CN111849981B - 基于PLIN1基因的sgRNA、质粒载体及其构建方法和应用 - Google Patents
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Abstract
本发明公开了一种基于PLIN1基因的sgRNA,所述sgRNA的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3所示。本发明还公开了扩增该sgRNA序列的引物对、基于PLIN1基因的CRISPR/Cas9敲除质粒载体及其应用。本发明的构建了控制猪PLIN1基因表达的载体及验证其相应的敲除效率。本发明还提供三种用于控制猪PLIN1表达的载体并验证其敲除效率并选出一种最优敲除载体,通过所述载体能够控制猪细胞中的PLIN1表达量,进而控制猪的肌肉的纤维组成,调节瘦肉率的变化。本发明还验证了该PLIN1基因敲除后的线粒体活性氧含量有所升高、线粒体膜电位检测升高、ATP含量降低、细胞周期发生阻滞,而细胞凋亡率大大降低。
Description
技术领域
本发明涉及生物技术领域,涉及基于PLIN1基因的sgRNA、质粒载体及其构建方法和应用。
背景技术
PLIN属于结构蛋白家族,最早由Greenberg等发现于小鼠的附睾脂肪细胞中有表达。围脂滴蛋白(Perilipin1,PLIN1)是PLIN家族中唯一一个通过与脂肪酶的相互作用在调节脂肪分解方面发挥特定作用的成员,这一分子仅在脂肪组织中表达,如棕色脂肪组织、白色脂肪组织和胆固醇等。PLIN1专门定位于脂肪细胞脂滴表面,调节脂肪甘油三酯的储存和水解。也有研究发现,在机体基础状态下PLIN1对脂滴存有屏障作用,可有效降低甘油三酯的解,当其含量降低或者发生磷酸化时,就可以加快甘油三酯的分解,是脂质代谢的一个重要环节。
越来越多研究表明,PLIN1基因对肌肉生长发育、能量代谢及线粒体功能有调控作用。目前的研究中,PLIN1基因均是和其他基因联合作用,例如PLIN1可以同线粒体融合蛋白Mfn2进行直接相互作用,线粒体融合蛋白2(mitofusin 2,Mfn 2)的表达和骨骼肌线粒体***的增强是胰岛素抵抗状态的标志。重要的是,mfn 2与perilipin 1直接相互作用,促进线粒体与脂质之间的相互作用。其次AMPK(amp依赖蛋白激酶)在FA释放时被激活,AMPK可以维持线粒体的FA氧化功能并能抑制ROS的产生,中、高浓度的ROS通过细胞氧化应激反应诱导细胞凋亡甚至导致其坏死,其中FA的释放需要磷酸化的PLIN1介导。目前还没有关于单独研究PLIN1基因功能的报道,因此关于PLIN1基因的单独的功能研究显得尤为重要。
gRNA长度约为80个核苷酸,包含两个区域:gRNA5’端前20个核苷酸对应于靶标DNA,能结合在靶标DNA上,其中含有一PAM结构,能与由任意核苷酸序列(N)+5’末端两个胞嘧啶核苷酸(-NCC)的DNA结合,即-NGG。剩余的约60个核苷酸形成一个发卡,此结构能帮助gRNA与Cas9结合,并由此指导与DNA的结合。
目前虽有一些研究将CRISPR/Cas9技术运用到PLIN1基因,然而大部分研究均为PLIN1基因对细胞脂解的影响,且少有研究目的细胞源为猪组织细胞,利用CRISPR/Cas9技术将PLIN1基因进行敲除并研究其对细胞线粒体功能的研究也鲜有所闻。目前并没有研究表明PLIN1基因能够直接对线粒体有直接功能,更没有证据表明PLIN1基因可以直接影响细胞的凋亡、以及线粒体活性等。因此针对PLIN1是否对线粒体功能存在影响的研究愈发重要。
发明内容
发明目的:本发明所要解决的技术问题是提供了一种基于PLIN1基因的sgRNA。
本发明还要解决的技术问题是提供了一种扩增所述的基于PLIN1基因的sgRNA的引物对。
本发明还要解决的技术问题是提供了一种基于PLIN1基因的CRISPR/Cas9敲除质粒载体。
本发明还要解决的技术问题是提供了含有所述的靶基因或所述的敲除质粒载体的重组菌或细胞。
本发明还要解决的技术问题是提供了一种控制动物细胞中PLIN1基因表达的方法。
本发明最后要解决的技术问题是提供了所述的sgRNA或所述的敲除质粒载体、或所述的重组菌或细胞、或所述的方法在动物育种中的应用。
技术方案:为了解决上述技术问题,本发明提供了一种基于PLIN1基因的sgRNA,所述sgRNA的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3所示。
本发明内容还包括一种扩增所述的基于PLIN1基因的sgRNA的引物对,所述引物对序列分别如SEQ ID NO:4和SEQ ID NO:5;或SEQ ID NO:6和SEQ ID NO:7所示;或SEQ IDNO:8或SEQ ID NO:9所示。
本发明内容还包括一种基于PLIN1基因的CRISPR/Cas9敲除质粒载体,所述载体包括所述的sgRNA。
其中,所述载体为pYSY-CMV-Cas9-U6--EFla-Puromycin。
本发明内容还包括含有所述的sgRNA或所述的敲除质粒载体的重组菌或细胞。
本发明内容还包括一种控制动物细胞中PLIN1基因表达的方法,通过任一所述的PLIN1基因的CRISPR/Cas9敲除质粒载体导入动物细胞中即可。
本发明内容还包括所述的sgRNA或所述的敲除质粒载体、或所述的重组菌或细胞、或所述的方法在动物育种中的应用。
其中,所述动物育种包括控制动物的肌肉的纤维组成或调节瘦肉率中的应用。
本发明内容还包括所述动物育种包括研究动物细胞群线粒体功能中的应用。
有益效果:本发明的构建了控制猪PLIN1基因表达的载体及验证其相应的敲除效率。本发明还提供三种用于控制猪PLIN1表达的载体并验证其敲除效率并选出一种最优敲除载体,通过所述载体能够控制猪细胞中的PLIN1表达量。本发明还验证了该PLIN1基因敲除后的线粒体活性氧含量有所升高、线粒体膜电位检测升高、ATP含量降低、细胞周期发生阻滞,而细胞凋亡率大大降低,说明PLIN1基因对线粒体存在一定的调节作用,为后续研究奠定基础。
附图说明
图1 PCR验证阳性克隆电泳图;自左向右第9泳道为DNAMarker:自左向右第1泳道为DNA Marker:从下向上依次为100bp、250bp、500bp、750bp、1000bp、2000bp、3000bp、5000bp;第2泳道到7泳道为PLIN1-KO-sgRNA1克隆;第8泳道到13泳道为PLIN1-KO-sgRNA2克隆;
图2 PCR验证阳性克隆电泳图;自左向右第9泳道为DNA Marker:自左向右第1泳道为DNAMarker:从下向上依次为100bp、250bp、500bp、750bp、1000bp、2000bp、3000bp、5000bp;第2泳道到7泳道为PLIN1-KO-sgRNA1克隆;第8泳道到13泳道为PLIN1-KO-sgRNA3克隆;
图3酶切验证电泳图;M:DNA标准分子量1kb;2、6、10:KpnI单酶切质粒PLIN1-KO-sgRNA(1-3);3、7、11:HindIII单酶切质粒PLIN1-KO-sgRNA(1-3);4、8、12:双酶切质粒PLIN1-KO-sgRNA(1-3);5、9、13:PLIN1-KO-sgRNA(1-3)环形质粒;
图4 K1组细胞DNA测序图;
图5 K2组细胞DNA测序图;
图6 K3组细胞DNA测序图;
图7 CON,K1-3组细胞cDNAqRT-PCR图;
图8 CON,K1-3组细胞Western blot及灰度分析图:A图分别为利用PLIN1及GAPDH抗体孵育蛋白Westernblot结果图;B图为利用Analysis软件测定杂交条带的灰度值分析图;
图9 CON组细胞凋亡图;
图10 K1组细胞凋亡图;
图11 K2组细胞凋亡图;
图12 K3组细胞凋亡图;
图13 CON,K1-3组细胞凋亡分析图;
图14 CON组线粒体活性氧图;
图15 K1组线粒体活性氧图;
图16 K2组线粒体活性氧图;
图17 K3组线粒体活性氧图;
图18 CON,K1-K3组细胞活性氧分析图;
图19 CON组线粒体膜电位图;
图20 K1组线粒体膜电位图;
图21 K2组线粒体膜电位图;
图22 K3组线粒体膜电位图;
图23 CON,K1-K3组线粒体膜电位分析图;
图24 CON,K1-3组线粒体ATP分析图;
图25 CON组细胞周期图;
图26 K1组细胞周期图;
图27 K2细胞周期图;
图28 K3组细胞周期图;
图29 CON,K1-3细胞周期分析图。
具体实施方式
实施例1:CRISPR/Cas9基因敲除质粒的设计、合成及鉴定
1.设计与合成互补引物
根据PLIN1序列(Genbank ID:654411)结构特点设计三对CRISPR序列,引物合成公司合成对应的互补引物。
PLIN1-1-Oligo序列:
F1-PLIN1:CACCGATGGTGTCCTTCAGCTCAG
R1-PLIN1:AAACCTGAGCTGAAGGACACCATC
PLIN1-2-Oligo序列:
F2-PLIN1:CACCGTTGTCCGCAGTGCTGGTGAC
R2-PLIN1:AAACGTCACCAGCACTGCGGACAAC
PLIN1-3-Oligo序列:
F3-PLIN1:CACCGAGCCAGCTAGAGCAGCACCG
R3-PLIN1:AAACCGGTGCTGCTCTAGCTGGCTC
2.引物退火
以上溶液分别混合于PCR管内,在PCR仪中以每分钟1.5℃逐渐从95℃降至22℃得到三组退火产物(sgRNA1~3)。
3.连接
以上溶液混合于1.5ml EP管中,室温孵育30min得到连接产物PLIN1-KO-sgRNA1、PLIN1-KO-sgRNA2、PLIN1-KO-sgRNA3。
4.转化
将10μl连接产物用50μl大肠杆菌DH5α感受态细胞(pfu≥108)(空斑形成单位(plaque forming unit),缩写pfu)进行转化,然后涂布于含氨苄(50μg/ml)(购自Solarbio公司,货号:A8180)的LB板,37℃下倒置培养过夜得到三组转化子。
5.转化子验证
5.1 PCR模板的快速制备
用10μl无菌枪头每组挑选6个克隆,放入含20μl LB的EP管中,合盖后快速涡旋混匀,用作PCR的模板。
5.2 PCR验证体系(10μl)
5.3 PCR反应条件
95℃3min;30×(95℃30s,55℃30s,72℃30s);72℃10min,4℃。
5.4 PCR验证阳性克隆电泳图
如图1所示,自左向右第9泳道为DNA Marker:自左向右第1泳道为DNA Marker:从下向上依次为100bp、250bp、500bp、750bp、1000bp、2000bp、3000bp、5000bp;第2泳道到7泳道为PLIN1-KO-sgRNA1克隆;第8泳道到13泳道为PLIN1-KO-sgRNA2克隆。如图2所示,自左向右第1泳道为DNA Marker:从下向上依次为100bp、250bp、500bp、750bp、1000bp、2000bp、3000bp、5000bp;第2泳道到7泳道为PLIN1-KO-sgRNA3克隆,图中约100bp条带的为阳性克隆。
5.5转化子的测序验证
将之前验证正确的阳性克隆菌液送至金斯瑞生物技术有限公司测序,测序比对结果如图所示,通过序列对比,结果表明目标质粒构建成功。
5.6 pYSY-CMV-Cas9-U6-PLIN1-sgRNA-EFla-Puromycin质粒在猪细胞内设计的sgRNA转录本序列为:(序列为质粒连接的sgRNA1~3片段在机体内进行转录后的RNA片段)PLIN1-KO-sgRNA1:
GAUGGUGUCCUUCAGCUCAGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuu
PLIN1-KO-sgRNA2:
GUUGUCCGCAGUGCUGGUGACguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuu
PLIN1-KO-sgRNA3:
GAGCCAGCUAGAGCAGCACCGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuu
6.CRISPR/Cas9基因敲除质粒的提取及鉴定
6.1质粒提取
配制含氨苄(100mg/ml)的液体LB及固体LB(1L加1mL氨苄溶液),步骤5.4阳性克隆菌液在固体LB上进行划线培养菌种,24h后挑取单个菌落到含有1mLLB的1.5EP管中,把EP管放入37℃的摇床(转速为200r/min)中进行振荡培养,18-24h后使用质粒小提试剂盒(购自天根公司,货号:DP103-02)提取PLIN1-KO-sgRNA(1-3)质粒。
6.2质粒酶切鉴定
利用KpnI(购自TAKARA公司,货号:1068A)和HindIII(购自TAKARA公司,货号:1060A)两种酶切酶对质粒进行酶切鉴定,如图3,其中,M:DNA标准分子量1kb;2、6、10:KpnI单酶切质粒PLIN1-KO-sgRNA(1-3);3、7、11:HindIII单酶切质粒PLIN1-KO-sgRNA(1-3);4、8、12:双酶切质粒PLIN1-KO-sgRNA(1-3);5、9、13:PLIN1-KO-sgRNA(1-3)环形质粒。接着将小摇菌液加入含有200mlLB的锥形瓶中,把锥形瓶放入37℃的摇床(转速为200r/min)中进行振荡培养,18-24h后使用Endo-free Plasmid Mini Kit II(购自OMEGA公司,货号:D6950-01)提取质粒,同时也利用KpnI和HindIII两种酶切酶对质粒进行酶切鉴定。结果证明,所得质粒即为目的质粒。
实施例2:PLIN1基因敲除细胞群的获得方法及分子水平鉴定
1.细胞培养及转染
1.1冻存细胞的复苏与培养
从液氮中取出装有猪PK15细胞(购自中国科学院细胞库)的冻存小管,立即投入37℃的温水中快速晃动,直至冻存液完全融解;将细胞悬液移入无菌的离心管,加入5mL培养液,轻轻吹匀;将细胞悬液800-1000r/min离心5min,弃上清;向含有细胞沉淀的离心管加入1mL DMEM高糖培养基(购自GIBCO公司,货号:12100-046),轻轻吹匀,将细胞悬液转入细胞培养瓶,加入适量的完全培养基(DMEM+10%南美胎牛血清+1%青链霉素混合液)进行培养。
1.2细胞转染
将猪PK15细胞系(购自中国科学院上海细胞库)细胞分为4组分别是正常细胞组及三个实验组PLIN1-KO-sgRNA1~3,每组3个重复。
转染前1天,向每孔含10%胎牛血清(购自GIBCO公司,货号:10270-106)的2mlDMEM高糖培养基(购自GIBCO公司,货号:12100-046)的6孔板中分别按细胞浓度1.5-2×105接种猪PK15细胞,使细胞在转染前达到80-90%的汇合;所用的Lipofectamine 3000Transfection Kit(购自Invitrogen公司,货号为L3000-015);用125μl的OPTI-MEM培养基(购自GIBCO公司,货号:31985-062)与7.5μl的Lipofectamine3000混匀,为A液,用125μl的OPTI-MEM培养基(购自GIBCO公司,货号:31985-062)与2.5μg质粒(PLIN1-KO-sgRNA1~3质粒)、5μl p3000(Lipofectamine 3000 Transfection Kit试剂)混匀,为B液,将A、B液等体积混合,室温孵育10-15分钟,将250μl的DNA脂质体复合物加入准备好的孔内,轻轻来回晃动培养板,置于37℃、饱和湿度、5%CO2的培养箱中培养,于36h后用含嘌呤霉素的DMEM高糖培养基(购自GIBCO公司,货号:12100-046)进行药物筛选。24h后撤去含药培养基,更换新鲜的DMEM高糖培养基,得到细胞K1~3。
2.敲除效率的鉴定
利用血液/细胞/组织基因组DNA提取试剂盒(购自天根公司,货号:DP304-02)提取K1~3组细胞基因组DNA,PCR扩增靶标区域。
3.1 PCR扩增引物序列如下,PLIN1-KO引物由合成公司合成:
PLIN1-KO引物:
F:GGCTTGACATGAGGCTTTC
R:GTGTTGGCGGCATATTCAG
3.2 PCR扩增体系
3.3 PCR扩增条件
95℃4min;30×(95℃30s,57℃30s,72℃30s);72℃5min,4℃。
4.各敲除细胞组PLIN1表达水平的测定
4.1细胞总DNA提取及测序
提取各K1~3组细胞的DNA用PLIN1-KO引物扩增,PCR产物纯化后连接至PGMT载体,转化DH5α后每组挑取50个菌落送往生工生物技术有限公司测序进行序列测定。三组送样测序结果为:K1组DNA突变率8%;K2组DNA突变率30%;K3组DNA突变率18%。如图4~图6,分别为PLIN1-KO-sgRNA1~3转染组。
4.2细胞总RNA提取及cDNA的制备
按照TRNZOL(购自天根公司,货号:DP424)提取法,提取上述各组细胞总RNA;凝胶电泳鉴定总RNA质量后,将提取的总RNA反转录成cDNA,(购自TAKARA公司,货号:RR047A)。
4.3 QRT-PCR检测PLIN1表达
以4.2中制备的cDNA为模板,以猪GAPDH基因为内参,进行QRT-PCR测定PLIN1表达量。
猪内参基因序列为:
GAPDH-RTL:AGCAATGCCTCCTGTACCAC,
GAPDH-RTR:AAGCAGGGATGATGTTCTGG
QRT-PCR PLIN1引物序列为:
PLIN1-RTL:GCAATCAACAAGGGCCTGAC,
PLIN1-RTR:CTTCCTTGGTGCTGGTGTAG
上样体系按照TAKARA定量试剂说明书(购自TAKARA公司,货号:RR420A)上样,反应体系如下:
QRT-PCR反应条件:
95℃30s;40×(95℃5s,60℃34s);95℃15s,60℃1min,95℃15s,20℃15s。
如图7所示为QRT-PCR检测CRISPR/Cas9质粒敲除后的猪PLIN1表达的荧光定量检测结果,图中control代表阴性对照,即正常细胞组,K1、K2、K3组。以正常细胞(CON)为基准值1,K2组的PLIN1表达是正常组的20%,最为明显。
4.3 CRISPR/Cas9质粒敲除猪PLIN1后目的蛋白检测
提取K1、K2、K3组敲除细胞以及正常细胞(CON)总蛋白,检测各组细胞中PLIN1蛋白表达量。如图8所示,Control:正常细胞对照组;K1、K2、K3组:PLIN1-KO-sgRNA1、PLIN1-KO-sgRNA2、PLIN1-KO-sgRNA3质粒转染的敲除细胞群。检测结果表明,利用Western blot检测各组细胞PLIN1蛋白表达,灰度分析表明以正常细胞(CON)为基准1,K2组的PLIN1表达是正常组的25%,最为明显。
实施例3 PLIN1基因敲除细胞群线粒体功能测定
1、细胞凋亡率检测
按照Annexin V-FITC细胞凋亡检测试剂盒(购自碧云天公司,货号:C1062L)说明处理CON组细胞及K1、K2、K3组细胞并进行流式检测。如图9~13所示,与CON组相比K1、K2、K3组细胞凋亡率均有所下降,其中CON组凋亡率为4.89±0.57,K2组凋亡率为2.61±0.25,相较于正常组下降46.62%。
2、线粒体活性氧含量检测
按照活性氧检测试剂盒(购自碧云天公司,货号:S0033S)说明处理CON组细胞及K1、K2、K3组细胞并进行流式检测。如图14~18所示,与CON组相比K1、K2、K3组线粒体活性氧含量均有所上升,其中CON组活性氧含量为54.37±2.76,K2组活性氧含量为56.77±2.86,K2组较CON组活性氧升高4.4%。
3、线粒体膜电位检测
按照检测试剂盒(购自碧云天公司,货号:S0033S)说明处理CON组细胞及K1、K2、K3组细胞并进行流式检测。如图19~23所示,与CON组相比K1、K2、K3组线粒体膜电位均有所下降,其中CON组膜电位为0.76±0.05,K2组膜电位为0.34±0.07,相较CON组下降55.26%。
4、ATP含量检测
将CON组细胞设置阳性组及测试组,按照活性氧检测试剂盒(购自碧云天公司,货号:S0033S)说明处理CON组细胞及K1、K2、K3组细胞并进行流式检测。如图24所示,与CON组相比K1、K2、K3组线粒体ATP含量均有所下降,其中CON组ATP含量为1.60±0.01,K2组ATP含量为1.23±0.05,相较于CON组下降23.13%。
5、细胞周期检测
将CON组细胞设置阳性组及测试组,按照活性氧检测试剂盒(购自碧云天公司,货号:S0033S)说明处理CON组细胞及K1、K2、K3组细胞并进行流式检测。如图25~29所示,与CON组相比K1、K2、K3组细胞均在G2期发生阻滞,其中CON组G2期为5.47±1.02,K2组G2期为26.97±2.32,相较于CON组升高了25.44%。
序列表
<110> 扬州大学
<120> 基于PLIN1基因的sgRNA、质粒载体及其构建方法和应用
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 102
<212> DNA
<213> sgRNA1(Artificial Sequence)
<400> 1
gatggtgtcc ttcagctcag gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 102
<210> 2
<211> 103
<212> DNA
<213> sgRNA2(Artificial Sequence)
<400> 2
gttgtccgca gtgctggtga cgttttagag ctagaaatag caagttaaaa taaggctagt 60
ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttt 103
<210> 3
<211> 103
<212> DNA
<213> sgRNA3(Artificial Sequence)
<400> 3
gagccagcta gagcagcacc ggttttagag ctagaaatag caagttaaaa taaggctagt 60
ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttt 103
<210> 4
<211> 24
<212> DNA
<213> F1- PLIN1(Artificial Sequence)
<400> 4
caccgatggt gtccttcagc tcag 24
<210> 5
<211> 24
<212> DNA
<213> R1- PLIN1(Artificial Sequence)
<400> 5
aaacctgagc tgaaggacac catc 24
<210> 6
<211> 25
<212> DNA
<213> F2- PLIN1(Artificial Sequence)
<400> 6
caccgttgtc cgcagtgctg gtgac 25
<210> 7
<211> 25
<212> DNA
<213> R2- PLIN1(Artificial Sequence)
<400> 7
aaacgtcacc agcactgcgg acaac 25
<210> 8
<211> 25
<212> DNA
<213> F3- PLIN1(Artificial Sequence)
<400> 8
caccgagcca gctagagcag caccg 25
<210> 9
<211> 25
<212> DNA
<213> R3- PLIN1(Artificial Sequence)
<400> 9
aaaccggtgc tgctctagct ggctc 25
<210> 10
<211> 102
<212> RNA
<213> PLIN1-KO-sgRNA1(Artificial Sequence)
<400> 10
gauggugucc uucagcucag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uu 102
<210> 11
<211> 103
<212> RNA
<213> PLIN1-KO-sgRNA2(Artificial Sequence)
<400> 11
guuguccgca gugcugguga cguuuuagag cuagaaauag caaguuaaaa uaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uuu 103
<210> 12
<211> 103
<212> RNA
<213> PLIN1-KO-sgRNA3(Artificial Sequence)
<400> 12
gagccagcua gagcagcacc gguuuuagag cuagaaauag caaguuaaaa uaaggcuagu 60
ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uuu 103
<210> 13
<211> 19
<212> DNA
<213> PLIN1-KO上游引物(Artificial Sequence)
<400> 13
ggcttgacat gaggctttc 19
<210> 14
<211> 19
<212> DNA
<213> PLIN1-KO下游引物(Artificial Sequence)
<400> 14
gtgttggcgg catattcag 19
<210> 15
<211> 20
<212> DNA
<213> GAPDH- RTL(Artificial Sequence)
<400> 15
agcaatgcct cctgtaccac 20
<210> 16
<211> 20
<212> DNA
<213> GAPDH- RTR(Artificial Sequence)
<400> 16
aagcagggat gatgttctgg 20
<210> 17
<211> 20
<212> DNA
<213> PLIN1- RTL(Artificial Sequence)
<400> 17
gcaatcaaca agggcctgac 20
<210> 18
<211> 20
<212> DNA
<213> PLIN1- RTR(Artificial Sequence)
<400> 18
cttccttggt gctggtgtag 20
Claims (1)
1. PLIN1基因在提高动物细胞群线粒体活性氧含量中的应用。
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