胞外及胞内Ca~(2+)共同参与拟南芥中茉莉酸诱导的钙动员

【目的】探究茉莉酸(Jasmonic acid,JA)诱导的钙动员的来源及钙调素(CaM)在JA信号通路中的作用。【方法】以拟南芥(Arabidopsis thaliana columbia)为材料,采用不同类型的离子通透性通道抑制剂(或拮抗剂)预处理拟南芥叶肉细胞,利用激光共聚焦显微镜检测其对JA诱导的[Ca2+]cyt升高的影响;同时还利用上述拮抗剂、钙调素(CaM)拮抗剂处理10d的拟南芥幼苗,采用Northern blot方法检测了其对JA诱导的VSP表达的影响。【结果】胞内及胞外钙离子共同参与JA诱导的钙动员,JA诱导的Ca2+通过CaM或CaM相关蛋白进一步传递JA信号。【结论】质外体钙离子的流入和胞内钙库中钙离子的释放均参与JA诱导的钙离子动员,Ca2+可能通过CaM或CaM相关蛋白传递JA信号,进一步诱导JA反应基因的表达。     

IP_3敏感的钙离子通透性通道参与茉莉酸诱导的钙动员

[目的]研究茉莉酸诱导的钙动员通往IP3敏感的钙离子通透性通道的作用。[方法]以低温导入法将钙离子荧光探针Fluo-3/AM导入拟南芥叶片细胞中,利用LASAF（Leica Application Suite-Advanced Fluorescence）软件记录肝素对茉莉酸（JA）诱导的胞内钙离子荧光强度的变化。[结果]经不同浓度的肝素预处理后,拟南芥叶细胞中胞内钙离子的荧光强度降低,再用100μmol/LJA处理时,其荧光强度升高,但仅与未经肝素处理的荧光强度相当。[结论]肝素预处理可抑制JA诱导的胞内钙离子浓度的升高。     

Extracellular and Intracellular Calcium both Involved in the Jasmonic Acid Induced Calcium Mobilization in Arabidopsis thaliana

The objective of this experiment is to evaluate the role of intracellular and extracellular Ca2＋ and calmodulin （CAM） in jasmonic acid （JA） signaling. The laser scanning microscopy was used to detect the changes of [Ca2＋]cyt of Arabidopsis thaliana leaf cells which pretreated with different types of calcium channel blocker. Moreover, the expression of VSP, one of JA response genes, was also investigated after pretreated with the above blocker and antagonist of CaM. The results showed that extracellular and intracellular calcium both involved in the JA-induced Ca2＋ mobilization, and then Ca2＋ exerted its functions through activating the CaM or CaM related proteins. The apoplast calcium influx and the calcium release from the calcium stores are both involved in the JA-induced calcium mobilization, then the JA-induced Ca2＋ transmited the JA signal through CaM or CaM related proteins, and regulated the JA responsive genes.     

茉莉酸对马铃薯苗期低温胁迫耐性的影响

[目的]研究不同茉莉酸(JA)施用浓度对马铃薯叶片活性氧(ROS)含量及其清除酶活性以及抗寒关键基因CBF表达的影响,初步确定提升马铃薯抗寒性的最适施用浓度,为马铃薯产业的发展提供一定的理论基础.[方法]以马铃薯品种费乌瑞它为研究对象,采用叶面喷施JA的方法,探究正常和低温胁迫条件下,植株叶片的活性氧含量,活性氧清除酶过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶SOD活性,以及抗寒关键基因CBF的表达.[结果]随着JA浓度的增加,马铃薯苗期叶片的活性氧含量逐渐降低,低温胁迫后活性氧含量能维持在较低水平,适宜浓度范围为100～400μmol/L,以200μmol/L JA处理效果最好,ROS含量降低47.6％.JA能够提升活性氧清除酶CAT和POD活性,且随着JA浓度增加效果越显著,低温胁迫后200μmol/L JA处理分别显著提升1.30、2.53倍.正常条件下,JA处理抑制SOD活性最低达70.7％;低温胁迫条件下,JA又可能通过其他途径提升SOD活性,最高达2.11倍.外源JA能诱导CBF1-3基因的表达,而低温胁迫能促进内源JA的合成,诱导CBF1-3基因的表达,提升马铃薯的抗寒性.[结论]马铃薯苗期的抗寒性与外源施加JA的浓度具有明显的相关性,适宜的JA浓度能够促进抗寒关键基因CBF1-3的表达,显著提升CAT、POD、SOD的活性,抑制低温胁胁迫诱导的活性氧生成,提高马铃薯抗寒性.适宜施用JA浓度为100～400μmol/L,以200μmol/L效果最佳.     

ABA诱导的玉米保卫细胞胞质钙离子浓度的变化

【目的】以玉米第二真叶下表皮为材料,研究ABA诱导的保卫细胞胞质Ca2+浓度的变化,并探讨Ca2+来源。【方法】通过数字激光共聚焦显微镜测定ABA诱导的胞质钙离子浓度变化,并通过异搏啶、EGTA[乙二醇双（2--氨基）乙基醚-N,N,N’,N’四乙酸]药理实验探讨钙离子的来源。【结果】ABA不能诱导所有的保卫细胞胞质钙离子浓度的增加,而可被ABA诱导的保卫细胞中又表现了不同的钙离子浓度变化形式。异搏啶预处理后,不能改变保卫细胞对ABA的反应,但EGTA预处理,则抑制了ABA诱导的胞质钙离子浓度升高。此外,气孔运动观察结果显示：ABA可以明显的诱导气孔关闭,但EGTA、异搏啶的预处理延缓了ABA诱导的气孔关闭速度。【结论】ABA作用下,保卫细胞胞质Ca2+浓度或不发生变化,或持续高浓度,或发生逐渐升高,形成不同的钙信号,最终造成气孔不同步关闭;ABA诱导的胞质钙离子升高主要源自胞外钙离子内流。     

环孢菌素A和苯磺酸氨氯地平对慢性缺氧致大鼠右心室肥厚的作用

目的探讨环孢菌素A（Cs A）和苯磺酸氨氯地平片（Norvasc）对大鼠慢性缺氧所致右心室肥厚的作用。方法48只SD大鼠随机分为慢性缺氧组、Cs A处理组、Norvasc处理组及正常对照组。慢性缺氧组、Cs A处理组、Norvasc处理组大鼠均置于缺氧仓内连续缺氧21 d,缺氧第1天始每天分别用生理盐水和Cs A10 mg/（kg·d）腹腔注射、Norvasc 30 mg/2＋（kg·d）灌胃,共21 d。检测右心室（RV）、左心室（LV）、室间隔（S）和体质量（BW）、Ca N和[Ca]i活性、以及Cn A、-MHC表达水平。结果慢性缺氧组RV/（LV＋S）、RV/BW比值、-MHC表达高于Cs A和Norvasc处理组、正常对照组（P〈0.01）;2＋Norvasc处理组[Ca]i、Ca N活性低于慢性缺氧组（P〈0.01）,而Cs A处理组仅有Ca N活性低于慢性缺氧组（P〈0.01）。结论2＋Cs A和Norvasc可能通过抑制[Ca]i、Ca N活性来防止慢性缺氧致大鼠右心室肥厚。     

汞胁迫下外源Ca2＋对蜈蚣草生物量和汞累积的影响

[目的]探讨汞胁迫下外源Ca2＋对蜈蚣草生物量和汞累积的影响。[方法]以蜈蚣草为材料,用含不同浓度汞（0、5、10mmol／L）和Ca2＋（0．03、2．50、5．00mmol／L）的Hoagland营养液处理,测定各处理的生物量及汞与Ca2＋含量。[结果]汞和Ca2＋处理降低了蜈蚣草的生物量;Ca2＋加速蜈蚣草对汞的吸收;汞抑制其对Ca2＋的吸收。[结论]外源Ca2＋可以加速蜈蚣草吸收环境中的汞。     

三角帆蚌钙调蛋白(CaM)基因的cDNA序列克隆与表达分析

[目的]研究三角帆蚌钙调蛋白(CaM)基因在育珠和非育珠蚌各组织中的表达和不同Ca2+浓度下外套膜中的表达变化。[方法]通过RACE技术,以我国重要的淡水珍珠贝——三角帆蚌为研究客体,克隆了CaM的cDNA全长,并通过Real-Time Q-PCR技术分析了该基因在育珠和非育珠蚌各组织中的表达和不同Ca2+浓度下外套膜中的表达变化。[结果]三角帆蚌CaM基因cDNA全长659bp,包括70 bp的5'UTR,299 bp的3'UTR和447 bp的ORF,编码149个氨基酸,预测其分子量为16.8 kDa,等电点为4.14。cDNA序列比对信息表明三角帆蚌与池蝶蚌、合浦珠母贝间均具有57%的相似度,而其蛋白具有高度的保守性,除斑马鱼外,各生物CaM相似率达97%。定量数据表明,CaM基因广泛表达于三角帆蚌各组织,外套膜内膜与腮中表达量显著升高(P0.05),其次是外套膜外膜和内脏团组织。插核育珠能增加该基因在各组织的表达,特别是在外套膜外膜和鳃组织中,育珠蚌该基因的表达显著高于非育珠蚌(P0.05)。此外,水体Ca2+浓度的增大对CaM基因表达有显著的影响,外套膜内膜该基因的表达随Ca2+浓度升高而增加,而外套膜外膜1.25 mmol/L Ca2+浓度下该基因的表达水平显著高于0.50 mmol/L、3.00 mmol/L组(P0.05)。[结论]为进一步深入研究钙调蛋白基因的在珍珠生长过程中的功能及其调控机理奠定了分子基础。     

不同处理对草莓成花过程中钙·钙调素及同化物含量的影响

[目的]探讨不同促控剂对草莓成花过程中Ca2+-CaM及同化物含量变化的影响。[方法]以草莓品种宝交早生的当年生子苗为试材,用10 mmol/LEGTA、5 mmol/LTFP和2μmol/LA2318(7清水为对照),对草莓植株进行叶面喷洒试验。[结果]叶片中的Ca2+含量在花芽分化始期时有一积累高峰,花序分化期再次积累成峰值;而顶芽中CaM的含量高峰与叶片Ca2+峰值同时出现或稍后。A23187处理可使植株Ca2+和CaM在花芽分化始期的高峰提前;EGTA处理使植株的Ca2+的含量下降,但CaM的含量变动规律性不大;而TFP可降低植株的CaM含量,但对植株的Ca2+影响不大。各处理植株的可溶性糖、还原糖和淀粉含量在成花前均处于高水平。植株的蛋白质含量在花序原始体出现时形成峰值,随后缓慢降低。[结论]该研究为阐明Ca2+-CaM在草莓花芽分化中的作用机制提供依据。     

The Maturation and Senescence in Relation to Ca2+ , CaM Content and Ca2+ -ATPase Activity and Active Oxygen Metabolism in Strawberry Fruits

The changes in content of Ca2 + and CaM, Ca2 + -ATPase activity and active oxygen metabolism during strawberry (Fragaria ananassa Duch. cv. Chunxing) fruits maturation and senescence were investigated in this study. The results showed that the soluble Ca2 + content and SOD activity in fruits tended to decline and O2.-production rate to increase, the Ca2+ -ATPase activity peaked at first and then declined during fruits maturation and senescence. There were the highest CaM content at white stage in preharvest fruits and at marked senescence stage in postharvest ones. The above biochemical changes in fruits stored at low temperature (4℃) were slower than those stored at normal temperature(25℃ ). Thus, it indicated that the stimulation of calcium messenger system and accumulation of active oxygen free radical were closely related to fruits maturation and senescence.     

Relationship Between Ca2+-CaM and Ethylene-Induced PG Activity in Tomato Fruit

Polygalacturonase (PG) was studied during ripening and senescence of postharvest tomatofruit at pink stage at low and normal temperature. The results showed that the PG activity increased, thendecreased during ripening and senescence of tomato. Low temperature inhibited but ethylene enhanced PGactivity. Ethylene also enhanced caimodulin content, which was dependent on Ca2+ concentration in cell.When EGTA(Ca2+ chelator), verapamil (Vp) and LaCl3 (Ca2+ channel blockers), trifluoperazine and chloro-promaize (two CaM antagonisms) were used to treat tomato fruit at green mature stage with ethylene, theycould reverse ethylene-induced increase in PG activity, but Vp, chloropromaize (CPZ), trifluoperazine(TFP) could not directly influence PG activity, which indirectly indicated that influx of Ca2+ from the ex-tracellular space including the cell wall via the Ca2+ channel localized in plasma membrane and CaM were re-quired for ethylene-induced PG activity increase and that ethylene signal transduction may be related to Ca2+- CaM messenger system.     

联合双基因表达载体的构建及其拟南芥转化

[目的]为了研究CBF3冷诱导转录因子和细胞渗透调节物质AtGloS3在抗寒中的相互作用,寻找理想的抗寒转基因方式。[方法]根据GenBank中拟南芥(Arabidopsis thaliana)基因组序列设计引物,通过PCR反应克隆包括CBF3(C-repeat Binding Factor 3)基因以及基因上游启动子的DNA序列:CP-CBF3,共2 075 bp。CP是CBF3基因的启动子,受低温诱导。用EcoRI和BamHI双酶切CP-CBF3并置于植物表达载体pCAMBIA1300中,构建CBF3基因冷诱导表达载体pCA-CP-CBF3。然后将带有组成型启动子的肌醇半乳糖苷酶基因AtG-loS3合并于pCA-CP-CBF3中,构建双基因植物表达载体pCA-CP-CBF3-35S-AtGloS3。用真空抽滤虑法将这两个表达载体转化到拟南芥中,并用潮霉素筛选出转基因植物苗。[结果]CBF3和AtGloS3基因均来自拟南芥,而CBF3不存在内含子,AtGloS3基因存在内含子,故以CB-1、CB-2为引物的非转基因植物中也能得到带,而以AT-1、AT-2为引物的非转基因植物不能得到带。[结论]该研究中pCA-CP-CBF3是1个对照,实验的后续工作需进一步进行。     

镉对菠菜中金属元素吸收影响的基因型差异研究

[目的]为了了解不同菠菜品种对镉处理的生理生化反应的差异和对镉积累的基因型背景差异。[方法]研究了镉对菠菜吸收金属元素的基因型差异及镉对菠菜生长的影响。[结果]10mg／L镉溶液可以显著降低菠菜生物学产量。经镉处理后,镉影响菠菜对金属元素的吸收、分配,地上部K、cu、Zn含量降低,根部K、Mg、Mn和Zn含量增加。无论地上部还是根部,Ca、Al、Mo和Fe含量都增加。[结论]该研究可以为培育无公害的菠菜品种提供理论依据。     

Effects of Ca2+/CaM Messenger System Inhibitors on Ethylene-Induced Increase in Lycopene Content

The changes of lycopene content during ripening and senescence of tomato fruit and the relationship between ethylene glycol-bis (EGTA, Ca2+ chelator), verapamil (Vp, Ca2+ channel blockers), trifluoperazine (TFP), chloropromaize (CPZ) (CaM antagonism) and ethylene-induced increase in lycopene content in tomato fruit were investigated. Lycopene content accumulated obviously during ripening and senescence of tomato fruit after harvest at pink stage. Low temperature inhibited but ethylene enhanced the lycopene content.Meanwhile, ethylene also promoted calmodulin (CaM) content in tomato fruit, which was related to the concentration of ethylene. When EGTA, Vp, TFP and CPZ with ethylene were used to treat tomato fruit, ethylene-induced increase in lycopene content could be reversed, indicating that blocking Ca2+ channel in plasma membrane or chelating extracellular Ca2+ or inhibiting the activity of CaM could decrease the action of ethylene, and suggesting that Ca2+-CaM messenger system may be involved in lycopene increase inducedby ethylene.