北海道黄杨叶片应答低温胁迫的蛋白质组分析

 采用双向电泳的蛋白质组学方法分析了北海道黄杨叶片蛋白质对低温胁迫的应答反应。北海道黄杨植株在室外种植,从2008年10月—2009年2月每月提取叶片总蛋白。在考马斯亮蓝染色的2-DE胶上发现36个表达量显著差异蛋白点,其中通过质谱鉴定出19个蛋白质,包括已知的和新鉴定的低温胁迫相关蛋白质。这些鉴定出的蛋白质涉及多个生理过程,如能量与代谢、调控、防御应答等,同时这些蛋白质的表达模式与生理生化变化模式相一致。     

热胁迫下辣椒叶肉细胞中Ca2+分布的变化

在适宜生长温度下,Ca^2 主要分布于辣椒叶肉细胞间隙及细胞壁中,热胁迫后细胞间隙及其壁中Ca^2 大量进入细胞质,叶绿体结构受到破坏后其中的Ca^2 也释放出来,使细胞质中Ca^2 浓度增高。热胁迫可能是通过诱导Ca^2 在细胞中分布的变化而导致其受到伤害。     

梨果实发育中Ca2+在果肉细胞的定位及变化研究*

 用焦锑酸钾沉淀的细胞化学方法, 研究了􀀂 幸水 梨果实发育中果肉细胞的焦锑酸Ca²⁺ 定位变化及其与细胞超微结构的关系。结果表明: ( 1) 在未受精之前, 果肉细胞内未检测到Ca²⁺ 沉淀颗粒, 细胞核内的染色质少且染色淡, 细胞质的细胞器数量也少; ( 2) 受精后果肉细胞呈现大量的Ca²⁺沉淀颗粒, 主要分布在细胞核、细胞质、质体以及叶绿体外膜上, 含Ca²⁺沉淀颗粒的质体非常膨大, 导管和初期发育的石细胞内也密集分布Ca2+ 沉淀颗粒; ( 3) 受精1 周后果肉细胞的Ca²⁺移向细胞之间的连接处; ( 4) 生理落果的细胞和导管中Ca²⁺没有或极少, 但有的细胞内沿液泡膜有Ca²⁺分布; ( 5) 受精3~ 4 周后, 果肉细胞中很难检测到Ca²⁺沉淀颗粒, 此状态一直持续到果实采收, 但果实腐烂前Ca²⁺沉淀颗粒沿果肉细胞 壁两侧出现。就Ca2+ 在果实发育中的作用及与细胞超微结构的关系等进行了讨论。     

外源Ca2+对低温胁迫下杨树幼苗抗寒相关生理指标的影响

以"陕林4号"和"07-69×青1"为试材,分别用浓度为0、5、10、15、20mmol/L的CaCl2溶液喷施杨树幼苗,4℃低温胁迫2d后测定杨树幼苗的膜伤害率、丙二醛(MDA)含量、抗氧化酶(SOD和POD)活性以及可溶性蛋白质含量等生理指标,以探究低温胁迫下不同浓度外源Ca2+对杨树幼苗抗寒相关生理指标的影响。结果表明:在喷施10mmol/L CaCl2的作用下,杨树幼苗的各项抗寒相关生理指标最优,可显著降低杨树幼苗的膜伤害率,减少丙二醛(MDA)含量,提高超氧化物歧化酶(SOD)活性和过氧化物酶(POD)活性,增加可溶性蛋白质含量。外源Ca2+可以有效提高杨树幼苗的抗寒能力,缓解低温胁迫对杨树幼苗的伤害。     

亚低温条件下外源褪黑素和Ca2+对西瓜幼苗生理特性的影响

以西瓜品种8424种子和幼苗为试材,利用人工气候室进行亚低温处理(昼/夜18℃/12℃)20 d,研究外源褪黑素(MT)和Ca²⁺浸种处理对亚低温条件下西瓜种子萌发,西瓜幼苗抗氧化酶SOD、POD和CAT等活性,渗透调节物质可溶性糖和脯氨酸含量的影响。结果表明,亚低温处理的西瓜种子发芽率和发芽势仅为46.5%和40.5%,外源100μmol·L^-1褪黑素和5 mmol·L^-1Ca²⁺复合浸种处理西瓜种子发芽率和发芽势分别达到62.3%和58.5%。外源褪黑素和Ca²⁺浸种处理显著提高了抗氧化酶SOD、POD、CAT和APX活性,促进了渗透调节物质可溶性糖和脯氨酸的积累,有效缓解亚低温对西瓜种子萌发和幼苗生长影响;褪黑素和Ca²⁺复合浸种西瓜幼苗在出苗第20天时植株鲜质量达到8.21 g·株^-1,达到对照处理的85.5%。综上,外源褪黑素和Ca²⁺能通过提高西瓜幼苗抗氧化酶活性和渗透调节能力等,缓解亚低温的不良影响,促进西瓜幼苗生长。     

盐胁迫下外源Ca2+添加对藤本月季光合及氮代谢相关酶活性的调控

以藤本月季“大游行”为试材,采用土壤喷施0、200 mmol·L^-1盐溶液的处理方法,探究不同浓度外源Ca²⁺(0、5、10、20 mmol·L^-1)对藤本月季光合以及氮代谢相关酶活性的影响,以期为外源Ca²⁺缓解盐胁迫对藤本月季的伤害提供参考依据。结果表明：一定浓度外源Ca²⁺能够增加藤本月季株高生长量、叶面积和地上生物量,促进光合作用,提高叶片中光合色素含量以及氮代谢相关酶活性来增强植物抗性。在200 mmol·L^-1盐处理下,与0 mmol·L^-1Ca²⁺处理浓度相比,添加10 mmol·L^-1Ca²⁺浓度处理下藤本月季的叶绿素a、叶绿素b、总叶绿素(a+b)以及叶黄素含量分别增加了143.9%、152.5%、151.9%和90.6%;光合参数中水分利用效率(WUE)、净光合速率(Pn)、蒸腾速率(Tr)、胞间CO₂浓度(Ci)和气孔导度(G...     

外源Ca2+对盐胁迫下唐古特白刺气孔形态的影响

以盐生植物唐古特白刺为试材,研究了不同浓度外源Ca2+(0、5、10、15、20mmol/L)对不同浓度NaCl(100、200、300、400mmol/L)胁迫下唐古特白刺的气孔形态变化的影响。结果表明:唐古特白刺气孔长度和宽度随盐浓度的升高均降低,NaCl浓度为200mmol/L处理下,气孔长度和宽度在Ca2+浓度为10mmol/L时出现最大值,NaCl浓度≥300mmol/L处理下,气孔长度和宽度在Ca2+浓度为15mmol/L时出现最大值。唐古特白刺表皮气孔密度随NaCl浓度升高,逐渐降低;随外源Ca2+浓度升高,气孔密度先增加后降低,NaCl浓度≤300mmol/L时,各盐处理在Ca2+浓度为5mmol/L时气孔密度达到最大值,NaCl浓度为400mmol/L时,虽然气孔密度增大,但是部分气孔发育不成熟,有些变形严重,不能行使其功能,说明唐古特白刺的最大耐盐浓度为300mmol/L。     

NaCl胁迫对南瓜幼苗Na~+及Ca~(2+)含量的影响

采用19个不同类型南瓜品种,研究了300 mmol/L NaCl下,幼苗地上部和根系Na+和Ca2+离子含量和Na+/Ca2+比值的变化。结果表明:NaCl胁迫8 d后,不同南瓜品种幼苗Na+含量均明显增加,离子平衡被打破;南瓜幼苗体内的Na+含量、地上部的Na+/Ca2+比值的变化反映植物对盐离子和营养元素相对的吸收情况;青栗(Q1)南瓜幼苗根系Na+含量和Na+/Ca2+比值明显高于黑蜜南瓜(H2)和黑籽南瓜(H3)。由此可知:不同南瓜品种幼苗体内Na+含量、地上部Na+/Ca2+比值变化趋势与NaCI胁迫下不同南瓜品种幼苗的盐害指数的结果基本一致;进一步验证了Q1耐盐性强与NaCl胁迫下地上部Na+/Ca2+比值较低,Ca2+离子含量较高有关;而品种H2和H3对盐敏感与NaCl胁迫下地上部Na+/Ca2+比值较高,Ca2+离子含量较低有关。     

Cu2+、Zn2+和Mn2+对低温胁迫下黄瓜幼苗叶片叶绿素荧光参数的影响

以黄瓜为对象,研究了低温胁迫下不同Cu²⁺、Zn²⁺和Mn²⁺浓度对其幼苗叶片叶绿素荧光参数的影响。结果表明：低温胁迫下,提高营养液Cu²⁺、Zn²⁺和Mn²⁺浓度能使黄瓜幼苗叶片Fv/Fm、ΦPSⅡ等叶绿素荧光参数明显增强,表明Cu²⁺、Zn²⁺和Mn²⁺浓度的提高能显著增强黄瓜叶片光合机构抗低温损伤的能力。     

Changes of myocardial nuclear membrane Ca2+-ATPase function in ischemia/reperfusion injury

AIM: The changes of myocardial nuclear membrane Ca²⁺ -ATPase function was investigated in ischemia/reperfusion injury. METHODS: The model of myocardial ischemia/reperfusion injury was established in rats. Myocardial nuclei were purified with sucrose density centrifugation, the activity of Ca²⁺ -ATPase was measured and calcium uptake was assayed with [⁴⁵ Ca²⁺ ] . RESULTS: Plasma levels of malondialdehyde (MDA) and free fatty acid (FFA) in myocardial ischemia/reperfusion injury increased significantly( P<0.01 vs control). Ca²⁺ -ATPase activity and [⁴⁵ Ca²⁺ ] uptake was lower than normal at below 10 μmol/L, while higher at 50 μmol/L. CONCLUSION:These data indicate dysfunction of nuclear menbrane calcium pump and [⁴⁵ Ca²⁺ ] uptake function in myocardial ischemia/reperfusion injury.     

Alteration of myocardial sarcoplasmic reticulum Ca2+ transport protein expression in neonatal hypothyroid rats

AIM: To investigate the alteration of sarcoplasmic reticulum (SR) Ca²⁺ transport proteins including sarcoplasmic reticulum Ca²⁺-ATPase 2a(SERCA2a) and phospholamban(PLB) mRNA expression as well as the alteration of myocardial SR Ca²⁺-ATPase activity in neonatal hypothyroid rats, and to explore the effect of levothyroxine(L-T₄) substitution therapy on the above indexes.METHODS: Hypothyroidism was induced by the administration of propylthiouracil (PTU, 50 mg/d) to the pregnant SD rats by gavage beginning on embryonic day 15 and continuing throughout the lactational period. A subgroup of neonatal hypothyroid rats were intraperitoneally injected with L-T₄ levothroxine (20 μg/kg BW daily), starting from the day of birth. Other pregnant SD rats received normal saline instead of PTU. The samples of the rats in all 3 groups were harvested at postnatal day 3, 5 and 7 respectively (n=10). After measurement of serum thyroid hormone levels, the hearts were removed and the ventricles were weighed (HW). The concentration of calcium in ventricular myocardium(ventricular myoCa²⁺) was detected by fluorospectrophotometry and the activity of SR Ca²⁺-ATPase was determined by the inorganic phosphorus method. The mRNA expression of SERCA2a and PLB was also detected by real-time PCR. RESULTS: Neonatal hypothyroid rats had a significant lower level of SERCA2a mRNA (P<0.05) and a higher level of PLB mRNA (P<0.01), and subsequent lower SERCA2a/PLB at each postnatal day (P<0.01) was observed. Compared with hypothyroid group, the mRNA expression of SERCA2a significantly increased (P<0.05) and that of PLB significantly decreased (P<0.05) in L-T₄ treatment group. The concentration of ventricular MyoCa²⁺ in hypothyroid group was significantly higher than that in control group (P<0.01), and that in L-T₄ treatment group showed a significant decrease as compared with hypothyroid group (P<0.05). The activity of sarcoplasmic reticulum Ca²⁺-ATPase in hypothyroid group was significantly lower than that in control group (P<0.01), and that in L-T₄ treatment group showed a significant increase as compared to hypothyroid group (P<0.05). CONCLUSION: The deficiency of thyroid hormone, resulting in decreased expression of SERCA2a mRNA as well as increased PLB mRNA, contributes to the reduction of SR Ca²⁺-ATPase activity in neonatal rats. This may be one of the most important mechanisms of myocardial systolic and diastolic dysfunctions.     

Effects of fructose-1,6-diphosphate on adriamycin-induced calcium and sarcoplosnic reticulum Ca2+-ATPase activity in cardiomyocytes of rats

AIM: To study the effects of fructose-1,6-diphosphate (FDP) on adriamycin (ADR)-induced calcium and sarcoplasmic reticulum Ca²⁺-ATPase activity in cardiomyocytes of rats. METHODS: Rats were treated with ADR by intraperitoneal injection (2.5 mg·kg^-1 body weight) once every two days for 11 days, and then ADR-treated rats were intervened by FDP at different dosages (ip) once every other day for 41 days. Enzyme linked immune absorption assay (ELISA) was employed to detect froponin I (CTnI). CK-MB was examined by monoclonal antibody. Intracellular free calcium concentration was measured on fluorescent spectrophotometry and SRCa²⁺-ATPase activity was examined by inorganic phosphate. RESULTS: FDP (300, 600, 1 200 mg·kg^-1) significantly reduced the levels of CTnI and CK-MB in serum. Decreased calcium and increased SRCa²⁺-ATPase activity in cardiomyocytes were also observed when ADR-treated rats were intervened by FDP (P＜0.01). CONCLUSION: FDP reduced the injury of cardiotoxicity induced by ADR via decreasing intracellular free calcium and increasing SRCa²⁺-ATPase activity in cardiomyocytes.     

Effects of benazepril on cardiac function,free oxygen radicals,sarcoplasmic reticulum Ca2+-ATPase following cardiac ischemia-reperfusion in spontaneously hypertensive rats

AIM: To investigate the effects of angiotensin converting enzyme inhibitor (ACEI), benazepril (B), on cardiac function , free oxygen radicals, sarcoplasmic reticulum(SR) Ca²⁺-ATPase following ischemia-reperfusion in sportaneously hypertensive rats (SHRs). METHODS: Thirty 10-week-old female SHRs were randomly assigned into two groups: group SHR was control; The animal in group SHR+B was given with 10 mg/kg of benazepril per day. Another 15 Wistar rats with the same age and sex were normal control (group Wistar). After 12 weeks of pretreatment, all rats in each group were subjected to 30 min of left anterior descending coronary artery occlusion and 30 min of reperfusion. Hemodynamic parameters, left heart-to-body weight ratio (LVW/BW), myocardial malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, and SR Ca²⁺-ATPase activity were measured. RESULTS: Compared to group Wistar, the rats in group SHR had higher blood pressure, LVW/BW and myocardial MDA concentration, more serious left cardiac function injury and lower myocardial SOD activity and SR Ca²⁺-ATPase activity; group SHR+B had lower myocardial MDA concentration, higher myocardial SOD activity, but no difference in blood pressure, LVW/BW, the degree of left cardiac function injury and myocardial SR Ca²⁺-ATPase activity. CONCLUSION: Benazepril can attenuate ischemia-reperfusion-induced cardiac function injury by regression of left ventricular hypertrophy (LVH), improving SR Ca²⁺-ATPase activity and decreasing oxygen free radicals injury in SHRs.     

The discovery of the interaction between augmenter of liver regeneration and Na+, K+-ATPase with yeast two-hybrid system

AIM:To screen the proteins interacting with human augmenter of liver regeneration (hALR) by yeast two-hybrid system and to study the mechanism of hALR action. METHODS:hALR bait plasmid was constructed by ligating the gene of hALR into pGBKT7, then transformed into yeast AH109. The yeast strain AH109 containing pGBKT7-hALR was mated with yeast Y187 containing human liver cDNA library plasmid. Diploid yeast was plated on SD/-trp-leu-his-ade (QDO) for screening and on QDO containing X-α-gal for further selection.The AD/library inserts were amplified by PCR and the PCR products were characterized by digesting with Sau3AⅠ and HaeⅢ restriction enzyme to eliminate the duplicates. After sequencing, the positive clones were analysed by bioinformatics. RESULTS:Several positive clones were obtaind. The sequencing and analysis shown that one of them is 669 bp DNA fragment encoding β subunit of Na⁺, K⁺-ATPase. The 224 bp 3'terminal DNA fragment is non-encoder region, and the 445 bp 5'terminal DNA encodes C-terminal 147 amino acid residues of Na⁺, K⁺-ATPase β subunit. CONCLUSION:The results of screening proteins using yeast two-hybrid system showed that hALR could interact directly with Na⁺, K⁺-ATPase in the yeast cell.     

柑橘果实总钙、可溶性Ca~(2+)含量及Ca~(2+)-ATPase活性的季节性变化

以单性结实的龟井和自花结实的鄂柑1号柑橘品种为材料,对果实的整个发育期的子房(幼果)、果皮和果肉的总钙、可溶性Ca2+含量和Ca2+-ATPase活性进行了测定。结果表明龟井花前至花期子房总钙含量就已很高,花后趋下降,而鄂柑1号花前至花期子房总钙含量相对较低,花后有一显著上升;龟井可溶性Ca2+含量在花期及花后有一明显下降,而对应鄂柑1号在花后却有一显著上升;龟井花前至花期Ca2+-ATPase活性较高,花后下降,而对应鄂柑1号花前较低,花期即始上升;果实增大期内两品种果皮的总钙均趋上升,对应果肉总钙却趋下降;而果皮和果肉的可溶性Ca2+含量和Ca2+-ATPase活性在增大期内均有一明显增长过程或居相对较高水平。     

Alterations in gene expression of sarcoplasmic reticulum Ca2+-ATPase and phospholamban detected by RNA array in spontaneously hypertensive rats

AIM: To explore the relationship between the alteration in gene expression of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) and phospholamban (PLB) in spontaneously hypertensive rats (SHR). METHODS: 294 samples of total RNA were obtained from the tissue of ventriculum , aortic smooth muscle, liver and kidney in SHR and normotensive rats (WKY). RNA array was used to determine the mRNA levels of SERCA and PLB. RESULTS: Compared with age-matched WKY rats, the systolic blood pressure increased higher in 6-week-old SHR (P<0.01). The cardiosomatic ratio was significantly higher in 10-week-old SHR (P<0.01), in cardiac sarcoplasmic reticulum, the mRNA levels of SERCA were significantly increased from 4 weeks (P<0.05 or P<0.01). In the aortic sarcoplasmic reticulum, the mRNA levels of SERCA were significantly increased from 4 weeks to 12 weeks (P<0.05 or P<0.01). There was no significant change in the expression of PLB between the two groups. The ratio of cardiac SERCA and PLB was significantly increased since 6-week-old (P<0.05 or P＜0.05)in SHR. The ratio of aortic SERCA and PLB in SHR was significantly increased since 4-week-old (P<0.05 or P<0.01) vs WKY. CONCLUSION: Our results provided the evidence that the abnormalities of intracellular Ca²⁺ hemostasis in SHR represent the progressive nature of essential hypertension.