本文主要研究内容
作者寇爽（2019）在《腐胺合成途径在马铃薯抗寒中的作用及机制解析》一文中研究指出：马铃薯(Solanum tuberosum L.)是世界上最重要的非谷类粮食作物,在保证粮食安全上起着重要作用。马铃薯不耐霜冻,也不具有低温驯化能力,我国不同产区均存在严重的霜冻威胁。实验室前期研究表明多胺代谢途径可能在马铃薯抗寒中起重要作用,但其抗寒机制仍不明晰。本研究通过外源施用、基因遗传转化及转录组测序等方法揭示了多胺途径中的腐胺合成在马铃薯驯化抗寒中的功能并进行机制解析,主要研究结果如下:1.在两组抗寒性差异的马铃薯基因型中对多胺途径上基因的表达量及主要代谢物的含量进行测定,结果表明精氨酸脱羧酶基因ADC1及其下游产物腐胺在低温处理后的三个抗寒基因型中特异上调,由此说明腐胺的合成可能在马铃薯抗寒中起着重要的作用。2.对低温敏感型品种鄂马铃薯3号(E3)外源施用腐胺,通过表型观察、损伤评级以及电解质渗透率测定的方法对腐胺处理植株及对照植株进行抗寒鉴定,结果表明在正常生长条件下,腐胺处理植株的抗寒能力与对照植株相比没有明显差别;而经过4°C 14 d驯化,又接着在-2°C处理8 d后,腐胺处理植株的损伤评级远远小于对照植株;腐胺处理植株叶片的电解质渗透率为47.12%,明显小于对照植株的69.69%;同时低温驯化后,经过水浴锅的程序降温,腐胺处理植株叶片在-3°C和-4°C的电解质渗透率也显著小于对照植株,因此腐胺处理植株的驯化抗寒能力明显强于对照植株,首次证明腐胺能够增强马铃薯的驯化抗寒能力。3.对马铃薯精氨酸脱羧酶基因ADC1进行克隆,发现在抗寒性基因型(S.acale,Aca)及敏感性基因型(S.tuberosum,Tub)中均有两个等位基因,CDS序列长度为2163bp,编码具有720个氨基酸的蛋白质,其中CDS的一致性为98.79%,氨基酸序列一致性达99.38%。将它们与不同物种的精氨酸脱羧酶进行蛋白序列聚类分析发现除了油菜中的三个ADC中羧基端的关键半胱氨酸C缺失外,其他ADC蛋白序列中的关键氨基端的赖氨酸K和羧基端的C均存在,因此ADC关键位点的保守性表明其在植物中具有重要的生物学功能。进一步对Aca和Tub中ADC1的CDS区上游2500bp的序列进行克隆,并对四条ADC1启动子上的顺式作用元件的进行预测,预测结果表明,ADC1启动子含有多种类型的响应元件,如响应ABA的ABRE元件,响应低温的LTR元件、响应光的p-box元件等,而在ADC1的CDS区ATG上游约600-1000bp处存在较大序列与元件差异,可能与不同基因型对低温的响应差异有关。随后对ADC1表达特性分析表明,ADC1在马铃薯的叶和花中表达量最高,在根、茎、匍匐茎和块茎中表达量相对较低;除响应低温外,ADC1还能够响应ABA诱导、干旱和盐胁迫;由此说明ADC1在马铃薯中可能具有多种生物学功能。4.为了明确ADC1在马铃薯抗寒中的作用,构建了SaADC1的超量表达载体,并将其遗传转化低温敏感型品种E3中。超量表达株系OE-ADC1-1和OE-ADC1-2中ADC1的表达量及腐胺含量与对照相比无论在正常生长条件还是驯化后均明显提高。抗寒性鉴定结果表明,正常生长条件下转基因株系的抗寒能力与野生植株相比没有明显差异,而驯化后转基因植株的抗寒能力明显强于野生型,首次证明了ADC1能够提高马铃薯的驯化抗寒能力。5.为了研究腐胺合成途径影响马铃薯驯化抗寒能力的作用机制,在腐胺处理植株及SaADC1超量表达株系中检测CBFs的表达,发现马铃薯中的四个CBFs的表达与对照相比均有不同程度的增强。转基因株系中检测CBFs下游CORs基因,也发现四个COR基因的表达与野生型植株相比得到增强。本研究在植物中首次发现ADC1介导的腐胺合成可能通过增强CBF途径上相关基因的表达来提高驯化抗寒性。为了探究腐胺合成途径是否影响ABA信号途径,检测了腐胺处理植株及转基因株系中的ABA含量。结果表明,与对照相比ABA的含量没有明显变化。因此腐胺合成途径不影响ABA的水平。ADC1启动上包含多个ABRE响应元件,同时ABA诱导ADC1的表达,推测ABA信号途径可能影响腐胺合成途径。在本氏烟中瞬时表达体系中检测马铃薯中的七个AREB/ABF/ABI5转录因子均能与ADC1启动子互作,增强报告基因的表达。同时,AREB3和ABI5受低温和ABA的诱导,依此推测在马铃薯中,低温通过ABA信号途径影响ADC1的表达可能与AREB3和ABI5有关。6.为了进一步研究腐胺合成途径影响马铃薯驯化抗寒的调控网络,本研究对OE-ADC1-1进行了转录组测序。差异基因的富集分析发现,4°C处理1 h后所富集的途径主要与物质转运及激素信号传导有关,而在4°C处理2 d后,则主要与糖类、氨基酸等物质的代谢相关,由此说明ADC1基因不仅能够影响低温信号途径,也影响体内物质的代谢,而这些信号途径与物质很可能与马铃薯驯化抗寒有关。进一步分析发现,4°C处理1 h后,在转基因株系中“oxidoreductase activity”途径上的5个脂肪酸去饱和酶和“Glutathione metabolism”途径上3个谷胱甘肽S转移酶基因的表达与野生型相比明显增强,暗示了腐胺合成途径在低温条件下影响马铃薯的氧化还原平衡。本研究揭示了腐胺合成途径在马铃薯抗寒中的功能及作用机制,为进一步的抗寒研究提供了一定的理论基础,为改善栽培种马铃薯不耐霜冻的特性提供了基因资源。
Abstract
ma ling shu (Solanum tuberosum L.)shi shi jie shang zui chong yao de fei gu lei liang shi zuo wu ,zai bao zheng liang shi an quan shang qi zhao chong yao zuo yong 。ma ling shu bu nai shuang dong ,ye bu ju you di wen xun hua neng li ,wo guo bu tong chan ou jun cun zai yan chong de shuang dong wei xie 。shi yan shi qian ji yan jiu biao ming duo an dai xie tu jing ke neng zai ma ling shu kang han zhong qi chong yao zuo yong ,dan ji kang han ji zhi reng bu ming xi 。ben yan jiu tong guo wai yuan shi yong 、ji yin wei chuan zhuai hua ji zhuai lu zu ce xu deng fang fa jie shi le duo an tu jing zhong de fu an ge cheng zai ma ling shu xun hua kang han zhong de gong neng bing jin hang ji zhi jie xi ,zhu yao yan jiu jie guo ru xia :1.zai liang zu kang han xing cha yi de ma ling shu ji yin xing zhong dui duo an tu jing shang ji yin de biao da liang ji zhu yao dai xie wu de han liang jin hang ce ding ,jie guo biao ming jing an suan tuo suo mei ji yin ADC1ji ji xia you chan wu fu an zai di wen chu li hou de san ge kang han ji yin xing zhong te yi shang diao ,you ci shui ming fu an de ge cheng ke neng zai ma ling shu kang han zhong qi zhao chong yao de zuo yong 。2.dui di wen min gan xing pin chong e ma ling shu 3hao (E3)wai yuan shi yong fu an ,tong guo biao xing guan cha 、sun shang ping ji yi ji dian jie zhi shen tou lv ce ding de fang fa dui fu an chu li zhi zhu ji dui zhao zhi zhu jin hang kang han jian ding ,jie guo biao ming zai zheng chang sheng chang tiao jian xia ,fu an chu li zhi zhu de kang han neng li yu dui zhao zhi zhu xiang bi mei you ming xian cha bie ;er jing guo 4°C 14 dxun hua ,you jie zhao zai -2°Cchu li 8 dhou ,fu an chu li zhi zhu de sun shang ping ji yuan yuan xiao yu dui zhao zhi zhu ;fu an chu li zhi zhu xie pian de dian jie zhi shen tou lv wei 47.12%,ming xian xiao yu dui zhao zhi zhu de 69.69%;tong shi di wen xun hua hou ,jing guo shui yu guo de cheng xu jiang wen ,fu an chu li zhi zhu xie pian zai -3°Che -4°Cde dian jie zhi shen tou lv ye xian zhe xiao yu dui zhao zhi zhu ,yin ci fu an chu li zhi zhu de xun hua kang han neng li ming xian jiang yu dui zhao zhi zhu ,shou ci zheng ming fu an neng gou zeng jiang ma ling shu de xun hua kang han neng li 。3.dui ma ling shu jing an suan tuo suo mei ji yin ADC1jin hang ke long ,fa xian zai kang han xing ji yin xing (S.acale,Aca)ji min gan xing ji yin xing (S.tuberosum,Tub)zhong jun you liang ge deng wei ji yin ,CDSxu lie chang du wei 2163bp,bian ma ju you 720ge an ji suan de dan bai zhi ,ji zhong CDSde yi zhi xing wei 98.79%,an ji suan xu lie yi zhi xing da 99.38%。jiang ta men yu bu tong wu chong de jing an suan tuo suo mei jin hang dan bai xu lie ju lei fen xi fa xian chu le you cai zhong de san ge ADCzhong suo ji duan de guan jian ban guang an suan Cque shi wai ,ji ta ADCdan bai xu lie zhong de guan jian an ji duan de lai an suan Khe suo ji duan de Cjun cun zai ,yin ci ADCguan jian wei dian de bao shou xing biao ming ji zai zhi wu zhong ju you chong yao de sheng wu xue gong neng 。jin yi bu dui Acahe Tubzhong ADC1de CDSou shang you 2500bpde xu lie jin hang ke long ,bing dui si tiao ADC1qi dong zi shang de shun shi zuo yong yuan jian de jin hang yu ce ,yu ce jie guo biao ming ,ADC1qi dong zi han you duo chong lei xing de xiang ying yuan jian ,ru xiang ying ABAde ABREyuan jian ,xiang ying di wen de LTRyuan jian 、xiang ying guang de p-boxyuan jian deng ,er zai ADC1de CDSou ATGshang you yao 600-1000bpchu cun zai jiao da xu lie yu yuan jian cha yi ,ke neng yu bu tong ji yin xing dui di wen de xiang ying cha yi you guan 。sui hou dui ADC1biao da te xing fen xi biao ming ,ADC1zai ma ling shu de xie he hua zhong biao da liang zui gao ,zai gen 、jing 、pu fu jing he kuai jing zhong biao da liang xiang dui jiao di ;chu xiang ying di wen wai ,ADC1hai neng gou xiang ying ABAyou dao 、gan han he yan xie pai ;you ci shui ming ADC1zai ma ling shu zhong ke neng ju you duo chong sheng wu xue gong neng 。4.wei le ming que ADC1zai ma ling shu kang han zhong de zuo yong ,gou jian le SaADC1de chao liang biao da zai ti ,bing jiang ji wei chuan zhuai hua di wen min gan xing pin chong E3zhong 。chao liang biao da zhu ji OE-ADC1-1he OE-ADC1-2zhong ADC1de biao da liang ji fu an han liang yu dui zhao xiang bi mo lun zai zheng chang sheng chang tiao jian hai shi xun hua hou jun ming xian di gao 。kang han xing jian ding jie guo biao ming ,zheng chang sheng chang tiao jian xia zhuai ji yin zhu ji de kang han neng li yu ye sheng zhi zhu xiang bi mei you ming xian cha yi ,er xun hua hou zhuai ji yin zhi zhu de kang han neng li ming xian jiang yu ye sheng xing ,shou ci zheng ming le ADC1neng gou di gao ma ling shu de xun hua kang han neng li 。5.wei le yan jiu fu an ge cheng tu jing ying xiang ma ling shu xun hua kang han neng li de zuo yong ji zhi ,zai fu an chu li zhi zhu ji SaADC1chao liang biao da zhu ji zhong jian ce CBFsde biao da ,fa xian ma ling shu zhong de si ge CBFsde biao da yu dui zhao xiang bi jun you bu tong cheng du de zeng jiang 。zhuai ji yin zhu ji zhong jian ce CBFsxia you CORsji yin ,ye fa xian si ge CORji yin de biao da yu ye sheng xing zhi zhu xiang bi de dao zeng jiang 。ben yan jiu zai zhi wu zhong shou ci fa xian ADC1jie dao de fu an ge cheng ke neng tong guo zeng jiang CBFtu jing shang xiang guan ji yin de biao da lai di gao xun hua kang han xing 。wei le tan jiu fu an ge cheng tu jing shi fou ying xiang ABAxin hao tu jing ,jian ce le fu an chu li zhi zhu ji zhuai ji yin zhu ji zhong de ABAhan liang 。jie guo biao ming ,yu dui zhao xiang bi ABAde han liang mei you ming xian bian hua 。yin ci fu an ge cheng tu jing bu ying xiang ABAde shui ping 。ADC1qi dong shang bao han duo ge ABRExiang ying yuan jian ,tong shi ABAyou dao ADC1de biao da ,tui ce ABAxin hao tu jing ke neng ying xiang fu an ge cheng tu jing 。zai ben shi yan zhong shun shi biao da ti ji zhong jian ce ma ling shu zhong de qi ge AREB/ABF/ABI5zhuai lu yin zi jun neng yu ADC1qi dong zi hu zuo ,zeng jiang bao gao ji yin de biao da 。tong shi ,AREB3he ABI5shou di wen he ABAde you dao ,yi ci tui ce zai ma ling shu zhong ,di wen tong guo ABAxin hao tu jing ying xiang ADC1de biao da ke neng yu AREB3he ABI5you guan 。6.wei le jin yi bu yan jiu fu an ge cheng tu jing ying xiang ma ling shu xun hua kang han de diao kong wang lao ,ben yan jiu dui OE-ADC1-1jin hang le zhuai lu zu ce xu 。cha yi ji yin de fu ji fen xi fa xian ,4°Cchu li 1 hhou suo fu ji de tu jing zhu yao yu wu zhi zhuai yun ji ji su xin hao chuan dao you guan ,er zai 4°Cchu li 2 dhou ,ze zhu yao yu tang lei 、an ji suan deng wu zhi de dai xie xiang guan ,you ci shui ming ADC1ji yin bu jin neng gou ying xiang di wen xin hao tu jing ,ye ying xiang ti nei wu zhi de dai xie ,er zhe xie xin hao tu jing yu wu zhi hen ke neng yu ma ling shu xun hua kang han you guan 。jin yi bu fen xi fa xian ,4°Cchu li 1 hhou ,zai zhuai ji yin zhu ji zhong “oxidoreductase activity”tu jing shang de 5ge zhi fang suan qu bao he mei he “Glutathione metabolism”tu jing shang 3ge gu guang gan tai Szhuai yi mei ji yin de biao da yu ye sheng xing xiang bi ming xian zeng jiang ,an shi le fu an ge cheng tu jing zai di wen tiao jian xia ying xiang ma ling shu de yang hua hai yuan ping heng 。ben yan jiu jie shi le fu an ge cheng tu jing zai ma ling shu kang han zhong de gong neng ji zuo yong ji zhi ,wei jin yi bu de kang han yan jiu di gong le yi ding de li lun ji chu ,wei gai shan zai pei chong ma ling shu bu nai shuang dong de te xing di gong le ji yin zi yuan 。
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论文作者分别是来自华中农业大学的寇爽，发表于刊物华中农业大学2019-07-15论文，是一篇关于马铃薯论文,抗寒性论文,冷驯化论文,腐胺论文,精氨酸脱羧酶基因论文,华中农业大学2019-07-15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，资料来自华中农业大学2019-07-15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：马铃薯论文; 抗寒性论文; 冷驯化论文; 腐胺论文; 精氨酸脱羧酶基因论文; 华中农业大学2019-07-15论文;