本文選題：開(kāi)花 + RNA結(jié)合蛋白　； 參考：《山東農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：開(kāi)花作為植物由營(yíng)養(yǎng)生長(zhǎng)向生殖生長(zhǎng)的轉(zhuǎn)變,既決定了植物能否成功地繁衍后代影響自身種群的延續(xù),同時(shí)也是一個(gè)可以影響到農(nóng)作物產(chǎn)量和質(zhì)量的重要農(nóng)藝性狀。植物的開(kāi)花時(shí)間受到復(fù)雜而精細(xì)的調(diào)控,涉及外界環(huán)境因子,如溫度和光照,以及植物自身內(nèi)在因子對(duì)開(kāi)花信號(hào)的識(shí)別與傳遞的共同作用。該過(guò)程包含了一系列基因的表達(dá)調(diào)控,從而確保植物在最合適的狀態(tài)下完成這一生物學(xué)過(guò)程的轉(zhuǎn)換。近期研究表明,除了四個(gè)經(jīng)典的開(kāi)花調(diào)控通路外,許多參與RNA轉(zhuǎn)錄后加工的RNA結(jié)合蛋白(RNA binding protein,RBP)或RNA加工因子參與植物開(kāi)花時(shí)間的調(diào)控。RNA結(jié)合蛋白在真核生物中廣泛存在,參與調(diào)控了RNA加工代謝的各個(gè)層面,包括5′-端加帽、可變剪接、RNA編輯、3′-末端多聚腺苷酸化、核質(zhì)穿梭、RNA穩(wěn)定性等過(guò)程,最終調(diào)控了基因的表達(dá)與生命體生長(zhǎng)發(fā)育。擬南芥基因組編碼約200個(gè)RNA結(jié)合蛋白,越來(lái)越多的研究表明,在開(kāi)花時(shí)間轉(zhuǎn)換、逆境響應(yīng)與染色質(zhì)修飾等過(guò)程中,RBP都扮演著重要的角色。前體mRNA 3′-末端的多聚腺苷酸化在真核生物基因表達(dá)與調(diào)控中具有重要的作用,該過(guò)程包含兩步緊密相聯(lián)的生化過(guò)程:首先在前體mRNA的多聚腺苷酸化位點(diǎn)(poly(A)sites,PAS)進(jìn)行切割,然后添加poly(A)尾巴。Poly(A)位點(diǎn)的選擇是多變的,該現(xiàn)象稱(chēng)之為選擇性多聚腺苷酸化(Alternative polyadenylation,APA),前體mRNA通過(guò)APA產(chǎn)生具有不同編碼序列或者是不同3′-UTR區(qū)域的轉(zhuǎn)錄本,增加了轉(zhuǎn)錄組的復(fù)雜性。大規(guī)模研究顯示,APA是一種在多物種間廣泛存在的基因表達(dá)調(diào)控機(jī)制,多數(shù)基因包含一個(gè)以上的多聚腺苷酸化位點(diǎn),其中酵母、哺乳動(dòng)物和擬南芥中高達(dá)70%以上的基因具有APA現(xiàn)象。APA介導(dǎo)的基因表達(dá)在控制機(jī)體發(fā)育、疾病發(fā)生、植物開(kāi)花與環(huán)境響應(yīng)等過(guò)程中具有重要的作用。APA受到了復(fù)雜而精細(xì)的調(diào)控,涉及到許多蛋白與核酸間的相互作用,然而APA在植物中的具體調(diào)控機(jī)制尚不清楚。本實(shí)驗(yàn)室前期在篩選植物開(kāi)花相關(guān)突變體時(shí)發(fā)現(xiàn)At5G40490的缺失突變可以導(dǎo)致晚花,該基因編碼一個(gè)RNA結(jié)合蛋白,進(jìn)化樹(shù)分析發(fā)現(xiàn)該蛋白與人類(lèi)中的hnRNP A1和酵母中的Hrp1高度同源,將其命名為HLP1。為了解析HLP1的作用機(jī)制,采用紫外交聯(lián)免疫沉淀結(jié)合大規(guī)模測(cè)序的技術(shù)在全基因組水平上鑒定HLP1所結(jié)合的靶RNA。生物信息分析顯示,HLP1在全基因組水平上具有廣泛的結(jié)合位點(diǎn),RIP-qRT-PCR有效證明了HLP1與靶RNA結(jié)合的真實(shí)性。進(jìn)一步的生物信息分析顯示,HLP1結(jié)合序列顯著富集在3′-UTR區(qū)域和內(nèi)含子區(qū)域,并且具有兩個(gè)明顯的序列特征,一個(gè)是富含腺嘌呤的5′-AGAAAA-3′,另外一個(gè)是富含尿嘧啶的5′-UUUUCU-3′,它們分布于轉(zhuǎn)錄終止位點(diǎn)附近�；蚬δ芨患治鲲@示,HLP1的結(jié)合基因開(kāi)花過(guò)程明顯相關(guān)。HLP1所結(jié)合的序列特征與poly(A)位點(diǎn)信號(hào)高度相似,暗示著HLP1可能參與APA的調(diào)控。poly(A)位點(diǎn)測(cè)序(poly(A)sites sequencing,PAS-seq)顯示,HLP1的缺失突變會(huì)導(dǎo)致許多基因的APA轉(zhuǎn)換。結(jié)合CLIP-seq分析發(fā)現(xiàn),很多發(fā)生APA轉(zhuǎn)換的基因是HLP1的直接靶RNA,暗示著HLP1可以直接參與APA調(diào)控。其中,HLP1可以結(jié)合在開(kāi)花促進(jìn)因子FCA的3′-UTR區(qū)域和第三個(gè)內(nèi)含子處,促進(jìn)FCA選擇遠(yuǎn)端poly(A)位點(diǎn),產(chǎn)生有功能的轉(zhuǎn)錄本,抑制FLC的表達(dá),促進(jìn)植物開(kāi)花。序列比對(duì)發(fā)現(xiàn),擬南芥中存在一個(gè)與HLP1高度同源的蛋白,二者蛋白結(jié)構(gòu)域類(lèi)似,均含有兩個(gè)RNA識(shí)別結(jié)構(gòu)域(RNA recognition motif,RRM),我們將其命名為HLP2。表型分析發(fā)現(xiàn),hlp2-1突變體中FLC的表達(dá)量上升,呈現(xiàn)晚花表型,暗示著HLP2對(duì)植物開(kāi)花時(shí)間的調(diào)控依賴(lài)于FLC。此外,我們沒(méi)有鑒定到hlp1-1與hlp2-1的雙突變體。進(jìn)一步研究表明,hlp1hlp2雙突變體胚胎致死,暗示了HLP1與HLP2在植物生長(zhǎng)發(fā)育過(guò)程中的重要作用。亞細(xì)胞定位與核質(zhì)分離實(shí)驗(yàn)顯示HLP1與HLP2均為細(xì)胞核定位蛋白,GUS染色分析表明HLP1與HLP2表達(dá)模式一致,暗示著二者存在功能上的相關(guān)性。由于HLP1參與調(diào)控APA,以At3G15450和At3G23030為例驗(yàn)證,發(fā)現(xiàn)HLP2的缺失突變導(dǎo)致與HLP1缺失突變類(lèi)似的poly(A)位點(diǎn)的轉(zhuǎn)換,暗示著HLP1與HLP2均參與了對(duì)poly(A)位點(diǎn)的選擇調(diào)控,并且二者在APA調(diào)控方面具有協(xié)同性。進(jìn)一步研究發(fā)現(xiàn),HLP1與HLP2均可以結(jié)合到FCA轉(zhuǎn)錄本的第三個(gè)內(nèi)含子和3′-UTR區(qū)域,協(xié)同促進(jìn)FCA轉(zhuǎn)錄本遠(yuǎn)端poly(A)位點(diǎn)的選擇,促進(jìn)植物開(kāi)花。生化分析顯示,HLP1與HLP2存在相互作用,暗示著植物體內(nèi)二者以異源二聚體的形式行使功能。有意思的是,HLP1可以通過(guò)調(diào)控HLP2的APA負(fù)調(diào)控HLP2的基因表達(dá)。HLP2前體mRNA具有APA現(xiàn)象,正常情況下,HLP1可以結(jié)合到HLP2的前體mRNA上,抑制選擇遠(yuǎn)端poly(A)位點(diǎn),即抑制功能型HLP2轉(zhuǎn)錄本產(chǎn)生,負(fù)調(diào)控HLP2的基因表達(dá)。綜上所述,該研究工作對(duì)擬南芥中兩個(gè)未知RNA結(jié)合蛋白HLP1與HLP2的功能研究發(fā)現(xiàn)HLP1與HLP2可以協(xié)同作用,通過(guò)直接結(jié)合到一些RNA分子上,調(diào)控靶RNA的APA,最終調(diào)控植物開(kāi)花。該工作為后續(xù)開(kāi)展RBP介導(dǎo)的RNA加工事件在植物發(fā)育調(diào)控中的作用機(jī)制提供了新的視野。
[Abstract]:As the transformation from vegetative growth to reproductive growth, flowering determines whether plants can successfully propagate their offspring to affect the continuity of their own population, but also an important agronomic character that can affect the yield and quality of crops. The flowering time of plants is regulated by complex and fine fines, involving environmental factors, such as temperature and Light, and the interaction of the plant's own intrinsic factors to the identification and transmission of the flowering signal. This process includes a series of gene expression regulation to ensure that the plant performs this biological process in the most appropriate state. Recent studies have shown that many of the RNA transcripts are involved in addition to the four classics of the flowering regulation pathway. The post processed RNA binding protein (RNA binding protein, RBP) or RNA processing factors participate in plant flowering time regulation.RNA binding proteins widely exist in eukaryotes, participating in the regulation of the various levels of RNA processing and metabolism, including 5 '- end caps, variable splicing, RNA editing, 3' - terminal polyadenylation, nuclear shuttle, RNA stability, etc. There are about 200 RNA binding proteins encoded in Arabidopsis thaliana genome. More and more studies have shown that RBP plays an important role in the process of flowering time conversion, adversity response and chromatin modification. The polyadenylation of the precursor mRNA 3 'terminal is acidified in eukaryotic genes. It plays an important role in expression and regulation, which includes two closely linked biochemical processes: first, the polyadenylation site (poly (A) sites, PAS) of the precursor mRNA is cut, and the selection of poly (A) tail.Poly (A) loci is changeable, which is called selective polyadenylation of adenosine (Alternative polyadenylatio). N, APA), precursor mRNA produces a transcriptional transcript with different coding sequences or different 3 '-UTR regions through APA, which increases the complexity of the transcriptional group. Large-scale studies show that APA is a regulatory mechanism for gene expression widely existed among multiple species. Most genes contain more than one polyadenosine acidification site, in which yeast, lactating More than 70% of the genes in the plant and Arabidopsis have APA.APA mediated gene expression in the process of controlling body development, disease, plant flowering and environmental response..APA is regulated by complex and fine regulation, involving the interaction between many proteins and nucleic acids. However, the specific modulation of APA in plants The control mechanism is not clear. In the early stage of screening plant flowering related mutants, the deletion mutation of At5G40490 was found to lead to late flowers. The gene encodes a RNA binding protein. Phylogenetic tree analysis found that the protein was highly homologous to the Hrp1 in human hnRNP A1 and yeast, and named it HLP1. to analyze HLP1. By using the technique of UV cross-linking immunoprecipitation and large-scale sequencing, the identification of target RNA. bioinformatics combined with HLP1 showed that HLP1 had a wide binding site at the whole genome level. RIP-qRT-PCR effectively demonstrated the true reality of the combination of HLP1 and target RNA. Further bioinformatics analysis showed that HLP1 The binding sequence is significantly enriched in the 3 '-UTR region and the intron, and has two distinct sequence characteristics. One is 5' -AGAAAA-3 'of adenine rich, and the other is 5' -UUUUCU-3 'rich in uracil. They are distributed near the transcriptional terminating site. Gene function enrichment analysis shows that the HLP1 binding gene has blooming over Cheng Ming The sequence characteristics associated with the explicit correlation.HLP1 are highly similar to the poly (A) site signals, suggesting that HLP1 may participate in the regulation of.Poly (A) site sequencing of APA (poly (A) sites sequencing, PAS-seq) shows that many genes are transformed by the deletion mutation. RNA, suggesting that HLP1 can be directly involved in APA regulation, in which HLP1 can combine the 3 '-UTR region of the flowering promoting factor FCA and third introns, promote the FCA selection of the distal poly (A) site, produce a functional transcript, inhibit the expression of FLC, and promote the flowering of the plants. The protein domain is similar to the two protein domain, which all contain two RNA identification domains (RNA recognition motif, RRM). We named it to HLP2. phenotypic analysis. The expression of FLC in the hlp2-1 mutant is rising, showing the late flower phenotype, suggesting that HLP2's regulation of flowering time of the plant depends on FLC. besides FLC.. We have not identified hlp1-1 and HLP. 2-1 of the double mutants. Further study showed that hlp1hlp2 double mutant embryos died, suggesting the important role of HLP1 and HLP2 in plant growth and development. Subcellular localization and nuclear qualitative separation experiments showed that both HLP1 and HLP2 were nuclear localizing proteins, and GUS staining analysis showed that HLP1 and HLP2 expression patterns were consistent, suggesting the existence of functions of the two. As HLP1 participates in the regulation of APA, as a result of At3G15450 and At3G23030, it is found that the deletion mutation of HLP2 leads to the transformation of the poly (A) site similar to the HLP1 deletion mutation, suggesting that both HLP1 and HLP2 are involved in the selection and regulation of poly (A) sites, and the two are synergistic in the regulation. The third introns and 3 '-UTR regions of the FCA transcript can be combined with HLP2 to promote the selection of the distal poly (A) loci of the FCA transcript and promote the flowering of plants. Biochemical analysis shows that there is interaction between HLP1 and HLP2, suggesting that two of the plants in the plant exercise their function in the form of a heterogenous two polymer. The gene expression of.HLP2 precursor mRNA of HLP2 controlled by the control of HLP2 has APA phenomenon. Under normal circumstances, HLP1 can bind to the precursor mRNA of HLP2, inhibit the selection of the distal poly (A) site, that is, the inhibition of functional HLP2 transcript production and negative regulation of gene expression. In summary, the research works on two unknown binding proteins in Arabidopsis thaliana. The functional study of P1 and HLP2 found that HLP1 and HLP2 can play a synergistic role in regulating the APA of target RNA by direct binding to some RNA molecules and ultimately regulating the flowering of plants. This work provides a new field of vision for the subsequent development of RBP mediated RNA processing events in the regulation of plant development.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2

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