CN116377104B - 一种快菜高温胁迫内参基因及其引物及应用 - Google Patents
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Abstract
本发明公开了一种快菜高温胁迫内参基因及其引物及应用。所述内参基因为BraSCE1,其核苷酸序列如SEQ ID NO.1所示。用于扩增BraSCE1基因的引物包括核苷酸序列如SEQ IDNO.2所示的正向引物和核苷酸序列如SEQ ID NO.3所示的反向引物。内参基因为BraSCE1及引物在快菜高温胁迫荧光定量PCR检测、快菜耐热性基因筛选或研究中的应用。本发明通过选取11个候选内参基因,利用实时荧光定量qRT‑PCR技术检测其在高温胁迫处理叶片中的表达水平,通过软件分析得到快菜高温胁迫下最适合的内参基因为BraSCE1,本发明为快菜高温胁迫基因表达研究奠定了基础。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种快菜高温胁迫内参基因及其引物及应用。
背景技术
快菜,也称早熟不结球白菜或速生白菜,食用部分主要为幼苗或半成株,原产中国,目前已是世界上广泛栽培和食用的叶菜类蔬菜。快菜喜冷凉气候,当遭受到高温天气时容易会出现出苗率低、生长缓慢、叶片变黄、产量下降等问题目前,实时荧光定量(Reversetranscription quantitative polymerase chain reaction,RT-qPCR)是最常用于检测基因转录水平的方法,但它依赖于合适的内参基因对功能基因的相对表达定量进行数据标准化。内参基因必须在特定的实验条件下满足稳定表达的要求。在快菜高温胁迫机理研究时,经常会运用实时荧光定量技术分析和验证基因的表达水平,而表达分析结果的准确性在很大程度上取决于内参基因的稳定性。理想的内参基因不受试验条件的影响,而在实际应用中,这样的理想内参基因很难存在。常用的内参基因有Actin、18sRNA、GAPDH等,但它们不适用于所有条件。因此筛选稳定表达的内参基因是实时荧光定量结果准确的关键因素。
目前,针对快菜高温胁迫条件下内参基因稳定性筛选的研究还未见报道,因此,急需筛选合适的高温胁迫内参基因,为后续快菜相关基因表达研究奠定基础。
发明内容
本发明的目的在于克服现有技术的缺点,提供一种快菜高温胁迫内参基因及其引物及应用。
本发明的目的通过以下技术方案来实现:一种快菜高温胁迫内参基因,所述内参基因为BraSCE1,其核苷酸序列如SEQ ID NO.1所示。
用于扩增BraSCE1基因的引物包括核苷酸序列如SEQ ID NO.2所示的正向引物和核苷酸序列如SEQ ID NO.3所示的反向引物。
BraSCE1基因在快菜高温胁迫荧光定量PCR检测中的应用。
BraSCE1基因在快菜耐热性基因筛选或研究中的应用。
用于扩增BraSCE1基因的引物在快菜高温胁迫荧光定量PCR检测中的应用。
用于扩增BraSCE1基因的引物在快菜耐热性基因筛选或研究中的应用。
本发明具有以下优点:本发明通过选取11个候选内参基因BraEF1B、BraEF1α、BraUBQ、BraCYP、BraTUA、BraTUB、Bra40sRPS14、Bra60sRPL10、BraActin、BraSCE1、BraRPT4A,各候选内参基因序列及其ID信息来源于BRAD数据库(http://brassicadb.cn/)中的白菜基因组数据(V1.5)。利用实时荧光定量qRT-PCR技术检测其在高温胁迫处理叶片中的表达水平,通过GeNorm、NormFinder、BestKeeper和ReFinder软件分析得到快菜高温胁迫下最适合的内参基因为BraSCE1,本发明为快菜高温胁迫基因表达研究奠定了基础。
具体实施方式
下面结合实施例对本发明做进一步的描述,本发明的保护范围不局限于以下所述:
1.试验材料及处理:
以苗期人工模拟高温胁迫和田间自然高温鉴定均表现为耐热的抗热5号大白菜(品种来自海南林忠民菜种行有限公司)和热敏的武蔬快菜(品种来自武汉蔬博农业科技有限公司)为试验材料。选择饱满的种子进行穴盘育苗,每穴2粒种子,穴盘规格为16孔。植株长出1片真叶时进行间苗,每孔留苗1株。当幼苗长至3叶1心时,移入温度25℃/18℃(昼/夜)、12h/12h(昼/夜)、光照80%、湿度70%的人工气候箱中预培养3d,之后进行高温处理。其中,将一半幼苗移入另一人工气候箱内,设置温度为37℃/27℃(昼/夜)进行高温胁迫处理,另一半仍然培养在25℃/18℃(昼/夜)的人工气候箱内(对照)。除温度环境不同外,高温胁迫处理和对照处理的其他环境条件均与预培养时保持一致。取37℃高温胁迫处理不同时间(0h、0.5h、1h、3h、6h、12h、24h、48h)的植株叶片。每个处理10株,设3次生物学重复,对照为未进行任何高温处理的材料。
2.试验方法:
参考Trizol试剂说明书(北京全式金生物技术有限公司)分别提取总RNA。利用cDNA合成试剂盒(北京全式金生物技术有限公司)去除基因组DNA 并反转录合成cDNA。cDNA稀释至100ng/μL作为RT-qPCR模板。参考Premix Ex TaqTM(Perfect Real Time)(TaKaRa,Japan)试剂盒进行实时荧光定量分析,引物组合见表1。
表1候选内参基因及引物信息
反应程序:94℃,30s,[94℃20s;56℃,30s,72℃,20s]×40个循环;溶解曲线程序:65℃加热至90℃,5s。每次反应要重复三次。PCR完成后,经自动分析,查看基因扩增情况,导出相应的域值循环数,即Ct值,详见表2。
表2候选内参基因在高温胁迫不同时间耐热和热敏快菜叶片中Ct平均值
表3高温胁迫不同时间候选内参基因在耐热和热敏快菜叶片中的表达稳定性
得到Ct值后,使用GeNorm、Normfinder、Bestkeeper和ReFinder软件对各候选内参基因稳定性进行分析。
3.实验结果
稳定性评价:GeNorm软件计算每个候选基因表达稳定度M值,M值越小,表明稳定性越高;反之M值越大,越不稳定。GeNorm软件稳定性分析由强到弱排序为:BraEF1α=BraSCE1>BraUBQ>BraTUB>Bra60sRPL10>Bra40sRPS14>BraRPT4A>BraActin>BraEF1B>BraCYP>BraTUA。
NormFinder软件结合组内方差与组间方差计算出各候选内参基因稳定值M,M值越小,表明内参基因越稳定;反之M值越高稳定性越差。NormFinder软件稳定性分析由强到弱排序为:BraSCE1>BraEF1α>Bra60sRPL10>BraTUB>BraUBQ>BraActin>BraEF1B>BraRPT4A>Bra40sRPS14>BraCYP>BraTUA。
Bestkeeper软件可直接输入Ct值,通过计算标准偏差和变异系数值的大小比较各内参基因的稳定性,标准差和变异系数越小,稳定性越好。Bestkeeper软件稳定性分析由强到弱排序为:BraSCE1>BraEF1α>Bra60sRPL10>BraTUB>BraEF1B>BraRPT4A>Bra40sRPS14>BraActin>BraUBQ>BraTUA>BraCYP。
采用ReFinder软件对GeNorm、NormFinder、Bestkeeper法分析得到的稳定性排名求几何平均值,得到综合指数排名,该指数越小,说明内参基因表达越稳定。结果如表3所示,高温胁迫中基因的稳定性为:BraSCE1>BraEF1α>Bra60sRPL10>BraTUB>BraUBQ>BraEF1B>BraActin>Bra40sRPS14=BraRPT4A>BraCYP>BraTUA。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都涵盖在本发明的保护范围之内。
Claims (3)
1.内参基因BraSCE1作为高温胁迫下荧光定量PCR检测快菜基因表达的内参基因的应用,其特征在于,所述内参基因为BraSCE1,其核苷酸序列如SEQ ID NO.1所示。
2.荧光定量PCR检测高温胁迫下快菜内参基因表达的引物,其特征在于:所述内参基因为BraSCE1,其核苷酸序列如SEQ ID NO.1所示,用于扩增BraSCE1基因的引物包括核苷酸序列如SEQ ID NO.2所示的正向引物和核苷酸序列如SEQ ID NO.3所示的反向引物。
3.根据权利要求2所述的引物在高温胁迫下荧光定量PCR检测快菜基因表达中的应用。
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