本文主要研究内容
作者李卉梓（2019）在《SIGLRs和SICaMs在调控番茄低温抗性中的机制研究》一文中研究指出：低温胁迫严重影响了番茄(Solanum lycopericum)等喜温作物的产量和品质,极大地限制了蔬菜的周年生产和供应。克服以番茄为代表的大宗蔬菜在冬春季节设施栽培中的低温胁迫障碍是当下亟待解决的问题。基于此,剖析蔬菜作物对低温的应答机制,挖掘低温响应的关键因子,进而通过基因编辑、植物生长调节剂、环境调控等手段,诱导蔬菜作物的低温抗性,对提高蔬菜产量、品质和经济效益,保障周年生产和供应具有十分重要的科学意义和现实意义。本论文以番茄为研究对象,利用遗传学、分子生物学、植物生理学、生物化学、转录组学等手段,明确了冷驯化及后续低温过程中的关键因子及其响应动态变化;筛选了冷驯化诱导的番茄低温抗性过程中类谷氨酸受体(Glutamate receptor-like,GLRs)家族中关键作用基因;探讨了 GLR3.3和GLR3.5以及关键因子H2O2和GSH的相互关系;筛选了低温过程中钙调素(Calmodulin,CaMs)家族中的关键作用基因,并利用转录组分析了低温过程中的差异基因及其功能。主要研究结果如下:第一,明确了冷驯化能够诱导番茄的低温抗性,且与H202和GSH积累有关。进一步细化了冷驯化及后续低温胁迫过程中H2O2和GSH/GSSG氧化还原状态的动态变化。结果显示,H202含量在冷驯化下显著积累,而后在低温胁迫过程中趋于稳定;而未经冷驯化直接低温处理下,H202含量持续增加。冷驯化也显著诱导了 GSH的积累和GSH/GSSG比例提升。同时,冷驯化也显著诱导了 NADPH氧化酶编码基因RBOH1及谷胱甘肽合成基因GSH1和GSH2的表达。以上结果表明,冷驯化诱导的番茄低温抗性与H202和GSH积累有关。第二,通过实时荧光定量PCR技术对已经鉴定的13个番茄GLRs基因在冷驯化及后续低温过程中的转录水平进行了检测,结果显示SlGLR3.3和SlGLR3.5的转录水平在冷驯化过程中显著上升。进一步利用病毒诱导的基因沉默(VIGS)技术和外源喷施实验,明确了SlGLR3.和SlGLR3.5介导了冷驯化诱导的番茄低温抗性。SlGLR3.3和SlGLR3.5沉默以及GLRs抑制剂处理均显著降低了冷驯化诱导的低温抗性,使得冷驯化诱导的NADPH氧化酶活性、RBOH1表达量、H2O2和GSH积累程度下调。以上结果表明,SlGLR3.3和SlGLR3.5介导了冷驯化诱导的番茄低温抗性,且冷驯化诱导的质外体H2O2积累和GSH/GSSG氧化还原状态变化受SlGLR3.3和SlGLR3.5调控。第三,研究了冷驯化诱导番茄低温抗性过程中GLRs、H202和GSH的相互关系。对GLR3.3/GLR3.5沉默植株外源喷施H202或GSH,利用rboh1和GSH1/2沉默植株进行外源喷施实验,研究GLR3.3/GLR3.5介导的冷驯化诱导的番茄低温抗性中H202和GSH的关系,结果表明:1)外源H202处理能够恢复GLR3.3/GLR3.5沉默植株冷驯化诱导的低温抗性,提高沉默植株中质外体H202含量、GSH含量以及GSH/GSSG 比例;2)外源GSH处理能够提高GLR3.3和GLR3.5的表达,诱导GLR3.3/GLR3.5沉默植株冷驯化诱导的低温抗性以及质外体H2O2的积累;3)在rboh1和GSH1/2沉默植株中,无论喷施GLRs激动剂Glu还是拮抗剂DNQX均不能诱导冷驯化下的低温抗性,冷驯化诱导的GSH积累和GSH/GSSG比例提高在rboh1和GSH1/2沉默植株中也消失。这些结果说明在冷驯化诱导的番茄低温抗性中,RBOH1介导的H2O2以及GSH1/2介导的GSH在冷驯化诱导的低温抗性中都起到重要作用,它们均作用于CLR3.3/GLR3.5的下游,且GSH又位于H202的下游。综上,GLRs-H202-GSH级联关系介导冷驯化诱导的番茄低温抗性。第四,利用qRT-PCR技术检测了番茄6个CaMs基因对冷驯化及后续低温胁迫的响应。结果显示,4℃处理12h时,无论前期是否经过冷驯化,SlCaM6的基因表达情况都显著上调。利用转基因技术构建了 SlCaM6过表达和CRISPR/Cas9材料,进行低温处理,结果表明,cam6表现为显著的低温耐受性,说明SlCaM6负调控番茄低温抗性。COR47-like基因的转录水平在SlCaM6过表达植株中显著下调。进而对CaM6和COR47-like进行亚细胞定位,结果表明CaM6在细胞核、细胞质中都有表达,而COR47-like定位在细胞膜上。利用双分子荧光互补、荧光素酶互补成像和膜蛋白分裂泛素化酵母双杂交技术进一步验证了 CaM6与COR47-like有相互作用。而COR47-like正调控番茄低温抗性。以上结果表明,SlCaM6可能通过与COR47-like互作,抑制其转录水平,进而负调控番茄的低温抗性。第五,利用RNA-seq技术对野生型、SlCaM6过表达以及CRISPR/Cas9植株常温、低温处理进行转录组研究。结果表明,与野生型低温处理相比,SlCaM6过表达植株低温下上调表达基因688个,下调表达677个;CRISPR/Cas9植株低温下上调表达基因1068个,下调表达761个。这些差异基因大致分为乙烯信号通路、油菜素内酯信号通路、逆境响应、光合作用相关以及代谢相关五组。差异基因GO富集结果表明,这些差异基因显著富集在逆境响应、防御响应以及DNA结合条目。基于以上研究结果,我们基本探明了SlGLR3.和SlGLR3.5介导了冷驯化诱导的番茄低温抗性,探讨了其下游的H202和GSH在冷驯化诱导番茄低温抗性中的作用和相互关系,初步探索了 SlCaM6通过与CORs互作负调控番茄低温抗性,挖掘了SlCaM 调控低温抗性中的相关差异基因,进一步完善了番茄低温胁迫过程中的信号转导网络,为合理利用基因改良、植物生长调节剂等手段来诱导番茄低温抗性提供科学依据。
Abstract
di wen xie pai yan chong ying xiang le fan jia (Solanum lycopericum)deng xi wen zuo wu de chan liang he pin zhi ,ji da de xian zhi le shu cai de zhou nian sheng chan he gong ying 。ke fu yi fan jia wei dai biao de da zong shu cai zai dong chun ji jie she shi zai pei zhong de di wen xie pai zhang ai shi dang xia ji dai jie jue de wen ti 。ji yu ci ,pou xi shu cai zuo wu dui di wen de ying da ji zhi ,wa jue di wen xiang ying de guan jian yin zi ,jin er tong guo ji yin bian ji 、zhi wu sheng chang diao jie ji 、huan jing diao kong deng shou duan ,you dao shu cai zuo wu de di wen kang xing ,dui di gao shu cai chan liang 、pin zhi he jing ji xiao yi ,bao zhang zhou nian sheng chan he gong ying ju you shi fen chong yao de ke xue yi yi he xian shi yi yi 。ben lun wen yi fan jia wei yan jiu dui xiang ,li yong wei chuan xue 、fen zi sheng wu xue 、zhi wu sheng li xue 、sheng wu hua xue 、zhuai lu zu xue deng shou duan ,ming que le leng xun hua ji hou xu di wen guo cheng zhong de guan jian yin zi ji ji xiang ying dong tai bian hua ;shai shua le leng xun hua you dao de fan jia di wen kang xing guo cheng zhong lei gu an suan shou ti (Glutamate receptor-like,GLRs)jia zu zhong guan jian zuo yong ji yin ;tan tao le GLR3.3he GLR3.5yi ji guan jian yin zi H2O2he GSHde xiang hu guan ji ;shai shua le di wen guo cheng zhong gai diao su (Calmodulin,CaMs)jia zu zhong de guan jian zuo yong ji yin ,bing li yong zhuai lu zu fen xi le di wen guo cheng zhong de cha yi ji yin ji ji gong neng 。zhu yao yan jiu jie guo ru xia :di yi ,ming que le leng xun hua neng gou you dao fan jia de di wen kang xing ,ju yu H202he GSHji lei you guan 。jin yi bu xi hua le leng xun hua ji hou xu di wen xie pai guo cheng zhong H2O2he GSH/GSSGyang hua hai yuan zhuang tai de dong tai bian hua 。jie guo xian shi ,H202han liang zai leng xun hua xia xian zhe ji lei ,er hou zai di wen xie pai guo cheng zhong qu yu wen ding ;er wei jing leng xun hua zhi jie di wen chu li xia ,H202han liang chi xu zeng jia 。leng xun hua ye xian zhe you dao le GSHde ji lei he GSH/GSSGbi li di sheng 。tong shi ,leng xun hua ye xian zhe you dao le NADPHyang hua mei bian ma ji yin RBOH1ji gu guang gan tai ge cheng ji yin GSH1he GSH2de biao da 。yi shang jie guo biao ming ,leng xun hua you dao de fan jia di wen kang xing yu H202he GSHji lei you guan 。di er ,tong guo shi shi ying guang ding liang PCRji shu dui yi jing jian ding de 13ge fan jia GLRsji yin zai leng xun hua ji hou xu di wen guo cheng zhong de zhuai lu shui ping jin hang le jian ce ,jie guo xian shi SlGLR3.3he SlGLR3.5de zhuai lu shui ping zai leng xun hua guo cheng zhong xian zhe shang sheng 。jin yi bu li yong bing du you dao de ji yin chen mo (VIGS)ji shu he wai yuan pen shi shi yan ,ming que le SlGLR3.he SlGLR3.5jie dao le leng xun hua you dao de fan jia di wen kang xing 。SlGLR3.3he SlGLR3.5chen mo yi ji GLRsyi zhi ji chu li jun xian zhe jiang di le leng xun hua you dao de di wen kang xing ,shi de leng xun hua you dao de NADPHyang hua mei huo xing 、RBOH1biao da liang 、H2O2he GSHji lei cheng du xia diao 。yi shang jie guo biao ming ,SlGLR3.3he SlGLR3.5jie dao le leng xun hua you dao de fan jia di wen kang xing ,ju leng xun hua you dao de zhi wai ti H2O2ji lei he GSH/GSSGyang hua hai yuan zhuang tai bian hua shou SlGLR3.3he SlGLR3.5diao kong 。di san ,yan jiu le leng xun hua you dao fan jia di wen kang xing guo cheng zhong GLRs、H202he GSHde xiang hu guan ji 。dui GLR3.3/GLR3.5chen mo zhi zhu wai yuan pen shi H202huo GSH,li yong rboh1he GSH1/2chen mo zhi zhu jin hang wai yuan pen shi shi yan ,yan jiu GLR3.3/GLR3.5jie dao de leng xun hua you dao de fan jia di wen kang xing zhong H202he GSHde guan ji ,jie guo biao ming :1)wai yuan H202chu li neng gou hui fu GLR3.3/GLR3.5chen mo zhi zhu leng xun hua you dao de di wen kang xing ,di gao chen mo zhi zhu zhong zhi wai ti H202han liang 、GSHhan liang yi ji GSH/GSSG bi li ;2)wai yuan GSHchu li neng gou di gao GLR3.3he GLR3.5de biao da ,you dao GLR3.3/GLR3.5chen mo zhi zhu leng xun hua you dao de di wen kang xing yi ji zhi wai ti H2O2de ji lei ;3)zai rboh1he GSH1/2chen mo zhi zhu zhong ,mo lun pen shi GLRsji dong ji Gluhai shi jie kang ji DNQXjun bu neng you dao leng xun hua xia de di wen kang xing ,leng xun hua you dao de GSHji lei he GSH/GSSGbi li di gao zai rboh1he GSH1/2chen mo zhi zhu zhong ye xiao shi 。zhe xie jie guo shui ming zai leng xun hua you dao de fan jia di wen kang xing zhong ,RBOH1jie dao de H2O2yi ji GSH1/2jie dao de GSHzai leng xun hua you dao de di wen kang xing zhong dou qi dao chong yao zuo yong ,ta men jun zuo yong yu CLR3.3/GLR3.5de xia you ,ju GSHyou wei yu H202de xia you 。zeng shang ,GLRs-H202-GSHji lian guan ji jie dao leng xun hua you dao de fan jia di wen kang xing 。di si ,li yong qRT-PCRji shu jian ce le fan jia 6ge CaMsji yin dui leng xun hua ji hou xu di wen xie pai de xiang ying 。jie guo xian shi ,4℃chu li 12hshi ,mo lun qian ji shi fou jing guo leng xun hua ,SlCaM6de ji yin biao da qing kuang dou xian zhe shang diao 。li yong zhuai ji yin ji shu gou jian le SlCaM6guo biao da he CRISPR/Cas9cai liao ,jin hang di wen chu li ,jie guo biao ming ,cam6biao xian wei xian zhe de di wen nai shou xing ,shui ming SlCaM6fu diao kong fan jia di wen kang xing 。COR47-likeji yin de zhuai lu shui ping zai SlCaM6guo biao da zhi zhu zhong xian zhe xia diao 。jin er dui CaM6he COR47-likejin hang ya xi bao ding wei ,jie guo biao ming CaM6zai xi bao he 、xi bao zhi zhong dou you biao da ,er COR47-likeding wei zai xi bao mo shang 。li yong shuang fen zi ying guang hu bu 、ying guang su mei hu bu cheng xiang he mo dan bai fen lie fan su hua jiao mu shuang za jiao ji shu jin yi bu yan zheng le CaM6yu COR47-likeyou xiang hu zuo yong 。er COR47-likezheng diao kong fan jia di wen kang xing 。yi shang jie guo biao ming ,SlCaM6ke neng tong guo yu COR47-likehu zuo ,yi zhi ji zhuai lu shui ping ,jin er fu diao kong fan jia de di wen kang xing 。di wu ,li yong RNA-seqji shu dui ye sheng xing 、SlCaM6guo biao da yi ji CRISPR/Cas9zhi zhu chang wen 、di wen chu li jin hang zhuai lu zu yan jiu 。jie guo biao ming ,yu ye sheng xing di wen chu li xiang bi ,SlCaM6guo biao da zhi zhu di wen xia shang diao biao da ji yin 688ge ,xia diao biao da 677ge ;CRISPR/Cas9zhi zhu di wen xia shang diao biao da ji yin 1068ge ,xia diao biao da 761ge 。zhe xie cha yi ji yin da zhi fen wei yi xi xin hao tong lu 、you cai su nei zhi xin hao tong lu 、ni jing xiang ying 、guang ge zuo yong xiang guan yi ji dai xie xiang guan wu zu 。cha yi ji yin GOfu ji jie guo biao ming ,zhe xie cha yi ji yin xian zhe fu ji zai ni jing xiang ying 、fang yu xiang ying yi ji DNAjie ge tiao mu 。ji yu yi shang yan jiu jie guo ,wo men ji ben tan ming le SlGLR3.he SlGLR3.5jie dao le leng xun hua you dao de fan jia di wen kang xing ,tan tao le ji xia you de H202he GSHzai leng xun hua you dao fan jia di wen kang xing zhong de zuo yong he xiang hu guan ji ,chu bu tan suo le SlCaM6tong guo yu CORshu zuo fu diao kong fan jia di wen kang xing ,wa jue le SlCaM diao kong di wen kang xing zhong de xiang guan cha yi ji yin ,jin yi bu wan shan le fan jia di wen xie pai guo cheng zhong de xin hao zhuai dao wang lao ,wei ge li li yong ji yin gai liang 、zhi wu sheng chang diao jie ji deng shou duan lai you dao fan jia di wen kang xing di gong ke xue yi ju 。
论文参考文献
论文详细介绍
论文作者分别是来自浙江大学的李卉梓，发表于刊物浙江大学2019-09-18论文，是一篇关于番茄论文,冷驯化论文,低温抗性论文,类谷氨酸受体论文,过氧化氢论文,还原型谷胱甘肽论文,钙调素论文,冷调节蛋白论文,相互作用论文,信号转导论文,浙江大学2019-09-18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，资料来自浙江大学2019-09-18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：番茄论文; 冷驯化论文; 低温抗性论文; 类谷氨酸受体论文; 过氧化氢论文; 还原型谷胱甘肽论文; 钙调素论文; 冷调节蛋白论文; 相互作用论文; 信号转导论文; 浙江大学2019-09-18论文;