大麦水孔蛋白基因HvTIP2;1及其启动子的表达特性分析

组织特异性启动子作为基因工程的一种重要调控元件,在生物反应器、转基因新品种培育等领域有重要的应用前景。基于芯片数据的基因数字化差异显示分析,筛选到一个根特异表达的大麦水孔蛋白基因Hv TIP2;1,利用实时荧光定量RT-PCR方法了解该基因在不同组织和处理条件下的表达特性,并对基因启动子及功能进行了研究。结果表明,Hv TIP2;1表达具有根特异性并受植物生长发育的调控,其在成株期的表达量明显高于苗期。ABA激素处理组,Hv TIP2;1表现先上升,处理10h后又下降的表达变化趋势;铝、锰有毒金属离子处理组,Hv TIP2;1表达量明显高于对照组,但表现出上升-下降-上升的波动变化特征;盐胁迫导致Hv TIP2;1表达量持续下降,而干旱诱导Hv TIP2;1表达量不断升高,处理24h后表达量迅速下降,低于对照水平。利用PCR方法克隆位于基因编码区上游的启动子序列Hv1310p,该序列含有多个与根特异表达和胁迫响应有关的顺式作用元件。通过5'端缺失法分别构建514bp和1 258bp启动子的融合GUS报告基因表达载体,转基因烟草的GUS活性检测表明两个启动子片段都具有根特异性的启动活性。     

HaSNPV的9个基因的转录谱分析

使用反转录和实时荧光定量PCR技术,我们对HaSNPv的几个预期的早期基因、早晚期基因、晚期基因、极晚期基因的转录时相进行分析,结果表明这些基因的起始转录时间与其自身的启动子类型基本是一致的.但是预期的早期基因pkip晚期才开始转录;预期的早晚期基因ha107早期不转录,仅在晚期转录.这些基因的转录水平一般都在病毒感染细胞72 h后达到最高,并且极晚期基因polyhedrin的转录水平明显高于其它基因.Iap2的转录水平仅次于polyhedrin,表明它可能是一个功能基因.与AcMNPV的p10不同,在HaSNPV/HzAM1系统中p10的转录水平并不高.     

梨树花芽休眠解除与活性氧代谢的关系

梨树(Pyrus bretschneideri Rehd.)自然休眠和休眠解除时,花芽的活性氧代谢发生变化.O2-·产生速率和H2O2的含量在休眠期间上升,在休眠后期下降.抗氧化系统中SOD活性在自然休眠期呈下降趋势,自然休眠结束活性上升.POD和CAT活性在自然休眠期上升.抗氧化物质AsA和GSH的含量随休眠进行而下降,休眠解除过程中重新升高.APX和GR的活性在休眠期间活性下降,休眠结束活性迅速上升.这些结果表明:花芽的休眠与活性氧的代谢有很大关系.     

HaSNPV的9个基因的转录谱分析

使用反转录和实时荧光定量PCR技术,我们对HaSNPV的几个预期的早期基因、早晚期基因、晚期基因、极晚期基因的转录时相进行分析,结果表明:这些基因的起始转录时间与其自身的启动子类型基本是一致的。但是预期的早期基因pkip晚期才开始转录;预期的早晚期基因ha107早期不转录,仅在晚期转录。这些基因的转录水平一般都在病毒感染细胞72h后达到最高,并且极晚期基因polyhedrin的转录水平明显高于其它基因。Iap2的转录水平仅次于polyhedrin,表明它可能是一个功能基因。与AcMNPV的p10不同,在HaSNPV/HzAM1系统中p10的转录水平并不高。     

桃叶片抗冷性对光周期诱导休眠的响应

以6年生“春捷”桃树为试材,以自然生长的桃树为对照,研究了长日照和短日照的休眠诱导效应和休眠诱导进程中叶片抗冷性对光周期的响应.结果表明: 在逐渐降低的自然环境温度下,长日照和短日照处理树体均能进入休眠诱导期,其中长日照处理延后1周而短日照处理提前1周进入.随着休眠程度的加深,各处理叶片的总含水量、自由水含量降低,束缚水含量、束缚水/自由水比值升高.超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性在休眠诱导期内均呈单峰曲线,高峰值出现在休眠诱导期的后期,过氧化物酶(POD)活性进入休眠诱导期后迅速下降,后期回升形成小高峰.休眠诱导期内可溶性蛋白含量稳步下降,脯氨酸和丙二醛(MDA)含量持续升高,伤害率逐渐增大.长日照可显著提高SOD、CAT活性及脯氨酸含量,减缓POD活性和可溶性蛋白含量的降幅,降低MDA和伤害率的增幅.这表明长日照处理叶片受伤害程度更轻,而短日照处理相应指标的变化则不同,尤其诱导期后期叶片的伤害率显著高于对照,表现出较低的抗冷性.如果环境温度允许,实际生产中可以适当延长光照时间来提高叶片的抗冷性.     

镉和铜对嗜热四膜虫金属硫蛋白基因的诱导表达

本文在荧光定量PCR优化的基础上,利用该技术考察了不同浓度的重金属镉和铜对嗜热四膜虫金属硫蛋白基因(MTT1)诱导表达的变化规律。结果表明MTT1基因的表达对镉离子的诱导更灵敏,且在一定阈值浓度(≤35.2μmol/L)范围内,镉离子浓度升高会增加MTT1基因表达量,超过该阈值后表达量迅速下降;镉与铜同时诱导时MTT1基因的表达情况与镉单独诱导的类似,但阈值浓度减小为22μmol/L,表明二者的联合毒性为协同作用。镉离子浓度低于22μmol/L时,与铜离子的共同作用会大大增加MTT1基因的表达量,从而增强了四膜虫的解毒能力。     

甘蔗金属硫蛋白基因(ScMT2-1-4)的克隆及表达分析

从甘蔗热带种Badila(Saccharum officinarum L.)中克隆获得1个2型金属硫蛋白基因的c DNA序列,命名为Sc MT2-1-4(Gen Bank登录号:KJ504375)。生物信息学分析显示,Sc MT2-1-4基因c DNA长459 bp,开放读码框为243 bp,编码80个氨基酸,富含14个半胱氨酸残基。推导的Sc MT2-1-4蛋白为亲水性蛋白,分子量为7.82 k D,等电点为5.59,该蛋白的二级结构由无规则卷曲和延伸链构成。实时荧光定量PCR检测结果显示,Sc MT2-1-4基因是重金属胁迫的快速响应基因,其对Cu2+、Zn2+和Cd2+胁迫的应答模式提示了:甘蔗不同组织中Sc MT2-1-4对Cu2+胁迫应答的分功不同;Sc MT2-1-4对甘蔗抵御Zn2+胁迫起积极的作用;但该基因不直接参与甘蔗对Cd2+的螯合和解毒的过程。研究结果有助于进一步深入探究MT2基因在甘蔗应答重金属胁迫过程中的作用,为阐明甘蔗富集和耐受重金属的分子机制研究奠定基础。     

棉花水孔蛋白基因的克隆及序列分析

采用RT-PCR的方法,根据GenBank上公布的棉花水孔蛋白基因序列设计引物,克隆得到4个棉花质膜水孔蛋白基因,分别为GhPIP1;1、GhPIP1;2、GhPIP2;1和GhPIP2;2,测序结果与公布序列的相似性都在98%以上,氨基酸序列在99%以上。这是首次从新陆早系列棉花(Gossypium hirsutum)的叶片中克隆得到,为以后研究棉花的质膜水孔蛋白提供参考。     

西洋梨水孔蛋白基因家族的全基因组鉴定及表达分析

水孔蛋白(AQPs,aquaporins)是高效转运水分子的膜内在蛋白,具有丰富的多样性,在调控植物的水分关系中有重要作用。本研究利用西洋梨(Pyrus communis L.‘Bartlett’)基因组数据库,通过生物信息学手段鉴定西洋梨PcAQPs基因家族成员;并利用MEGA 6.0.5软件,采用邻接法构建系统发育树;利用GSDS 2.0软件进行基因结构分析,MEME程序进行Motif分析,AgBase v2.00程序进行GO分析;采用半定量RT-PCR技术研究PcAQPs基因组织表达情况。结果表明,西洋梨基因组中共有54个PcAQPs家族成员,均含有AQP特征结构域和保守的Motif基序,根据基因结构及系统进化分析可分为PIP、TIP、NIP和SIP等4个亚家族。不同基因间结构差别较大,但聚类关系较近的基因其结构类似。GO分析发现,多数PcAQPs基因具有转运蛋白活性,参与物质转运、应激反应、发育和代谢等生物学过程,但不同亚家族成员构成的细胞组分及参与的生物学过程具有明显差异。半定量结果表明,大多数PcAQPs基因在根、茎、叶和果实中均有表达,而且不同基因家族、不同基因间的组织表达模式存在差异。该研究为今后西洋梨PcAQPs基因的克隆和功能分析奠定了基础。     

丹参中病程相关蛋白基因SmSTH-2的生物信息学分析

对丹参cDNA文库的表达序列标签(EST)序列进行BLAST分析显示,其中一条序列与病程相关蛋白基因STH-2有较高的同源性。该序列全长691bp,包含1个长483bp的开放阅读框(ORF),编码160个氨基酸,命名为SmSTH-2。生物信息学分析显示:SmSTH-2所编码蛋白的分子质量为17990Da,等电点为5.15,富含谷氨酸、赖氨酸、甘氨酸、丝氨酸,无信号肽,属于稳定类蛋白。与NCBI注册的其他6种植物来源的病程相关蛋白基因编码的氨基酸序列的同源性在42%～46%之间。实时荧光定量PCR的方法检测丹参不同组织部位中SmSTH-2表达和病原菌对该基因诱导表达的影响的结果表明:SmSTH-2在植物的根、茎、叶中均有不同程度的表达,其表达丰度为根>叶>茎;丹参叶片接种黄瓜细菌性角斑病病原菌后,4d内可诱导该基因的表达量持续增加。用PCR方法从基因组水平克隆到SmSTH-2的DNA序列,测序表明SmSTH-2的编码序列在DNA水平上含有一个71bp的内含子,DNA序列注册号为EF621486.     

Dormancy in peach (Prunus persica L.) flower buds

Morphological studies were carried out with peach flower buds collected monthly in 1989 and 1990, from two months before leaf fall (7 March) until two to three weeks before bloom (7/8 August). Chilled (2–4°C for 30 days) and unchilled buds were exposed to 20 to 25°C, 100% RH and continuous light. Gibberellin A₃ (3 ng or 30 ng) was applied to some of the non-chilled cuttings at three days intervals. Then, 12, 19, and 26 days after they were planted, the buds were sampled and processed for histological studies. Cultured flower buds (chilled or unchilled) had accelerated anther and gynoecium morphogenesis after 12 days under controlled conditions, compared to buds processed immediately after collection from the field. Chilling treatment augmented the bud culture effect, while Gibberellin A₃ applications to the excised buds retarded bud morphogenesis to a stage comparable to that of buds collected directly from the field. This, suggests that the comparatively high levels of Gibberellin A_1/3 we previously found in mid winter [15, 18] could be at least one of the factors that controls floral bud dormancy by retarding anther and gynoecium development.     

黄瓜花芽启始分化的形态解剖研究

苗龄 6d的黄瓜幼苗 ,在第一节位叶腋处花芽原基开始启动分化。花芽分化时间早、速度快、节位低、同步性好。诱导黄瓜开花的因素可能不是光和夜低温 ,其开花特性类似于自主开花植物     

Modelling compensatory regrowth with bud dormancy and gradual activation of buds

Many plants show compensatory regrowth after herbivory and dormant buds often have an important role in compensatory responses. Theoretical models have shown that herbivore damage may select for a bud bank, i.e., a pool of dormant buds that are protected from herbivory and that are activated after herbivore damage. Earlier models assumed that undamaged plants cannot activate their dormant buds without damage, although they apparently have sufficient resources for successful seed production through the additional shoots dormant buds could produce. However, many plants are able to gradually activate buds over an extended period of time without any cue from damage. The aim of this study was to analyze how herbivory imposes selection for gradual mobilization of the bud bank. I assume that selection pressures that affect the fraction of buds active at each time point include damage by herbivores, time left to the end of season, and the opportunity costs of dormant buds. I modelled bud dynamics with gradual activation when there is a single damage event and (i) when the seed set of a shoot is not dependent on the time it is active, or (ii) when the seed set of a shoot diminishes with later activation. In addition, I analyzed how (iii) risk of repeated herbivory affects selection for gradual activation. Under these models, gradual activation is optimal over a wide range of herbivory pressures. Selection appears to favour activation of all buds at the beginning of the season only when herbivore pressure is weak and when early shoots have a higher seed set than late shoots. Alternatively, strong herbivore pressure and late damage may select for a large bud bank throughout the growing season, without gradual activation; the bud bank is only mobilized after damage. In this case, damaged plants can overcompensate, i.e. they have a higher seed set than undamaged plants with the same bud activation pattern. Selection for overcompensation demands a stronger herbivore pressure in this current model than in earlier bud bank models. The model never predicts selection for overcompensation when there is a risk of repeated herbivory. This revised version was published online in July 2006 with corrections to the Cover Date.     

Endogenous IAA levels and development of coffee flower buds from dormancy to anthesis

Dormant coffee (Coffea arabica L.) flower buds require water stress to stimulate regrowth. A xylem specific water-soluble dye, azosulfamide, was used to quantify water uptake of buds after their release from dormancy by water stress. In non-stressed flower buds, the rate of water uptake was generally slower and variable compared to stressed flower buds, where the rate of uptake tripled from 1 to 3 days after rewatering and preceded the doubling of fresh and dry weight of buds. Free, ester and amide IAA levels of developing flower buds were measured by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution technique with [¹³C₆]IAA as an internal standard. Throughout development, the majority of IAA was present as amide IAA. The proportions of amide and free IAA increased one day after plants were released from water stress, and preceded the doubling of fresh and dry weight. Free and conjugated IAA content per bud remained stable during the period of rapid flower growth until one day before anthesis.Abbreviations FW fresh weight - IAA indole 3-acetic acid - HPLC high performance liquid chromatography - GC-MS-SIM gas chromatography-mass spectrometry selected ion monitoring - NAA naphthalene acetic acid - IBA indole butyric acid     

Programmed accumulation of LEA-like proteins during desiccation and cold acclimation of overwintering grape buds

Molecular investigation of the process of cold acclimation in woody plants has been limited by the superimposition of dormancy-related events on the process of cold tolerance development. To address this limitation, we have used the grape Vitis labruscana L. ev. Concord to develop a system in which the developmental programme of dormancy can be induced seperately from cold acclimation. Using this system we have characterized differential accumulation of several proteins in grape buds during the normally superimposed endodormancy and cold acclimation programmes, and in buds which have entered only the endodormancy programme. A set of 47 kD proteins accumulated during endodormancy without cold acclimation to a level similar to that found in endodormant and cold-acclimated buds, but without any associated increase in bud cold-acclimation level. However, a 27 kD LEA-like protein accumulated only in cold acclimated buds. We conclude that expression of the 47 kD glycoprotein is endodormancy-related, but is not strictly related to the development of cold acclimation, while the 27 kD protein appears to be more specific to cold acclimation. In addition to strengthening the association of LEA-like proteins with cold acclimation, this system allows more specific assessment of cold acclimation-associated phenomena in overwintering buds.     

Expressed sequence tags of young floral buds and characterization of a bud- preferential lectin- like cDNA fromPharbitis nil

To identify the genes involved in flower development, we analyzed 207 expressed sequence tags (ESTs) from a young floral bud cDNA library ofPharbitis nil. Of these, 87 clones (42%) showed significant homology to known protein sequences in the NCBI database. Four of these had not been reported previously in the plant kingdom, indicating that 1.9% of the ESTs were newly identified in plants. Functional categorization revealed that the genes involved in metabolic pathways, such as glycolysis and photosynthesis, were most abundant Reverse-northern and northern analyses showed that one clone,PnFP161, was expressed preferentially in floral buds. DNA sequence analysis indicated that this clone contained 147 bp of 5′-UTR, 264 bp of -UTR, and an open reading frame of 233 amino acids, thereby sharing 33% identity with a lectin fromCalystegia sepium. The C-terminal regionof PnFPI61 had well-conserved residue with that of the lectins. Southern blot analysis demonstrated thatPnFPI61 exists as a multigene family.     

Abscisic acid,abscisic acid-esters and phaseic acid in vegetative terminal buds of Acer pseudoplatanus during emergence from winter dormancy

Levels of free-abscisic acid and boundabscisic acid (alkaline hydrolyzable abscisic acidesters) in replicated samples of terminal vegetative buds of sycamore trees were measured during natural emergence from winter dormancy by gas chromatographic methods together with isotope dilution estimation of recovery rates. Not until after the buds had been released from true dormancy in January by winter chilling did any clear change occur in either abscisic acid (ABA) fraction, or in total ABA, on any basis of comparison. The percentage of total ABA present as the free acid declined at the end of true dormancy to approximately two-thirds of its value in the earlier winter months. It is concluded that glucosylation of ABA is unlikely to play a major part in the mechanism of release from dormancy in vegetative sycamore buds. At the end of true dormancy there was a large transient increase in what appeared to be phaseic acid, but this was not accompanied by any marked decrease in either free- or bound-ABA.Abbreviations ABA abscisic acid - TLC thin layer chromatography - GLC gas chromatography     

Freezing exposure releases bud dormancy in Betula pubescens and B. pendula

Bud dormancy in woody plants is released by long-term exposure to non-freezing chilling temperatures, whereas freezing temperatures have been considered to have little or no effect. However, the present results demonstrate that short-term exposure to freezing can release bud dormancy in Betula pubescens (Ehrh.) and B. pendula (Roth). Short-term freezing during the dormancy induction phase improved the release of bud dormancy only if an adequate level of dormancy had been reached. In fully dormant or chilled plants both the percentage and the speed of bud-burst increased, the more so the lower the temperature. Our results rule out the possibility that endogenous abscisic acid could be directly involved in the physiological control of bud dormancy release. The fast, easily applicable method presented here for bud dormancy release could further investigations into the biochemical and biophysical background to the process. The mechanisms of bud dormancy release and its relationship to cold acclimation are discussed in the light of these results, as also are the implications of the findings for modelling of bud dormancy.