拟南芥核膜孔蛋白Nup160基因的突变体表型分析

植物开花受环境条件的影响,其分子调控机制在转录、翻译和表观遗传等层次已有较广泛而深入的研究;核膜孔复合物是真核细胞中细胞质与细胞核之间物质交换的重要通道。筛选到拟南芥核膜孔蛋白Nup（Nucleoporin） 160基因的3个突变体nup160-1/-2/-3,均表现出发育的多种表型,最显著的表型为早花,而且是不依赖于环境条件（光周期和温度）的早花。但是,突变体在长日下的开花时间依然早于短日条件下的开花时间,暗示突变体仍然对光周期有反应。结果表明,在拟南芥中Nup160作为一个植物开花的负调控因子,主要在温敏途径和光周期途径中参与拟南芥开花时间的调节。     

利用EMS筛选豆科模式植物百脉根的根瘤突变体及突变部位

豆科植物百脉根是研究共生固氮体系的模式植物之一,被根瘤菌感染后形成根瘤,根瘤菌固定氮素变换成铵态氮,植物利用铵态氮进行营养生长,而根瘤菌利用植物供给的碳酸同化产物为能源。为获得根瘤的成熟、维持的基因,本研究利用化学诱变试剂EMS处理百脉根MG-20得到多种突变体,筛选根瘤成熟、维持有异常的突变植株,利用SSR标记和dCAPS标记进行基因定位。结果表明:约10万M2百脉根植株中获得nup85突变体2株,nup133突变体4株,pollux突变体6株,ccamk、symrk、castor、nin、nfr1、nfr5突变体各1株等不结瘤突变体(Nod~-)18株;结无效根瘤突变体(Fix~-)6株并确定了突变体的突变部位。     

核孔蛋白Atnup98-like降低拟南芥对细菌和干旱的抗性

用HrpNEa处理拟南芥(Arabidopsis)时发现核孔蛋白AtlG59660基因的表达被抑制.因该基因编码的蛋白质与哺乳动物中的核孔蛋白(nucleoporin98)具有相似性质,具有FG(phenylalanine-glycine)重复基序,故命名为Atnup98-like.同时对野生型和Atnup98-like突变体植株分别进行病原细菌Psyringae syringae pv.tomato DC3000接种及干旱处理,结果发现突变体植株对DC3000的抗性明显增强,而且突变体中抗病相关基因PR1a的表达明显强于野生型;同时,突变体植株比野生型植株抗旱性增强,脯氨酸含量明显升高,且干旱诱导的效应基因RD29B的表达水平强于野生型.上述结果表明,核孔蛋白基因Atnup98-like的突变增强了拟南芥对DC3000和干旱的抗性,因此,其可能是拟南芥抗性反应中一个重要的负向调节因子.     

转录中介体复合物的研究进展

转录中介体复合物(Mediator complex)广泛存在于真核生物中,在进化上高度保守,是一个具有模块化组织的大复合物,参与转录因子与RNA聚合酶Ⅱ之间的信息传递。中介体复合物的主要功能是通过其特定亚基与不同信号通路激发的转录因子相互作用,调节下游基因的表达。此外,中介体复合物还与各种辅因子相互作用,参与转录延伸、mRNA输出及选择性剪切等过程,从而调控细胞的各种生理功能。中介体复合物与疾病的密切联系使得将中介体作为靶标研究疾病的治疗方法成为可能。以中介体复合物的结构为基础,重点介绍了中介体复合物在基因表达调控中的分子机制及功能,以期为后续研究提供参考。     

拟南芥非特异性磷脂酶C2的磷脂酰胆碱特异性磷脂酶C活性研究

在拟南芥中有6个非特异性磷脂酶C(NPC1~NPC6)。GUS染色显示NPC2在根中主要是在根尖分生区和伸长区表达,在成熟区主要是在维管组织中表达;在叶中,NPC2主要在发育的叶片中表达,随着叶片成熟进程,NPC2基因的表达量逐渐降低。npc2-1突变体的PC-PLC活性比野生型降低47.7%,互补的植物能不同程度地恢复突变体的PC-PLC酶活性。这说明NPC2具有PC-PLC酶活性。NPC2基因在侧根形成位点的表达虽然与IAA诱导的侧根形成紧密相关,但外源IAA处理野生型和突变体之间的侧根密度、主根长度和侧根总长无明显区别。     

稻瘟病菌分泌复合物的生物信息学分析及MoSec15定位研究

利用稻瘟病菌分泌途径相关的调控因子分泌复合物进行生物信息学分析及相关基因定位初探．经同源比对获得稻瘟病菌中分泌复合物8个亚基的同源蛋白,通过在线工具分析蛋白结构域、构建系统进化树、预测亚细胞定位;利用公共数据库获得其在稻瘟病菌不同组织和发育阶段的表达情况;此外,亚细胞定位分析表明,稻瘟病菌分泌复合物亚基MoSec15可能定位于菌丝体的内膜结构中．     

雷竹PvJ3基因克隆及功能分析

拟南芥Arabidopsis thaliana中J3基因参与花期的调节,调控开花时间。为了探究竹类植物中J3基因的功能,根据植物中J同源基因的高度保守性,采用同源克隆技术从雷竹Phyllostachys vilascens中克隆出1个J3基因,命名为PvJ3。该基因的开放阅读框（ORF）区长为1 260 bp,编码419个氨基酸。通过同源比对和系统进化树分析,可知雷竹PvJ3蛋白和其他物种中J3同源蛋白的氨基酸序列同源性很高。通过实时定量聚合酶链式反应（qRT-PCR）,发现PvJ3基因在同一竹鞭的笋、花芽、花,开花与不开花雷竹的茎秆、成叶、幼叶中都有表达,且开花雷竹茎秆中表达量最高,表明PvJ3基因在雷竹各组织部位都有表达。亚细胞定位分析结果显示,PvJ3蛋白定位在细胞质和细胞核。通过转化拟南芥对其功能进行了初步分析,表型统计结果显示,PvJ3转基因拟南芥与野生型相比,开花时间明显提早,而且出现多主茎现象,表明PvJ3可以促使植物提前开花并且参与植物茎秆的发育。     

水稻晚开花突变体lft1的鉴定与基因克隆

[目的]通过对水稻晚开花突变体lft1(late flowering time)的鉴定及基因定位,解析水稻响应光周期调控开花的分子机制。[方法]从‘日本晴’T-DNA插入突变体库中筛选得到一个在长日照下晚开花的突变体lft1。调查了lft1/日本晴F_2分离群体的开花期表型,分析晚开花表型与T-DNA插入之间的相关性。比较日本晴/9311 F_2群体和lft1/9311 F_2群体的开花期分布,选取群体中具有突变效应的极端晚开花单株进行基因定位。比较突变体lft1和野生型‘日本晴’中已知开花期基因的表达水平,分析基因在调控网络中的作用。[结果]相比于野生型‘日本晴’,突变体lft1在长日照条件下晚开花20 d,粒长和千粒质量均显著增加。对lft1/日本晴F_2群体调查发现,晚开花性状由1对隐性基因控制,且与T-DNA插入呈现非共分离。相比于对照日本晴/9311 F_2群体,在lft1/9311 F_2群体中出现了极端晚开花的个体,从中选取了58株极端个体用于基因的初定位。利用‘日本晴’和‘9311’之间的多态性分子标记对极端晚开花个体进行了图位克隆,将基因限定在第9染色体短臂上,分子标记RM219与RM3912之间,物理距离为2.94 Mb的范围内。在开发标记的过程中,发现分子标记SJ9-32能够在‘日本晴’和‘9311’中实现扩增,但是在lft1及其后代的极端个体中均不能有效扩增。通过进一步设计标记,我们最终验证了在lft1中存在DNA片段的缺失,该缺失区段覆盖了SDG724基因。等位性测验进一步证实lft1的晚开花表型是由于SDG724基因的缺失而引起的。q RT-PCR结果表明,SDG724可以调节Os MADS51和RFT1的表达水平。[结论]突变体lft1中由于SDG724基因的缺失,导致Os MADS51和RFT1的表达水平下调,从而引起晚开花性状。通过调控SDG724基因的表达水平来调节水稻开花期,这在生产上将具有重要意义。     

捕光色素蛋白复合物的研究进展

高等植物体内的2种光合系统PSⅠ和PSⅡ,分别由各自独立的核心复合物和捕光色素蛋白复合物组成。对PSⅠ和PSⅡ的各组分主要捕光色素蛋白复合物的结构特点和功能研究新进展进行了综述。     

拟南芥18srRNA参与隐花色素介导的信号传导途径的研究

通过筛选以拟南芥蓝光受体隐花色素双突变体cry1cry2为遗传背景的激活标签突变体库,得到一株SCC98D株系,该突变体具有早开花,下胚轴变短,并具有花瓣数目增加等表型,彻底恢复了cry1cry2的晚开花和长下胚轴表型.通过TailPCR的方法克隆得到SCC98D突变体中激活标签插入位点的侧翼序列,通过对该侧翼序列测序发现插入位点在18srRNA的1751bp处,运用RTPCR方法分析插入位点周围基因的表达,发现只有18srRNA的表达被激活,由此证实了18srRNA参与隐花色素介导的信号传导途径.     

闽西地区蝴蝶兰高山催花技术研究

探讨海拔高度、上山处理时间、光照强度、施肥配比等因素对蝴蝶兰花芽分化的影响。试验结果表明:海拔高度直接影响蝴蝶兰的高山催花效果,上山催花时期的选择是蝴蝶兰高山催花技术的关键,充足的光照和增施磷钾肥有利于蝴蝶兰的花芽分化和发育。在闽西地区,苗龄16个月左右、叶冠幅达(30±2)cm、长势良好的健壮蝴蝶兰的高山催花适宜条件是在8月中下旬,将蝴蝶兰置于海拔1 100～1 200 m的高山,光照强度为20 000～25 000 lx,施用N∶P2O5∶K2O为9∶45∶15的1 000倍花肥,高山低温处理45～50 d,最有利于蝴蝶兰花芽的分化和发育,高山催花效果最好,花芽萌发率达96%以上,且整齐度高,花梗长度可达10 cm。     

The pseudo-response regulator Ppd-H1 provides adaptation to photoperiod in barley

Plants commonly use photoperiod (day length) to control the timing of flowering during the year, and variation in photoperiod response has been selected in many crops to provide adaptation to different environments and farming practices. Positional cloning identified Ppd-H1, the major determinant of barley photoperiod response, as a pseudo-response regulator, a class of genes involved in circadian clock function. Reduced photoperiod responsiveness of the ppd-H1 mutant, which is highly advantageous in spring-sown varieties, is explained by altered circadian expression of the photoperiod pathway gene CONSTANS and reduced expression of its downstream target, FT, a key regulator of flowering.     

Nuclear pores form de novo from both sides of the nuclear envelope

Nuclear pore complexes are multiprotein channels that span the double lipid bilayer of the nuclear envelope. How new pores are inserted into the intact nuclear envelope of proliferating and differentiating eukaryotic cells is unknown. We found that the Nup107-160 complex was incorporated into assembly sites in the nuclear envelope from both the nucleoplasmic and the cytoplasmic sides. Nuclear pore insertion required the generation of Ran guanosine triphosphate in the nuclear and cytoplasmic compartments. Newly formed nuclear pore complexes did not contain structural components of preexisting pores, suggesting that they can form de novo.     

Ordered and dynamic assembly of single spliceosomes

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.     

Unmanned aerial vehicle canopy reflectance data detects potassium deficiency and green peach aphid susceptibility in canola

There is growing evidence that potassium deficiency in crop plants increases their susceptibility to herbivorous arthropods. The ability to remotely detect potassium deficiency in plants would be advantageous in targeting arthropod sampling and spatially optimizing potassium fertilizer to reduce yield loss due to the arthropod infestations. Four potassium fertilizer regimes were established in field plots of canola, with soil and plant nutrient concentrations tested on three occasions: 69 (seedling), 96 (stem elongation), and 113 (early flowering) days after sowing (DAS). On these dates, unmanned aerial vehicle (UAV) multi-spectral images of each plot were acquired at 15 and 120 m above ground achieving spatial (pixel) resolutions of 8.1 and 65 mm, respectively. At 69 and 96 DAS, field plants were transported to a laboratory with controlled lighting and imaged with a 240-band (390–890 nm) hyperspectral camera. At 113 DAS, all plots had become naturally infested with green peach aphids (Hemiptera: Aphididae), and intensive aphid counts were conducted. Potassium deficiency caused significant: (1) increase in concentrations of nitrogen in youngest mature leaves, (2) increase in green peach aphid density, (3) decrease in vegetation cover, (4) decrease in normalized difference vegetation indices (NDVI) and decrease in canola seed yield. UAV imagery with 65 mm spatial resolution showed higher classification accuracy (72–100 %) than airborne imagery with 8 mm resolution (69–94 %), and bench top hyperspectral imagery acquired from field plants in laboratory conditions (78–88 %). When non-leaf pixels were removed from the UAV data, classification accuracies increased for 8 mm and 65 mm resolution images acquired 96 and 113 DAS. The study supports findings that UAV-acquired imagery has potential to identify regions containing nutrient deficiency and likely increased arthropod performance.     

利用CRISPR/Cas9技术研究玉米ZmFKF1在开花过程中的作用

【目的】FKF1是多种植物开花途径中发挥重要作用的关键基因。为研究玉米FKF1功能,利用CRISPR/Cas9基因编辑技术将ZmFKF1进行定点编辑,获得ZmFKF1编辑突变体。同时以此为材料,通过表型分析及关键开花基因的表达量变化分析,明确ZmFKF1在玉米开花途径中的作用,为玉米分子育种及遗传改良提供理论依据。【方法】以玉米B104为材料,克隆ZmFKF1,通过序列比对明确ZmFKF1的基因结构。以ZmFKF1为靶标基因,根据CRISPR/Cas9技术原理设计靶点,将设计的靶点序列在玉米参考基因组中进行比对分析,排除非特异性靶位点,最终筛选出在ZmFKF1第1外显子上的靶位点ZmFKF1-T1,构建CRISPR/Cas9基因编辑表达载体。利用农杆菌介导法,转化玉米B104幼胚,通过抗性筛选获得抗性愈伤组织,之后诱导出芽和生根,获得T₀代ZmFKF1编辑阳性植株,并利用cas9特异引物进行验证。利用靶位点扩增测序法,明确T₁代ZmFKF1编辑植株在ZmFKF1预期靶标位点是否发生突变及突变的类型,筛选获得ZmFKF1定点突变纯合株系。获得这些材料后,以野生型B104为对照,统计和分析开花表型。同时,利用实时荧光定量PCR技术检测上述材料中与玉米开花途径相关的关键基因的表达量变化,对表型进行进一步的验证。【结果】在玉米FKF1的第1外显子上设计靶点构建基因编辑表达载体,通过遗传转化获得的转基因株系实现了对ZmFKF1的定点突变,共获得T₀代ZmFKF1编辑阳性株系18株,在预期靶标位点上发生突变的有6株,2种不同的突变类型：单碱基插入和多碱基缺失。通过开花时间统计和分析,与野生型B104相较,3个T₂代ZmFKF1编辑纯合突变体的开花时间延迟,显著（P＜0.05）晚于B104。进一步对这些材料中的开花关键基因ZmGI、conz1和ZmZCN8进行表达量检测,发现突变体中这些基因的表达明显（P＜0.05）低于野生型B104,与晚花表型相符。【结论】可利用CRISPR-Cas9技术对ZmFKF1进行定点编辑获得基因编辑突变体,且突变体开花时间明显延迟。     

Photoreceptor regulation of CONSTANS protein in photoperiodic flowering

Many plants flower in response to seasonal fluctuations in day length. The CONSTANS (CO) gene of Arabidopsis promotes flowering in long days. Flowering is induced when CO messenger RNA expression coincides with the exposure of plants to light. However, how this promotes CO activity is unknown. We show that light stabilizes nuclear CO protein in the evening, whereas in the morning or in darkness the protein is degraded by the proteasome. Photoreceptors regulate CO stability and act antagonistically to generate daily rhythms in CO abundance. This layer of regulation refines the circadian rhythm in CO messenger RNA and is central to the mechanism by which day length controls flowering.