RAPD技术在广西产绞股蓝遗传特征及鉴别中的应用研究
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摘要
采用RAPD分子标记技术对广西区不同产地、不同来源的7份绞股蓝材料进行了遗传多样性分析,并在DNA分子水平上鉴别绞股蓝及其伪品乌蔹莓的新鲜品、干品,从而为绞股蓝资源的分析收集、分类研究、多样性保护、伪品鉴别,进而为药用植物资源的育种工作和临床安全用药提供技术保障及分子生物学依据。主要研究结果如下:
1.采用改进的CTAB法成功提取了绞股蓝和乌蔹莓新鲜品、干品DNA。
2.优化了绞股蓝的RAPD-PCR反应体系:主要以校园采集的绞股蓝DNA为模板,采用随机引物WGS001进行PCR扩增,对反应体系包括模板、Mg~(2+)、Taq酶、牛血清白蛋白(BSA)、退火温度进行优化。优化的反应体系总体积25μL,含MgCl_2 2 mmol/L、dNTP 0.2 mmol/L、引物0.4μmol/L、模板60 ng、Taq酶1 U、BSA 2μg/μL。反应程序:预变性94℃3min,然后94℃30 s,58℃30 s,72℃1 min 20 s,40个循环,72℃延伸10 min。
3.采用优化了的RAPD-PCR反应体系,从10条含20个碱基的随机引物中筛选出了2条具有多态性高且重复性好的引物对广西区不同产地、不同来源的7份绞股蓝新鲜品材料进行RAPD指纹图谱研究。两条引物共扩增出33个位点,多态位点30个,占90.91%,说明绞股蓝种内具有丰富的遗传多样性。应用SPSS16.0软件计算Jaccard遗传相似系数并进行聚类分析。绞股蓝植物间的相似性系数分布较广,范围为0.217～0.688。聚类树状图表明绞股蓝亲缘关系与地理距离相关,地理分布距离越近的样品,在聚类树状图上的距离越近,说明它们之间的遗传背景差异越小,亲缘关系越近,反之越远。
4.应用筛选的两条引物对广西不同产地绞股蓝及其伪品乌蔹莓的新鲜品和干品进行RAPD鉴别。根据DNA图谱上的差异性条带可以将绞股蓝及其伪品乌蔹莓很好的区分开来。且两条引物均能扩增出所有绞股蓝新鲜品和干品共有而乌蔹莓没有的特异DNA片段,WGS001扩增的特异片段约550bp,WGS004扩增的特异片段约500 bp。初步认为可将这两条特异DNA片段作为鉴别广西产绞股蓝与乌蔹莓的依据。
5.对用引物WGS004扩增7份绞股蓝新鲜品共有特异DNA片段(约500 bp)进行克隆、测序、生物信息学分析。进行生物信息学分析后确定所克隆的7条DNA序列在NCBI GenBank数据库中未收录,为新发现的序列。
本研究首次采用RAPD技术对广西产绞股蓝进行遗传特征研究,为研究绞股蓝遗传多样性提供了分子生物学依据。首次报道采用RAPD技术能在DNA分子水平上鉴别对绞股蓝及其伪品乌蔹莓的新鲜品和干品。这一方法克服了传统的鉴别方法易受到外界环境因素影响的困难,为绞股蓝的鉴别提供了新的思路。
The RAPD method was applied to analyze the genetic diversity of seven samples of medicinal plant Gynostemma pentaphyllum which were collected from different regions and different sources in Guang Xi province.This method also was used to identify the resources of Gynostemma pentaphylum and its spurious breed Cayratia japonica at level of DNA.The results would offer the technological guarantee and molecular biology basis for Gynostemma pentaphyllum resources collection and analysis,classification research, biodiversity protection,genuine drug identification,and further,it would be of great benefit to breeding of medicinal plant resources and safe clinical medication.The main results of this study were as follows:
1.The genomic DNA of fresh samples and dry samples of Gynostemma pentaphyllum and Cayratia japonica were extracted successfully by modified CTAB method.
2.The reaction system of RAPD-PCR was optimized:With the DNA of Gynostemma pentaphyllum collected from campus as template,the random primer WGS001 for PCR has been done meanwhile RAPD reaction system has been optimized so that it could be determined for the suitable annealing temperature and concentrations of template,Mg~(2+),primer,Taq DNA polymerase, BSA.In the optimized system of 25μL,MgCl_2 2 mmol/L,primer 0.4μmol/L, template 60 ng,Taq DNA polymerase 1 U,BSA 2μg/μL.The reaction procedure was 94℃in 3 min,then 40 cycle times with 94℃in 30 s,58℃in 30 s,72℃in 80 s,lastly 72℃in 10 min,and preserved at 4℃.
3.Based on the optimized system,ten random primers(20 bp)were used for the PCR amplification of genomic DNA.Two of these primers(WGS001, WGS004)could amplify bands which have good reproducibility and high polymorphism.With this two screened random primers,PCR was done for the above mentioned seven fresh samples of Gynostemma pentaphyllum.The RAPD fingerprints were obtained,thirty of thirty-three amplified sites were polymorphic,accounts for 90.91%.It revealed that Gynostemma pentaphyllum existed abundant genetic diversity.The software SPSS16.0 was used to calculate the Jaccard coefficient of similarity and do cluster analysis.The coefficient of similarity of Gynostemma pentaphyllum was in the range of 0.217-0.688.The dendrogram of RAPD cluster analysis indicated that the smaller the geographic distance between two Gynostemma pentaphyllum samples,the smaller their genetic difference,vice versa.
4.The fresh samples and dry samples of Gynostemma pentaphyllum and Cayratia japonica from different regions in Guang Xi province were authenticated by two screened primers.According to the different bands of DNA fingerprints,Gynostemma pentaphyllum and Cayratia japonica could be distinguished obviously.Besides,these two primers could amplify characteristic fragments which were common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica.One common characteristic DNA fragment was about 550 bp by primer WGS001,other was about 500 bp by primer WGS004.The preliminary view was that these two common characteristic DNA fragments could be taken as basis for distinction molecular marker for Gynostemma pentaphyllum and Cayratia japonica in Guang Xi province.
5.These seven characteristic DNA fragments(500 bp)amplified by WGS004 were cloned,sequenced and analyzed for bioinformatics.Through bioinformatics analyze we could know these seven DNA sequences had not been recorded in NCBI GenBank before.They were new sequences.
It was the first time to apply the RAPD analysis to study hereditary feature of Gynostemma pentaphyllum(Thunb.)Makino in Guang Xi province.This research's result could supply molecular biology basis for genetic diversity of Gynostemma pentaphyllum.It also was the first time to report that RAPD technology could identify both of fresh samples and dry samples between Gynostemma pentaphyllum and its spurious breed Cayratia japonica.The Chinese traditional medicine identification was so easily to be affected by external factors that leaded to inaccurate conclusion.This RAPD method could overcome difficulties in Chinese traditional medicine identification and provide a new thread for identification of Gynostemma pentaphyllum.
1.采用改进的CTAB法成功提取了绞股蓝和乌蔹莓新鲜品、干品DNA。
2.优化了绞股蓝的RAPD-PCR反应体系:主要以校园采集的绞股蓝DNA为模板,采用随机引物WGS001进行PCR扩增,对反应体系包括模板、Mg~(2+)、Taq酶、牛血清白蛋白(BSA)、退火温度进行优化。优化的反应体系总体积25μL,含MgCl_2 2 mmol/L、dNTP 0.2 mmol/L、引物0.4μmol/L、模板60 ng、Taq酶1 U、BSA 2μg/μL。反应程序:预变性94℃3min,然后94℃30 s,58℃30 s,72℃1 min 20 s,40个循环,72℃延伸10 min。
3.采用优化了的RAPD-PCR反应体系,从10条含20个碱基的随机引物中筛选出了2条具有多态性高且重复性好的引物对广西区不同产地、不同来源的7份绞股蓝新鲜品材料进行RAPD指纹图谱研究。两条引物共扩增出33个位点,多态位点30个,占90.91%,说明绞股蓝种内具有丰富的遗传多样性。应用SPSS16.0软件计算Jaccard遗传相似系数并进行聚类分析。绞股蓝植物间的相似性系数分布较广,范围为0.217～0.688。聚类树状图表明绞股蓝亲缘关系与地理距离相关,地理分布距离越近的样品,在聚类树状图上的距离越近,说明它们之间的遗传背景差异越小,亲缘关系越近,反之越远。
4.应用筛选的两条引物对广西不同产地绞股蓝及其伪品乌蔹莓的新鲜品和干品进行RAPD鉴别。根据DNA图谱上的差异性条带可以将绞股蓝及其伪品乌蔹莓很好的区分开来。且两条引物均能扩增出所有绞股蓝新鲜品和干品共有而乌蔹莓没有的特异DNA片段,WGS001扩增的特异片段约550bp,WGS004扩增的特异片段约500 bp。初步认为可将这两条特异DNA片段作为鉴别广西产绞股蓝与乌蔹莓的依据。
5.对用引物WGS004扩增7份绞股蓝新鲜品共有特异DNA片段(约500 bp)进行克隆、测序、生物信息学分析。进行生物信息学分析后确定所克隆的7条DNA序列在NCBI GenBank数据库中未收录,为新发现的序列。
本研究首次采用RAPD技术对广西产绞股蓝进行遗传特征研究,为研究绞股蓝遗传多样性提供了分子生物学依据。首次报道采用RAPD技术能在DNA分子水平上鉴别对绞股蓝及其伪品乌蔹莓的新鲜品和干品。这一方法克服了传统的鉴别方法易受到外界环境因素影响的困难,为绞股蓝的鉴别提供了新的思路。
The RAPD method was applied to analyze the genetic diversity of seven samples of medicinal plant Gynostemma pentaphyllum which were collected from different regions and different sources in Guang Xi province.This method also was used to identify the resources of Gynostemma pentaphylum and its spurious breed Cayratia japonica at level of DNA.The results would offer the technological guarantee and molecular biology basis for Gynostemma pentaphyllum resources collection and analysis,classification research, biodiversity protection,genuine drug identification,and further,it would be of great benefit to breeding of medicinal plant resources and safe clinical medication.The main results of this study were as follows:
1.The genomic DNA of fresh samples and dry samples of Gynostemma pentaphyllum and Cayratia japonica were extracted successfully by modified CTAB method.
2.The reaction system of RAPD-PCR was optimized:With the DNA of Gynostemma pentaphyllum collected from campus as template,the random primer WGS001 for PCR has been done meanwhile RAPD reaction system has been optimized so that it could be determined for the suitable annealing temperature and concentrations of template,Mg~(2+),primer,Taq DNA polymerase, BSA.In the optimized system of 25μL,MgCl_2 2 mmol/L,primer 0.4μmol/L, template 60 ng,Taq DNA polymerase 1 U,BSA 2μg/μL.The reaction procedure was 94℃in 3 min,then 40 cycle times with 94℃in 30 s,58℃in 30 s,72℃in 80 s,lastly 72℃in 10 min,and preserved at 4℃.
3.Based on the optimized system,ten random primers(20 bp)were used for the PCR amplification of genomic DNA.Two of these primers(WGS001, WGS004)could amplify bands which have good reproducibility and high polymorphism.With this two screened random primers,PCR was done for the above mentioned seven fresh samples of Gynostemma pentaphyllum.The RAPD fingerprints were obtained,thirty of thirty-three amplified sites were polymorphic,accounts for 90.91%.It revealed that Gynostemma pentaphyllum existed abundant genetic diversity.The software SPSS16.0 was used to calculate the Jaccard coefficient of similarity and do cluster analysis.The coefficient of similarity of Gynostemma pentaphyllum was in the range of 0.217-0.688.The dendrogram of RAPD cluster analysis indicated that the smaller the geographic distance between two Gynostemma pentaphyllum samples,the smaller their genetic difference,vice versa.
4.The fresh samples and dry samples of Gynostemma pentaphyllum and Cayratia japonica from different regions in Guang Xi province were authenticated by two screened primers.According to the different bands of DNA fingerprints,Gynostemma pentaphyllum and Cayratia japonica could be distinguished obviously.Besides,these two primers could amplify characteristic fragments which were common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica.One common characteristic DNA fragment was about 550 bp by primer WGS001,other was about 500 bp by primer WGS004.The preliminary view was that these two common characteristic DNA fragments could be taken as basis for distinction molecular marker for Gynostemma pentaphyllum and Cayratia japonica in Guang Xi province.
5.These seven characteristic DNA fragments(500 bp)amplified by WGS004 were cloned,sequenced and analyzed for bioinformatics.Through bioinformatics analyze we could know these seven DNA sequences had not been recorded in NCBI GenBank before.They were new sequences.
It was the first time to apply the RAPD analysis to study hereditary feature of Gynostemma pentaphyllum(Thunb.)Makino in Guang Xi province.This research's result could supply molecular biology basis for genetic diversity of Gynostemma pentaphyllum.It also was the first time to report that RAPD technology could identify both of fresh samples and dry samples between Gynostemma pentaphyllum and its spurious breed Cayratia japonica.The Chinese traditional medicine identification was so easily to be affected by external factors that leaded to inaccurate conclusion.This RAPD method could overcome difficulties in Chinese traditional medicine identification and provide a new thread for identification of Gynostemma pentaphyllum.
引文
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