Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae

Fusions of the glycolytic genes TPI1, PGK1, ENO1, PYK1, PDC1, and ADH1 with the lacZ reporter gene of Escherichia coli and a lacZ fusion construct of a 390-bp fragment from the promoter of the HXT7 gene were assayed for β-galactosidase activity. The glycolytic promoters were induced after addition of glucose to ethanol-grown cells, whereas the HXT7 promoter fragment showed a constitutive β-galactosidase expression on both carbon sources. The genes coding for the seven enzymes of lower glycolysis Tdh, Pgk, Gpm, Eno, Pyk, Pdc, and Adh were simultaneously put under the control of the same strong promoter, a truncated HXT7 promoter that is constitutively active on ethanol as well as on glucose medium. Genomic expression of the glycolytic genes under the control of this promoter, resulted in an at least 2-fold overexpression. The gene MSG5 was isolated, coding for a protein phosphatase normally involved in cell cycle regulation, as a factor that possibly influences the expression of the HXT7 gene. However, overexpression of MSG5 had no effect on the expression of the HXT7/lacZ fusion, whereas a deletion of this gene resulted in a decreased expression of β-galactosidase.     

异源过表达水稻OsSAPP3基因促进拟南芥叶片衰老

蛋白磷酸酶催化的蛋白质可逆磷酸化反应是叶片衰老的关键环节。该研究筛选并克隆了1个新的参与水稻(Oryza sativa)叶片衰老调控的PP2C基因OsSAPP3。研究表明, OsSAPP3的启动子在Pro_OsSAPP3-GUS转基因拟南芥(Arabidopsis thaliana)的莲座叶中有活性, 并且活性以依赖叶龄方式增加。利用CaMV 35S启动子驱动组成型异源过表达OsSAPP3导致转基因拟南芥无法正常生长。用可诱导型启动子GVG系统驱动OsSAPP3异源过表达导致转基因拟南芥出现莲座叶变小、数量增加、叶片早衰及抽薹开花提前等早衰表型。外源诱导OsSAPP3基因异源过表达后, 利用实时荧光定量PCR检测到SAG12、WRKY6和NAC2等衰老标志基因显著上调表达。研究结果表明, OsSAPP3是参与水稻叶片衰老的正向调控因子。     

AtCPL1调控拟南芥开花的机制

RNA聚合酶II CTD磷酸化酶1 (CPL1)作为影响RNA聚合酶II磷酸化水平的重要因子, 在植物逆境响应、离子吸收以及成花诱导等生命过程中扮演重要角色。为深入探究CPL1参与植物开花时间调控的作用机制, 以拟南芥(Arabidopsis thaliana) AtCPL1突变体cpl1-3和fry2-1为研究材料, 观察了长日照条件下突变体与野生型的开花时间, 并利用荧光定量PCR技术对突变体中开花相关基因的表达情况进行了检测。结果表明: 在长日照条件下, 突变体cpl1-3和fry2-1抽薹时的莲座叶数目均显著多于野生型, 且表现出明显的开花时间延迟现象; 荧光定量PCR分析显示, 突变体cpl1-3和fry2-1中开花抑制因子miR156a、TOEs和SMZ基因的表达量较野生型显著升高, 而开花促进因子FT、FD、CO和miR172基因的表达量则显著降低。由此推测, AtCPL1通过调控miR156和miR172的表达水平进而影响下游开花相关基因TOEs、SMZ、FT、FD及CO等的表达, 从而实现对拟南芥开花时间的调控。     

拟南芥金属蛋白酶FtSH4通过生长素与活性氧调控叶片衰老

植物金属蛋白酶Ft SH基因家族在拟南芥(Arabidopsis thaliana)中有12个成员,目前各基因的功能还不清楚。该文利用细胞生物学和遗传学方法初步分析了拟南芥FtSH4在叶片衰老中的功能。ftsh4-4突变体叶片中H_2O_2含量及细胞死亡率增加,叶绿素含量降低;此外,突变体中过氧化物酶基因表达上调,过氧化物酶活性增加,出现早衰表型。外源抗氧化剂As A、内源和外源生长素能够通过降低ftsh4-4体内H_2O_2含量、过氧化物酶基因的表达及过氧化物酶活性,恢复ftsh4-4叶片的衰老表型。ftsh4-4突变体中生长素响应因子基因ARF2和ARF7上调表达,外源生长素和抗氧化剂能够降低ARF2和ARF7的表达,并且ARF2突变能够降低ftsh4-4的H_2O_2含量并恢复其早衰表型。以上结果表明,FtSH4基因通过生长素与活性氧在调控植物叶片衰老中起重要作用。     

Two Pax2/5/8-binding sites in Engrailed2 are required for proper initiation of endogenous mid-hindbrain expression

During early brain development mouse Engrailed2 (En2) is expressed in a broad band across most of the mid-hindbrain region. Evidence from gene expression data, promoter analysis in transgenic mice and mutant phenotype analysis in mice and zebrafish has suggested that Pax2, 5 and 8 play a critical role in regulating En2 mid-hindbrain expression. Previously, we identified two Pax2/5/8-binding sites in a 1.0 kb En2 enhancer fragment that is sufficient to directed reporter gene expression to the early mid-hindbrain region and showed that the two Pax2/5/8-binding sites are essential for the mid-hindbrain expression in transgenic mice. In the present study we have examined the functional requirements of these two Pax2/5/8-binding sites in the context of the endogenous En2 gene for directing mid-hindbrain expression. The two Pax2/5/8-binding sites were deleted from the En2 locus and replaced with the bacterial neo gene by homologous recombination in mouse embryonic stem cells. After transmitting the mutation into mice, the neo gene was removed by breeding with transgenic mice expressing cre from a CMV promoter. Embryos homozygous for this En2 Pax2/5/8-binding site deletion mutation had a mild reduction in En2 expression in the presumptive mid-hindbrain region at the 5-7 somite stage, when En2 expression is normally initiated. However, from embryonic day 9.0 onwards, the mutant embryos showed En2 expression indistinguishable from that seen in wild type embryos. Furthermore, the mutants did not show the cerebellar defect seen in mice with a null mutation in En2. This result demonstrates that the two Pax2/5/8-binding sites that were deleted, while being required for mid-hindbrain expression in the context of a 1.0 kb En2 enhancer, are only required for proper initiation of expression of the endogenous En2 gene. Interestingly, a comparison of the lacZ RNA and protein expression patterns directed by the 1.0 kb enhancer fragment revealed that lacZ protein was acting as a lineage marker in the mid-hindbrain region by persisting longer than the mRNA. The transgene expression directed by the 1.0 kb enhancer fragment therefore does not mimic the entire broad domain of En2 expression. Taken together, these two studies demonstrate that DNA binding sites in addition to the two Pax2/5/8-binding sites must be necessary for En2 mid-hindbrain expression.     

sly-miR399在番茄抗晚疫病中的初步探究*

背景: 番茄(Solanum lycopersicum)是广泛种植的最具价值的果蔬之一,番茄晚疫病会导致其产量和品质降低。microRNAs(miRNAs)是一类内源性非编码RNA,广泛参与基因转录后调控。已有研究表明miR399家族可参与调控植物抗病过程。目的:探究番茄miR399(sly-miR399)对番茄抗晚疫病的影响。方法:构建sly-miR399的过表达和沉默载体,利用农杆菌介导在番茄叶片中瞬时表达;实时荧光定量PCR(qRT-PCR)技术检测相关基因表达水平;台盼蓝染色并统计分析瞬时过表达和瞬时沉默sly-miR399的番茄叶片的病斑情况。结果:瞬时过表达sly-miR399导致其靶基因UBC24的表达量下降了75%,病程相关蛋白(pathogenesis related proteins,PRs)SlPR1、SlPR2、SlPR3和SlPR5的表达量分别上升了3.6倍、2.2倍、2.3倍和6.4倍,茉莉酸(JA)信号通路相关基因SlJA1、SlLOX1和SlLOX2的表达量分别上升了1.3倍、2.5倍和1.5倍,JA转录抑制因子SlJAZ1的表达量下降了50%,接种晚疫病菌后,叶片上相对病斑面积明显减小;瞬时沉默导致其靶基因的表达量上升了1.8倍,PRs基因的表达量分别下降了65%、82%、52%和80%,SlJA1、SlLOX1和SlLOX2的表达量分别下降了84%、50%和65%,SlJAZ1的表达量上升了1.8倍,病菌接种后叶片上相对病斑面积明显变大。结论:初步揭示了sly-miR399在番茄抗晚疫病过程中的正调控因子作用。     

陆地棉GST基因家族全基因组分析

谷胱甘肽转移酶(glutathione-S-transferase, GST)是一种普遍存在的具有多功能的超家族蛋白,在植物初次生代谢、逆境胁迫、胞间信号传递等方面具有重要作用;同时,作为配体其在植物激素代谢以及物质转运方面也发挥作用。为了解析陆地棉(Gossypium hirsutum L.) GST基因家族的信息,本研究对该基因家族成员的种类、进化关系、物理定位、基因结构和保守基序以及表达模式进行了分析。结果显示,在陆地棉全基因组中共含有70个GST基因,进化树和基因结构分析将该家族分为U族、F族、T族、Z族、EF1Bγ族和TCHQD族。基因定位分析发现,除了AD/At2、AD/At4、AD/At5、AD/Dt5、AD/Dt10号染色体上没有GST基因外,其他染色体上都有GST基因,并且在AD/At9、AD/Dt7、AD/Dt12、AD/Dt13这4条染色体上出现基因簇。对F族(Phi类) 9个GST基因进行荧光定量分析,结果表明,除GhGSTF1可能为假基因外,GhGSTF2~9等8个基因在陆地棉根、茎、叶以及各个发育时期的纤维中均有表达;结合生物信息学分析,推测GhGSTF8可能参与原花青素/花青素的转运和积累;GhGSTF4、GhGSTF6和GhGSTF9可能在调节陆地棉的生长和胁迫反应中起作用,而GhGSTF2、GhGSTF3、GhGSTF5和GhGSTF7的功能还有待进一步研究。本研究为陆地棉GST基因家族的分子进化及功能研究提供了理论依据。     

萱草叶片响应低温胁迫的转录组分析

低温胁迫是萱草（Hemerocallis fulva）生长过程中经常会遭遇的一种非生物胁迫。比较了萱草叶片在低温处理（10、5、0 ℃）下转录组与对照（15 ℃）数据的差异,共筛选出差异表达基因2 457个,其中上调基因1 253个,下调基因1 204个。差异表达基因主要富集在细胞过程、代谢过程和催化活性等49个GO过程,代谢途径、次生代谢产物的生物合成、植物激素信号转导等42条KEGG代谢途径中。其中参与植物激素信号转导通路的差异表达基因发生了不同程度的变化,GH3.10基因上调至对照组的13.624倍,IAA1基因下调0.120倍;参与可溶性糖合成通路的差异基因发生了0.076~28.114倍不同程度的变化。随后对3个低温处理组共有的29个差异表达基因进行热图和网络调控分析,基于基因在网络调控中的位置,对ABCF5、OFPs和SWEETs等基因在冷应答的作用进行了分析。本研究结果为进一步挖掘萱草低温响应的关键基因及耐寒萱草种质开发、分子育种提供了一定的理论支撑。     

Establishment of a tissue-specific RNAi system in C. elegans

In C. elegans, mosaic analysis is a powerful genetic tool for determining in which tissue or specific cells a gene of interest is required. For traditional mosaic analysis, a loss-of-function mutant and a genomic fragment that can rescue the mutant phenotype are required. Here we establish an easy and rapid mosaic system using RNAi (RNA mediated interference), using a rde-1 mutant that is resistant to RNAi. Tissue-specific expression of the wild type rde-1 cDNA in rde-1 mutants limits RNAi sensitivity to a specific tissue. We established hypodermal-and muscle-specific RNAi systems by expressing rde-1 cDNA under the control of the lin-26 and hlh-1 promoters, respectively. We confirmed tissue-specific RNAi using two assays: (1) tissue-specific knockdown of GFP expression, and (2) phenocopy of mutations in essential genes that were previously known to function in a tissue-specific manner. We also applied this system to an essential gene, ajm-1, expressed in hypodermis and gut, and show that lethality in ajm-1 mutants is due to loss of expression in hypodermal cells. Although we demonstrate tissue-specific RNAi in hypodermis and muscle, this method could be easily applied to other tissues.     

决明GRAS基因家族全基因组鉴定及其在盐和干旱胁迫条件下的表达分析

目的: 基于决明(Senna tora L.)全基因组数据,对GRAS家族成员、理化性质、基因结构、进化关系以及胁迫条件下的表达模式进行鉴定和分析。方法: 将决明基因组蛋白数据与拟南芥GRAS成员进行比对,分别利用TBtools、MEGA-X、CLUSTALW、MEME等生物信息学软件和工具,对决明GRAS基因家族成员进行分析。利用qRT-PCR(quantitative real-time PCR)检测干旱和盐胁迫条件下决明根中GRAS基因的表达情况。结果: 50个StGRAS分为9个亚家族,不均等地分布在13条染色体上。结构分析表明,StGRAS34和StGRAS12分别与蒺藜苜蓿(Medicago truncatula)结瘤信号蛋白NSP1和NSP2高度同源。StGRAS的启动子区域多含有与胁迫响应、激素调节等相关的响应元件。qRT-PCR结果表明,在盐胁迫条件下,StGRAS表达具有明显差异;在干旱胁迫条件下,绝大多数检测基因能够快速响应,表达显著升高;两种胁迫条件下,StGRAS28和StGRAS29表达趋势互补,具有协同调控关系。结论: GRAS基因家族能够广泛参与胁迫响应,其中StGRAS28与StGRAS29可能共同参与介导决明根的盐与干旱胁迫应答,StGRAS34和StGRAS12分别作为决明共生结瘤的NSP1和NSP2,可能与增强结瘤因子信号诱导相关,这为进一步挖掘和研究GRAS基因在决明响应胁迫和共生固氮过程所发挥的作用提供了基础。