CN103981257B - 用于蝇蛹金小蜂多样性分析的微卫星引物及其用途和分析方法 - Google Patents
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Abstract
本发明涉及一种用于蝇蛹金小蜂多样性分析的微卫星引物及其用途和分析方法,分析方法包括如下步骤:(1)基因组DNA提取;(2)TP-M13-SSR分子标记检测;(3)进行遗传结构分析。该组引物是从样本中提取DNA,筛选微卫星位点,从而设计引物。该组引物PCR扩增产物稳定、多态性好,可用于多样性及群体遗传结构的分析。
Description
技术领域
本发明涉及昆虫进化领域,具体涉及一种用于蝇蛹金小蜂多样性分析的微卫星引物及其用途和分析方法,可用于蝇蛹金小蜂群体遗传多样性分析。
背景技术
寄生蜂是害虫的重要天敌组成,在控制害虫种群密度中起着重要的调控作用。在生产实践中,应用并适当维护寄生蜂的密度,可长期将害虫种群密度控制在一定阈值以内。充分发挥生物防治不污染环境,不杀伤天敌,且具有持续控灾效果的优势,可以达到保护生态环境,保护生物多样性,持续控制虫害的目标。蝇蛹金小蜂Pachycrepoideusvindemmiae(Rondani)是家蝇、实蝇和果蝇等蝇类蛹期常见寄生蜂种类,属于小蜂总科Chalcidoidea,金小蜂科Pteromalidae,金小蜂亚科Pteromalinae,在对蝇类害虫的生物防治上起着非常重要的作用,具有很大的利用潜力。家蝇是重要的卫生害虫,在我国广泛分布,严重威胁食品和卫生安全;果蝇和实蝇是樱桃、杨梅、柑橘、苦瓜等果蔬上的重要害虫,严重影响到果蔬的安全生产和销售。
微卫星DNA又称简单序列重复(SimpleSequenceRepeats,SSR)和短串连重复(ShortTandemRepeats,STRs),一般以2-6个碱基为核心序列,首尾相连组成的串联重复序列。微卫星DNA序列被分为3种类型:单一型(pure)、复合型(compound)和间断型(interrupted)。重复序列两侧的DNA序列保守性很强。这种串联重复序列存在于几乎所有真核生物的基因组中,且呈随机均匀分布。它们不仅大量分布于基因的间隔区和内含子中,而且还分布于基因的外显子和调控区(如启动子、增强子)。例如,完整的人类基因组序列显示,微卫星标记约占3%。在染色体上,除着丝粒及端粒区域外,其它区域均广泛分布有微卫星位点。其重复单位数目的改变可以引起相当高的多态性,但突变率仅为0.5×10-4—5.0×10-4,在谱系中可以稳定地遗传,是一种很好的遗传标志。到目前为止,国际上还没有一套用以分析蝇蛹金小蜂遗传结构的微卫星引物。
发明内容
本发明的目的在于提供一种用于蝇蛹金小蜂多样性分析的微卫星引物及其用途和分析方法,并进一步给出了应用该引物的PCR测定方法。具体技术方案如下:
一种用于蝇蛹金小蜂多样性分析的微卫星引物,其核苷酸序列为:
BL1F:GGGGTGTGTGAGATAACGC,BL1R:GGAGGCGAAATTTGTTGCT;
BL2F:CGTCCATTGGAGAGACATCG,BL2R:TGTATATCGCGCACGGACC;
BL3F:AGCCATCGTGATTTCTCCG,BL3R:CGATACACGCGCGTCTATTC;
BL4F:AGCGAGCAAAGGTAGTGGTG,BL4R:CACCGACCATCGTCATTATCT;
BL5F:CGACGTAAAAAGTCATTACGAGTC,BL5R:ATTTCCAATTTGGCACGGTC;
BL6F:GCATCGTCTCCTGAACAATC,BL6R:GATACCACTCGCACGAGGT;
BL7F:GCGAAGGTGAAACGGAGAA,BL7R:ATAGGCATACGTGCGACAGC;
BL8F:CGTTTCTGTTTGTCATCGACAG,BL8R:AGATGGTTCGGCGATAAAGA;
BL9F:TCAGCATTAAATGGCGTCG,BL9R:CACTTGAGCGCGTTCAATC;
BL10F:GTCTGCCTCGTCTCGGATT,BL10R:AGCGAGCGAGTGAGAGAGTAAG。
进一步地,
引物BL1重复基序为(TC)12,溶解温度Tm为55.5℃,扩增片段大小为162-174;
引物BL2重复基序为(CT)11,溶解温度Tm为55.7℃,扩增片段大小为156-169;
引物BL3重复基序为(TC)13,溶解温度Tm为55.4℃,扩增片段大小为166-182;
引物BL4重复基序为(AG)10TA(AG)7,溶解温度Tm为51.7℃,扩增片段大小为211-215;
引物BL5重复基序为(AG)8,溶解温度Tm为52.9℃,扩增片段大小为130-149;
引物BL6重复基序为(AG)11,溶解温度Tm为51.8℃,扩增片段大小为128-174;
引物BL7重复基序为(AG)9,溶解温度Tm为53.7℃,扩增片段大小为157-167;
引物BL8重复基序为(AG)10,溶解温度Tm为53.0℃,扩增片段大小为155-177;
引物BL9重复基序为(GA)11,溶解温度Tm为54.4℃,扩增片段大小为172-184;
引物BL10重复基序为(GA)11,溶解温度Tm为55.7℃,扩增片段大小为282-292。
上述用于蝇蛹金小蜂多样性分析的微卫星引物的用途,用于蝇蛹金小蜂群体遗传结构分析。
上述用于蝇蛹金小蜂多样性分析的微卫星引物的用途的分析方法,包括如下步骤:
(1)基因组DNA提取;
(2)TP-M13-SSR分子标记检测;
(3)进行遗传结构分析。
进一步地,步骤(1)中具体包括如下步骤:
1)从样本中取出个体;
2)容器中研磨成粉末,加入BufferDigestion,震荡混匀;
3)水浴至细胞完全裂;
4)加入BufferPA,充分颠倒混匀;
5)离心,将上清液转移到新的容器;
6)加入等体积的异丙醇,充分混匀,室温放置;
7)离心,弃上清;
8)加入乙醇,颠倒漂洗,离心弃上清;
9)开盖室温倒置至残留的乙醇完全挥发;
10)得到的DNA用TEBuffer溶解;
11)收集DNA,保存。
进一步地,步骤(1)中采用动物基因组DNA抽提试剂盒进行DNA提取。
进一步地,步骤(3)中用popgen3.2进行等位基因数、有效等位基因数、基因多样性等分析。
进一步地,步骤1)中从样本中取出60个体,用双蒸水浸泡清洗。
进一步地,步骤2)中加到1.5ml离心管中,研磨成粉末,加入400μLBufferDigestion,震荡混匀。
进一步地,步骤3)65℃水浴1小时至细胞完全裂;步骤4)加入200μLBufferPA,充分颠倒混匀,置于-20℃冰箱放置5min;步骤5)室温10000rpm离心5min,将500-550μL上清液转移到新的1.5ml离心管中;步骤6)加入等体积的异丙醇,颠倒5-8次使之充分混匀,室温放置2-3min;步骤7)室温10000rpm离心5min,弃上清;步骤8)加入1ml75%乙醇,颠倒漂洗1-3min,10000rpm离心2min,弃上清,且此步骤8)重复2次;步骤9)开盖室温倒置5-10min至残留的乙醇完全挥发;步骤10)得到的DNA用20μLTEBuffer溶解;步骤11)收集DNA,-20℃保存。
与目前现有技术相比,本发明引物是从样本中提取DNA,筛选微卫星位点,从而设计引物。该组引物PCR扩增产物稳定、多态性好,可用于多样性及群体遗传结构的分析。
具体实施方式
下面对本发明进行详细描述,其为本发明多种实施方式中的一种优选实施例。
一组用于蝇蛹金小蜂多样性分析的微卫星引物,进一步地微卫星引物的核苷酸序列及指标参数如表1所示:
表1蝇蛹金小蜂微卫星引物及对应的指标参数
其中,所述的微卫星引物用于蝇蛹金小蜂群体遗传结构分析。包括以下步骤:基因组DNA提取;TP-M13-SSR分子标记检测;用popgen3.2进行等位基因数、有效等位基因数、基因多样性等分析。
实施例1
1、引物合成:构建蝇蛹金小蜂基因组文库,筛选出多态性丰富的微卫星引物,送上海生工合成10对引物,序列如下:
2、基因组DNA提取:采用上海生工生物工程技术服务有限公司的动物基因组DNA抽提试剂盒进行DNA提取:
1)从样本中取出60个体,用双蒸水浸泡清洗。
2)加到1.5ml离心管中,研磨成粉末,加入400μLBufferDigestion,震荡混匀
3)65℃水浴1小时至细胞完全裂。
4)加入200μLBufferPA,充分颠倒混匀,置于-20℃冰箱放置5min。
5)室温10000rpm离心5min,将上清液(500-550μL)转移到新的1.5ml离心管中。
6)加入等体积的异丙醇,颠倒5-8次使之充分混匀,室温放置2-3min。
7)室温10000rpm离心5min,弃上清。
8)加入1ml75%乙醇,颠倒漂洗1-3min,10000rpm离心2min,弃上清(此步骤重复2次)。
9)开盖室温倒置5-10min至残留的乙醇完全挥发。
10)得到的DNA用20μLTEBuffer(75℃预热)溶解。
11)收集DNA,-20℃保存。
3、TP-M13-SSR技术
以提取的DNA为模板,进行PCR扩增,反应体系包括:
反应程序:
94℃预变性5min;
94℃变性30s,筛选的温度30s,72℃延伸30s,27个循环;
94℃变性30s,53℃30s,72℃延伸30s,10个循环;
72℃终延伸8min。
所得产物按不同荧光信号组合后送上海点晶生物科技有限公司,以ABI3700测序仪进行毛细管电泳。
对个体分型得到的结果用Genescan3.7分析软件并以LIZ500为内标进行个体微卫星基因型判定。参数信息见表2。
表210个微卫星位点的相关信息
用本发明的引物对60个蝇蛹金小蜂基因组DNA进行扩增,遗传多样性分析结果表明每个微卫星位点的等位基因数目从3到7不等,平均等位基因数目为5.5个,期望杂合度的范围从0.369到0.775。由此说明,本发明的微卫星引物可以用于蝇蛹金小蜂多样性和群体遗传结构分析。本发明提供了一组应用于蝇蛹金小蜂多样性分析的微卫星引物,具有PCR扩增结果稳定,多态性高等特点,有很好的应用价值。
上面对本发明进行了示例性描述,显然本发明具体实现并不受上述方式的限制,只要采用了本发明的方法构思和技术方案进行的各种改进,或未经改进直接应用于其它场合的,均在本发明的保护范围之内。
Claims (9)
1.一种用于蝇蛹金小蜂多样性分析的微卫星引物,其特征在于,其核苷酸序列为:BL1F:GGGGTGTGTGAGATAACGC,BL1R:GGAGGCGAAATTTGTTGCT;BL2F:CGTCCATTGGAGAGACATCG,BL2R:TGTATATCGCGCACGGACC;BL3F:AGCCATCGTGATTTCTCCG,BL3R:CGATACACGCGCGTCTATTC;BL4F:AGCGAGCAAAGGTAGTGGTG,BL4R:CACCGACCATCGTCATTATCT;BL5F:CGACGTAAAAAGTCATTACGAGTC,BL5R:ATTTCCAATTTGGCACGGTC;BL6F:GCATCGTCTCCTGAACAATC,BL6R:GATACCACTCGCACGAGGT;BL7F:GCGAAGGTGAAACGGAGAA,BL7R:ATAGGCATACGTGCGACAGC;BL8F:CGTTTCTGTTTGTCATCGACAG,BL8R:AGATGGTTCGGCGATAAAGA;BL9F:TCAGCATTAAATGGCGTCG,BL9R:CACTTGAGCGCGTTCAATC;BL10F:GTCTGCCTCGTCTCGGATT,BL10R:AGCGAGCGAGTGAGAGAGTAAG;
引物BL1重复基序为(TC)12,溶解温度Tm为55.5℃,扩增片段大小为162-174;
引物BL2重复基序为(CT)11,溶解温度Tm为55.7℃,扩增片段大小为156-169;
引物BL3重复基序为(TC)13,溶解温度Tm为55.4℃,扩增片段大小为166-182;
引物BL4重复基序为(AG)10TA(AG)7,溶解温度Tm为51.7℃,扩增片段大小为211-215;
引物BL5重复基序为(AG)8,溶解温度Tm为52.9℃,扩增片段大小为130-149;
引物BL6重复基序为(AG)11,溶解温度Tm为51.8℃,扩增片段大小为128-174;
引物BL7重复基序为(AG)9,溶解温度Tm为53.7℃,扩增片段大小为157-167;
引物BL8重复基序为(AG)10,溶解温度Tm为53.0℃,扩增片段大小为155-177;
引物BL9重复基序为(GA)11,溶解温度Tm为54.4℃,扩增片段大小为172-184;
引物BL10重复基序为(GA)11,溶解温度Tm为55.7℃,扩增片段大小为282-292。
2.如权利要求1所述用于蝇蛹金小蜂多样性分析的微卫星引物的用途,其特征在于,用于蝇蛹金小蜂群体遗传结构分析。
3.如权利要求2所述用于蝇蛹金小蜂多样性分析的微卫星引物的用途的分析方法,其特征在于,包括如下步骤:
(1)基因组DNA提取;
(2)TP-M13-SSR分子标记检测;
(3)进行遗传结构分析。
4.如权利要求3所述用于蝇蛹金小蜂多样性分析的微卫星引物的用途的分析方法,其特征在于,步骤(1)中具体包括如下步骤:
1)从样本中取出个体;
2)容器中研磨成粉末,加入BufferDigestion,震荡混匀;
3)水浴至细胞完全裂解;
4)加入BufferPA,充分颠倒混匀;
5)离心,将上清液转移到新的容器;
6)加入等体积的异丙醇,充分混匀,室温放置;
7)离心,弃上清;
8)加入乙醇,颠倒漂洗,离心弃上清;
9)开盖室温倒置至残留的乙醇完全挥发;
10)得到的DNA用TEBuffer溶解;
11)收集DNA,保存。
5.如权利要求4所述用于蝇蛹金小蜂多样性分析的微卫星引物的用途的分析方法,其特征在于,步骤(1)中采用动物基因组DNA抽提试剂盒进行DNA提取。
6.如权利要求4或5所述用于蝇蛹金小蜂多样性分析的微卫星引物的用途的分析方法,其特征在于,步骤(3)中用popgen3.2进行等位基因数、有效等位基因数、基因多样性等分析。
7.如权利要求4或5所述用于蝇蛹金小蜂多样性分析的微卫星引物的用途的分析方法,其特征在于,步骤1)中从样本中取出60个体,用双蒸水浸泡清洗。
8.如权利要求4或5所述用于蝇蛹金小蜂多样性分析的微卫星引物的用途的分析方法,其特征在于,步骤2)中加到1.5ml离心管中,研磨成粉末,加入400μLBufferDigestion,震荡混匀。
9.如权利要求4或5所述用于蝇蛹金小蜂多样性分析的微卫星引物的用途的分析方法,其特征在于,步骤3)65℃水浴1小时至细胞完全裂解;步骤4)加入200μLBufferPA,充分颠倒混匀,置于-20℃冰箱放置5min;步骤5)室温10000rpm离心5min,将500-550μL上清液转移到新的1.5ml离心管中;步骤6)加入等体积的异丙醇,颠倒5-8次使之充分混匀,室温放置2-3min;步骤7)室温10000rpm离心5min,弃上清;步骤8)加入1ml75%乙醇,颠倒漂洗1-3min,10000rpm离心2min,弃上清,且此步骤8)重复2次;步骤9)开盖室温倒置5-10min至残留的乙醇完全挥发;步骤10)得到的DNA用20μLTEBuffer溶解;步骤11)收集DNA,-20℃保存。
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