CN109517904A - circRNA_14759及其互作基因的应用 - Google Patents
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Abstract
本发明涉及circRNA_14759及其互作基因的应用,具体的涉及circRNA_14759及其互作基因在检测肌内脂肪中的应用。本发明选择典型的瘦肉型大白猪和典型高肌内脂肪含量的莱芜猪进行高通量测序及数据分析,寻找到调控猪脂肪沉积的circRNA_14759及其互作基因ssc‑miR‑370,本发明为选育具有不同肉质品质的种猪提供理论基础和机理指导。
Description
技术领域
本发明涉及生物技术领域,具体涉及circRNA_14759及其互作基因ssc-miR-370应用,更具体的涉及circRNA_14759及其互作基因ssc-miR-370在检测肌内脂肪中的应用。
背景技术
circRNA是一类由特殊选择性剪切产生的环形内源性分子,呈共价闭合的环形结构,没有5'和3'极性,不能被核糖核酸酶降解。在哺乳动物细胞中,circRNA分布广泛,含量丰富,且具有稳定性,保守性和组织特异性。已经有研究发现circRNA与多种疾病相关。但是,circRNA是否参与脂肪沉积与代谢,目前仍不清楚。miRNAs是一类长度在21nt左右的RNA,它们可以通过碱基互补配对直接与mRNA靶标相结合,从而起到抑制mRNA翻译的作用。有研究显示,环状RNA拥有miRNA海绵结合位点,可以通过吸附特定的miRNA,以竞争性抑制剂的形式抑制miRNA与靶标结合的能力。目前研究circRNA与miRNA相互作用机制较少,还需要更多的探索。
肌内脂肪是影响肉质的重要因素,多年来在以高瘦肉率和快速生长为目标的选育过程中,猪肉品质下降,主要原因是脂肪的减少,尤其是肌内脂肪沉积减少。但脂肪沉积过多,又会导致肥胖和机体能量代谢失衡,并引发一系列肥胖相关性疾病,如二型糖尿病、动脉粥样硬化、高血压以及血脂异常等,严重威胁人类健康。有研究发现某些生物活性物质如共轭亚油酸在提高肌内脂肪含量的同时,却降低或维持了肌内脂肪的沉积量不变,由此推测,肌内脂肪可能具有不同于皮下脂肪的生物学功能以及分子调控机制。因此,了解猪肉肌内脂肪沉积的分子机制有助于改善猪肉品质。
大白猪属于典型的瘦肉型猪种,肌内脂肪含量低,莱芜猪肉色鲜红,肌内脂肪含量高;二者在脂肪沉积方面的显著差异为脂肪代谢的机制研究提供了良好模型。因此本研究利用生物信息学方法对莱芜猪和大白猪肌内脂肪中circRNA、miRNA及他们的相互作用关系进行分析,以找出调控猪脂肪沉积的circRNAs、miRNA,进一步了解脂肪沉积和脂代谢的作用机理,为选育具有不同肉质品质的种猪提供理论基础和机理指导。
发明内容
一种与猪肌内脂肪相关的环状RNA,命名为circRNA_14759,序列与SEQ ID NO.1具有95%以上序列同源性。
优选的,circRNA_14759序列为SEQ ID NO.1。
一种检测肌内脂肪的试剂,检测circRNA_14759及其互作基因的表达水平,互作基因选自下列中的一种或几种:ssc-miR-326、ssc-miR-140-3p、ssc-miR-328、ssc-miR-370、ssc-miR-7144-3p。
进一步,采用测序技术、核酸杂交技术或核酸扩增技术检测样品中circRNA_14759或ssc-miR-370的表达水平。
优选的,采用高通量测序技术、探针杂交技术、基因芯片技术或荧光定量PCR技术检测样品中circRNA_14759的表达水平。
优选的,试剂包含检测circRNA_14759的探针或用于荧光定量PCR的引物。
优选的,荧光定量PCR检测引物序列为SEQ ID NO.2和SEQ ID NO.3。
优选的,试剂包含特异性扩增ssc-miR-370的引物。优选的,特异性扩增ssc-miR-370的引物序列为SEQ ID NO.4。
进一步,样品为组织,优选的,样品为肌内脂肪组织,更优选的,样品为猪肌内脂肪组织。
进一步,所述核酸扩增技术选自聚合酶链式反应(PCR)、逆转录聚合酶链式反应(RT-PCR)、转录介导的扩增(TMA)、连接酶链式反应(LCR)、链置换扩增(SDA)和基于核酸序列的扩增(NASBA)。其中,PCR需要在扩增前将RNA逆转录成DNA(RT-PCR),TMA和NASBA直接扩增RNA。
本发明中的核酸杂交技术包括但不限于原位杂交(ISH)、微阵列和Southern或Northern印迹。
本发明的目的在于提供下述任意一项应用:
circRNA_14759在预测或辅助预测猪肉品质中的应用;
circRNA_14759在制备预测或辅助预测猪肉品质试剂中的应用;
circRNA_14759在选育具有不同肉质品质猪中的应用;
上述试剂在预测或辅助预测猪肉品质中的应用;
上述试剂在制备预测或辅助预测猪肉品质试剂中的应用;
上述试剂在选育具有不同肉质品质猪中的应用。
一种检测猪肌内脂肪含量的方法,包括:(1)选取猪肌内脂肪组织;(2)检测样品中circRNA_14759和/或其互作基因的表达量。
优选的,互作基因为ssc-miR-370。
定义:
术语“同源”是主要是指序列上的同源,也就是用来说明两个或多个RNA或DNA序列具有相同的祖先。同源的序列一般有相似的功能。一般来说,当相似程度高于50%时,常推测检测序列和目标序列可能是同源序列;当相似性程度低于20%时,就难以确定其是否具有同源性。
环状RNA(circular RNA,circRNA),亦称环形RNA,是最近几年研究确认的一种新型的非编码RNA(noncoding RNA,ncRNA)分子。根据RNA构成的不同,环状RNA可分为三类:外显子环状RNA(exon circular RNA,ecircRNA)、内含子环状RNA(circular intronic RNAs,ciRNAs)和外显子-内含子circRNA(exon-intron circRNA,EIciRNA)。
本发明中“探针”指能与另一分子的特定序列或亚序列或其它部分结合的分子。除非另有指出,术语“探针”通常指能通过互补碱基配对与另一多核苷酸结合的多核苷酸探针。根据杂交条件的严谨性,探针能和与该探针缺乏完全序列互补性的靶多核苷酸结合。探针可作直接或间接的标记,其范围包括引物。
所述探针具有与靶点基因的特定的碱基序列互补的碱基序列。这里,所谓“互补”,只要是杂交即可,可以不是完全互补。这些多核苷酸通常相对于该特定的碱基序列具有90%以上、更优选95%以上、特别优选100%的同源性。这些探针可以是DNA,也可以是RNA,另外,可以为在其一部分或全部中核苷酸通过PNA(Polyamide nucleic acid,肽核酸)、LNA(注册商标,locked nucleic acid,Bridged Nucleic Acid,交联化核酸)、ENA(注册商标,2′-O,4′-C-Ethylene-bridged nucleic acids)、GNA(Glycerol nucleic acid,甘油核酸)、TNA(Threose nucleic acid,苏糖核酸)等人工核酸置换得到的多核苷酸。
本发明中的术语“杂交”用于指代互补核酸的配对。杂交和杂交强度受诸如以下的因素影响:核酸之间的互补程度、所涉及的条件的严格性、形成的杂交体的Tm和核酸内的G:C比率。
测序技术主要为高通量测序技术(High-throughput sequencing),又称下一代测序技术(next generation sequencing),一次对几十万到几百万条DNA分子进行序列测定,极大提高了测序效率。高通量测序平台的代表是罗氏公司(Roche)的454测序仪(RochGSFLX sequencer),Illumina公司的Solexa基因组分析仪(Illumina Genome Analyzer)和ABI的SOLiD测序仪(ABI SOLiD sequencer)。
附图说明
图1是肌内脂肪差异表达circRNA分类图:A为上调的差异表达circRNA分类图,B为下调的差异表达circRNA分类图
图2是circRNA_14759-miRNA互作网络图
图3是荧光定量验证候选基因差异表达图
具体实施方式
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1circRNA测序及差异表达circRNA挑选
1.1实验动物:
该试验以脂肪沉积存在明显差异的大白猪和莱芜猪为材料,饲养于莱芜市大千农牧有限公司,在相同饲养条件和环境下生长育肥,参照营养需要标准(National ResearchCouncil,NRC,1998)饲喂日粮,选择180日龄、种内体重接近的大白猪(约100kg)和莱芜猪(约35kg)各3只,且健康、体质优良。
1.2样品采集:
屠宰实验猪后,迅速将肌内脂肪组织分离。为减少RNA的降解,所有过程在冰上进行。之后用消毒剪刀将组织剪成小块,分别装入5mL冻存管,放入液氮冷冻,后转移至-80℃冰箱保存,用于提取总RNA。实验设置分为2组,分别对大白猪肌内脂肪组织(D_JN)与莱芜猪肌内脂肪组织(L_JN)中的circRNA进行鉴定及分析,每个样本设置3个生物学重复。
1.3样品总RNA的提取及质控:
取出等量低温保存的脂肪组织,使用QIAGEN抽提试剂盒提取RNA,提取的总RNA保存于-80℃冰箱。用NanoDrop 2000紫外分光光度计测定RNA样品的浓度和OD260nm/OD280nm比值,比值在1.9~2.1间,Bioanalyzer 2100检测总RNA的质量,且RIN≥7、28S/18S≥0.7,用RNase-free DNaseⅠ除去基因组DNA污染。
1.4 circRNA测序建库:
(1)Ribo-zero kit去除rRNA及RNA的片段化;(2)核糖核酸酶R去除线性RNA;(3)双链cDNA的合成与纯化;(4)末端修复,加A碱基;(5)测序接头连接;(6)DNA片段富集纯化;(7)文库的质检;(8)本试验共建立6个cDNA文库(大白猪和莱芜猪肌内脂肪组织cDNA文库),分别为D_JN_1、D_JN_2、D_JN_3、L_JN_1、L_JN_2、L_JN_3,文库质检合格后,运用IlluminaHiSeqTM2500测序平台进行双端测序,对文库进行测序分析,下机得到的数据是原始测序数据。
1.5原始数据质控及过滤:
主要应用NGSQCToolkit进行质控并去除接头,后续的分析以clean reads为基础。具体步骤如下:
(1)首先过滤低质量的reads,质量阈值设置为20,过滤长度阈值设置为70%。
(2)然后从5’端及3’端去除低质量碱基,质量阈值设置为20。
(3)最后切除reads中含N部分序列,长度阈值设置为35bp。
1.6 circRNA鉴定:
使用BWA软件与参考基因序列比对,生成SAM文件,对文件中的CIGAR值进行分析,并从SAM文件扫描PCC信号(paired chiastic clipping signals)。CIGAR值在junctionread的特征为xS/HyM或者xMyS/H,其中x,y代表碱基数目,M是mapping,S是soft clipping,H是hard clipping。关于双端Reads,CIRI算法考虑一对reads,其中一条mapping到circRNA上,另一条也需mapping到circRNA的区间内。关于单外显子成环,或“长外显子1-短外显子-长外显子2”成环,CIGAR值应该是xS/HyMzS/H以及(x+y)S/HzM或者xM(y+z)S/H,CIRI软件能够将这两种情况分开。关于splicing信号(GT,AG)CIRI会考虑其它弱splicing信息例如(AT-AC)。算法:从GTF/GFF文件中抽取外显子边界位置,用已知边界来过滤假阳性。
1.7 circRNA注释:
将circRNA与基因元件进行比较,获取circRNA在基因组上的位置信息。利用circRNA位置信息与已知数据库中的蛋白编码基因注释信息,对circRNA进行注释,获取序列信息,详细处理过程如下:
(1)利用intersectBed软件,根据环化位点是否落在转录本区域的数目及剪切位点之间的外显子数目,获取与circRNA在基因组位置上有最大重叠区域的蛋白编码转录本;
(2)如果circRNA的反向剪切位点落在或远离最大重叠转录本的外显子,则对相应的外显子进行相应的截断或延伸,作为circRNA的边界外显子,同时取最大重叠转录本与circRNA重叠区域的其他外显子,作为circRNA的中间外显子;在该过程中需要保证circRNA的反向剪切位点不变
(3)如果circRNA与外显子没有重叠区域,则认为该circRNA是一个单外显子circRNA,取circRNA的反向剪切位点之间的序列作为外显子序列。
1.8 circRNA分布统计:
统计各个组织中circRNA的数目情况。统计预测到的所有circRNA长度分布情况,在基因组染色体上的分布情况,circRNA外显子数目情况。
1.9 circRNA表达水平定量:
利用RPM(spliced reads per millon reads)对circRNA进行定量,其中RPM计算公式如下:RPM=number of circular reads/number of total reads(units inmillion)其中:number of circular reads表示比对到circRNA的back-splicedjunctions区域的reads数,该数值来源于circRNA预测软件中提供的支持circRNA成环的reads数目;number of total reads(units in million)表示每个样本测序数据中(clean_data)reads数目(单位为millon)。
1.10生物学重复样品间相关性分析:
样品间circRNA表达水平相关性可以用来检验实验可靠性和样本选择合理性。相关系数越接近于1,说明样品间表达模式相似度则越高。当样本数目较多时(≥3),circRNA的表达量情况,采用R语言中的cor函数及corrplot包,计算样本间的pearson相关系数,并绘制热图进行展示。
1.11样品组间差异circRNA筛选:
由于实验有生物学重复,运用DESeq包(Huber&Anders,2014),是基于reads count进行差异分析的R包,用负二项分布检验的方式对reads数进行差异的显著性检验,估算转录本表达量的方式采用basemean值来估算表达量,计算差异倍数。筛选差异的条件为p<0.05且|FoldChange|>2。
在莱芜猪和大白猪肌内脂肪组织(L_JN vs D_JN)中共鉴定得到283个差异表达circRNAs(101个上调,182个下调),其中具有2倍以上差异的circRNAs约占35.7%。在101个上调的circRNA中,有26个intergenic circRNA,1个intronic circRNA,73个sense-overlapping circRNA和1个antisense circRNA;在182个下调的circRNA中,有1个exoniccircRNA,29个intergenic circRNA,1个intronic circRNA,149个sense-overlappingcircRNA和2个antisense circRNA(图1)。其中,circRNA_14759进入后续研究范围。
实施例2 circRNA深度分析
2.1差异表达circRNA功能分析:
得到差异表达circRNA之后,利用circRNA来源基因的信息,对差异表达circRNA进行GO富集分析。统计每个GO条目中所包括的差异circRNA个数,并用超几何分布检验方法计算每个GO条目中差异circRNA富集的显著性。KEGG是与Pathway相关的重要公共数据库,利用KEGG数据库对差异circRNA来源基因进行Pathway分析,可以找到富集差异circRNA来源基因的Pathway条目,寻找潜在的不同样品的差异circRNA可能和哪些细胞通路的改变有关。大白猪和莱芜猪肌内脂肪沉积存在显著差异,通过GO分析,差异表达circRNA来源基因显著富集于脂质代谢与细胞分化的生物学过程,表明二者在肌内脂肪沉积、代谢的分子机制存在差异,受到不同的基因调控。GO富集还显示circRNA来源基因富集于与脂肪代谢相关的疾病过程,如胰岛素抵抗,氧化应激,流体剪切力,炎症反应等。
2.2 circRNA-miRNA互作网络图
选择在莱芜肌内脂肪组织上调的circRNA_14759进行后续的分析,结合生物学信息学分析和本实验室前期做过的大白猪和莱芜猪肌内脂肪组织miRNA的测序结果进行整合分析,挑选出了与circRNA_14759结合的5个miRNA结合位点,将分析的数据进行整理,构建了circRNA-miRNA互作网络图(见图2)。
实施例3实时荧光定量PCR验证
选择circRNA_14759,circRNA_14759靶基因中ssc-miR-370,每个设置20个生物学重复(大白猪和莱芜猪各20例),分别以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因和5S rRNA为内参,qRT-PCR方法用于验证基因的表达水平。
3.1引物设计:
circRNA_14759扩增:
正向引物:5’-CTGGGGAGACGCCAAGACA-3’(SEQ ID NO.2)
反向引物:5’-ACGATGTCCGTGAAGTCCG-3’(SEQ ID NO.3)
ssc-miR-370:
特异引物:5’-GCCTGCTGGGGTGGAACCTG-3’(SEQ ID NO.4)
3.2反转录体系:
将下列物质加入0.5ml的离心管中:RNA 5μl;Oligo(dT)2μl;dd H2O(DEPC处理)4.5μl;
以上首先70℃孵育5min,然后迅速放在冰上。
5×Buffer 5μl;dNTP(10mM)2μl;Ribonuclease inhibitor 0.5μl;M-MLV RT 1μl;以上20μl体系42℃孵育60min,然后70℃10min。
3.3Real-time RCR反应
(1)将所得的cDNA与下列物质配成20μl体系加入0.2ml的离心管中混匀。
Real-Time PCR体系:cDNA 2μl;引物1(10pmol/μl)0.5μl;引物2(10pmol/μl)0.5μl;SYBR mix 10μl;dd H2O 7μl。
(2)Real-Time PCR程序设置:94℃预变性10min;94℃15s,60℃60s,45个循环;72℃10min。
3.4结果统计分析:
为了验证测序分析结果,选取目标分子circRNA_14759、ssc-miR-370,进行验证。2-△△Ct法分析每组样本间基因的相对表达情况,用t-检验统计分析相对表达量,数据结果展示为平均数±标准差(Mean±SD),P<0.05为差异显著。
结果显示,circRNA_14759在瘦肉型猪大白猪肌内脂肪组织中显著低表达,在脂肪含量较高的莱芜猪肌内脂肪组织中显著高表达,ssc-miR-370在瘦肉型猪大白猪肌内脂肪组织显著高表达,在脂肪含量较高的莱芜猪肌内脂肪组织显著低表达,具体结果见图3。荧光定量PCR结果与测序结果一致。显示circRNA_14759、ssc-miR-370可以作为猪肌内脂肪含量检测的分子标志物,在预测或辅助预测猪肉品质、选育具有不同肉质品质猪等领域具有很好的应用前景。例如,在选育瘦肉型猪的时候考虑circRNA_14759表达量较低的个体或者ssc-miR-370基因表达量高的个体。由于大多数环状RNA拥有多个miRNA海绵结合位点,故而circRNA-miRNA互作网络比较复杂,存在很多circRNA靶向同一个miRNA,或者很多miRNA结合到同一个circRNA上。对于环状RNA的功能的研究、环状RNA作用机制的研究还需要不断探索。
序列表
<110> 中国农业科学院北京畜牧兽医研究所
<120> circRNA_14759及其互作基因的应用
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caagagcggc ggccaccgca gcccttactg cacttgagcc gggcctcagc aggcacgtct 840
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gaggagctgg gaggacagag gagggccacc ccggctgctg cccctcaggt gtcaggagct 1260
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Claims (10)
1.一种与猪肌内脂肪相关的环状RNA,命名为circRNA_14759,序列与SEQ ID NO.1具有95%以上序列同源性。
2.一种检测肌内脂肪的试剂,其特征在于,试剂检测circRNA_14759和/或其互作基因的表达水平,互做基因为ssc-miR-370。
3.权利要求2所述的试剂,其特征在于,试剂采用测序技术、核酸杂交技术或核酸扩增技术检测样品中circRNA_14759或ssc-miR-370的表达水平。
4.权利要求2所述的试剂,其特征在于,试剂采用高通量测序技术、探针杂交技术、基因芯片技术或荧光定量PCR技术检测样品中circRNA_14759或ssc-miR-370的表达水平。
5.权利要求3所述的试剂,其特征在于,试剂包含检测circRNA_14759的探针或用于荧光定量PCR的引物,优选的,荧光定量PCR检测引物序列为SEQ ID NO.2和SEQ ID NO.3。
6.权利要求3所述的试剂,其特征在于,试剂包含特异性扩增ssc-miR-370的引物;优选的,特异性扩增ssc-miR-370的引物序列为SEQ ID NO.4。
7.根据权利要求2-6任意一项所述的试剂,其特征在于,样品为肌内脂肪组织。
8.下述任意一项应用:
circRNA_14759在预测或辅助预测猪肉品质中的应用;
circRNA_14759在制备预测或辅助预测猪肉品质试剂中的应用;
circRNA_14759在选育具有不同肉质品质猪中的应用。
权利要求2-6任一项所述试剂在预测或辅助预测猪肉品质中的应用;
权利要求2-6任一项所述试剂在制备预测或辅助预测猪肉品质试剂中的应用;权利要求2-6任意一项所述的试剂在选育具有不同肉质品质猪中的应用。
9.一种检测猪肌内脂肪含量的方法,包括:(1)选取猪肌内脂肪组织;(2)检测样品中circRNA_14759和/或其互作基因的表达量。
10.根据权利要求9所述的方法,其特征在于,互作基因选自下列中的一种或几种:ssc-miR-326、ssc-miR-140-3p、ssc-miR-328、ssc-miR-370、ssc-miR-7144-3p。
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