【作者】 谢卡斌；

【导师】 熊立仲；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 水稻是全球最重要的粮食作物之一。水稻遗传改良的关键是寻找能调节水稻株高、分蘖数、抽穗期、穗的大小和育性等重要农艺性状的基因。以MicmRNA为代表的内源small RNA分子是生物生长发育关键调节因子,为水稻的遗传改良提供了新的基因资源。本研究包含三方面的内容:一是构建水稻全长cDNA文库,得到13000个以上的全长cDNA克隆,同时构建用于酵母双杂交筛选的cDNA文库;二是系统的分析水稻中的OsSPL（Oryza Sativa SQUAMOSA Promoter-binding Like）基因,特别是miR156的靶基因;三是以筛选控制水稻重要农艺性状的small RNA基因为目标,重点研究miR156和miR164在水稻生长发育中的调节机制。cDNA文库的构建的结果如下:1.利用改良的Oligo-capping方法构建了全长比例在92%以上的水稻幼穗的cDNA文库,并对3000个随机挑选的克隆测序。此外,从本实验室已有的cDNA文库中筛选了10828个包含完整读码框的cDNA克隆。总共得到了13650个全长cDNA克隆。2.构建了两个用于酵母双杂交的cDNA文库,在本实验室已成功用于转录因子、mRNA剪切因子、未知基因等不同类型基因的互作蛋白的筛选,假阳性率在10%以下。miR156及靶基因OsSPL基因分离鉴定的主要结果如下:1.分析了水稻中miR156的靶基因OsSPL家族。主要包括水稻中18个OsSPL基因的分离、植物中SPL基因的进化分析、保守结构域的分析,并分析了OsSPL基因在水稻的13不同发育时期的组织中的表达水平。对8个OsSPL基因进行了超表达。2.序列分析表明水稻中11个OsSPL基因含miR156的结合位点（M156BR,miR156 binding region）。通过Northern blot的结果表明,OsSPL12和OsSPL14的mRNA分子被miR156-RISC切断。3.RLM-RACE分析OsSPL14和OsSPL17被miR156-RISC切断的位置。miR156-RISC在M156BR的第7和8个核酸之间的位置切断靶基因,正好位于M156BR与miR156错配的核酸的位置。4.对miR156的一个靶基因OsSPL14进行了定点诱变,得到不受miR156调节的基因OsSPL14ml和OsSPL14m2,并对OsSPL14m2进行了超表达。5.通过Y2H筛选穗的cDNA文库,得到了OsSPL14的6个互作蛋白。其中三个是与ubiquitin途径相关的RING finger类蛋白,表明OsSPL14除了受miR156调节外,蛋白的稳定性可能还受ubiquitin途径的调节。miR156和miR164功能研究主要结果如下:1.在玉米Ubiquitin启动子的作用下超表达了miR156和miR164,得到了miR156的两个前体（pri-miR156b和pri-miR156h）的超表达植株（Md和Mh）和miR164一个前体（pri-miR164b）的超表达植株（MI7）。2.分析了Md和Mh的T₄代植株与WT（wild type）在生长和发育上的差异。在苗期（四叶期以前）Md和Mh与WT无差异。在第四叶生长出以后,Md和Mh的叶片和分蘖发生的速度是WT的3倍以上。到灌浆期,Md和Mh植株叶片的数目是WT的100倍以上,有效分蘖的数目是WT的50倍以上。在大田种植条件下（武汉7月至10月）Md和Mh的营养生长延长,抽穗期推迟7天以上。Md和Mh植株高度只有WT的50%,穗的二次枝梗和颖花数变少。通过比较Md和Mh的叶片大小、叶片表皮细胞的发育情况和SAM（shoot apical meristem）的大小,推断miR156超表达改变了水稻的发育时间。3.通过small RNA gel blot分析miR156在不同“年龄”的叶片中的表达水平表明miR156的高低与叶片发育时间正相关,miR156是叶片发育时间的Marker基因。4.利用水稻的全基因组芯片分析了Md/Mh和WT植株的老叶和新叶中基因的表达情况,并据此分析miR156的下游基因以及叶片发育时间相关的基因。5.比较了含M156BR的OsSPL基因在Md/Mh和WT不同器官中的表达量,结果表明miR156-OsSPL基因的互作受其他因子的调节,可能的调节因子有DRG12和已报道的PLA2。6.在叶片的生长发育过程中miR164表达模式与miR156正好相反,说明两者在叶片的发育过程中存在联系。同时在small RNA gel blot中还检测到一个表达模式与成熟miR164相反的未知前体,表明miR164的水平受启动子和microRNA形成过程的双重调节。7.序列分析表明水稻中仅有OsNAC1和OsNAC2含M164BR（miR164 bindregion）。RLM-RACE分析也证明OsNAC1和OsNAC2的mRNA在M164BR第10个核酸的位置被切断。OsNAC1和OsNAC2的转录本在不同发育时间的叶片中,表达模式与miR164的表达模式相反。8.miR164能调节水稻叶片的边界。在miR164超表达的MI7植株中,叶鞘边缘融合、部分叶片融合或扭曲,在一些极端的植株中有类似拟南芥cup-shaped的结构形成。MI7叶片发育的缺陷随植株生长时间的增加而加剧。9.miR164调节水稻的生殖生长。MI7的花药形态发育异常,花粉完全不育。MI7虽能正常开花,但胚和胚乳不能正常发育。正常抽穗开花的MI7主分蘖的倒数第二个节（穗颈节的前一个节）未伸长。MI7的叶片中,一个Flowers Locus T（FT）的同源基因（MI7D1）被抑制。在长日照条件下,OsNAC1、OsNAC2和MI7D1的表达与日照有关,OsNAC1和OsNAC2表达的最高峰在黑暗时间段的正中点。10.miR164的超表达改变了水稻体内的激素水平。通过外施生长素和细胞分裂素可以使MI7生长发育恢复正常。在MI7植株中部分生长素合成、运输和应答的基因被抑制。11.通过比较不同植物中miR156和miR164的表达,发现miR156-miR164在禾本科作物中保守,但在油菜叶片中存在不同的表达模式。12.根据以上结果,可以推断出以下结论:miR156是控制水稻发育特别是叶片发育的异时性基因。miR164在水稻中除了调节器官边界外,还具有与拟南芥中不同的功能—调节水稻的生殖生长。最后,一个miR164-miR156Paradigm的模型用来解释miR156和miR164在协同调节水稻器官发生、生长和成熟过程中的作用。MicroRNA表达模式的改变和靶基因数目的变化是miR156-miR164进化的重要组成部分。更多还原

【Abstract】 Rice（Oryza Sativa L.） is one of the most important crops worldwide.Identification of genes,which control plant height,heading date,number of tillers and panicle size,is essential for rice genetic improvement.Endogenous small RNAs,especially microRNAs, are key regulators for plant development.MicroRNAs are novel candidate genes for rice genetic improvement.There are three objectives of this research:1) construction of cDNA libraries including full-length cDNA libraries and yeast two hybrid cDNA libraries;2) identification and analysis of the OsSPL genes from rice,specifically for miR156 targeted OsSPL genes;and 3) studying the functions of miR156 and miR164 in rice.In the first part,the main results are as follows:1.One full-length cDNA library has been constructed from young panicles（stage 3, 4,5）.The sequences of 3,000 random selected clones indicated that more than 92% clones are full-length cDNA.A total of 10,828 cDNA clones,which contain full length open reading frames,have identified from the conventional cDNA library.Thus,13,650 full-length cDNA clones have been identified in this research.2.Two cDNA libraries have been constructed for yeast two hybrid（Y2H）.The cDNA library have been used for Y2H screening for identification of interacting proteins of intested proteins in our group with less than 10%false positive clones.The main results of the second part of this research:1.OsSPL gene family in rice was systematically analyzed.18 OsSPL genes have been identified.The phylogenetic relation ship of plant SPL genes,conserved protein motifs,and expression pattern in 13 different tissues were studied in this research.2.There are 11 OsSPL genes containing M156BR（miR156 binding region） based on sequence analysis.The miR156-directed mRNA cleavage of OsSPL12 and OsSPL14 can be detected when they were over expressed in plants.3.The cleavage sites of OsSPL14 and OsSPL17 were mapped by RLM-RACE. The cleavage sites of miR156 targets were located between the seventh and eighth nucleotide of M156BR,where is the mismatch site of M156BR and miR156.4.Two point mutations of OsSPL14 gene,OsSPL14m1 and OsSPL14m2,were generated and OsSPL14m2 was over-expressed in rice.5.OsSPL14-interact-proteins were identified by Y2H screen of a panicle cDNA library.Three of them encode a RING finger protein,which is involved in ubiquitin pathway,suggesting that OsSPL14 may involved in the regulation of protein turnover.In the third part of this research,the functions of miR156 and miR164 was analyzed. The results list as follows:1.Two precursors of miR156（pri-miR156d and pri-miR156h） and one of miR164 （pri-miR164b） were transformed rice under the control of maize ubiquitin promoter.The transgenic plants named Md（pri-miR156d）,Mh（pri-miR156h）,and MI7（pri-miR164b） respectively.2.The developmental difference of Md and Mh were investigated.Md and Mh plants showed distinct developmental differences to WT after the fourth leaves emerged. The rate of leaf and tiller initiation of Md and Mh are 300%of that in WT.The heading dates of Md and Mh delayed more than 7 days.Compared to WT,the number of leaves increased more than one hundred folds and the number of tillers increased more than 50 folds.However,the plant height of the Md and Mh plants decreased 50%.Though the branches of panicle and the spikelet number of Md and Mh decreased dramatically,the yield and biomass are not changed.The shape and epidermis of leaves indicate that the developmental time of rice was changed in Md and Mh.3.The expression level of miR156 increased gradually parallel to the development of leaves.It is concluded that the expression level of miR156 can reflect the developmental time of rice leaf as a marker gene.4.The up-and down-regulated genes in mature or immature leaves of Md and Mh were identified by microarray assay.5.The interaction of miR156 and OsSPL may be regulated by other regulators.Two candidates including a no-coding RNA gene（DRG12） and PLA2 were identified in this research. 6.The expression of miR164 showed reverse pattern to miR156 during the leaf development.An unknown primary transcript of miR164 has been identified in small RNA gel blot.It is suggested that the expression of miR164 is regulated by its promoter and microRNA processing.7.miR164 targets OsNAC1 and OsNAC2 in rice.Cleavage site mapping indicated that miR164 cleavages the 10^th nucleotide of M164BR.The transcript levels of OsNAC1 and OsNAC2 were decreased when miR164 was increased in different leaves.8.miR164 may regulate the organ boundary.The defects of MI7 leaf include fused leaf sheath and twist leaf blade.At reproductive stages,the abnormal development of MI7 pistil turns out sterile.9.The defects of MI7 can be rescued by applying hormones.The genes involved in auxin synthesis,transport and responses were down-regulated in MI7.Moreover,DRG12 is down-regulated in MI7.10.miR164 plays an essential regulation role in rice reproductive growth.One of the internodes of MI7 main culm did not elongate.In MI7 leaves,a LFY/FT homologous gene（MI7D1） was down-regulated.In addition,the expression levels of OsNAC1,OsNAC2,and MI7D1 were changed in association with photoperiodic rhythm: all the genes showed peak expression in the middle of dark phase.11.The expression pattern of miR156-miR164 is conserved in gramineae crops, but different in Brassica napus.12.A paradigm model of miR156-miR164 action has been concluded to explain the regulatory role of miR156 and miR164 in rice.更多还原