本文主要研究内容
作者王丹丹(2019)在《黄瓜CsMPC1和CsE1α基因在线粒体介导植物PCD中的作用研究》一文中研究指出:水杨酸(SA)是植物防御的关键信号因子,可以诱导某些抗病基因的表达,在植物抗病中起重要作用。课题组前期研究证实,SA能够诱导黄瓜叶片发生超敏反应(HR),是一个细胞程序性死亡(PCD)的过程。为进一步探究SA的作用机制,对SA处理前后的黄瓜叶片进行了转录组分析,得到1756个差异表达基因。本研究选择与线粒体功能相关的显著上调表达的CsMPC1和CsE1α基因,研究其表达特性,利用超表达拟南芥和突变体拟南芥研究基因的功能,为揭示基因在线粒体介导的PCD中的作用奠定基础。研究结果如下:1.用10mM SA和0.2%MeJA分别处理黄瓜叶片,在营养器官根、茎、叶和MeJA处理叶片进行CsMPC1和CsE1α基因的qRT-PCR表达分析,并对根、茎、叶和SA和MeJA处理叶片进行相应指标丙酮酸(PA)含量和丙酮酸脱氢酶(PDH)活性测定。CsMPC1和CsE1α基因在叶片中具有较高表达量,在MeJA处理介导PCD过程皆呈上调表达调控;CsMPC1丙酮酸转运体基因水平改变引起其转运PA含量的变化,比较发现两者呈负相关;CsE1α丙酮酸脱氢酶基因水平的改变影响PDH活性的变化,比较发现两者呈正相关。但MeJA和SA处理发生PCD过程中PDH活性无明显变化。2.根据mRNA开放读码区序列设计引物,从黄瓜中克隆CsMPC1和CsE1α基因。CsMPC1基因cDNA全长311bp,蛋白由103个氨基酸构成。CsE1α基因cDNA全长1199bp,蛋白由399个氨基酸构成。通过NCBI BlastX比对测序结果显示CsMPC1属于MPC超家族,CsE1α属于TPP超家族。3.构建CsMPC1和CsE1α基因的超表达载体,通过农杆菌介导的浸花法将质粒转入到野生型拟南芥植株中,获得了转CsMPC1和CsE1α基因拟南芥植株。利用三引物法将从拟南芥生物资源中心获得MPC1基因(SALK089763.42.10.x)和E1α基因(SALK056967.56.00.x)T-DNA插入突变体进行PCR鉴定获得纯合株系。4.对筛选出的MPC1和E1α的超表达拟南芥植株(OE-1和OE#-1)和突变体拟南芥植株(mpc1-1和E1α-1)进行表型鉴定、生理指标测定和PCD鉴定。结果如下:(1)突变体植株mpc1-1对ABA敏感,具有抗旱的功能;超表达植株OE-1转运PA水平升高,表明PA转运体行使功能;突变体植株mpc1-1转运PA水平降低,表明PA转运受到阻碍,导致线粒体功能下降,糖酵解途径增强,植株生长受到抑制,但在超表达和突变体植株中均未检测到PCD现象。说明MPC1基因超表达与敲除时,PA代谢发生变化但并不影响植物PCD的发生,这是因为在MPC1基因敲除时MPC家族其他转运蛋白仍在行驶功能,MPC1基因超表达时植株正常生长没有出现细胞死亡现象。(2)超表达植株OE#-1正常生长,并且PDH活性与WT无明显差异,没有出现细胞死亡的现象;在突变体植株E1α-1中PDH活性受到抑制,生物体的有氧代谢受到阻碍,严重阻碍细胞的呼吸,导致ROS大量积累,可能引起植物PCD发生,从而影响植株正常生长发育。这说明E1α基因敲除可能引起植物PCD的发生。
Abstract
shui yang suan (SA)shi zhi wu fang yu de guan jian xin hao yin zi ,ke yi you dao mou xie kang bing ji yin de biao da ,zai zhi wu kang bing zhong qi chong yao zuo yong 。ke ti zu qian ji yan jiu zheng shi ,SAneng gou you dao huang gua xie pian fa sheng chao min fan ying (HR),shi yi ge xi bao cheng xu xing si wang (PCD)de guo cheng 。wei jin yi bu tan jiu SAde zuo yong ji zhi ,dui SAchu li qian hou de huang gua xie pian jin hang le zhuai lu zu fen xi ,de dao 1756ge cha yi biao da ji yin 。ben yan jiu shua ze yu xian li ti gong neng xiang guan de xian zhe shang diao biao da de CsMPC1he CsE1αji yin ,yan jiu ji biao da te xing ,li yong chao biao da ni na gai he tu bian ti ni na gai yan jiu ji yin de gong neng ,wei jie shi ji yin zai xian li ti jie dao de PCDzhong de zuo yong dian ding ji chu 。yan jiu jie guo ru xia :1.yong 10mM SAhe 0.2%MeJAfen bie chu li huang gua xie pian ,zai ying yang qi guan gen 、jing 、xie he MeJAchu li xie pian jin hang CsMPC1he CsE1αji yin de qRT-PCRbiao da fen xi ,bing dui gen 、jing 、xie he SAhe MeJAchu li xie pian jin hang xiang ying zhi biao bing tong suan (PA)han liang he bing tong suan tuo qing mei (PDH)huo xing ce ding 。CsMPC1he CsE1αji yin zai xie pian zhong ju you jiao gao biao da liang ,zai MeJAchu li jie dao PCDguo cheng jie cheng shang diao biao da diao kong ;CsMPC1bing tong suan zhuai yun ti ji yin shui ping gai bian yin qi ji zhuai yun PAhan liang de bian hua ,bi jiao fa xian liang zhe cheng fu xiang guan ;CsE1αbing tong suan tuo qing mei ji yin shui ping de gai bian ying xiang PDHhuo xing de bian hua ,bi jiao fa xian liang zhe cheng zheng xiang guan 。dan MeJAhe SAchu li fa sheng PCDguo cheng zhong PDHhuo xing mo ming xian bian hua 。2.gen ju mRNAkai fang dou ma ou xu lie she ji yin wu ,cong huang gua zhong ke long CsMPC1he CsE1αji yin 。CsMPC1ji yin cDNAquan chang 311bp,dan bai you 103ge an ji suan gou cheng 。CsE1αji yin cDNAquan chang 1199bp,dan bai you 399ge an ji suan gou cheng 。tong guo NCBI BlastXbi dui ce xu jie guo xian shi CsMPC1shu yu MPCchao jia zu ,CsE1αshu yu TPPchao jia zu 。3.gou jian CsMPC1he CsE1αji yin de chao biao da zai ti ,tong guo nong gan jun jie dao de jin hua fa jiang zhi li zhuai ru dao ye sheng xing ni na gai zhi zhu zhong ,huo de le zhuai CsMPC1he CsE1αji yin ni na gai zhi zhu 。li yong san yin wu fa jiang cong ni na gai sheng wu zi yuan zhong xin huo de MPC1ji yin (SALK089763.42.10.x)he E1αji yin (SALK056967.56.00.x)T-DNAcha ru tu bian ti jin hang PCRjian ding huo de chun ge zhu ji 。4.dui shai shua chu de MPC1he E1αde chao biao da ni na gai zhi zhu (OE-1he OE#-1)he tu bian ti ni na gai zhi zhu (mpc1-1he E1α-1)jin hang biao xing jian ding 、sheng li zhi biao ce ding he PCDjian ding 。jie guo ru xia :(1)tu bian ti zhi zhu mpc1-1dui ABAmin gan ,ju you kang han de gong neng ;chao biao da zhi zhu OE-1zhuai yun PAshui ping sheng gao ,biao ming PAzhuai yun ti hang shi gong neng ;tu bian ti zhi zhu mpc1-1zhuai yun PAshui ping jiang di ,biao ming PAzhuai yun shou dao zu ai ,dao zhi xian li ti gong neng xia jiang ,tang jiao jie tu jing zeng jiang ,zhi zhu sheng chang shou dao yi zhi ,dan zai chao biao da he tu bian ti zhi zhu zhong jun wei jian ce dao PCDxian xiang 。shui ming MPC1ji yin chao biao da yu qiao chu shi ,PAdai xie fa sheng bian hua dan bing bu ying xiang zhi wu PCDde fa sheng ,zhe shi yin wei zai MPC1ji yin qiao chu shi MPCjia zu ji ta zhuai yun dan bai reng zai hang shi gong neng ,MPC1ji yin chao biao da shi zhi zhu zheng chang sheng chang mei you chu xian xi bao si wang xian xiang 。(2)chao biao da zhi zhu OE#-1zheng chang sheng chang ,bing ju PDHhuo xing yu WTmo ming xian cha yi ,mei you chu xian xi bao si wang de xian xiang ;zai tu bian ti zhi zhu E1α-1zhong PDHhuo xing shou dao yi zhi ,sheng wu ti de you yang dai xie shou dao zu ai ,yan chong zu ai xi bao de hu xi ,dao zhi ROSda liang ji lei ,ke neng yin qi zhi wu PCDfa sheng ,cong er ying xiang zhi zhu zheng chang sheng chang fa yo 。zhe shui ming E1αji yin qiao chu ke neng yin qi zhi wu PCDde fa sheng 。
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论文作者分别是来自哈尔滨师范大学的王丹丹,发表于刊物哈尔滨师范大学2019-07-01论文,是一篇关于水杨酸论文,线粒体论文,基因表达论文,克隆论文,哈尔滨师范大学2019-07-01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,资料来自哈尔滨师范大学2019-07-01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:水杨酸论文; 线粒体论文; 基因表达论文; 克隆论文; 哈尔滨师范大学2019-07-01论文;