拟南芥ABA不敏感型突变体及其ABA结合特性

拟南芥的脱落醚（ABA）不敏感型突变体abi2,在对ABA的敏感性、气孔开度及种子休眠方面,与野生型有明显差异。通过3H－ABA与野生型对的亚细胞组分的结合分析,表明38000×g组分特异结合活性最高,结合最适温度为20℃,最适保温时间：20℃时为70min;0℃时为90min。由饱和曲线的Scatchard分析表明：abi2存在一种ABA结合位点,野生型有两种ABA结合位点。对3H－ABA结合的38000×g组分的SDS－PAGE电泳分析表明：野生型有3个结合活性峰,而abi2只有1个结合活性峰。     

拟南芥光周期开花突变体的筛选和基因的鉴定

以双子叶模式植物拟南芥（Arabidopsis thaliana）突变体crylcry2为实验材料,用舍有激活标记质粒DSK1015的农杆菌浸花进行转化,构建了拟南芥T-DNA插入突变体库．通过筛选和观察分析,获得了一些开花时间比crylcry2明显延迟或明显提早的突变体．采用IPCR（inverse PCR）和TAIL-PCR（thermal asymmetric interlaced PCR）等方法,鉴定了这些突变体T-DNA插入位点的基因组旁邻序列,并采用半定量RT-PCR对插入位点两侧基因的mRNA水平进行了分析,初步鉴定了与开花相关的候选基因,为进一步研究其功能,深入研究隐花素调节光周期开花的作用机制奠定了基础．     

激活标签法构建拟南芥突变体库及其表型分析

以拟南芥(ArabMopsis thaliana)野生生态型(Columbia)植株为实验材料,以含有激活标记双元质粒pCB260的农杆菌进行转化,并以抗除草剂Basta为筛选标记,构建了拟南芥激活标签突变体库,所用pCB260双元质粒含有两个Ds位点、一个GFP标记基因与一个抗basta标记基因,可以方便高效地筛选转基因植物.目前经初步筛选获得了约10 000个独立转化株系(T1代),其中约50个株系具有明显的表型变化,包括花期改变、株型变异、叶形特异、育性降低、花发育异常、种子颜色变浅等.运用TAIL-PCR技术,成功获得了其中10个表型特异株系的T-DNA侧翼序列,分别分布于拟南芥基因组的5条染色体上.     

拟南芥网状突变体E-210基因精细定位

通过甲基磺酸乙酯(EMS)诱变与遗传分析,从拟南芥(Arabidopsis thaliana)中筛选到一株隐性单基因控制的网状突变体E-210.该突变体植株生长缓慢,叶脉呈绿色,叶肉呈黄色.通过透射电镜观察,发现野生型植株和突变植株在叶绿体结构上差异不大,猜测该突变体E-210基因与叶绿体的发育可能没有直接关系,而很可能同叶绿素或叶绿体的生物合成有关.通过图位克隆的方法,将该突变体的突变基因定位在第5条染色体上的MRBl7和MBG8-5的分子标记之间,精确到87.130 kb.对MRB17和MBG8-5的分子标记之间的22个基因进行了分析,预测突变体E-210基因可能是At5g54770,编码THI1,即噻唑合成酶.     

拟南芥叶边缘锯齿状突变体的分离与鉴定

叶的极性建立直接决定叶的平展性发育,极性改变导致叶形态异常,影响植物体的各种正常生理活动。利用反向遗传学方法,从拟南芥基因激活标签突变体库中分离到一个叶片边缘锯齿状表型的突变体（命名为pCB1294）,该突变体同时表现出叶表皮腺毛形态发育异常。通过TailPCR方法成功定位突变基因为At5g41663,该基因编码miR319b基因。Real time PCR显示,pCB1294突变体植株中miR319b基因的表达量是野生型（col）植株的11倍多。所得结果为进一步研究miRNA调控叶极性的分子机制和进一步分析miR319b与叶形态发生的关系奠定了基础。     

拟南芥雄性不育突变体fne的遗传与定位分析

在T-DNA插入突变体Salk_118481株系的群体中,筛选到一株雄性不育突变体,用T-DNA序列上的一对引物进行PCR鉴定表明其基因组中没有T DNA插入。通过背景纯化与遗传分析发现该雄性不育突变体是由单个隐性基因控制的,引起不育的主要原因是在花药发育的第13~14期,花丝不能伸长以完成授粉,故该突变体命名为fne （filament no elongation）。利用图位克隆的方法对FNE基因进行了定位,结果表明FNE基因位于第五条染色体上分子标记MBD2和MMG4之间的97kb区间内。目前该区间内尚未见到控制花丝伸长基因的报道,因此,FNE基因是一个控制花丝伸长的新基因。     

拟南芥雄性不育突变体ms1142的遗传定位与功能分析

经EMS诱变野生型拟南芥（Arabidopsis thaliana）群体筛选得到一株雄性不育突变体ms1142,突变体的果荚短小,不含种子。细胞学观察和扫描电镜结果表明,突变体花药发育过程中,花药中小孢子外壁异常、破裂,最后没有花粉形成。遗传分析表明,该突变体为隐性单核基因突变所致;利用图位克隆的方法将MS1142基因定位于第1条染色体的BAC克隆F16P17上44kb区间内,目前尚未见该区间内有雄性不育基因的报道。以上结果结合生物信息学分析表明,MS1142是一个新的调控花药发育的关键基因。该工作为花药发育关键基因MS1142的克隆及功能分析奠定了基础。     

Isolation and Characterization of a Mutant of Arabidopsis thaliana Resistant to alpha-Methyltryptophan

Mutants of Arabidopsis thaliana have been selected for resistance to growth inhibition at the seedling stage by α-methyltryptophan (aMT). One mutant, amt-1 has been characterized in detail. The appearance and growth rate of the mutant in the absence of the inhibitor are similar to wild type, both as plants and callus. However, mutant plant growth is unaffected by 25 micromolar aMT and mutant callus growth by 50 micromolar aMT, concentrations that completely inhibit the growth of wild-type plants and callus, respectively. Tryptophan levels in mutant and wild-type plants are 24.3 ± 2.7 and 4.7 ± 1.2 micrograms per gram fresh weight, respectively, and in the corresponding callus 64.0 ± 2.6 and 31.8 ± 8.4 micrograms per gram fresh weight, respectively. Anthranilate synthase (AS) activity levels in crude extracts from whole plants are 3.09 ± 0.54 nanomoles per milligram protein per hour in amt-1 and 1.32 ± 0.21 nanomoles per milligram protein per hour in wild-type plants. In crude extracts from callus, anthranilate synthase levels are 11.54 ± 2.05 nanomoles per milligram protein per hour and 7.74 ± 1.58 in amt-1 and wild type, respectively. Enzyme extracts are inhibited by l-tryptophan; the concentrations required for 50% inhibition (I₅₀) are 3.9 and 1.9 micromolar for amt-1 and for wild type, respectively. The mutation segregates as a single nuclear allele and shows incomplete dominance. The concomitant increases in both AS activity and its I₅₀ for tryptophan suggest that the mutation amt-1 either resides in one of the AS structural genes or causes increased expression of an AS isoform with an I₅₀ greater than the average for the entire extract.     

拟南芥叶绿体分裂突变体pdl37的基因鉴定与分析

pd137是经甲基磺酸乙脂（ethyl methane sulphonate, EMS）诱变并通过筛选得到的一个拟南芥叶绿体分裂突变体。该突变体的叶绿体表型与野生型相比有很大差异： 叶绿体面积显著增大, 细胞中叶绿体数量明显减少。遗传分析显示pd137的突变表型受隐性单基因控制。本研究通过遗传作图将该突变基因粗定位于拟南芥2号染色体的分子标记CH2-13.70和CH2-16.0区间内。该区间内已知的与叶绿体分裂相关的基因只有FtsZ2-1。对FtsZ2-1基因的测序结果显示pd137突变体的FtsZ2-1基因第505位碱基发生了无义突变, 使蛋白质翻译提前终止。该突变还严重影响了FtsZ2-1基因的mRNA水平。转基因互补实验进一步验证了该突变体表型是由于FtsZ2-1基因突变引起。本项工作为研究叶绿体分裂的机制提供了新材料和一些有用的线索。     

拟南芥叶绿体分裂突变体cpd111的基因定位与分析

通过EMS(ethyl methane sulphonate)诱变从拟南芥(Arabidopsis thaliana)突变体库中筛选到一个叶绿体分裂突变体(c)hloro(p)last (d)ivision 111 (cpd111).遗传学分析表明,该突变体的表型是单基因控制的隐性性状.与野生型相比,突变体植物细胞的叶绿体数量少,叶绿体形态和大小多样化,并且细胞体积与叶绿体数量之间无相关性.利用图位克隆的方法确定cpd111的突变基因为FtsZ1.进一步的分析表明,该突变影响FtsZ7基因mRNA的正常剪切和稳定性,使蛋白质翻译提前终止,最终导致叶绿体分裂异常.该工作为研究FtsZ1在叶绿体分裂中的作用提供了新的材料和线索.     

拟南芥黄化突变体chlm-4的基因定位与分析

叶绿素是光合作用必需的重要色素,其合成需要Mg-原卟啉Ⅸ甲基转移酶等一系列酶的催化。我们通过筛选拟南芥EMS（乙基磺酸甲酯,ethyl-methane sulphonate）突变体库,分离到一个黄化突变体。该突变体叶绿素含量显著减少,叶绿体内垛叠的基粒缺失。遗传学分析表明,该突变体的黄化表型是由单基因控制的隐性性状。利用图位克隆的方法最终将基因定位在第IV条染色体分子标记F13M23和T30C3之间114kb的区间内,其中包含编码Mg-原卟啉Ⅸ甲基转移酶的CHLM基因。通过测序及等位分析表明该突变体是chlm的等位突变体,命名为chlm-4。在chlm-4中,CHLM蛋白的Gly59突变成Glu59,说明Gly59对于Mg-原卟啉Ⅸ甲基转移酶功能的行使是必需的。     

拟南芥雄性不育突变体ms1502的遗传及定位分析

通过EMS诱变、背景纯化与遗传分析,从拟南芥（Arabidopsis thaliana）中筛选到了一棵隐性单基因控制的雄性不育突变体ms1502。细胞学观察发现,突变体在小孢子从四分体释放出后花药绒毡层过早衰亡,小孢子的内容物不正常地凝聚,最终无法形成正常的花粉粒。利用图位克隆的方法对该基因MSl502进行了定位,结果表明MS1502位于第4条染色体上分子标记F25124和T12H20之间105kb区间内。目前该区间内尚未见到花药发育必需基因（不育基因）的报道,因此MS1502是一个控制花粉发育的新基因。     

Molecular Cloning and Characterization of a Calmodulin Gene from Arabidopsis thaliana

The calcium binding protein, calmodulin Is involved in regulating various cellular and biochemical processes. A gene tor calmodulin (CaM) has been Isolated from a genomic library of Arabidopsis thaliana constructed in ; EMBL-4 using a heterologous cDNA probe from electric eel. One of the positive clones was characterized and the region containing the calmodulin gene sequences was Identified, excised using appropriate restriction enzymes and subcloned Into a plasmid vector. The genomic clone contains a complete copy of the calmodulin gene. A comparison of the nucleotide sequence of the part of the clone with those of the other plant and animal systems confirms that the clone In fact contains the calmodulin gene sequences. Southern hybridization ulling the calmodulin gene sequences as a probe reveals the presence of more than one copy of the calmodulin gene. The results of this investigation taken together with those Iff the other. indicate that the calmodulin gene belongs to a small mutigene family consisting of atieast four member. In the Arabidopsis genome.     

Discovery and Characterization of an Arabidopsis thaliana N-Acylphosphatidylethanolamine Synthase

N-Acylethanolamines (NAEs) are lipids involved in several physiological processes in animal and plant cells. In brain, NAEs are ligands of endocannabinoid receptors, which modulate various signaling pathways. In plant, NAEs regulate seed germination and root development, and they are involved in plant defense against pathogen attack. This signaling activity is started by an enzyme called N-acylphosphatidylethanolamine (NAPE) synthase. This catalyzes the N-acylation of phosphatidylethanolamine to form NAPE, which is most likely hydrolyzed by phospholipase D β/γ isoforms to generate NAE. This compound is further catabolized by fatty amide hydrolase. The genes encoding the enzymes involved in NAE metabolism are well characterized except for the NAPE synthase gene(s). By heterologous expression in Escherichia coli and overexpression in plants, we characterized an acyltransferase from Arabidopsis thaliana (At1g78690p) catalyzing the synthesis of lipids identified as NAPEs (two-dimensional TLC, phospholipase D hydrolysis assay, and electrospray ionization-tandem mass spectrometry analyses). The ability of free fatty acid and acyl-CoA to be used as acyl donor was compared in vitro with E. coli membranes and purified enzyme (obtained by immobilized metal ion affinity chromatography). In both cases, NAPE was synthesized only in the presence of acyl-CoA. β-Glucuronidase promoter experiments revealed a strong expression in roots and young tissues of plants. Using yellow fluorescent protein fusion, we showed that the NAPE synthase is located in the plasmalemma of plant cells.N-Acylethanolamines (NAEs)² are bioactive lipids composed of an ethanolamine headgroup amide-linked to an acyl chain varying in length and degree of saturation. In animals, NAEs are involved in different physiological processes, such as neuroprotective action (1), embryo development (2), cell proliferation (3), apoptosis (4), nociception, anxiety, inflammation, appetite/anorexia, learning, and memory (for review, see Ref. 5). Most studies carried out with animal cells/tissues have focused on N-arachidonoylethanolamine (anandamide, NAE20:4), which is synthesized in brain neurons but also, under certain conditions, in macrophage cells (6). NAE20:4 binds CB1 cannabinoid receptors located in brain neurons (7) and also acts as ligand of vanilloid receptors for pain modulation (8). In addition, it has been shown that NAE20:4 also promotes food intake, whereas NAE18:0 and NAE18:1 exert anorexic effects by increasing satiety (9–11). NAE16:0 is accumulated during inflammation and has several anti-inflammatory effects (for a review, see Ref. 12).In plants, NAEs are thought to be involved in various physiological functions. For example, because NAE levels observed in various dry seeds decline rapidly after imbibition, a possible role of these compounds in the regulation of seed germination has been proposed (13). It was further observed that the addition of 25 μm NAE12:0 to growth medium of Arabidopsis thaliana leads to a decrease in the size of the main and lateral roots and in root hair formation. This reduction in growth was associated with a modification of cytoskeletal organization (14). NAE12:0 is also able to delay cut Dianthus caryophyllus (carnation) senescence by decreasing oxidative damage and enhancing antioxidant defense (15), whereas NAE14:0 inhibits the elicitor-induced medium alkalinization and activates phenylalanine ammonia lyase gene expression involved in plant defense against pathogen attack (16).Both in plant and animal cells (for a review, see Ref. 17), NAEs are formed by the hydrolysis (by PLDs) of N-acylphosphatidylethanolamine (NAPE). NAPE is an unusual derivative of phosphatidylethanolamine (PE) with a third fatty acid linked to the amine position of the ethanolamine headgroup. In animals, the formation of NAEs is catalyzed by a PLD with a high specificity toward NAPE (NAPE-PLD). In plants, PLDβ and PLDγ isoforms, but not PLDα, hydrolyzed NAPE into NAE in vitro, and this is thought to operate in response to several biotic and abiotic stresses. Both in animals and in plants, NAEs signaling is terminated by the action of fatty acid amide hydrolases, which hydrolyze NAEs to free fatty acid and ethanolamine. FAAH has been identified and characterized in mammals and plants (for a review, see Ref. 17). In Arabidopsis, FAAH has been shown to modulate NAE content. Moreover, lines overexpressing FAAH displayed enhanced seedling growth as well as increased cell size (18) and were also more susceptible to bacterial pathogens (19).Although the role of NAEs and their catabolism have been extensively investigated, little is known about their precursors, the NAPEs. NAPEs represent a minor phospholipid class but are present in all tissues of plants and animals. The principal function of NAPEs is to serve as a precursor for the production of lipid mediator NAEs, but it has also been suggested that NAPEs could serve as a membrane stabilizer to maintain cellular compartmentalization during tissue damage (20). More recently, N-palmitoyl-PE was proposed to act as an inhibitor of macrophage phagocytosis through inhibition of the activation of Rac1 and Cdc42 (21).In the animal and plant kingdoms, therefore, the signaling events mediated by NAEs appear to be involved in many physiological processes that have been extensively studied. The genes encoding the enzymes involved in the synthesis (from NAPEs) and the degradation of NAEs have been cloned and characterized. By contrast, little is known about the physiological roles of NAPEs or about the first step of this lipid signaling pathway, namely the N-acylation of PE to form NAPEs. In animals, the synthesis of NAPEs is catalyzed by an N-acyltransferase, where the O-linked acyl unit from a phospholipid donor is transferred to the ethanolamine headgroup of PE (22). Recently, a rat LRAT-like protein 1 or RLP1 was shown to display such an activity, but according to the authors, RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca²⁺-dependent N-acyltransferase (23). However, a different situation is observed in plants. NAPE synthase activity was shown to directly acylate PE with free fatty acids (24, 25), but a gene encoding a NAPE synthase activity remained unidentified until now. The present work shows that the A. thaliana acyltransferase At1g78690p catalyzes the synthesis of NAPEs from PE and acyl-CoAs in vitro as well as in vivo when this enzyme is expressed in E. coli and overexpressed in plants.     

Characterization of an acyl-CoA synthetase from Arabidopsis thaliana

One of the major goals of modern plant biotechnology is to manipulate lipid metabolism in oilseed crops to produce new and improved edible and industrial vegetable oils. Lipids constitute the structural components of cellular membranes and act as sources of energy for the germinating seed and are therefore essential to plant cell function. Both de novo synthesis and modification of existing lipids are dependent on the activity of acyl-CoA synthetases (ACSs). To date, ACSs have been recalcitrant to traditional methods of purification due to their association with membranes. In our laboratory, several isoforms of ACSs have been identified in Arabidopsis thaliana. Reverse genetics allowed us to identify a mutant containing a transfer DNA-interrupted ACS gene. Results will be presented that describe the isolation and characterization of this mutant. The elucidation of the specific roles of ACSs will lead to a greater understanding of plant lipid metabolism.     

拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制。最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制。通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf l,budl)突变体。突变体植株的表型包括顶端优势丧失、株型矮小,表明budl突变体存在生长素代谢、运输或信号传导的缺陷。一个对生长素特异反应的启动子驱动的报告基因在budl中表达模式改变。生长素敏感性和运输能力的测定表明这两个过程在budl中均正常。以上结果显示budl表型是生长素代谢缺陷的结果。遗传分析表明BUDI为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离。