本文主要研究内容
作者牛丽丽（2019）在《激素诱导对银香菊组培再生苗中挥发性成分合成代谢影响机制研究》一文中研究指出：银香菊(Santolina chamaecyparissus L.)为多年生草本芳香植物,多生长于地中海沿岸以及长江以南地区,含有多种挥发性活性成分,具有显著的抗菌、抗肿瘤、抗病毒等药理作用。在我国,银香菊多被用于制作成驱虫避蚊药草或作为园林景观装饰使用,虽然银香菊具有非常丰富的药理活性成分,但由于其多数为野生,缺乏专业的栽培与种质资源保护,草本形态易受气候变化、病虫害等诸多不利因素影响,使得银香菊植物资源与活性成分的深入研究一直被忽视,导致银香菊植物产业化开发与利用进展缓慢。为了解决上述问题,本文建立了银香菊组织培养再生技术体系,通过激素诱导促进挥发性成分合成增量,利用转录组测序技术对激素诱导后银香菊再生苗中差异性表达基因进行功能注释,总结银香菊挥发性成分生物合成通路并对其候选酶基因的表达进行验证。本论文的主要研究内容及结果如下:1、建立了银香菊挥发性成分GC-MS检测分析方法色谱条件:所用毛细管柱为DB-5MS 30 m x 0.25 mm x 0.25 μm;载气为高纯氦气,流速为1mL/min。不分流模式,进样口温度为250℃。柱温箱的升温程序为:40℃保持2 min,然后以5℃/min速度升至180℃,保持2 min,继而以5℃/min速度升至240℃,保持 5 min。质谱条件:电离方式EI,离子能量70 eV;质谱接口温度250℃,离子源温度230℃,质量扫描范围为35-550 U。结果表明在银香菊中共鉴定得到48个挥发性物质,富含倍半萜类化合物,本方法重复性好、精确度高,适用于后续对银香菊中挥发性成分的分析检测。2、建立了银香菊组织培养再生体系并对其挥发性成分进行检测分析(1)银香菊外植体最佳消毒方式为依次使用75%乙醇与1%次氯酸钠溶液对银香菊茎段进行消毒,接种于MS培养基培养10天后,统计银香菊外植体存活率为95%。(2)银香菊愈伤组织最佳诱导培养基为MS+0.2 mg L-1 NAA+1.0 mg L-1 BA+30 g L-1蔗糖+8 g L-1琼脂,调节培养基pH为5.8,统计愈伤组织诱导率为82%。(3)银香菊最佳丛生芽诱导培养基为MS+0.04 mg L-1 BA+0.1 mg L-1 NAA,+30 g L-1蔗糖+8gL-1琼脂,调节培养基pH为5.8,在25±1℃的冷白荧光灯,光照强度为1800 Lx,16 h/天的光照下培养6周可得到生长状态最佳及数量最多的银香菊丛生芽。(4)以丛生芽生根的长度、数量、生根诱导率和生物量为指标考察培养基中添加不同浓度的NAA对银香菊丛生芽生根的影响。实验结果表明银香菊最佳生根诱导培养基为MS+0.1mg L-1NAA+30g L-1蔗糖+8gL-1琼脂,调节培养基pH为5.8,在25±1℃的冷白荧光灯,光照强度为1800 Lx,16 h/天光照。在上述最佳条件下,银香菊丛生芽生根率达到最大。(5)将银香菊再生苗转移至砾石与沙石与珍珠岩体积比为1:1:1(v/v)的无菌基质中进行炼苗,炼苗培养30天后,将温室中生长状态良好的银香菊再生植株转移至田间自然环境生长,2个月后,统计田间生长的银香菊再生苗存活率为100%。(6)通过GC-MS分析方法对银香菊组培再生苗与田间生长植株中挥发性成分进行比较与评估发现银香菊组培再生苗中的单萜和倍半萜成分积累量均高于田间生长的植株。3、激素诱导对银香菊组培再生苗中挥发性成分的影响(1)使用吲哚-3-乙酸(IAA)对银香菊再生苗进行喷施诱导处理考察IAA对银香菊组培再生苗中挥发性成分的影响。实验结果表明通过1 mmol/L的IAA诱导处理,银香菊再生苗中挥发性成分积累增多,其中倍半萜类物质生物合成受到显著影响,长叶蒎烯化合物,经过IAA诱导刺激后含量迅速积累。(2)使用褪黑素对银香菊再生苗进行喷施诱导处理考察褪黑素对银香菊组培再生苗中挥发性成分的影响。实验结果可知,0.5 mmol/L褪黑素诱导处理对银香菊再生苗中挥发性成分含量积累和组成都产生更加显著的影响,其中delta-榄香烯倍半萜化合物经过褪黑素诱导后大量合成积累。4、激素诱导处理银香菊组培再生苗的转录组测序及数据分析(1)本研究利用Illumina测序技术获得了植物激素IAA与褪黑素喷施处理后银香菊组培再生植株的转录组数据,组装后获得转录本共756826条,最小转录本长度为200 bp。(2)通过比对IAA激素处理前后银香菊再生苗转录组数据,共筛选获得22326个差异表达基因,其中上调表达的基因个数为10373个,下调表达的基因个数为11953个。通过GO分类功能分析发现所有差异表达基因主要被分布到细胞组分,分子功能与生物过程;通过KEGG分析共注释到了 3747个差异表达基因于20个显著富集的KEGG Pathway上,此外,银香菊萜类物质合成通路中关键酶基因会受到外源激素的显著诱导与调控,IAA激素诱导后有20个关键酶基因表达量发生变化。(3)通过比对褪黑素处理前后银香菊再生苗转录组数据,共筛选获得24387个差异表达基因,其中上调表达的基因个数为11705个,下调表达的基因个数为12682个。褪黑素处理银香菊再生苗中获得的差异表达基因GO分类功能分析表明差异表达基因主要富集在结合功能、催化活性、细胞生命过程、代谢过程与单细胞形成过程;KEGG代谢通路富集分析共注释到了 3495个差异表达基因,褪黑素诱导后银香菊萜类骨架合成通路中有19个关键酶基因的表达量发生变化。5、激素诱导处理银香菊组培再生苗中挥发性成分生物合成途径关键调控基因的表达验证(1)使用IAA激素诱导处理银香菊再生苗12 h时,除了HMGS基因以外的所有基因相对表达水平均显著提高,其中,乙酰基转移酶、甲羟戊酸激酶和香叶基焦磷酸合成酶基因在IAA激素诱导银香菊再生苗中萜类物质生物合成过程中响应效果显著,分别为空白对照组的50.7倍、41.6倍及60.8倍,表明银香菊再生苗中萜类生物合成酶基因能够显著响应IAA激素的诱导刺激发生转录激活作用。(2)使用褪黑素诱导处理银香菊再生苗6 h时,HMGS1基因、HMGR基因、MK基因、PMVK基因、MVD基因、IDI基因及FDPS基因的相对表达水平达到最大值,分别为空白对照处理组的21.5倍、8.2倍、20.2倍、27.1倍、20.6倍、11.5倍及22.0倍,表明银香菊再生苗中萜类生物合成酶基因能够显著响应褪黑素的诱导刺激发生转录激活作用。本研究建立的优化的生产挥发性成分的银香菊组培再生体系,为未来利用该离体器官培养体系工业化生产挥发性活性成分奠定理论基础。利用诱导子技术增强了银香菊组培再生苗中挥发性成分的积累,通过高通量转录组测序初步明确了银香菊组培再生苗中调控挥发性成分生物合成的一些关键酶基因并对其基因表达进行验证,为利用诱导子技术调控其他植物组织培养体系高效生产次生代谢活性成分以及阐明其生物合成关键调控酶基因提供了重要的理论依据。
Abstract
yin xiang ju (Santolina chamaecyparissus L.)wei duo nian sheng cao ben fang xiang zhi wu ,duo sheng chang yu de zhong hai yan an yi ji chang jiang yi na de ou ,han you duo chong hui fa xing huo xing cheng fen ,ju you xian zhe de kang jun 、kang zhong liu 、kang bing du deng yao li zuo yong 。zai wo guo ,yin xiang ju duo bei yong yu zhi zuo cheng qu chong bi wen yao cao huo zuo wei yuan lin jing guan zhuang shi shi yong ,sui ran yin xiang ju ju you fei chang feng fu de yao li huo xing cheng fen ,dan you yu ji duo shu wei ye sheng ,que fa zhuan ye de zai pei yu chong zhi zi yuan bao hu ,cao ben xing tai yi shou qi hou bian hua 、bing chong hai deng zhu duo bu li yin su ying xiang ,shi de yin xiang ju zhi wu zi yuan yu huo xing cheng fen de shen ru yan jiu yi zhi bei hu shi ,dao zhi yin xiang ju zhi wu chan ye hua kai fa yu li yong jin zhan huan man 。wei le jie jue shang shu wen ti ,ben wen jian li le yin xiang ju zu zhi pei yang zai sheng ji shu ti ji ,tong guo ji su you dao cu jin hui fa xing cheng fen ge cheng zeng liang ,li yong zhuai lu zu ce xu ji shu dui ji su you dao hou yin xiang ju zai sheng miao zhong cha yi xing biao da ji yin jin hang gong neng zhu shi ,zong jie yin xiang ju hui fa xing cheng fen sheng wu ge cheng tong lu bing dui ji hou shua mei ji yin de biao da jin hang yan zheng 。ben lun wen de zhu yao yan jiu nei rong ji jie guo ru xia :1、jian li le yin xiang ju hui fa xing cheng fen GC-MSjian ce fen xi fang fa se pu tiao jian :suo yong mao xi guan zhu wei DB-5MS 30 m x 0.25 mm x 0.25 μm;zai qi wei gao chun hai qi ,liu su wei 1mL/min。bu fen liu mo shi ,jin yang kou wen du wei 250℃。zhu wen xiang de sheng wen cheng xu wei :40℃bao chi 2 min,ran hou yi 5℃/minsu du sheng zhi 180℃,bao chi 2 min,ji er yi 5℃/minsu du sheng zhi 240℃,bao chi 5 min。zhi pu tiao jian :dian li fang shi EI,li zi neng liang 70 eV;zhi pu jie kou wen du 250℃,li zi yuan wen du 230℃,zhi liang sao miao fan wei wei 35-550 U。jie guo biao ming zai yin xiang ju zhong gong jian ding de dao 48ge hui fa xing wu zhi ,fu han bei ban tie lei hua ge wu ,ben fang fa chong fu xing hao 、jing que du gao ,kuo yong yu hou xu dui yin xiang ju zhong hui fa xing cheng fen de fen xi jian ce 。2、jian li le yin xiang ju zu zhi pei yang zai sheng ti ji bing dui ji hui fa xing cheng fen jin hang jian ce fen xi (1)yin xiang ju wai zhi ti zui jia xiao du fang shi wei yi ci shi yong 75%yi chun yu 1%ci lv suan na rong ye dui yin xiang ju jing duan jin hang xiao du ,jie chong yu MSpei yang ji pei yang 10tian hou ,tong ji yin xiang ju wai zhi ti cun huo lv wei 95%。(2)yin xiang ju yu shang zu zhi zui jia you dao pei yang ji wei MS+0.2 mg L-1 NAA+1.0 mg L-1 BA+30 g L-1zhe tang +8 g L-1qiong zhi ,diao jie pei yang ji pHwei 5.8,tong ji yu shang zu zhi you dao lv wei 82%。(3)yin xiang ju zui jia cong sheng ya you dao pei yang ji wei MS+0.04 mg L-1 BA+0.1 mg L-1 NAA,+30 g L-1zhe tang +8gL-1qiong zhi ,diao jie pei yang ji pHwei 5.8,zai 25±1℃de leng bai ying guang deng ,guang zhao jiang du wei 1800 Lx,16 h/tian de guang zhao xia pei yang 6zhou ke de dao sheng chang zhuang tai zui jia ji shu liang zui duo de yin xiang ju cong sheng ya 。(4)yi cong sheng ya sheng gen de chang du 、shu liang 、sheng gen you dao lv he sheng wu liang wei zhi biao kao cha pei yang ji zhong tian jia bu tong nong du de NAAdui yin xiang ju cong sheng ya sheng gen de ying xiang 。shi yan jie guo biao ming yin xiang ju zui jia sheng gen you dao pei yang ji wei MS+0.1mg L-1NAA+30g L-1zhe tang +8gL-1qiong zhi ,diao jie pei yang ji pHwei 5.8,zai 25±1℃de leng bai ying guang deng ,guang zhao jiang du wei 1800 Lx,16 h/tian guang zhao 。zai shang shu zui jia tiao jian xia ,yin xiang ju cong sheng ya sheng gen lv da dao zui da 。(5)jiang yin xiang ju zai sheng miao zhuai yi zhi li dan yu sha dan yu zhen zhu yan ti ji bi wei 1:1:1(v/v)de mo jun ji zhi zhong jin hang lian miao ,lian miao pei yang 30tian hou ,jiang wen shi zhong sheng chang zhuang tai liang hao de yin xiang ju zai sheng zhi zhu zhuai yi zhi tian jian zi ran huan jing sheng chang ,2ge yue hou ,tong ji tian jian sheng chang de yin xiang ju zai sheng miao cun huo lv wei 100%。(6)tong guo GC-MSfen xi fang fa dui yin xiang ju zu pei zai sheng miao yu tian jian sheng chang zhi zhu zhong hui fa xing cheng fen jin hang bi jiao yu ping gu fa xian yin xiang ju zu pei zai sheng miao zhong de chan tie he bei ban tie cheng fen ji lei liang jun gao yu tian jian sheng chang de zhi zhu 。3、ji su you dao dui yin xiang ju zu pei zai sheng miao zhong hui fa xing cheng fen de ying xiang (1)shi yong yin duo -3-yi suan (IAA)dui yin xiang ju zai sheng miao jin hang pen shi you dao chu li kao cha IAAdui yin xiang ju zu pei zai sheng miao zhong hui fa xing cheng fen de ying xiang 。shi yan jie guo biao ming tong guo 1 mmol/Lde IAAyou dao chu li ,yin xiang ju zai sheng miao zhong hui fa xing cheng fen ji lei zeng duo ,ji zhong bei ban tie lei wu zhi sheng wu ge cheng shou dao xian zhe ying xiang ,chang xie pai xi hua ge wu ,jing guo IAAyou dao ci ji hou han liang xun su ji lei 。(2)shi yong tui hei su dui yin xiang ju zai sheng miao jin hang pen shi you dao chu li kao cha tui hei su dui yin xiang ju zu pei zai sheng miao zhong hui fa xing cheng fen de ying xiang 。shi yan jie guo ke zhi ,0.5 mmol/Ltui hei su you dao chu li dui yin xiang ju zai sheng miao zhong hui fa xing cheng fen han liang ji lei he zu cheng dou chan sheng geng jia xian zhe de ying xiang ,ji zhong delta-lan xiang xi bei ban tie hua ge wu jing guo tui hei su you dao hou da liang ge cheng ji lei 。4、ji su you dao chu li yin xiang ju zu pei zai sheng miao de zhuai lu zu ce xu ji shu ju fen xi (1)ben yan jiu li yong Illuminace xu ji shu huo de le zhi wu ji su IAAyu tui hei su pen shi chu li hou yin xiang ju zu pei zai sheng zhi zhu de zhuai lu zu shu ju ,zu zhuang hou huo de zhuai lu ben gong 756826tiao ,zui xiao zhuai lu ben chang du wei 200 bp。(2)tong guo bi dui IAAji su chu li qian hou yin xiang ju zai sheng miao zhuai lu zu shu ju ,gong shai shua huo de 22326ge cha yi biao da ji yin ,ji zhong shang diao biao da de ji yin ge shu wei 10373ge ,xia diao biao da de ji yin ge shu wei 11953ge 。tong guo GOfen lei gong neng fen xi fa xian suo you cha yi biao da ji yin zhu yao bei fen bu dao xi bao zu fen ,fen zi gong neng yu sheng wu guo cheng ;tong guo KEGGfen xi gong zhu shi dao le 3747ge cha yi biao da ji yin yu 20ge xian zhe fu ji de KEGG Pathwayshang ,ci wai ,yin xiang ju tie lei wu zhi ge cheng tong lu zhong guan jian mei ji yin hui shou dao wai yuan ji su de xian zhe you dao yu diao kong ,IAAji su you dao hou you 20ge guan jian mei ji yin biao da liang fa sheng bian hua 。(3)tong guo bi dui tui hei su chu li qian hou yin xiang ju zai sheng miao zhuai lu zu shu ju ,gong shai shua huo de 24387ge cha yi biao da ji yin ,ji zhong shang diao biao da de ji yin ge shu wei 11705ge ,xia diao biao da de ji yin ge shu wei 12682ge 。tui hei su chu li yin xiang ju zai sheng miao zhong huo de de cha yi biao da ji yin GOfen lei gong neng fen xi biao ming cha yi biao da ji yin zhu yao fu ji zai jie ge gong neng 、cui hua huo xing 、xi bao sheng ming guo cheng 、dai xie guo cheng yu chan xi bao xing cheng guo cheng ;KEGGdai xie tong lu fu ji fen xi gong zhu shi dao le 3495ge cha yi biao da ji yin ,tui hei su you dao hou yin xiang ju tie lei gu jia ge cheng tong lu zhong you 19ge guan jian mei ji yin de biao da liang fa sheng bian hua 。5、ji su you dao chu li yin xiang ju zu pei zai sheng miao zhong hui fa xing cheng fen sheng wu ge cheng tu jing guan jian diao kong ji yin de biao da yan zheng (1)shi yong IAAji su you dao chu li yin xiang ju zai sheng miao 12 hshi ,chu le HMGSji yin yi wai de suo you ji yin xiang dui biao da shui ping jun xian zhe di gao ,ji zhong ,yi xian ji zhuai yi mei 、jia qiang wu suan ji mei he xiang xie ji jiao lin suan ge cheng mei ji yin zai IAAji su you dao yin xiang ju zai sheng miao zhong tie lei wu zhi sheng wu ge cheng guo cheng zhong xiang ying xiao guo xian zhe ,fen bie wei kong bai dui zhao zu de 50.7bei 、41.6bei ji 60.8bei ,biao ming yin xiang ju zai sheng miao zhong tie lei sheng wu ge cheng mei ji yin neng gou xian zhe xiang ying IAAji su de you dao ci ji fa sheng zhuai lu ji huo zuo yong 。(2)shi yong tui hei su you dao chu li yin xiang ju zai sheng miao 6 hshi ,HMGS1ji yin 、HMGRji yin 、MKji yin 、PMVKji yin 、MVDji yin 、IDIji yin ji FDPSji yin de xiang dui biao da shui ping da dao zui da zhi ,fen bie wei kong bai dui zhao chu li zu de 21.5bei 、8.2bei 、20.2bei 、27.1bei 、20.6bei 、11.5bei ji 22.0bei ,biao ming yin xiang ju zai sheng miao zhong tie lei sheng wu ge cheng mei ji yin neng gou xian zhe xiang ying tui hei su de you dao ci ji fa sheng zhuai lu ji huo zuo yong 。ben yan jiu jian li de you hua de sheng chan hui fa xing cheng fen de yin xiang ju zu pei zai sheng ti ji ,wei wei lai li yong gai li ti qi guan pei yang ti ji gong ye hua sheng chan hui fa xing huo xing cheng fen dian ding li lun ji chu 。li yong you dao zi ji shu zeng jiang le yin xiang ju zu pei zai sheng miao zhong hui fa xing cheng fen de ji lei ,tong guo gao tong liang zhuai lu zu ce xu chu bu ming que le yin xiang ju zu pei zai sheng miao zhong diao kong hui fa xing cheng fen sheng wu ge cheng de yi xie guan jian mei ji yin bing dui ji ji yin biao da jin hang yan zheng ,wei li yong you dao zi ji shu diao kong ji ta zhi wu zu zhi pei yang ti ji gao xiao sheng chan ci sheng dai xie huo xing cheng fen yi ji chan ming ji sheng wu ge cheng guan jian diao kong mei ji yin di gong le chong yao de li lun yi ju 。
论文参考文献
论文详细介绍
论文作者分别是来自东北林业大学的牛丽丽，发表于刊物东北林业大学2019-11-18论文，是一篇关于银香菊论文,组培再生体系论文,挥发性成分论文,诱导增量论文,转录组测序论文,东北林业大学2019-11-18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，资料来自东北林业大学2019-11-18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：银香菊论文; 组培再生体系论文; 挥发性成分论文; 诱导增量论文; 转录组测序论文; 东北林业大学2019-11-18论文;