豆梨植物络合素合酶PcPCS1基因克隆及其表达分析

以梨砧木豆梨（Pyrus calleryana Dcne.）为试材,克隆其植物络合素合酶基因（PcPCS1）的cDNA全长序列,并研究该基因的表达特点。结果表明PcPCS1 cDNA 序列长度为1 970 bp,编码497个氨基酸,推导的氨基酸序列由两个典型的植物络合素亚家族结构域组成,具有3个相邻的Cys-Cys元件（331-332、351-352和369-370位氨基酸）和植物络合素合酶蛋白的特征位点（Cys-56、Cys-90/91和Cys-109）。它与豆科植物的PCS1蛋白处于系统发生树的同一分支,且与百脉根LjPCS1蛋白的同源性最高（84%）。荧光定量RT-PCR分析结果显示：PcPCS1基因为组成型表达,各器官表达量存在差异,在20 mmol · L-1 CdSO4胁迫条件下,其表达量迅速上升,并且在豆梨叶中的表达量高于根;不同重金属离子对其上调表达的诱导能力为Cd2+ > Zn2+ > Cu2+。200 mmol · L-1 γ–谷氨酰半胱氨酸合成酶抑制剂丁胱亚磺酰亚胺能够显著抑制该基因的表达,补充200 mmol · L-1谷胱甘肽后,其相对表达量恢复或超过单一重金属离子处理的表达水平,即豆梨植物络合素以谷胱甘肽为底物,通过γ–谷氨酰半胱氨酸合成酶途经合成。     

湖北海棠植物络合素合酶MhPCS基因克隆及表达分析

【目的】为了探明植物络合素酶参与植物解毒重金属的作用,【方法】以湖北海棠(Malus hupehensis)为试材,克隆其植物络合素合酶基因(MhPCS)编码区序列,并研究该基因的表达特点。【结果】结果表明MhPCS基因编码区全长为1 494 bp,编码497个氨基酸。序列分析表明,MhPCS编码的氨基酸序列由2个典型的植物络合素合酶亚家族结构域组成,具有3个相邻的Cys-Cys元件(331-332、351-352和369-370位氨基酸)和植物络合素合酶蛋白的特征位点(Cys-56、Cys-90/91和Cys-109)。【结论】湖北海棠MhPCS与杜梨PbPCS蛋白同源性最高(94%),属于系统发生树的同一分支。MhPCS基因为组成型表达,各器官表达水平存在差异,其中根的表达量最高;100μmol.L-1CdSO4处理48 h内,MhPCS基因的表达量迅速上升;不同金属离子对其上调表达的诱导能力为:Cd2+>Cu2+>Pb2+。这为揭示重金属胁迫下湖北海棠环境适应的分子机制奠定了基础。     

莲种子防御素基因序列分析及表达载体构建

从莲种子发育中期胚cDNA文库中克隆得到的防御素基因cDNA全长561bp,GenBank登录号为EF421192。分析发现该基因234bp的开放读码框编码77个氨基酸的多肽序列,除信号肽序列外与其它不同来源的植物防御素基因有较高同源性,将其命名为NnDefensin。NnDefensin蛋白带有30个氨基酸的信号肽,具有8个半胱氨酸的高度保守结构等典型的植物防御素特征,成熟肽部分含有γ–硫素蛋白功能结构域。在系统进化上NnDefensin与单子叶植物中的粳稻和小麦的同源蛋白,以及车前草同源蛋白亲缘关系较为接近。通过PCR扩增克隆莲胚防御素基因片段并连接到载体pBI121和带有花生油体蛋白种子特异启动子的载体pAhOleo17.8︰GUS中,成功构建了35S启动子控制的植物表达双元载体pBI121-NnDef和种子特异表达双元载体pAhOleo-NnDef,为该基因的功能鉴定及通过基因工程方法提高转基因植物以及种子的抗病能力奠定基础。     

豆梨NAC转录因子基因PcNAC1的克隆、亚细胞定位及功能初探

根据豆梨(Pyrus calleryana Dence)接种黑斑病菌后的转录组测序结果,筛选出一个上调表达的NAC转录因子基因。从豆梨中获得了该基因的全长片段,并将其命名为PcNAC1。该基因的开放阅读框为792 bp,编码263个氨基酸。将PcNAC1蛋白序列与其他物种蛋白序列进行对比,并构建系统进化树后发现,其与小麦TaNAC4及拟南芥ATAF1具有较高同源性。通过实时荧光定量分析PcNAC1在豆梨不同组中的表达发现,PcNAC1在茎、叶和根中表达量较高,在花和果中表达量较低。构建了亚细胞定位载体,瞬时侵染本氏烟,通过激光共聚焦显微镜观察融合蛋白在细胞内的分布情况,发现该基因行使功能的主要区域定位在细胞核。进一步在本氏烟中瞬时表达PcNAC1,接种烟草疫霉病菌后发现PcNAC1可以通过调控植物激素通路防卫反应基因的表达来增强植物的抗病性。     

豆梨中谷胱甘肽还原酶基因的分离、表达特点及酶活性分析

【目的】测定谷胱甘肽还原酶活性变化,结合其催化产物含量、编码基因表达特点分析,明确该酶是否参与豆梨应对镉胁迫的调控过程。【方法】采用紫外/可见分光光度法检测谷胱甘肽还原酶(glutathione reductase,GR)活性、谷胱甘肽池组成及过氧化氢(hydrogen peroxide,H_2O_2)含量,RT-PCR和PCR克隆PcGRchl和PcGRcyt的cDNA和DNA序列,利用生物信息学方法进行序列比较分析,荧光定量PCR检测它们在镉胁迫下转录水平变化。【结果】镉胁迫情况下,豆梨叶片GR活性上升,谷胱甘肽池中总谷胱甘肽(total glutathione,T-GSH)和还原型谷胱甘肽(reduced glutathione,GSH)减少、氧化型谷胱甘肽(oxidized glutathione,GSSG)上升,H_2O_2积累增加。豆梨叶绿体PcGRchl和胞质PcGRcyt的序列长度、基因结构及所编码蛋白特征各不相同,它们在豆梨叶片中的表达量上调以响应镉胁迫信号,以PcGRchl的转录占主导。外源GSH预处理有助于豆梨预先储存GSH,减缓镉胁迫下H_2O_2的积累,与Cd(2 mmol·L~(-1)CdCl2·2.5H_2O+Hoagland营养液)组相比较,GC(2 mmol·L~(-1),GSH预处理12 h转入2 mmol·L~(-1)CdCl2·2.5H_2O+Hoagland营养液)组PcGRchl和PcGRcyt的表达水平没有太大变化,但GR活性部分受抑制;外源BSO预处理抑制植株叶片GSH合成,镉胁迫下BC(2 mmol·L~(-1)丁硫氨酸-亚砜亚胺预处理12 h转入2 mmol·L~(-1)CdCl2·2.5H_2O+Hoagland营养液)组H_2O_2产生加剧,GR活性上升幅度变大,但PcGRchl和PcGRcyt的转录不受影响。上述结果表明,GSH预处理或BSO预处理,可改变豆梨叶片GSH池的组成,从而在蛋白质水平上反馈调节镉胁迫情况下GR活性。【结论】豆梨GR参与应对镉胁迫的调控过程。镉处理后,豆梨叶片中GSH减少,促使GR酶活性上升,以补充植株应对逆境所需GSH,这一过程主要通过叶绿体PcGRchl的转录上调来实现;GSH或BSO预处理,改变植株GSH含量,从而影响GR活性,减缓或加剧镉胁迫下H_2O_2的产生,这一过程主要是在GR基因翻译后的蛋白水平上进行反馈调节。     

番茄植物络合素合酶基因全长cDNA的克隆及其表达特点

为从分子水平上揭示PCS基因在番茄重金属螯合解毒过程中的作用机制,该研究以番茄（Lycopersicon esculen turn Mill．）品种苏红2003幼苗cDNA为模板,根据NCBI上登录的其他植物的植物络合素合酶基因保守区设计简并引物,利用RT-PCR和RACE技术,克隆番茄植物络合素合酶基因cDNA全长,     

肥料和硫处理对镉胁迫下茭白植物络合素（PCs）含量的影响

以单季茭品种蒋墅茭和双季茭品种葑红早为试材,土壤为栽培介质,对100 mg?L-1 Cd2+胁迫进行不同肥料和不同浓度的硫处理,研究了茭白根系和叶片中的非蛋白巯基、谷胱甘肽及植物络合素含量变化。结果表明：供硫能促进茭白根系和叶片中非蛋白巯基、谷胱甘肽及植物络合素含量的增加,能增强茭白对镉胁迫的抗性;无机肥（三元复合肥）处理的谷胱甘肽、植物络合素含量高于有机肥（菜籽饼）处理;根系中的非蛋白巯基、谷胱甘肽及植物络合素含量显著高于叶片;品种间则以蒋墅茭高于葑红早。     

白菜S位点糖蛋白基因的克隆与表达分析

 以白菜自交不亲和系为材料,采用RT-PCR技术,用特异引物对SLG基因进行克隆,获得SLG基因cDNA序列长度为1 324 bp,命名为BcSLG。序列分析表明：所获得的白菜BcSLG基因cDNA序列包含一完整的编码框,编码432个氨基酸,含有12个保守的半胱氨酸残基和7个N-糖基化位点。序列比对和系统进化分析表明：BcSLG与其它植物的SLG基因氨基酸序列具有较高的同源性,与大白菜和甘蓝亲缘关系最近。荧光定量PCR分析表明：BcSLG基因在自交不亲和系的柱头中表达量最高,其次是花蕾,叶片中表达最低, 在自交亲和系的柱头、花蕾和叶片中相对表达较低。     

多年生黑麦草P5CS基因的cDNA克隆、表达及亚细胞定位

以多年生黑麦草(Lolium perenne)‘Derby'为试材,采用RT-PCR结合RACE技术,克隆获得1个Δ1–吡咯啉–5–羧酸合成酶基因(P5CS)的cDNA序列,全长2528bp,推断其编码716个氨基酸,命名为LpP5CS。序列分析表明:LpP5CS基因与小麦TaP5CS和水稻OsP5CS基因核苷酸序列的相似性分别为92.05%和85.82%,氨基酸序列的相似性分别为93.99%和87.99%。半定量PCR结果表明,LpP5CS基因在多年生黑麦草根、茎,叶均有表达。盐处理下,表达量高于对照,其中叶片中表达量最高,根中最低。200mmol·L-1NaCl处理下,LpP5CS基因表达量随处理时间延长,有先升高后降低的趋势。洋葱鳞茎表皮细胞的瞬时表达显示,LpP5CS蛋白定位于细胞膜和细胞核。     

苹果铁结合蛋白基因的克隆及表达特性

 根据已报道的植物铁结合蛋白基因( ferritin) 的保守区域设计引物, 用RT2PCR 的方法从‘嘎拉’苹果叶片中获得铁结合蛋白基因的全长序列。该基因cDNA共有1 043个碱基, 其中编码区834bp, 编码277个氨基酸残基的蛋白质, 终止密码子后有209 bp左右的3’端尾随序列区。Southern杂交结果显示ferritin基因在‘嘎拉’苹果基因组中至少有7个拷贝存在。0.5 mmol·L ^{- 1}的柠檬酸铁处理‘嘎拉’苹果试管苗不同时间后的定量RT-PCR分析表明, ‘嘎拉’苹果的铁结合蛋白基因随着诱导时间的延长表达量增加, 说明该基因可能在转录水平上受铁诱导表达。     

Fruit growth,cuticle deposition,water uptake,and fruit cracking in jostaberry,gooseberry, and black currant

Fruit cracking after rain limits the production of a number of crops, including some Ribes species. To gain a better understanding of the factors involved in cracking, fruit growth, deposition of the cuticular membrane (CM), water uptake and fruit cracking were studied in black currant (Ribes nigrum L. cv. Zema), gooseberry (Ribes uva-crispa L. cv. Rote Triumph), and jostaberry (Ribes nidigrolaria B. cv. Jostine). Fruit surface area and fresh mass increased continuously throughout development, whereas deposition of the CM was biphasic. CM mass per fruit increased rapidly up to 42, 41, and 49 days after full bloom (DAFB) in black currant, gooseberry, and jostaberry, respectively. Thereafter, CM mass per fruit remained constant in gooseberry and jostaberry or increased at a lower rate in black currant. The cessation of or reduced rate of CM deposition resulted in a decrease in CM mass per unit area in all berries. Elastic strain of the CM at maturity averaged 23.8% and 19.5% in gooseberry and jostaberry, respectively, and only 8.2% in black currant. Microcracks in the CM were observed first in gooseberry and jostaberry 64 DAFB, whereas there were no microcracks in black currant. Water uptake into mature detached berries was linear over 2 h of incubation. Rates of uptake were highest in gooseberry followed by black currant and jostaberry. Relative uptake was similar via the cut end of the pedicel (32.1%), the apex of the fruit (34.7%) and the fruit surface (33.2%). Rates of water uptake through the fruit surface were positively related to surface area. Average fruit water potential for black currant, gooseberry, and jostaberry was −2.14 ± 0.17, −1.24 ± 0.03, and −1.89 ± 0.20 MPa, while the permeability for osmotic water uptake was 7.7 ± 0.4 × 10⁻⁸, 5.2 ± 0.1 × 10⁻⁸, and 3.3 ± 0.3 × 10⁻⁸ m s⁻¹. Incubating whole fruit in deionized water for 72 h resulted in more cracked jostaberries (94%) than black currants (74%) or gooseberries (50%). A comparison of our findings in Ribes berries with published data for the sweet cherry drupe revealed that the berries fitted the relationships established in sweet cherry among fruit growth, cuticle deposition, strain of the cuticle, microcracking, permeability for osmotic water uptake, frequency of stomata and cracking. The Ribes berries were less susceptible to cracking than sweet cherry.     

Similitude,similarity, and scaling

An exceptionally rich and colorful literature, drawn in almost equal parts from pure mathematics, from the sciences, and from the technologies, has grown up over the years, which bear in different ways on the topics under discussion. It is the intent of the present paper to survey this far-flung literature, point out some of the commonalities and interrelationships which underlie it, and briefly indicate how it has been and can be applied. To my knowledge, this kind of review has not been attempted before.     

Climatic, landform, microtopographic, and overstory canopy controls of tree invasion in a subalpine meadow landscape, Oregon Cascades, USA

Tree invasions have been documented throughout Northern Hemisphere high elevation meadows, as well as globally in many grass and forb-dominated ecosystems. Tree invasions are often associated with large-scale changes in climate or disturbance regimes, but are fundamentally driven by regeneration processes influenced by interactions between climatic, topographic, and biotic factors at multiple spatial scales. The purpose of this research was to quantify spatiotemporal patterns of meadow invasion; and how climate, larger landforms, topography, and overstory trees have interactively influenced tree invasion. We combined airborne Light Detection and Ranging (LiDAR) characterizations of landforms, topography, and overstory vegetation with historical climate, field measurements of snow depth, tree abundance, and tree ages to reconstruct spatial and temporal patterns of tree invasion over five decades in a subalpine meadow complex in the Oregon Cascade Range, USA. Proportion of meadow occupied by trees increased from 8?% in 1950 to 35?% in 2007. Larger landforms, topography, and tree canopies interactively mediated regional climatic controls of tree invasion by modifying depth and persistence of snow pack, while tree canopies also influenced seed source availability. Landscape context played an important role mediating snow depth and tree invasion; on glacial landforms tree invasion was negatively associated with spring snowfall, but on debris flows tree invasion was not associated with snow fall. The importance of snow, uncertain climate change impacts on snow, and mediation of snow by interacting and context dependent factors in complex mountain terrain poses substantial hurdles for understanding how these ecotones may respond to future climate conditions.     

Wetlands, carbon, and climate change

Wetland ecosystems provide an optimum natural environment for the sequestration and long-term storage of carbon dioxide (CO₂) from the atmosphere, yet are natural sources of greenhouse gases emissions, especially methane. We illustrate that most wetlands, when carbon sequestration is compared to methane emissions, do not have 25 times more CO₂ sequestration than methane emissions; therefore, to many landscape managers and non specialists, most wetlands would be considered by some to be sources of climate warming or net radiative forcing. We show by dynamic modeling of carbon flux results from seven detailed studies by us of temperate and tropical wetlands and from 14 other wetland studies by others that methane emissions become unimportant within 300 years compared to carbon sequestration in wetlands. Within that time frame or less, most wetlands become both net carbon and radiative sinks. Furthermore, we estimate that the world’s wetlands, despite being only about 5–8 % of the terrestrial landscape, may currently be net carbon sinks of about 830 Tg/year of carbon with an average of 118 g-C m^?2 year^?1 of net carbon retention. Most of that carbon retention occurs in tropical/subtropical wetlands. We demonstrate that almost all wetlands are net radiative sinks when balancing carbon sequestration and methane emissions and conclude that wetlands can be created and restored to provide C sequestration and other ecosystem services without great concern of creating net radiative sources on the climate due to methane emissions.     

Up-regulation of mRNA expressions of fas,bcl-2, bim,bax, caspase-3, caspase-9, and bcl2l12 in K562 treated with bortezomib

AIM: To detect the treatment of K562 leukemia cells with bortezomib altering the expression of genes fas, bcl-2, bcl2l12, bim, bax, caspase-9 and caspase-3.METHODS: MTT assay was used to detect the inhibition of proliferation. Apoptosis was detected by Annexin-V staining and mitochondrial transmembrane potential (Δψm). RT-PCR was used to analyze the mRNA expressions of fas, bcl-2, bcl2l12, bim, bax, caspase-3 and caspase-9.RESULTS: Bortezomib caused a time- and dose-dependent inhibition of cell proliferation and IC50 of 24 h and 48 h were 161.41 nmol/L and 96.33 nmol/L, respectively. At the concentration of 104 nmol/L, bortezomib induced apoptosis in a time-dependent manner, including increasing annexin-V positivity and decreasing the Δψm. RT-PCR showed that bortezomib up-regulated the mRNA expression of fas, bcl2l12, caspase-9 and caspase-3, but mRNA expressions of bcl-2, bim and bax did not changed obviously.CONCLUSION: Bortezomib inhibits the proliferation of K562 and induces apoptosis, in which fas, bcl2l12, caspase-9 or caspase-3 gene is one of the main genes taking part in.     

氮磷钾硼肥配施对青花菜养分吸收分配及产量和品质的影响

研究了不施肥处理,氮磷钾配施和增施硼肥对青花菜养分吸收分配及产量和品质的影响。结果表明,现蕾期至花球膨大期,青花菜对氮、磷、钾的吸收量最大,整个生长期对钾的吸收最多,氮次之,磷最少。氮磷钾主要分配在叶片中,现蕾后逐渐向花球转移,从现蕾到采收时,叶片中氮、磷、钾分配率分别降低了19.6%、10%和9.0%,花球中氮磷钾分配率分别提高了23.1%、15.9%和11.1%。不施氮、磷、钾肥显著降低了花球产量、维生素C含量和成品率,增施硼肥处理的花球成品率较氮磷钾肥处理提高了4.8%,不施氮肥和钾硫肥(K₂SO₄)显著降低了花球中的硫代葡萄糖苷含量。相关性分析表明,青花菜产量与植株内氮、钾积累量显著正相关,成品率与植株内磷营养显著正相关。     

Principles for forcing tulips,hyacinths, daffodils,Easter lilies and Dutch irises

Systematic investigations on the forcing of tulips, hyacinths, daffodils, Easter lilies and Dutch iris have produced a fairly complete set of scientific principles. In this review, these principles have been classified into a 3-phase concept of forcing. These are: production, programming, and greenhouse. The production phase is defined as all processes which occur during bulb production and it terminates with the harvesting of the bulbs. The programming phase comprises all handling of the bulbs from harvesting until they are placed under greenhouse conditions. The greenhouse phase is the accelerated development of the bulbs until anthesis or marketing of the plants. These phases have been discussed relative to floral and root development and the basic environmental requirements of the bulb species.