CN110878360A - 基于asp基因表达量差异鉴定清溪乌鳖品系和日本品系的方法 - Google Patents
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Abstract
本发明公开了一种基于ASP基因表达量差异鉴定中华鳖清溪乌鳖品系和日本品系的方法,该方法通过对待鉴定中华鳖腹部皮肤ASP基因的表达量进行检测,若ASP基因相对表达量值≥0.04,则确定为日本品系,若ASP基因相对表达量值<0.04,则确定为清溪乌鳖品系。本发明提供了一种快速鉴定中华鳖日本品系和清溪乌鳖品系的方法。可用于中华鳖的遗传育种中,特别是乌质性状的选育。
Description
技术领域
本发明属于分子生物学领域,具体涉及一种基于ASP基因表达量差异鉴定清溪乌鳖品系和日本品系的方法。
背景技术
中华鳖(Pelodiscus sinensis)是我国重要的名特优水产养殖品种,具有高蛋白、低脂肪的营养特点,富含多种维生素和微量元素,营养价值高。中华鳖种质资源是其产业发展的源头,中华鳖日本品系(品种登记号:GS03-001-2007)和清溪乌鳖(品种登记号:GS01-003-2008)是经全国水产原种和良种审定委员会认定并经农业部公告的国家水产新品种。这2个中华鳖新品种具有各自独特的外部形态与生产性能特征,既是养殖生产的优良品种,也是中华鳖种质持续改良的宝贵种质资源。其中中华鳖日本品系背甲似青苔,底板似白玉,生长特别快,是目前全国广泛饲养的品种;清溪乌鳖最大特征是独特的乌黑腹部体色,口味、品质和营养价值远胜于普通鳖,富含具有食用价值和药用价值的黑色素,是中华鳖遗传育种研究的优良材料,已成为中华鳖各品系中珍贵的种苗品种,其乌质性状对提升中华鳖养殖效益、促进中华鳖品质改良具有重要的意义。
对于含乌质性状的物种来说,其食用价值主要在于体内丰富的黑色素,乌质性状表型更是市场经济价值提高的关键,也是种质资源改良的目标。目前,对乌鳖黑色素合成的调控研究尚未见报道,乌鳖乌质性状的分子机理有待深入研究。
发明内容
本发明所要解决的技术问题为:如何提供一种中华鳖中清溪乌鳖品系和日本品系的鉴定方法,从而为改善乌质性状育种提供依据。
本发明的技术方案为:基于ASP基因表达量差异鉴定清溪乌鳖品系和日本品系的方法,该方法通过对待鉴定中华鳖腹部皮肤ASP基因的表达量进行检测,若ASP基因相对表达量值≥0.04,则确定为日本品系,若ASP基因相对表达量值<0.04,则确定为清溪乌鳖品系。
进一步地,鉴定方法为:提取待鉴定中华鳖腹部皮肤组织RNA进行反转录,得到cDNA,然后以cDNA为模板进行qRT-PCR扩增,以GAPDH基因作为内参,其中ASP基因扩增的引物对为:ASP-F:5′-TCTCTGCTGCATTTTGCTTTCA-3′,ASP-R:5′-AGATGGGTGGAAGATCTGGGA-3′;GAPDH基因扩增的引物对为:GAPDH-F:5′-CCTGGTATGACAATGAGTT-3′,GAPDH-R:5′-GTGCCTGGTTTATTCCTT-3′;ASP基因相对于GAPDH基因的表达水平计算使用2-ΔCt,当ASP基因相对表达量值≥0.04可确定为日本品系,当ASP基因相对表达量值在0.04以下的为清溪乌鳖品系。
进一步地,qRT-PCR扩增的条件为:
反应体系:6.25μL SYBR预混染料、1μL cDNA模板、0.25μL各引物对、4.25μLddH2O,总体积为12μL;
扩增参数:95℃30s预变性,95℃15s变性、55℃30s退火、72℃30s延伸,40个循环后从65℃升温到95℃获得溶解曲线。
本发明通过转录组测序分析发现,腹部体色性状差异明显的中华鳖日本品系和清溪乌鳖品系中,黑色素合成主要调控途径α-MSH–MC1R–cAMP–TYR信号通路中反向调控因子ASP表达差异显著。而通过荧光定量PCR验证,其中中华鳖日本品系的相对表达量2-ΔCt值为0.2089±0.0269,清溪乌鳖的相对表达量2-ΔCt值为0.0201±0.0080,日本品系比清溪乌鳖的ASP相对表达量高达10.42倍。ASP作为黑色素合成的关键信号通路α-MSH–MC1R–cAMP–TYR的反向调控因子,在通路中拮抗MC1R与α-MSH的结合阻抑cAMP激活的级联通路、抑制真黑素的形成,其中清溪乌鳖腹部皮肤中的基因表达量显著低于在日本品系中的表达量,表明其抑制黑色素合成的作用降低,与清溪乌鳖腹部乌质性状的形成呈正相关。因此,ASP基因表达量可作为中华鳖这两个腹部体色性状截然不同的品系乌质性状鉴定的指标进行应用。
与现有技术相比,本发明具有以下有益效果:
本发明提供了一种快速鉴定中华鳖日本品系和清溪乌鳖品系的方法。可用于中华鳖的遗传育种中,特别是乌质性状的选育。
具体实施方式
实施例1
取冻存提取的中华鳖两品系腹部皮肤组织RNA样本进行反转录,取2μg的RNA按TAKARA PrimescriptTMⅡ1st strand cDNA synthesis Kit说明书进行cDNA反转录。反转录好的cDNA稀释10倍作为模板进行qRT-PCR检测,甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因作为内参,采用Takara的SYBR Green I real-timePCR Master Mix进行反应,在12μL反应混合物加入6.25μL SYBR预混染料、1μL cDNA模板、0.25μL各引物和4.25μL ddH2O。混合均匀后在Bio-Rad CFX96上进行扩增,扩增条件为95℃30s预变性,95℃15s变性、55℃30s退火、72℃30s延伸,40个循环后从65℃升温到95℃获得溶解曲线。
基因检测的引物序列如下:
ASP-F:5′-TCTCTGCTGCATTTTGCTTTCA-3′,
ASP-R:5′-AGATGGGTGGAAGATCTGGGA-3′;
GAPDH-F:5′-CCTGGTATGACAATGAGTT-3′,
GAPDH-R:5′-GTGCCTGGTTTATTCCTT-3′。
ASP和GAPDH扩增片段大小分别为113bp和100bp。ASP相对于GAPDH的表达水平计算使用2-ΔCt(ΔCt=目的基因ASP的Ct值-内参基因GAPDH的Ct值),执行三个重复实验的获得平均值±标准差为,并采用单因素方差分析进行统计学分析,用IBM SPSS Statistics20软件进行显著性差异检验,所得结果显示黑色素合成通路中反向调控因子ASP基因表达量具有显著性差异(p<0.01,n=3),其中中华鳖日本品系的相对表达量2-ΔCt值为0.2089±0.0269,清溪乌鳖的相对表达量2-ΔCt值为0.0201±0.0080,日本品系比清溪乌鳖的ASP相对表达量高达10.42倍。ASP作为黑色素合成的关键信号通路α-MSH–MC1R–cAMP–TYR的反向调控因子,在通路中拮抗MC1R与α-MSH的结合阻抑cAMP激活的级联通路、抑制真黑素的形成,其中清溪乌鳖腹部皮肤中的基因表达量显著低于在日本品系中的表达量,表明其抑制黑色素合成的作用降低,与清溪乌鳖腹部乌质性状的形成呈正相关。因此,ASP基因表达量可作为中华鳖这两个腹部体色性状截然不同的品系乌质性状鉴定的指标进行应用,经本实施方法测定的ASP相对表达量值≥0.04可确定为日本品系,在0.04以下的为清溪乌鳖品系。
表1中华鳖日本品系和清溪乌鳖品系腹部皮肤ASP相对表达量
以上所述实施例仅表达了本申请的具体实施方式,其描述较为具体和详细,但并不能因此而理解为对本申请保护范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本申请技术方案构思的前提下,还可以做出若干变形和改进,这些都属于本申请的保护范围。
序列表
<110> 浙江万里学院
<120> 基于ASP基因表达量差异鉴定清溪乌鳖品系和日本品系的方法
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Claims (3)
1.基于ASP基因表达量差异鉴定清溪乌鳖品系和日本品系的方法,其特征在于,该鉴定方法通过对待鉴定中华鳖腹部皮肤ASP基因的表达量进行检测,若ASP基因相对表达量值≥0.04,则确定为日本品系,若ASP基因相对表达量值<0.04,则确定为清溪乌鳖品系。
2.根据权利要求1所述的方法,其特征在于,提取待鉴定中华鳖腹部皮肤组织RNA进行反转录,得到cDNA,然后以cDNA为模板进行qRT-PCR扩增,以GAPDH基因作为内参,其中ASP基因扩增的引物序列如SEQ ID No.1和SEQ ID No.2所示;GAPDH基因扩增的引物序列如SEQID No.3和SEQ ID No.4所示;ASP基因相对于GAPDH基因的表达水平计算使用2-ΔCt,当ASP基因相对表达量值≥0.04可确定为日本品系,当ASP基因相对表达量值在0.04以下的为清溪乌鳖品系。
3.根据权利要求2所述的方法,其特征在于,qRT-PCR扩增的条件为:
反应体系:6.25μL SYBR预混染料、1μL cDNA模板、0.25μL各引物对、4.25μL ddH2O,总体积为12μL;
扩增参数:95℃30s预变性,95℃15s变性、55℃30s退火、72℃30s延伸,40个循环后从65℃升温到95℃获得溶解曲线。
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