本文主要研究内容
作者刘祥政（2019）在《拟南芥MYB6调控茎顶端生长点大小的机制研究》一文中研究指出：在植物中,干细胞聚集在一起形成特化的组织,被称为分生组织。分生组织的规律活动使植物在整个生命周期内能够不断产生新的组织和器官。在地上部分,干细胞主要集中于茎顶端分生组织内,产生植物地上部分的所有器官,包括叶子及花,并在植物的生长和繁殖过程中起到关键作用。在茎顶端分生组织中,干细胞的分裂和分化受到复杂分子调控网络的精密调控,以使茎顶端按照精确的时空模式形成新的器官。目前,茎顶端分生组织中的分子调控网络已基本构建完成,但其中一些调控因子,尤其是在离体再生中起到重要作用的调控因子在茎顶端分生组织中的作用及调控机制仍不清楚。MYB转录因子家族庞大,功能多样,普遍存在于所有的真核细胞生物内。作为拟南芥中最为庞大的一个转录因子家族,MYB家族成员在植物体内的多种生物过程中起到重要作用,包括生物胁迫以及非生物胁迫响应、细胞分化、激素响应、植物防御反应及昼夜节律调节等方面。至今尚未见MYB调控茎顶端分生组织维持及分化中的功能的报道。本实验室前期工作中通过对两种再生能力不同的愈伤组织的基因表达谱芯片结果进行分析,筛选得到一个调控离体苗再生的MYB家族转录因子MYB6。在此基础上,本研究通过分析MYB6相关突变体植株表型,发现MYB6的过表达突变体MYB6-OX无法正常抽薹,由此推断其茎顶端生长点维持与分化异常,并就此展开研究。首先利用myb6(MYB6缺失突变体),myb4(同源基因MYB4缺失突变体),myb4/6(双突变体)、MYB6-OX及WT(野生型对照)进行整体表型观察,发现MYB6-OX植株主枝无法正常发育,其在萌发2-3个月后抽出侧枝,且抽薹具有较高的随机性,而其他三个突变体与野生型相比无明显差别。对以上材料茎顶端进行石蜡切片分析,以从细胞学水平对茎顶端进行更为细致的观察,结果显示,myb4、myb6及myb4/6突变体茎顶端宽度平均值分别比野生型增大了6.02%,26.00%及28.12%,而MYB6-OX突变体茎顶端宽度平均值比野生型减少了19.52%。利用GUS报告基因观察两者在茎顶端中的表达模式,发现MYB4及MYB6都在茎顶端范围内表达,其中MYB4表达位置集中在距离茎顶端较远的叶原基内,MYB6的表达位置集中茎顶端分生组织的边界区细胞内。由此得出结论,MYB4和MYB6均在茎顶端中表达,为茎顶端生长点大小的负调控因子,其中MYB6的作用明显大于MYB4。进一步将WUSpro:GUS及CLV3pro:GUS植株分别与不同突变体杂交,以观察这两个重要的干细胞调控因子在不同突变体中的表达模式变化。实验结果发现WUS及CZV3在myb4、myb6及myb4/6突变体中表达范围及表达量均较野生型有明显上升。其中,myb4/6中表达的变化最明显,myb6中表达的变化次之,同时,WUS和CZV3在MYB6-OX突变体中表达量及表达范围下降,表明MYB4和MYB6均抑制WUS和CLV3的表达。由于MYB6-OX无法正常抽薹,且生长点体积减小,为了探究二者之间的关系,将clv3-7突变体(生长点增大)与MYB6-OX杂交,并观察杂交系的抽薹表型,结果显示,clv3-7无法恢复MYB6-OX表型,结合MYB4和MYB6不在干细胞区表达的实验结果,表明虽然MYB4及MYB6表达量的变化引起了茎顶端中WUS和CZV3表达的变化,但MYB4和MYB6并未通过直接调控WUS和CLV3来调控茎顶端生长点的维持和分化。为了研究MYB6如何调控茎顶端生长点的维持与分化,提取myb6、WT茎顶端RNA进行转录组测序。对转录组测序结果进行分析发现,差异基因在生长素信号通路及ABA信号通路有富集。利用实时定量方法对myb6突变体及野生型茎顶端中AUX/IAA家族和ARF家族基因的表达量进行检测,发现大部分的AUX/IAA家族和ARF家族基因在突变体中的表达量发生了显著变化。进一步构建DR5:GUS载体并获得myb6突变体及野生型背景下的转基因株系,GUS染色观察发现,myb6突变体中生长素含量与野生型相比明显下降。通过EMSA和LUC实验检测MYB6是否能够与预测的生长素通路基因的调控序列直接结合,实验结果显示MYB6能够与ASA1、ARF5、FKD1、IAA9、IAA19等五个基因的DNA直接结合,并调节其在茎顶端中的表达量。以上结果表明MYB6可直接调节生长素相关基因表达,从而调节茎顶端生长素的响应。为验证MYB6是否通过生长素通路调控茎顶端生长点的维持和分化,将生长素响应因子突变体 mARFx与MYB6-OX进行杂交,观察杂交后代表型恢复情况。实验结果显示,mARFx×MYB6-OX杂交系能够正常抽薹,表明MYB6调控茎顶端生长素信号通路调控,从而对茎顶端生长点的维持与分化进行间接调控。综上所述,MYB6通过直接调节生长素通路基因的表达来调节茎顶端中生长素的分布,同时通过调节ABA响应基因来对外界刺激做出反应。两种作用相互结合,共同调节茎顶端生长点的维持与分化。
Abstract
zai zhi wu zhong ,gan xi bao ju ji zai yi qi xing cheng te hua de zu zhi ,bei chen wei fen sheng zu zhi 。fen sheng zu zhi de gui lv huo dong shi zhi wu zai zheng ge sheng ming zhou ji nei neng gou bu duan chan sheng xin de zu zhi he qi guan 。zai de shang bu fen ,gan xi bao zhu yao ji zhong yu jing ding duan fen sheng zu zhi nei ,chan sheng zhi wu de shang bu fen de suo you qi guan ,bao gua xie zi ji hua ,bing zai zhi wu de sheng chang he fan shi guo cheng zhong qi dao guan jian zuo yong 。zai jing ding duan fen sheng zu zhi zhong ,gan xi bao de fen lie he fen hua shou dao fu za fen zi diao kong wang lao de jing mi diao kong ,yi shi jing ding duan an zhao jing que de shi kong mo shi xing cheng xin de qi guan 。mu qian ,jing ding duan fen sheng zu zhi zhong de fen zi diao kong wang lao yi ji ben gou jian wan cheng ,dan ji zhong yi xie diao kong yin zi ,you ji shi zai li ti zai sheng zhong qi dao chong yao zuo yong de diao kong yin zi zai jing ding duan fen sheng zu zhi zhong de zuo yong ji diao kong ji zhi reng bu qing chu 。MYBzhuai lu yin zi jia zu pang da ,gong neng duo yang ,pu bian cun zai yu suo you de zhen he xi bao sheng wu nei 。zuo wei ni na gai zhong zui wei pang da de yi ge zhuai lu yin zi jia zu ,MYBjia zu cheng yuan zai zhi wu ti nei de duo chong sheng wu guo cheng zhong qi dao chong yao zuo yong ,bao gua sheng wu xie pai yi ji fei sheng wu xie pai xiang ying 、xi bao fen hua 、ji su xiang ying 、zhi wu fang yu fan ying ji zhou ye jie lv diao jie deng fang mian 。zhi jin shang wei jian MYBdiao kong jing ding duan fen sheng zu zhi wei chi ji fen hua zhong de gong neng de bao dao 。ben shi yan shi qian ji gong zuo zhong tong guo dui liang chong zai sheng neng li bu tong de yu shang zu zhi de ji yin biao da pu xin pian jie guo jin hang fen xi ,shai shua de dao yi ge diao kong li ti miao zai sheng de MYBjia zu zhuai lu yin zi MYB6。zai ci ji chu shang ,ben yan jiu tong guo fen xi MYB6xiang guan tu bian ti zhi zhu biao xing ,fa xian MYB6de guo biao da tu bian ti MYB6-OXmo fa zheng chang chou tai ,you ci tui duan ji jing ding duan sheng chang dian wei chi yu fen hua yi chang ,bing jiu ci zhan kai yan jiu 。shou xian li yong myb6(MYB6que shi tu bian ti ),myb4(tong yuan ji yin MYB4que shi tu bian ti ),myb4/6(shuang tu bian ti )、MYB6-OXji WT(ye sheng xing dui zhao )jin hang zheng ti biao xing guan cha ,fa xian MYB6-OXzhi zhu zhu zhi mo fa zheng chang fa yo ,ji zai meng fa 2-3ge yue hou chou chu ce zhi ,ju chou tai ju you jiao gao de sui ji xing ,er ji ta san ge tu bian ti yu ye sheng xing xiang bi mo ming xian cha bie 。dui yi shang cai liao jing ding duan jin hang dan la qie pian fen xi ,yi cong xi bao xue shui ping dui jing ding duan jin hang geng wei xi zhi de guan cha ,jie guo xian shi ,myb4、myb6ji myb4/6tu bian ti jing ding duan kuan du ping jun zhi fen bie bi ye sheng xing zeng da le 6.02%,26.00%ji 28.12%,er MYB6-OXtu bian ti jing ding duan kuan du ping jun zhi bi ye sheng xing jian shao le 19.52%。li yong GUSbao gao ji yin guan cha liang zhe zai jing ding duan zhong de biao da mo shi ,fa xian MYB4ji MYB6dou zai jing ding duan fan wei nei biao da ,ji zhong MYB4biao da wei zhi ji zhong zai ju li jing ding duan jiao yuan de xie yuan ji nei ,MYB6de biao da wei zhi ji zhong jing ding duan fen sheng zu zhi de bian jie ou xi bao nei 。you ci de chu jie lun ,MYB4he MYB6jun zai jing ding duan zhong biao da ,wei jing ding duan sheng chang dian da xiao de fu diao kong yin zi ,ji zhong MYB6de zuo yong ming xian da yu MYB4。jin yi bu jiang WUSpro:GUSji CLV3pro:GUSzhi zhu fen bie yu bu tong tu bian ti za jiao ,yi guan cha zhe liang ge chong yao de gan xi bao diao kong yin zi zai bu tong tu bian ti zhong de biao da mo shi bian hua 。shi yan jie guo fa xian WUSji CZV3zai myb4、myb6ji myb4/6tu bian ti zhong biao da fan wei ji biao da liang jun jiao ye sheng xing you ming xian shang sheng 。ji zhong ,myb4/6zhong biao da de bian hua zui ming xian ,myb6zhong biao da de bian hua ci zhi ,tong shi ,WUShe CZV3zai MYB6-OXtu bian ti zhong biao da liang ji biao da fan wei xia jiang ,biao ming MYB4he MYB6jun yi zhi WUShe CLV3de biao da 。you yu MYB6-OXmo fa zheng chang chou tai ,ju sheng chang dian ti ji jian xiao ,wei le tan jiu er zhe zhi jian de guan ji ,jiang clv3-7tu bian ti (sheng chang dian zeng da )yu MYB6-OXza jiao ,bing guan cha za jiao ji de chou tai biao xing ,jie guo xian shi ,clv3-7mo fa hui fu MYB6-OXbiao xing ,jie ge MYB4he MYB6bu zai gan xi bao ou biao da de shi yan jie guo ,biao ming sui ran MYB4ji MYB6biao da liang de bian hua yin qi le jing ding duan zhong WUShe CZV3biao da de bian hua ,dan MYB4he MYB6bing wei tong guo zhi jie diao kong WUShe CLV3lai diao kong jing ding duan sheng chang dian de wei chi he fen hua 。wei le yan jiu MYB6ru he diao kong jing ding duan sheng chang dian de wei chi yu fen hua ,di qu myb6、WTjing ding duan RNAjin hang zhuai lu zu ce xu 。dui zhuai lu zu ce xu jie guo jin hang fen xi fa xian ,cha yi ji yin zai sheng chang su xin hao tong lu ji ABAxin hao tong lu you fu ji 。li yong shi shi ding liang fang fa dui myb6tu bian ti ji ye sheng xing jing ding duan zhong AUX/IAAjia zu he ARFjia zu ji yin de biao da liang jin hang jian ce ,fa xian da bu fen de AUX/IAAjia zu he ARFjia zu ji yin zai tu bian ti zhong de biao da liang fa sheng le xian zhe bian hua 。jin yi bu gou jian DR5:GUSzai ti bing huo de myb6tu bian ti ji ye sheng xing bei jing xia de zhuai ji yin zhu ji ,GUSran se guan cha fa xian ,myb6tu bian ti zhong sheng chang su han liang yu ye sheng xing xiang bi ming xian xia jiang 。tong guo EMSAhe LUCshi yan jian ce MYB6shi fou neng gou yu yu ce de sheng chang su tong lu ji yin de diao kong xu lie zhi jie jie ge ,shi yan jie guo xian shi MYB6neng gou yu ASA1、ARF5、FKD1、IAA9、IAA19deng wu ge ji yin de DNAzhi jie jie ge ,bing diao jie ji zai jing ding duan zhong de biao da liang 。yi shang jie guo biao ming MYB6ke zhi jie diao jie sheng chang su xiang guan ji yin biao da ,cong er diao jie jing ding duan sheng chang su de xiang ying 。wei yan zheng MYB6shi fou tong guo sheng chang su tong lu diao kong jing ding duan sheng chang dian de wei chi he fen hua ,jiang sheng chang su xiang ying yin zi tu bian ti mARFxyu MYB6-OXjin hang za jiao ,guan cha za jiao hou dai biao xing hui fu qing kuang 。shi yan jie guo xian shi ,mARFx×MYB6-OXza jiao ji neng gou zheng chang chou tai ,biao ming MYB6diao kong jing ding duan sheng chang su xin hao tong lu diao kong ,cong er dui jing ding duan sheng chang dian de wei chi yu fen hua jin hang jian jie diao kong 。zeng shang suo shu ,MYB6tong guo zhi jie diao jie sheng chang su tong lu ji yin de biao da lai diao jie jing ding duan zhong sheng chang su de fen bu ,tong shi tong guo diao jie ABAxiang ying ji yin lai dui wai jie ci ji zuo chu fan ying 。liang chong zuo yong xiang hu jie ge ,gong tong diao jie jing ding duan sheng chang dian de wei chi yu fen hua 。
论文参考文献
论文详细介绍
论文作者分别是来自山东大学的刘祥政，发表于刊物山东大学2019-07-16论文，是一篇关于茎顶端分生组织论文,转录因子论文,生长素论文,干细胞论文,山东大学2019-07-16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，资料来自山东大学2019-07-16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：茎顶端分生组织论文; 转录因子论文; 生长素论文; 干细胞论文; 山东大学2019-07-16论文;