植物GT元件和GT因子的研究进展

GT元件是位于植物基因启动子区域中的串联重复DNA序列, 是启动子中富含T和A的顺式作用元件。迄今已经在不同植物中鉴定了一些GT元件并分析了其功能。GT因子是以三螺旋基序为DNA结合域, 与GT元件特异结合的一类转录因子。到目前为止, 仅在植物中发现有GT因子。GT因子与GT元件相互作用调节相关基因的转录, 进而提高植物的抗性或影响植物形态建成。文章就植物中不同的GT元件及GT因子的发现、结构及二者相互作用等方面进行了综述。     

CYP72B1基因和AUR3基因响应光、生长素和油菜素内酯的转录调控机制研究

为研究光、生长素和油菜素内酯在基因层次上的互作机制,开发了转录调控元件识别工具OCMMat,其中,在对共表达基因信息和直系同源基因信息进行整合时,利用了转录调控元件在直系同源基因启动子中的富集性.利用该方法发现,CYP7281基因和AUR3基因启动子含有3个相同的调控模序GAGACA、AAGAAAAA、ATCATG,它们分别承担了AuxRE元件、GT元件和GT辅助元件的功能.其中,ATCATG模序是目前尚未报道过的调控元件,与AAGAAAAA模序的距离相对恒定.基于调控元件识别结果,构建了CYP7281基因和AUR3基因响应光、生长素和油菜素内酯的转录调控模型,模型显示:光信号和生长素、油菜素内酯信号在CYP72B1基因和AUR3基因的转录调控元件上相互交叠,而生长素和油菜素内酯信号则在转录因子ARF水平上相交.     

杜氏盐藻DCA1启动子内GT重复序列在盐诱导调控中的作用

为了研究杜氏盐藻双拷贝碳酸酐酶（DCA1）启动子中高度重复的GT序列在盐诱导表达时的调控作用,设计不同的引物,通过PCR法获得6条不同长度的DCA1启动子片段,分别与gus报告基因融合后构建6个表达载体;电击法转化杜氏盐藻细胞。组织化学染色和荧光定量法检测GUS在不同盐浓度下的瞬时表达。结果显示,DCA1启动子内高度重复的GT序列无论与其上游、下游或上下游片段同时结合均能驱动gus基因的表达,并且其表达受氯化钠浓度调控,其中和上下游均结合时活性最强;无GT重复序列的融合片段及GT 重复的下游片段也能驱动gus基因的表达,但其表达不受氯化钠浓度调控;而GT重复的上游片段不能驱动gus基因的表达。结果提示:盐藻DCA1启动子中高度重复的GT序列在盐诱导调控中起重要作用,可能为一种新型的盐诱导元件。     

原生动物基因组转座元件的研究进展

转座元件是一类广泛分布于真核生物的可移动的遗传因子, 可以引起基因重组和变异, 在物种进化及遗传改良中起着重要作用。针对近年来原生动物全基因组序列中大量发现的转座元件, 文章着重比较了转座元件在锥虫、利什曼虫、微孢子虫、变形虫和滴虫基因组序列中的存在种类、分布特征及其功能意义。原生动物转座元件以LINE 和SINE为主, 其次是DNA转座元件和LTR反转座元件, 部分转座元件在高A+T含量区富集, 预示着转座元件与基因组序列A+T含量有着紧密联系。根据不同种微孢子虫基因组之间转座元件的差异, 推测在微孢子虫基因组进化过程中, 至少经历了一次转座元件的丢失事件。最后对转座元件在原生动物寄生虫的进一步研究和应用作了展望。     

海巴戟ACO基因启动子的克隆及功能初步验证

为了进一步明确海巴戟(Morinda citrifolia Linn.)ACO基因(GenBank登录号:ARB05681.1)功能,本研究通过染色体步移法克隆得到2 066 bp启动子.该区域除了含有大量TATA-box、CAAT-box等启动子核心元件外,还含有乙烯等植物激素响应元件、胁迫和防御相关元件、光相关元件等,转录起始位点预测在-96 bp处.为了验证启动子序列中的植物激素响应元件,分别用乙烯利、茉莉酸甲酯、赤霉素、生长素、脱落酸分别处理海巴戟叶片,荧光定量PCR结果 表明,5种激素均能上调ACO基因表达,与启动子中存在相应的元件一致,暗示着这些元件的存在能够调控海巴戟ACO基因的表达;此外,ACO受乙烯诱导表达最强,表明启动子中确实存在乙烯应答元件.     

西瓜果实AGPL1基因启动子的序列分析

利用PCR技术从西瓜嫩叶中扩增出1.2 kb AGPL1基因启动子,通过PlantCARE和PLACE数据库对启动子序列进行生物信息学分析。结果表明,AGPL1启动子具有多个典型的TATA-Box和CAAT-Box等基本元件,以及高水平转录顺式作用元件5UTR Py-rich stretch,光响应元件ACE、ATCC-motif、Box 4、I-Box、Sp1、TCCC-motif、GAG-motif、MNF1,2个赤霉素响应元件GARE-motif、P-box,参与蛋白代谢调节的顺式作用元件O2-site,参与水杨酸反应的顺式作用元件TCA-element,厌氧诱导所需的顺式作用元件ARE、逆境胁迫响应元件TC-rich repeats、生理昼夜节律控制元件circadian等,为研究外源基因在果实中的特异表达提供参考。     

真核蛋白编码基因核心启动子元件

核心启动子（core promoter,CP）是位于转录起始位点附近由100个左右核苷酸所构成的功能区域,负责与转录因子结合同其它顺式作用元件如增强子、沉默子一起对转录进行调控。目前所发现的真核蛋白编码基因核心启动子元件包括：TATA盒、上游启动子元件BRE、起始子Initiator（Inr）、下游启动子元件DPE以及新发现的位于DPE附近的十基序元件MTE。上述核心启动子元件普遍存在于真核蛋白编码基因之中,对真核生物的生长、发育、适应环境起着重要作用。     

水稻胁迫相关基因OsPM1启动子的克隆与分析

依据NCBI数据库OsPM1的序列信息,采用PCR技术扩增获取OsPM1的2 100bp的启动子序列。利用PLACE预测启动子的顺式作用元件分析表明,启动子内含有大量与胁迫相关的顺式作用元件,主要有ABA响应相关元件、脱水响应元件、低温响应元件、热激响应元件和转录因子结合元件。构建OsPM1的启动子和GUS基因融合表达载体,转入拟南芥。组织化学染色分析结果显示,非生物胁迫处理前,幼苗中GUS基因表达水平很低;干旱、低温、高盐等胁迫处理后,GUS基因表达量显著升高。研究表明,OsPM1的启动子能够显著提高在干旱、高盐和低温处理后下游基因的表达水平。     

生物元件库国内外研究进展

生物元件库是对元件数据和实物进行收集、整理和共享的重要平台,对合成生物学的研究和应用具有非常重要的支撑作用。本文对国内外生物元件库的研究进展进行了介绍和综述。国外先后出现了标准生物元件登记库等多个元件库,通过制定OpenMTA协议实现了元件实物的免费分发共享,并通过制定“合成生物学开放语言”（SBOL）实现了多个元件库之间的数据交换;通过bionet项目对元件数据和实物的去中心化管理作了进一步尝试。近年来我们与国内多家科研单位合作建设了合成生物学元件与数据库（RDBSB,https：//www.biosino.org/rdbsb/or https：//www.biosino.org/npbiosys/）。在建设过程中,我们首先建立了与SBOL语言兼容的《催化元件数据标准》、多种重要元件的标准功能测试方法以及安全高效的元件提交系统,并在此基础上完成了元件数量最多且信息最为全面的催化元件数据库的构建。目前,收集了30多万个催化元件,包括7万多个具有文献支持功能表征信息的催化元件,并保藏了1万个以上的实物元件和5000个底盘菌株,通过网站实现了数据和实物的公开与共享。上述元件库已经对国内合成生物学的研究和应用产生了积极地推动作用,访问量超过100万次/年,提供元件和底盘共享服务大于100次/年,并为多家企业和研究单位提供了元件和底盘的定制服务。虽然我们的元件库建设取得了巨大的进步,但是仍需在调控元件的收集整理和共享机制创新等方面取得更多的突破,在数据有源、多层审核、资源共享、信息公开、信息安全和授权访问的基础上,加速生物元件数据、实物和设计工具的汇聚,并进一步服务合成生物学研究。     

恒河猴基因组SINEs和Alu元件的数量和分布

哺乳动物的基因组中富含各种类型的重复序列,其中的微卫星作为分子标记在相关研究中得到了广泛的应用。而重复序列中的SINEs元件,在各类群物种的分子系统发育、遗传多样性等方面的研究也得到了使用。其中,灵长类物种特有的SINEs元件、Alu元件,也在灵长类物种的进化研究中得到了成功的使用。本研究对于重要的医学模式动物恒河猴的基因组中SINEs和Alu元件进行了搜索,并进一步统计分析其分布规律、长度等信息。在恒河猴基因组的20条常染色体上共发现了Alu元件1 093 185个,在性染色体X上发现了45 215个。长度为200 bp至300 bp区间的Alu元件分布最多;Alu元件中75%的分化值至少都为10,而只有6.2%左右的元件分化值能达到至少20,这一结果表明绝大部分的Alu元件都比较年轻。本研究的统计结果为后续应用SINEs和Alu元件作为分子标记的研究提供了重要的信息。     

Combinatorial interplay of promoter elements constitutes the minimal determinants for light and developmental control of gene expression in Arabidopsis.

Higher plants are able to integrate environmental and endogenous signals to regulate gene expression for optimal development. To define the minimal sequence requirement sufficient to integrate light and developmental signals in controlling promoter activity, we carried out a systematic analysis of the roles of four well-conserved 'light-responsive elements (LREs)' common to many nuclear-encoded photosynthetic genes. A gain-of-function assay using basal promoter-reporter fusions in stable transgenic Arabidopsis was employed to demonstrate that pairwise combinations of the LREs, but not the individual elements alone, can confer light-inducible expression to the reporter gene independently of the basal promoter context and the light-triggered morphological changes. The activity of the synthetic promoters with the paired LREs can be modulated at least by the phytochrome system. Further, those synthetic light-regulated promoters confer a photosynthetic cell-specific expression pattern and respond to the chloroplast development state. Our data suggest that distinct combinatorial interactions of LREs can serve as minimal autonomous promoter determinants which integrate light and developmental signals and modulate promoter activity.     

Drosha mediates destabilization of Lin28 mRNA targets

Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.     

Drosha mediates destabilization of Lin28 mRNA targets

Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.     

An RNA-binding Protein,Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates

Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.