水稻Os05g0442400基因启动子分析以及由Os05g0442400基因启动子引导GUS报告基因在转基因水稻中的表达

采用Plant CARE和PLACE软件分析预测水稻Os05g0442400基因启动子序列中可能存在的顺式作用元件.结果显示,在起始密码子ATG上游1500bp区域内,除了启动子基本的核心作用元件外,还存在一些与植物抵御非生物胁迫过程有关的作用元件、脱落酸应答元件、光诱导启动子作用元件、病原菌诱发因子作用元件,以及根特异性结合位点.采用根癌农杆菌介导法成功将Os05g0442400 promoter::gus构建导入"中花11"水稻.由不同组织部位GUS染液检测表明:在水稻苗期,内源Os05g0442400基因可能主要在根部表达;随着水稻生殖期的延续,水稻内源Os05g0442400基因在颖壳中的表达区域由上向下面积增大,并在抽穗后达到最大表达区域.这些结果可能与Os05g0442400基因启动子上分别存在根特异性结合位点和光诱导启动子作用元件具有一定的联系.     

OsNAC2转录因子介导生长素代谢通路调节水稻早期根的发育

NAC家族是植物特有的一类转录因子,在植物的生长发育和逆境胁迫等方面具有重要的功能.研究发现,OsNAC2基因的过表达(ON11)和RNAi(RNAi31)转基因株系,早期主根的长度有显著变化的特征.与野生型日本晴水稻(WT)相比,ON11植株的主根变短,而RNAi31的主根变长.通过对早期OsNAC2pro∶∶GUS株系根尖染色结果表明OsNAC2在根尖具有时序性表达.不同激素诱导OsNAC2表达谱和OsNAC2pro∶∶GUS株系对激素响应结果显示OsNAC2受生长素显著性诱导.结合芯片数据和qRT-PCR分析,OsNAC2转基因株系中生长素合成代谢及信号通路相关基因表达量变化较为明显.同时,ON11根尖淀粉粒发育及其向重力性受到抑制.因此,推测OsNAC2可能通过抑制生长素的合成代谢及信号通路相关基因的表达,降低生长素含量,并参与生长素响应通路,最终影响水稻根的生长.     

茉莉酸对水稻根系生长素合成及运输的调控

以水稻中花11和DR5∷GUS转基因植株为实验材料,观察茉莉酸(Jasmonic acid,JA)对水稻根系中生长素积累和分布的影响.结果表明,JA负调控水稻主根、冠根和侧根的生长,抑制DR5∷GUS在水稻根系形态学下端的表达.实时定量PCR结果显示,在JA处理的水稻根系中,7个水稻生长素合成基因OsYUCCAs(OsYUCCA1～7)全部显著下调表达,4个生长素输入载体基因OsAUX1,OsAUX3～5显著上调表达,4个生长素输出载体基因OsPIN1b、OsPIN1c、OsPIN2、OsPIN9显著下调表达.     

一个水稻类受体蛋白激酶OsRPK2的过量表达分析

为了解类受体蛋白激酶Os RPK2在水稻发育中的作用,采用反向遗传学方法构建了该蛋白激酶基因的过表达载体,将其导入野生型水稻中获得转基因植株后,观察植株的表型变化以及该基因在水稻植株不同部位的表达情况.结果发现,Os RPK2的转基因植株产生白化现象,q RT-PCR分析表明白化植株中Os RPK2的表达量明显增加,说明水稻转基因植株的白化表型是由Os RPK2基因的过表达造成的.Os RPK2的组织特异性表达结果表明,Os RPK2在水稻的不同组织器官中均有表达,但在水稻幼嫩的分生组织和幼龄叶片中表达量较高,反映了Os RPK2在水稻植株发育及建成中具有重要作用.     

NAC1启动子驱动GFP表达的组织特征及对生长素和赤霉素的反应

将拟南芥中转录因子基因NAC1上游调控区1kb克隆到pRD420质粒，构建由NAC1启动子驱动绿色荧光蛋白基因（GFP）表达的植物表达载体，并以此载体转化烟草，对阳性转基因植株研究表明，GFP在根部有较高水平的表达.进一步用生长素、生长素拮抗剂NPA和赤霉素处理，荧光强度的变化表明：该调控区受生长素作用的同时也受赤霉素诱导.初步说明NAC1基因不仅是一个生长素反应基因，同时可能也是一个赤霉素反应基因.     

NACl启动子驱动GFP表达的组织特征及对生长素和赤霉素的反应

将拟南芥中转录因子基因NACl上游调控区1kb克隆到pRD420质粒，构建由NACl启动子驱动绿色荧光蛋白基因（GFP）表达的植物表达载体，并以此载体转化烟草，对阳性转基因植株研究表明，GFP在根部有较高水平的表达．进一步用生长素、生长素拮抗剂NPA和赤霉素处理，荧光强度的变化表明：该调控区受生长素作用的同时也受赤霉素诱导．初步说明NACl基因不仅是一个生长素反应基因，同时可能也是一个赤霉素反应基因．     

AhHDA1影响花生毛状根生长的研究

花生组蛋白去乙酰化酶AhHDA1转到花生毛状根中超表达后,短侧根的毛状根比例增加. 基因表达检测结果显示,AhHDA1超表达后上调了细胞周期相关基因AhCYCD4和生长素信号转导相关基因AhIAA28的表达水平,而AhARF19的表达水平被显著抑制. 进一步通过LUC实验发现, AhHDA1激活AhCYCD4和AhIAA28启动子的活性. 说明AhHDA1可能通过调控生长素信号转导和细胞分裂影响花生毛状根侧根的生长.     

基于位置关联性打分方程的人类转录因子结合位点的预测(英文)

转录因子结合位点的识别是阐明基因转录调控机制的重要环节,准确的转录因子结合位点的预测算法将有助于人们识别转录因子的目标基因,进而研究转录因子结合位点在上游调控区中的位置对转录调控的影响.然而,目前存在的预测转录因子结合位点的算法所得结果的特异性普遍较低,因此有必要提出一种新的有效的预测转录因子结合位点的算法.本文利用JASPAR数据库上的数据,在深入分析转录因子结合位点生物学特征的基础上,构建了考虑位点保守性和伪计数的位置关联性打分方程.预测结果表明,在最佳阈值之下,该算法对转录因子结合位点的假阳率低于0.01%.     

基于位置关联性打分方程的果蝇转录因子结合位点的预测

转录因子结合位点的识别是阐明基因转录调控机制的重要环节,准确的转录因子结合位点的预测算法将有助于人们识别转录因子的目标基因,进而研究转录因子结合位点在上游调控区中的位置对转录调控的影响.然而,目前存在的预测转录因子结合位点的算法所得结果的特异性普遍较低,因此有必要提出一种新的有效的预测转录因子结合位点的算法.本文利用JASPAR数据库上的数据,在深入分析转录因子结合位点生物学特征的基础上,构建了考虑位点保守性和伪计数的位置关联性打分方程,并对果蝇转录因子结合位点进行预测,预测结果的假阳率均低于0.02%.     

Identification of auxin responsive genes in Arabidopsis by cDNA array

The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.     

Subcellular localization and functional analyses of structural domains of COP1 in transgenic tobacco

Plants have evolved an extremely exquisite light signal regulatory network to adapt to the changing ambient light conditions, in which COP1 plays a critical role of the light signal transduction. Based on the cloned pea COP1 cDNA sequence and its protein structure, four individual gene fragments encoding different structural domains of the COP1 were designed to fuse to the GFP gene. The plant expression vectors containing these fusion genes as well as the COP1GFP fusion gene were constructed and used to transform tobacco by Agribacterium as confirmed by Southern analyses. Antibodies were raised against the recombinant GFP-COP1 overproduced in Escherichia coli. Immunoblotting results demonstrated that all of the fusion genes were constitutively expressed in transgenic tobacco plants. We systematically investigated the different subcellular localization of these fusion proteins and the resulting phenotypic characteristics of these transgenic plants under light and dark conditions. Our data show that (1) the molecular mass of the tobacco endogenous COP1 protein is 76 kD. It is constitutively expressed in all of the tested tissues and the total cellular content of COP1 protein is not noticeably affected by light conditions. (2) The nuclear localization signal of COP1 plays a critical role in regulation of its nuclear-cytoplasmic partitioning. The subcellular localization of the COP1 protein containing nuclear localization signal is regulated by light in the epidermal cells of leaves, but, it is located in nucleus constitutively in root cells. (3) The coiled-coil domain is very critical to the function of COP1 protein, while the zinc binding RING finger domain only plays a supportive role. (4) The WD-40 repeats domain is essential to the COP1 function, but this domain alone does not affect photomorphogenesis. (5) Overexpression of COP1 protein not only inhibits the photomorphogenesis of the stems and leaves of the transgenic tobacco, but also results in the generation of short and clustered roots. In contrast, overexpression of COP1 protein without WD-40 repeats domain promotes the photomorphogenesis process in the stems and leaves and lead to root elongation and lack of lateral roots. The COP1-COP1 interaction happens not only in the nucleus, but also in cytoplasm.     

拟南芥钙依赖蛋白激酶CPK11与ABA响应元件结合因子ABF4相互作用的研究

为研究钙依赖蛋白激酶CPK11参与ABA信号通路的方式,本文利用体外酵母双杂实验(Y2H)以及体内双分子荧光互补实验(BiFC)分析CPK11与ABA响应元件结合因子(ABA-responsive element binding factors, ABFs)ABF4之间的关系.酵母双杂交实验表明CPK11与ABF4存在体外相互作用,双分子荧光互补实验表明CPK11与ABF4存在体内相互作用.以上实验共同证明CPK11与ABF4存在直接相互作用.作为CPK11的同源蛋白CPK4与转录因子ABF4在植物体内也存在相互作用.以上实验表明CPK11及其同源蛋白CPK4可能通过与转录因子ABF4相互作用从而参与钙离子介导的ABA信号通路.     

A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants

The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes.     

Secretive expression of Aspergillus fumigatus phytase in tobacco improves phosphorus nutrition in plant

To generate transgenic plants capable of utilizing exogenous phytate,an Aspersgillus fumigatus phytase gene(fphyA) was constitutively expressed in tobacco and recombinant enzyme was secreted from plant roots into the rhizosphere using the signal sequence from tobacco calretieulin.After 40 days of plant growth in hydroponic media,phytase activities in leaves,stems,roots and growth media of transgenic plants were 8.6-fold,7.4-fold,12.6-fold and 14.3-fold higher than those of wild-type plants.Signifi-cant improvements in plant growth and phosphoms(P)utilization were observed in the transgenic plants.When phytate was supplied as the sole P source.45-day-old transgenic tobaccos accumulated 1.0-fold and 0.5-fold more shoot and root biomass than wild-type tobaccos.with a concomitant of 1.7-fold increase in total P concentration.These results indicate that secretive expression of the A.fumigatus phytase improves acquisition and use of P from phytate in plants.     

Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions

Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication-a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.     

植物抗盐碱、耐干旱基因工程研究进展

综述了近年来国内外克隆、鉴定的抗盐、耐旱基因中细胞相容性物质合成基因及其来源、作用机制,及它们在转基因植物中的遗传稳定性及转基因植物的抗盐、耐旱性等;同时对抗盐;耐旱的信号传导和转录因子等的研究作了概述。     

Over-expression of an Arabi-dopsisd δ-OAT gene enhances salt and drought tolerance in transgenic rice

δ-OAT, ornithine-δ-aminotransferase, is the key enzyme involved in proline biosynthesis. In this study the Arabidopsis δ-OAT gene was transferred into rice (Oryza sativa L. ssp japonica cv. Zhongzuo 321), whose successful integration was demonstrated by PCR and Southern blot analysis. The over-expression of the gene in transgenic rice was also confirmed. Biochemical analysis showed that, under salt or drought stress conditions, proline contents in the leaves and roots in transgenic rice plants were 5- to 15-fold of those in non-transgenic controls. Under stress conditions, germinating rate of transgenic lines is higher than that of controls. Although the growth of rice plants tested were more and more retarded with the increasing of NaCI concentration, the transgenic plants grow faster compared to the controls under the same stress condition. Meanwhile, the resistance to KCl and MgSO4 stresses was also found enhanced in transgenie rice. Furthermore, the over-expression of δ-OAT also improved the yield of transgenic plants under stress conditions. The average yield per plant of transgenic lines increases about 12%--41% more than that of control line sunder 0.1 mol/L NaCI stress. These data indicated that the over-expression of δ-OAT, with the accumulation of proline, resulted in the enhancement of salt and drought tolerance and an increase of rice yield, which is of significance in agriculture.