本文主要研究内容
作者尹彦超（2019）在《基于毛状根培养体系的甘草CHS及CHI基因功能研究》一文中研究指出：甘草是我国最常用的大宗药材之一,素有“十方九草”之誉。近些年来,栽培甘草普遍存在品质退化和有效成分含量低等问题,已成为制约甘草资源可持续发展的关键问题。大量文献报道显示:毛状根可稳定高效的生产药用植物的次生代谢产物,因此利用毛状根培养体系对甘草的次生代谢途径开展研究具有重要价值。甘草苷是甘草中最重要的活性成分之一,查尔酮合酶(chalcone synthase,CHS)及查尔酮异构酶(chalcone isomerase,CHI)是甘草苷生物合成途径中起关键调控作用的限速酶。因此,本论文拟在前期克隆获得甘草CHS及CHI基因的基础上,构建植物双元表达载体及发根农杆菌工程菌,利用毛状根培养体系对甘草CHS及CHI基因进行功能研究,揭示其影响黄酮类化合物生物合成的分子机制。本课题的研究结果还将进一步筛选甘草苷高水平积累的毛状根体系,为甘草苷的离体合成奠定理论基础和提供技术支持。本文根据课题组前期对于甘草黄酮类化合物含量与CHS、CHI基因多态性相关关系的分析结果筛选出黄酮高含量对应的特异CHS、CHI基因单倍型;采用基因融合的方法构建甘草转CHS、CHI基因植物双元表达载体;通过电转法将甘草转CHS、CHI基因植物双元表达载体导入发根农杆菌;用转基因发根农杆菌工程菌侵染甘草外植体诱导转基因毛状根,并进行PCR和测序验证;建立同时测定甘草毛状根中四种黄酮成分含量的UPLC方法,并对培养得到的转CHS、CHI基因甘草毛状根进行含量测定;通过qRT-PCR对转CHS、CHI基因毛状根中目标基因的拷贝数进行测定,分析目标基因拷贝数与毛状根中黄酮类化合物含量的相关性。本论文共取得了如下研究结果:(1)根据黄酮类化合物含量与CHS、CHI基因多态性相关关系的分析结果,筛选出黄酮类化合物高含量甘草样品对应的特异CHI-1(GenBank注册号:KY115232),CHS-60(GenBank注册号:KY810356)单倍型。(2)选择Bgl Ⅱ和Spe Ⅰ作为酶切位点,pCAMBIA1305.1为植物双元表达载体,采用基因融合的方法,成功构建甘草转CHI、CHS基因植物双元表达载体pCA-CHI、pCA-CHS。(3)采用电转法(电转条件:C:25μF;PC:200Ω;U:2400V)分别将携带有甘草CHI和CHS基因的双元表达载体pCA-CHI和pCA-CHS导入到发根农杆菌ACCC10060中,构建得到转甘草CHI、CHS的发根农杆菌工程菌。(4)利用转CHI、CHS基因发根农杆菌工程菌侵染甘草外植体胚轴及子叶,通过PCR验证及测序验证,成功获得了转CHI、CHS基因甘草毛状根。(5)建立了同时测定甘草毛状根中4种主要黄酮类化合物含量的UPLC方法,并采用此方法对54份甘草毛状根样品中的黄酮类化合物进行含量测定。统计学分析表明,黄酮总含量在转CHI基因甘草毛状根和空白甘草毛状根样品、转CHS基因甘草毛状根和空白甘草毛状根样品中均存在显著性差异,通过比较秩均数,黄酮含量满足:转CHI基因甘草毛状根>转CHS基因甘草毛状根>空白甘草毛状根,从而证实过表达CHS、CHI基因能够增加甘草毛状根中黄酮类成分的含量。(6)利用qRT-PCR对15份转CHI基因和转CHS基因毛状根中目标基因拷贝数进行了测定,结果显示8份过表达CHI基因甘草毛状根中CHI基因拷贝数分别为1、2或5个,7份过表达CHS基因毛状根中CHS基因拷贝数分别为7、9、10、11、13或18个。(7)根据目标基因拷贝数及黄酮类化合物含量测定结果,最终优选出拷贝数为9的转CHS基因甘草毛状根以及拷贝数为5的转CHI基因甘草毛状根作为进一步离体积累黄酮类化合物的甘草毛状根材料,为将来扩大培养奠定基础。
Abstract
gan cao shi wo guo zui chang yong de da zong yao cai zhi yi ,su you “shi fang jiu cao ”zhi yu 。jin xie nian lai ,zai pei gan cao pu bian cun zai pin zhi tui hua he you xiao cheng fen han liang di deng wen ti ,yi cheng wei zhi yao gan cao zi yuan ke chi xu fa zhan de guan jian wen ti 。da liang wen suo bao dao xian shi :mao zhuang gen ke wen ding gao xiao de sheng chan yao yong zhi wu de ci sheng dai xie chan wu ,yin ci li yong mao zhuang gen pei yang ti ji dui gan cao de ci sheng dai xie tu jing kai zhan yan jiu ju you chong yao jia zhi 。gan cao gan shi gan cao zhong zui chong yao de huo xing cheng fen zhi yi ,cha er tong ge mei (chalcone synthase,CHS)ji cha er tong yi gou mei (chalcone isomerase,CHI)shi gan cao gan sheng wu ge cheng tu jing zhong qi guan jian diao kong zuo yong de xian su mei 。yin ci ,ben lun wen ni zai qian ji ke long huo de gan cao CHSji CHIji yin de ji chu shang ,gou jian zhi wu shuang yuan biao da zai ti ji fa gen nong gan jun gong cheng jun ,li yong mao zhuang gen pei yang ti ji dui gan cao CHSji CHIji yin jin hang gong neng yan jiu ,jie shi ji ying xiang huang tong lei hua ge wu sheng wu ge cheng de fen zi ji zhi 。ben ke ti de yan jiu jie guo hai jiang jin yi bu shai shua gan cao gan gao shui ping ji lei de mao zhuang gen ti ji ,wei gan cao gan de li ti ge cheng dian ding li lun ji chu he di gong ji shu zhi chi 。ben wen gen ju ke ti zu qian ji dui yu gan cao huang tong lei hua ge wu han liang yu CHS、CHIji yin duo tai xing xiang guan guan ji de fen xi jie guo shai shua chu huang tong gao han liang dui ying de te yi CHS、CHIji yin chan bei xing ;cai yong ji yin rong ge de fang fa gou jian gan cao zhuai CHS、CHIji yin zhi wu shuang yuan biao da zai ti ;tong guo dian zhuai fa jiang gan cao zhuai CHS、CHIji yin zhi wu shuang yuan biao da zai ti dao ru fa gen nong gan jun ;yong zhuai ji yin fa gen nong gan jun gong cheng jun qin ran gan cao wai zhi ti you dao zhuai ji yin mao zhuang gen ,bing jin hang PCRhe ce xu yan zheng ;jian li tong shi ce ding gan cao mao zhuang gen zhong si chong huang tong cheng fen han liang de UPLCfang fa ,bing dui pei yang de dao de zhuai CHS、CHIji yin gan cao mao zhuang gen jin hang han liang ce ding ;tong guo qRT-PCRdui zhuai CHS、CHIji yin mao zhuang gen zhong mu biao ji yin de kao bei shu jin hang ce ding ,fen xi mu biao ji yin kao bei shu yu mao zhuang gen zhong huang tong lei hua ge wu han liang de xiang guan xing 。ben lun wen gong qu de le ru xia yan jiu jie guo :(1)gen ju huang tong lei hua ge wu han liang yu CHS、CHIji yin duo tai xing xiang guan guan ji de fen xi jie guo ,shai shua chu huang tong lei hua ge wu gao han liang gan cao yang pin dui ying de te yi CHI-1(GenBankzhu ce hao :KY115232),CHS-60(GenBankzhu ce hao :KY810356)chan bei xing 。(2)shua ze Bgl Ⅱhe Spe Ⅰzuo wei mei qie wei dian ,pCAMBIA1305.1wei zhi wu shuang yuan biao da zai ti ,cai yong ji yin rong ge de fang fa ,cheng gong gou jian gan cao zhuai CHI、CHSji yin zhi wu shuang yuan biao da zai ti pCA-CHI、pCA-CHS。(3)cai yong dian zhuai fa (dian zhuai tiao jian :C:25μF;PC:200Ω;U:2400V)fen bie jiang xie dai you gan cao CHIhe CHSji yin de shuang yuan biao da zai ti pCA-CHIhe pCA-CHSdao ru dao fa gen nong gan jun ACCC10060zhong ,gou jian de dao zhuai gan cao CHI、CHSde fa gen nong gan jun gong cheng jun 。(4)li yong zhuai CHI、CHSji yin fa gen nong gan jun gong cheng jun qin ran gan cao wai zhi ti pei zhou ji zi xie ,tong guo PCRyan zheng ji ce xu yan zheng ,cheng gong huo de le zhuai CHI、CHSji yin gan cao mao zhuang gen 。(5)jian li le tong shi ce ding gan cao mao zhuang gen zhong 4chong zhu yao huang tong lei hua ge wu han liang de UPLCfang fa ,bing cai yong ci fang fa dui 54fen gan cao mao zhuang gen yang pin zhong de huang tong lei hua ge wu jin hang han liang ce ding 。tong ji xue fen xi biao ming ,huang tong zong han liang zai zhuai CHIji yin gan cao mao zhuang gen he kong bai gan cao mao zhuang gen yang pin 、zhuai CHSji yin gan cao mao zhuang gen he kong bai gan cao mao zhuang gen yang pin zhong jun cun zai xian zhe xing cha yi ,tong guo bi jiao zhi jun shu ,huang tong han liang man zu :zhuai CHIji yin gan cao mao zhuang gen >zhuai CHSji yin gan cao mao zhuang gen >kong bai gan cao mao zhuang gen ,cong er zheng shi guo biao da CHS、CHIji yin neng gou zeng jia gan cao mao zhuang gen zhong huang tong lei cheng fen de han liang 。(6)li yong qRT-PCRdui 15fen zhuai CHIji yin he zhuai CHSji yin mao zhuang gen zhong mu biao ji yin kao bei shu jin hang le ce ding ,jie guo xian shi 8fen guo biao da CHIji yin gan cao mao zhuang gen zhong CHIji yin kao bei shu fen bie wei 1、2huo 5ge ,7fen guo biao da CHSji yin mao zhuang gen zhong CHSji yin kao bei shu fen bie wei 7、9、10、11、13huo 18ge 。(7)gen ju mu biao ji yin kao bei shu ji huang tong lei hua ge wu han liang ce ding jie guo ,zui zhong you shua chu kao bei shu wei 9de zhuai CHSji yin gan cao mao zhuang gen yi ji kao bei shu wei 5de zhuai CHIji yin gan cao mao zhuang gen zuo wei jin yi bu li ti ji lei huang tong lei hua ge wu de gan cao mao zhuang gen cai liao ,wei jiang lai kuo da pei yang dian ding ji chu 。
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论文详细介绍
论文作者分别是来自北京中医药大学的尹彦超，发表于刊物北京中医药大学2019-07-01论文，是一篇关于查尔酮合酶论文,查尔酮异构酶论文,甘草论文,黄酮论文,毛状根论文,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：查尔酮合酶论文; 查尔酮异构酶论文; 甘草论文; 黄酮论文; 毛状根论文; 北京中医药大学2019-07-01论文;