CN114703273A - Star及其调控基因的用途 - Google Patents
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Abstract
本发明涉及STAR及其调控基因的用途,具体涉及STAR及其调控基因MSTRG.9543在制备检测卵巢发育成熟的试剂中的应用。羊卵巢的发育和成熟是影响羊养殖场经济的重要因素,本申请通过高通量测序技术研究卵巢发育和成熟相关的基因,探索影响卵巢发育和成熟的分子机制,为畜牧生物育种提供研究基础。
Description
技术领域
本发明属于细胞工程和基因工程技术领域,具体涉及STAR及其调控基因的 用途。
背景技术
卵巢发育情况复杂,其中某一阶段出现问题就可能引发相关疾病的产生,影 响生育与繁殖,且现阶段发生卵巢相关疾病的概率变大,探究影响卵巢卵泡生长 发育等方面的机制也是非常重要的。已有研究表明许多非编码RNA如miRNA 参与调控卵巢发育过程,影响动物的繁殖能力并与卵巢组织相关疾病有很大关系, 例如,miRNA-124被证实参与卵巢分化,在雌性动物性腺中低的SOX9水平与 性腺分化成卵巢有关,在性腺分化过程中miRNA-124上调表达,靶SOX9基因 表达下调。
非编码RNA在开始被发现的一段时间内一直被认为是“转录噪音”,近年关 于非编码RNA的研究逐渐增多,其功能也越来越多的被发现,例如lncRNA、 microRNA、tRNA、snoRNA等。已有研究发现lncRNA虽然不具有编码蛋白质 的性质,但是能在转录和转录后水平影响基因发挥功能,其作为一类新的重要调 节因子广泛参与各种生理和病理过程。对卵母细胞分化和成熟、卵巢内各种细胞 发育、以及激素分泌和卵巢功能的发挥具有重要的调控作用。此外还与多囊卵巢 综合征(PCOS)等卵巢发育相关疾病的产生和治疗有关,因此lncRNA表达失 调常常导致多种疾病。
本申请选取中国本土湖羊品种为实验对象,采用RNA-seq技术对卵巢组织中 的lncRNA进行测序分析,比较1、3、8三个月龄的湖羊卵巢组织中lncRNA的 差异表达情况,利用生物信息学方法筛选出各阶段差异表达的lncRNA及其调控 基因,从而筛选出调控卵巢组织发育的lncRNA及其调控基因,探索lncRNA影 响卵巢发育的调控机制,为提高卵巢发育水平、增强繁殖能力和繁殖速度提供有 力依据。
发明内容
为了克服现有技术的缺点与不足,本发明的目的在于提供STAR在制备检测 卵巢发育水平试剂中的应用。
优选的,通过测序技术、核酸杂交技术或核酸扩增技术检测STAR基因的表 达水平。
优选的,核酸扩增技术采用一对特异性引物扩增STAR基因;核酸杂交包括 与STAR基因的核酸序列杂交的探针。
优选的,通过免疫方法检测STAR基因表达产物的表达水平。
优选的,通过ELISA检测试剂盒和/或胶体金检测试剂盒检测STAR基因达产 物的表达水平。
优选的,卵巢为羊卵巢。
一种与羊卵巢发育相关的lncRNA,所述lncRNA为MSTRG.9543,序列与 SEQ IDNO.1具有90%以上序列同源性。
优选的,MSTRG.9543序列与SEQ ID NO.1具有95%以上序列同源性;更 优选的,长链非编码RNA序列为SEQ ID NO.1。
MSTRG.9543用于制备检测卵巢发育水平试剂中的应用。
优选的,通过测序技术、核酸杂交技术或核酸扩增技术检测MSTRG.9543基 因的表达水平。
优选的,核酸扩增技术采用一对特异性引物扩增MSTRG.9543基因;核酸杂 交包括与STAR基因的核酸序列杂交的探针。
一种检测羊卵巢发育的试剂,试剂包含用于核酸扩增的引物对,检测 MSTRG.9543基因的表达水平。
优选的,用于核酸扩增的引物对序列为SEQ ID NO.2和SEQ ID NO.3。
优选的,试剂检测的样本为组织。
附图说明
图1为总RNA琼脂糖凝胶电泳结果;
图2为卵巢组织差异表达mRNA KEGG pathway富集分析(H1vs H3)图;
图3为卵巢组织差异表达mRNA KEGG pathway富集分析(H3vs H8)图
图4为1月龄VS3月龄卵巢组织差异表达lncRNA和mRNA共表达网络图,三角形 节点代表lncRNA,圆形节点代表mRNA;
图5为3月龄VS8月龄卵巢组织差异表达lncRNA和mRNA共表达网络图,三角形 节点代表lncRNA,圆形节点代表mRNA;
图6为差异表达基因qRT-PCR验证结果图。
具体实施方式
下面结合具体实施例,进一步阐述本发明,仅用于解释本发明,而不能理解 为对本发明的限制。本领域的普通技术人员可以理解:在不脱离本发明的原理和 宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范 围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法,通常 按照常规条件或按照厂商所建议的条件实施检测。
实施例1样品的采集剂RNA提取
1.1样品的采集
将三个月龄的羊屠宰后,将两侧的卵巢组织摘除,剪成小块组织样后迅速放 入冻存管中并投入液氮中保存,组织样温度维持低温后放入-80℃冰箱中保存, 整个过程保持无菌环境,所有器具消毒处理。试验设置为3组,采集1、3、8三 个月龄的湖羊卵巢组织,进行1月龄湖羊卵巢组织与3月龄湖羊卵巢组织(H1vs H3)、1月龄湖羊卵巢组织与8月龄湖羊卵巢组织(H3vs H8)差异的lncRNA 和mRNA的鉴定,每个组安排3个生物学重复。
1.2样本RNA的提取和质量检测
每个样品分别取出等量的卵巢组织用来提取RNA,保证无菌环境,并将得到 的所有RNA在-80℃低温保存。
提取RNA后,用NanoDrop 2000紫外分光光度计对所提RNA的纯度和浓度 进行检测,在1.5%琼脂糖凝胶上监测RNA降解和污染,尤其是DNA污染,测 定RNA的完整性。使用Bioanalyzer 2100检测样品RNA的质量情况,测定RIN 值。建库测序的要求是RNA总量不少于2ug,OD260nm和OD280nm两者比值 在1.9-2.1的范围内、RIN>=7且28S/18S>=0.7,浓度≥100ng/μL,以确保使用 合格的样品进行转录组测序,同时满足以上条件的样品可以进行下一步实验。
结果如图1表示,可以看出28S和18S条带清晰可见,各个样品结果中RIN ≥7、28S/18S≥1且OD260/280两者比值在1.9-2.1的范围内,表明RNA较完整, 污染较少,样品合格,符合测序要求。
实施例2测序及数据分析
构建cDNA文库:建立9个cDNA文库,分别为H1O1、H1O2、H1O3(1 月龄湖羊卵巢组织cDNA文库),H3O2、H3O3、H3O4(3月龄湖羊卵巢组织cDNA 文库),H8O1、H8O2、H8O3(8月龄湖羊卵巢组织cDNA文库)。
文库质检合格后,可以进行上机测序。利用IlluminaHiSeq平台进行对检测 合格的cDNA文库进行测序,对下机之后的原始数据(raw data)进行分析。
预测lncRNA:预测lncRNA首先需要完成基本筛选,再检测是否具有潜在 编码能力,经过这两部分筛选后得到的是lncRNA。首先筛选包含intergenic lncRNA,introniclncRNA,sense-lncRNA和anti-sense lncRNA等不同类型的转录 本。预测得到的四种lncRNA,鉴定得到37309个lncRNA,通过与已知lncRNA 数据库(lncRNAdb,NONCODE等)比对,鉴定到样本中的已知lncRNA 33924个, 其中lncRNA数量最多,达23072条,占总lncRNA数量的62.82%,3239个 antisense-lncRNA,9139个intronic-lncRNA和1859个sense-lncRNA。
表达水平分析及差异表达基因筛选:对样品中的Mapped Reads的数目和转 录本长度进行归一化,采用FPKM法(Fragments Per Kilobase of transcript per Millionfragments mapped)作为衡量mRNA和lncRNA表达水平的指标,在差异 基因筛选过程中(H1vs H3、H3vs H8),将Fold Change≥2且FDR<0.01作 为筛选标准。
1月龄湖羊卵巢基本未发育,3月龄湖羊卵巢未发育成熟,通过对两个阶段 (H1vsH3)的卵巢组织进行比较分析,将log2|Fold Change|≥2且FDR≤0.01 作为筛选标准。结果在1月龄和3月龄湖羊卵巢组织中共,筛选出6716个差异 表达的mRNAs(3377个上调,3339个下调)和1972个差异lncRNAs(1093个 上调,879个下调)。通过对3月(卵巢未发育成熟)、8月(卵巢发育成熟)龄 两个阶段的卵巢组织(H3vs H8)中表达的mRNA和lncRNA进行比对分析,经 过差异分析筛选出2903个差异表达mRNAs(1736个上调,1167个下调)和636 个差异lncRNAs(256个上调,380个下调)。
GO注释(Gene Ontology)包括生物过程(BP)、分子功能(MF)和细胞成 分(CC)三个方面,可以描述基因或基因产物功能的特定方面。
通过对1月龄和3月龄湖羊卵巢组织差异mRNA进行GO富集,以P≤0.05 筛选显著富集的通路,并使用R语言画图,发现在生物学过程方面差异基因主 要富集在黄体酮代谢过程(progesterone metabolic process)、卵巢卵泡发育(ovarian follicledevelopment)、类固醇激素介导的信号通路(steroid hormone mediated signalingpathway)、生殖细胞发育(germ cell development)、调节类固醇激素的 生物合成过程(regulation of steroid hormone biosynthetic process)、对促性腺激素 反应(response to gonadotropin)等条目;在分子功能方面,主要富集在生长因子 结合(growth factor binding)、胰岛素样生长因子受体结合(insulin-like growth factorreceptor binding)、雌激素受体结合(estrogen receptor binding)、细胞周期 蛋白依赖性蛋白丝氨酸/苏氨酸激酶活性(cyclin-dependent protein serine/threonine kinaseactivity)等条目;在细胞组分方面,主要富集在小核糖体亚基(small ribosomalsubunit)、电压门控钙通道复合物(voltage-gated calcium channel complex)、线粒体膜部分(mitochondrial membrane part)等条目。
通过对3、8月龄差异表达的mRNA进行GO注释,发现在生物学过程方面, 差异基因主要富集在雌激素代谢过程(estrogen metabolic process)、卵巢卵泡排 卵(ovulationfrom ovarian follicle)、减数分裂I(meiosis I)、卵母细胞发育(oocyte development)、卵子发生(oogenesis)、调节胰岛素样生长因子受体信号通路 (regulation of insulin-like growth factor receptor signaling pathway)、转化生长因 子β生产(transforming growth factor beta production)、排卵周期过程(ovulation cycleprocess)等条目;在分子功能方面,主要富集在转化生长因子β结合 (transforminggrowth factor beta binding)、胰岛素样生长因子结合(insulin-like growth factorbinding)、雌二醇17-β-脱氢酶活性(estradiol 17-beta-dehydrogenase activity)、钙依赖性ATP酶活性(calcium-dependent ATPase activity)等条目;在 细胞组分方面,主要富集在高尔基体相关的囊泡膜(Golgi-associated vesicle membrane)、层粘连蛋白复合物(laminin complex)等条目。
KEGG是通过生物信号途径对完全测序的基因组进行生物学解释的综合数 据库资源。对差异表达mRNA进行通路分析,结果富集在卵巢类固醇生成 (Ovariansteroidogenesis)、TGF-β信号通路、雌激素信号通路(Estrogen signaling pathway)、MAPK信号通路(MAPK signaling pathway)、NF-κB信号通路 (NF-kappa B signalingpathway)等通路中,每个通路对应的差异基因数目如图 所示。
KEGG通路富集分析表明,1、3月龄湖羊卵巢组织中差异表达mRNA参与 到多个与卵巢卵泡发育相关的通路中,富集在这些通路中的mRNA可能参与调 控湖羊两个阶段之间卵巢各部分的发育,以及影响性成熟等一系列现象的出现, 对于本研究中挖掘相关mRNA的功能具有重要的指导意义,结果见图2。
对3、8月龄湖羊卵巢中差异表达的mRNA进行KEGG富集分析,如图3所 示,差异表达基因富集在细胞周期(Cell cycle)、卵巢类固醇生成(Ovarian steroidogenesis)、p53信号通路(p53signaling pathway)、孕酮介导的卵母细胞成 熟(Progesterone-mediatedoocyte maturation)、VEGF信号通路(VEGF signaling pathway)等信号通路,其中富集在卵巢类固醇生成通路的显著性值 q-value=0.0092,且有11个基因富集在此通路中。孕酮介导的卵母细胞成熟通路 的q-value=0.019,14个基因富集在此通路。通过KEGG分析,发现差异mRNA 富集在多个与卵母细胞成熟、类固醇激素生成等通路中。
组间差异表达lncRNA靶基因预测:lncRNA靶基因预测共有两种方式,一 种是基于位置关系的cis靶基因预测,另一种是基于表达量关系的trans靶基因预 测。lncRNA能够调控其邻近基因的表达,主要根据lncRNA和基因的位置预测, 在lncRNA基因组位置上下游100kb范围内的mRNA预测为cis作用靶基因即顺 式调控靶基因。通过分析lncRNA和mRNA的表达量是否具有相关性来确定 lncRNA靶向的远距离蛋白编码基因,即trans反式作用靶基因。采用Pearson相 关系数法分析样本间lncRNA与mRNA的相关性,以相关性绝对值|PCC|≥0.9 和显著性p≤0.01为阈值筛选反式作用靶基因。
将差异mRNA进行功能注释后,筛选出与卵巢发育相关通路的GO条目,这 些条目中对应的差异mRNA即可能参与卵巢生长过程的调节,也是研究卵巢发 育的关键基因。将差异表达lncRNA的靶基因进行功能注释,并筛选差异lncRNA 对应的差异靶基因,两者均差异表达。结合差异mRNA和差异lncRNA靶基因 功能注释结果,经过筛选发现几个与卵巢发育密切相关的基因差异表达,1月龄 和3月龄差异表达的基因参与cAMP信号通路、卵巢类固醇生成通路、TGF-β 信号通路、PI3K-Akt信号通路、孕酮介导的卵母细胞成熟等通路和卵泡发育过程,并筛选到有几个lncRNA通过cis和trans调控作用靶向其中的一些基因,结 果如图4所示。3月龄和8月龄差异表达基因参与减数分裂I、卵母细胞发育、 卵子发生、胰岛素样生长因子结合等过程和胰岛素样生长因子受体信号通路、 Jak-STAT信号通路等途径,并筛选到几个lncRNA通过cis和trans调控作用靶向 其中的一些基因,结果如图5所示。MSTRG.9543及其调控的基因STAR成为我 们的候选基因,二者既参与了卵巢的生长过程(1月到3月),又参与了卵巢的 成熟过程(3月到8月)。
实施例3qRT-PCR检测验证
3.1测序结果的可靠性验证
本实验在两个比较组(H1vs H3、H3vs H8)之间随机选择差异表达的lncRNA 和mRNA进行PCR检测,每个基因3个重复,验证每个lncRNA和mRNA的表 达趋势,主要方法如下:
反转录
将低温保存的RNA样品取出,解冻后在PCR管中配置反转录体系。在体系中加 入以下试剂:
RNA样品 0.5μg
4×gDNA wiper Mix 2μl
Nuclease-free H2O 5.5μl
在42℃条件下反应2min,再加入5×HiScript II Q RT SuperMix IIa,2μl,以上共10μl。设置反应条件为25℃10min,50℃30min,85℃5min,全部完成后低 温-20℃保存备用。
PCR反应设置
PCR反应体系如下表,按步骤加入如下组分进行反应
表1 PCR体系中组分和体积
PCR数据分析及结果
利用2-△△Ct法计算所有基因在各个样本间的相对表达量,数据表示为平均数 ±标准差(Mean±SD),P<0.05表示该基因在两组间差异显著。结果表明,STAR、 CYP11A1、FSHR在3月龄卵巢中显著上调表达,MSTRG.283534.2显著下调; 在8月龄卵巢组织中,FST、INHBA、INHA、MSTRG.123289.5显著下调,STAR、 MSTRG.77987.3在显著上调,说明与测序结果一致,测序结果可靠。
表2. 1月龄和3月龄湖羊卵巢差异表达基因验证结果
表3 3月龄和8月龄湖羊卵巢差异表达基因验证结果
3.2候选基因的验证
本实验收集18只1月龄、18只3月龄、18只8月龄的湖羊,在两个比较组 (H1vs H3、H3vs H8)之间对差异表达的lncRNA和mRNA进行PCR检测, 每个基因3个重复,验证MSTRG.9543及其调控的基因STAR差异表达情况, 实验步骤同3.1。
PCR数据分析及结果,利用2-△△Ct法计算MSTRG.9543及基因STAR在各个 样本间的相对表达量,数据表示为平均数±标准差(Mean±SD),P<0.05表示该 基因在两组间差异显著。结果表明,相对于1月龄组,STAR在3月龄组卵巢中 显著上调表达,MSTRG.9543显著上调表达。并且,相对于3月龄组,MSTRG.9543 及基因STAR在8月龄卵巢组织中表现相同的趋势。
STAR是急性调节阶段中不可缺少的成分,其介导细胞中胆固醇从线粒体的 外膜到内膜的转移,形成第一种类固醇,在性激素合成过程中发挥重要调节作用, 表明差异lncRNA可能通过影响STAR介导卵巢激素合成。与1、3月龄相同, STAR基因在3、8月龄卵巢之间差异表达,表明STAR在整个卵巢发育中发挥 持续的调节作用,并受到lncRNA的调节,对卵巢发育具有重要的作用。
序列表
<110> 中国农业科学院北京畜牧兽医研究所
<120> STAR及其调控基因的用途
<130> P2022F0099-DJL
<150> 2021102450205
<151> 2021-03-05
<160> 21
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aaggttctgg aaggcatca 19
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aagggcacct gattatggta 20
<210> 11
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
acgctgttct gagtatcg 18
<210> 12
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gtctgtgcca gtgacaat 18
<210> 13
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cggagtgctt cacttcca 18
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gcaaggtcaa catttgctgt a 21
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tcacagtagt tggcgtggta 20
<210> 16
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
acctcggatg gaggttac 18
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ggattccctt agatgcaagc 20
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tttcagcatt agaggcacct ac 22
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gtgaagtcac acgccttatt 20
<210> 20
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atggagctgc aaagaatcg 19
<210> 21
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
taggtcatgc tgagtgagtt ag 22
Claims (10)
1.STAR在制备检测卵巢发育水平试剂中的应用。
2.根据权利要求1所述的应用,其特征在于,通过测序技术、核酸杂交技术或核酸扩增技术检测STAR基因的表达水平。
3.根据权利要求1所述的,其应用特征在于,核酸扩增技术采用一对特异性引物扩增STAR基因;核酸杂交包括与STAR基因的核酸序列杂交的探针。
4.根据权利要求1所述的应用,其特征在于,通过免疫方法检测STAR基因表达产物的表达水平。
5.一种与羊卵巢发育相关的lncRNA,所述lncRNA为MSTRG.9543,其特征在于,所述lncRNA序列与SEQ ID NO.1具有90%以上序列同源性。
6.MSTRG.9543用于制备检测卵巢发育水平试剂中的应用。
7.根据权利要求6所述的应用,其特征在于,通过测序技术、核酸杂交技术或核酸扩增技术检测MSTRG.9543基因的表达水平。
8.根据权利要求6所述的应用,其特征在于,酸扩增技术采用一对特异性引物扩增MSTRG.9543基因;核酸杂交包括与STAR基因的核酸序列杂交的探针。
9.一种检测羊卵巢发育或成熟的试剂,试剂包含用于核酸扩增的引物对,检测MSTRG.9543基因的表达水平。
10.根据权利要求9所述的试剂,其特征在于,核酸扩增的引物对序列为SEQ ID NO.2和SEQ ID NO.3。
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