CN100547082C - 鉴别黄独性别的引物、片段与方法 - Google Patents
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Abstract
本发明属于分子标记领域,具体公开了鉴别黄独性别的引物、片段与方法。该片断核苷酸序列如SEQ ID NO.1所示,该组引物的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。使用该组引物进行PCR反应可以从黄独雄株中获得长度为677bp的扩增片段,而雌株中没有获得任何扩增产物,说明该引物为雄株特异,从而表明该引物可以用于黄独雌雄株的性别鉴定。
Description
技术领域:
本发明属于分子标记技术领域,是一组鉴别黄独性别的引物、片段与方法。
背景技术:
黄独Dioscorea bulbifera L.为薯蓣科Dioscoreaceae薯蓣属Dioscorea L.植物,世界广布种,散布于非洲、美洲和亚洲等地。黄独花单性,雌雄异株,且性别表现稳定。黄独块茎和零余子(珠芽)富含淀粉和糖,在非洲、东南亚和玻利尼西亚等地广泛栽培和食用。在我国黄独块茎为常用中药,又名黄药子,其性凉,味苦,有散结消瘦、清热解毒、凉血止血、抗癌的功效。与黄独同属的盾叶薯蓣Dioscorea zingiberensis C.H.Wright,其根茎中含有薯蓣皂苷元,是甾体激素类药物的重要药源植物,且有研究表明,其雄株根茎中的薯蓣皂苷元含量高于雌株,因此生长上早期筛选到盾叶薯蓣雄性植株具有重要的经济价值。但是在栽培条件下,盾叶薯蓣的性别表现比较复杂,没有同属的黄独稳定,因此从分子水平找到一种DNA标记来鉴别发育早期的黄独植株具有重要的应用价值,对盾叶薯蓣植株早期雌雄鉴别具有重要的实践意义。
自从1990年Williams和Welsh创立RAPD(Random Amplified polymorphie DNA)技术以来,该技术已成功用于植物的性别差异研究。SCAR(sequence characterized amplified region)分子标记,是根据RAPD分子标记的序列分析结果用20-28个碱基的特异引物进行PCR扩增得到的,对基因组限定区域可重复高特异性的扩增。目前RAPD-SCAR标记技术已在很多植物的性别研究中获得成功,如大麻(Cannabis sativa L.)与Y染色体连锁的RAPD-SCAR雄性标记、番木瓜(Carica papaya L.)RAPD-SCAR雄性标记物。
目前国内外尚无有关分子标记鉴别黄独性别方法的报道,本发明填补了该领域的空白。
发明内容:
本发明目的是提供鉴别黄独性别的一组引物和一段DNA片段;
本发明的另外一个目的是提供一种鉴别黄独性别的方法。
本发明运用RAPD方法在雄性黄独基因组中扩增出一条雄性特异性条带,通过克隆和测序获得了一段长度为681bp的DNA序列(SEQ ID NO:1),并根据此序列设计了一组鉴别黄独性别的引物,其序列如SEQ ID NO:2和SEQ ID NO:3所示,根据该对引物在黄独基因组中能够扩增得到一条雄性特异性序列,其长度为677bp。
本发明提供一种鉴别黄独性别的方法,其特征在于方法是根据样品的PCR反应产物琼脂糖凝胶电泳结果鉴定样品的性别。该方法中特异性引物为SEQ ID NO:2和SEQ ID NO:3。该方法中PCR产物电泳结果出现长度为677bp的条带,该样品为雄株,面相同部位没有出现条带的样品为雌株。
PCR反应体系为20μl,反应液组成为10×PCRbuffer,2.5mmol/L的dNTPs,25mmol/L的MgCl2,1u的Taq DNA聚合酶,0.4μmol/L随机引物,20-50ng模板DNA。在PE-9600PCR仪上按下面的循环程序进行PCR扩增:94℃预变性3min;94℃变性45s,38℃退火30s,72℃延伸90s,35个循环;最后72℃延伸5min,4℃保存。
本发明一共实验了100个RAPD随机引物,购自上海英骏生物有限公司,在雄性黄独基因组中扩增出一条681bp雄性特异性条带(其DNA序列为SEQ ID NO:1),含有多个终止子,属非编码序列,将该序列在Genebank上进行序列比对,未发现与之同源的序列。根据该特异性条带测序结果,设计出一对特异性引物SEQ ID NO:2和SEQ ID NO:3。
根据全序列设计出一对SCAR特异引物:
SEQ ID NO:25′-GGCTTCTGTCACTACATGGG-3′
SEQ ID NO:35′-GGCTTCTGTCCAGTGCATCT-3′
通过已知性别的35个黄独个体检验这对引物的有效性,SCAR反应表明在所有13个黄独雄株个体的DNA电泳结果中均出现了677bp条带,而22个黄独雌性个体中相应部位没有出现该条带(图1,2)。
综上所述,本发明提供了一段长度为681bp的黄独雌雄特异性的DNA序列,可以用于黄独的雌雄性别鉴定。检验结果证明了本发明中引物和PCR方法可以准确鉴别出黄独雌雄的性别。
附图说明:
图1RAPD-SCAR标记单株检测结果1,其中13个雄株(1-13)的电泳条带上有一个特异条带。M:100bp Ladder DNA分子量标记。箭头1所示为黄独雄性相关SCAR标记,箭头2所示为引物二聚体。
图2RAPD-SCAR标记单株检测结果2,22个雌株(14-35)的677bp位置上没有条带。M:100bp Ladder DNA分子量标记。箭头2所示为引物二聚体。
具体实施方式:
1.植物基因组DNA和RAPD-SCAR标记的获得
采用CTAB法从黄独幼叶中提取DNA,提取的DNA用北京莱博生物实验材料研究所生产的DNA纯化回收试剂盒纯化后,溶解于灭菌双蒸水中,-20℃储藏,备用。取雌、雄株齐全的四川峨眉、广西田林、云南蒙自3个居群野生黄独雄性和雌性各1株,将单株DNA样品等量混合,分别建成雄性或雌性DNA池(DNA pools),以提供具有相同遗传背景的雌、雄性DNA样品。
随机引物筛选采用Michelmore的混合分组分析法(BSA)进行。扩增反应体系为20μl,反应液组成为10×PCRbuffer,2.5mmol/L的dNTPs,25mmol/L的MgCl2,1u的Taq DNA聚合酶,0.4μmol/L随机引物,20-50ng模板DNA。在PE-9600PCR仪上按下面的循环程序进行PCR扩增:94℃预变性3min;94℃变性45s,38℃退火30s,72℃延伸90s,35个循环;最后72℃延伸5min。1.2%琼脂糖凝胶电泳(80V,1h)检测PCR扩增结果,溴化乙锭(EB)染色,紫外凝胶成像系统拍照,100bp DNA Ladder作分子量标记。
PCR产物经纯化、转质粒后,挑选1个克隆测序,其DNA序列为SEQ ID NO:1。根据DNA序列分析结果,设计出一对20个碱基的特异性引物。本发明中这对引物为SEQ IDNO:2和SEQ ID NO:3。
2.PAPD-SCAR分子标记的验证实验
将SEQ ID NO:2和SEQ ID NO:3这对引物,以12个居群共35个单株DNA为模板进行PCR扩增。扩增反应体系为20μl,反应液组成为10×PCRbuffer,2.5mmol/L的dNTPs,25mmol/L的MgCl2,1u的Taq DNA聚合酶,上下游引物各0.3μmol/L,50ng模板DNA。在PE-9600PCR仪上按下面的循环程序进行PCR扩增:94℃预变性3min;94℃变性45s,62℃退火30s,72℃延伸90s,35个循环;最后72℃延伸5min。1.2%琼脂糖凝胶电泳(80V,1h)检测PCR扩增结果,溴化乙锭(EB)染色,紫外凝胶成像系统拍照,100bp DNALadder作分子量标记。
结果如图1,2所示,图1中13个雄株(1-13)的电泳条带677bp位置上有一个特异条带,而在图2中22个雌株(14-35)的677bp位置上没有条带。这表明本发明设计的引物和方法能够用于黄独雌雄性别鉴定。
序列表
<110>江苏省中国科学院植物研究所
<120>鉴别黄独性别的引物、片段与方法
<160>3
<210>1
<211>681
<212>DNA
<213>来源于黄独(Dioscorea bulbifera L.)
<400>1
ggcttctgtc actacatggg atgaggtggt agaggctttt ctcgcatgat acttcttgtt 60
tggaaaacca gcaaagcata acaatgaaat cttgtcctat gtacaaaata agcgggagtc 120
cttatttgag acttgggaga aatttaagga tctcttgtgg cggtgccccc aacatggatt 180
tctccattgg atggtagctc acacattttc ttatgggctc aatttgagca ctaggtaact 240
cttagatgtc attgcaagag gtaatttggg taacaagacc tagaagatgc tagacagctc 300
atagagtgtc acgcctggac ccgccgacat ggcacacaac ataccgccat gacaacccaa 360
cgcaaagtga acaccgccaa gaccatgagt tatcgtaagg atagcatatt tcctgtttac 420
aaacactggt ataactggga aagataacaa ctcatgatga tatattctca agaaaatata 480
tatacgcata aggtaataca tgtcctcaat acgagtttta atacaacagt ttgacattaa 540
caagcttaga atttccaaga tagaagaaca aaatgcaaga aatccaaatg aatacagtaa 600
tggatcccat gactacatgt cacaacagat aagaagacct acacaatgca atacaagaga 660
tgcactggac agaagccaat c 681
<210>2
<211>20
<212>DNA
<213>人工引物
<400>2
ggcttctgtc actacatggg 20
<210>3
<211>20
<212>DNA
<213>人工引物
<400>3
ggcttctgtc cagtgcatct 20
Claims (4)
1.黄独药材DNA分子鉴定性别的特异性片断,其序列为SEQ ID NO:1所示。
2.黄独药材DNA分子鉴定性别的一组引物,该组引物由SEQ ID NO:1所示序列设计合成的,其序列为SEQ ID NO:2和SEQ ID NO:3所示。
3.黄独药材DNA分子鉴定性别的方法,其特征在于该方法是利用序列为SEQ ID NO:2和SEQ ID NO:3所示的引物对鉴定样品进行PCR反应,然后通过PCR反应产物的琼脂糖凝胶电泳结果鉴定样品,PCR产物电泳的结果中出现一段677bp条带的样品为雄株,而相同部位没有出现条带的样品为雌株。
4.据权利要求3所述的方法,其特征在于该反应体系为20μl,反应液组成为:10×PCRbuffer,2.5mmol/L的dNTPs,25mmol/L的MgCl2,lu的Taq DNA聚合酶,上下游引物各0.3μmol/L,50ng模板DNA;在PCR仪上按下面的循环程序进行PCR扩增:94℃预变性3min;94℃变性45s,62℃退火30s,72℃延伸90s,35个循环;最后72℃延伸5min;在电压为80V下,经1.2%琼脂糖凝胶电泳1h检测PCR扩增结果,溴化乙锭(EB)染色,紫外凝胶成像系统拍照,100bp DNA ladder作分子量标记;该方法中PCR产物电泳结果中出现一段677bp条带的样品为雄性,而相同部位没有出现条带的样品为雌株。
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US6037128A (en) * | 1997-03-31 | 2000-03-14 | Council Of Scientific & Industrial Research | Process for the preparation of semisynthetic amplicon useful for sex determination of the papaya plant |
US6180345B1 (en) * | 1998-03-31 | 2001-01-30 | Counsel Of Scientific & Industrial Research | Process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination |
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US6037128A (en) * | 1997-03-31 | 2000-03-14 | Council Of Scientific & Industrial Research | Process for the preparation of semisynthetic amplicon useful for sex determination of the papaya plant |
US6180345B1 (en) * | 1998-03-31 | 2001-01-30 | Counsel Of Scientific & Industrial Research | Process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination |
Non-Patent Citations (4)
Title |
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A male and hermaphrodite specific RAPD marker for papaya(Carica papaya L.). N. Urasaki · M et al.Theor Appl Genet,Vol.104 . 2002 |
A male and hermaphrodite specific RAPD marker for papaya(Carica papaya L.). N. Urasaki · M et al.Theor Appl Genet,Vol.104 . 2002 * |
Molecular markers for sex determination in papaya (Caricapapaya L.). J. C. Deputy · R et al.Theor Appl Genet,Vol.106 . 2002 |
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