小麦叶片暗诱导衰老期间内肽酶的特性

研究了小麦(Triticum aestivum L.cv.Yangmai 158)叶片暗诱导衰老期间内肽酶同工酶的变化及其部分生化特性,发现叶片衰老期间,内肽酶活性升高,同时出现5种新的内肽酶同工酶(EP1、EP2、EP4、EP5、EP6)。6-苄氨基嘌呤(6-BA)处理能延缓这些同工酶的出现,而脱落酸(ABA)处理则加速它们的表达。衰老期间小麦叶片内的6种内肽酶同工酶(EP1-EP6)中的EP1、EP2、EP4、EP5、EP6呈现活性的pH及温度范围较窄,而EP3有活性的pH范围和温度范围均较宽,且EP3在嫩叶、老叶中均有活性。另外,EP3、EP5、EP6对热不太敏感。蛋白酶抑制剂实验表明,EP1、EP2是需金属离子的半胱氨酸型内肽酶,EP4是丝氨酸型内肽酶。     

紫萍叶状体衰老过程中的内肽酶谱变化和外源L-丝氨酸对内肽酶谱的影响

采用双向凝胶电泳技术分析紫萍叶状体衰老期间内肽酶同工酶的变化以及外源L-丝氨酸对内肽酶影响的结果表明,在衰老的紫萍叶状体中检测到9种内肽酶同工酶,丝氨酸内肽酶EP3可能在叶状体衰老的早期起作用,而半胱氨酸内肽酶EP9在第6天出现,是衰老后期活性最强的内肽酶。培养液中加入外源的L-丝氨酸后,叶状体蛋白质含量下降加剧,与衰老相关的内肽酶EP3、EP4和EP9的活性明显增强或提前出现。     

人类免疫缺陷病毒(HIV-1)整合酶抑制剂筛选及其活性测定

整合酶作用的病毒DNA整合进宿主DNA的过程是反转录病毒在宿主细胞中增殖的关键步骤.由于在正常人类细胞中不存在相似的功能蛋白,其抑制剂对人体的副作用可能很小,相对于经典AIDS治疗药物的毒副作用,整合酶抑制剂理论上要具有优势.在线性七肽库中筛选与整合酶有特异结合作用的噬菌体展示肽,选取TPSHSSR和HPERATL 2条肽,它们可以竞争性地抑制展示相应肽段的噬菌体与整合酶的结合,同时它们对整合酶的整合活性也有一定程度的抑制,半数抑制率分别为IC50=(54.56±5.18)μmol/L,IC50=(28.29±1.32)μmol/L.这些多肽可用于治疗艾滋病新药的开发应用及整合酶结构及作用机制的研究.     

外源脱落酸对小麦幼苗抗镉胁迫能力的影响

以小麦(Triticum aestivum L.)为试材,采用水培法研究了100mg/L镉(Cd2+)胁迫条件下施用外源脱落酸(ABA)对小麦幼苗生长及某些生理生化指标的影响。结果表明:(1)100mg/L Cd2+胁迫下,小麦叶片膜脂过氧化产物丙二醛(MDA)含量显著提高,植株生长受到抑制;(2)外源ABA能够明显提高Cd2+胁迫小麦幼苗的根系活力,增加其叶片SOD、CAT和POD活性,促进其脯氨酸积累,降低MDA的含量,并以5.0μmol/L ABA的效果最明显;(3)1.0～5.0μmol/L外源ABA不同程度地缓解Cd2+胁迫对小麦幼苗生长的抑制作用,且5.0μmol/L时效果最明显,其株高、根长、总干重分别比单一Cd2+胁迫处理显著提高6.73%、149%和10.52%,而10.0μmol/LABA反而加重了Cd2+对小麦幼苗生长的伤害。因此,适宜浓度的外源ABA能够通过增加体内保护酶活性和脯氨酸含量来缓解Cd2+胁迫对小麦幼苗生长的抑制作用,增强小麦幼苗的抗Cd2+胁迫能力,并以5.0μmol/L ABA处理效果最好。     

第25届国际生物学奥林匹克竞赛试题 理论A-3

正18.赤霉酸(GA)和脱落酸(ABA)对分离的大麦糊粉层细胞的影响用α-淀粉酶活性和转录响应进行测量评估。糊粉层细胞用1μmol/L GA3和50μmol/L ABA处理15 h。用麦芽糖作为标准测定α-淀粉酶的活性(图A)。编码高等电点淀粉酶(Contig3952)和低等电点淀粉酶(Contig7087)的     

外源ABA对低温不同光周期下四季秋海棠叶片花色素苷的诱导机理

以‘超级奥林匹克’四季秋海棠(‘Super Olympia’ Begonia semperflorens)为材料,于常温（25 ℃/15 ℃）等日照条件下用0、5、10、50、100、500 μmol/L脱落酸(ABA)和低温（15 ℃/6 ℃）不同光周期下用10 μmol/L ABA分别喷施各处理植株,对不同处理下植株的色素含量、内源激素含量及其酶活性进行比较分析,探讨ABA对其叶片花色素苷合成的调控作用及其机理。结果显示：（1）常温等日照条件下,四季秋海棠叶片在5和10 μmol/L ABA处理后的第3~5天有明显变红趋势,且花色素苷含量和内源ABA含量显著增加,而内源赤霉素（GA）含量的下降幅度较为显著,相应的花色素苷合成关键酶和运输酶活性也显著提高。（2）低温条件下,四季秋海棠叶片花色素苷积累量与光周期密切相关,短日照处理的花色素苷积累量最大并显著高于等日照和长日照,但等日照与长日照下花色素苷积累量无显著差异;外源10 μmol/L ABA处理均可显著提高低温不同光周期下四季秋海棠叶片的花色素苷含量。（3）外施10 μmol/L ABA增加了低温下长日照和等日照处理中DFR（二氢黄酮醇 4 还原酶）还原反应中H供体NADPH的含量,促使DFR和UFGT（糖苷转移酶）的活性上调进而增加了花色素苷的含量;外施ABA处理均能够提高低温不同光周期处理组的内源ABA含量,降低内源GA的含量,与花色素苷的生成量相一致。研究表明,外源ABA能够通过调节花色素苷合成的关键酶来影响花色素苷的合成,外施适宜浓度ABA能够促进四季秋海棠叶片花色素苷的积累,可用于实际生产中的叶片着色调控管理。     

黄瓜花期高温胁迫对叶片衰老特性和内源激素的影响

为了研究黄瓜花期高温胁迫对植株叶片衰老特性及内源激素的影响,本研究以黄瓜‘南杂2号’(Nan ZaⅡ)品种为试材,利用人工气候箱设计3个温度处理:昼温/夜温分别为36℃/26℃、39℃/29℃、42℃/32℃,以昼温/夜温24℃/18℃为对照(CK),持续时间设计1、3、5和7 d。处理完成后分别测定黄瓜叶片叶绿素含量、抗氧化酶活性、MDA含量和顶芽内源激素含量。结果表明:42℃/32℃处理7 d时黄瓜叶片叶绿素a、叶绿素b、类胡萝卜素含量,POD、SOD活性,顶芽IAA、GA3、ZT含量分别较CK降低了16.61%、23.17%、73.33%、17.89%、56.90%、94.16%、54.32%、90.68%;而叶片MDA含量、CAT活性和顶芽ABA含量分别较CK增加了62.66%、28.06%和294.35%;短期高温胁迫增加了黄瓜叶片的叶绿素含量,随着温度的升高,胁迫时间的延长叶片叶绿素b含量逐渐降低;短期高温胁迫增加了叶片抗氧化酶活性,随着高温胁迫的加剧,抗氧化酶活性表现出逐渐降低的趋势;高温胁迫下黄瓜顶芽ABA含量表现出增加的趋势,而IAA、GA3和ZT含量的变化则相反。本研究揭示了黄瓜花期高温胁迫下叶片衰老特性和内源激素含量的变化规律,为黄瓜栽培的温度管理提供科学依据。     

吲哚丁酸通过蛋白磷酸化激活湖北海棠根系Ca2+-ATP酶

以湖北海棠(Malus hupehensis Rhed.)实生苗为试材,通过在砂培液中加入吲哚丁酸(IBA)和蛋白激酶抑制剂3,3’,4’,5,7-五羟黄酮(quercetin)研究了IBA对根系膜蛋白磷酸化和Ca2 -ATPase活性的影响.试验表明根系膜蛋白磷酸化反应主要发生在丝氨酸残基上100 μmol/L的IBA使蛋白激酶和Ca2 -ATPase活性在2～3h内升高数十倍,之后很快下降,蛋白激酶活性变化明显早于Ca2 -ATPase;蛋白激酶抑制剂quercetin不仅抑制根系膜蛋白的磷酸化,也显著削弱IBA对Ca2 -ATPase的激活作用.结果显示,在对IBA响应中Caa2 -ATPase是信号转导途径中的成员,IBA可能通过蛋白磷酸化激活根系Ca2 -ATPase而起作用.     

ABA和别嘌呤醇对黄化黄瓜子叶脂氧合酶活性的影响

脂氧合酶(LOX,EC1.13.11.12)是植物组织中唯一催化不饱和脂肪酸氢过氧化作用的酶,植物衰老时,其活性增高。Leshem等(1982)认为别嘌呤醇抑制黄嘌呤氧化酶反应产生自由基和抑制脂氧合酶活性。以黄瓜(Cucuis sativus)刺春品种的黄化幼苗子叶分别浸入5ppm ABA,5ppm ABA+5ppm α-生育酚和5ppm α-生育酚溶液及水中,24小时后切取子叶,经预冷后加入50m mol/L磷酸盐缓冲液(pH 7.0)和1％PVP(w/v),在研钵中研磨,匀浆通过过滤和3000×g 4℃下离心15分钟。根据Peterman等方法,以Clark氧电极测定上清液酶活性。参照Surry方法,以Beckman DU-7型分光光度计测定脂质氢过氧化物     

镉胁迫对旱柳光合作用和内肽酶变化的影响

通过水培方法,添加不同浓度CdCl2(0、5、25、50 μmol·L^-1)处理14 d,测定叶绿素含量、核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)和磷酸烯醇式丙酮酸羧化酶(PEPC)活性、游离氨基酸含量及内肽酶活性,调查Cd对旱柳光合作用和内肽酶变化活性影响。结果发现：Cd处理降低了总叶绿素、叶绿素a、b含量; Rubisco活性随着介质中Cd浓度增加而降低;Cd抑制根和叶PEPC活性;同对照相比,根中游离氨基酸含量没有显著变化,而叶中游离氨基酸含量增加;不同浓度Cd处理降低根的内肽酶活性,高浓度Cd使叶内肽酶活力增加。这些结果表明,Cd通过降低叶绿素含量,促进叶内肽酶活性和抑制了CO₂羧化酶活性来影响旱柳光合作用。     

Endopeptidase Isoenzyme Characteristics in Cucumis sativus Leaves During Dark-induced Senescence

The changes and characteristics of endopeptidase （EP） isoenzymes in cucumber （Cucumis sativus L.） leaves during dark-induced senescence were investigated by activity staining after gradient-polyacrylamide gel electrophoresis （G-PAGE） containing co-polymerized gelatin as substrate. The results showed that both the chlorophyll and the protein contents of leaves were decreased, and the protein degradation was correlated with the increase of proteolytic activity during the course of leaf senescence. Meanwhile, nine cucumber endopeptidases isoenzymes （CEP） with 140, 120, 106, 94, 76, 55, 46, 39 and 35 kDa molecular weights were detected. Four of these, CEP2, 3, 4 and CEP9 appeared all the time, but the changes of the activity were different during incubation. Another four CEPs （CEP5, 6, 7 and CEP8） whose activities increased with dark-induced time were only detected in senescent leaves. Furthermore, the biochemical properties of these nine CEP were also characterized. All the CEPs had high activities from 35 ℃ to 45 ℃, and the optimum temperature was found to be 40 ℃. However, the activities of CEPs were not detected below 25 ℃ or over 60 ℃. The activity bands appeared at a wide range of pH from 5.0 to 9.0, but the optimum pH was found at 7.0. No CEPs were detected at pH 4 or pH 10. By inhibition analysis we concluded that CEP2, 3, 4 and CEP9 were serine endopeptidases and CEP6 was a kind of cysteine protease. It is suggested that serine endopeptidases might play a major role in cucumber leaf senescence, and for the first time, six senescencerelated endopeptidases （CEP1, 5, 6, 7, 8 and 9） were found in cucumber leaves.     

6-BA延缓大豆叶片衰老的作用与膜蛋白磷酸化状态的关系

蛋白激酶（proteinkinase,PK）和蛋白磷酸酯酶（pIDt6inphOSpha～,PP）是生物体内催化蛋白质磷酸化／脱磷酸化过程的两种重要酶类。目前已有越来越多的实验证据表明：这种可逆的磷酸化／脱磷酸化过程所导致的蛋白质（酶）活性的改变是生物体内信号传导过程中的重要环节（Hunter1995）。已有一些实验系统涉及了植物激素对于植物蛋白磷酸化过程的影响（Mi－zogUchi等1994,Sano和Youssefian1994）,并有一些与此相关的蛋白激酶和蛋白磷酸酯酶的基因被克隆（kleber等1993,temp等1994）。细胞分裂素延缓植物叶片衰老的作用早已被各种实…     

棉花叶片衰老过程中激素和膜脂过氧化的关系

以陆地棉品种辽棉9号的去根幼苗为材料,对其进行暗诱导衰老培养.在培养液中分别加入6-BA、ABA、GSH、H2O2、CaCl2、A23187 和A23187 CaCl2,测定在不同培养条件下棉花去根幼苗叶片内源激素、SOD酶活性和MDA含量的变化.结果表明:棉花叶片衰老表现为细胞分裂素含量的下降和ABA含量的上升.6-BA、GSH和钙离子均延缓叶片的衰老,ABA和H2O2促进叶片的衰老.     

Influence of senescence and of carbohydrate levels on the pattern of leaf proteases in purple nutsedge (Cyperus rotundus)

Cyperus rotundus L. is a monocotyledonous perennial weed, which forms large numbers of tubers during its vegetative growth. Since these tubers represent major sinks, source/sink interactions are more complex, and leaf senescence and proteolytic processes in this species may be different from the situation in the well‐investigated annual crop plants characterized by monocarpic senescence. Judged by native PAGE and by inhibitor studies, three different aminopeptidases, one iminopeptidase, two or more carboxypeptidases and two or more different endopeptidases were present in mature green leaves. Exo‐ and endoproteolytic activities increased during the senescence of excised leaf segments. A marked change was observed in the endopeptidase pattern, since a cysteine proteinase activity was strongly induced during senescence of the segments. This endopeptidase was also found in naturally senescing leaves and may, therefore, participate in nitrogen salvage from these organs. An increase of different protease activities was demonstrated in leaf segments of C. rotundus in the presence of high carbohydrate levels. The mechanisms involved, and the importance of this phenomenon for the interaction between source/sink relations and senescence, remain to be demonstrated.     

盐胁迫对露花叶片的脱落酸含量及磷酸烯醇式丙酮酸羧化酶昼夜调节特性的影响

兼性CAM植物在转为CAM型后,CAM代谢的关键酶磷酸烯醇式丙酮酸（PEP）核化酶会出现昼夜调节特性的变化（Osmond1978）。关于PEP梭化酶昼夜调节特性的机理存在两种观点：1．PEPK化酶昼夜聚合度发生了变化,白天为二聚体PEPK化酶,对苹果酸抑制敏感;而夜间为四聚体,对苹果酸抑制不敏感（U和Wedding1985）。2·nsv＄化酶昼夜磷酸化状态发生变化,夜间PEPW化酶磷酸化,对苹果酸抑制不敏感;而白天PEP＄化酶脱磷酸化,对苹果酸抑制敏感（Nimmo等1986）。植物生长调节物质如ABA和细胞分裂素对兼性CAM植物PEP＆化酶的表达有诱…     

Effects of ABA on Senescence and Cellulase Activity of Detached Rice Leaves

The relationship of cellulase to detached leaf senescence of rice seedlings was investigated by examining the effect of ABA and 6-BA on changes in the level of cellulase of leaf segments during senescence. It was shown that the rise in cellulase activity increased with declining chlorophyll content, which was used as the senescence indicator during the senescence of detached rice leaves caused by ABA. The action of ABA took place only after a 48h lag period. ABA enhanced the cellulase secretion and increased the permeability of plasma membrane. A high level of cellulase activity in cell wall closely related to membrane permeability changes. The action of cellulase in the cell wall may cause depolymerization of β-1, 4-glucan in situ, thus speeding senescence. The 6-BA reverses completely or partly the increase in cellulase activity and tile permeability caused by ABA during the first two day, }) ut it antagonized hardly any of the ABA effect from the third day on, suggesting the onset of an irreversible stage in the senescence of detached rice leaves.     

A high molecular weight glutamyl endopeptidase and its endogenous inhibitors from cucumber leaves

We purified a glutamyl endopeptidase that is a major foliar endopeptidase in cucumber. The endopeptidase had a molecular mass of 400 kDa, consisted of four subunits of 97 kDa, and was inactivated by SH-modifying reagents. Its optimum pH and optimum temperature were 8.0 and 30-37 degrees C, respectively. An internal amino acid sequence of the endopeptidase was highly homologous to a partial sequence of unidentified proteins deduced from genetic information for Arabidopsis thaliana, soybean and rice, but not to the sequences of bacterial glutamyl endopeptidases or animal proteases. Therefore, the unidentified proteins might be glutamyl endopeptidases and be widely distributed only among plant species. The activity of the cucumber glutamyl endopeptidase was inhibited by at least three inhibitors existing in cucumber leaves. One of the inhibitors was a competitive inhibitor of 25 kDa, which did not significantly inhibit commercial endopeptidases derived from animals and microorganisms. This suggests that the cucumber glutamyl endopeptidase might be controlled by endogenous inhibitors in vivo.     

两个大豆类受体蛋白激酶基因的克隆及其结构和功能的初步分析

利用高等植物类受体蛋白激酶基因的保守域设计简并引物的通过RT-PCR方法,从大豆叶片中克隆到两个新的,可能的类受体蛋白激酶基因的部分cDNA片段。对其基因结构的分析表明：在RLPK2的激酶保守域Vib与Ⅸ之间有一个407bp长的内含子。利用RT-PCR方法对它们的表达特性进行初步研究。发现这两个基因可能参与了对大豆叶片衰老和/或细胞分裂素延缓衰老过程的调节机制。     

Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis)

Chinese flowering cabbage is one of the main leafy vegetables produced in China. They have a rapid leaf yellowing due to chlorophyll degradation after harvest that limits their marketing. In the present study, leaf senescence of the cabbages was manipulated by ethylene and 6-benzyl aminopurine (6-BA) treatment to investigate the correlation of leaf senescence and chlorophyll degradation related to gene expression/activities in the darkness. The patterns of several senescence associated markers, including a typical marker, the expression of senescence-associated gene SAG₁₂, demonstrated that ethylene accelerated leaf senescence of the cabbages, while 6-BA retarded this progress. Similar to the trends of BrSAG₁₂ gene expression, strong activation in the expression of three chlorophyll degradation related genes, pheophytinase (BrPPH), pheophorbide a oxygenase (BrPAO) and red chlorophyll catabolite reductase (BrRCCR), was detected in ethylene treated and control leaves during the incubation, while no evident increase was recorded in 6-BA treated leaves. The overall dynamics of Mg-dechelatase activities in all treatments displayed increasing trends during the senescence process, and a delayed increase in the activities was observed for 6-BA treated leaves. However, chlorophyllase activity as well as the expression of BrChlase1 and BrChlase2 decreased with the incubation in all treatments. Taken together, the expression of BrPPH, BrPAO and BrRCCR, and the activity of Mg-dechelatase was closely associated with the chlorophyll degradation during the leaf senescence process in harvested Chinese flowering cabbages under dark conditions.