCTATTTATATTTACTAAGGTATCAAGTGAAATTCCTTTATTGTCAAAGTCATTACAAAGAGCTAATGCCTGATGAACCTTAAACACACTCATCATAGGATAAACACTCTTATTATTGACCTTAACCGTATCTCTGTTATTAACAATAACCGCCACACCAATTTCGCCAGGACAAGCTGAGACAATTTGAGAAATGCTATCAGTCAAAACATTTGTTAAAGGAGGATTTGCGCTATCTTTTGTCGCTGATTTATGGAACAATGAAAATACCAAGATGAAAATGCAAACTAAAGCTATACTCAAAACTACGATTTGTTTTTTTCTGTTTTTTTCCATGTTTtATATTATTTATATTTGTTTGACGAGAATATCTTTATTTGCCGACAAAGGTACATAACTAAAGTTTCCCACCCAAATAAATAGATAGAAAAATAACAGTTTGTCGAATTTTCTTTGTAAATTAGTAATCGCTAAAGAACTGATTTTGGGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAaGAGTATGAGTATTCaACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAaGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGA。AAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTCGCCTGATGCGGTATTTTCTCCTTACGCATATCGACATCCGCCCTCACCGCCAGGAACGCAACCGCAGCCTCATCACGCCGGCGCTTCTTGGCCGCGCGGGATTCAACCCACTCGGCCAGCTCGTCGGTGTAGCTCTTTGGCATCGTCTCTCGCCTGTCCCCTCAGTTCAGTAATTTCCTGCATTTGCCTGTTTCCAGTCGGTAGATATTCCACAAAACAGCAGGGAAGCAGCGCTTTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGGGTTCGTGTAGACTTTCCTTGGTGTATCCAACGGCGTCAGCCGGGCAGGATAGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCCGGCTACCGCCGGCGTAACAGATGAGGGCAAGCGGATGGCTGATGAAACCAAGCCAACCAGGAAGGGCAGCCCACCTATCAAGGTGTACTGCCTTCCAGACG AACGAAGAGCGATTGAGGAAAAGGCGGCGGCGGCCGGCATGAGCCTGTCGGCCTACCTGCTGGCCGTCGGCCAGGGCTACAAAATCACGGGCGTCGTGGACTATGAGCACGTCCGCGAGCTGGCCCGCATCAATGGCGACCTGGGCCGCCTGGGCGGCCTGCTGAAACTCTGGCTCACCGACGACCCGCGCACGGCGCGGTTCGGTGATGCCACGATCCTCGCCCTGCTGGCGAAGATCGACTCTAGGCATGCCTATTTAGGCATTAGCCCTCTTTTTAACCTGACTTAAAAAATAATTAAATCATAAGATATTAAATATCAAGTGATTATGATTTACTATGCTATACATAATATCACCCAGTGTTAGAAAATTCTTGCTATGCTTTTTGTTTGTTTTTTTTCATTCGTGAACTTTTAGAATACTTTTAAAATAAACTCAAATTTATAAACTATTGATTTTCAATATTATACAAAACCAAACTTATTAACATTAAACTTATTTCTATTACCTTTAAAATAAACTACTAAAAGACAAATCTACCAATCCTCGGAACTATTTAATAAATAGGAAGTTATATAAAAATAATGAACTTTTAGAATACTTTATTAGTCTTTTTAGCTCCAAAAATGAACTTTTAGAATACTTTTTTTGAACTTTTAGAATACTTTTTTAAACTTGCTTTTTAGTAAGTTCATTTTTTATATATTTGTATTCTAAAAGTGCATTTTATATTCATAACTCTCTATATATGGCAAAAGTAGATAATAAACTAATGTTTCAGTCCTATATTTTTACTACGGCTAAATATGATTTTTCAGTCTATGAAAAGCGTATACTATATAGGCAAATAGAAATAGAACAGGCATTGCTAAATAATGAGGCGTTGAAAGATTGTATCAAAATAGATACTAACCTATGGGGGGATAAAAAATATACAATTCCAATTTCTATGCTGTTAGCTAATGATGAAGATAAAAATTATTATAAAATTAAAAAAG CATTCAAAGACCTAAAATCAAAGAACATTGAGTACGAAGATGAAAAAGAATACGCTTGCTTTGGTGTTATTGATAAGTTTTGGATTGATAAATATGGAAGAAATGTTACTTGGCAATCTGACAAAAGAATTGTTGAAGCGGTAATGGATTTTACAAGAGGATGGAGAAAATATGAATTGAAGATTGCTATGCAGTTTGAGACTATTTATGCAATGCGTTTTTACGAACTGATAGCTAATAAAACCAAACCAATAACATATGAAGTGTCATTTCTTGAAAAAATGTTTCAATTGGAAAATAAGTACAGGAACCCTCAGGGTAATCTTAATATCAGTATGTTTAAACGCCGTGTATTAGACCCCGCTAAAAACGAGTTAGATAAATGTTCTCCATACACTTTTGATTATACTCTTTCAAAGGATAAAAAAATTATTTCTCTAATTCCTATTTTCCAAGAACAATTTGCAGATGAAAGTATGAAGATGATCAAAATAGAAGAGGAAAAAGATATACACTCTGTATTAACTCAAAGAGAAATTGATGTTTTtACAAACGAATTTGGTTTTACACAACAAGGTTTATTAAACAACTATGCCCTATTCAAAGATTGTAAGGCTACACTTCCATCAAACTATTCTTTCTTCCTTTTTCAAGAGATAAGAAAAAGTCGGACTAAGAAAGGAAAAATATCTCCTGCTTATGTGATTGGGATTATAAGAAATATGCTTAAAGATTTCAAAAAAGAACAACAATAGGCTTATGAAAAAATATACAACAAAAGAAATCGCAGAGCTTTTTGGCGTCAGCGAACGGACAATCCAGCGACACATAACGACACTAAGCGACAACATATCAAATACCGACAGAAAGGGCTTTTCAATACCAGAAGACATTGTTTTCTTTCTTGCCGAGCGACACCATTACGACATTTTAGCGACAACGAACGACAACGAACGACAGGATAAAAATGAAGAATTTCCTCATATTGAATACTTCACAG AAGAGGAATACCAAGAGTTTAAAAAAAGGATCACAGAGTACCCTTTTTTGAAAGAGCAAATTAAACTGTCTAATGAACATTTGGATACTCTAAAAACACAAATAGAATATTTCAGACTATCTTACAATAAGCAATTGGATATTCACGAAAAATTAATCGGAGCGTTCCGAGAAAGAAACTTTATTGAAGCTAAAGAAAAGGGGATGGATAAATAGCTCCTAATAATTAAAGAAGTATTAGAAAAAAAATGCTATATAAAAACTTAATCGAGCTCTATACGAGATCTGTTCCAATGCATTGGCTGCAGGTCGACGTCGGCCATATTTCAAAAATTTAACTTAAACCACTGATTTACAAAAATGTTTATTTGCAAAACTATTTTACAAAGATATTACAATAATTTTTCTAGCACAAATTTATTTCTCACCATATCTTTGCCTTGTTCAAGTGAGTGAACAGAGTAACAAACAAAAAAAGAAACAAATTTAAAAAAATTATTTAAAATTCCGCGGGCTAGCAGGCCTGCGCGCTGCTCTAGAACTCGAGCATCATCATCATCATCATTGAACTAGTGGAGTTGCTTCTCTTTTCGGGAGTGGAAAACTGAAAGAACTGGAACGTGCCAACGAAAAACTGCAAGACGAGGTTTCAAAACGGAACACCAATATTGAAAAATTGCAGAGCCAAGTACAGCAGATGCAGAAACAGCATGATACGCAAATCCACAATCTCAGAGAAATGCACAGGCAGGAACTTGACATGAAAGAAAAAGAACTGTCACGGCTCGCCAGAATCATAGACAAGGCTTTCAGGTGGTTTCCGATGTTCAGGGAAATGCTGCGCATGGAAAAGTTTTGTGCCATGCTGGGATTCTCTAAAGAAATGACTGAAAGTCTTATAGTCAAAAAAGAAGCCCTGAAATGTAGCGGTAAAATCTATTCCGAGCAACACAGGCGGAACTTTGATATAAAGGATGATATTTTAAGGGTGGAAAATGACC CTGACGATGAAAGCAGGCTGAACCTGACAATAAACAGGAAGCCGATTGCCGACTGGTTCAGGGAGCAATGGCACAGGCTTAGATATGGAGCAAGAGTGCCGCAACAGGAAGAAAGAAAAAGTAGAGGATTCAAATTATAATAGAAGCAATTTGATTAGTAATCTAAAAGCACTCCGATAACGATTAGAGTGCTTTTAGATTGTTTATCATTAATTATCAAAGCAAGTGCAGTTTAAGATTTTACTGAAGTTTGCATTAATAAAGAATATACTACAGCTGATATATGCGCAACATATTGTGACGCTTGTGATTTATTTCCCTTGAAATCCTTAACAAATACCGCTAAGGTATAACTGATATTATTAGGCAGACATATATAGGCAACATCATTGTGAGCTGCAAGAACACCATTTTCATTAACATAACCTGAACCTGTCTTATGCGCTATAACAACCCCTTCTTTATCAAGAAGTGGAGCTGCTATCCTATCTACACCTGTTTTGCATTCTTTTAACGTATTCTTAATGAAACTTTGTTTCTCATCATCGATAAGACCTTCAGTAAACAAACGATTCATCAACATTGCAGCACCAAGAGGAGATGTATAGTTAGAGTAAGCCTTGTTATGGTCAGCCGACATTTCCTCTTCCGTATAAGCTATCTGAAAACTTGAACGAGGAATGAGTGTGGCTATAAAACTATCTGTTTGAGCGACATTAACCATATCCTTAAACATAAGGTTGCTTGCATTGTTGTCACTCTGAGTAAGAGTATAACGCAGCAAATCTCTCACTGTCAATGATATGACTGGCCCTGAATAATCTTTCAGCATAGGACTCCAAGTCTTTGGGTCAAGTTTATCCCTATTTATATTTACTAAGGTATCAAGTGAAATTCCTTTATTGTCAAAGTCATTACAAAGAGCTAATGCCTGATGAACCTTAAACACACTCATCATAGGATAAACACTCTTATTATTGACCTTAACCGTATCTCTGTT ATTAACAATAACCGCCACACCAATTTCGCCAGGACAAGCTGAGACAATTTGAGAAATGCTATCAGTCAAAACATTTGTTAAAGGAGGATTTGCGCTATCTTTTGTCGCTGATTTATGGAACAATGAAAATACCAAGATGAAAATGCAAACTAAAGCTATACTCAAAACTACGATTTGTTTTTTTCTGTTTTTTTCCATGTTTtATATTATTTATATTTGTTTGACGAGAATATCTTTATTTGCCGACAAAGGTACATAACTAAAGTTTCCCACCCAAATAAATAGATAGAAAAATAACAGTTTGTCGAATTTTCTTTGTAAATTAGTAATCGCTAAAGAACTGATTTTGGGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAaGAGTATGAGTATTCaACATTTCCGTGTCGCCC TTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAaGGATC TTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGA。
实施例4：Example 4:
验证pFY02的稳定性和高效性Verify the stability and efficiency of pFY02
首先制备E.coli S17.1感受态细胞，将质粒pFY02转化至E.coli S17.1中，用加有100μg/ml氨苄西林的LB固体培养基培养细菌，分离长出的单菌落，用PCR的方法鉴定出阳性克隆(鉴定oriT+pRA0726 ori片段，约2800bp)，结果如图7。First prepare E.coli S17.1 competent cells, transform the plasmid pFY02 into E.coli S17.1, culture the bacteria with LB solid medium added with 100 μg/ml ampicillin, isolate the grown single colony, and use PCR The method identified positive clones (identification oriT+pRA0726 ori fragment, about 2800bp), the results are shown in Figure 7.
申请人使用大肠杆菌(供体菌)—鸭疫里默氏杆菌(受体菌)结合转移的方法，将质粒pFY02导入鸭疫里默氏杆菌中，即将E.coli S17.1+pFY02和鸭疫里默氏杆菌RAATCC11845(BMC genomics.2014；15:479)分别用LB和TSB摇菌至对数生长期，用7000rmp离心10min，去掉上清，用10mM的MgSO4分别将菌重悬，再次用7000rmp离心10min，去掉上清，用10mM的MgSO4分别将菌重悬，然后按1OD鸭疫里默氏杆菌和5OD大肠杆菌的比例混合两种菌，用孔径为0.22μm的滤膜过滤细菌，将滤膜贴于血平板(加有脱纤维羊血的LB固体培养基)上，30℃培养10～12h，取出滤膜，用10ml的10mM的MgSO4洗下滤膜上的菌，取100μl菌液涂于加有50μg/ml卡那霉素和1μg/ml头孢西丁的血平板上，37℃培养过夜，第二天即可看到长出的单菌落。分离长出的单菌落，用PCR的方法鉴定(鉴定oriT+pRA0726 ori片段，约2800bp)，结果如图8。将鉴定为阳性的菌传十代，用PCR的方法鉴定质粒的稳定性，结果如图9。可以看出质粒能稳定的存在于鸭疫里默氏杆菌中。与普通的穿梭质粒pLMF01(Genbank:KU963002)相比，进行结合转移效率提高了很多(图10)。The applicant used the Escherichia coli (donor bacterium)-Rimerella anatipestifer (recipient bacterium) combined transfer method to introduce the plasmid pFY02 into Riemerella anatipestifer, that is, E.coli S17.1+pFY02 and duck R. epiphytes RAATCC11845 (BMC genomics.2014; 15:479) was shaken with LB and TSB to the logarithmic growth phase respectively, centrifuged at 7000rmp for 10min, removed the supernatant, resuspended the bacteria with 10mM MgSO4 , and again Centrifuge at 7000rmp for 10min, remove the supernatant, resuspend the bacteria with 10mM MgSO4 respectively, then mix the two bacteria according to the ratio of 1OD Riemerella anatipestifer and 5OD Escherichia coli, filter the bacteria with a filter membrane with a pore size of 0.22μm , paste the filter membrane on a blood plate (LB solid medium with defibrated sheep blood), incubate at 30°C for 10-12 hours, take out the filter membrane, wash down the bacteria on the filter membrane with 10ml of 10mM MgSO4 , take Apply 100 μl of bacterial solution on a blood plate added with 50 μg/ml kanamycin and 1 μg/ml cefoxitin, incubate overnight at 37°C, and a single colony can be seen the next day. The grown single colonies were isolated and identified by PCR (identification of oriT+pRA0726 ori fragment, about 2800 bp), the results are shown in Figure 8. The bacteria identified as positive were propagated for ten generations, and the stability of the plasmid was identified by the PCR method. The results are shown in Figure 9. It can be seen that the plasmid can stably exist in Riemerella anatipestifer. Compared with the common shuttle plasmid pLMF01 (Genbank: KU963002), the combined transfer efficiency is much improved (Fig. 10).
质粒pFY02可以用于缺失基因的功能回补验证Plasmid pFY02 can be used for functional complementation verification of deleted genes
我们选择鸭疫里默氏杆菌tonB2缺失株(PloS one 10,e0127506,(2015)来验证穿梭质粒pFY02的基因回补能力。根据鸭疫里默氏杆菌tonB2基因序列(NCBI中的序号为RA0C_1209)设计两条PCR引物P1和P2，上游引物P1：AAGGCCTTATGTCAGATGAAAATTTAGG，如SEQ IDNO.8所示；下游引物P2：GCTCTAGAGCTTAATACTCAAAATTCATTGCCAC，如SEQ ID NO.9所示。以RAATCC11845(BMC genomics.2014；15:479)菌株为模板，P1和P2为引物用PCR扩增tonB2基因序列，扩增程序为：98℃30S，(98℃10S，55℃30S，72℃50S)×30个循环，72℃7min，22℃∞。用DNA纯化回收试剂盒回收PCR产物tonB2，用限制性内切酶StuI和XbaI酶切质粒pFY02和tonB2片段，用DNA纯化回收试剂盒回收长片段酶切产物。用DNA连接试剂盒连接载体片段pFY02与基因片段tonB2，并制备E.coli DH5α感受态细胞，将连接产物转化至E.coli DH5α，用加有100μg/ml氨苄西林的LB固体培养基培养细菌，分离长出的单菌落，用PCR的方法鉴定出阳性克隆(鉴定tonB2片段，约850bp)，结果如图14。用LB液体培养基扩大培养阳性克隆菌株，用质粒小提试剂盒提取质粒，即pFY02::tonB2构建成功。We selected the Riemerella anatipestifer tonB2 deletion strain (PloS one 10, e0127506, (2015)) to verify the gene complementation ability of the shuttle plasmid pFY02. According to the Riemerella anatipestifer tonB2 gene sequence (the sequence number in NCBI is RA0C_1209) Design two PCR primers P1 and P2, upstream primer P1: AAGGCCTTATGTCAGATGAAAATTTAGG, as shown in SEQ ID NO.8; downstream primer P2: GCTCTAGAGCTTAATACTCAAAATTCATTGCCAC, as shown in SEQ ID NO.9. RAATCC11845 (BMC genomics.2014; 15:479) The strain is used as a template, P1 and P2 are used as primers to amplify the tonB2 gene sequence by PCR, the amplification program is: 98°C 30S, (98°C 10S, 55°C 30S, 72°C 50S) × 30 cycles, 72°C 7min, 22°C ∞. Recover the PCR product tonB2 with the DNA purification and recovery kit, digest the plasmid pFY02 and tonB2 fragments with restriction enzymes StuI and XbaI, and recover the long fragment digestion product with the DNA purification and recovery kit. Connect the vector with the DNA ligation kit Fragment pFY02 and gene fragment tonB2, and prepare E.coli DH5α competent cells, transform the ligation product into E.coli DH5α, use LB solid medium with 100 μg/ml ampicillin to culture the bacteria, and isolate the grown single colony, Positive clones were identified by PCR (identification of the tonB2 fragment, about 850bp), and the results are shown in Figure 14. The positive clone strain was expanded and cultured with LB liquid medium, and the plasmid was extracted with a plasmid mini-extraction kit, that is, pFY02::tonB2 was successfully constructed.
将构建好的质粒pFY02::tonB2用结合转移的方法(如上所述)导入RA ATCC tonB2缺失株中，分离长出的单菌落，用PCR的方法鉴定(鉴定tonB2片段，约850bp)，结果如图15。即重组质粒pFY02::tonB2成功转移至RA ATCC tonB2缺失株中。The constructed plasmid pFY02::tonB2 was introduced into the RA ATCC tonB2 deletion strain by the combined transfer method (as described above), and the grown single colonies were isolated and identified by PCR (identification of the tonB2 fragment, about 850bp), and the results were as follows Figure 15. That is, the recombinant plasmid pFY02::tonB2 was successfully transferred to the RA ATCC tonB2 deletion strain.
在3个均加有20mLTSB的离心管中分别加入过夜培养的RA ATCC野生株、tonB2缺失株及tonB2回补株，并控制初始OD为0.1，放入37℃摇床培养，每隔2个小时测一次OD，结果如图16所示。结果显示，与野生株相比，缺失株生长明显受损，然而回补株可以恢复缺失株的生长。Add RA ATCC wild strain, tonB2 deletion strain and tonB2 replenishment strain cultivated overnight to three centrifuge tubes each with 20mL of TSB, and control the initial OD to 0.1, put them into a shaker at 37°C for culture, every 2 hours Measure the OD once, and the result is shown in Figure 16. The results showed that compared with the wild strain, the growth of the deletion strain was significantly impaired, but the complementation strain could restore the growth of the deletion strain.
以上实验可证明穿梭质粒可以用于鸭疫里默氏杆菌基因敲除株的回补。The above experiments can prove that the shuttle plasmid can be used for the complementation of the Riemerella anatipestifer gene knockout strain.
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 四川农业大学<110> Sichuan Agricultural University
<120> 一种用于大肠杆菌-鸭疫里默氏杆菌的高效穿梭质粒<120> A high-efficiency shuttle plasmid for Escherichia coli-Rimerella anatipestifer
<130><130>
<160> 9<160> 9
<170> PatentIn version 3.3<170> PatentIn version 3.3
<210> 1<210> 1
<211> 7590<211> 7590
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> pFY02<223>pFY02
<400> 1<400> 1
aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 60aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 60
tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 120tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 120
ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 180ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 180
aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 240aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 240
ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 300ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 300
agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 360agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 360
gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc 420gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc 420
ttcgcctgat gcggtatttt ctccttacgc atatcgacat ccgccctcac cgccaggaac 480ttcgcctgat gcggtatttt ctccttacgc atatcgacat ccgccctcac cgccaggaac 480
gcaaccgcag cctcatcacg ccggcgcttc ttggccgcgc gggattcaac ccactcggcc 540gcaaccgcag cctcatcacg ccggcgcttc ttggccgcgc gggattcaac ccactcggcc 540
agctcgtcgg tgtagctctt tggcatcgtc tctcgcctgt cccctcagtt cagtaatttc 600agctcgtcgg tgtagctctt tggcatcgtc tctcgcctgt cccctcagtt cagtaatttc 600
ctgcatttgc ctgtttccag tcggtagata ttccacaaaa cagcagggaa gcagcgcttt 660ctgcatttgc ctgtttccag tcggtagata ttccacaaaa cagcagggaa gcagcgcttt 660
tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt 720tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt 720
ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa 780ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa 780
cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc 840cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc 840
actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg 900actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg 900
gctaccgccg gcgtaacaga tgagggcaag cggatggctg atgaaaccaa gccaaccagg 960gctaccgccg gcgtaacaga tgagggcaag cggatggctg atgaaaccaa gccaaccagg 960
aagggcagcc cacctatcaa ggtgtactgc cttccagacg aacgaagagc gattgaggaa 1020aagggcagcc cacctatcaa ggtgtactgc cttccagacg aacgaagagc gattgaggaa 1020
aaggcggcgg cggccggcat gagcctgtcg gcctacctgc tggccgtcgg ccagggctac 1080aaggcggcgg cggccggcat gagcctgtcg gcctacctgc tggccgtcgg ccagggctac 1080
aaaatcacgg gcgtcgtgga ctatgagcac gtccgcgagc tggcccgcat caatggcgac 1140aaaatcacgg gcgtcgtgga ctatgagcac gtccgcgagc tggcccgcat caatggcgac 1140
ctgggccgcc tgggcggcct gctgaaactc tggctcaccg acgacccgcg cacggcgcgg 1200ctgggccgcc tgggcggcct gctgaaactc tggctcaccg acgacccgcg cacggcgcgg 1200
ttcggtgatg ccacgatcct cgccctgctg gcgaagatcg actctaggca tgcctattta 1260ttcggtgatg ccacgatcct cgccctgctg gcgaagatcg actctaggca tgcctattta 1260
ggcattagcc ctctttttaa cctgacttaa aaaataatta aatcataaga tattaaatat 1320ggcattagcc ctctttttaa cctgacttaa aaaataatta aatcataaga tattaaatat 1320
caagtgatta tgatttacta tgctatacat aatatcaccc agtgttagaa aattcttgct 1380caagtgatta tgattacta tgctatacat aatatcaccc agtgttagaa aattcttgct 1380
atgctttttg tttgtttttt ttcattcgtg aacttttaga atacttttaa aataaactca 1440atgctttttg tttgttttttttcattcgtg aacttttaga atacttttaa aataaactca 1440
aatttataaa ctattgattt tcaatattat acaaaaccaa acttattaac attaaactta 1500aatttataaa ctattgattt tcaatattat acaaaaccaa acttattaac attaaactta 1500
tttctattac ctttaaaata aactactaaa agacaaatct accaatcctc ggaactattt 1560tttctattac ctttaaaata aactactaaa agacaaatct accaatcctc ggaactattt 1560
aataaatagg aagttatata aaaataatga acttttagaa tactttatta gtctttttag 1620aataaatagg aagttatata aaaataatga acttttagaa tactttatta gtctttttag 1620
ctccaaaaat gaacttttag aatacttttt ttgaactttt agaatacttt tttaaacttg 1680ctccaaaaat gaacttttag aatacttttt ttgaactttt agaatacttt tttaaacttg 1680
ctttttagta agttcatttt ttatatattt gtattctaaa agtgcatttt atattcataa 1740ctttttagta agttcatttt ttatatattt gtattctaaa agtgcatttt atattcataa 1740
ctctctatat atggcaaaag tagataataa actaatgttt cagtcctata tttttactac 1800ctctctatat atggcaaaag tagataataa actaatgttt cagtcctata tttttactac 1800
ggctaaatat gatttttcag tctatgaaaa gcgtatacta tataggcaaa tagaaataga 1860ggctaaatat gatttttcag tctatgaaaa gcgtatacta tataggcaaa tagaaataga 1860
acaggcattg ctaaataatg aggcgttgaa agattgtatc aaaatagata ctaacctatg 1920acaggcattg ctaaataatg aggcgttgaa agattgtatc aaaatagata ctaacctatg 1920
gggggataaa aaatatacaa ttccaatttc tatgctgtta gctaatgatg aagataaaaa 1980gggggataaa aaatatacaa ttccaatttc tatgctgtta gctaatgatg aagataaaaa 1980
ttattataaa attaaaaaag cattcaaaga cctaaaatca aagaacattg agtacgaaga 2040ttattataaa attaaaaaag cattcaaaga cctaaaatca aagaacattg agtacgaaga 2040
tgaaaaagaa tacgcttgct ttggtgttat tgataagttt tggattgata aatatggaag 2100tgaaaaagaa tacgcttgct ttggtgttat tgataagttt tggattgata aatatggaag 2100
aaatgttact tggcaatctg acaaaagaat tgttgaagcg gtaatggatt ttacaagagg 2160aaatgttact tggcaatctg acaaaagaat tgttgaagcg gtaatggatt ttacaagagg 2160
atggagaaaa tatgaattga agattgctat gcagtttgag actatttatg caatgcgttt 2220atggagaaaa tatgaattga agattgctat gcagtttgag actatttatg caatgcgttt 2220
ttacgaactg atagctaata aaaccaaacc aataacatat gaagtgtcat ttcttgaaaa 2280ttacgaactg atagctaata aaaccaaacc aataacatat gaagtgtcat ttcttgaaaa 2280
aatgtttcaa ttggaaaata agtacaggaa ccctcagggt aatcttaata tcagtatgtt 2340aatgtttcaa ttggaaaata agtacaggaa ccctcagggt aatcttaata tcagtatgtt 2340
taaacgccgt gtattagacc ccgctaaaaa cgagttagat aaatgttctc catacacttt 2400taaacgccgt gtattagacc ccgctaaaaa cgagttagat aaatgttctc catacacttt 2400
tgattatact ctttcaaagg ataaaaaaat tatttctcta attcctattt tccaagaaca 2460tgattatact ctttcaaagg ataaaaaaat tatttctcta attcctattt tccaagaaca 2460
atttgcagat gaaagtatga agatgatcaa aatagaagag gaaaaagata tacactctgt 2520atttgcagat gaaagtatga agatgatcaa aatagaagag gaaaaagata tacactctgt 2520
attaactcaa agagaaattg atgtttttac aaacgaattt ggttttacac aacaaggttt 2580attaactcaa agagaaattg atgtttttac aaacgaattt ggttttacac aacaaggttt 2580
attaaacaac tatgccctat tcaaagattg taaggctaca cttccatcaa actattcttt 2640attaaacaac tatgccctat tcaaagattg taaggctaca cttccatcaa actattcttt 2640
cttccttttt caagagataa gaaaaagtcg gactaagaaa ggaaaaatat ctcctgctta 2700cttccttttt caagagataa gaaaaagtcg gactaagaaa ggaaaaatat ctcctgctta 2700
tgtgattggg attataagaa atatgcttaa agatttcaaa aaagaacaac aataggctta 2760tgtgattggg attataagaa atatgcttaa agatttcaaa aaagaacaac aataggctta 2760
tgaaaaaata tacaacaaaa gaaatcgcag agctttttgg cgtcagcgaa cggacaatcc 2820tgaaaaaata tacaacaaaa gaaatcgcag agctttttgg cgtcagcgaa cggacaatcc 2820
agcgacacat aacgacacta agcgacaaca tatcaaatac cgacagaaag ggcttttcaa 2880agcgacacat aacgacacta agcgacaaca tatcaaatac cgacagaaag ggcttttcaa 2880
taccagaaga cattgttttc tttcttgccg agcgacacca ttacgacatt ttagcgacaa 2940taccagaaga cattgttttc tttcttgccg agcgacacca ttacgacatt ttagcgacaa 2940
cgaacgacaa cgaacgacag gataaaaatg aagaatttcc tcatattgaa tacttcacag 3000cgaacgacaa cgaacgacag gataaaaatg aagaatttcc tcatattgaa tacttcacag 3000
aagaggaata ccaagagttt aaaaaaagga tcacagagta cccttttttg aaagagcaaa 3060aagaggaata ccaagagttt aaaaaaagga tcacagagta cccttttttg aaagagcaaa 3060
ttaaactgtc taatgaacat ttggatactc taaaaacaca aatagaatat ttcagactat 3120ttaaactgtc taatgaacat ttggatactc taaaaacaca aatagaatat ttcagactat 3120
cttacaataa gcaattggat attcacgaaa aattaatcgg agcgttccga gaaagaaact 3180cttacaataa gcaattggat attcacgaaa aattaatcgg agcgttccga gaaagaaact 3180
ttattgaagc taaagaaaag gggatggata aatagctcct aataattaaa gaagtattag 3240ttattgaagc taaagaaaag gggatggata aatagctcct aataattaaa gaagtattag 3240
aaaaaaaatg ctatataaaa acttaatcga gctctatacg agatctgttc caatgcattg 3300aaaaaaaatg ctatataaaa acttaatcga gctctatacg agatctgttc caatgcattg 3300
gctgcaggtc gacgtcggcc atatttcaaa aatttaactt aaaccactga tttacaaaaa 3360gctgcaggtc gacgtcggcc atatttcaaa aatttaactt aaaccactga tttacaaaaa 3360
tgtttatttg caaaactatt ttacaaagat attacaataa tttttctagc acaaatttat 3420tgtttatttg caaaactatt ttacaaagat attacaataa tttttctagc acaaatttat 3420
ttctcaccat atctttgcct tgttcaagtg agtgaacaga gtaacaaaca aaaaaagaaa 3480ttctcaccat atctttgcct tgttcaagtg agtgaacaga gtaacaaaca aaaaaagaaa 3480
caaatttaaa aaaattattt aaaattccgc gggctagcag gcctgcgcgc tgctctagaa 3540caaatttaaa aaaattattt aaaattccgc gggctagcag gcctgcgcgc tgctctagaa 3540
ctcgagcatc atcatcatca tcattgaact agtggagttg cttctctttt cgggagtgga 3600ctcgagcatc atcatcatca tcattgaact agtggagttg cttctctttt cgggagtgga 3600
aaactgaaag aactggaacg tgccaacgaa aaactgcaag acgaggtttc aaaacggaac 3660aaactgaaag aactggaacg tgccaacgaa aaactgcaag acgaggtttc aaaacggaac 3660
accaatattg aaaaattgca gagccaagta cagcagatgc agaaacagca tgatacgcaa 3720accaatattg aaaaattgca gagccaagta cagcagatgc agaaacagca tgatacgcaa 3720
atccacaatc tcagagaaat gcacaggcag gaacttgaca tgaaagaaaa agaactgtca 3780atccacaatc tcagagaaat gcacaggcag gaacttgaca tgaaagaaaa agaactgtca 3780
cggctcgcca gaatcataga caaggctttc aggtggtttc cgatgttcag ggaaatgctg 3840cggctcgcca gaatcataga caaggctttc aggtggtttc cgatgttcag ggaaatgctg 3840
cgcatggaaa agttttgtgc catgctggga ttctctaaag aaatgactga aagtcttata 3900cgcatggaaa agttttgtgc catgctggga ttctctaaag aaatgactga aagtcttata 3900
gtcaaaaaag aagccctgaa atgtagcggt aaaatctatt ccgagcaaca caggcggaac 3960gtcaaaaaag aagccctgaa atgtagcggt aaaatctatt ccgagcaaca caggcggaac 3960
tttgatataa aggatgatat tttaagggtg gaaaatgacc ctgacgatga aagcaggctg 4020tttgatataa aggatgatat tttaagggtg gaaaatgacc ctgacgatga aagcaggctg 4020
aacctgacaa taaacaggaa gccgattgcc gactggttca gggagcaatg gcacaggctt 4080aacctgacaa taaacaggaa gccgattgcc gactggttca gggagcaatg gcacaggctt 4080
agatatggag caagagtgcc gcaacaggaa gaaagaaaaa gtagaggatt caaattataa 4140agatatggag caagagtgcc gcaacaggaa gaaagaaaaa gtagaggatt caaattataa 4140
tagaagcaat ttgattagta atctaaaagc actccgataa cgattagagt gcttttagat 4200tagaagcaat ttgattagta atctaaaagc actccgataa cgattagagt gcttttagat 4200
tgtttatcat taattatcaa agcaagtgca gtttaagatt ttactgaagt ttgcattaat 4260tgtttatcat taattatcaa agcaagtgca gtttaagatt ttactgaagt ttgcattaat 4260
aaagaatata ctacagctga tatatgcgca acatattgtg acgcttgtga tttatttccc 4320aaagaatata ctacagctga tatatgcgca acatattgtg acgcttgtga tttatttccc 4320
ttgaaatcct taacaaatac cgctaaggta taactgatat tattaggcag acatatatag 4380ttgaaatcct taacaaatac cgctaaggta taactgatat tattaggcag acatatatag 4380
gcaacatcat tgtgagctgc aagaacacca ttttcattaa cataacctga acctgtctta 4440gcaacatcat tgtgagctgc aagaacacca ttttcattaa cataacctga acctgtctta 4440
tgcgctataa caaccccttc tttatcaaga agtggagctg ctatcctatc tacacctgtt 4500tgcgctataa caaccccttc tttatcaaga agtggagctg ctatcctatc taacccctgtt 4500
ttgcattctt ttaacgtatt cttaatgaaa ctttgtttct catcatcgat aagaccttca 4560ttgcattctt ttaacgtatt cttaatgaaa ctttgtttct catcatcgat aagaccttca 4560
gtaaacaaac gattcatcaa cattgcagca ccaagaggag atgtatagtt agagtaagcc 4620gtaaacaaac gattcatcaa cattgcagca ccaagaggag atgtatagtt agagtaagcc 4620
ttgttatggt cagccgacat ttcctcttcc gtataagcta tctgaaaact tgaacgagga 4680ttgttatggt cagccgacat ttcctcttcc gtataagcta tctgaaaact tgaacgagga 4680
atgagtgtgg ctataaaact atctgtttga gcgacattaa ccatatcctt aaacataagg 4740atgagtgtgg ctataaaact atctgtttga gcgacattaa ccatatcctt aaacataagg 4740
ttgcttgcat tgttgtcact ctgagtaaga gtataacgca gcaaatctct cactgtcaat 4800ttgcttgcat tgttgtcact ctgagtaaga gtataacgca gcaaatctct cactgtcaat 4800
gatatgactg gccctgaata atctttcagc ataggactcc aagtctttgg gtcaagttta 4860gatatgactg gccctgaata atctttcagc ataggactcc aagtctttgg gtcaagttta 4860
tccctattta tatttactaa ggtatcaagt gaaattcctt tattgtcaaa gtcattacaa 4920tccctattta tatttactaa ggtatcaagt gaaattcctt tattgtcaaa gtcattacaa 4920
agagctaatg cctgatgaac cttaaacaca ctcatcatag gataaacact cttattattg 4980agagctaatg cctgatgaac cttaaacaca ctcatcatag gataaacact cttattattg 4980
accttaaccg tatctctgtt attaacaata accgccacac caatttcgcc aggacaagct 5040accttaaccg tatctctgtt attaacaata accgccaacac caatttcgcc aggacaagct 5040
gagacaattt gagaaatgct atcagtcaaa acatttgtta aaggaggatt tgcgctatct 5100gagacaattt gagaaatgct atcagtcaaa atatttgtta aaggaggatt tgcgctatct 5100
tttgtcgctg atttatggaa caatgaaaat accaagatga aaatgcaaac taaagctata 5160tttgtcgctg atttatggaa caatgaaaat accaagatga aaatgcaaac taaagctata 5160
ctcaaaacta cgatttgttt ttttctgttt ttttccatgt tttatattat ttatatttgt 5220ctcaaaacta cgatttgttt ttttctgttt ttttccatgt tttatattat ttatatttgt 5220
ttgacgagaa tatctttatt tgccgacaaa ggtacataac taaagtttcc cacccaaata 5280ttgacgagaa tatctttattgccgacaaa ggtacataac taaagtttcc cacccaaata 5280
aatagataga aaaataacag tttgtcgaat tttctttgta aattagtaat cgctaaagaa 5340aatagataga aaaataacag tttgtcgaat tttctttgta aattagtaat cgctaaagaa 5340
ctgattttgg ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc 5400ctgattttgg ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc 5400
gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 5460gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 5460
ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 5520ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 5520
tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 5580tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 5580
accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 5640accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 5640
gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 5700gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 5700
acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 5760acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 5760
cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 5820cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 5820
taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 5880taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaacccccta 5880
tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 5940tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 5940
aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 6000aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 6000
ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 6060ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 6060
aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 6120aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 6120
acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 6180acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 6180
ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6240ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6240
gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 6300gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 6300
atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 6360atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 6360
acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 6420acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 6420
tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 6480tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 6480
ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 6540ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 6540
aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 6600aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 6600
aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 6660aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 6660
ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 6720ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 6720
atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 6780atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 6780
aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 6840aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 6840
accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 6900accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 6900
tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 6960tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 6960
tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 7020tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 7020
tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 7080tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 7080
cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 7140cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 7140
caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 7200caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 7200
cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 7260cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 7260
cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7320cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7320
gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 7380gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 7380
acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 7440acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 7440
atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 7500atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 7500
cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7560cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7560
gatgctcgtc aggggggcgg agcctatgga 7590gatgctcgtc aggggggcgg agcctatgga 7590
<210> 2<210> 2
<211> 31<211> 31
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> oriT上游引物<223> oriT upstream primer
<400> 2<400> 2
cccaagcttc gcctgatgcg gtattttctc c 31cccaagcttc gcctgatgcg gtattttctc c 31
<210> 3<210> 3
<211> 31<211> 31
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> oriT下游引物<223> oriT downstream primer
<400> 3<400> 3
acatgcatgc ctagagtcga tcttcgccag c 31acatgcatgc ctagagtcga tcttcgccag c 31
<210> 4<210> 4
<211> 30<211> 30
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> pRA0726 ori上游引物<223> pRA0726 ori upstream primer
<400> 4<400> 4
acatgcatgc ctatttaggc attagccctc 30acatgcatgc ctatttaggc attagccctc 30
<210> 5<210> 5
<211> 44<211> 44
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> pRA0726 ori下游引物<223> pRA0726 ori downstream primer
<400> 5<400> 5
aaaactgcag ccaatgcatt ggaacagatc tcgtatagag ctcg 44aaaactgcag ccaatgcatt ggaacagatc tcgtatagag ctcg 44
<210> 6<210> 6
<211> 42<211> 42
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> High EXP上游引物<223> High EXP upstream primer
<400> 6<400> 6
acgcgtcgac gtcggccata tttcaaaaat ttaacttaaa cc 42acgcgtcgac gtcggccata tttcaaaaat ttaacttaaa cc 42
<210> 7<210> 7
<211> 62<211> 62
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> High EXP下游引物<223> High EXP downstream primer
<400> 7<400> 7
tgctctagag cagcgcgcag gcctgctagc ccgcggaatt ttaaataatt tttttaaatt 60tgctctagag cagcgcgcag gcctgctagc ccgcggaatt ttaaataatt tttttaaatt 60
tg 62tg 62
<210> 8<210> 8
<211> 28<211> 28
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> tonB2上游引物<223> tonB2 upstream primer
<400> 8<400> 8
aaggccttat gtcagatgaa aatttagg 28aaggccttat gtcagatgaa aatttagg 28
<210> 9<210> 9
<211> 34<211> 34
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> tonB2下游引物<223> tonB2 downstream primer
<400> 9<400> 9
gctctagagc ttaatactca aaattcattg ccac 34gctctagagc ttaatactca aaattcattg ccac 34
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611038010.XACN106497960A (en) | 2016-11-23 | 2016-11-23 | A kind of efficient shuttle plasmid for Escherichia coli riemerella anatipestifer |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611038010.XACN106497960A (en) | 2016-11-23 | 2016-11-23 | A kind of efficient shuttle plasmid for Escherichia coli riemerella anatipestifer |
| Publication Number | Publication Date |
|---|---|
| CN106497960Atrue CN106497960A (en) | 2017-03-15 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611038010.XAPendingCN106497960A (en) | 2016-11-23 | 2016-11-23 | A kind of efficient shuttle plasmid for Escherichia coli riemerella anatipestifer |
| Country | Link |
|---|---|
| CN (1) | CN106497960A (en) |
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| CN106520818A (en)* | 2016-12-07 | 2017-03-22 | 四川农业大学 | Method for rapidly complementing missing gene of Riemerella anatipestifer |
| CN108841850A (en)* | 2018-04-23 | 2018-11-20 | 四川农业大学 | For the carrier and method in riemerella anatipestifer genome insertion foreign gene |
| CN108841849A (en)* | 2018-04-23 | 2018-11-20 | 四川农业大学 | Carrier and method for riemerella anatipestifer genome point mutation |
| CN116254284A (en)* | 2023-01-17 | 2023-06-13 | 华南农业大学 | pCasRA plasmid and preparation method and application thereof |
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| CN103361376A (en)* | 2012-04-09 | 2013-10-23 | 中国农业科学院上海兽医研究所 | Riemerella anatipestifer-escherichia coli shuttle expression vector as well as construction method and application thereof |
| CN105567721A (en)* | 2016-01-19 | 2016-05-11 | 四川农业大学 | Shuttle plasmid and construction method and application thereof |
| CN105755032A (en)* | 2016-04-12 | 2016-07-13 | 四川农业大学 | Method and application of shuttle plasmid pMY03 in interference of target gene expression of riemerella anatipestifer |
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| CN103361376A (en)* | 2012-04-09 | 2013-10-23 | 中国农业科学院上海兽医研究所 | Riemerella anatipestifer-escherichia coli shuttle expression vector as well as construction method and application thereof |
| CN105567721A (en)* | 2016-01-19 | 2016-05-11 | 四川农业大学 | Shuttle plasmid and construction method and application thereof |
| CN105755032A (en)* | 2016-04-12 | 2016-07-13 | 四川农业大学 | Method and application of shuttle plasmid pMY03 in interference of target gene expression of riemerella anatipestifer |
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| MANUELA ET AL: "Genetic tools for studying Capnocytophaga canimorsus", 《APPLIED AND ENVIRONMENT MICROBIOLOGY》* |
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| CN106520818A (en)* | 2016-12-07 | 2017-03-22 | 四川农业大学 | Method for rapidly complementing missing gene of Riemerella anatipestifer |
| CN108841850A (en)* | 2018-04-23 | 2018-11-20 | 四川农业大学 | For the carrier and method in riemerella anatipestifer genome insertion foreign gene |
| CN108841849A (en)* | 2018-04-23 | 2018-11-20 | 四川农业大学 | Carrier and method for riemerella anatipestifer genome point mutation |
| CN116254284A (en)* | 2023-01-17 | 2023-06-13 | 华南农业大学 | pCasRA plasmid and preparation method and application thereof |
| CN116254284B (en)* | 2023-01-17 | 2025-01-28 | 华南农业大学 | A pCasRA plasmid and its preparation method and application |
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| Date | Code | Title | Description |
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| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
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| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication | Application publication date:20170315 |