本文主要研究内容
作者段存英（2019）在《拟南芥KETCH1通过介导核糖体蛋白RPL27a的核积累调控配子体发育》一文中研究指出：开花植物有性生殖的主要特征是精细胞与卵细胞及中央细胞的双受精过程。雌雄配子体的发育对于双受精过程的顺利进行至关重要。其中小孢子母细胞与大孢子母细胞经过一系列的减数分裂及有丝分裂过程分别产生雄配子与雌配子。研究表明,有丝分裂过程的异常往往会导致细胞死亡,这暗示着有丝分裂的调控对于配子体的发育过程至关重要。在核糖体合成异常的突变体中,许多配子体致死都是由于有丝分裂缺陷导致。人们根据同源比对在植物中已经发现了大量的核糖体蛋白(Ribosomal Proteins,RPs),但是关于这些蛋白是如何被调控的,比如动态的亚细胞靶向过程,目前还尚不清楚。在本研究中,我们鉴定了一个拟南芥importinβ蛋白家族成员KETCH1,通过介导核糖体蛋白L27a(RPL27a)的核积累,参与调控配子体的发育。本论文的主要研究成果和结论如下:(1)KETCH1功能缺失导致雌雄配子体传代率下降已有研究表明,KETCH1功能缺失会造成纯合致死,得不到纯合体植株(Zhang et al.,2017)。我们用野生型Col-0作为对照,对ketch1-2/+进行育性分析,发现杂合突变体角果中有47%的败育胚珠,败育率显著高于胚致死的25%。此外,ketch1-2/+中小而皱缩的胚珠是未受精的,胚囊中含有不规则分布的细胞核。正反交实验结果显示,ketch1-2的雌雄配子体传代率均显著降低,表明KETCH1对于雌雄配子体的功能至关重要。(2)KETCH1功能缺失导致单细胞小孢子和功能大孢子发育停滞为了确定雄配子体传代率降低的原因,我们通过亚历山大染色、DAPI染色以及扫描电镜观察对花粉的活性、核发育及外壁形态进行了观察。与野生型相比,ketch1-2/+中有32%左右的花粉败育。我们通过半薄切片观察了不同发育时期的花药。在发育的第10期,突变体与野生型相比,花粉发育并没有任何异常。然而第11期开始出现异常,突变体小孢子只含有一个细胞核并且有许多异常的大液泡存在,暗示了ketch1-2小孢子的第一次有丝分裂出现异常。为了确定雌配子体发育异常,我们通过组织光学切片对不同发育时期的胚珠进行观察。ketch1-2/+在3-I时期可以正常形成功能大孢子,但是有30%左右的功能大孢子有丝分裂过程出现异常,导致突变体成熟胚囊中细胞核数目呈现不规则状态,从1个到6个不等。这些结果证明,KETCH1对于单细胞小孢子和功能大孢子发育过程中的第一次有丝分裂极为重要。(3)KETCH1组成型表达,并且在生殖组织中高量表达荧光定量PCR结果以及KETCH1启动子驱动GUS报告基因的转基因株系均显示KETCH1是组成型表达,并且在生殖组织如花序、胚珠及花粉中高量表达。通过分析KETCH1启动子驱动核靶向荧光蛋白YFP的转基因株系,表明KETCH1在配子体发育过程中的生殖细胞中高量表达。(4)KETCH1在配子体发育过程中的功能并不依赖HYL1之前的研究报道显示,KETCH1能够介导miRNA的关键调控因子HYL1的核质穿梭过程。尽管HYL1功能缺失突变会导致植株育性下降,但是我们的结果表明,hyl1-2雌雄配子体传代率与野生型相比没有差异,这说明HYL1并不参与KETCH1介导的配子体发育过程。(5)RPL27aC在细胞核中的积累需要KETCH1的运输根据动物细胞同源蛋白IPO5的研究结果显示,KETCH1可能参与核糖体蛋白的核输入过程。通过BiFC和体外pull-down实验,证明KETCH1与RPL27aC互作。而且我们的实验结果也证明了核输入蛋白与底物分子的结合是特异的。并且在ketch1-2背景下,RPL27aC在花粉及花粉管的细胞核中的积累量显著降低,同样的在KETCH1-RNAi植株的叶片原生质体中,RPL27aC核积累量也是降低的,这表明RPL27aC是KETCH1的底物蛋白。已有研究表明,RPL27a功能缺失突变会导致雌雄配子体传代率显著下降,这与它在KETCH1调控的配子发生中的功能相一致。因此,KETCH1很有可能是通过介导RPL27a的核积累来调控核糖体生物发生过程,进而调控配子发生期间的有丝分裂过程。核转运蛋白是一类介导核质穿梭的分子伴侣。本论文结果显示,拟南芥核转运蛋白KETCH1功能缺失导致减数分裂后的雌雄配子体发育停滞。同时我们证明了KETCH1在配子体发育过程中的功能并不依赖已报道的底物HYL1。相反的,KETCH1对RPL27a的核积累至关重要,并且RPL27a的突变导致了类似的配子体缺陷。因此我们推测,核糖体蛋白核积累的减少可能导致了细胞翻译能力下降,进而在配子发生过程中触发有丝分裂细胞周期的停滞。
Abstract
kai hua zhi wu you xing sheng shi de zhu yao te zheng shi jing xi bao yu luan xi bao ji zhong yang xi bao de shuang shou jing guo cheng 。ci xiong pei zi ti de fa yo dui yu shuang shou jing guo cheng de shun li jin hang zhi guan chong yao 。ji zhong xiao bao zi mu xi bao yu da bao zi mu xi bao jing guo yi ji lie de jian shu fen lie ji you si fen lie guo cheng fen bie chan sheng xiong pei zi yu ci pei zi 。yan jiu biao ming ,you si fen lie guo cheng de yi chang wang wang hui dao zhi xi bao si wang ,zhe an shi zhao you si fen lie de diao kong dui yu pei zi ti de fa yo guo cheng zhi guan chong yao 。zai he tang ti ge cheng yi chang de tu bian ti zhong ,hu duo pei zi ti zhi si dou shi you yu you si fen lie que xian dao zhi 。ren men gen ju tong yuan bi dui zai zhi wu zhong yi jing fa xian le da liang de he tang ti dan bai (Ribosomal Proteins,RPs),dan shi guan yu zhe xie dan bai shi ru he bei diao kong de ,bi ru dong tai de ya xi bao ba xiang guo cheng ,mu qian hai shang bu qing chu 。zai ben yan jiu zhong ,wo men jian ding le yi ge ni na gai importinβdan bai jia zu cheng yuan KETCH1,tong guo jie dao he tang ti dan bai L27a(RPL27a)de he ji lei ,can yu diao kong pei zi ti de fa yo 。ben lun wen de zhu yao yan jiu cheng guo he jie lun ru xia :(1)KETCH1gong neng que shi dao zhi ci xiong pei zi ti chuan dai lv xia jiang yi you yan jiu biao ming ,KETCH1gong neng que shi hui zao cheng chun ge zhi si ,de bu dao chun ge ti zhi zhu (Zhang et al.,2017)。wo men yong ye sheng xing Col-0zuo wei dui zhao ,dui ketch1-2/+jin hang yo xing fen xi ,fa xian za ge tu bian ti jiao guo zhong you 47%de bai yo pei zhu ,bai yo lv xian zhe gao yu pei zhi si de 25%。ci wai ,ketch1-2/+zhong xiao er zhou su de pei zhu shi wei shou jing de ,pei nang zhong han you bu gui ze fen bu de xi bao he 。zheng fan jiao shi yan jie guo xian shi ,ketch1-2de ci xiong pei zi ti chuan dai lv jun xian zhe jiang di ,biao ming KETCH1dui yu ci xiong pei zi ti de gong neng zhi guan chong yao 。(2)KETCH1gong neng que shi dao zhi chan xi bao xiao bao zi he gong neng da bao zi fa yo ting zhi wei le que ding xiong pei zi ti chuan dai lv jiang di de yuan yin ,wo men tong guo ya li shan da ran se 、DAPIran se yi ji sao miao dian jing guan cha dui hua fen de huo xing 、he fa yo ji wai bi xing tai jin hang le guan cha 。yu ye sheng xing xiang bi ,ketch1-2/+zhong you 32%zuo you de hua fen bai yo 。wo men tong guo ban bao qie pian guan cha le bu tong fa yo shi ji de hua yao 。zai fa yo de di 10ji ,tu bian ti yu ye sheng xing xiang bi ,hua fen fa yo bing mei you ren he yi chang 。ran er di 11ji kai shi chu xian yi chang ,tu bian ti xiao bao zi zhi han you yi ge xi bao he bing ju you hu duo yi chang de da ye pao cun zai ,an shi le ketch1-2xiao bao zi de di yi ci you si fen lie chu xian yi chang 。wei le que ding ci pei zi ti fa yo yi chang ,wo men tong guo zu zhi guang xue qie pian dui bu tong fa yo shi ji de pei zhu jin hang guan cha 。ketch1-2/+zai 3-Ishi ji ke yi zheng chang xing cheng gong neng da bao zi ,dan shi you 30%zuo you de gong neng da bao zi you si fen lie guo cheng chu xian yi chang ,dao zhi tu bian ti cheng shou pei nang zhong xi bao he shu mu cheng xian bu gui ze zhuang tai ,cong 1ge dao 6ge bu deng 。zhe xie jie guo zheng ming ,KETCH1dui yu chan xi bao xiao bao zi he gong neng da bao zi fa yo guo cheng zhong de di yi ci you si fen lie ji wei chong yao 。(3)KETCH1zu cheng xing biao da ,bing ju zai sheng shi zu zhi zhong gao liang biao da ying guang ding liang PCRjie guo yi ji KETCH1qi dong zi qu dong GUSbao gao ji yin de zhuai ji yin zhu ji jun xian shi KETCH1shi zu cheng xing biao da ,bing ju zai sheng shi zu zhi ru hua xu 、pei zhu ji hua fen zhong gao liang biao da 。tong guo fen xi KETCH1qi dong zi qu dong he ba xiang ying guang dan bai YFPde zhuai ji yin zhu ji ,biao ming KETCH1zai pei zi ti fa yo guo cheng zhong de sheng shi xi bao zhong gao liang biao da 。(4)KETCH1zai pei zi ti fa yo guo cheng zhong de gong neng bing bu yi lai HYL1zhi qian de yan jiu bao dao xian shi ,KETCH1neng gou jie dao miRNAde guan jian diao kong yin zi HYL1de he zhi chuan suo guo cheng 。jin guan HYL1gong neng que shi tu bian hui dao zhi zhi zhu yo xing xia jiang ,dan shi wo men de jie guo biao ming ,hyl1-2ci xiong pei zi ti chuan dai lv yu ye sheng xing xiang bi mei you cha yi ,zhe shui ming HYL1bing bu can yu KETCH1jie dao de pei zi ti fa yo guo cheng 。(5)RPL27aCzai xi bao he zhong de ji lei xu yao KETCH1de yun shu gen ju dong wu xi bao tong yuan dan bai IPO5de yan jiu jie guo xian shi ,KETCH1ke neng can yu he tang ti dan bai de he shu ru guo cheng 。tong guo BiFChe ti wai pull-downshi yan ,zheng ming KETCH1yu RPL27aChu zuo 。er ju wo men de shi yan jie guo ye zheng ming le he shu ru dan bai yu de wu fen zi de jie ge shi te yi de 。bing ju zai ketch1-2bei jing xia ,RPL27aCzai hua fen ji hua fen guan de xi bao he zhong de ji lei liang xian zhe jiang di ,tong yang de zai KETCH1-RNAizhi zhu de xie pian yuan sheng zhi ti zhong ,RPL27aChe ji lei liang ye shi jiang di de ,zhe biao ming RPL27aCshi KETCH1de de wu dan bai 。yi you yan jiu biao ming ,RPL27agong neng que shi tu bian hui dao zhi ci xiong pei zi ti chuan dai lv xian zhe xia jiang ,zhe yu ta zai KETCH1diao kong de pei zi fa sheng zhong de gong neng xiang yi zhi 。yin ci ,KETCH1hen you ke neng shi tong guo jie dao RPL27ade he ji lei lai diao kong he tang ti sheng wu fa sheng guo cheng ,jin er diao kong pei zi fa sheng ji jian de you si fen lie guo cheng 。he zhuai yun dan bai shi yi lei jie dao he zhi chuan suo de fen zi ban lv 。ben lun wen jie guo xian shi ,ni na gai he zhuai yun dan bai KETCH1gong neng que shi dao zhi jian shu fen lie hou de ci xiong pei zi ti fa yo ting zhi 。tong shi wo men zheng ming le KETCH1zai pei zi ti fa yo guo cheng zhong de gong neng bing bu yi lai yi bao dao de de wu HYL1。xiang fan de ,KETCH1dui RPL27ade he ji lei zhi guan chong yao ,bing ju RPL27ade tu bian dao zhi le lei shi de pei zi ti que xian 。yin ci wo men tui ce ,he tang ti dan bai he ji lei de jian shao ke neng dao zhi le xi bao fan yi neng li xia jiang ,jin er zai pei zi fa sheng guo cheng zhong chu fa you si fen lie xi bao zhou ji de ting zhi 。
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论文详细介绍
论文作者分别是来自山东农业大学的段存英，发表于刊物山东农业大学2019-07-05论文，是一篇关于配子发生论文,有丝分裂论文,花粉管导向论文,核质穿梭论文,核糖体生物发生论文,山东农业大学2019-07-05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，资料来自山东农业大学2019-07-05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：配子发生论文; 有丝分裂论文; 花粉管导向论文; 核质穿梭论文; 核糖体生物发生论文; 山东农业大学2019-07-05论文;