转录因子FaNAC56在草莓果实成熟中的功能分析

[目的]研究NAC转录因子FaNAC56在草莓果实成熟中的作用.[方法]根据前期果实成熟过程中的蛋白组数据,以八倍体红颜草莓为试材,克隆FaNAC56基因及其启动子.在蛋白水平,利用MEGA构建FaNAC56系统进化树,并通过生物信息学预测其编码蛋白的二级结构,以及利用烟草进行亚细胞定位;在转录调控水平,利用预测网站分析FaNAC56启动子区顺式作用元件以及下游靶基因,并通过RT-qPCR分析FaNAC56的时空表达模式.最后,利用果实圆片温育方法分析FaNAC56基因受外源激素诱导情况.[结果]FaNAC56基因全长1035 bp,编码344个氨基酸,具有NAC转录因子NAM保守结构域.系统发育分析表明FaNAC56与月季NAC56同源性最高.亚细胞定位显示FaNAC56定位于细胞核内.FaNAC56在果实中高度表达并随果实成熟表达量急剧增加.FFaNAC56启动子区含有ABA、赤霉素、生长素、乙烯等激素和胁迫相关响应元件,其表达水平受这些激素诱导.靶基因分析发现FaNAC56可能响应多种激素、花色苷和蔗糖调控元件.[结论]FaNAC56可能通过多种激素调控草莓果实的发育和成熟.     

扁桃AcCBF1转录因子的克隆及表达分析

【目的】分离和克隆新疆栽培扁桃CBF1转录因子基因,了解其在扁桃响应环境胁迫和逆境相关激素诱导的表达情况。【方法】以新疆扁桃幼苗为试验材料,对其进行低温、干旱、盐及ABA处理,使用PCR技术从新疆扁桃‘双果’克隆得到CBF1转录因子基因,通过农杆菌将35S-Ac CBF1-GFP融合质粒转化洋葱表皮细胞后在激光共聚焦显微镜下观察结果,并通过实时荧光定量PCR技术分析了SG-Ac CBF1转录因子基因在不同非生物逆境胁迫下的表达情况。【结果】序列分析表明,该基因编码框为729 bp,编码242个氨基酸,蛋白质分子量为27.405 ku,命名为SG-Ac CBF1,GenBank登录号为KM245571。氨基酸序列同源比对分析证实,SG-Ac CBF1属于CBF转录因子家族基因。系统发育树分析表明,SG-Ac CBF1与甜樱桃Pa DREB1的亲缘关系最近。亚细胞定位分析显示,SG-Ac CBF1蛋白定位于细胞核中。实时荧光定量PCR分析结果表明,低温、干旱、盐及ABA均能诱导SG-Ac CBF1基因表达。【结论】SG-Ac CBF1转录因子具有核定位功能,并参与了植物对逆境胁迫的调控。由于该转录因子属于家族基因,因此其对环境胁迫响应的强弱还有待探讨。     

黄瓜促分裂原活化蛋白激酶基因的生物信息学分析及原核表达

利用RT-PCR技术结合RACE技术,从NO₃^-胁迫下的黄瓜根中克隆出促分裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）基因的同源序列,命名为CsNMAPK,GenBank注册号为DQ812086。生物信息学分析表明,该基因全长1 636 bp,开放阅读框（ORF）1 113 bp,编码370个氨基酸。亚细胞定位预测其编码蛋白可能定位于细胞质;跨膜结构分析表明该基因有一个强的跨膜螺旋结构;PlantCare分析结果显示该基因序列具有脱落酸诱导、茉莉酸诱导、赤霉素诱导、水杨酸诱导、伤害诱导等顺式作用元件。成功构建原核表达载体,并转化E.coli BL21（DE3）,SDS-PAGE检测结果显示表达了一个约46 kD的蛋白。为进一步揭示黄瓜MAPK基因在NO3-胁迫中的作用奠定基础。     

草莓八氢番茄红素脱氢酶基因pds的克隆及特征分析

 采用RT-PCR和RACE技术从草莓果实中克隆到草莓类胡萝卜素合成途径中关键基因pds,该cDNA全长2 043 bp,具有一个1 704 bp的完整开放阅读框（ORF）,编码568个氨基酸。序列分析表明,pds编码的氨基酸序列与其它植物的PDS蛋白有很高的相似性。系统进化树分析显示,草莓PDS与杏的PDS蛋白亲缘关系比较近。原核表达结果表明pds基因在大肠杆菌中获得高效表达。利用半定量RT-PCR技术进行组织表达模式分析发现,pds基因在草莓的花、叶片和果实中均有表达,表达量为红果＞粉红果＞白果＞花＞青果＞叶。     

森林草莓FvbHLH130转录因子调控植株提前开花

分析草莓碱性螺旋—环—螺旋（basichelix-loop-helix,bHLH）转录因子家族,鉴定到森林草莓（Fragaria vesca）花特异表达的转录因子基因FvbHLH130。FvbHLH130编码区序列长度为960 bp,编码319个氨基酸,保守结构域预测结果显示第247～297 aa是bHLH结合域,其属于bHLH家族成员。FvbHLH130启动子含有生长素等激素响应、逆境胁迫和低温响应元件,推测基因表达可能受到生物和非生物胁迫诱导。FvbHLH130蛋白氨基酸序列与月季（Rosa chinensis）的相似性高达93.70%,与其他蔷薇科bHLH蛋白聚在一个分支上,与拟南芥AtFBH4是同源蛋白。农杆菌介导的FvbHLH130拟南芥转基因株系提前7 d开花,且促进开花相关基因AtAP1、AtFT、AtFUL和AtCO的表达。酵母双杂交实验结果表明FvbHLH130与FvARF4和FvARF6互作,可能作为蛋白复合体共同参与开花调控。     

垂枝与直枝梅花PmTAC1启动子序列差异与垂枝性状的关系初探

在前期转录组数据的基础上对梅花（Prunus mume）垂枝候选基因PmTAC1(TILLER ANGLE CONTROL1)进行了克隆及相关分析。PmTAC1编码区全长915bp，编码304个氨基酸，且在梅花垂枝和直枝品种中无序列差异。氨基酸序列比对进一步证实Pm TAC1具备IGT家族典型结构域，进化树分析表明PmTAC1与桃（Prunus persica）的PpeTAC1亲缘关系最近，亚细胞定位预测该蛋白定位于细胞核和叶绿体，蛋白疏水性分析显示其具有亲水性。PmTAC1在茎中表达最高，叶和顶芽次之。PmTAC1在垂枝品种的1年生枝条中的表达显著高于直枝品种，且在两种枝型枝条近远轴侧均有差异。垂枝品种PmTAC1的启动子序列长度为1 608 bp，直枝品种启动子序列长度为1 379 bp。启动子顺式元件预测到光响应、赤霉素合成、脱落酸响应顺式元件的数量在两种枝型品种间具有差异。推测PmTAC1在垂枝和直枝梅花品种中的启动子序列及表达量差异可能与垂枝性状形成有关。     

金线莲ArLBD1基因克隆、亚细胞定位及表达分析

以金线莲(Anoectochilus roxburghii)叶片的转录组数据为基础,采用RT-PCR技术克隆到了金线莲ArLBD1转录因子基因,采用生物信息学、原核表达、Western blot、亚细胞定位及qPCR等方法对该基因功能进行初步分析,以期为进一步研究其功能提供参考依据。结果表明：ArLBD1基因CDS长度864 bp,编码287个氨基酸。ArLBD1蛋白分子量为30.62 kD,理论等电点为6.25,不稳定系数55.55,属于不稳定蛋白。ArLBD1蛋白含有LOB superfamily保守结构域,第7～28氨基酸为C基序,第36～84氨基酸为GAS基序,第89～107氨基酸为类亮氨酸拉链基序,具有完整的类亮氨酸拉链基序,属于ClassⅠ。系统进化分析表明ArLBD1与拟南芥AtLBD36的亲缘关系最近,其次是AtLBD6。SDS-PAGE电泳及Western blot结果显示,ArLBD1基因在大肠杆菌中成功诱导表达。亚细胞定位分析发现ArLBD1蛋白定位在细胞核。qPCR分析结果表明,ArLBD1基因在金线莲叶、茎和根中都能检测到表达,在叶片表达量最高,其次是根,茎的...     

西瓜ClWRKY54基因的克隆、亚细胞定位及表达分析

WRKY转录因子是植物中所特有的一类转录因子,以基因家族形式存在,并在植物的生长发育和逆境响应等过程中发挥重要的作用。采用RT-PCR的方法从西瓜中分离得到一条西瓜CIRKY54基因。序列分析表明,该基因开放阅读框全长1 428 bp,编码475个氨基酸。其编码蛋白分子量约为51.82 KD,等电点为6.17。该基因蛋白序列中包含2个WRKY保守结构域,锌指结构为C2H2型,属于典型的1类WRKY基因。进化树分析显示,该基因蛋白序列同葫芦科作物中的甜瓜、黄瓜、西葫芦等的WRKY26具有较高的同源性,处于同一分支上。亚细胞定位分析显示,该基因编码蛋白定位于细胞核中。对该基因进行荧光定量PCR分析研究其表达特性,结果显示该基因在西瓜根、茎、叶中均有表达,但是在叶片中表达量最高;H_2O_2可以诱导该基因的表达,而乙烯处理可以抑制该基因的表达,这说明该基因可能参与逆境下H_2O_2和乙烯所介导的信号途径。在模拟干旱下,该基因表达无变化,说明该基因同干旱胁迫抗性不相关。本研究的结果将为进一步的解析ClWRKY54的功能奠定基础。     

草莓FaWRKY31的克隆、亚细胞定位及表达特性分析

为明确草莓FaWRKY31的序列特征、表达特性和亚细胞定位情况,通过同源克隆的方法从八倍体草莓‘红颜’中得到3条长度为1 644 bp、1条长度为1 669 bp的cDNA序列和两条长度约为2 000bp的启动子序列。生物信息学分析表明:长度为1669bp的FaWRKY31-like因插入了一段25bp的短序列导致开放阅读框提前终止,缺失了WRKY结构域和C2H2锌指结构。启动子的序列分析结果表明:FaWRKY31与森林草莓FvWRKY31的启动子序列存在较大差异,相似性仅为70%,FaWRKY31的启动子序列发生了多个片段的缺失和插入,可能导致其与森林草莓FvWRKY31有不同的诱导特性。qRT-PCR结果表明:FaWRKY31在草莓的根、茎、叶、花及果实中均有表达,根中的表达量最高,全红期果实中的最低。FaWRKY31能同时响应红光和蓝光的调控,抑制其在草莓果实中的表达。此外,FaWRKY31能在不同程度上响应低钾、低磷、低温、ABA、盐害、干旱这6种非生物胁迫,且低温、ABA及干旱都显著抑制FaWRKY31的表达。亚细胞定位结果显示FaWRKY31蛋白定位于细胞核内。     

光皮桦BBX类基因BlCOL13的克隆与表达分析

利用RACE、RT-PCR以及基因组步移技术克隆得到光皮桦BlCOL13全长cDNA（GenBank登录号：KP663425）、全长DNA及其启动子序列。该基因DNA全长2 808 bp,具有4个外显子,3个内含子;cDNA全长1 370 bp,包含1 140 bp的开放阅读框,编码含379个氨基酸的蛋白。BlCOL13蛋白N、C端分别有两个B-box和一个CCT结构域,属于典型的BBX蛋白。以拟南芥中BBX蛋白的分类为参照,通过聚类分析表明BlCOL13属于Ⅱ型BBX蛋白;克隆得到的BlCOL13启动子序列长685 bp,含有多个与成花、光响应、激素信号转导以及逆境胁迫相关的顺式作用元件。亚细胞定位试验结果表明该基因主要在细胞核中表达。不同组织中该基因都有一定程度的表达,但在雄花中表达量最高。低温、干旱、ABA和盐胁迫等非生物逆境都在一定程度上诱导BlCOL13的表达,盐胁迫的影响最为明显,表明该基因在光皮桦逆境响应中可能发挥比较重要的作用。     

Effects of radiation from 188Re on smooth muscle cell proliferation

AIM: Although endovascular radiotherapy inhibits neointimal hyperplasia, the exact alterations induced by β-particles irradiation remain to be elucidated. The objective of this study was to investigate the ability and the cellular mechanism of local β^-particles emission from ¹⁸⁸Re to inhibit vascular smooth muscle cells (SMCs). METHODS: The SMCs in vitro were irradiated by ¹⁸⁸Re with single doses of 2.6 Gy-25.8 Gy. The effects of β-particles on SMCs, such as effective irradiate doses, the period of inhibition for SMCs proliferation, the changes of cell proliferation rate and DNA synthesis rate, cell cycle progression and related gene expression, were investigated by cell count, [³H]-TdR incorporation, cell cycle progression analysis, cell viability and immunocytochemistry, respectivecy. RESULTS: β-particles irradiation with dose of 5.2 Gy could inhibit significantly SMCs proliferation. At dose of 20.6 Gy DNA synthesis inhibitory rate was 92%, SMCs proliferation rate was only 3%. Renoval of ¹⁸⁸Re did not abolish the inhibitory effects of β-particles on SMCs proliferation. The expression of P53 was up regulation and PCNA was down regulation after irradiation. CONCLUSION: β-particles from ¹⁸⁸ Re was significantly effective and permanent in inhibiting SMCs proliferation, and inhibitory effect was in dose-dependet manner ED50was 5 Gy, the best dose to inhibit SMCs proliferation was 20 Gy. β-particles irradiation induced SMCs to occur G₀/G₁ arrest, damaged the ability of SMCs reproliferation and led to cell clonogenic death. P53 and PCNA had regulatiory effects on SMCs proliferation after β-particles irradiation.     

Effect of L-Arg on plasma content of endothelin and expression of c-fos mRNA in the left ventricle of rats with renovascular hypertensive hypertrophy

AIM:To study the effect of L-Arg on plasma content of endothelin (ET) and the expression of proto-oncogene c-fos mRNA in the left ventricle of rats with renovascular hypertensive hypertrophy. METHODS: The level of c-fos mRNA were measured by in situ hybridization. The ET in plasma were measured by radioimmunoassay. RESULTS:After eight weeks of treatment with L-Arg, the expression of c-fos decreased markedly (P＜0.01). The ET content in plasma also decreased significantly by L-Arg(P＜0.01).CONCLUSION: Plasma ET content and the expression of c-fos in the left ventricle of rats with renovascular hypertensive hypertrophy could be decreased by L-Arg administration.     

Relationship between renal hypertension and cardiac α1-adrenoceptor autoantibodies in rats

AIM: To examine the autoantibody against α₁-adrenoceptor and its biologic activities during the development of renal hypertension. METHODS: Renal hypertension of rat was achieved by clipped renal artery, the titre of autoantibody to α₁-adrenoceptor was detected using ELISA immunoassay. Furthermore, the biological offects of these autoantibodies on cultured cardiomyocytes were also examined. RESULTS: After two weeks of clipping renal arteries, both the frequency of occurrence and the titre of autoantibodies to cardiac α₁-adrenergic receptor were significantly increased as compared with the control of pre-treatment. The increased autoantibodies lasted for several weeks and then automatically decreased gradually to the pre-clipping level at 12 weeks. The biological effects of these autoantibodies displayed an "agonistic-like" activities on the beating frequency of cultured neonatal cardiomyocytes. CONCLUSION: Autoantibodies against α₁-adrenoceptor may play a role in the elevation of peripheral vascular resistance and in the development of cardiac hypertrophy in rats with renal hypertension.     

Compositional and Functional Properties of Saskatoon Berry and Blueberry

Abstract

Saskatoon berry (Amelanchier alnifolia Nutt., Rosaceae) and blueberry (Vaccinium corymbosum L., Ericaceae) are substantially equivalent in all characteristics that are important to the consumer, including fruit color, shape, size, nutrition, texture, and uses. In addition, both fruits are native to North America and they have practically identical historical uses and known health benefits. Their composition, processing, nutritional value and metabolism, intended uses, and levels of undesirable substances are compared.     

Influence of Mineral Nutrients on Strawberry Fruit Quality and Their Accumulation in Plant Organs

Abstract

This review is based partly on complete articles and partly on abstracts. Three of the 60 articles deal with the total uptake of elements in strawberry plant organs in two different strawberry production systems, both considered as optimal concerning amount and balance of elements. The effect on fruit quality may be dramatic if the level of a particular element is outside this range, but there may also be effects initiated by differences within the optimal range of elements. Most articles refer to product oriented quality, but some focus on consumer oriented quality, as discussed by Shewfelt (1999). The discussion here is on a general basis, so one should keep in mind that there are cultivar differences and that specification of nutrition ideally should mirror the needs of a single cultivar, or a group of cultivars with similar requirements. Also, to get a complete understanding of the subject future reviews should embrace a broader access of information including the effect on plant development of individual elements, such as the role of calcium in fruit firmness and its importance in cell wall structure. However, the intention here is to narrow the information to results that suggest a direct connection between nutrient uptake and fruit quality.     

Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia

The objective of this study was to establish a cryopreservation protocol for hawthorn shoot apices (Crataegus pinnatifida Bge.). Cryopreservation was carried out via encapsulation–dehydration, vitrification, and encapsulation–vitrification on shoot apices excised from in vitro cultures. We began by showing that cold-acclimation enhanced the regrowth of cryopreserved apices from 10.0 to 65.5% in encapsulation–dehydration. We then decided that the encapsulation–dehydration method was an optimal cryopreservation method for hawthorn shoot apices in terms of its high recovery after cryopreservation as well as its ease of use compared with vitrification and encapsulation–vitrification. In encapsulation–dehydration, the protocol leading to optimal regrowth was as follows: after cold-acclimation at 5 °C in the dark for 2 weeks, excised shoot tips were pretreated for 24 h at 25 °C on hormone-free Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497] (MS) basal medium with 0.4 mol/L sucrose, then encapsulated and precultured in liquid MS medium with 0.8 mol/L sucrose for 16 h at 25 °C. Precultured beads were dehydrated for 6 h at 25 °C in the dessicator containing 50 g silica gel to a moisture content of 15.3% (fresh-weight basis) before cryostorage for 1 h. In addition, we examined the effect of adding glycerol to both the alginate beads and loading solution to enhance regrowth after cryopreservation in encapsulation–dehydration. In the present study, it was shown that adding 0.5 mol/L glycerol resulted in high regrowth percentages (82.5–90.0%) in four Crataegus species.     

多效唑对猕猴桃离体试管苗生长及内源激素的影响

多效唑（ＰＰ３３３）处理猕猴桃试管苗，降低了其生长强度；植株体内的ＧＡ３、ＩＡＡ和ＺＴ含量下降，ＡＢＡ的含量上升，乙烯释放率增加；并且能降低外源的ＧＡ３和ＩＡＡ促进生长的作用，而外源的ＧＡ３和ＩＡＡ又能不同程度地逆转多效唑的抑制作用，使植株恢复生长。     

Proteomic analysis between pathological scars and normal skin

AIM: To investigate and screen the sensitive proteins in the formation mechanism of pathological scars by comparing the results of differential proteomic analysis between pathological scars and normal skin.METHODS: Two-dimensional gel electrophoresis was used to detect the protein expression profiles in 8 keloid patients, 8 hypertrophic scar patients and 3 matched normal skin patients.The proteins that showed differential expression of over 4-fold change were cut and analyzed by MALDI-TOF/TOF mass spectrometry.RESULTS: A two-dimensional protein profiling comparison between pathological scars and normal skin was successfully established.On average, 2 978 spots in keloid, 2 975 spots in hypertrophic scar and 3 053 spots in normal skin were identified using gel analysis software.Compared with normal skin, there were totally 36 differentially-expressed proteins in keloid and hypertrophic scar identified from the spots of over 4-fold change, including 16 proteins in both keloid and hypertrophic scar (8 up-regulated and 8 down-regulated), 11 only in keloid (9 up-regulated and 2 down-regulated) and 9 only in hypertrophic scar (4 up-regulated and 5 down-regulated).CONCLUSION: Proteomic analysis can identify the proteins with variance of pathological scars versus normal skin, thus providing probable new clues to reveal the formation mechanism of pathological scars.     

Mixed-severity fire regime in a high-elevation forest of Grand Canyon,Arizona, USA

Fire regime characteristics of high-elevation forests on the North Rim of the Grand Canyon, Arizona, were reconstructed from fire scar analysis, remote sensing, tree age, and forest structure measurements, a first attempt at detailed reconstruction of the transition from surface to stand-replacing fire patterns in the Southwest. Tree densities and fire-/non-fire-initiated groups were highly mixed over the landscape, so distinct fire-created stands could not be delineated from satellite imagery or the oldest available aerial photos. Surface fires were common from 1700 to 1879 in the 4,400 ha site, especially on S and W aspects. Fire dates frequently coincided with fire dates measured at study sites at lower elevation, suggesting that pre-1880 fire sizes may have been very large. Large fires, those scarring 25% or more of the sample trees, were relatively infrequent, averaging 31 years between burns. Four of the five major regional fire years occurred in the 1700s, followed by a 94-year gap until 1879. Fires typically occurred in significantly dry years (Palmer Drought Stress Index), with severe drought in major regional fire years. Currently the forest is predominantly spruce-fir, mixed conifer, and aspen. In contrast, dendroecological reconstruction of past forest structure showed that the forest in 1880 was very open, corresponding closely with historical (1910) accounts of severe fires leaving partially denuded landscapes. Age structure and species composition were used to classify sampling points into fire-initiated and non-fire-initiated groups. Tree groups on nearly 60% of the plots were fire-initiated; the oldest such groups appeared to have originated after severe fires in 1782 or 1785. In 1880, all fire-initiated groups were less than 100 years old and nearly 25% of the groups were less than 20 years old. Non-fire-initiated groups were significantly older (oldest 262 years in 1880), dominated by ponderosa pine, Douglas-fir, or white fir, and occurred preferentially on S and W slopes. The mixed-severity fire regime, transitioning from lower-elevation surface fires to mixed surface and stand-replacing fire at higher elevations, appeared not to have been stable over the temporal and spatial scales of this study. Information about historical fire regime and forest structure is valuable for managers but the information is probably less specific and stable for high-elevation forests than for low-elevation ponderosa pine forests.This revised version was published online in May 2005 with corrections to the Cover Date.     

Metallothionein inhibits homocysteine-induced proliferation of rat vascular smooth muscle cells

AIM:To investigate the effect of metallothionein(MT) on proliferation of rat vascular smooth muscle cells (VSMCs) stimulated by homocysteine and its mechanism. METHODS:VSMCs proliferation was measured by [³-H]-TdR incorporation, mitogen-activated protein kinase(MAPK)activity were determined by immunoprecipitation method, the intracellular contents of MT and malondialdehyde (MDA)were assayed by -hemoglobin saturation method and TBA reaction, respectively, and lactate dehydrogenase (LDH) leakage was measured by NADH oxidation. RESULTS:Hcy(10^-6-10^-4 mmol/L) stimulated [³-H]-TdR incorporation by the VSMCs in a concentration-dependent manner. Compared with control, [³-H]-TdR incorporation in VSMCs treated with 0.1 mmol/L Hcy was increased by 4.2 fold (P＜0.01). Meanwhile, Hcy enhanced MAPK activity, MDA formation and LDH release (P＜0.01)in a concentration-dependent manner. Treatment of VSMCs with MT alone did not change above parameters, compared with control. However, MT (10^-6-10^-4 mol/L)attenuated significantly Hcy-stimulated proliferation of VSMCs (P＜0.01)in a concentration-dependent manner. And MT inhibited obviously Hcy-induced activation of MAPK activity, MDA formation and LDH release. Preincubation of VSMCs with 0.5 mmol/L ZnCl₂ for 6 h induced an increase cellular MT content by 5.7-fold (P＜0.01). The MT-overexpressed VSMCs resisted Hcy-stimulating action on MAPK activity, MDA formation and LDH leakage (P＜0.01). CONCLUSION:These results show that MT has an inhibitory effect on Hcy-induced VSMCs proliferation, and that MT could inhibit Hcy-stimulated MAPK activity and lipid peroxidation.