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
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.
引文
[1]Yamamoto Yutaka, Majima Takami, Saiki Ikuo. Pharmaceutical Evaluation of Glycyrrhiza uralen-sis Roots Cultivated in Eastern Nei-Meng-Gu of China[J]. Biol. Pharm. Bull,2003,26(8):1144.
[2]赵则海,杨逢建,曹建国,等.野生与栽培乌拉尔甘草不同部位甘草酸含量分析[J].植物研究,2005,25(4)：444.
[3]刘春生,王文全.栽培甘草不同部位甘草酸含量的单株分析[J].中国中药杂志,2007,32(24)：2660.
[4]牛小宇,刘春生.栽培甘草群体中不同单株甘草酸的含量差异研究[J].时珍国医国药,2009.20(9)：2121.
[5]Redon, Ishikawa, Fitch, et al. Global variation in copy number in the human genome [J]. Nature, 2006,444(7118):444.
[6]Gonzalez, Kulkarni, Bolivar, et al. The influence of CCL3L1 gene-containing segmental dupli-cations on HIV-1/AIDS susceptibility [J]. Science,2005,307(5714):1434.
[7]Hong-Yu Lu, Jing-Mei Liu, Hai-Chao Zhang, et al. Ri-mediated Transformation of Glycyrrhiza uralensis with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin[J]. Plant Mol Biol Rep,2008,26:1.
[8]Masferrer, Aroo, Manazano, et all. Overexpression of Arabidopsis thaliana famesyl diphosphate synthase (FPA1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cy-tokinin lecels[J]. Plant,2002,30:123.
[9]Oliver, Andreas, Hur-Song Chang. Crosstalk between cytosolic and plastidial pathways of isopren-oid biosynthesis in Arabidopsis thaliana[J]. PNAS,2003,11:6867.
[10]Harwood, Quinn. Sterols and Isoprenoids[J]. Biochemical Society Transactions,2000,28:785.
[11]Michihara, Shimatani, Mortia, et al. Reduction of Mevalonate Pyrophosphate Decarboxylase in Mouse [J]. Health Science,2010,56(3):355.
[12]Michihara, Mortia, Toda. Mevalonate Pyrophosphate. Decarboxylase is Predominantly. Located in the Cytosol of both. B16 and B16F10 Cells in Mouse [J]. Health Science,2008,54 (2):216.
[13]Montserrat Enjuto, Liuis Balcells, Narciso Campos. Arabidopsis thaliana contains two differentia-lly expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the en-zyme [J]. Plant Biology,1994,91:927.
[14]Andrea Hemmerlin, Thomas J, Bach. Farnesol-Induced Cell Death and Stimulation of 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Activity in Tobacco cv Bright Yellow-2 Cells [J]. Plant Physiol,2000,123:1257.
[15]陈大华.马铃薯HMGR基因的克隆、序列分析及其表达特征[J].植物学报.2000,42(7)：724.
[16]许巧仙,刘春生.甘草HMGR基因克隆及其多态性对酶促效率的影响研究[D].北京中医药大学：硕士论文,2010.
[17]Giinel, Kuntz, Arda, et all. Metabolic Engineering for production of Geranylgeranyl [J]. Biotech-nol,2006,3:76.
[18]Tao-Hsin Changa, Po-Huang Lianga, Andrew H. Structure of Geranylgeranyl Pyrophosphate Synthase from Saccharomyces cerevisiae-Mechanism of Product Chain Length Determination of tra-nsprenyltransferases[J]. Life Science,2006,281(21):14991.
[19]Timothy P. Devarenne, Anirban Ghosh, Joe Chappell. Regulation of Squalene Synthase, a Key Enzyme of Sterol Biosynthesis in Tobacco. Plant Physiol[J],2002,129(3):1095.
[20]Shechter I, The road to squalene synthase[J]. Biochem Biophys Res Commun,2002,202:1261.
[21]卢虹玉,刘敬梅.甘草鲨烯合成酶基因的分离及植物表达载体的构建[J].药物生物技术,2007,14(4)：255.
[22]张宁,刘春生.甘草sQs基因克隆及其多态性对酶促效率的影响研究[D].北京中医药大学：硕士论文,2010.
[23]赵明文,钟家禹,王南,等.鲨烯合酶的研究进展[J].微生物学报,2003,43(5)：676.
[24]隋春,魏建和,战晴晴,等.北柴胡鲨烯合酶基因及其编码区cDNA克隆与序列分析[J].园艺学报,2010,37(2)：283.
[25]Haralampidis, Bryan, Qi, et all. A new class of oxidosqualene cyclases directs synthesis of antimic-robial phytoprotectants in monocots [J]. PNAS,2001,98(23):13431.
[26]Mi-Hyun Lee, Jae-Hun Jeong, Jin-Wook Seo, et al. Enhanced Triterpene and Phytosterol Biosynthesis in Panax ginseng Overexpressing Squalene Synthase Gene[J], Plant Cell Physiol,2004, 45(8):976.
[27]Daisuke Umeno, Alexander, Frances. Evolution of the C30 Carotenoid Synthase CrtM for Function in a C40 Pathway [J]. Bacteriology,2002,184(23):6690.
[28]吴琼,周应群,孙超,等.人参皂苷生物合成和次生代谢工程[J].中国生物工程杂志,2009,29(10)：102.
[29]邢朝斌,王一曼,陈正恒,等.三萜皂苷的生物合成[J].生命的化学,2005,25(5)：420.
[30]沈湛云,刘春生,王学勇.甘草β-香树酯醇合成酶的编码区克隆与序列分析[J].中国中药杂志.2009,34(19)：21.
[31]何阳花,俞英,张沅.拷贝数变异与疾病的关系及其在动物抗病育种中的应用前景[J].遗传,2008,30(11)：1385.
[32]Timothy, Aitman, Rong Dong, et all. Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans [J]. Nature,2006,16(439):851.
[33]Aitman TJ, Dong R, Vyse TJ, et al. Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans [J]. Nature,2006,439:851.
[34]Gonzalez E, Kulkarni H, Bolivar H, et al. The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility [J]. Scienc,2005,307(5714):1434.
[35]Sebat J, Lakshmi B, Troge J, et al. Large-scale copy number polymorphism in the human genome[J]. Science,2004,305:525.
[36]Ying Qiao, Xudong Liu. Chansonette Harvard. Large-scale copy number variants (CNVs): Distribution in normal subjects and FISH/real time qPCR analysis [J]. BMC Genomics,2007,8:167.
[37]Guan M, liu L, Zhao X, et all. Copy Number Variations of EphA3 Are Associated With Multiple Types of Hematologic Malignancies [J]. Clin Lymphoma Myeloma LeuK,2011,1(1):50.
[38]Timothy, Graubert, Patrick Cahan, et al. A High-Resolution Map of Segmental DNA Copy Number Variation in the Mouse Genome [J]. PLos Genetics,2007,3:21.
[39]Liu, Van Tassel, Sonstegard, et al. Detection of germline and somatic copy number variations in cattle [J]. Dev Biol (Basel),2008,132:231.
[40]George, Perry, Fengtang Yang, et al. Copy number variation and evolution in humans and chimpanzees [J]. Genome Research,2008,18:1698.
[41]George, Yali Hou, Bin Zhu, et all. Analysis of copy number variations among diverse cattle bree ds [J]. Genome Research,2010,4,20:693.
[42]Luz, Shawn, Cokus, et al. Copy number variation influences gene expression and metabolic traits in mice [J]. Human Molecular Genetics,2009,18(21):4118.
[43]Clare, Sreekrishna, Romanos. Expression of tetanus toxin fragment C[J]. Methods Mol Biol, 1998,103:193.
[44]Masferrer, Arro, Manzano, et all. Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cyt-okinin levels [J]. Plant,2002,30(2):123.
[45]Besumbes, Sauret-Gueto, Phillips MA, et all. Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol [J]. Biotechnol Bioeng, 2004,88(2):168.
[46]夏铁骑.荧光原位杂交技术及其在微生物生态学中的应用[J].新乡学院学报(自然科学版),2009,26(2)：52.
[47]钟梅,黎静,丁彦青.应用荧光原位杂交技术检测卵巢浆液性囊腺癌中ZNF217基因拷贝数[J].中华医学遗传学杂志,2006,23(6)：5665.
[48]吴爱华,孟金萍,张爱东,等.荧光原位杂交技术产前诊断唐氏综合征[J].中国优生与遗传杂志,2005,3(5)：33.
[49]卢建,章钧,何蕴韶.荧光原位杂交技术及其临床应用[J].分子诊断与治疗杂志,2009,1(1)：38.
[50]王忠华,李旭晨,夏英武.荧光原位杂交技术在植物学中的应用[J].植物学通报,2001,18(1)：40.
[51]张树辉,魏泓,史景泉.比较基因组杂交技术在肿瘤病理研究中的应用[J].临床与实验病理学杂志,1999,15(4)：330.
[52]牛超.比较基因组技术及其应用[J].遗传学分册,1997,4：21.
[53]张开立.比较基因组杂交新进展Array-based CGH[J].国外医学遗传学分册,2003,26(1)：17.
[54]秦艳茹,王立东,邝丽芸.利用比较基因组杂交技术分析食管癌组织中染色体基因组的变化特征[J].中华病理学杂志,2005,34(2)：80.
[55]韩瀚,刘敬忠,王清涛.多重连接探针扩增技术的研究进展及其应用[J].诊断学理论与实践,2007,6(4)：380.
[56]刘和平,王宏,陆祖宏,等.一种基于寡核苷酸微阵列芯片的多重可扩增探针杂交技术[J].遗传学报,2004,31(2)：119.
[57]胡晓,李汶,卢光琇.多重连接依赖性探针扩增技术及其应用进展[J].现代生物医学进展,2010,2：55.
[58]张宏,盛剑秋,耿洪刚.多重连接依赖的探针扩增技术检测中国人遗传性非息肉病性结直肠癌错配修复基因大片段缺失[J].中国医学科学院学报,2006.23(6)：837.
[59]武雪梅,肖华胜.人类基因组结构变异检测研究进展[J].中国科学,2009,39(3)：237.
[60]魏惠平,伍治平,谢建生,等.多重连接依赖式探针扩增技术及其在医学上的应用[J].中国实用医药,2009,4(12)：1.
[61]周大文,许淼,颜景斌,等.应用MLPA-微阵列技术分析基因组内拷贝数变异的初步探讨[J].现代诊断与治疗,2008,19(2)：98.
[62]周大文,管翌华,许淼,等.MLPA-微阵列技术在Y染色体异常检测中的初步应用[J].遗传,2008,30(2)：1629.
[63]饶友生,张细权.人类基因组结构变异检测研究展[J].生物多样性,2008,16(4)：399.
[64]董园园,盛海辉,杨茜,等.一种新的基于单碱基延伸的SNP芯片技术[J].遗传,2009,31(4)：439.
[65]董园园,盛海辉,陈光,等.SNP芯片技术进展[J].中华医药杂志,2008,8(6)：512.
[66]陆长勇,施春雷,张春秀,等.基于单碱基延伸标签反应的常见食源性致病菌基因芯片检测方法的建立[J].生物工程学报,2009,25(4)：554.
[67]曾朝阳,熊炜,周艳宏,等.高密度全基因组单核苷酸多态芯片研究进展及其在肿瘤研究中的应用[J].癌症,2006,25(11)：1454.
[68]胡彬,陈军,刘红雨,等.利用单核苷酸多态性芯片全基因组检测人大细胞肺癌细胞株的杂合性缺失和拷贝数变异[J].中国肺癌杂志,2008,11(3)：327.
[69]林连捷,林艳,郑长青,等.单核苦酸多态性芯片分析膜腺癌基因组杂合性缺失[J].第四军医大学学报,2008,29(11)：988.
[69]何永蜀,张闻,杨照青.人类基因组结构变异[J].遗传,2009,31(8)：771.
[70]杨凤秋,朱正歌.实时定量PCR技术及应用[J].生物学杂志,2006,23(3)：44.
[71]韩俊英,曾瑞萍.荧光定量PCR技术及其应用[J].国外医学遗传学分册,2000,23(3)：117.
[72]王爱民.实时荧光定量PCR(TaqMan)法测定外源基因的拷贝数[J].广西植物,2009,29(3)：408.
[73]王江,李琳,宛新杉.含Ds转座因子的T-DNA在水稻染色体上的分布研究[J].中国科学C辑,2004,34(1)：47
[74]卢春斌.转基因拟南芥中外源基因的Southern blot检测[J].种子,2007,26(3)：23.
[75]杨英军,周鹏.葡萄无核基因的SCAR标记及Southern-blot分析[J].西北农林科技大学学报：自然科学版,2002,30(6)：77.
[76]陈琳,徐秀英.组织型纤溶酶原激活剂突变体细胞t-PA基因拷贝数测定及分析[J].生物技术,1998,8(2)：16.
[77]Tae-Wook Kang, Yeo-Jin Jeon, Eunsu Jang, et all. Copy number variations (CNVs) identified in Korean individuals [J]. BMC Genomics,2008,9:492.
[78]杨全,王文全,魏胜利,等.不同变异类型甘草中甘草苷及甘草酸量比较研究[J].中草药,2007,38(7)：1087.
[79]Hikaru Seki, Kiyoshi Ohyama, Satoru Sawai, et all. Licorice β-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin[J]. PNAS,2008,105(37) :14204.
[80]廖一凡.五环三萜皂苷的合成途径及生物活性研究进展[J].湖北中医学院学报,2010,12(3)：60.