【作者】 罗育；

【导师】 吴耀生；

【作者基本信息】 广西医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 前言1.绞股蓝的研究现状绞股蓝（Gynostemma pentaphyllum（Thunb.）Makino）是葫芦科绞股蓝属多年生草质藤本植物,是五加科以外的植物中发现有人参皂苷成分的药用植物,对绞股蓝的化学成分研究证实,绞股蓝含有100多种绞股蓝皂苷,均为四环三萜达玛烷型;其中绞股蓝皂苷Ⅲ、Ⅳ、Ⅷ、Ⅻ、绞股蓝皂苷元Ⅴ-AH分别与人参皂苷Rb₁、Rb₃、Rd、F₂、Rg₃为同一化合物,还有多种绞股蓝皂苷水解产生的次级苷、苷元和次级苷元,与人参皂苷水解产物相同,故被誉为“南方人参”,而绞股蓝只含二醇组皂苷而无三醇组皂苷,故无人参皂苷作用的双重性。全世界已发现绞股蓝16-17种2个变种,我国共有14种2个变种,主产于我国秦岭山区和江南各省,尤其是西南地区,绞股蓝资源极为丰富。经国内近年来对绞股蓝的药理和临床方面的研究证明,其有如下诸方面的药理作用:①抗肿瘤;②抗应激作用;③抗心肌缺血、缺氧作用;④降低血脂、抑制肥胖作用;⑤清除皮质激素副作用,增强机体免疫功能;⑥护肝作用;⑦镇静、止痛、抗紧张;⑧抗突变作用;⑨抗衰老、延长寿命等作用。2.绞股蓝生态学特征绞股蓝为多年生草质藤本,全株长1～1.5m,根状茎细长横走,长50～100cm,直径粗者可达1cm,有分枝或不分枝,节上生须根。茎细长柔弱,节部具疏生柔毛或无毛,干燥后表面棕色或暗棕色,具纵沟。卷须分2叉或不分叉,腋生。叶膜质,互生,通常有5枚小叶组成鸟趾状复叶,有时为3或7枚;小叶卵状长椭圆形或披针形,有小叶柄,长约2～7cm,中间小叶长3～9cm,宽2～3cm,先端圆钝或短尖,基部楔形;叶边缘有锯齿,背面或沿两面叶脉有短刚毛或近无毛,两侧小叶成对,较中间叶小,着生于同一小柄上。夏季开黄绿色花,圆锥花序,腋生,疏松,总花梗细长,约10～20cm;花单性,雌雄异株,花梗短,苞片钻形,花萼裂片三角形,长约0.5 mm;花冠裂片披针形,先端尾状长尖,长约2.5 mm;雄蕊5,花丝极短,花药卵形;子房球形,2～3室,花柱3,柱头2裂。果实球形,浆果,直径5～8 mm,绿黑色,上半部具一横纹,种子1～3粒,种子宽卵形或长椭圆形,长约4 mm,两面有小疣状凸起或皱纹。花期7～8月,果期9～10月。3.绞股蓝与其混淆品研究绞股蓝主要混淆品为乌蔹莓（Cayratia japonica（Thunb.）Gagnep）,早在明代的《救荒本草》和《本草纲目》中就有可作为野菜食用和药用的记载。绞股蓝和乌蔹莓的生长环境相似,而且全草有许多相同或相似的形态特征,不论是鲜草还是干草都较难分辨。豫东南地区民间有采绞股蓝煮茶饮用的习惯,而且常常把乌蔹莓误认为绞股蓝,甚至某些书籍上也把乌蔹莓误说成是绞股蓝。目前,已有大量的文献资料对绞股蓝与乌蔹莓这两种药用植物进行了鉴别,如刘天学等从植物形态特征、化学成分与药理作用、采集加工与简要用法等方面对绞股蓝与乌蔹莓进行了鉴别;马成亮从分类归属、性状特征、名称、分布区域等方面进行鉴别;程存归等使用FTIR法直接对二者进行鉴定;王太霞等对绞股蓝与乌蔹莓药用部分的进行显微鉴别;杜丽君等从性状、解剖结构、现代研究等方面对绞股蓝与乌蔹莓进行鉴定。4.本研究的目的对绞股蓝种类鉴定及与其混淆品乌蔹莓的鉴别当前仅限于生态学和显微学方面的研究,鲜见分子生物学水平上的鉴定,仅见庞敏等应用DNA分子标记RAPD技术从分子生物学水平上对绞股蓝不同居群的亲缘关系作了鉴定。因此,本研究应用RAPD技术从分子生物学水平上对不同来源绞股蓝及其它药用植物进行遗传特征鉴定。首先,建立能排除植物次级代谢产物干扰的DNA提取方法;然后,使用正交设计法对RAPD反应体系进行优化,筛选合适引物（组）,摸索出最佳反应条件,期望优化后的RAPD反应体系能用于多种植物RAPD指纹分析,如绞股蓝、乌蔹莓、积雪草、烟草等。最后,对不同来源的绞股蓝DNA再行PCR扩增,找出特有的DNA片段,对其进行基因克隆测序;根据序列特征,设计特异性引物,进行PCR扩增,以期建立能对药用植物绞股蓝进行分子遗传特征鉴定的基本标准,达到鉴别绞股蓝与其混淆品乌蔹莓的目的。5.本研究的方法本研究采用RAPD方法,首先使用改良CTAB法提取基因组DNA,得到纯度好的DNA后,进行PCR扩增,并使用正交设计法筛选出最佳的10碱基随机引物组,同时,进行RAPD优化体系的摸索;重复RAPD扩增2-3次后,对扩增产物进行分析,初步建立不同种属药用植物的RAPD指纹图谱。然后,使用最优的RAPD反应体系对不同来源的绞股蓝再行扩增,找出其特有DNA条带,对其进行基因克隆测序及比对分析,确定特征条带的长度,运用Blast等分子生物学信息分析软件,在GenBank查询同源序列,分析序列特征;根据序列特征,运用Primer Premier 5.0、Oligo等软件设计特异引物,增加模板数量,进行PCR扩增,建立遗传特征图谱。综上所述,本研究分成2个部分:（1）摸索基因组DNA提取方法,使用正交设计法优化RAPD反应体系,初步构建遗传图谱;（2）对绞股蓝特征条带基因克隆、测序,并比对后,确定特征条带长度;根据序列特征,设计特异性引物进行PCR扩增,构建绞股蓝属遗传特征图谱,完成绞股蓝与其混淆品乌蔹莓的鉴别。目的:从分子生物学水平上研究不同来源绞股蓝的遗传特征,并与其混淆品乌蔹莓及其它药用植物进行初步鉴别。方法:使用Primer Premier 5.0软件设计20条10碱基随机引物,并进行随机分组,每组5条引物,共有4组;应用RAPD技术,首先用改良CTAB提取药用植物基因组DNA,并结合正交设计法对RAPD反应体系进行优化,筛选合适引物组,摸索最佳反应体系。结果:运用改良的DNA提取方法—CTAB法,提取不同来源的绞股蓝及乌蔹莓、积雪草、烟草等的基因组DNA,均能得到纯度好,质量高的基因组DNA。采用正交设计法对RAPD反应体系进行优化,摸索出了最佳的反应条件,并扩增出了绞股蓝与其混淆品乌蔹莓及其它药用植物的区别条带,同时得到了不同来源绞股蓝的共有特征条带。结论:建立了能排除植物次级代谢产物干扰的DNA提取方法,保证所提取的药用植物基因组DNA质量,并能够进行RAPD扩增。运用正交设计法对RAPD反应体系进行优化,不仅能节约材料和时间,使实验更简便,而所得到的最佳反应体系亦可用于多种药用植物RAPD指纹分析,如本研究中不同来源的绞股蓝,乌蔹莓,积雪草及烟草等。目的:对所获得的不同来源绞股蓝基因组DNA,经RAPD扩增得到的共有特征条带进行克隆、测序及比对分析,确定特征条带片段长度,根据序列特征,设计出特异性引物,构建遗传特征图谱。方法:采用上一篇经优化的RAPD反应体系,对不同来源的绞股蓝DNA再行PCR扩增;所得电泳条带进一步分析后,对所获得共有特征条带经胶回收处理,目的条带连接、转化、提质粒并纯化后,送交生物公司测序及使用软件Vector NTI比对分析,并运用Blast等分子生物学信息分析软件,在GenBank查询同源序列。结果:经Vector NTI软件比对分析后发现,有六种不同来源的绞股蓝基因组DNA经PCR扩增后均可得到一条约826 bp的条带,与计算所得条带长度相近（832 bp）,而仅有一种绞股蓝基因组DNA扩增条带长度为811bp,需要进一步分析;不同来源的绞股蓝特征片段比对结果同源性均在99%以上。结论:以上述测序所得的绞股蓝特征条带为依据,运用Primer Premier5.0及Oligo等软件设计特异性引物,增加模板数,如其混淆品乌蔹莓及其它不同科属药用植物积雪草、烟草等进行特异PCR扩增,最终构建出绞股蓝与其混淆品乌蔹莓及其它药用植物能进行鉴别的遗传特征图谱,并完成了鉴别绞股蓝与其混淆品乌蔹莓的目的。更多还原

【Abstract】 Objetive: To study the variety of Gynostemma pentaphylum and distinguish the counterfeit plant, Cayratia japonica from Gynostemma Pentaphyllum on the Molecular Biology.Methods: The software of Primer Premier 5.0 was used to work out the random primes that the number was 20, and then they were divided into four groups. With the RAPD technique, the improved CTAB method was used to extract the genomic DNA from the medicinal plants that were gathered in distinct places. At the same time, the method of orthogonal design was applied to optimize the reaction system of RAPD. The optimum group of primers was screened and the best reaction system was groped, too.Results: With the improved CTAB method, the extracted genomic DNA from Gynostemma Pentaphyllum, Cayratia japonica, Tobacco and Herba Centellae in distinct places, respectively presented better quality and could be used to progress the amplification of RAPD. The best reaction system of RAPD was groped from the table of orthogonal design. After analyzing the amplification, there were amplified strips to differ Gynostemma Pentaphyllum with the counterfeit plant, Cayratia japonica and other medicinal plants. In the amplified strips of Gynostemma Pentaphyllum from the different places, there were characteristic strips to be amplified, too.Conclusion: The method of extracting the genomic DNA was founded to get rid of the secondary metabolite. The extracted genomic DNA presented better quality and could be used to progress the amplification of RAPD. After optimizing the reaction system of RAPD with the method of orthogonal design, not only we could save material and time so that the experiment was more convenient, but also the optimum reaction system could help us establish the RAPD finger prints of extracted genomic DNA from the medicinal plants. Objetive: To clone the characteristic strips that were progressed with RAPD technique from the genomic DNA that were extracted from the varieties of Gynostemma Pentaphyllum, then align and analyze the feature of sequences. After determining the length of sequence with Vector NTI, we designed 20-mer characteristic primers. At last, construct the finger printing of hereditary feature about the varieties of Gynostemma Pentaphyllum.Methods: The extracted genomic DNA from the seven varieties of Gynostemma Pentaphyllum were progressed by PCR with the optimum reaction system of RAPD again. The progressed characteristic strips were analyzed and then sliced from agarose gel. The objective strips were ligated with the vector pTG19-T, and then the ligated vectors were transformated into Bacterium coli JM109. After purified, the recombinant plasmids were send to the bio-company and the result was aglined and analyzed with Vector NTI. At last, the homologous sequence was inquested with Blast-the software of molecular biology-on GenBank.Results: After aligning and analyzing the sequences with Vector NTI, we could know the extracted genomic DNA from six varieties of Gynostemma Pentaphyllum could be progressed about 826 bp fragment by PCR. And the fragment was close to our calculation (832 bp). But only one Gynostemma Pentaphyllum could be progressed 811 bp fragment and need more study. The homologization of aligning characteristic fragment from the seven varieties of Gynostemma Pentaphyllum was more than 99%.Conclusion: The 20-mer characteristic primers were designed from thecharacteristic sequences of Gynostemma Pentaphyllum. And then the templates that included the seven varieties of Gynostemma Pentaphyllum and other medicinal plants (as Cayratia japonica, Tobacco, Herba Centellae and so on), were progressed by PCR with the 20-mer primers. At last, we could construct the finger printing that could be differed the counterfeit plant, Cayratia japonica and other medicinal plants from Gynostemma Pentaphyllum on molecular biology.更多还原