CN116287372B - 一种鉴定圆果杜英的dna条形码、鉴定方法与应用 - Google Patents
一种鉴定圆果杜英的dna条形码、鉴定方法与应用 Download PDFInfo
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Abstract
本发明提供了一种鉴定圆果杜英的DNA条形码、鉴定方法与应用,属于物种鉴定技术领域,所述DNA条形码包括ycf1、trnS‑atpA和ITS基因序列中的一种或多种。本发明首次提供的鉴定圆果杜英的DNA条形码有利于实现圆果杜英的分子鉴定,有效缩短鉴定的时间,且本发明利用ycf1、trnS‑atpA、ITS的联合基因片段鉴定效率高,鉴定效率为100%,极大地提高了圆果杜英分子鉴定的成功率。本发明的鉴定方法利用检测引物或试剂盒进行PCR扩增获得圆果杜英ycf1、trnS‑atpA、ITS序列,然后将扩增序列应用于构建杜英属的系统发育树,实现对圆果杜英的精准鉴定,且鉴定方法简单、快速、效率高、重复性好。
Description
技术领域
本发明属于物种鉴定技术领域,尤其涉及一种鉴定圆果杜英的DNA条形码、鉴定方法与应用。
背景技术
圆果杜英是杜英科杜英属植物。圆果杜英的种子(包括内果皮)是串珠(金刚菩提)的重要原材料,由其串在一起制作成念珠,是东方宗教的重要元素。因其较高的经济价值,圆果杜英被列入濒危野生动植物种国际贸易公约(CETS),是我国粤港澳海关检疫执法常见送检植物。但是市场上金刚菩提的来源极其复杂,如杜英属的毛果杜英,褐毛杜英的果实也被当做圆果杜英来销售。因此,急需开发有效的圆果杜英鉴定方法。然而传统的鉴定方法均存在不同程度的局限,如性状鉴定主要依据叶片的形状,子房几室来区分物种。不同物种间的性状会有重叠,种间特征性状界限模糊,分类鉴定较为困难。其次鉴定人员受物种采摘的生境变化、物种生长状况差异和个人鉴定经验的影响,其鉴定结果常常缺乏准确性和可重复性。因此开发一种更简便、快捷、准确、高效的圆果杜英鉴别方法迫在眉睫。
DNA条形码(DNA barcoding)是指生物体内能够代表该物种的、标准的、易扩增且相对较短的DNA片段,该段DNA序列能快速、准确的鉴定物种。2003年加拿大动物学家PaulHebert首次提出DNA条形码的概念,之后DNA条形码逐渐应用于植物体中。目前我国已有大量的学者相继对植物进行了DNA条形码序列筛选及鉴定研究,其中国际生命条形码联盟植物工作组建议将rbcL和matK基因片段作为植物的核心条形码,叶绿体psbA-trnH片段和核基因ITS片段为补充条形码。2015年Phoon利用上述标准条形码片段以及trnL-trnF和trnV-ndhC基因片段构建系统发育树,但是构建的系统发育树建树结果不理想。因此选择一个更全面、更实用的圆果杜英DNA条形码鉴定体系仍是亟需解决的问题。
发明内容
有鉴于此,本发明的目的在于提供一种鉴定圆果杜英的DNA条形码、鉴定方法与应用,本发明利用圆果杜英的DNA条形码实现了对圆果杜英的精准鉴定,且鉴定方法简单、快速、效率高、重复性好。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种鉴定圆果杜英的DNA条形码,所述DNA条形码包括ycf1、trnS-atpA和ITS基因序列中的一种或多种;所述ycf1的核苷酸序列如SEQ IDNo.1所示,所述trnS-atpA的核苷酸序列如SEQ ID No.2所示,所述ITS的核苷酸序列如SEQ ID No.3所示。
本发明还提供了一种鉴定圆果杜英的引物组,所述引物组包括ycf1引物、trnS-atpA引物和ITS引物中的一种或多种;所述ycf1的引物序列如SEQ ID No.4和SEQ ID No.5所示,所述trnS-atpA的引物序列如SEQ ID No.6和SEQ ID No.7所示,所述ITS的引物序列如SEQ ID No.8和SEQ ID No.9所示。
本发明还提供了一种鉴定圆果杜英的试剂盒,所述试剂盒包括上述的引物组。
优选的,所述试剂盒还包括PCR扩增反应体系,所述PCR扩增反应体系包括DNA模板2μL,10×Dream Taq Greenbuffer 5μL,dNTP Mix 5μL,DMSO 2μL,BSA 0.5μL,正向引物2μL,反向引物2μL,Dream Taq DNAPloymrerase 1.5μL,双蒸水加至50μL。
本发明还提供了一种上述的DNA条形码或引物组或试剂盒在鉴定圆果杜英中的应用。
本发明还提供了一种圆果杜英的鉴定方法,包括以下步骤:
提取样品的总DNA,以总DNA为模板,PCR扩增ycf1、trnS-atpA、ITS基因片段,然后对ycf1、trnS-atpA、ITS基因片段测序结果进行拼接,获得联合基因片段,构建样品的联合基因片段BI树,根据BI树判断该样品是否为圆果杜英。
优选的,如果待测样品联合基因片段单独分类出一个大分支,则该样品为圆果杜英。
优选的,所述PCR扩增反应体系包括DNA模板2μL,10×Dream Taq Green buffer 5μL,dNTP Mix 5μL,DMSO 2μL,BSA 0.5μL,正向引物2μL,反向引物2μL,Dream TaqDNAPloymrerase 1.5μL,双蒸水加至50μL;所述PCR扩增程序为:95℃预变性3分钟;95℃变性30秒,52℃退火30秒,72℃扩增1分钟,共30个循环;72℃终扩增10分钟。
相对于现有技术,本发明具有如下有益效果:
本发明提供了一种鉴定圆果杜英的DNA条形码、鉴定方法与应用,本发明首次提供的鉴定圆果杜英的DNA条形码有利于实现圆果杜英的分子鉴定,有效缩短鉴定圆果杜英的时间,且本发明利用ycf1、trnS-atpA、ITS的联合基因片段鉴定效率高,鉴定效率为100%,极大地提高了圆果杜英分子鉴定的成功率。本发明首次提供了一种鉴定圆果杜英的引物组,实现了对圆果杜英的快速鉴定。本发明的鉴定方法利用检测引物或试剂盒进行PCR扩增获得圆果杜英ycf1、trnS-atpA、ITS序列,然后将扩增序列应用于构建杜英属的系统发育树,实现对圆果杜英的精准鉴定,且鉴定方法简单、快速、效率高、重复性好。
附图说明
图1~图14为Phylosuite软件中使用MAFFT比对完的联合基因片段序列矩阵结果;
图15为实施例1中通过联合基因片段构建的圆果杜英及相近种的BI系统发育树。
具体实施方式
本发明提供了本发明提供了一种鉴定圆果杜英的DNA条形码,所述DNA条形码包括ycf1、trnS-atpA和ITS基因序列中的一种或多种;
所述ycf1的核苷酸序列为:GAATCATCCCATCTTAATTGAAAACGTAAATACCGTTTTAGGAAGAAATCAAGCTCTGCTTTCGTGTTACTCTTGTATTTCTTTTTCTTTCTACGTTTTTTCATGTCTGGGTCTGATTCCCCATAATCTTCTTCAATATCTTTTTCGTGGTCAATATCTTTTTCGTGGTCAATATCTTTTTCGTGGTTGGAGCAAACAACTGATCCAAGGGCCCCTTGGCCCTCGAATTCTTTTTCTTCTTGATTTTGATTCTCTAATTCAAGAGATTTTTTTTCATGCGATGATATAAAAAGATTCCACTTTTTCTTTCTGTTGATGTTTTTATTTTTACTAACATTTCTATTAAAATTAAAAAAAAGTAATTTGATTGGGATGATCCATGGCTTAATCTTATATACACCGTAAAGTACCACAAATTTTTGAAATAGCCAAAGTTCCAGATTTGAGATGGAATGGTTTAGTATTTCTTTGTTCATCCCCATCCAATCAAACCAATCAAAAAAGGTTTTTTTTTGATTGGTTTGATTGGATGGGTTGATTTCTTGATCTTGATGAATTGTGAAATAAAAAAGATCTTTCGTATCAATTTTATCAATTATTTGATAATTATTAATTCCTGTTTTAGTATTTTTATTTCTCTTGTTTCCTGTATCGATCCAGGACTTAATATCAATTTTCTTTCTAAAACAAAAATTGAGAATTCTCCAATCAAAATCTTTTTTATCCGGATTTTTCTCCATATCCATAATATCATCTTTTCCTAGATAATGATTGATAGGAATACCTTCCAGCATATAAAATAAATTGCGTTTACGTGTGTTGTAATTATAAAAAACCTCTTGGTTATTAGTTATTTGTAATGGTGATTCATAAACGTATGAGTTCTTCTTATCTTCATAATTAATAGATTTATATGCTAAAAAATTATATTTATAGTGTTTTTTAAATTTTTCTTTTTGATTTAGTAATGAGTCTGCTTTAAAATGAA(SEQ ID No.1);
所述trnS-atpA的核苷酸序列为:TACGGATTCTTGCACAATTCATTTTGAACTTAAGTAGTTTTGTCAAGCCATATCCGTCAAAAACCCTCCAAAAAAGAAAGAACTTTTGATTTAGTTATTTAAACGAGCCCTCTTTTCCTAATCTCATTAGATTCCCTAATGAAATACCTCGCCGCTCTAATTTTCATGATTCTTTTCTGATTCTATATTGGTTTCTCCTGATTACCTTACTCGACAAAAGGCCCTTATTATATACTTATATACAATTATATACAATAATGTTATATACTTATATACAATTATATACAATAATAATACAATTATATACAATAATGAATTATACAATTATATACAATAATGGTACCGCAGCGGGTATAGTTTAGTGGTAAAAGTGTGATTCGTTCTATTAATCCCCTTAATAGTTAAGGGGTCCCTCGGTTTGATTCATATTCCGATCAAAAATTTTATTTCTTAAAAGGATTTAATCCTTTACCTTTCAATGAAAGACTCGAGGAAGAACATACATTCTCGTGATTTGTATCCAAGAGTCAATTAAAAATTGGATTTGGATTATTAAATTAAATTACGAAACATAATTTTATTGAATTGAATCAATACTTCCAATTGAATGAGTATGAATAAAGAATCCATGGATAAAGATAGAAAAGTGTATTTCTAATCGTAACTAAATCTTCAACTTTTGGATTTTGGAATAGGGGAGATTGAAGCAAAATAGCCATTAAACGATGCTTTTGGTTTACTAGAGACATCGACATATTGTTTTAGCTCGGTGGAAACAACATCCGTTTCCTAAGGATTCTCTCAAATAGCAATAGAGAACGAAGTAATTAGAAAGATTGTTAAAACCACCCTCTTCTAGAGGGATCATCTAGAAAGCGAGGTGTTTTGAAT(SEQ ID No.2);
所述ITS的核苷酸序列为:AATCGGGGTGTCCGCCTGACCTGGGGTCGCGATGCGGACAGGTTAGGGTCGCGGAGAACCACCTCGGTGGCGACGGGCGCGCGCGACGGGTGTATCGAGGTCTTTGGATCAGGCCAACCACCGTTTGTCATGGCGCTCGACGCCGTGGGACTCGATTTTGGGCCAACCGCGGGCAGCGAGGCGCACGGGAGGCCAGTATCCGCCCCCCTCGGGCACCGCCCCGCGTGGCGAGGGGTATTTTTTGGGGGGCGACGATGCGTGACACCCAGGCAGACGTGCCCTCGGCCTAATGGCTTAGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTTTGGATCTTAAAGCAAGAGTTCGGCCGCCCCCGTGCGCGCGCGGCGACGGCGGAGCGATCTCTTCGTTGTAGTTCCTTGGCGCAGATAGCGCCGGGGTTCGTTGTTATGTCAGGGGGGGGAACACGGGGACCGCCGGGGCGGGGACCCGCGCTTTTCCCCCCGACCGAATCGTTTGGTTTCTTAACACGTTCACGGGTCGTTCTGCTAGGCAGGTTTCGACAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTAC(SEQ ID No.3)。
在本发明中,所述DNA条形码优选的包括ycf1、trnS-atpA和ITS基因序列。
本发明还提供了一种鉴定圆果杜英的引物组,所述引物组包括ycf1引物、trnS-atpA引物和ITS引物中的一种或多种;所述ycf1的引物序列如下:
正向ycf1-2F:5’-GAATCATCCCATCTTAATTG-3’(SEQ ID No.4)反向ycf1-2R:5’-TTCATTTTAAAGCAGACTCA-3’(SEQ ID No.5)所述trnS-atpA的引物序列如下:
正向trnS-2F:5’-TACGGATTCTTGCACAATTC-3’(SEQ ID No.6)
反向trnS-2R:5’-ATTCAAAACACCTCGCTTTC-3’(SEQ ID No.7)
所述ITS的引物序列如下:
正向ITS4-1F:5’-TCCTCCGCTTATTGATATGC-3’(SEQ ID No.8)反向ITS5-1R:5’-GGAAGTAAAAGTCGTAACAAGG-3’(SEQ ID No.9)。
在本发明中,所述引物组优选的包括ycf1引物、trnS-atpA引物和ITS引物。
本发明还提供了一种鉴定圆果杜英的试剂盒,所述试剂盒包括上述的引物组。
在本发明中,所述试剂盒还包括PCR扩增反应体系,所述PCR扩增反应体系包括DNA模板2μL,10×Dream Taq Greenbuffer 5μL,dNTP Mix 5μL,DMSO2μL,BSA 0.5μL,正向引物2μL,反向引物2μL,Dream Taq DNAPloymrerase1.5μL,双蒸水加至50μL。
本发明还提供了一种上述的DNA条形码或引物组或试剂盒在鉴定圆果杜英中的应用。
本发明还提供了一种圆果杜英的鉴定方法,包括以下步骤:
提取样品的总DNA,以总DNA为模板,PCR扩增ycf1、trnS-atpA、ITS基因片段,然后对ycf1、trnS-atpA、ITS基因片段测序结果进行拼接,获得联合基因片段,构建样品的联合基因片段BI树,根据BI树判断该样品是否为圆果杜英。
在本发明中,提取样品的总DNA,所述样品可以为新鲜采集的带果皮的果实或新鲜采用干燥保存的叶片。本发明的DNA提取方法没有特殊限定,采用本领域常规的方法即可,如改良CTAB法。本发明所获得的植物基因组总DNA均用1%琼脂糖凝胶进行电泳检测,检测其DNA浓度以及是否降解。
在本发明中,PCR扩增ycf1、trnS-atpA、ITS基因片段,所述PCR扩增的引物采用上述ycf1引物、trnS-atpA引物和ITS引物。所述PCR扩增反应体系优选的包括DNA模板2μL,10×Dream Taq Greenbuffer 5μL,dNTPMix 5μL,DMSO2μL,BSA 0.5μL,正向引物2μL,反向引物2μL,Dream Taq DNAPloymrerase1.5μL,双蒸水加至50μL;所述PCR扩增程序优选为:95℃预变性3分钟;95℃变性30秒,52℃退火30秒,72℃扩增1分钟,共30个循环;72℃终扩增10分钟。在本发明中,如果待测样品联合基因片段单独分类出一个大分支,则该样品为圆果杜英。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1圆果杜英的分子鉴定
实验材料:
本实施例所涉及实验材料为申请人2014~2021年间赴江西、云南、广西和海南出差期间,于当地采集的杜英科材料,其余部分材料从英国皇家植物园购买。具体信息见表1。
表1杜英科采集物种及其凭证标本汇总表
实验步骤:
步骤一:DNA提取
剪取干燥叶片的样品100mg,尽可能剪成碎片或碎块,用震荡磨样机磨成粉末,用改良CTAB法提取总DNA。所获得的植物基因组总DNA用1%琼脂糖凝胶进行电泳检测有明显条带。
步骤二:PCR扩增ycf1、trnS-atpA、ITS基因片段及测序
PCR引物:
(1)正向trnS-2F:5’-TACGGATTCTTGCACAATTC-3’
反向trnS-2R:5’-ATTCAAAACACCTCGCTTTC-3’
(2)正向ycf1-2F:5’-GAATCATCCCATCTTAATTG-3’
反向ycf1-2R:5’-TTCATTTTAAAGCAGACTCA-3’
(3)正向ITS4-1F:5’-TCCTCCGCTTATTGATATGC-3’
反向ITS5-1R:5’-GGAAGTAAAAGTCGTAACAAGG-3’;
PCR反应体系为:DNA模板2μL,10×Dream Taq Green buffer 5μL,dNTP Mix 5μL,DMSO(二亚甲基亚砜)2μL,BSA(牛血清蛋白)0.5μL,正向引物2μL,反向引物2μL,Dream TaqDNA Ploymrerase 1.5μL,双蒸水加注至50μL。PCR扩增流程为:95℃预变性3分钟;95℃变性30秒,52℃退火30秒,72℃扩增1分钟,共30个循环;最终72℃终扩增10分钟。PCR扩增后,用1%琼脂糖凝胶电泳检测扩增产物的浓度,浓度合格进行双向测序。
步骤三:序列校正与比对
将DNA测序返回的序列文件导入MEGA7.0软件内进行正反向序列校正并命名,并保存为后缀名FASTA的序列文件。使用PhyloSuite软件中的MAFFT功能对序列进行比对,然后使用GBlock删除两端不可信区域,将获得的序列填加到杜英属植物联合基因片段标准数据库中。
PCR扩增得到上述编号1~32的基因序列,然后使用SequenceMatrix将3个基因序列连接成联合基因序列,如SEQ ID No.10~41所示,最后使用Phylosuite软件中使用MAFFT比对完的联合基因片段序列矩阵结果见图1~14。
步骤四:构建BI树
使用PhyloSuite软件ModelFinder进行模型分析,并使用MrBayes构建贝叶斯法系统发育树。Tree文件使用软件FigTree v1.3读取。
步骤五:鉴定判断
待测样品联合基因片段单独分类出一个大分支,该分支为圆果杜英。
由图15可知,本发明的鉴定方法属内分组划分清晰,且能从杜英科材料成功鉴定出圆果杜英。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种鉴定圆果杜英的DNA条形码,其特征在于,所述DNA条形码由ycf1、trnS-atpA和ITS基因序列组成;所述ycf1的核苷酸序列如SEQ ID No.1所示,所述trnS-atpA的核苷酸序列如SEQ ID No.2所示,所述ITS的核苷酸序列如SEQ ID No.3所示。
2.一种鉴定圆果杜英的引物组,其特征在于,所述引物组由ycf1引物、trnS-atpA引物和ITS引物组成;所述ycf1的引物序列如SEQ ID No.4和SEQ ID No.5所示,所述trnS-atpA的引物序列如SEQ ID No.6和SEQ ID No.7所示,所述ITS的引物序列如SEQ ID No.8和SEQID No.9所示。
3.一种鉴定圆果杜英的试剂盒,其特征在于,所述试剂盒包括权利要求2所述的引物组。
4.权利要求1所述的DNA条形码、权利要求2所述的引物组或权利要求3所述的试剂盒在鉴定圆果杜英中的应用。
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