本文主要研究内容
作者张悦（2019）在《刚毛柽柳ThSZF3基因耐盐功能分析》一文中研究指出：锌指蛋白(ZFP,Zinc Finger Proteins)是植物一大类重要的转录调控因子,参与调控一些重要的生理过程,如:形态建成、花粉发育、胁迫应答、胚胎发育等[1]。在前期研究中发现刚毛柽柳ThZFP基因可能是耐盐基因,因此本研究进一步对ThZFP基因的耐盐功能进行了研究,并对其耐盐调控机制进行了初步的分析。本研究从抗旱耐盐的木本植物刚毛柽柳转录组数据中鉴定了一条盐胁迫诱导表达的锌指蛋白(salt-induciblezincfinger)基因,命名为ThSZF3。ThSZF3基因全长1176bp,发现该基因编码391个氨基酸,分子量为97.26 kDa,理论等电点为5.00。亚细胞定位结果表明ThSZF3蛋白在细胞核中表达。构建了ThSZF3基因过表达载体pROKII-ThSZF3及RNAi载体pFGC5941-ThSZF3,将其与空pROKII载体瞬时转化到刚毛柽柳中,分别获得过表达(OE)、抑制表达(RNAi)及对照(CON)刚毛柽柳转基因植株并对转基因刚毛柽柳进行NaCl胁迫。分析比较了三种转基因刚毛柽柳中的DAB、NBT及Evans Blue染色以及POD、SOD、PRO、MDA、H2O2含量及相对电导率等生理指标以快速鉴定ThSZF3基因的功能。结果显示,在三种转基因植株中,过表达刚毛柽柳的POD、SOD活性和脯氨酸(PRO)含量最高,H2O2、MDA含量和相对电导率最低,细胞膜完整性最好,而ThSZF3抑制表达的植株POD、SOD活性和PRO含量最低,O2-、H2O2和MDA最高,相对电导率最高,细胞膜损伤最严重。结果表明ThSZF3能够提高柽柳细胞内抗氧化酶活性,促进活性氧清除,降低细胞膜损伤,从而使柽柳适应高盐环境。为了进一步验证刚毛柽柳中瞬时表达ThSZF3基因的结果,将ThSZF3基因过表达载体转入野生型拟南芥,筛选获得T3代纯合株系。对转基因及野生型拟南芥进行150mM NaCl胁迫,分析比较盐胁迫前后转基因和对照拟南芥的种子萌发率、根长、鲜重及长势等生长指标,结果显示盐胁迫后过表达株系的种子萌发率、根长和鲜重都明显高于对照,长势也优于对照,表明转基因转系的耐盐能力显著提高,过表达ThSZF3基因能提高转基因拟南芥的耐盐能力。进一步分析比较了盐胁迫前后转基因及野生型对照株系的 DAB、NBT、Evans blue 染色以及 POD、SOD、PRO、MDA、H2O2含量、相对电导率等生理指标,结果显示与瞬时转化刚毛柽柳结果一致,盐胁迫后转基因拟南芥株系的染色更浅,POD、SOD、PRO含量最高,MDA、H2O2含量和相对电导率值明显低于对照。表明ThSZF3基因能通过减少转基因植株细胞内的活性氧积累,保护细胞膜的完整性,从而提高转基因植物的耐盐能力。利用以转录因子为中心的酵母单杂交技术(TF-centered Y1H),鉴定ThSZF3识别和结合的顺式作用元件。结果表明ThSZF3能够特异性的识别4个DNA序列,包括一个未知的新元件 SBS(SZF Binding Site)和 3 个已知元件(Dof、CAAT-box、G-box)。通过酵母单杂交验证得出SBS元件核心序列为“CTGCC”。利用转录组测序鉴定ThSZF3过表达、抑制表达及对照柽柳之间的差异表达基因。ThSZF3过表达柽柳与对照之间上调表达的基因有22个;ThSZF3抑制表达柽柳与对照之间下调表达的基因140个。两组共有的基因有6个。经过序列比对发现“CTGCC”元件存在于其中4条基因启动子序列中,选择其中一个柽柳乙烯不敏感蛋白ThEIN2(Ethylene-insensitive protein 2)的启动子序列进行探究,酵母单杂交结果显示ThSZF3能够与ThEIN2包含SBS元件的启动子片段结合,推测可能为ThSZF3所调控的下游基因。
Abstract
xin zhi dan bai (ZFP,Zinc Finger Proteins)shi zhi wu yi da lei chong yao de zhuai lu diao kong yin zi ,can yu diao kong yi xie chong yao de sheng li guo cheng ,ru :xing tai jian cheng 、hua fen fa yo 、xie pai ying da 、pei tai fa yo deng [1]。zai qian ji yan jiu zhong fa xian gang mao cheng liu ThZFPji yin ke neng shi nai yan ji yin ,yin ci ben yan jiu jin yi bu dui ThZFPji yin de nai yan gong neng jin hang le yan jiu ,bing dui ji nai yan diao kong ji zhi jin hang le chu bu de fen xi 。ben yan jiu cong kang han nai yan de mu ben zhi wu gang mao cheng liu zhuai lu zu shu ju zhong jian ding le yi tiao yan xie pai you dao biao da de xin zhi dan bai (salt-induciblezincfinger)ji yin ,ming ming wei ThSZF3。ThSZF3ji yin quan chang 1176bp,fa xian gai ji yin bian ma 391ge an ji suan ,fen zi liang wei 97.26 kDa,li lun deng dian dian wei 5.00。ya xi bao ding wei jie guo biao ming ThSZF3dan bai zai xi bao he zhong biao da 。gou jian le ThSZF3ji yin guo biao da zai ti pROKII-ThSZF3ji RNAizai ti pFGC5941-ThSZF3,jiang ji yu kong pROKIIzai ti shun shi zhuai hua dao gang mao cheng liu zhong ,fen bie huo de guo biao da (OE)、yi zhi biao da (RNAi)ji dui zhao (CON)gang mao cheng liu zhuai ji yin zhi zhu bing dui zhuai ji yin gang mao cheng liu jin hang NaClxie pai 。fen xi bi jiao le san chong zhuai ji yin gang mao cheng liu zhong de DAB、NBTji Evans Blueran se yi ji POD、SOD、PRO、MDA、H2O2han liang ji xiang dui dian dao lv deng sheng li zhi biao yi kuai su jian ding ThSZF3ji yin de gong neng 。jie guo xian shi ,zai san chong zhuai ji yin zhi zhu zhong ,guo biao da gang mao cheng liu de POD、SODhuo xing he fu an suan (PRO)han liang zui gao ,H2O2、MDAhan liang he xiang dui dian dao lv zui di ,xi bao mo wan zheng xing zui hao ,er ThSZF3yi zhi biao da de zhi zhu POD、SODhuo xing he PROhan liang zui di ,O2-、H2O2he MDAzui gao ,xiang dui dian dao lv zui gao ,xi bao mo sun shang zui yan chong 。jie guo biao ming ThSZF3neng gou di gao cheng liu xi bao nei kang yang hua mei huo xing ,cu jin huo xing yang qing chu ,jiang di xi bao mo sun shang ,cong er shi cheng liu kuo ying gao yan huan jing 。wei le jin yi bu yan zheng gang mao cheng liu zhong shun shi biao da ThSZF3ji yin de jie guo ,jiang ThSZF3ji yin guo biao da zai ti zhuai ru ye sheng xing ni na gai ,shai shua huo de T3dai chun ge zhu ji 。dui zhuai ji yin ji ye sheng xing ni na gai jin hang 150mM NaClxie pai ,fen xi bi jiao yan xie pai qian hou zhuai ji yin he dui zhao ni na gai de chong zi meng fa lv 、gen chang 、xian chong ji chang shi deng sheng chang zhi biao ,jie guo xian shi yan xie pai hou guo biao da zhu ji de chong zi meng fa lv 、gen chang he xian chong dou ming xian gao yu dui zhao ,chang shi ye you yu dui zhao ,biao ming zhuai ji yin zhuai ji de nai yan neng li xian zhe di gao ,guo biao da ThSZF3ji yin neng di gao zhuai ji yin ni na gai de nai yan neng li 。jin yi bu fen xi bi jiao le yan xie pai qian hou zhuai ji yin ji ye sheng xing dui zhao zhu ji de DAB、NBT、Evans blue ran se yi ji POD、SOD、PRO、MDA、H2O2han liang 、xiang dui dian dao lv deng sheng li zhi biao ,jie guo xian shi yu shun shi zhuai hua gang mao cheng liu jie guo yi zhi ,yan xie pai hou zhuai ji yin ni na gai zhu ji de ran se geng jian ,POD、SOD、PROhan liang zui gao ,MDA、H2O2han liang he xiang dui dian dao lv zhi ming xian di yu dui zhao 。biao ming ThSZF3ji yin neng tong guo jian shao zhuai ji yin zhi zhu xi bao nei de huo xing yang ji lei ,bao hu xi bao mo de wan zheng xing ,cong er di gao zhuai ji yin zhi wu de nai yan neng li 。li yong yi zhuai lu yin zi wei zhong xin de jiao mu chan za jiao ji shu (TF-centered Y1H),jian ding ThSZF3shi bie he jie ge de shun shi zuo yong yuan jian 。jie guo biao ming ThSZF3neng gou te yi xing de shi bie 4ge DNAxu lie ,bao gua yi ge wei zhi de xin yuan jian SBS(SZF Binding Site)he 3 ge yi zhi yuan jian (Dof、CAAT-box、G-box)。tong guo jiao mu chan za jiao yan zheng de chu SBSyuan jian he xin xu lie wei “CTGCC”。li yong zhuai lu zu ce xu jian ding ThSZF3guo biao da 、yi zhi biao da ji dui zhao cheng liu zhi jian de cha yi biao da ji yin 。ThSZF3guo biao da cheng liu yu dui zhao zhi jian shang diao biao da de ji yin you 22ge ;ThSZF3yi zhi biao da cheng liu yu dui zhao zhi jian xia diao biao da de ji yin 140ge 。liang zu gong you de ji yin you 6ge 。jing guo xu lie bi dui fa xian “CTGCC”yuan jian cun zai yu ji zhong 4tiao ji yin qi dong zi xu lie zhong ,shua ze ji zhong yi ge cheng liu yi xi bu min gan dan bai ThEIN2(Ethylene-insensitive protein 2)de qi dong zi xu lie jin hang tan jiu ,jiao mu chan za jiao jie guo xian shi ThSZF3neng gou yu ThEIN2bao han SBSyuan jian de qi dong zi pian duan jie ge ,tui ce ke neng wei ThSZF3suo diao kong de xia you ji yin 。
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张悦，发表于刊物东北林业大学2019-11-18论文，是一篇关于刚毛柽柳论文,瞬时侵染论文,盐胁迫论文,酵母单杂交论文,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：刚毛柽柳论文; 瞬时侵染论文; 盐胁迫论文; 酵母单杂交论文; 东北林业大学2019-11-18论文;