油菜素甾醇信号通路中BKI1互作蛋白ERD7的筛选与鉴定

油菜素甾醇(Brassinosteroid,BR)是一类重要的调控植物生长发育的激素,BKI1在BR信号转导通路中具有正负调控的双重功能.为了深入研究BK11正/负调控BR信号通路的分子机制,本研究使用BKI1作为诱饵蛋白对拟南芥cDNA文库进行酵母双杂交筛选,获得了一段新的与BR信号通路组分BKI1相互作用的肽段,BLAST搜索显示该肽段与拟南芥ERD7的N端序列一致.生物信息学分析表明ERD7是一个植物特异表达基因,在单子叶、双子叶、以及低等植物中保持很高的一致性.酵母双杂交方法证实全长ERD7与BKI1能够相互作用,Pull-down分析证实这两个基因在植物体内直接相互作用,烟草细胞瞬时表达显示ERD7和BKI1能够共定位于细胞质和细胞膜.BKI1和ERD7相互作用的发现为进一步研究BKI1的调控机制奠定了基础.     

油菜素甾醇信号通路中BKI1互作蛋白Hsp70的筛选与鉴定

植物激素油菜素甾醇(Brassinosteroids,BRs)在植物生长发育过程中起着重要作用.BKI1和14-3-3蛋白都具有正负调控BR信号通路的双重功能.为了进一步研究BKI1调控BR信号通路的分子机制,本研究用BKI1的过表达植株35S∶∶BKI1-FLAG进行液相色谱-串联质谱法(LC-MS/MS)分析BKI1复合体组分,发现BKI1复合体中含有分子伴侣Hsp70.BKI1与Hsp70共定位在细胞膜和细胞质上.FoldIndex工具预测发现BKI1存在内在无序区域(Intrinsically Disordered Regions,IDR),表明在细胞中BKI1可能需要Hsp70帮助其正确折叠.半体内pull-down和双分子荧光互补实验(BiFC)表明BKI1可以与Hsp70在植物体内互作.同时,截短实验结果表明BKI1与Hsp70的底物结合结构域相互作用,是Hsp70的底物.     

抑制bri1表型的ABI突变体的筛选

油菜素甾醇(BR)是一类重要的调控植物生长发育的激素.脱落酸(ABA)是一类重要的帮助植物适应逆境的激素.之前的研究表明BR和ABA信号对萌发率的调控存在拮抗作用,BR合成或信号缺失的突变体在萌发率上对ABA更加敏感.为了进一步研究BR与ABA信号通路互作的分子机制,采用化学诱变剂甲基磺酸乙酯(EMS)诱变bri1-301(BR受体BRI1的一个弱突变体),0.75μmol/L ABA的培养基上筛选对ABA不敏感的突变体.经过多次连续世代筛选,获得了大约40个稳定的株系,发现其中8个株系与abi1、abi5 ABA不敏感突变体的萌发率相似.通过利用PCR扩增ABI1、ABI2、ABI3、ABI4和ABI5基因并测序,鉴定到bri1-301背景下的4个ABA不敏感突变体abi2-8、hab2-1、abi3-21和abi4-13,表明ABA与BR信号的互作可以不依赖BR受体调控种子的萌发,为ABA与BR信号互作提供了遗传的证据.     

植物富亮氨酸重复类受体蛋白激酶的研究进展

介绍了植物富亮氨酸重复类受体蛋白激酶(LRR-RLK)的结构与分类;分别以CLAVATA1,BRI1,FLS2为例,阐述了LRR-RLK基因在植物生长发育、激素信号转导和植物抗病防御等方面的生理功能及作用机制;总结了LRR-RLK作用模型、信号通路间的交互作用及展望.     

甘蓝型油菜BnBRI1基因的克隆及表达分析

以拟南芥油菜素内酯受体BRI1基因序列设计同源简并引物,利用同源克隆技术,克隆了甘蓝型油菜中编码油菜素内酯受体基因全长cDNA序列,命名为BnBRI1(GenBank:JX871217),该基因开放阅读框(ORF)大小为3591bp,与拟南芥中BRI1基因(GenBank:NM_120100)相似性为94.21%,编码1196个氨基酸,与拟南芥中BRI编码的氨基酸相似性为89.23%.采用实时定量PCR分析表明,BnBRI1基因在根、茎、叶中都有表达,但表达量存在显著差异.在两叶一心期和花期的根中表达量最高,四叶一心期和抽薹期的叶中表达量最高.     

油菜素甾族化合物对植物干旱适应性的影响及调控机理

总结了油菜素甾族化合物对植物渗透系统、保水性、抗氧化系统及生长和发育的生理效应的最新进展,并概括了这些现象的生理和分子调控机制.综述了BR调节植物干旱适应性的基因应答路径,这些路径与其他主要干旱胁迫激素(如脱落酸)之间存在复杂的"信号交互"效应,共同对植物生长发育起到系统调控作用.该方面的创新研究将是BR在生产应用上取得突破的理论基础.     

ISDN BRI及 NT1+在远程抄表系统中的应用

介绍了N-ISDN NT1^ 在一种自主研制的远程抄表系统中的应用．着重阐述了抄表主机通信卡的软、硬件结构和充分利用ISDN BRI接口NT1^ 功能的设计特点．提出的实现远程抄表通信与采集器初始化数据设置单机一体化完成的综合解决方案，可大大降低抄表中心的设备投入费用．     

油菜素内酯对仙人掌愈伤组织诱导的影响

研究了不同浓度的油菜素内酯(Brassinolide,BR)对仙人掌外植物体愈伤组织诱导的影响.结果表明,BR促进仙人掌嫩芽、茎、幼叶等外植体愈伤组织的诱导和芽增殖.最适培养基为MS BR 0.2 mg·L-1.这对仙人掌组织培养快繁技术的改进和对同属其他植物的快速繁殖具有积极意义.     

免疫共沉淀与质谱联用检测拟南芥SIK1/Hippo互作蛋白

Hippo通路是迄今为止发现的最重要的负调控动物器官大小的信号通路之一,其核心由两对级联的激酶-脚手架蛋白复合物构成.本实验室在前期工作中首次发现了拟南芥Hippo(Hpo)的同功能基因SIK1,并建立了与Hpo-Mats对应的SIK1-MOB1模块,发现其通过促进退出细胞分裂与细胞延展,参与植物侧生器官大小的调控.为进一步探究SIK1调控器官大小的分子机制,构建了SIK1-GFP转基因株系,用GFP-Trap免疫共沉淀方法捕获与SIK1内源互作的蛋白,并进行质谱分析,获得了SIK1的候选互作蛋白.研究结果将为后续建立完整的植物Hippo信号通路打下基础.     

利用嫁接技术研究植物固醇的长距离运输

植物固醇是真核生物细胞膜系统的重要组成成分,在植物生长发育过程中起了重要的作用.植物固醇是植物重要的代谢产物之一,并且近年来的研究表明植物固醇可能作为一种信号分子,调控植物的生长发育.信号分子和代谢产物往往能进行长距离运输,但是目前关于植物固醇能否进行长距离运输尚未有报道.本研究以植物固醇合成途径关键基因CPI1的突变体cpi1-1为研究材料,利用嫁接技术探究植物固醇长距离运输问题.研究结果发现植物固醇不能在根和地上部之间进行长距离运输.     

Structural insight into brassinosteroid perception by BRI1

Brassinosteroids are essential phytohormones that have crucial roles in plant growth and development. Perception of brassinosteroids requires an active complex of BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE 1 (BAK1). Recognized by the extracellular leucine-rich repeat (LRR) domain of BRI1, brassinosteroids induce a phosphorylation-mediated cascade to regulate gene expression. Here we present the crystal structures of BRI1(LRR) in free and brassinolide-bound forms. BRI1(LRR) exists as a monomer in crystals and solution independent of brassinolide. It comprises a helical solenoid structure that accommodates a separate insertion domain at its concave surface. Sandwiched between them, brassinolide binds to a hydrophobicity-dominating surface groove on BRI1(LRR). Brassinolide recognition by BRI1(LRR) is through an induced-fit mechanism involving stabilization of two interdomain loops that creates a pronounced non-polar surface groove for the hormone binding. Together, our results define the molecular mechanisms by which BRI1 recognizes brassinosteroids and provide insight into brassinosteroid-induced BRI1 activation.     

Structural basis of steroid hormone perception by the receptor kinase BRI1

Polyhydroxylated steroids are regulators of body shape and size in higher organisms. In metazoans, intracellular receptors recognize these molecules. Plants, however, perceive steroids at membranes, using the membrane-integral receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Here we report the structure of the Arabidopsis thaliana BRI1 ligand-binding domain, determined by X-ray diffraction at 2.5?? resolution. We find a superhelix of 25 twisted leucine-rich repeats (LRRs), an architecture that is strikingly different from the assembly of LRRs in animal Toll-like receptors. A 70-amino-acid island domain between LRRs 21 and 22 folds back into the interior of the superhelix to create a surface pocket for binding the plant hormone brassinolide. Known loss- and gain-of-function mutations map closely to the hormone-binding site. We propose that steroid binding to BRI1 generates a docking platform for a co-receptor that is required for receptor activation. Our findings provide insight into the activation mechanism of this highly expanded family of plant receptors that have essential roles in hormone, developmental and innate immunity signalling.     

BRI1 is a critical component of a plasma-membrane receptor for plant steroids

Most multicellular organisms use steroids as signalling molecules for physiological and developmental regulation. Two different modes of steroid action have been described in animal systems: the well-studied gene regulation response mediated by nuclear receptors, and the rapid non-genomic responses mediated by proposed membrane-bound receptors. Plant genomes do not seem to encode members of the nuclear receptor superfamily. However, a transmembrane receptor kinase, brassinosteroid-insensitive1 (BRI1), has been implicated in brassinosteroid responses. Here we show that BRI1 functions as a receptor of brassinolide, the most active brassinosteroid. The number of brassinolide-binding sites and the degree of response to brassinolide depend on the level of BRI1 protein. The brassinolide-binding activity co-immunoprecipitates with BRI1, and requires a functional BRI1 extracellular domain. Moreover, treatment of Arabidopsis seedlings with brassinolide induces autophosphorylation of BRI1, which, together with our binding studies, shows that BRI1 is a receptor kinase that transduces steroid signals across the plasma membrane.     

A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence

Plants sense potential microbial invaders by using pattern-recognition receptors to recognize pathogen-associated molecular patterns (PAMPs). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref. 2) and elongation factor Tu receptor (EFR) (ref. 3) act as pattern-recognition receptors for the bacterial PAMPs flagellin and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucine-rich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellin-triggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.     

Brassinosteroid signal transduction: An emerging picture

Brassinosteroids (BRs) are plant steroid hormones that have similar structures and functions with Brassino-lide (BL). BR signaling is complicated, and the input of BR signal is under fine control. The active BR level is regulated at the levels of BR biosynthesis and metabolic inactivation[1,2]. BR biosynthetic pathway consists of a metabolic grid, and the expression of several genes for BR biosynthesis is feedback regulated by BR signaling[3,4]. The sequential events of BR action inclu…     

Combretastatin B-1的化学合成及结构衍生化研究

近来,Combretastatin B-1（CB1）由于在抗肿瘤活性尤其在靶向恶性肿瘤血管的选择性方面引起了医学界广泛关注.该化合物提取自药用植物Combretum caffrum,其抑制肿瘤细胞生长,抑制血管生成和微管蛋白聚合方面的应用前景激发了人们对CB1化学合成的极大兴趣.CB1及其衍生物是一类具有广泛生物活性和良好应用前景的化合物,因此CB1的化学合成及结构衍生化研究显得极为重要.     

拟南芥RING finger结构域ABRv1基因的功能初步研究

拟南芥ABscisic acid responsive RING-v protein 1基因（ABRv1）编码一个含有RING finger结构域的E3连接酶。为了分析ABRv1的基因功能,我们首先构建了高表达载体pBI121- ABRv1,经农杆菌转化,花序浸染后得到T0代转基因种子,并经卡那霉素板筛选得到转基因苗,再经过两代的筛选获取了纯合的高表达株系ABRv1。通过DNA及RNA水平鉴定了该基因的T –DNA插入突变体abrv1。对突变体、高表达转基因株系及野生型进行ABA诱导处理,检测其萌发率、气孔关闭情况进行,发现ABA处理后突变体的萌发率降低,过表达株系萌发率升高;突变体株系气孔几乎未关闭,过表达株系气孔关闭非常明显。以上结果表明ABRv1基因在拟南芥的ABA信号应答响应中可能起负调控作用,为深入分析该基因的功能打下基础。