甘草HMGR、SQS、β-AS合酶基因CNVs与产地、形态的相关性研究

【作者】 刘东吉；

【导师】 刘春生；

【作者基本信息】 北京中医药大学 ， 中药学， 2011， 硕士

【摘要】 甘草(Glycyrrhiza uralensis Fisch.)是常用的大宗药材,是调解百药的首选佳品,有“十药九甘草”和“药中国老”之称。甘草酸是甘草中最重要的活性成分,具有抗炎、抗癌、增强免疫功能等作用。近年来野生甘草禁止采挖,栽培甘草已经成为主流商品,但栽培甘草存在品质退化及甘草酸含量低等问题,因此能否提高栽培甘草的质量,是制约甘草资源可持续发展的“瓶颈”。基因组拷贝数变异(copy number variations, CNVs)是指与基因组参考序列相比,基因组中≥1kb的DNA片段的缺失、插入、重复和复杂多位点的变异,也称为拷贝数多态性(copy number polymorphisms,CNPs)。目前已发现不少人类复杂性状疾病和CNVs有密切关系。随着临床表型-基因分型关联研究的深入,目前已明确功能基因的CNVs直接通过剂量效应改变特定基因的表达量。进一步研究结果表明改变功能基因的拷贝数对于提高有效成分含量具有重要意义。因此,本论文以甘草酸生物合成的三个重要功能基因HMGR.SQS.β—AS合酶基因为研究对象,筛选其CNVs个体,探讨CNVs与甘草产地、形态特征及甘草酸的相关性。本论文主要目标是建立甘草HMGR.SQS.β—AS合酶基因的Southern blot和real timePCR方法,并利用real time PCR方法筛选栽培甘草HMGR.SQS.β-AS合酶基因的CNVs,为深入研究甘草功能基因CNVs对甘草酸含量的影响奠定基础,对于甘草资源的可持续发展具有重要的意义,同时对其他药用植物CNVs的研究也具有重要的借鉴意义。本论文的主要结论如下：1、Southern杂交测定甘草HMGR、SQS、β-AS合酶基因拷贝数的结果表明,只有PCR产物杂交出条带,DNA样品无条带。该方法操作复杂,耗时长,不适于甘草HMGR、SQS1、β-AS合酶基因CNVs研究。2、建立了real time PCR测定甘草HMGR、SQS1、β-AS合酶基因拷贝数的方法,基因定量校正曲线的R2在0.994～0.999之间,所建立的定量方法操作简便、重复性好。为了保证质粒标准品与目的基因的高度同源性,本实验根据乌拉尔甘草SQS1基因的序列设计引物进行下游实验,因此本实验所得SQS的拷贝数是SQS1的拷贝数。3、甘草3个功能基因的拷贝数多态性。HMGR合酶基因的拷贝数有3种类型,其中以两拷贝为主,占52%,是单拷贝的1.2倍,是三拷贝的3倍,多态性范围较广泛。SQS1合酶基因的拷贝数有2种,其中以单拷贝为主,占90%,是两拷贝的9倍,多态性范围较小β-AS合酶基因均为单拷贝,不存在拷贝数多态性。4、甘草3个功能基因的拷贝数组合多态性。根据HMGR, SQS1,β-AS合酶基因的拷贝数组合将其分类：2+1+1(A型)、1+1+1(B型)、3+2+1型(C型)、2+2+1型(D型)、3+1+1型(E型)。3个基因的拷贝数组合类型以A型和B型为主,分别占46%和42%,其中C型和E型所占比例最少,为3%,D型所占比例为6%。5、甘草3个功能基因的拷贝数多态性及组合多态性与产地相关性,可能成为解析道地药材形成机制的关键因素之一。宁夏盐池的拷贝数组合类型变异最大,包括A、B、C、D共4种拷贝数组合型,其主流组合型是B型,占56%,是A型和C型的5倍,是D型的1.3倍；其次是甘肃民勤,包括3种拷贝数组合类型,其主流组合型是A型,占57%,是B型的2倍,是E型的4倍；内蒙古赤峰包括A型和B型,主流组合型是A型,是B型的3倍；内蒙古杭锦旗包括A型和B型,其中B型所占比例略高于A型,是A型的1.3倍。6、甘草3个功能基因的拷贝数多态性及组合多态性与形态的相关性,为甘草优良种质资源的筛选奠定基础。根据甘草的形态特征将其分类：Ⅰ型(绿茎茎光滑叶平展)、Ⅱ型(绿茎茎有刺毛叶皱缩)、Ⅲ型(绿茎茎有刺毛叶平展)、Ⅳ型(紫红茎茎光滑叶平展)、V型(紫红茎茎有刺毛叶皱缩)、Ⅵ型(紫红茎茎光滑叶皱缩)、Ⅶ型(紫红茎茎有刺毛叶平展)、Ⅷ型(绿茎茎光滑叶皱缩)。Ⅲ型、Ⅵ型、Ⅷ型甘草包括3种拷贝数组合类型,其中Ⅲ型甘草包括A型、B型、C型；Ⅵ型甘草包括A型、B型、D型；Ⅷ型甘草包括B型、D型、E型。其余类型甘草则包括两种拷贝数组合类型,为A型和B型。7、甘草3个功能基因的拷贝数组合多态性与甘草酸含量的相关性。参考文献《不同变异类型甘草中甘草苷及甘草酸量比较研究》,将Ⅰ型和Ⅱ型甘草定义为高含量组,Ⅲ型和Ⅳ型定义为对照组。其中A型是高含量组甘草的主要拷贝数组合类型,占63%,是对照组的2.2倍。HMGR的拷贝数多态性对甘草酸含量影响较大,SQS基因由于本文只测定了SQS1的拷贝数,其对甘草酸含量的影响尚需继续研究,p-AS拷贝数没有多态性。本文的创新性如下：(1)建立real timePCR测定甘草HMGR, SQS1,β-AS合酶基因拷贝数的方法,并进一步筛选出3个基因的CNVs个体,为研究甘草功能基因CNVs与甘草酸含量的相关性奠定基础。(2)分析甘草功能基因CNVs与产地、形态及甘草酸含量的相关性,为甘草优良种质的筛选及道地药材形成机制的解析奠定基础,并为其他药用植物的研究提供重要的借鉴更多还原

【Abstract】 Licorice root has an impressive list of well documented uses and is probably one of the most over-looked of all herbal remedies. The herb’s key therapeutic compound, glycyrrhizin (which is 50 times sweeter than sugar) exerts numerous beneficial effects on the body, making licorice a valuable herb for treating a host of ailments. It seems to prevent the breakdown of adrenal hormones such as cortisol (the body’s primary stress-fighting adrenal hormone), making these hormones more available to the body. Glycyrrhizin is the most important active ingredient, which has function of anti-inflammatory, anti-cancer and so on.Gene copy number variations (CNVs) is one of the hot genetic research in recent years. CNVs are alterations of the DNA of a genome that results in the cell having an abnormal nu-mber of copies of one or more sections of the DNA. CNVs correspond to relatively large regi-ons of the genome that have been deleted or duplicated on certain chromosomes. HMGR, SQS,β-AS synthase gene is the key enzyme genes of Licorice biosynthetic pathway, therefore, we study these three genes to explore their glycyrrhizic acid synthesis pathway.We used Real time PCR method for screening of cultivated licorice HMGR, SQS1,β-AS synthase gene copy number variation. We also analyze the correlations between place of orig-in and morphologicy of Licorice.The main conclusions of this topic are as follows: 1, The results of Southern blot show that only PCR product have a hybrid band, DNA samples have no bands. The method is complicated, time-consuming, not suitable for licorice HMGR, SQS1,β-AS Research synthase gene CNVs. 2, We havd established a methods for determination of glycyrrhizic HMGR, SQS1,β-AS synthase gene copy number. The Correlation coefficient of Real time PCR is between 0.994 and 0.999. This method is simple and repeatable. 3, The CNVs of glycyrrhizic three functional gene. HMGR synthase gene copy number has 3 types, of which one copy has 42% and two copie have 52%. SQS1 synthase gene copy num-ber of 2 species, of which one copy have 90% and two copies has 10%.β-AS synthase genes are single copy, there is no copy number polymorphisms. 4, The combination of CNVs for glycyrrhizic three functional gene.According to three gene copies divided into:2+1+1 (A type),1+1+1 (B type),3+2+1 type (C type),2+2+1 type (D type),3+1+1-type (E type). Combination of the three types of gene copy number to the main A-and B-type, respectively,46% and 42%, of which C-and E-based proportion of the least, to 3%, D-type accounted for 6%.5,The correlations between place of origin and the CNVs.The combination of CNVs for NINGXIA including A, B, C, D types. There is 56% for B type,which is the A type and C type’s 5 times. Minqin includes three types of CNVs, the main type is A type, accounting for 57%, which is 2 times of B-type and 4 times of E-type; Inner Mongolia, including A type and B type, the main type is type A,which is 3 times of A-types; Inner Hangjinqi includs A type and B type, in which B type is slightly higher than A type.6, The correlations between morphology and the CNVs. According to morphological characteristics of licorice Category:Ⅰtype:green stem and leaf stems smooth flat;Ⅱtype: green stem and leaf stalks are bristles shrinkage;Ⅲtype:green stem and leaf stem with bristles flat;Ⅳtype:purple stems smooth,flat leaves;Ⅴtype:purple stem and leaf stalks are bristles shrinkage;Ⅵtype:purple stem and leaf stems smooth shrinkage;Ⅶtype: purple stem and leaf stem with bristles flat;Ⅷtype:smooth green leaf stem stem shrinkage TypeⅢ,Ⅵtype,Ⅷtype consists of three copies, respectively, licorice combination of types, which include licorice typeⅢA, B type, C type;Ⅵtype of licorice, including A type and B type, D type;Ⅷlicorice, including Btype, D type, E type. Other types including two copies of licorice are A type and B type.更多还原