CN110055200A - 一株变形假单胞菌clpV基因沉默菌株 - Google Patents
一株变形假单胞菌clpV基因沉默菌株 Download PDFInfo
- Publication number
- CN110055200A CN110055200A CN201910013464.9A CN201910013464A CN110055200A CN 110055200 A CN110055200 A CN 110055200A CN 201910013464 A CN201910013464 A CN 201910013464A CN 110055200 A CN110055200 A CN 110055200A
- Authority
- CN
- China
- Prior art keywords
- clpv
- bacterial strain
- gene
- pseudomonad
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 33
- 230000030279 gene silencing Effects 0.000 title claims abstract description 25
- 238000012226 gene silencing method Methods 0.000 title claims abstract description 18
- 241000251468 Actinopterygii Species 0.000 claims abstract description 12
- 241000248484 Ichthyophthirius Species 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 210000001835 viscera Anatomy 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 238000005516 engineering process Methods 0.000 claims description 10
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 239000004055 small Interfering RNA Substances 0.000 claims description 7
- 238000010276 construction Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 238000013518 transcription Methods 0.000 claims description 4
- 230000035897 transcription Effects 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 230000005611 electricity Effects 0.000 claims description 2
- 238000012358 sourcing Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 2
- 238000011160 research Methods 0.000 abstract description 5
- 229940031567 attenuated vaccine Drugs 0.000 abstract description 3
- 230000008506 pathogenesis Effects 0.000 abstract description 3
- 230000002265 prevention Effects 0.000 abstract description 3
- 230000003247 decreasing effect Effects 0.000 abstract description 2
- 230000007918 pathogenicity Effects 0.000 abstract description 2
- 241000357444 Epinephelus coioides Species 0.000 description 10
- 208000015181 infectious disease Diseases 0.000 description 7
- 230000001018 virulence Effects 0.000 description 7
- 108091030071 RNAI Proteins 0.000 description 6
- 230000009368 gene silencing by RNA Effects 0.000 description 6
- 210000004185 liver Anatomy 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011304 droplet digital PCR Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000357439 Epinephelus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241001596950 Larimichthys crocea Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001223182 Pseudomonas plecoglossicida Species 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241001417495 Serranidae Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 101150013736 gyrB gene Proteins 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 238000003012 network analysis Methods 0.000 description 1
- 101150012629 parE gene Proteins 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- General Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Oncology (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明提供一株变形假单胞菌clpV基因沉默菌株，所述菌株为Pseudomonas plecoglossicida clpV‑RNAi，已于2018年11月12日于中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2018772。所构建的变形假单胞菌clpV基因稳定沉默菌株对海水鱼的致病力及显著下降，不仅可用于研究变形假单胞菌的致病机理，还可用于研制变形假单胞菌的弱毒疫苗并为海水鱼“内脏白点病”的预防和治疗寻找新的靶点。
Description
技术领域
本发明属于微生物技术领域，更具体涉及一株变形假单胞菌clpV基因沉默菌株。
背景技术
变形假单胞菌(Pseudomonas plecoglossicida)是大黄鱼、斜带石斑鱼等海水养殖鱼类“内脏白点病”的病原菌，每年造成的直接经济损失超亿元。
在先前的研究中，与野生型变形假单胞菌（在18℃下培养）的纯培养相比，在感染后24、48、72和96 小时，所有差异表达基因都被用于加权基因共表达网络分析并作为不同的基因共表达模块鉴定。所有模块均用KEGG富集分析。用这种方法，筛选出clpV基因被选择为 在VI 型分泌***（T6SS）中的一个候选关键毒力基因。
本发明通过比较转录组学分析技术，结合RNA干扰这一定向高效的基因沉默技术，构建一株变形假单胞菌clpV基因稳定沉默菌株，并对该菌株的毒力效果进行检测。所构建的变形假单胞菌clpV基因稳定沉默菌株对海水鱼的致病力及显著下降，不仅可用于研究变形假单胞菌的致病机理，还可用于研制变形假单胞菌的弱毒疫苗并为海水鱼“内脏白点病”的预防和治疗寻找新的靶点。
发明内容
本发明的目的在于提供一株变形假单胞菌clpV基因稳定沉默菌株及其功能。
为实现上述目的，本发明采用如下技术方案：
一株变形假单胞菌clpV基因沉默菌株，所述菌株为Pseudomonas plecoglossicidaclpV-RNAi，已于2018年11月12日于中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2018772，地址为武汉，武汉大学。
本发明提供的这株变形假单胞菌clpV基因稳定沉默菌株是通过下面的技术手段进行构建，
（1）通过比较转录组学分析技术，将筛选致病功能基因目标锁定clpV基因；
（2）合成shRNA，退火后连入pCM130/tac载体，通过电转技术导入变形假单胞菌感受态细胞，构建变形假单胞菌clpV基因稳定沉默菌株；利用QPCR技术，对沉默效果进行检验；最终获得变形假单胞菌clpV基因沉默菌株。所述的pCM130/tac载体是利用质粒pCM130加上启动子tac构成。
（3）利用人工感染实验，研究变形假单胞菌野生株、clpV基因沉默株的毒力，以及clpV基因沉默对变形假单胞菌及斜带石斑鱼基因表达的影响。
通过基因测序和比对，本发明提供的菌株为一株变形假单胞菌clpV基因稳定沉默菌株。
所述clpV基因序列如SEQ ID NO.1所示。
本发明的优点在于：
人工感染实验结果表明，变形假单胞菌clpV基因稳定沉默菌株的毒力显著低于变形假单胞菌野生株的毒力，在同等感染条件下不会引起斜带石斑鱼的死亡，具有研制减毒疫苗的潜力。
Dual RNA-seq分析结果表明，clpV基因的稳定沉默不仅能够显著影响变形假单胞菌的转录组表达，而且也能显著影响斜带石斑鱼的转录组表达，这说明变形假单胞菌clpV基因稳定沉默菌株可用于研究变形假单胞菌的致病机理，还可用于为海水鱼“内脏白点病”的预防和治疗寻找新的靶点。
附图说明
图1为斜带石斑鱼感染变形假单胞菌后的存活率图。
图2为斜带石斑鱼感染后脾脏组织的病症变化图。
图3为clpV基因沉默株和野生株在组织器官分布的差异倍数动态变化图。
具体实施方式
实施例1
本发明实施例提供的变形假单胞菌clpV基因稳定沉默菌株的构建方法包括以下步骤：
S101：通过比较转录组学分析技术，对变形假单胞菌基因表达水平进行检测；利用生物信息学技术，极大的缩小研究范围挑选出最具有研究价值的致病功能基因，用KEGG富集分析这种方法，筛选出clpV基因被选择为 在VI 型分泌***（T6SS）中的一个候选关键毒力基因。故将目标锁定clpV基因；
S102：利用Thermo-fisher Scientific 公司在线shRNA设计工具（http://rnaidesigner.thermofisher.com/rnaiexpress/setOption.dodesignOption=shrna&pid=708587103220684543），设计并合成shRNA，然后使用退火缓冲液退火形成双链shRNA。shRNA序列F:5’-TGCAGTGTTCAAACGCATTTCCTTCAAGAGAGGAAATGCGTTTGAACACTGCTTTTTTT-3’
R:5’-GTACAAAAAAAGCAGTGTTCAAACGCATTTCCTCTCTTGAAGGAAATGCGTTTGAACACTGCATGCA-3’，从百奥迈科公司购买原始质粒pCM130加上去的启动子tac，构成pCM130/tac质粒。买使用限制性内切酶BsrGI和 NsiI双酶切pCM130/tac质粒使其线性化，然后使用T4连接酶将线性化质粒和双链shRNA连接。将重组质粒先热击转化进大肠杆菌DH5α感受态细胞中，37℃扩大培养，测序成功后提取重组质粒，电击转化进变形假单胞菌感受态细胞中，18℃条件下扩大培养。此菌株即为变形假单胞菌clpV沉默突变株。所述变形假单胞菌clpV沉默突变株为Pseudomonas plecoglossicida clpV-RNAi，已于2018年11月12日于中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2018772，地址为武汉，武汉大学。
使用qRT-PCR技术，对沉默效果进行检验，上游引物序列（5’-3’’）为：CGAGGTCATTAAAGTTGCCCAATC，下游引物序列（5’-3’’）为：CGCCGAAACCGACATACCCT；检验结果与转录组测序结果一致，感染后clpV基因表达量上调；
S103：利用人工感染实验，对变形假单胞菌野生株和clpV基因沉默株的毒力进行对比。
clpV基因沉默株（clpV-RNAi株）、变形假单胞菌野生株和PBS（NaCl 0.8g、KCl0.02g、Na2HPO4 0.36g、 KH2PO4 0.024g、H2O 1L）分别对三组斜带石斑鱼进行胸腔注射感染，菌株感染浓度为103cfu/g，每条鱼注射0.2 mL，每组20尾鱼，然后继续正常暂养，并记录每天各组鱼的存活状况。
结果发现，对注射clpV-RNAi株和PBS的斜带石斑鱼，在注射后未观察到长达20 天都没观察到死亡。注射含有野生菌株的斜带石斑鱼在注射7 天内死亡(图1)。注射含有野生菌株的鱼的脾脏中观察到大量白色结节，表现出与自然疾病相同的症状。相反，在注射clpV-RNAi株和PBS的鱼中没有观察到白斑点(图2)。此外，所有接受clpV-RNAi菌株的斜带石斑鱼的脾脏、肾脏和肝脏都变得红肿，但在注射5-8天时未能形成可见的结节，肿胀逐渐消失。
变形假单胞菌的看家基因 gyrB 的 DNA 拷贝数被用来评估该病原菌在感染组织中的丰度。把 gyrB基因序列与 NCBI 数据库比对后，筛选出种间特异性高和种内变异率小的序列，使用 Primer Express 3.0 软件进行引物和探针的设计。为了便于与试剂盒配套使用，引物和探针由美国 Life Technologies 公司合成。
dPCR 引物和探针的序列
正向引物 5'-CAGACCTACGTGCACGGTGTT-3'
反向引物 5'-GGATTCACTGTCGCCCACTAC-3'
探针 5'-VIC-AGGCTCCAATGGC-MGB-3'
用dPCR方法检测感染了clpV-RNAi株在斜带石斑鱼的脾脏、肝脏、头肾、体肾和血液中的动态分布。图3显示了clpV-RNAi株和野生株分布的差异倍数动态变化。在感染后的大部分时间点，斜带石斑鱼中的脾脏和血液中的clpV-RNAi菌株丰度明显低于野生菌株。clpV-RNAi株在在感染后72到120小时头肾和体肾中的丰度表现为急剧下降。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 集美大学
<120> 一株变形假单胞菌clpV基因沉默菌株
<130> 8
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 2466
<212> DNA
<213> 人工序列
<400> 1
ttgctgaccg agtgggtgca ggatgcctgg ttgatcagct cgctggtttt gggctgtgga 60
cgtatccgcg gcggatcgct gctgttggcg ctggtgtcgc gcgcaagcta ctacaccgcc 120
ggtgctcgtt acagtgcagt gttcaaacgc atttccgtag acgcgttgac cgccgaattc 180
gccgcgctgg tggccagctc aagcgaaggg cccttggaca ttttgcacag tgcggccggg 240
gccgccaacc ctgtcgaggg cgctgtcgca gaaaaccaaa tcgcacgctt ttgcgaagac 300
cttaccgaga aggcccggca ggggaaaatc gaccccgtct tcgggcgcga tgacgaagtg 360
cgtcagatga tcgatattct cgcgcgtcgt cgcaagaaca accctattgc agtgggtgag 420
ccgggtgttg gcaagtcggc agtggtcgag ggcttggcgc tgttgatcaa tgagggcaac 480
gtgccggcgt ttctccaggg cacacgcctg ctcagcctgg atctgggggc acttgaggca 540
ggcgccagcg tcaagggtga gttcgaaaac cgtctgcgtg gtgtcattga ggagatcaag 600
gcctcgacca caccggtggt cttgtttatc gatgaagcac acatgttgat cggcgcaggc 660
ggggcggcgg gtggctcgga tgcggccaac ctgctcaagc ctgcgttggc ccgtggtgaa 720
ctgcgcacca tcgccgcaac aacctggtcg gaatataaaa agtatttcga gaaggacccg 780
gcgctggcca gacgctttca gctggtcaag ctggatgagc ccagtgtcgg cactgcagtg 840
cacattctgc gagggctcaa agggcattat gaaaaggtcc atggggtcag cgtgcgtgat 900
gatgccatcg ttgcggcggc tgagttgtcc gatcgataca tcaccggccg gttgctaccg 960
gacaaggcgg tggacctgct cgatacggcc agtgcccgcg tgcgcattgg ccttggtatc 1020
aaacctgcgc aactggaacg tctggagcga cagctttcgg gcctgatgcg tgaacgggcg 1080
gcgttggagc gggaccaggc cgacggcttc ccgatcgaac ctcagcgcct ggaaaccata 1140
gccgatgagc aagcgcacat tgagggggct gtggatgctc tggaggcgcg ctgggccaat 1200
gaacggcagg cggcgatgcg ggtgctggag gcgcgcacca acctcgcaac gctcaagcag 1260
gccggtgacg gcagcacagc gctgggctcg ctcgacgcac accagatgga gctcgaacag 1320
gctcgcgatg cactggcgac gttgcagggc gacgagcctt tgctctttac cgaggtcggt 1380
cccgacacga ttgcccaggt ggtctcggac tggacgggga taccgttagg taaggttcag 1440
cgtgatgcct ctgatggcct gctggcgctg gccagccacc tcaagcagcg tatctgcggc 1500
caggaagccg ccgtggagca gattgccgag gtcattaaag ttgcccaatc gggcttgcgt 1560
gaccctcagc agccgttggg ggtcttcttg ctggtcgggc cctcgggagt cggcaagacc 1620
gaaacagcgc tggcgctggc agagcaattg ttcggcggcg agcaggcgtt gattaccgtg 1680
aacatgagcg agtatcaaga aaagcacacc gtcagccgtt tggtcggttc gcctgcaggg 1740
tatgtcggtt tcggcgaggg tggcatgttg accgaagccg tgcgcaaacg cccttattcg 1800
gtcgtgttgc tggatgaagt ggaaaaggcg cacttggaag tagtgaatat tttctaccag 1860
gtattcgaca agggcttttt gacagatggc gaggggcggc agatcgactt cagaaacacc 1920
gtgattctgc tgaccagcaa cttggccagt gatgaaattc aagcgcgttg cgcaggcgca 1980
cgtccagacg cggaagcgct ggtcgagcat gttcgcccac ggctgtctca atacttcaag 2040
cctgcattat tggcgcgaat gacgattgtg ccctatttca ccctggcggg cgcgcaattg 2100
gaagagattg tgcggctgaa gttggcgcgt ctgaccgaac gcctttggtg gacatcgcgg 2160
gtcaacctga gcttcgccga agaggtcgcc acggtcattg ccgagcgctg tacggaagtg 2220
gaaagtggtg cgcgcaatat cgattacatc ttgcgtaaaa gtctcacccc gcggctttcc 2280
gaggccctgt tggcggccat tgcgcagcag cgaccgctgt gcacgctgac ggttgcagtg 2340
agcccgacgg gcgagtggtt gatcacggct gtcgagcgcg tcgatccggt tgttgtggag 2400
gccgacaatg caggcgttgt caccgctagc cctgcagcaa atgcagagac agtgacgccc 2460
gtctga 2466
<210> 2
<211> 59
<212> DNA
<213> 人工序列
<400> 2
tgcagtgttc aaacgcattt ccttcaagag aggaaatgcg tttgaacact gcttttttt 59
<210> 3
<211> 67
<212> DNA
<213> 人工序列
<400> 3
gtacaaaaaa agcagtgttc aaacgcattt cctctcttga aggaaatgcg tttgaacact 60
gcatgca 67
<210> 4
<211> 24
<212> DNA
<213> 人工序列
<400> 4
cgaggtcatt aaagttgccc aatc 24
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<400> 5
cgccgaaacc gacataccct 20
<210> 6
<211> 21
<212> DNA
<213> 人工序列
<400> 6
cagacctacg tgcacggtgt t 21
<210> 7
<211> 21
<212> DNA
<213> 人工序列
<400> 7
ggattcactg tcgcccacta c 21
<210> 8
<211> 13
<212> DNA
<213> 人工序列
<400> 8
aggctccaat ggc 13
Claims (5)
1.一株变形假单胞菌clpV基因沉默菌株，其特征在于：所述菌株为Pseudomonas plecoglossicida clpV-RNAi，已于2018年11月12日于中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2018772。
2.如权利要求1所述的一株变形假单胞菌clpV基因沉默菌株的构建方法，其特征在于：所述方法包括如下：
（1）通过比较转录组学分析技术，将筛选致病功能基因目标锁定clpV基因；
（2）合成shRNA，退火后连入pCM130/tac载体，通过电转技术导入变形假单胞菌感受态细胞，构建变形假单胞菌clpV基因稳定沉默菌株；利用QPCR技术，对沉默效果进行检验，最终获得变形假单胞菌clpV基因沉默菌株。
3.根据的权利要求2所述的一株变形假单胞菌clpV基因沉默菌株的构建方法，其特征在于：所述clpV基因序列如SEQ ID NO.1所示。
4.根据的权利要求2所述的一株变形假单胞菌clpV基因沉默菌株的构建方法，其特征在于：所述的pCM130/tac载体是利用质粒pCM130加上启动子tac构成。
5.如权利要求1所述的一株变形假单胞菌clpV基因沉默菌株在制备预防和治疗海水鱼内脏白点病制剂中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910013464.9A CN110055200B (zh) | 2019-01-07 | 2019-01-07 | 一株变形假单胞菌clpV基因沉默菌株 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910013464.9A CN110055200B (zh) | 2019-01-07 | 2019-01-07 | 一株变形假单胞菌clpV基因沉默菌株 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110055200A true CN110055200A (zh) | 2019-07-26 |
| CN110055200B CN110055200B (zh) | 2020-09-08 |
Family
ID=67315580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910013464.9A Expired - Fee Related CN110055200B (zh) | 2019-01-07 | 2019-01-07 | 一株变形假单胞菌clpV基因沉默菌株 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110055200B (zh) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454872A (zh) * | 2020-03-16 | 2020-07-28 | 扬州大学 | 禽致病性大肠杆菌VI型分泌***clpV基因缺失株及其构建方法和应用 |
| CN112625996A (zh) * | 2021-02-04 | 2021-04-09 | 集美大学 | 一株变形假单胞菌znuA基因稳定沉默菌株及应用 |
| CN112662608A (zh) * | 2021-02-04 | 2021-04-16 | 集美大学 | 一株变形假单胞菌exbB基因稳定沉默菌株及应用 |
| CN113151134A (zh) * | 2021-05-12 | 2021-07-23 | 集美大学 | 一株变形假单胞菌fliG基因沉默菌株及其应用 |
| CN113528407A (zh) * | 2021-05-31 | 2021-10-22 | 集美大学 | 一株变形假单胞菌tonB基因沉默菌株及其应用 |
| CN114703115A (zh) * | 2022-04-22 | 2022-07-05 | 集美大学 | 一种变形假单胞菌fliS基因沉默菌株和用途 |
| CN116287330A (zh) * | 2022-12-30 | 2023-06-23 | 中国科学院生态环境研究中心 | 一种可检测多种毒力基因的高通量实时荧光定量pcr芯片及其检测方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047783A (zh) * | 2016-05-25 | 2016-10-26 | 浙江万里学院 | 杀香鱼假单胞菌ExoU基因敲除突变株及其应用 |
| CN106834199A (zh) * | 2017-02-27 | 2017-06-13 | 山东省农业科学院畜牧兽医研究所 | 铜绿假单胞菌pyoS5基因敲除突变株及构建方法及应用 |
-
2019
- 2019-01-07 CN CN201910013464.9A patent/CN110055200B/zh not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047783A (zh) * | 2016-05-25 | 2016-10-26 | 浙江万里学院 | 杀香鱼假单胞菌ExoU基因敲除突变株及其应用 |
| CN106834199A (zh) * | 2017-02-27 | 2017-06-13 | 山东省农业科学院畜牧兽医研究所 | 铜绿假单胞菌pyoS5基因敲除突变株及构建方法及应用 |
Non-Patent Citations (2)
| Title |
|---|
| 肖何: "罗尔斯通氏菌中 Reut_A2805 蛋白调节 VI 型分泌***抗氧化功能的研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 胡娇等: "大黄鱼(Pseudosciaena crocea)内脏白点病", 《海洋与湖沼》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454872A (zh) * | 2020-03-16 | 2020-07-28 | 扬州大学 | 禽致病性大肠杆菌VI型分泌***clpV基因缺失株及其构建方法和应用 |
| CN112625996B (zh) * | 2021-02-04 | 2022-06-10 | 集美大学 | 一株变形假单胞菌znuA基因稳定沉默菌株及应用 |
| CN112625996A (zh) * | 2021-02-04 | 2021-04-09 | 集美大学 | 一株变形假单胞菌znuA基因稳定沉默菌株及应用 |
| CN112662608A (zh) * | 2021-02-04 | 2021-04-16 | 集美大学 | 一株变形假单胞菌exbB基因稳定沉默菌株及应用 |
| CN112662608B (zh) * | 2021-02-04 | 2022-06-10 | 集美大学 | 一株变形假单胞菌exbB基因稳定沉默菌株及应用 |
| CN113151134A (zh) * | 2021-05-12 | 2021-07-23 | 集美大学 | 一株变形假单胞菌fliG基因沉默菌株及其应用 |
| CN113151134B (zh) * | 2021-05-12 | 2023-09-12 | 集美大学 | 一株变形假单胞菌fliG基因沉默菌株及其应用 |
| CN113528407A (zh) * | 2021-05-31 | 2021-10-22 | 集美大学 | 一株变形假单胞菌tonB基因沉默菌株及其应用 |
| CN113528407B (zh) * | 2021-05-31 | 2023-01-10 | 集美大学 | 一株变形假单胞菌tonB基因沉默菌株及其应用 |
| CN114703115A (zh) * | 2022-04-22 | 2022-07-05 | 集美大学 | 一种变形假单胞菌fliS基因沉默菌株和用途 |
| CN114703115B (zh) * | 2022-04-22 | 2023-09-29 | 集美大学 | 一种变形假单胞菌fliS基因沉默菌株和用途 |
| CN116287330A (zh) * | 2022-12-30 | 2023-06-23 | 中国科学院生态环境研究中心 | 一种可检测多种毒力基因的高通量实时荧光定量pcr芯片及其检测方法 |
| CN116287330B (zh) * | 2022-12-30 | 2023-12-05 | 中国科学院生态环境研究中心 | 一种可检测多种毒力基因的高通量实时荧光定量pcr芯片及其检测方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110055200B (zh) | 2020-09-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110055200A (zh) | 一株变形假单胞菌clpV基因沉默菌株 | |
| Georgoulis et al. | Genome-wide identification of tomato xylem sap fitness factors for three plant-pathogenic Ralstonia species | |
| LeGault et al. | Temporal shifts in antibiotic resistance elements govern phage-pathogen conflicts | |
| O’Connor et al. | Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk | |
| Links et al. | De novo sequence assembly of Albugo candida reveals a small genome relative to other biotrophic oomycetes | |
| Bobik et al. | FixJ: a major regulator of the oxygen limitation response and late symbiotic functions of Sinorhizobium meliloti | |
| Bellafiore et al. | Nematode effectors and plant responses to infection | |
| CN109694840B (zh) | 一株变形假单胞菌abc转运蛋白基因沉默菌株 | |
| Morimoto et al. | Cooccurrence of broad-and narrow-host-range viruses infecting the bloom-forming toxic cyanobacterium Microcystis aeruginosa | |
| Carretero-Paulet et al. | Genome-wide analysis of adaptive molecular evolution in the carnivorous plant Utricularia gibba | |
| Yang et al. | Prophage-related gene VpaChn25_0724 contributes to cell membrane integrity and growth of Vibrio parahaemolyticus CHN25 | |
| Dong et al. | Novel viroid‐like RNAs naturally infect a filamentous fungus | |
| Rispe et al. | De novo transcriptome assembly of the grapevine phylloxera allows identification of genes differentially expressed between leaf-and root-feeding forms | |
| Bidle et al. | Proteomic analysis of Haloferax volcanii reveals salinity-mediated regulation of the stress response protein PspA | |
| Okamura et al. | Molecular signatures of selection associated with host plant differences in Pieris butterflies | |
| Zhang et al. | Genome analysis of two newly emerged potato late blight isolates sheds light on pathogen adaptation and provides tools for disease management | |
| Lu et al. | Comparative gene expression analysis reveals mechanism of Pinus contorta response to the fungal pathogen Dothistroma septosporum | |
| Rafiqi et al. | Endophytic fungi related to the ash dieback causal agent encode signatures of pathogenicity on European ash | |
| Takemura et al. | The transcription regulator ChpA affects the global transcriptome including quorum sensing‐dependent genes in Ralstonia pseudosolanacearum strain OE1‐1 | |
| Huang et al. | Reference nodule transcriptomes for Melilotus officinalis and Medicago sativa cv. Algonquin | |
| Cheng et al. | Cadmium stress triggers significant metabolic reprogramming in Enterococcus faecium CX 2–6 | |
| US20220333120A1 (en) | Bactericidal phage vectors | |
| CN114470209A (zh) | Trim2在防治猪流行性腹泻病毒感染中的应用 | |
| Goodwin et al. | Comparative analysis of expressed sequence tags from Malva pusilla, Sorghum bicolor, and Medicago truncatula infected with Colletotrichum species | |
| Pachauri et al. | Comparative Phenotypic, Genomic, and Transcriptomic Analyses of Two Contrasting Strains of the Plant Beneficial Fungus Trichoderma virens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200908 |