CN117431246A - 一种抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的siRNA及其应用 - Google Patents
一种抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的siRNA及其应用 Download PDFInfo
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Abstract
本发明公开了一种抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的siRNA及其应用,其正义链的序列为5’‑GACAGACUAUUCAACUAUA‑3’,反义链的序列为5’‑UAUAGUUGAAUAGUCUGUC‑3’。同时,本发明还公开了含有编码特异性抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的siRNA的核苷酸序列的重组表达载体、转基因细胞系或重组病毒。本发明的siRNA通过特异性抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达来鉴定基因功能,可应用于优质鸡的遗传育种以及为人类疾病研究提供参考数据,具有很大的经济价值和科研价值。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的siRNA及其应用。
背景技术
长期以生长速度为重点的肉鸡育种方式,导致肉鸡生产中存在肉品质明显下降的问题。如何提高鸡肉品质已经成为当今肉鸡育种工作者面临的重要理论和实践问题。
由不同类型肌纤维组成的骨骼肌是动物体内最大的糖代谢组织。位于线粒体基质中的丙酮酸脱氢酶激酶1(pyruvate dehydrogenase kinase 1, PDK1)是细胞糖酵解中关键限制酶。在糖代谢过程中,PDK1通过磷酸化丙酮酸脱氢酶(PDH),从而阻断丙酮酸转化成乙酰辅酶A进入线粒体参加三羧酸循环,在调节细胞氧化和酵解代谢转换以及线粒体的三磷酸腺苷(ATP)合成中发挥核心作用。在低氧诱导条件下,大鼠骨骼肌PDK1表达的提高,导致富含糖酵解型MyHC Πb蛋白的纤维增加;在C2C12肌管中过表达 PDK1,不仅促进糖酵解型MyHC Πb的增加,还促进MyHC Πa和 MyHC Πx蛋白的增加;PDK1可能在肌肉代谢和肌纤维类型组成中发挥纽带作用,从而导致肌肉功能特性的改变。
在鸡上,本课题组在前期研究中发现,鸡骨骼肌中PDK1蛋白的表达与肌肉的肌纤维类型组成密切相关,快白肌纤维占比越高的肌肉中PDK1表达越高。但目前对鸡PDK1的功能还不是很清楚。在鸡肌肉组织和成肌细胞中,PDK1蛋白表达主要定位于细胞质,因此,借助转录后水平调控靶基因表达的siRNA开展鸡PDK1基因功能研究显得十分迫切。
发明内容
针对目前对鸡PDK1的功能还不是很清楚的问题,本发明提供了一种抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的siRNA及其应用,该siRNA能够通过特异性敲降鸡丙酮酸脱氢酶激酶1基因PDK1的表达来鉴定基因功能,揭示鸡骨骼肌生长发育和肌纤维类型形成的调控机制。
为了实现上述目的,本发明第一方面提供一种抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的siRNA,所述siRNA的正义链的序列为5’-GACAGACUAUUCAACUAUA-3’(SEQ NO.1),反义链的序列为5’-UAUAGUUGAAUAGUCUGUC-3’(SEQ NO.2)。
进一步地,所述正义链和所述反义链的3’端还添加有两个垂悬碱基dT。
本发明第二方面提供一种上述的siRNA在制备抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的试剂盒中的应用。
所述试剂盒还包括用于检测鸡丙酮酸脱氢酶激酶1基因PDK1表达的引物组。
本发明第三方面提供一种上述的抑制鸡丙酮酸脱氢酶激酶1基因PDK1的siRNA的核苷酸序列的重组表达载体、转基因细胞系或重组病毒。
本发明第四方面提供一种上述的siRNA、上述的重组表达载体、转基因细胞系或重组菌在鉴定鸡丙酮酸脱氢酶激酶1基因PDK1功能中的应用。
通过上述技术方案,本发明实现了以下有益效果:
本发明相对于现有技术而言,提供了一种能够靶向丙酮酸脱氢酶激酶1基因PDK1的siRNA,将其转入鸡成肌细胞后可以有效降低丙酮酸脱氢酶激酶1基因PDK1的表达,抑制效率接近50%,可见其敲降效率很高。本发明的siRNA通过特异性敲降鸡丙酮酸脱氢酶激酶1基因PDK1的表达来鉴定基因功能,揭示鸡骨骼肌生长发育和肌纤维类型形成的调控机制,不仅对提升我国家禽产品的市场竞争力具有重要意义,而且对于揭示人类骨骼肌代谢功能异常引起的肌肉相关疾病的分子机理也具有很好的借鉴价值。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式描述中所需要使用的附图作简单地介绍。
图1是用Western blot检测PDK1在鸡肌肉组织和成肌细胞中胞质和胞核表达,泳道1是11胚龄鸡腿肌组织胞浆蛋白,泳道2是11胚龄鸡胸肌组织胞浆蛋白,泳道3是成年鸡腿肌组织胞浆蛋白,泳道4是成年鸡胸肌组织胞浆蛋白,泳道5是分化的鸡成肌细胞胞浆蛋白,泳道6是11胚龄鸡腿肌组织胞核蛋白,泳道7是11胚龄鸡胸肌组织胞核蛋白,泳道8是成年鸡腿肌组织胞核蛋白,泳道9是成年鸡胸肌组织胞核蛋白,泳道10是分化的鸡成肌细胞胞核蛋白;
图2是不同siRNA在原代成肌细胞增殖期和分化期干扰鸡丙酮酸脱氢酶激酶1基因PDK1表达的效率,其中,ns表示干扰组与NC对照组相比,差异不显著(P>0.05);**表示干扰组与NC对照组相比,差异极显著(P<0.01);
图3是Western blot检测siRNA 3在原代成肌细胞分化期干扰鸡PDK1蛋白表达的效率,其中,*表示干扰组与NC对照组相比,差异显著(P<0.05);
图4是分化的鸡成肌细胞中PDK1基因表达敲低后mRNA-seq差异共表达基因的KEGG富集分析;
图5是PDK1基因在鸡和其它物种中的同源性比较。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。以下实施例中如无特别说明,均为常规方法,所用的试剂均为市售试剂。
实施例1Western blot检测鸡PDK1基因胞质胞核表达定位
用Nuclear Extract Kit(Active Motiff,40010)试剂盒提取肌肉组织样和鸡成肌细胞样的胞质和胞核蛋白,ProStatainTM蛋白质定量试剂盒(Active Motiff,15001)测定蛋白浓度,分别用β-Actin(ERWAN, ER001, 1:10000)和Histone H3(ERWAN, EAB02004, 1:1000)做一抗检测胞质内参蛋白β-Actin和胞核内参蛋白Histone H3,用PDK1(NOVUS,NB100-2383SS, 1:2000)作一抗检测胞质和胞核中的PDK1蛋白表达。由图1可见,胞质内参蛋白β-Actin在鸡肌肉组织和成肌细胞的胞质和胞核中均表达,胞核内参蛋白Histone H3仅在鸡肌肉组织和成肌细胞胞核中均表达,PDK1蛋白主要表达于鸡肌肉组织和成肌细胞胞质中。提示PDK1在鸡肌肉组织和成肌细胞中的表达主要定位于细胞质,可用在转录后水平发挥调控靶基因表达的siRNA来调控鸡PDK1的表达。
实施例2靶向鸡PDK1基因siRNA最佳序列筛选
2.1 siRNA设计
根据NCBI在线数据库,获得鸡PDK1基因mRNA序列信息(登录号:NM_001031352.4),采用优化的siCatchTM siRNA设计软件,设计3对靶向鸡PDK1基因CDS区的siRNA。设计时从靶基因起始密码子AUG下游100bp的核苷酸开始搜寻理想的siRNA,避开外显子与外显子的交界区域。为了增强siRNA双链复合体的稳定性,在每对siRNA的正义链和反义链3’端添加2个垂悬碱基dT,由广州锐博生物科技有限公司合成。设计获得的3对siRNA的序列如表1所示,3对siRNA靶向的序列如表2所示,靶点分别起始于280、685、1021。
表13对siRNA的序列
表23对siRNA的靶向序列
2.2 siRNA转染原代鸡成肌细胞
从动物组织中分离提取的原代细胞保持了细胞在体内许多重要生物学特征和功能,因此原代细胞不仅被广泛应用于分子、细胞生物学和生物医学基础研究,而且在生物医药领域更具有不可替代性的作用。但相对于细胞系,原代细胞难培养、外源基因转染效率低。本实施例以从11胚龄鸡胚腿肌分离提取的原代鸡成肌细胞为转染细胞,分别在细胞增殖期和分化期转染PDK1 siRNA。
(1)增殖期转染
将分离提取的原代鸡成肌细胞以1×105个细胞/孔的密度接种24孔培养板,培养过夜后,在细胞处于增殖期时,借助转染试剂Lipofectamine 3000将3对靶向鸡PDK1基因的siRNA转染和阴性对照siRNA NC分别转染入鸡成肌细胞,每组设4个重复孔,转染浓度为100nmol/L。
(2)分化期转染
将分离提取的原代鸡成肌细胞以3×105个细胞/孔的密度接种24孔培养板,待细胞密度生长至70-90%时,用含有5%马血清的培养基替代含有20%胎牛血清的培养基,诱导细胞分化,培养1d后,借助转染试剂Lipofectamine 3000将3对靶向鸡PDK1基因的siRNA转染和阴性对照siRNA NC分别转染入鸡成肌细胞,每组设4个重复孔,转染浓度为100 nmol/L。
2.3 RNA提取和cDNA制备
转染48h后,每孔用PBS清洗2次,选择南京诺唯赞生物科技股份有限公司提取细胞总RNA的试剂RNA isolater Total RNA Extraction Reagent收集和提取转染细胞的总RNA。核酸定量仪测定RNA浓度。按照南京诺唯赞生物科技股份有限公司的HiScript Ⅲ RTSuperMix for qPCR说明书进行cDNA合成。
2.4 实时荧光定量PCR检测siRNA干扰效率
选择南京诺唯赞生物科技股份有限公司的HiScript Ⅲ RT SuperMix for qPCR(+gDNA wiper)试剂采用SYBR Green Ι法进行实时荧光定量PCR检测siRNA特异性干扰鸡丙酮酸脱氢酶激酶1基因PDK1表达的效率。实时荧光定量PCR反应的体系如表3所示。每个样品设置3个重复。
表3 实时荧光定量PCR反应的体系
检测鸡PDK1基因表达的引物对核酸序列如下:
上游引物:5’-CAGCTGGTGCAGAGTTGGTA-3’(SEQ ID NO.10)
下游引物:5’-TCGTTGTGTCGATTGCGTAT-3’(SEQ ID NO.11)
检测鸡内参ACTB基因表达的引物对核酸序列如下:
上游引物:5’- TGCTGTGTTCCCATCTATCG-3’(SEQ ID NO.12)
下游引物:5’- TTGGTGACAATACCGTGTTCA-3’(SEQ ID NO.13)
3对siRNA特异性干扰鸡丙酮酸脱氢酶激酶1基因PDK1表达的效率结果见图2。由图2可见,3对siRNA对增殖期、分化期鸡原代细胞中PDK1基因表达的抑制效率,均是siRNA 3干扰效率最好,与NC对照组相比,siRNA 3的抑制效率均接近50%,极显著(P<0.01)抑制鸡成肌细胞中鸡PDK1基因的表达,后续实验选择siRNA 3用于研究鸡PDK1基因功能。siRNA 3的正义链为SEQ ID NO.1所示的碱基序列5’-GACAGACUAUUCAACUAUA-3’,所述siRNA的反义链为SEQ ID NO.2所示的碱基序列5’-UAUAGUUGAAUAGUCUGUC-3’,正义链和反义链的3’端还添加有两个垂悬碱基dT。
2.5 Western blot检测siRNA干扰效率
转染72h后,每孔用PBS清洗2次,选择赛默飞RIPA细胞裂解液提取细胞总蛋白,BCA法测定提取的蛋白浓度后,分别用PDK1(NOVUS, NB100-2383SS, 1:2000)和β-Actin(ERWAN, ER001, 1:10000)作一抗和羊抗兔IgG(Invitrogen, A32732, 1:5000)和羊抗鼠IgG(Invitrogen, A32723, 1:5000)作二抗进行Western blot实验。由图3可见,与NC对照组相比,siRNA 3能显著(P<0.05)抑制PDK1蛋白的表达。
实施例3鸡成肌细胞中敲低PDK1基因表达,RNA-seq筛选差异基因并进行功能分析
在诱导分化的鸡成肌细胞中转染siRNA3,同时设siRNA NC对照组,48h收集细胞,用RNA-seq技术筛选差异表达基因,P<0.05为筛选标准,结果共筛到2283个差异表达基因,PDK1敲低表达组中,线粒体载体同源蛋白2基因MTCH2、辅肌动蛋白ɑ4基因ACTN4、肌分化标志基因MyoG、脂肪酸结合蛋白基因FABP3和FABP7、转录因子基因FOXO6、钙蛋白酶1基因CAPN1、慢肌肌钙蛋白1基因TNNI1等表达下调,而快肌肌纤维类型标志基因MYH1E、MYH1F、MYH1D、MYH1C、MYH1B、MYH1A和PDK家族同源蛋白PDK3基因表达上调。提示在细胞糖酵解发挥关键作用的酶PDK1能够调节细胞分化和肌纤维类型转换,有促进慢肌纤维形成的作用。
对差异的表达基因基因KEGG富集分析,以P<0.05为筛选标准,结果共显著富集到15个信号通路,结果见图4。由图4可见,与肌肉发育相关的regulation of actincytoskeleton、tight junction、MAPK signaling pathway等信号通路被显著富集。
实施例4 鸡PDK1基因的mRNA序列与其它物种PDK1基因mRNA序列同源性分析
通过NCBI在线比对软件blastn,将鸡丙酮酸脱氢酶激酶1基因PDK1 mRNA序列与人、小鼠、大鼠、鸭及鹅物种中PDK1基因mRNA序列进行同源性分析,结果如图5所示,鸡PDK1基因mRNA序列与人PDK1基因mRNA序列相比,仅29%的区域能用于比对,比对的同源性为81.88%;鸡PDK1基因mRNA序列与小鼠PDK1基因mRNA序列相比,仅29%的区域能用于比对,比对的同源性为80.28%;鸡PDK1基因mRNA序列与大鼠PDK1基因mRNA序列相比,仅29%的区域能用于比对,比对的同源性为80.54%;鸡PDK1基因mRNA序列与鸭PDK1基因mRNA序列相比,60%的区域能用于比对,比对的同源性为89.01%。可见,鸡丙酮酸脱氢酶激酶1基因PDK1 mRNA序列与其他物种中的PDK1基因mRNA序列可比对的区域比较低,本发明的siRNA序列大小只有19bp,在针对鸡鸡丙酮酸脱氢酶激酶1基因PDK1 mRNA选择靶向位点以及siRNA设计过程中,上述物种的PDK1基因mRNA序列并没有参考价值。
以上结合实施例详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
Claims (5)
1.一种抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的siRNA,其特征在于,所述siRNA的正义链的序列为5’-GACAGACUAUUCAACUAUA-3’(SEQ NO.1),反义链的序列为5’-UAUAGUUGAAUAGUCUGUC-3’(SEQ NO.2)。
2.根据权利要求1所述的siRNA,其特征在于,所述正义链和所述反义链的3’端还添加有两个垂悬碱基dT。
3.权利要求1或2所述的siRNA在制备抑制鸡丙酮酸脱氢酶激酶1基因PDK1表达的试剂盒中的应用。
4.含有编码权利要求1或2所述的抑制鸡丙酮酸脱氢酶激酶1基因PDK1的siRNA的核苷酸序列的重组表达载体、转基因细胞系或重组病毒。
5.权利要求1或2所述的siRNA、权利要求4所述的重组表达载体、转基因细胞系或重组菌在鉴定鸡丙酮酸脱氢酶激酶1基因PDK1功能中的应用。
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