本文主要研究内容
作者熊雨飞（2019）在《水稻种子特异表达核因子Y（NF-Y）转录因子的功能和分子机理研究》一文中研究指出：稻米是全球近一半人口的主食。提高水稻产量和改善稻米品质是水稻基础研究和生产实践中的两个关键科学问题。水稻种子大小/粒形直接影响水稻的产量,胚乳中淀粉和贮藏蛋白的含量与稻米的品质息息相关。近年来,影响水稻粒形和种子贮藏物质积累的基因时有报道,然而这两个重要性状的转录调控机制和调控网络仍不明确。核因子Y(NF-Y)作为一类转录因子,参与调控植物生长发育的多个过程。本研究选取5个水稻种子/胚乳特异表达的NF-Y基因(NF-YA8,NF-YB1,NF-YB9,NF-YC10和NF-YC12),通过创建基因表达抑制、基因功能缺失突变体和超量表达等遗传材料来鉴定它们在水稻种子/胚乳发育中的功能,并对其调控机理进行了解析。主要结果如下:1.通过分析5个胚乳特异表达的NF-Y基因在水稻中花11中的表达模式,发现它们的表达具有胚乳特异性,此结果初步表明:水稻胚乳特异NF-Y基因可能参与水稻胚乳发育的调控。本研究中,我们利用RNAi技术和CRISPR技术创建了这5个胚乳特异NF-Y基因的转基因遗传材料,其中有2个基因的RNAi转基因材料和5个基因的基因敲除突变体;同时还创建了2个基因的超表达材料。2.水稻种子特异表达因子NF-YC12是水稻贮藏物质积累的关键调节因子。NFYC12编码一个NF-Y转录因子C亚基,qRT-PCR结果显示其在受精后7–14天的种子中高表达,原位杂交实验表明NF-YC12主要在种子糊粉层和淀粉胚乳部位表达。NF-YC12突变导致粒重显著降低,种子垩白增加。扫描电镜观察显示nf-yc12突变体成熟种子的淀粉颗粒形态异常、呈不规则排列。透射电镜观察结果表明nf-yc12突变体中淀粉颗粒的形成受到影响。此外,nf-yc12突变体中淀粉、直链淀粉和醇溶蛋白的含量均显著降低,谷蛋白含量有所升高;淀粉的理化性质(如支链淀粉的链长分布和淀粉的糊化特性)也发生改变。超表达NF-YC12能显著提高种子粒形和粒重,同时超表达材料中种子贮藏物质的积累也发生了变化。RNA-seq分析显示,nf-yc12突变体中下调的差异表达基因与淀粉生物合成和能量储备代谢过程相关,qRT-PCR结果显示,参与水稻种子淀粉合成和蛋白合成的基因在nf-yc12中的表达量明显低于野生型,以上表明NF-YC12在水稻种子贮藏物质的合成和积累过程中起作用。结合ChIP-seq和RNA-seq结果分析,通过ChIP-qPCR和酵母单杂交等实验证明了NF-YC12能直接结合OsSUT1(Sucrose Transporter)、OsGS1;3(Glutamine Synthetase1)和FLO6(FLOURY ENDOSPERM6)的启动子。同时qRT-PCR结果显示在nf-yc12突变体中,这3个基因的表达出现明显的下调,表明OsGS1;3、OsSUT1和FLO6是NF-YC12直接作用的靶基因。NF-YC12和NF-YB1在体内/外均能发生互作,结合两者的表达模式的异同,我们解析了围绕NF-YC12-NF-YB1参与调控胚乳发育和贮藏物质积累过程的转录调控网络。在糊粉层中,NF-YC12与NF-YB1互作,协同调控蔗糖转运蛋白OsSUT1的功能,此外NF-YC12通过直接结合并调控FLO6和OsGS1;3的表达,来调节胚乳中贮藏物质的合成和积累。3.水稻种子糊粉层特异表达因子NF-YB1调控胚乳发育和水稻灌浆。NF-YB1编码NF-Y转录因子B亚基,在水稻种子糊粉层特异表达。NF-YB1表达下调或上调均导致种子垩白的增加,说明NF-YB1表达的改变影响稻米的品质。NF-YB1表达的改变不影响种子糊粉层的发育。NF-YB1表达下调导致种子淀粉颗粒形态发生异常。与野生型相比,nf-yb1突变体(利用CRISPR创建的NF-YB1 knock-out突变体)种子粒重显著降低,垩白率明显增加。此外,nf-yb1突变体中总淀粉和直链淀粉的含量明显降低,支链淀粉的链长分布发生改变。以上结果表明,NF-YB1在水稻胚乳发育和籽粒灌浆过程中发挥重要的作用。4.水稻种子优势表达基因NF-YC10调节水稻种子大小。NF-YC10具有胚乳优势表达的模式,qRT-PCR和原位杂交实验结果显示NF-YC10在受精后5–10天的胚乳中高表达。NF-YC10蛋白定位于细胞核中,且该蛋白具有转录激活活性。利用CRISPR基因敲除NF-YC10或RNAi抑制NF-YC10表达均产生小粒种子,粒重和粒宽明显降低/减少。NF-YC10调控粒形可能是通过调节粒形相关基因的表达来完成的。我们的研究鉴定了一个调控水稻粒形的新因子NF-YC10,其具有提高水稻种子大小和产量的潜力。5.水稻NF-YA8基因的表达模式和功能研究。NF-YA8启动子融合GUS报告基因实验表明NF-YA8只在胚乳最外围的糊粉层中表达。qRT-PCR结果显示,NF-YA8在种子发育的中后期高表达,在受精后7–10天达到峰值,与GUS染色分析的结果相一致,这说明NF-YA8是一个糊粉层特异表达的基因。NF-YA8-GFP蛋白定位在细胞核中。酵母双杂交结果显示NF-YA8与NF-YB1和NF-YC12均不发生互作。NF-YA8突变之后种子外观没有发生异常。
Abstract
dao mi shi quan qiu jin yi ban ren kou de zhu shi 。di gao shui dao chan liang he gai shan dao mi pin zhi shi shui dao ji chu yan jiu he sheng chan shi jian zhong de liang ge guan jian ke xue wen ti 。shui dao chong zi da xiao /li xing zhi jie ying xiang shui dao de chan liang ,pei ru zhong dian fen he zhu cang dan bai de han liang yu dao mi de pin zhi xi xi xiang guan 。jin nian lai ,ying xiang shui dao li xing he chong zi zhu cang wu zhi ji lei de ji yin shi you bao dao ,ran er zhe liang ge chong yao xing zhuang de zhuai lu diao kong ji zhi he diao kong wang lao reng bu ming que 。he yin zi Y(NF-Y)zuo wei yi lei zhuai lu yin zi ,can yu diao kong zhi wu sheng chang fa yo de duo ge guo cheng 。ben yan jiu shua qu 5ge shui dao chong zi /pei ru te yi biao da de NF-Yji yin (NF-YA8,NF-YB1,NF-YB9,NF-YC10he NF-YC12),tong guo chuang jian ji yin biao da yi zhi 、ji yin gong neng que shi tu bian ti he chao liang biao da deng wei chuan cai liao lai jian ding ta men zai shui dao chong zi /pei ru fa yo zhong de gong neng ,bing dui ji diao kong ji li jin hang le jie xi 。zhu yao jie guo ru xia :1.tong guo fen xi 5ge pei ru te yi biao da de NF-Yji yin zai shui dao zhong hua 11zhong de biao da mo shi ,fa xian ta men de biao da ju you pei ru te yi xing ,ci jie guo chu bu biao ming :shui dao pei ru te yi NF-Yji yin ke neng can yu shui dao pei ru fa yo de diao kong 。ben yan jiu zhong ,wo men li yong RNAiji shu he CRISPRji shu chuang jian le zhe 5ge pei ru te yi NF-Yji yin de zhuai ji yin wei chuan cai liao ,ji zhong you 2ge ji yin de RNAizhuai ji yin cai liao he 5ge ji yin de ji yin qiao chu tu bian ti ;tong shi hai chuang jian le 2ge ji yin de chao biao da cai liao 。2.shui dao chong zi te yi biao da yin zi NF-YC12shi shui dao zhu cang wu zhi ji lei de guan jian diao jie yin zi 。NFYC12bian ma yi ge NF-Yzhuai lu yin zi Cya ji ,qRT-PCRjie guo xian shi ji zai shou jing hou 7–14tian de chong zi zhong gao biao da ,yuan wei za jiao shi yan biao ming NF-YC12zhu yao zai chong zi hu fen ceng he dian fen pei ru bu wei biao da 。NF-YC12tu bian dao zhi li chong xian zhe jiang di ,chong zi e bai zeng jia 。sao miao dian jing guan cha xian shi nf-yc12tu bian ti cheng shou chong zi de dian fen ke li xing tai yi chang 、cheng bu gui ze pai lie 。tou she dian jing guan cha jie guo biao ming nf-yc12tu bian ti zhong dian fen ke li de xing cheng shou dao ying xiang 。ci wai ,nf-yc12tu bian ti zhong dian fen 、zhi lian dian fen he chun rong dan bai de han liang jun xian zhe jiang di ,gu dan bai han liang you suo sheng gao ;dian fen de li hua xing zhi (ru zhi lian dian fen de lian chang fen bu he dian fen de hu hua te xing )ye fa sheng gai bian 。chao biao da NF-YC12neng xian zhe di gao chong zi li xing he li chong ,tong shi chao biao da cai liao zhong chong zi zhu cang wu zhi de ji lei ye fa sheng le bian hua 。RNA-seqfen xi xian shi ,nf-yc12tu bian ti zhong xia diao de cha yi biao da ji yin yu dian fen sheng wu ge cheng he neng liang chu bei dai xie guo cheng xiang guan ,qRT-PCRjie guo xian shi ,can yu shui dao chong zi dian fen ge cheng he dan bai ge cheng de ji yin zai nf-yc12zhong de biao da liang ming xian di yu ye sheng xing ,yi shang biao ming NF-YC12zai shui dao chong zi zhu cang wu zhi de ge cheng he ji lei guo cheng zhong qi zuo yong 。jie ge ChIP-seqhe RNA-seqjie guo fen xi ,tong guo ChIP-qPCRhe jiao mu chan za jiao deng shi yan zheng ming le NF-YC12neng zhi jie jie ge OsSUT1(Sucrose Transporter)、OsGS1;3(Glutamine Synthetase1)he FLO6(FLOURY ENDOSPERM6)de qi dong zi 。tong shi qRT-PCRjie guo xian shi zai nf-yc12tu bian ti zhong ,zhe 3ge ji yin de biao da chu xian ming xian de xia diao ,biao ming OsGS1;3、OsSUT1he FLO6shi NF-YC12zhi jie zuo yong de ba ji yin 。NF-YC12he NF-YB1zai ti nei /wai jun neng fa sheng hu zuo ,jie ge liang zhe de biao da mo shi de yi tong ,wo men jie xi le wei rao NF-YC12-NF-YB1can yu diao kong pei ru fa yo he zhu cang wu zhi ji lei guo cheng de zhuai lu diao kong wang lao 。zai hu fen ceng zhong ,NF-YC12yu NF-YB1hu zuo ,xie tong diao kong zhe tang zhuai yun dan bai OsSUT1de gong neng ,ci wai NF-YC12tong guo zhi jie jie ge bing diao kong FLO6he OsGS1;3de biao da ,lai diao jie pei ru zhong zhu cang wu zhi de ge cheng he ji lei 。3.shui dao chong zi hu fen ceng te yi biao da yin zi NF-YB1diao kong pei ru fa yo he shui dao guan jiang 。NF-YB1bian ma NF-Yzhuai lu yin zi Bya ji ,zai shui dao chong zi hu fen ceng te yi biao da 。NF-YB1biao da xia diao huo shang diao jun dao zhi chong zi e bai de zeng jia ,shui ming NF-YB1biao da de gai bian ying xiang dao mi de pin zhi 。NF-YB1biao da de gai bian bu ying xiang chong zi hu fen ceng de fa yo 。NF-YB1biao da xia diao dao zhi chong zi dian fen ke li xing tai fa sheng yi chang 。yu ye sheng xing xiang bi ,nf-yb1tu bian ti (li yong CRISPRchuang jian de NF-YB1 knock-outtu bian ti )chong zi li chong xian zhe jiang di ,e bai lv ming xian zeng jia 。ci wai ,nf-yb1tu bian ti zhong zong dian fen he zhi lian dian fen de han liang ming xian jiang di ,zhi lian dian fen de lian chang fen bu fa sheng gai bian 。yi shang jie guo biao ming ,NF-YB1zai shui dao pei ru fa yo he zi li guan jiang guo cheng zhong fa hui chong yao de zuo yong 。4.shui dao chong zi you shi biao da ji yin NF-YC10diao jie shui dao chong zi da xiao 。NF-YC10ju you pei ru you shi biao da de mo shi ,qRT-PCRhe yuan wei za jiao shi yan jie guo xian shi NF-YC10zai shou jing hou 5–10tian de pei ru zhong gao biao da 。NF-YC10dan bai ding wei yu xi bao he zhong ,ju gai dan bai ju you zhuai lu ji huo huo xing 。li yong CRISPRji yin qiao chu NF-YC10huo RNAiyi zhi NF-YC10biao da jun chan sheng xiao li chong zi ,li chong he li kuan ming xian jiang di /jian shao 。NF-YC10diao kong li xing ke neng shi tong guo diao jie li xing xiang guan ji yin de biao da lai wan cheng de 。wo men de yan jiu jian ding le yi ge diao kong shui dao li xing de xin yin zi NF-YC10,ji ju you di gao shui dao chong zi da xiao he chan liang de qian li 。5.shui dao NF-YA8ji yin de biao da mo shi he gong neng yan jiu 。NF-YA8qi dong zi rong ge GUSbao gao ji yin shi yan biao ming NF-YA8zhi zai pei ru zui wai wei de hu fen ceng zhong biao da 。qRT-PCRjie guo xian shi ,NF-YA8zai chong zi fa yo de zhong hou ji gao biao da ,zai shou jing hou 7–10tian da dao feng zhi ,yu GUSran se fen xi de jie guo xiang yi zhi ,zhe shui ming NF-YA8shi yi ge hu fen ceng te yi biao da de ji yin 。NF-YA8-GFPdan bai ding wei zai xi bao he zhong 。jiao mu shuang za jiao jie guo xian shi NF-YA8yu NF-YB1he NF-YC12jun bu fa sheng hu zuo 。NF-YA8tu bian zhi hou chong zi wai guan mei you fa sheng yi chang 。
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论文作者分别是来自华中农业大学的熊雨飞，发表于刊物华中农业大学2019-07-15论文，是一篇关于水稻论文,因子论文,种子特异表达论文,贮藏物质积累论文,种子大小论文,胚乳发育论文,转录调控论文,华中农业大学2019-07-15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，资料来自华中农业大学2019-07-15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：水稻论文; 因子论文; 种子特异表达论文; 贮藏物质积累论文; 种子大小论文; 胚乳发育论文; 转录调控论文; 华中农业大学2019-07-15论文;