本文主要研究内容
作者孙宇峰(2019)在《甘草酸生物合成途径中GuGDHs与GuCPR基因的克隆及GuGDHs的活性分析》一文中研究指出:甘草具有补脾益气,清热解毒,祛痰止咳,缓急止痛等功效,甘草酸是甘草的主要活性成分,甘草酸的含量是《中国药典》列入的衡量甘草药材品质优劣的主要指标。甘草酸药用价值与工业价值显著,不仅运用于临床治疗,还广泛应用于化妆品、食品、保健品等行业。目前,仅靠种植甘草无法满足其巨大市场需求,同时,种植甘草周期长,品质优劣不一,过渡采挖甘草造成土地沙化、土壤贫瘠、生态环境破坏等严重问题,因此,采用新兴科学技术获得甘草酸已成为甘草资源研究的热点问题。近年来,随着合成生物学技术的飞速发展,人们对于甘草酸生物合成途径的研究日趋成熟,因此,运用合成生物学技术人工合成甘草酸有望成为缓解甘草酸市场供不应求,改善甘草种质资源的重要方法。目前,甘草酸生物合成途径中的关键基因多数已被成功表征,甘草酸的生物合成通路已基本打通,科学家已在酵母中合成了甘草酸的前体——甘草次酸。然而,甘草酸中下游途径中的UDP-葡萄糖脱氢酶(GuGDHs)与细胞色素P450还原酶(GuCPR)仍未被成功解析。甘草酸糖基转移酶需在UDP-葡萄糖脱氢酶(GuGDHs)催化作用下,方可催化甘草次酸生成终产物甘草酸;甘草酸细胞色素P450还原酶(GuCPR)为细胞色素P450的催化过程提供电子,是细胞色素P450超家族酶中的重要成员,因此,解析甘草酸UDP-葡萄糖脱氢酶(GuGDHs)与甘草酸细胞色素P450还原酶(GuCPR)对甘草酸的生物全合成至关重要。因此,本文研究内容及主要研究成果如下:1.通过本课题组前期转录组数据库与NCBI上报道的同源基因比对,筛选出了3条候选甘草酸UDP-葡萄糖脱氢酶(GuGDHs),命名为GuGDH1、GuGDH2、GuGDH3;筛选出了1条甘草酸细胞色素P450还原酶(命名为GuCPR)全长编码序列,并以cDNA为模板,基于PCR(Polymerase Chain Reaction,聚合酶链式反应)技术,通过特异性引物扩增,克隆了GuGDH1、GuGDH2、GuGDH3与GuCPR的完整编码区(CDS),得到了3条1000bp左右和1条2000bp左右的条带,并通过连接pEASY-Blunt载体得到GuGDH1、GuGDH2、GuGDH3与GuCPR的克隆重组质粒。基于蛋白结构预测与系统进化树分析,显示GuGDH1、GuGDH2、GuGDH3归属于UDP-葡萄糖脱氢酶超家族,GuCPR归属于细胞色素P450还原酶超家族。此外,利用相关生物信息学分析方法分析了GuGDHs与GuCPR的基本特性。2.通过同源重组方法构建了GuGDH1、GuGDH2、GuGDH3的pET-30a(+)原核表达载体,并在大肠杆菌E.coliBL21(DE3)中异源表达。通过IPTG(异丙基硫代半乳糖苷,Isopropy1 β-D-Thiogalactoside)诱导表达,获得GuGDH1、GuGDH2、GuGDH3重组蛋白。3.通过酶促反应催化实验,检测NADPH在340nm处吸光度的增加来确定甘草酸UDP-葡萄糖脱氢酶(GuGDHs)的活性,证明了GuGDH1、GuGDH2具有活性,而GuGDH3不具有活性。本文研究成果可为下一步解析甘草酸生物合成途径奠定基础。
Abstract
gan cao ju you bu pi yi qi ,qing re jie du ,qu tan zhi hai ,huan ji zhi tong deng gong xiao ,gan cao suan shi gan cao de zhu yao huo xing cheng fen ,gan cao suan de han liang shi 《zhong guo yao dian 》lie ru de heng liang gan cao yao cai pin zhi you lie de zhu yao zhi biao 。gan cao suan yao yong jia zhi yu gong ye jia zhi xian zhe ,bu jin yun yong yu lin chuang zhi liao ,hai an fan ying yong yu hua zhuang pin 、shi pin 、bao jian pin deng hang ye 。mu qian ,jin kao chong zhi gan cao mo fa man zu ji ju da shi chang xu qiu ,tong shi ,chong zhi gan cao zhou ji chang ,pin zhi you lie bu yi ,guo du cai wa gan cao zao cheng tu de sha hua 、tu rang pin ji 、sheng tai huan jing po huai deng yan chong wen ti ,yin ci ,cai yong xin xing ke xue ji shu huo de gan cao suan yi cheng wei gan cao zi yuan yan jiu de re dian wen ti 。jin nian lai ,sui zhao ge cheng sheng wu xue ji shu de fei su fa zhan ,ren men dui yu gan cao suan sheng wu ge cheng tu jing de yan jiu ri qu cheng shou ,yin ci ,yun yong ge cheng sheng wu xue ji shu ren gong ge cheng gan cao suan you wang cheng wei huan jie gan cao suan shi chang gong bu ying qiu ,gai shan gan cao chong zhi zi yuan de chong yao fang fa 。mu qian ,gan cao suan sheng wu ge cheng tu jing zhong de guan jian ji yin duo shu yi bei cheng gong biao zheng ,gan cao suan de sheng wu ge cheng tong lu yi ji ben da tong ,ke xue jia yi zai jiao mu zhong ge cheng le gan cao suan de qian ti ——gan cao ci suan 。ran er ,gan cao suan zhong xia you tu jing zhong de UDP-pu tao tang tuo qing mei (GuGDHs)yu xi bao se su P450hai yuan mei (GuCPR)reng wei bei cheng gong jie xi 。gan cao suan tang ji zhuai yi mei xu zai UDP-pu tao tang tuo qing mei (GuGDHs)cui hua zuo yong xia ,fang ke cui hua gan cao ci suan sheng cheng zhong chan wu gan cao suan ;gan cao suan xi bao se su P450hai yuan mei (GuCPR)wei xi bao se su P450de cui hua guo cheng di gong dian zi ,shi xi bao se su P450chao jia zu mei zhong de chong yao cheng yuan ,yin ci ,jie xi gan cao suan UDP-pu tao tang tuo qing mei (GuGDHs)yu gan cao suan xi bao se su P450hai yuan mei (GuCPR)dui gan cao suan de sheng wu quan ge cheng zhi guan chong yao 。yin ci ,ben wen yan jiu nei rong ji zhu yao yan jiu cheng guo ru xia :1.tong guo ben ke ti zu qian ji zhuai lu zu shu ju ku yu NCBIshang bao dao de tong yuan ji yin bi dui ,shai shua chu le 3tiao hou shua gan cao suan UDP-pu tao tang tuo qing mei (GuGDHs),ming ming wei GuGDH1、GuGDH2、GuGDH3;shai shua chu le 1tiao gan cao suan xi bao se su P450hai yuan mei (ming ming wei GuCPR)quan chang bian ma xu lie ,bing yi cDNAwei mo ban ,ji yu PCR(Polymerase Chain Reaction,ju ge mei lian shi fan ying )ji shu ,tong guo te yi xing yin wu kuo zeng ,ke long le GuGDH1、GuGDH2、GuGDH3yu GuCPRde wan zheng bian ma ou (CDS),de dao le 3tiao 1000bpzuo you he 1tiao 2000bpzuo you de tiao dai ,bing tong guo lian jie pEASY-Bluntzai ti de dao GuGDH1、GuGDH2、GuGDH3yu GuCPRde ke long chong zu zhi li 。ji yu dan bai jie gou yu ce yu ji tong jin hua shu fen xi ,xian shi GuGDH1、GuGDH2、GuGDH3gui shu yu UDP-pu tao tang tuo qing mei chao jia zu ,GuCPRgui shu yu xi bao se su P450hai yuan mei chao jia zu 。ci wai ,li yong xiang guan sheng wu xin xi xue fen xi fang fa fen xi le GuGDHsyu GuCPRde ji ben te xing 。2.tong guo tong yuan chong zu fang fa gou jian le GuGDH1、GuGDH2、GuGDH3de pET-30a(+)yuan he biao da zai ti ,bing zai da chang gan jun E.coliBL21(DE3)zhong yi yuan biao da 。tong guo IPTG(yi bing ji liu dai ban ru tang gan ,Isopropy1 β-D-Thiogalactoside)you dao biao da ,huo de GuGDH1、GuGDH2、GuGDH3chong zu dan bai 。3.tong guo mei cu fan ying cui hua shi yan ,jian ce NADPHzai 340nmchu xi guang du de zeng jia lai que ding gan cao suan UDP-pu tao tang tuo qing mei (GuGDHs)de huo xing ,zheng ming le GuGDH1、GuGDH2ju you huo xing ,er GuGDH3bu ju you huo xing 。ben wen yan jiu cheng guo ke wei xia yi bu jie xi gan cao suan sheng wu ge cheng tu jing dian ding ji chu 。
论文参考文献
论文详细介绍
论文作者分别是来自北京中医药大学的孙宇峰,发表于刊物北京中医药大学2019-07-01论文,是一篇关于甘草酸论文,活性分析论文,基因克隆论文,生物合成途径论文,葡萄糖脱氢酶论文,细胞色素还原酶论文,北京中医药大学2019-07-01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,资料来自北京中医药大学2019-07-01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。
标签:甘草酸论文; 活性分析论文; 基因克隆论文; 生物合成途径论文; 葡萄糖脱氢酶论文; 细胞色素还原酶论文; 北京中医药大学2019-07-01论文;