本文主要研究内容
作者赵力（2019）在《桑叶经霜前后绿原酸生物合成途径差异表达基因分析及关键酶基因功能验证》一文中研究指出：桑叶为桑科植物桑（Morus alba L.）的干燥叶,是我国传统中药之一,味甘、苦,性寒,归肝、肺经,具有疏散风热,清肺润燥,清肝明目的功效。桑叶药用历史悠久,历来有经霜采收的要求,且中医认为霜桑叶品质上乘,但现有结果对桑叶经霜采收的科学内涵尚未完全阐明。课题组前期对桑叶经霜前后主要活性成分的动态变化进行了研究,发现经霜后绿原酸和黄酮醇苷类成分含量升高,已经初步探究了经霜对黄酮醇苷类成分影响的分子机制,但尚未对经霜前后绿原酸成分差异的分子机制进行研究。本论文采用高效液相色谱法研究桑叶不同生长期绿原酸含量动态变化规律,选择绿原酸含量差异显著的样本进行转录组测序,利用比较转录组学技术筛选差异表达基因,分析差异基因表达水平与绿原酸含量的相关性,挖掘与绿原酸生物合成相关的关键酶基因,并对其进行克隆及功能分析,为阐明经霜前后绿原酸含量差异的分子机制奠定基础,同时也为研究桑叶绿原酸生物合成途径提供依据。主要研究内容如下:1.桑叶经霜前后绿原酸含量动态变化研究采用高效液相色谱法对桑叶经霜前后绿原酸含量动态变化进行研究,结果表明,绿原酸含量在7、8月份较低,霜降（10月23日）后含量逐渐升高于11月份达到最大值。绿原酸含量在7月31日为1.35?0.16 mg/g,11月15日达2.43?0.08 mg/g,两个样本中的绿原酸含量呈显著性差异（P<0.01）,该结果为转录组测序桑叶样本的选择提供了依据。2.桑叶经霜前后绿原酸生物合成途径差异表达基因研究采用Illumina HiSeq 2500测序平台对桑叶绿原酸含量差异显著（经霜前和经霜后）的样本进行转录组测序,通过转录组注释结果,并结合KEGG代谢通路分析,共有58条基因与绿原酸的生物合成相关,编码5种酶,分别为苯丙氨酸解氨酶（Phenylalanine ammonia-lyase,PAL）、4-香豆酸CoA连接酶（4-Coumarate-CoA ligase,4CL）、肉桂酸4-羟化酶（Cinnamic acid 4-hydroxylase,C4H）、莽草酸/奎宁酸羟基肉桂酰转移酶（Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase,HCT）和香豆酸-3’-羟化酶（Coumaroyl ester 3’-hydroxylase,C3’H）。其中17条为差异表达基因,包括12条上调表达基因,5条下调表达基因。分析不同生长期差异基因表达水平与绿原酸含量的相关性,结果表明c34446g1、c82179g1、c46108g1和c31168g1基因表达水平与绿原酸含量变化呈显著正相关,这4条基因分别编码PAL、4CL、HCT和C3’H。推测这4条基因为绿原酸生物合成途径的关键酶基因,经霜后气候温度的下降可能诱导c34446g1、c82179g1、c46108g1和c31168g1基因的表达,有利于绿原酸的积累。3.桑叶莽草酸/奎宁酸羟基肉桂酰转移酶基因克隆、表达及功能分析从桑叶中克隆得到MaHCT（c46108g1）基因,GenBank登录号为MH476577。MaHCT基因的开放阅读框长度为1320 bp,编码439个氨基酸。序列同源性分析表明MaHCT蛋白包含酰基转移酶家族的保守域HXXXD和DFGWG。构建了重组质粒pET-30a（+）/MaHCT,并在大肠杆菌中诱导表达获得重组蛋白,重组蛋白为部分可溶性蛋白,分子量约为53 kDa。对MaHCT蛋白进行性质表征,结果表明反应条件为37℃、pH=7.4时MaHCT酶活力最大;温度为4℃和25℃时,酶活性残留大于85%;pH值在6.4-7.4之间时,酶活性残留大于89%;Fe3+和Zn2+对酶活力起抑制作用,而Cu2+对酶活力具有增强作用。体外酶促反应结果表明,MaHCT蛋白能有效地催化生成对香豆酰莽草酸、对香豆酰奎宁酸和绿原酸。酶动力学常数结果表明,当对香豆酰CoA作为酰基供体时,MaHCT对奎宁酸的亲和力高于莽草酸,并且对奎宁酸的催化效率高于莽草酸。当奎宁酸作为酰基受体时,MaHCT对对香豆酰CoA的亲和力高于咖啡酰CoA,并且对对香豆酰CoA的催化效率约为咖啡酰CoA的32倍。推测MaHCT更倾向于以对香豆酰CoA为酰基供体,奎宁酸为酰基受体,生成对香豆酰奎宁酸。4.桑叶香豆酸-3’-羟化酶基因克隆、表达及功能分析从桑叶中克隆得到MaC3’H（c31168g1）基因,GenBank登录号为MK738016。MaC3’H基因的开放阅读框长度为1530bp,编码509个氨基酸。序列同源性分析表明MaC3’H蛋白属于p450 super family,包含4个CYP保守结构域:富含脯氨酸的膜铰链PPGP、I螺旋参与氧结合和激活GGXDTT、PERF和半胱氨酸结构域PFGAGRRXCP。构建了重组质粒pET-28a（+）/MaC3’H,并在大肠杆菌中诱导表达获得重组蛋白,最佳诱导条件:IPTG浓度为1 mmol/L,诱导温度为30℃,诱导时间6 h。重组蛋白为包涵体蛋白,分子量约为58 kDa,进行变性复性后获得可溶的MaC3’H蛋白。体外酶促反应结果表明,MaC3’H蛋白能有效地催化对香豆酰莽草酸、对香豆酰奎宁酸与NADPH生成咖啡酰莽草酸和绿原酸,表明MaC3’H具有3’位羟基化作用。但MaHCT不能催化咖啡酰莽草酸生成咖啡酰CoA。因此初步推测了桑叶中绿原酸可能的主要生物合成途径。
Abstract
sang xie wei sang ke zhi wu sang （Morus alba L.）de gan zao xie ,shi wo guo chuan tong zhong yao zhi yi ,wei gan 、ku ,xing han ,gui gan 、fei jing ,ju you shu san feng re ,qing fei run zao ,qing gan ming mu de gong xiao 。sang xie yao yong li shi you jiu ,li lai you jing shuang cai shou de yao qiu ,ju zhong yi ren wei shuang sang xie pin zhi shang cheng ,dan xian you jie guo dui sang xie jing shuang cai shou de ke xue nei han shang wei wan quan chan ming 。ke ti zu qian ji dui sang xie jing shuang qian hou zhu yao huo xing cheng fen de dong tai bian hua jin hang le yan jiu ,fa xian jing shuang hou lu yuan suan he huang tong chun gan lei cheng fen han liang sheng gao ,yi jing chu bu tan jiu le jing shuang dui huang tong chun gan lei cheng fen ying xiang de fen zi ji zhi ,dan shang wei dui jing shuang qian hou lu yuan suan cheng fen cha yi de fen zi ji zhi jin hang yan jiu 。ben lun wen cai yong gao xiao ye xiang se pu fa yan jiu sang xie bu tong sheng chang ji lu yuan suan han liang dong tai bian hua gui lv ,shua ze lu yuan suan han liang cha yi xian zhe de yang ben jin hang zhuai lu zu ce xu ,li yong bi jiao zhuai lu zu xue ji shu shai shua cha yi biao da ji yin ,fen xi cha yi ji yin biao da shui ping yu lu yuan suan han liang de xiang guan xing ,wa jue yu lu yuan suan sheng wu ge cheng xiang guan de guan jian mei ji yin ,bing dui ji jin hang ke long ji gong neng fen xi ,wei chan ming jing shuang qian hou lu yuan suan han liang cha yi de fen zi ji zhi dian ding ji chu ,tong shi ye wei yan jiu sang xie lu yuan suan sheng wu ge cheng tu jing di gong yi ju 。zhu yao yan jiu nei rong ru xia :1.sang xie jing shuang qian hou lu yuan suan han liang dong tai bian hua yan jiu cai yong gao xiao ye xiang se pu fa dui sang xie jing shuang qian hou lu yuan suan han liang dong tai bian hua jin hang yan jiu ,jie guo biao ming ,lu yuan suan han liang zai 7、8yue fen jiao di ,shuang jiang （10yue 23ri ）hou han liang zhu jian sheng gao yu 11yue fen da dao zui da zhi 。lu yuan suan han liang zai 7yue 31ri wei 1.35?0.16 mg/g,11yue 15ri da 2.43?0.08 mg/g,liang ge yang ben zhong de lu yuan suan han liang cheng xian zhe xing cha yi （P<0.01）,gai jie guo wei zhuai lu zu ce xu sang xie yang ben de shua ze di gong le yi ju 。2.sang xie jing shuang qian hou lu yuan suan sheng wu ge cheng tu jing cha yi biao da ji yin yan jiu cai yong Illumina HiSeq 2500ce xu ping tai dui sang xie lu yuan suan han liang cha yi xian zhe （jing shuang qian he jing shuang hou ）de yang ben jin hang zhuai lu zu ce xu ,tong guo zhuai lu zu zhu shi jie guo ,bing jie ge KEGGdai xie tong lu fen xi ,gong you 58tiao ji yin yu lu yuan suan de sheng wu ge cheng xiang guan ,bian ma 5chong mei ,fen bie wei ben bing an suan jie an mei （Phenylalanine ammonia-lyase,PAL）、4-xiang dou suan CoAlian jie mei （4-Coumarate-CoA ligase,4CL）、rou gui suan 4-qiang hua mei （Cinnamic acid 4-hydroxylase,C4H）、mang cao suan /kui ning suan qiang ji rou gui xian zhuai yi mei （Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase,HCT）he xiang dou suan -3’-qiang hua mei （Coumaroyl ester 3’-hydroxylase,C3’H）。ji zhong 17tiao wei cha yi biao da ji yin ,bao gua 12tiao shang diao biao da ji yin ,5tiao xia diao biao da ji yin 。fen xi bu tong sheng chang ji cha yi ji yin biao da shui ping yu lu yuan suan han liang de xiang guan xing ,jie guo biao ming c34446g1、c82179g1、c46108g1he c31168g1ji yin biao da shui ping yu lu yuan suan han liang bian hua cheng xian zhe zheng xiang guan ,zhe 4tiao ji yin fen bie bian ma PAL、4CL、HCThe C3’H。tui ce zhe 4tiao ji yin wei lu yuan suan sheng wu ge cheng tu jing de guan jian mei ji yin ,jing shuang hou qi hou wen du de xia jiang ke neng you dao c34446g1、c82179g1、c46108g1he c31168g1ji yin de biao da ,you li yu lu yuan suan de ji lei 。3.sang xie mang cao suan /kui ning suan qiang ji rou gui xian zhuai yi mei ji yin ke long 、biao da ji gong neng fen xi cong sang xie zhong ke long de dao MaHCT（c46108g1）ji yin ,GenBankdeng lu hao wei MH476577。MaHCTji yin de kai fang yue dou kuang chang du wei 1320 bp,bian ma 439ge an ji suan 。xu lie tong yuan xing fen xi biao ming MaHCTdan bai bao han xian ji zhuai yi mei jia zu de bao shou yu HXXXDhe DFGWG。gou jian le chong zu zhi li pET-30a（+）/MaHCT,bing zai da chang gan jun zhong you dao biao da huo de chong zu dan bai ,chong zu dan bai wei bu fen ke rong xing dan bai ,fen zi liang yao wei 53 kDa。dui MaHCTdan bai jin hang xing zhi biao zheng ,jie guo biao ming fan ying tiao jian wei 37℃、pH=7.4shi MaHCTmei huo li zui da ;wen du wei 4℃he 25℃shi ,mei huo xing can liu da yu 85%;pHzhi zai 6.4-7.4zhi jian shi ,mei huo xing can liu da yu 89%;Fe3+he Zn2+dui mei huo li qi yi zhi zuo yong ,er Cu2+dui mei huo li ju you zeng jiang zuo yong 。ti wai mei cu fan ying jie guo biao ming ,MaHCTdan bai neng you xiao de cui hua sheng cheng dui xiang dou xian mang cao suan 、dui xiang dou xian kui ning suan he lu yuan suan 。mei dong li xue chang shu jie guo biao ming ,dang dui xiang dou xian CoAzuo wei xian ji gong ti shi ,MaHCTdui kui ning suan de qin he li gao yu mang cao suan ,bing ju dui kui ning suan de cui hua xiao lv gao yu mang cao suan 。dang kui ning suan zuo wei xian ji shou ti shi ,MaHCTdui dui xiang dou xian CoAde qin he li gao yu ga fei xian CoA,bing ju dui dui xiang dou xian CoAde cui hua xiao lv yao wei ga fei xian CoAde 32bei 。tui ce MaHCTgeng qing xiang yu yi dui xiang dou xian CoAwei xian ji gong ti ,kui ning suan wei xian ji shou ti ,sheng cheng dui xiang dou xian kui ning suan 。4.sang xie xiang dou suan -3’-qiang hua mei ji yin ke long 、biao da ji gong neng fen xi cong sang xie zhong ke long de dao MaC3’H（c31168g1）ji yin ,GenBankdeng lu hao wei MK738016。MaC3’Hji yin de kai fang yue dou kuang chang du wei 1530bp,bian ma 509ge an ji suan 。xu lie tong yuan xing fen xi biao ming MaC3’Hdan bai shu yu p450 super family,bao han 4ge CYPbao shou jie gou yu :fu han fu an suan de mo jiao lian PPGP、Iluo xuan can yu yang jie ge he ji huo GGXDTT、PERFhe ban guang an suan jie gou yu PFGAGRRXCP。gou jian le chong zu zhi li pET-28a（+）/MaC3’H,bing zai da chang gan jun zhong you dao biao da huo de chong zu dan bai ,zui jia you dao tiao jian :IPTGnong du wei 1 mmol/L,you dao wen du wei 30℃,you dao shi jian 6 h。chong zu dan bai wei bao han ti dan bai ,fen zi liang yao wei 58 kDa,jin hang bian xing fu xing hou huo de ke rong de MaC3’Hdan bai 。ti wai mei cu fan ying jie guo biao ming ,MaC3’Hdan bai neng you xiao de cui hua dui xiang dou xian mang cao suan 、dui xiang dou xian kui ning suan yu NADPHsheng cheng ga fei xian mang cao suan he lu yuan suan ,biao ming MaC3’Hju you 3’wei qiang ji hua zuo yong 。dan MaHCTbu neng cui hua ga fei xian mang cao suan sheng cheng ga fei xian CoA。yin ci chu bu tui ce le sang xie zhong lu yuan suan ke neng de zhu yao sheng wu ge cheng tu jing 。
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论文详细介绍
论文作者分别是来自江苏大学的赵力，发表于刊物江苏大学2019-09-19论文，是一篇关于桑叶论文,经霜论文,绿原酸论文,莽草酸论文,奎宁酸羟基肉桂酰转移酶论文,香豆酸羟化酶论文,江苏大学2019-09-19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，资料来自江苏大学2019-09-19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：桑叶论文; 经霜论文; 绿原酸论文; 莽草酸论文; 奎宁酸羟基肉桂酰转移酶论文; 香豆酸羟化酶论文; 江苏大学2019-09-19论文;