玉米胚发育过程中热激蛋白的合成

35S-Met标记玉米胚蛋白合成结果表明,热激处理(42℃)与对照(25℃)的蛋白合成趋势相近,热激抑制16 DAP的蛋白合成,增加22和34 DAP蛋白合成.SDS-PAGE自显影图谱表明,热激诱导16DAP的胚合成86.4、80.0、73.2 kD等3种分子量较高的热激蛋白,22DAP后热激诱导合成86.4、80.0、73.2、24.4、18.2、16.8和13.6 kD等7种分子量的热激蛋白.2D-PAGE自显影图谱进一步显示,热激诱导22和28 DAP的胚合成近20种热激蛋白,其中超过10种为小分子热激蛋白.特异热激蛋白BiP(HsP70)、PDI(HsP60)Western blot表明,这2种热激蛋白在玉米胚发育过程均有高水平的表达,热激对其合成影响不明显.     

热激对水稻幼苗耐冷性及热激蛋白合成的诱导

萌发的水稻种子经42℃热激处理后其幼苗的耐冷性明显增强,膜伤害程度降低,脯氨酸含量增加,超氧物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性和抗氧化物质抗坏血酸含量增加,而膜脂过氧化的关键酶脂氧合酶(LOX)活性及其产物丙二醛(MDA)含量下降.并且热激诱导萌发的水稻胚合成78、70、64、60、46、38、24、17、16kD的热激蛋白(HSP),其中属于HSP70的内质网结合蛋白(BiP)的合成与水稻幼苗耐寒性的提高有关.     

不同活力玉米种子胚萌发期间热激蛋白的合成

玉米（Ｚｅａ ｍａｙｓ Ｌ．）种子在萌发期间热激处理（４２℃）时蛋白质合成率低于对照（２５℃）;高活力种子胚热激蛋白的合成率最高。在４２℃热激处理时玉米种胚子合成的热激蛋白的分子量分别为７３、６５、６２、５４、１８ｋＤ等５种。高活力种胚合成的热激蛋白在最上高于低活力种子,高活力种胚合成的特异性热激蛋白５６ｋＤ可以作为衡量种子活力的指标。双向电泳表明高低活力种子间热激蛋白的合成有更多质上的差异。     

寄生虫热激蛋白的研究进展

热激蛋白(Heat shock proteins,HSPs)是所有原核生物和真核生物在发育过程中,或为适应不同环境中合成的一组起关键作用的蛋白质家族.为全面了解寄生虫热激蛋白的研究情况,介绍了熟激蛋白的发现、功能、分类及寄生虫(原虫、线虫、吸虫)热激蛋白(HSP70,HSP90,HSP60,sHSP)的研究进展,并对寄生虫热激蛋白的研究意义作了探讨.     

玉米胚发育过程中内源ABA和GA3的含量变化及其与脱水耐性形成的关系

以发育的玉米肌为材料。用酶联免疫吸附检测技术测定了玉米胚发育和脱水过程中内源ABA和GA2含量的动态变化,以及它们与胚的脱水耐性形成的关系。结果表明．玉米胚在发育过程中其ABA含量峰值出现在GA2含量峰值之前,而且ABA峰值出现在授粉后28d,晚于胚的含水量下降;胚在脱水过程中ABA和GA2的含量都呈先上升后下降的变化。可以认为在脱水过程中ABA含量的增加是胚对脱水胁迫应激反应的结果．有保护胚的作用;但GA2含量增加的生理机制及其作用尚不清楚。在玉米胚的发育过程中．内源ABA和GA3之间的相对平衡可能主要调控胚的发育进程和萌发力的形成;胚的ABA含量变化可能不足以引起其脱水耐性的形成。     

昆虫小分子量热激蛋白的研究进展

昆虫小分子量热激蛋白(Small heat shock proteins,s HSPs)是最早被发现的热激蛋白,但是有关它们的研究相对较少。本文对昆虫小分子量热激蛋白的最新研究成果进行了总结,旨在引起人们对该类蛋白的关注,以便进一步研究其功能,探讨其可能的应用前景。目前研究表明:昆虫小分子量热激蛋白是其所有热激蛋白中最不保守的家族。同时,它们通常拥有一个α-晶状体结构域;分子量范围一般在12~43 ku;具有分子伴侣的活性。每种昆虫体内拥有多种s HSPs,而且其功能也各不相同。这些热激蛋白在昆虫的生长发育、生殖以及滞育等重要生命活动中起着重要的作用;同时在抵御不良环境以及适应性进化中也具有重要意义。随着研究的深入,还将会有更多的昆虫s HSPs被鉴定,它们更多的功能也将被逐渐发掘。     

温度,热激蛋白与高粱育性的变化

高粱不育系3A在热激(43～45℃)诱导下结出了种子,由不育系转变为可育系。比较3A和3B线粒体在热激条件的热激蛋白得知,它们的热激蛋白是由核编码的,在细胞质中合成后才运到线粒体中,热激2h,3A出现70、31、24、18和16kDa 5条蛋白带,3B除出现上述5条蛋白带外还多出现96、94kDa 2条,而且70kDa含量比3A大。热激4h,3B的96、94kDa消失,两系趋于一致。此时,3A和3B线粒体总蛋白比热激前大量增加。此后HSPs急剧降低。热激8h,3B线粒体仅有70、31、24和16kDa 4条蛋白带,70kDa特别明显,而3A则全部消失。从而表明,HSPs在3B中是稳定的,在3A中是缺乏或不稳定的,这些差异可能与3B育性稳定性及3A不育性有关。     

几种热激蛋白在细胞凋亡信号通路中的调控作用

热激蛋白(heat shock proteins, HSPs)作为进化保守的蛋白家族 之一,普遍存在于各种生物体中,并在生物体内发挥着重要的生理功能.大 量的实验证据表明,热激蛋白与细胞凋亡密切相关,参与细胞凋亡信号通 路的多个环节. 近年来有关该领域的研究已获得了重要的突破与进展.一方 面,热激蛋白主要起着抑制细胞凋亡、促进细胞存活的作用;另一方面, 某些热激蛋白又能够作为凋亡蛋白的分子伴侣,促进细胞凋亡,比如HSP70 能够激活DNase来促使细胞凋亡,线粒体内HSP60能够促进caspase依赖的细 胞凋亡途径.本文在阐明细胞凋亡信号通路的基础上,综述了近年来几种不 同热激蛋白家族（HSP90、 HSP70 、HSP60和小分子HSPs）在细胞凋亡调控 中作用的研究进展,重点阐述了几种主要热激蛋白与细胞凋亡信号通路上 相关因子的相互作用,并绘制了热激蛋白在细胞凋亡信号通路中的调控图 ,为进一步完善细胞凋亡调控网络研究提供一定的参考.     

过量表达叶绿体小分子热激蛋白提高番茄的抗寒性

小分子热激蛋白与植物耐寒性提高有相关性,但是没有直接的实验证据能证明小分子热激蛋白的存在增加植物抗寒性.我们克隆了番茄叶绿体(定位)小分子热激蛋白cDNA,并将35SCaMV启动子驱动的番茄叶绿体小分子热激蛋白cDNA植物表达构架导入番茄,测定转基因番茄和未转基因番茄的抗寒性水平.低温处理后,转基因番茄的冷害症状轻于未转基因的番茄;转基因番茄细胞电解质外渗较少、花青素和MDA累积量较低;净光合速率和叶绿体含量高于对照.这些实验结果说明叶绿体小分子热激蛋白的过量表达提高了植物抗寒性.     

果蝇热激蛋白的研究进展

热休克蛋白(heat shock proteins,HSPs)是生物体受到应激刺激时诱导产生的一组保守性蛋白,普遍存在于各种生物体中。近年来,果蝇Drosophila作为生命科学与人类疾病研究的重要模式生物,其热激蛋白的研究取得了许多新的进展。文章对果蝇热激蛋白的类别、热激蛋白基因的表达调控机制、热激蛋白的分子伴侣功能、调节细胞存亡和影响发育及寿命等相关生物学功能进行综述,并对热激蛋白在神经退行性疾病治疗中的应用前景作展望。     

Activity and distribution of nitrogen-metabolism enzymes in the developing maize kernel

The activities of glutamine synthetase (EC 6.3.1.2), glutamate dehydrogenase (EC 1.4.1.2), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and soluble protein content in the developing endosperm and embryo of normal (Oh-43) and mutant (Oh-430₂) maize were investigated. Maize inbred lines were grown under field conditions and all plants were self-pollinated. Ears for experiments were harvested over the period of 15 lo 45 days after pollination. After pollination kernel capacity for soluble protein synthesis is located mainly in the endosperm. This progressively decreases and about 40 days after pollination soluble protein synthesis is taken over by the embryo. Comparative data on the activity of the investigated enzymes in the embryo and endosperm indicate that the capacity for synthesis of glutamine and glutamate predominates in the embryo tissue, whereas transamination processes at the initial stages of the embryo development are less intensive than their counterparts in the endosperm. The roles of embryo and endosperm subsequently interchange. Biosynthetic processes of soluble precursors for protein synthesis in the embryo and endosperm of the developing kernel are mutually coordinated.     

Increases in binding protein (BiP) accompany changes in protein body morphology in three high-lysine mutants of maize

Summary A maize 75 kDa protein recently has been identified as a plant homolog of the mammalian binding protein (BiP). To better understand the function of BiP in protein body formation in maize endosperm, immunomicroscopy studies were conducted on three maize endosperm mutants, floury-2, Mucronate, and Defective endosperm-B 30, in which the level of BiP is highly elevated. Our results showed that protein body morphology in all three mutants was altered. In addition, BiP was localized in both the ER and peripheral regions of the abnormal protein bodies. The degree to which protein body morphology differed from normal was positively correlated with increased amounts of BiP. In addition, the accumulation of BiP in abnormal protein bodies increased with protein body maturation. In the three endosperm mutants, the arrangement of zeins within protein bodies had been perturbed, yet none of the specific zein subclasses exhibited the staining pattern found for BiP. The association of BiP with abnormal packaging of proteins in protein bodies may reflect a biological function to mediate protein folding and assembly in maize endosperm.Abbreviations BiP binding protein - BSA bovine serum albumin - De*-B 30 Defective endosperm B 30 - DAP day after pollination - ER endoplasmic reticulum - fl 2 floury-2 - hsp 70 70 kDa heat shock protein - Mc Mucronate - TBST 20mM Tris-HCl, pH8.2 at 20°C, 500mM NaCl, 0.3% Tween 20 - TBST-B TBST with 1% (w/v) BSA     

小叶兜兰的果实生长和雌配子体发育

为了解濒危兰科植物小叶兜兰(Paphiopedilum barbigerum Tang et Wang)胚珠和雌配子体的发育过程,采用常规石蜡切片技术对其果实的生长动态进行了研究。结果表明,授粉后60~75 d的蒴果内种子数量迅速增加,到授粉后120 d时种子充满整个蒴果。授粉后40 d的胎座上分化形成多数由1层表皮细胞包被1列细胞的胚珠原基;授粉后60 d时位于胎座指状结构末端处紧靠表皮细胞下方的孢原细胞分化为大孢子母细胞。之后,大孢子母细胞经过减数分裂和有丝分裂最终形成成熟胚囊;授粉后135 d胚囊发育成熟,附着在胎座上的种子个体分化明显。小叶兜兰胚囊的发育类型为双孢子葱型,胚珠为倒生胚珠,薄珠心,单珠被,成熟胚囊为8核。这为小叶兜兰的生殖生物学及繁殖体系的建立提供理论依据。     

Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis.     

不同类型玉米籽粒的营养品质及其与籽粒质地的关系

测定普通玉米、爆裂玉米、糯玉米和甜玉米4种类型玉米籽粒不同发育时期的直链淀粉、支链淀粉、总淀粉、可溶性糖和蛋白的含量,分析这些营养物质与角质率的关系。结果表明：灌浆期间4种类型玉米的直链淀粉、支链淀粉和总淀粉含量呈上升趋势,总蛋白含量呈下降趋势,可溶性糖含量变化规律不明显。爆裂玉米的直链淀粉含量始终最高（4．7％-23．1％）,甜玉米（1．4％-4．6％）和糯玉米（2．3％-4．9％）的始终较低;甜玉米的支链淀粉含量一直最低（15．7％-35．5％）,除授粉10d以外,糯玉米的支链淀粉含量一直最高（65．5％-69．8％）;甜玉米总淀粉含量始终最低（17．1％36.1％）、总蛋白含量（15．2％-26．9％）和授粉30d后的可溶性糖含量最高（14．2％-17．6％）。高蛋白含量可能是爆裂玉米和甜玉米角质率高的原因,糯玉米的角质率低可能与支链淀粉含量高和蛋白积累少有关。     

Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant

A maize protein disulfide isomerase (PDI, EC 5.3.4.1) cDNA clone was isolated and characterized. The deduced amino acid sequence contains two regions characteristic of the active sites for PDI and a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, Lys-Asp-Glu-Leu. Southern blot analysis indicated the maize PDI is encoded by a single gene that maps to the short arm of chromosome 4. When isolated from the cisternal and protein body ER, the PDI protein resolves into a fast and a slow form on SDS-PAGE. During endosperm development, the PDI RNA level increases between 10 and 14 days after pollination. In floury-2 (fl2) endosperm, which contains an abnormally processed -zein protein, PDI expression is significantly increased, and the level of PDI protein and RNA is positively correlated with the dosage of fl2 alleles. The increase of PDI in fl2 occurs mainly in the cisternal ER fraction, whereas the most dramatic increase of binding protein (BiP) is in the protein body ER. We propose that the induction of PDI in the fl2 mutant reflects its role as a molecular chaperone, and that PDI functions in concert with BiP at different stages of zein processing and assembly into protein bodies.     

A Rapid Increase in the Level of Binding Protein (BiP) is Accompanied by Synthesis and Degradation of Storage Proteins in Pumpkin Cotyledons

The binding protein (BiP) has been implicated in cotranslationalfolding of nascent polypeptides, and in the recognition anddisposal of aberrant polypeptides. To elucidate the involvementof BiP in the biosynthesis of vacuolar proteins, we have characterizedthe protein in pumpkin cotyledons during seed maturation andseedling growth. Isolated microsomes from maturing pumpkin cotyledonscontained a significant amount of BiP, protein-disulfide isomeraseand calreticulin. We have purified a 70-kDa protein; sequencesof the N-terminus and internal fragments of this protein exhibiteda high identity to the sequence of soybean BiP. Immunoblot analysiswith specific antibodies raised against the purified BiP showedthat the amount of BiP in a cotyledon increased markedly atthe middle stages and then decreased. The increase was accompaniedby the synthesis of storage proteins and the development ofthe endoplasmic reticulum in the cotyledons at the middle stageof seed maturation. Most of these storage proteins degradeddramatically between 2 and 5 days after seed germination, andthe degradation was also accompanied by a rapid increase inthe level of BiP. Subcellular fractionation of the 4-day-oldcotyledons showed a high accumulation of BiP in the endoplasmicreticulum. It is possible that BiP might be involved in thesynthesis of seed storage proteins during maturation and inthe synthesis of hydrolytic enzymes responsible for the degradationof the storage proteins during seed germination. (Received September 18, 1996; Accepted January 8, 1997)     

Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell-free system

The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.     

Fertilization and Embryo Development of Populus lasiocarpa Oliv

The double fertilization and embryo development of P. lasiocarpa were studied using cytochemical method for DNA, RNA, polysaccharides and proteins. Stigmas were covered by secretion stained positively with PAS and mercuric bromphenol blue at receptive stage. The pollen grains germinated on the stigma in large quantities 6 hours after pollination. Fertilization started at the 6th days after pollination. The sperm nucleus fused with the secondary nucleus faster than the sperm nucleus with the egg nucleus. The syngamy belonged to peremitotic type. A great deal of starch grains in the embryo sac disappeared during fertilization. The endosperm was nuclear type and becomed a cel 31 days after pollination. The endosperm was characterized by a dense cytoplasm rich in protein. No discrete starch grains were observed in endosperm. Afterwards, the endosperm was consumed by the developing embryo, thus the mature seeds were non-endospermous. The zygote was dormant for 6–8 days. During the dormency, many striking changes took place, and then, the zygotes showed more pronounced polarity. These changes included the shinkage of the large vacuole, the reduced size, the reappearance of large vacuole, the enlarging of the size. The embryogenesis conformed to the Solanad type. The ovules matured into seeds successively 44 days after pollination. The mature embryo was straight. Two cotyledons folded each other.