本文主要研究内容
作者张华北（2019）在《半枝莲、夏枯草萜类生物合成相关基因的鉴定及功能研究》一文中研究指出：半枝莲Scutella barbata 和夏枯草Prunella vulgaris属于唇形科植物。半枝莲是一种重要的药用植物,广泛应用于中国、韩国、印度等亚洲国家。新克罗烷型二萜类化合物是半枝莲中主要的活性成分,对多种癌细胞具有良好的细胞毒性。夏枯草是唇形科分布最广泛的物种,是一种常用的中药材及草药制剂。三萜类化合物是夏枯草的主要活性成分。本论文通过对半枝莲和夏枯草不同组织进行Illumina测序及分析,系统克隆萜类化合物生物合成途径中的基因,并对部分二萜合酶基因进行了功能鉴定,为唇形科植物萜类化合物多样性的产生机制以及特定萜类化合物的生物合成途径研究奠定了基础。主要研究结果如下:1、首先对半枝莲和夏枯草的不同组织进行转录组测序及组装。提取半枝莲的根(BZLR)、茎叶(BZLS)、花(BZLF)三个组织的RNA;夏枯草的根(XKCR)、茎叶(XKCS)、花穗(XKCF)以及未成熟的种子(XKCZ)四个组织的RNA。每个组织分别进行两次重复。将半枝莲中121,958,564条clean RNA-sequence reads 组装成 88,980 个转录本,共计 52,211 个 unigenes。unigenes平均长度为1370 nt,N50长度为2144 nt;夏枯草中121,809,359条clean RNA-sequence reads 组装成 108,672 个转录本,共计 57,985 个 unigenes。Unigene 平均长度为1,120 nt,N50长度为1,843 nt。通过搜索常用公共数据库如Swiss-Prot、TrEMBL、Pfam(HMM)、Gene Ontology 和 KEGG 等,结果半枝莲中有36659 个 unigenes(70.23%),夏枯草中有 33323 个 unigenes(54.47%)得到了注释。2、利用NCBI公共数据库和丹参、拟南芥等已知基因序列系统鉴定参与半枝莲和夏枯草中萜类backbone生物合成的基因。半枝莲和夏枯草分别注释71个unigenes和53个unigenes。共计鉴定33个基因(32个具有全长cDNA)和35个(35个具有全长cDNA)分别参与半枝莲和夏枯草中萜类backbone的生物合成。对照丹参全基因组鉴定的backbone基因,我们鉴定了半枝莲和夏枯草中全部17个参与萜类backbone生物合成的基因家族。包括MVA途径的AACT,HMGS,HMGR,MVK,PMK,MDD 基因;MEP 途径的 DXS,DXR,MCT,CMK,MDS,HDS,HDR和 IDI,IPK基因;异戊烯转移酶 FPPS,GGPPS,GPPS.SSU基因。发现10个基因家族在三个物种中非常保守,分别为AACT,HMGS,MK,PMK,MDD,DXS,DXR,MCT,CMK 和 MDS。而另外 7 个基因家族,则在不同物种中表现出一定程度的扩张和收缩。如HMGR基因家族在夏枯草和丹参中均有4个基因,而在半枝莲中只有3个。DXS在半枝莲和丹参中均有5个基因,而在夏枯草中有6个,以及IDI,FPPS,HDR和GGPPS基因家族均略有差异。3、在半枝莲和夏枯草中分别鉴定了 14个和17个萜类合酶基因。通过聚类分析显示半枝莲和夏枯草中分别有1个和2个基因属于TPS-b基因家族,推测参与单萜的生物合成。分别有5个和9个基因属于TPS-a基因家族,推测参与半枝莲和夏枯草中倍半萜的生物合成。半枝莲和夏枯草中分别有8个和6个二萜合酶基因。由于夏枯草富含三萜类化合物,因此对夏枯草中的三萜合成相关基因进行了鉴定。共计鉴定了9个基因,包括2个角鲨烯合成酶(SQS),2个角鲨烯单加氧酶(SM),以及5个氧化角鲨烯环化酶(OSC)。OSC基因家族负责多样化的三萜骨架形成,聚类分析显示PvOSC2,PvOSC3以及半枝莲的SbOSC2与人参中的达玛烷型dammarenediol II合酶聚为一类,后者参与人参中的人参皂甙的生物合成,推测该类OSC基因在唇形科中也普遍存在,其催化功能值得进一步探索。为进一步揭示上述萜类生物合成相关基因可能的生物学功能以及它们之间的关系,我们分别对半枝莲和夏枯草的表达谱数据进行基于log-2转化的FPKM值的聚类分析。结果表明,半枝莲的二萜合酶基因TPS8,TPS2以及HMGR3主要在根和花中高表达。DXP途径中的基因在不同组织均表现出高水平的表达,这与二萜类化合物主要通过DXP途径合成的认知相符。DXS1和二萜合酶基因TPS11特异地在地上部分的茎叶中高表达,因此,该基因有可能参与半枝莲中新克罗烷型二萜化合物的生物合成。单萜和倍半萜合酶在不同组织中均具有较低的表达水平。因此,二萜类生物合成途径在半枝莲中占有优势地位。发现夏枯草中高表达的基因主要来源于MVA途径,如HMGR1,HMGR3,IDI1,AACT1,FPPS1,及HMGS与三萜合酶相关基因SQS1,OSC2和OSC3,这也与三萜合成主要来源于MVA途径和夏枯草富含三萜类化合物的报道一致。4、对半枝莲和夏枯草中二萜合酶基因的克隆和体外功能鉴定研究。从半枝莲和夏枯草根cDNA中分别克隆到3个和2个二萜合酶基因,其序列与转录组组装序列一致。SbTPS8,SbTPS9和SbTPS12的cDNA长度分别为2793bp,2690bp 和 3102bp,蛋白长度分别为 803aa,810aa 和 594aa;PvTPS14 和PvTPS16的CDNA长度分别为2403bp和1785bp,蛋白长度分别为800aa和593aa。SbTPS8,SbTPS9和PvTPS14具有保守的结构域是DxDD结构与,为Class Ⅱ二萜合酶。SbTPS12和PvTPS16具有保守的DDxxD结构域,为Class I二萜合酶。将半枝莲,夏枯草与唇形科中52个已知功能二萜合酶进行系统进化树分析显示,SbTPS9,PvTPS15与参与ent-CPP形成的二萜合酶聚为一类。SbTPS8和PvTPS14与唇形科中特化的CPP/LDPP合酶聚类。进一步大肠杆菌原核表达发现SbTPS8,SbTPS9,SbTPS12重组蛋白可溶性好,纯化后目的蛋白的条带大小与预测的一致。而PvTPS14和PvTPS在相同条件下可溶蛋白表达量低。对其诱导温度及纯化条件进行优化,依然没有得到可溶性目的蛋白。以GGPP为底物,将纯化后的pET32-CPS重组蛋白分别与丹参的SmKSL1和拟南芥中的AtKS进行体外酶促反应。SmKSL1特异作用于normal-CPP,而AtKS特异作用于ent-CPP。结果表明,SbTPS8只与SmKSL1共催化,产物与丹参中SmCPSl(normal-CPP合酶)和SmKSL1共催化产物一致,为miltiradiene,确定其为normal-CPP合酶。SbTPS9只与AtKS共催化,产物与丹参中SmCPS5(ent-CPP合酶)和AtKS共催化产物一致,为ent-kaurene,因此确定SbTPS9为ent-CPP合酶。SbTPS12与SbTPS8共催化产生miltiradiene,确定SbTPS12为miltiradiene合酶。体外验证PvTPS 14和PvTPS 16无明显的稳定活性。5、利用高产GGPP的大肠杆菌工程菌体系验证Class I二萜合酶的底物杂泛性的研究。首先利用南欧丹参Salvia sclarea的香紫苏醇合酶(SsSS)来验证己报道大肠杆菌工程菌体系的重复性和稳定性。SsSS分别与12种class Ⅱ型二萜合酶共同发酵,结果SsSS与9种二萜合酶共同发酵能检测到明显的与文献报道一致的产物,而与CLPP(12)和ent-CLPP(13)共同发酵的产物量低。由于terpentedienyl diphosphate(9)载体的抗性特殊,不便于高通量操作,因此,本实验利用该体系测试了SbTPS12和PvTPS16与11种class II型二萜合酶的反应情况。结果表明,SbTPS12可以与两种类型的底物反应,SbTPS12与Copaly1 diphosphate(3)共催化产物为miltiradiene;SbTPS12与ent-kolavenyl diphosphate(12)共催化产物为ent-kaurene。PvTPS16可以与四种类型的底物反应,与Copalyl diphosphate(3)和8α-hydroxy-CPP(7)的产物为miltiradiene。与endo-CPP(6)共催化的产物推测为 13R-(+)-manoyl oxide。说明 SbTPS12,PvTPS16具有不同程度的底物杂泛性,不同的KSL可以催化的底物类型是不同的。综上,本研究利用Illumina测序、数字表达谱、大肠杆菌体外酶促鉴定及大肠杆菌工程菌等体系对半枝莲、夏枯草中参与萜类生物合成的相关基因进行了系统鉴定和分析。半枝莲DXP途径基因的高水平表达,二萜合酶基因的扩张以及在根、地上部分的不同表达模式,推测二萜类生物合成途径为半枝莲的优势生物合成途径。而夏枯草中MVA途径以及三萜生物合成的一系列酶基因在不同组织呈现较高表达,从而推测三萜生物合成途径为夏枯草中的优势生物合成途径。4个二萜合酶基因功能的鉴定和底物杂泛性的研究,明确了相关基因的功能及其催化反应特性。这些结果使我们对半枝莲和夏枯草的萜类生物合成有了全面的认识,从分子水平上阐释了半枝莲和夏枯草中萜类化合物多样性的成因,并为通过基因工程技术提高萜类化合物产量奠定了基础。
Abstract
ban zhi lian Scutella barbata he xia ku cao Prunella vulgarisshu yu chun xing ke zhi wu 。ban zhi lian shi yi chong chong yao de yao yong zhi wu ,an fan ying yong yu zhong guo 、han guo 、yin du deng ya zhou guo jia 。xin ke luo wan xing er tie lei hua ge wu shi ban zhi lian zhong zhu yao de huo xing cheng fen ,dui duo chong ai xi bao ju you liang hao de xi bao du xing 。xia ku cao shi chun xing ke fen bu zui an fan de wu chong ,shi yi chong chang yong de zhong yao cai ji cao yao zhi ji 。san tie lei hua ge wu shi xia ku cao de zhu yao huo xing cheng fen 。ben lun wen tong guo dui ban zhi lian he xia ku cao bu tong zu zhi jin hang Illuminace xu ji fen xi ,ji tong ke long tie lei hua ge wu sheng wu ge cheng tu jing zhong de ji yin ,bing dui bu fen er tie ge mei ji yin jin hang le gong neng jian ding ,wei chun xing ke zhi wu tie lei hua ge wu duo yang xing de chan sheng ji zhi yi ji te ding tie lei hua ge wu de sheng wu ge cheng tu jing yan jiu dian ding le ji chu 。zhu yao yan jiu jie guo ru xia :1、shou xian dui ban zhi lian he xia ku cao de bu tong zu zhi jin hang zhuai lu zu ce xu ji zu zhuang 。di qu ban zhi lian de gen (BZLR)、jing xie (BZLS)、hua (BZLF)san ge zu zhi de RNA;xia ku cao de gen (XKCR)、jing xie (XKCS)、hua sui (XKCF)yi ji wei cheng shou de chong zi (XKCZ)si ge zu zhi de RNA。mei ge zu zhi fen bie jin hang liang ci chong fu 。jiang ban zhi lian zhong 121,958,564tiao clean RNA-sequence reads zu zhuang cheng 88,980 ge zhuai lu ben ,gong ji 52,211 ge unigenes。unigenesping jun chang du wei 1370 nt,N50chang du wei 2144 nt;xia ku cao zhong 121,809,359tiao clean RNA-sequence reads zu zhuang cheng 108,672 ge zhuai lu ben ,gong ji 57,985 ge unigenes。Unigene ping jun chang du wei 1,120 nt,N50chang du wei 1,843 nt。tong guo sou suo chang yong gong gong shu ju ku ru Swiss-Prot、TrEMBL、Pfam(HMM)、Gene Ontology he KEGG deng ,jie guo ban zhi lian zhong you 36659 ge unigenes(70.23%),xia ku cao zhong you 33323 ge unigenes(54.47%)de dao le zhu shi 。2、li yong NCBIgong gong shu ju ku he dan can 、ni na gai deng yi zhi ji yin xu lie ji tong jian ding can yu ban zhi lian he xia ku cao zhong tie lei backbonesheng wu ge cheng de ji yin 。ban zhi lian he xia ku cao fen bie zhu shi 71ge unigeneshe 53ge unigenes。gong ji jian ding 33ge ji yin (32ge ju you quan chang cDNA)he 35ge (35ge ju you quan chang cDNA)fen bie can yu ban zhi lian he xia ku cao zhong tie lei backbonede sheng wu ge cheng 。dui zhao dan can quan ji yin zu jian ding de backboneji yin ,wo men jian ding le ban zhi lian he xia ku cao zhong quan bu 17ge can yu tie lei backbonesheng wu ge cheng de ji yin jia zu 。bao gua MVAtu jing de AACT,HMGS,HMGR,MVK,PMK,MDD ji yin ;MEP tu jing de DXS,DXR,MCT,CMK,MDS,HDS,HDRhe IDI,IPKji yin ;yi wu xi zhuai yi mei FPPS,GGPPS,GPPS.SSUji yin 。fa xian 10ge ji yin jia zu zai san ge wu chong zhong fei chang bao shou ,fen bie wei AACT,HMGS,MK,PMK,MDD,DXS,DXR,MCT,CMK he MDS。er ling wai 7 ge ji yin jia zu ,ze zai bu tong wu chong zhong biao xian chu yi ding cheng du de kuo zhang he shou su 。ru HMGRji yin jia zu zai xia ku cao he dan can zhong jun you 4ge ji yin ,er zai ban zhi lian zhong zhi you 3ge 。DXSzai ban zhi lian he dan can zhong jun you 5ge ji yin ,er zai xia ku cao zhong you 6ge ,yi ji IDI,FPPS,HDRhe GGPPSji yin jia zu jun lve you cha yi 。3、zai ban zhi lian he xia ku cao zhong fen bie jian ding le 14ge he 17ge tie lei ge mei ji yin 。tong guo ju lei fen xi xian shi ban zhi lian he xia ku cao zhong fen bie you 1ge he 2ge ji yin shu yu TPS-bji yin jia zu ,tui ce can yu chan tie de sheng wu ge cheng 。fen bie you 5ge he 9ge ji yin shu yu TPS-aji yin jia zu ,tui ce can yu ban zhi lian he xia ku cao zhong bei ban tie de sheng wu ge cheng 。ban zhi lian he xia ku cao zhong fen bie you 8ge he 6ge er tie ge mei ji yin 。you yu xia ku cao fu han san tie lei hua ge wu ,yin ci dui xia ku cao zhong de san tie ge cheng xiang guan ji yin jin hang le jian ding 。gong ji jian ding le 9ge ji yin ,bao gua 2ge jiao sha xi ge cheng mei (SQS),2ge jiao sha xi chan jia yang mei (SM),yi ji 5ge yang hua jiao sha xi huan hua mei (OSC)。OSCji yin jia zu fu ze duo yang hua de san tie gu jia xing cheng ,ju lei fen xi xian shi PvOSC2,PvOSC3yi ji ban zhi lian de SbOSC2yu ren can zhong de da ma wan xing dammarenediol IIge mei ju wei yi lei ,hou zhe can yu ren can zhong de ren can zao dai de sheng wu ge cheng ,tui ce gai lei OSCji yin zai chun xing ke zhong ye pu bian cun zai ,ji cui hua gong neng zhi de jin yi bu tan suo 。wei jin yi bu jie shi shang shu tie lei sheng wu ge cheng xiang guan ji yin ke neng de sheng wu xue gong neng yi ji ta men zhi jian de guan ji ,wo men fen bie dui ban zhi lian he xia ku cao de biao da pu shu ju jin hang ji yu log-2zhuai hua de FPKMzhi de ju lei fen xi 。jie guo biao ming ,ban zhi lian de er tie ge mei ji yin TPS8,TPS2yi ji HMGR3zhu yao zai gen he hua zhong gao biao da 。DXPtu jing zhong de ji yin zai bu tong zu zhi jun biao xian chu gao shui ping de biao da ,zhe yu er tie lei hua ge wu zhu yao tong guo DXPtu jing ge cheng de ren zhi xiang fu 。DXS1he er tie ge mei ji yin TPS11te yi de zai de shang bu fen de jing xie zhong gao biao da ,yin ci ,gai ji yin you ke neng can yu ban zhi lian zhong xin ke luo wan xing er tie hua ge wu de sheng wu ge cheng 。chan tie he bei ban tie ge mei zai bu tong zu zhi zhong jun ju you jiao di de biao da shui ping 。yin ci ,er tie lei sheng wu ge cheng tu jing zai ban zhi lian zhong zhan you you shi de wei 。fa xian xia ku cao zhong gao biao da de ji yin zhu yao lai yuan yu MVAtu jing ,ru HMGR1,HMGR3,IDI1,AACT1,FPPS1,ji HMGSyu san tie ge mei xiang guan ji yin SQS1,OSC2he OSC3,zhe ye yu san tie ge cheng zhu yao lai yuan yu MVAtu jing he xia ku cao fu han san tie lei hua ge wu de bao dao yi zhi 。4、dui ban zhi lian he xia ku cao zhong er tie ge mei ji yin de ke long he ti wai gong neng jian ding yan jiu 。cong ban zhi lian he xia ku cao gen cDNAzhong fen bie ke long dao 3ge he 2ge er tie ge mei ji yin ,ji xu lie yu zhuai lu zu zu zhuang xu lie yi zhi 。SbTPS8,SbTPS9he SbTPS12de cDNAchang du fen bie wei 2793bp,2690bp he 3102bp,dan bai chang du fen bie wei 803aa,810aa he 594aa;PvTPS14 he PvTPS16de CDNAchang du fen bie wei 2403bphe 1785bp,dan bai chang du fen bie wei 800aahe 593aa。SbTPS8,SbTPS9he PvTPS14ju you bao shou de jie gou yu shi DxDDjie gou yu ,wei Class Ⅱer tie ge mei 。SbTPS12he PvTPS16ju you bao shou de DDxxDjie gou yu ,wei Class Ier tie ge mei 。jiang ban zhi lian ,xia ku cao yu chun xing ke zhong 52ge yi zhi gong neng er tie ge mei jin hang ji tong jin hua shu fen xi xian shi ,SbTPS9,PvTPS15yu can yu ent-CPPxing cheng de er tie ge mei ju wei yi lei 。SbTPS8he PvTPS14yu chun xing ke zhong te hua de CPP/LDPPge mei ju lei 。jin yi bu da chang gan jun yuan he biao da fa xian SbTPS8,SbTPS9,SbTPS12chong zu dan bai ke rong xing hao ,chun hua hou mu de dan bai de tiao dai da xiao yu yu ce de yi zhi 。er PvTPS14he PvTPSzai xiang tong tiao jian xia ke rong dan bai biao da liang di 。dui ji you dao wen du ji chun hua tiao jian jin hang you hua ,yi ran mei you de dao ke rong xing mu de dan bai 。yi GGPPwei de wu ,jiang chun hua hou de pET32-CPSchong zu dan bai fen bie yu dan can de SmKSL1he ni na gai zhong de AtKSjin hang ti wai mei cu fan ying 。SmKSL1te yi zuo yong yu normal-CPP,er AtKSte yi zuo yong yu ent-CPP。jie guo biao ming ,SbTPS8zhi yu SmKSL1gong cui hua ,chan wu yu dan can zhong SmCPSl(normal-CPPge mei )he SmKSL1gong cui hua chan wu yi zhi ,wei miltiradiene,que ding ji wei normal-CPPge mei 。SbTPS9zhi yu AtKSgong cui hua ,chan wu yu dan can zhong SmCPS5(ent-CPPge mei )he AtKSgong cui hua chan wu yi zhi ,wei ent-kaurene,yin ci que ding SbTPS9wei ent-CPPge mei 。SbTPS12yu SbTPS8gong cui hua chan sheng miltiradiene,que ding SbTPS12wei miltiradienege mei 。ti wai yan zheng PvTPS 14he PvTPS 16mo ming xian de wen ding huo xing 。5、li yong gao chan GGPPde da chang gan jun gong cheng jun ti ji yan zheng Class Ier tie ge mei de de wu za fan xing de yan jiu 。shou xian li yong na ou dan can Salvia sclareade xiang zi su chun ge mei (SsSS)lai yan zheng ji bao dao da chang gan jun gong cheng jun ti ji de chong fu xing he wen ding xing 。SsSSfen bie yu 12chong class Ⅱxing er tie ge mei gong tong fa jiao ,jie guo SsSSyu 9chong er tie ge mei gong tong fa jiao neng jian ce dao ming xian de yu wen suo bao dao yi zhi de chan wu ,er yu CLPP(12)he ent-CLPP(13)gong tong fa jiao de chan wu liang di 。you yu terpentedienyl diphosphate(9)zai ti de kang xing te shu ,bu bian yu gao tong liang cao zuo ,yin ci ,ben shi yan li yong gai ti ji ce shi le SbTPS12he PvTPS16yu 11chong class IIxing er tie ge mei de fan ying qing kuang 。jie guo biao ming ,SbTPS12ke yi yu liang chong lei xing de de wu fan ying ,SbTPS12yu Copaly1 diphosphate(3)gong cui hua chan wu wei miltiradiene;SbTPS12yu ent-kolavenyl diphosphate(12)gong cui hua chan wu wei ent-kaurene。PvTPS16ke yi yu si chong lei xing de de wu fan ying ,yu Copalyl diphosphate(3)he 8α-hydroxy-CPP(7)de chan wu wei miltiradiene。yu endo-CPP(6)gong cui hua de chan wu tui ce wei 13R-(+)-manoyl oxide。shui ming SbTPS12,PvTPS16ju you bu tong cheng du de de wu za fan xing ,bu tong de KSLke yi cui hua de de wu lei xing shi bu tong de 。zeng shang ,ben yan jiu li yong Illuminace xu 、shu zi biao da pu 、da chang gan jun ti wai mei cu jian ding ji da chang gan jun gong cheng jun deng ti ji dui ban zhi lian 、xia ku cao zhong can yu tie lei sheng wu ge cheng de xiang guan ji yin jin hang le ji tong jian ding he fen xi 。ban zhi lian DXPtu jing ji yin de gao shui ping biao da ,er tie ge mei ji yin de kuo zhang yi ji zai gen 、de shang bu fen de bu tong biao da mo shi ,tui ce er tie lei sheng wu ge cheng tu jing wei ban zhi lian de you shi sheng wu ge cheng tu jing 。er xia ku cao zhong MVAtu jing yi ji san tie sheng wu ge cheng de yi ji lie mei ji yin zai bu tong zu zhi cheng xian jiao gao biao da ,cong er tui ce san tie sheng wu ge cheng tu jing wei xia ku cao zhong de you shi sheng wu ge cheng tu jing 。4ge er tie ge mei ji yin gong neng de jian ding he de wu za fan xing de yan jiu ,ming que le xiang guan ji yin de gong neng ji ji cui hua fan ying te xing 。zhe xie jie guo shi wo men dui ban zhi lian he xia ku cao de tie lei sheng wu ge cheng you le quan mian de ren shi ,cong fen zi shui ping shang chan shi le ban zhi lian he xia ku cao zhong tie lei hua ge wu duo yang xing de cheng yin ,bing wei tong guo ji yin gong cheng ji shu di gao tie lei hua ge wu chan liang dian ding le ji chu 。
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论文作者分别是来自中国中医科学院的张华北，发表于刊物中国中医科学院2019-10-28论文，是一篇关于半枝莲论文,夏枯草论文,转录组分析论文,萜类骨架生物合成论文,萜类合酶论文,二萜合酶论文,大肠杆菌工程菌论文,中国中医科学院2019-10-28论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自中国中医科学院2019-10-28论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
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