柑橘全爪螨代谢抗性相关基因表达差异分析

【目的】为了明确谷胱甘肽S-转移酶(GST)基因、羧酸酯酶(CarE)基因,过氧化氢酶(CAT)基因在柑橘全爪螨Panonychus citri抗性中的作用,【方法】在室内用噻螨酮对柑橘全爪螨进行抗性选育,进一步构建抗/敏品系数字基因表达谱,采用RPKM法对柑橘全爪螨敏感品系和噻螨酮抗性品系3种代谢抗性相关基因进行表达差异分析。【结果】经过20代抗性选育,获得了柑橘全爪螨噻螨酮抗性品系,与敏感品系比较,柑橘全爪螨对噻螨酮的抗性倍数达到3 532.12倍。基因差异性分析发现,抗性品系中有11条GST基因、17条CarE基因和6条CAT基因表达上调;14条GST基因、24条CarE基因和3条CAT基因表达下调。上调倍数最高的GST基因、CarE基因和CAT基因分别为Unigene31530[log2 ratio(RS/SS)=1.05]、Unigene23121[log2 ratio(RS/SS)=2.05]和Unigene31477[log2 ratio(RS/SS)=10.04]。进一步对Unigene31477进行荧光定量PCR分析发现,抗性和敏感品系基因表达水平没有显著差异。【结论】根据柑橘全爪螨抗/敏性品系基因表达差异推断,GST、CarE和CAT基因可能与柑橘全爪螨对噻螨酮产生的抗性没有密切关系。     

等钳蠊螨联苯肼酯抗敏品系对柑橘全爪螨的捕食作用

【目的】探究不同温度下等钳蠊螨(Blattisocius dentriticus Berlese)联苯肼酯抗性品系和敏感品系对柑橘全爪螨(Panonychus citri McGregor)的捕食能力差异。【方法】采用室内生物测定的方法,研究了在16、20、24、28、32℃条件下等钳蠊螨联苯肼酯抗性品系和敏感品系对柑橘全爪螨卵和雌成螨的捕食作用。【结果】等钳蠊螨联苯肼酯抗性品系和敏感品系对柑橘全爪螨卵和雌成螨的捕食量没有显著差异。【结论】联苯肼酯抗性获得并未影响等钳蠊螨对柑橘全爪螨的捕食功能,因此,等钳蠊螨有发展成为生防天敌的潜力。     

橘全爪螨不同品系γ氨基丁酸受体基因差异性分析

【目的】为明确γ氨基丁酸受体基因与橘全爪螨抗性的关系,【方法】通过BLAST搜索,对GABA受体基因进行鉴定;进一步通过序列比对和Sanger测序,对橘全爪螨敏感品系和噻螨酮抗性品系GABA受体基因SNPs进行分析和真实性验证;采用RPKM法对橘全爪螨敏感品系和噻螨酮抗性品系GABA受体基因进行表达差异分析。【结果】从橘全爪螨转录组中获得了19条GABA受体基因。通过敏感品系和抗性品系GABA受体基因序列比较和sanger测序发现Unigene11199_All有2个SNP位点。对GABA受体基因表达差异分析发现,相对于敏感品系,抗性品系中部分GABA受体基因表达量发生了不同程度的变化,抗性品系中有3条GABA受体基因表达上调,13条GABA受体基因表达下调,Unigene24440_All下调倍数最高[log2 Ratio(RS/SS)=-10.452479]。【结论】由此推断,橘全爪螨抗性产生可能是一个比较复杂的过程,而Unigene11199_All和Unigene24440_All可能是橘全爪螨对噻螨酮产生抗性的重要原因。     

柑桔全爪螨噻螨酮抗性与敏感品系GST基因表达差异分析

为了明确谷胱甘肽S-转移酶（glutathion S-transferases,GSTs）基因与柑桔全爪螨Panony-chus citri抗性的关系,通过BLAST搜索,对柑桔全爪螨转录组数据库的GST基因进行鉴定,进一步采用RPKM法分析柑桔全爪螨噻螨酮敏感品系（SS）和抗性品系（RS）的GST基因表达差异。从柑桔全爪螨转录组中获得了30条GST基因,11条基因属于Delta家族,10条属于Mu家族,6条属于Kappa家族,2条属于Omega家族,1条属于Zeta家族,同一家族的基因聚在同一进化分支上;两个品系有5条GST基因表达没有差异,此外,抗性品系中有16条发生了下调,有9条GST基因发生了上调;抗性品系上调倍数最高的3个GST基因分别是GSTd6、GSTm5和GSTm4,log₂(RPKM_RS/RPKM_SS)分别仅为1.05、0.74和0.71,荧光定量PCR分析测得上调倍数分别仅为1.13、1.42和1.21,上调倍数均不高。推断,GST基因上调可能不是柑桔全爪螨对噻螨酮产生抗性的重要原因。     

柑橘全爪螨对4种杀螨剂的抗性监测及增效作用

为探明近年来柑橘全爪螨田间种群对常用杀螨剂抗性的变化趋势,采用玻片浸渍法于2008—2009年连续2a监测了柑橘全爪螨重庆种群对甲氰菊酯、哒螨酮、阿维菌素和甲维盐4种常用杀螨剂的抗性,同时测定了磷酸三苯酯(TPP)、顺丁烯二酸二乙酯(DEM)和增效醚(PBO)的增效作用。结果表明,柑橘全爪螨对甲氰菊酯的抗性上升速度较快,抗性增长倍数达5.1倍,哒螨酮次之,抗性增长倍数为2.2倍。柑橘全爪螨对阿维菌素的抗性两年间变化很小,相反对甲维盐的敏感性有所升高。增效作用研究表明,TPP对甲氰菊酯、阿维菌素和甲维盐的增效倍数分别为3.2倍、2.0倍和3.8倍。DEM对哒螨酮和甲维盐的增效倍数分别为3.3倍和2.6倍。PBO对甲氰菊酯、阿维菌素、哒螨酮和甲维盐的增效倍数分别为2.7倍、1.7倍、2.4倍和7.2倍。而TPP对哒螨酮以及DEM对甲氰菊酯和阿维菌素均未表现出明显的增效作用。     

17.5%唑螨酯·噻嗪酮ME防治柑橘橘全爪螨试验

柑橘全爪螨(Panonychus citri)又称柑橘红蜘蛛,是柑橘生产中分布广,为害最严重的害虫,浙江省1年发生约16代,繁殖速度快,易对药剂产生抗性,为此需要不断研制新的制剂进行轮换使用以延缓害虫抗药性压力。2008年受宁波中北生物科技发展股份有限公司委托,对17.5%唑螨酯·噻嗪酮ME     

牛蒡子杀柑橘全爪螨活性成分的分离与鉴定

【目的】为了探明牛蒡子杀柑橘全爪螨的活性成分,【方法】采用杀螨活性追踪、溶剂萃取、硅胶柱层析、紫外吸收、颜色反应、气相色谱-质谱联用(GC-MS)等方法研究牛蒡子乙醇提取物对柑橘全爪螨的抑制作用,并分离和鉴定其杀螨活性成分。【结果】结果表明,牛蒡子组分L1-2杀柑橘全爪螨的活性最高,触杀毒力高达0.002 5 g.L-1;组分L1-2中含有共轭双键或三键化合物,主要包括十六碳烷酸甲酯、十八烷酸甲酯、三十烷酸甲酯、三十一烷酸甲酯、十八碳二烯酸甲酯等9种主要成分,总含量达到85.32%,还有少量醛类、烯醇、烯烃类物质。【结论】牛蒡子杀柑橘全爪螨的活性成分主要为脂肪酸类物质。     

苦参碱防治柑橘红蜘蛛的效果

柑橘全爪螨（Panonychus citri McGregor）是常山柑橘的主要虫害之一，生产上选择高效、经济、安全的药剂进行有效控制是当前柑橘生产的关键。为探索植物源农药苦参碱水剂对柑橘全爪螨的防治效果、安全性和应用技术，在2005年进行了田间小区效果和应用试验，得出了该药剂防治柑橘全爪螨具有安全、高效、经济的特点，尤其是在绿色、有机柑橘生产上具有广阔应用前景。     

17．5％唑螨酯·噻嗪酮ME防治柑橘橘全爪螨试验

柑橘全爪螨（Panonychuscitri）又称柑橘红蜘蛛，是柑橘生产中分布广，为害最严重的害虫，浙江省1年发生约16代，繁殖速度快，易对药剂产生抗性，为此需要不断研制新的制剂进行轮换使用以延缓害虫抗药性压力。2008年受宁波中北生物科技发展股份有限公司委托，对17．5％唑螨酯·噻嗪酮ME进行了防治柑橘全爪螨的田间药效试验，获得了一定的防治效果，现将试验情况报告如下。     

0.1%藜芦根茎提取物复配矿物油防治柑橘全爪螨的研究

中国是世界上柑橘产量最多的国家之一[1],资源丰富,优良品种众多。柑橘富含糖、酸、蛋白质、氨基酸等营养物质和生物活性成分[2]。柑橘全爪螨是一种真螨目叶螨科全爪螨属昆虫,广泛分布在中国各柑橘产区。常群集叶片、嫩梢、果皮上吸汁危害,引致落叶、落果,尤以叶片受害为重,严重影响树势和产量[3]。柑橘全爪螨类个体小、繁殖快、适应性强和易产生抗药性,是公认的难以防治的有害生物群落[4]。     

Interaction between human augmenter of liver regeneration and Na+-K+-ATPase in vitro

AIM: To check the physical interaction between GST- Na+-K+-ATPase domain and recombinant human augmenter of liver regeneration (rhALR) by GST pull down assay. METHODS: With PCR and genetic recombinant techniques, the coding region of β subunit of Na+-K+-ATPase was cloned into expressing plasmid pGEX-4T and identified by endonuclease digestion and sequencing methods. Under the inducing of 0.1 mmol/L IPTG, the fusion protein GST- Na+-K+-ATPase domain was highly expressed by E.coli DH-5α. After hypersound quassating, the GST- Na+-K+-ATPase domain was purified by glutathione agarose beads and the physical interaction with rhALR was checked by GST pull down assay. RESULTS: Analysis by SDS-PAGE showed the rhALRs of monomer and dimmer in GST- Na+-K+-ATPase domain lane. The Western blotting of the GST-pull down assay showed the same results as well. CONCLUSION: The Na+-K+-ATPase domain is associated with rhALR specifically in vitro.     

Dexamethasone pretreatment attenuates reperfusion arrhythmia in rats

AIM: To investigate the effect of dexamethasone (DEX) preconditioning on reperfusion arrhythmia. METHODS: 46 Sprague-Dawley rats were divided randomly into DEX and control (CON) group, the rats were pretreated with DEX or sodium chloride before their hearts were separated for Langendorff perfusion and for ischemia/reperfusion. The reperfusion arrhythmias were observed dynamically after 60 min reperfusion following 30 min ischemia. The expression of HSP72 in myocardium was examined by Western blotting and immunohistochemistry at reperfusion 60 min. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and the activities of Na+-K+-ATP ase, Ca2+-Mg2+-ATPase on myocardial plasma membrane were detected. RESULTS: Compared with control group, the accumulated points and persistence time of ventricular arrhythmia were reduced significantly in DEX group (P<0.05), the expression of HSP72 was significant upregulated (P<0.05), the level of MDA was reduced significantly, the activities of SOD, CAT, GSH-Px and Na+-K+-ATPase were significantly higher (P<0.05). CONCLUSION: Dexamethasone pretreatment markedly reduces the reperfusion ventricular arrhythmias in rats, which may be attributed to upregulation of HSP72, SOD, CAT, GSH-Px , Na+-K+-ATPase and inhibition of lipid peroxidation.     

Effects of hypotensive or aggressive resuscitation on tissue ATPase activity in pregnant rabbits with uncontrolled hemorrhagic shock

AIM: To compare the effects of hypotensive and aggressive resuscitation strategies on blood loss, fluid requirements, hematocrit (Hct), tissue Na＋-K＋-ATPase and Ca2＋-Mg2+-ATPase activities in a clinically relevant model of uncontrolled hemorrhagic shock in pregnancy rabbits. METHODS: Thirty anesthetized New Zealand white rabbits at mid and late gestation underwent uncontrolled hemorrhagic shock by transecting a small artery of mesometrium, followed by bleeding via carotid artery to mean arterial pressure (MAP) of 40-45 mmHg. Animals were randomly divided into five groups (n=6 each): sham shock (SS); shock without resuscitation (SH); aggressive resuscitation in pre-hospital phase with 4 mL/kg normal saline, followed by Ringer’s solution to MAP of 80 mmHg (NS), hypotensive resuscitation with 4 mL/kg of normal saline (NH) or hypertonic hydroxyl ethyl starch (7.5% NaCl + hydroxy ethyl starch, HHES, HHH) followed by Ringer’s solution to MAP of 60 mmHg. Finally, all the resuscitated animals received hemorrhage controlled and fully resuscitated to MAP of 80 mmHg. At the end of the experiment, survivors were sacrificed, skeletal muscle, cardiac muscle, liver, kidney, lung and ileum were harvested for determination of Na＋-K＋-ATPase and Ca2＋-Mg2+-ATPase activities. RESULTS: Total blood loss and infused volume were compared between NH［(4.3±0.2)mL/kg, (47.2±4.1)mL/kg］ and HHH［(4.1±0.3)mL/kg,(44.9±4.3)mL/kg］ groups, both were significantly less than NS［(5.5±0.2)mL/kg, (65.5±3.8）mL/kg］　group. Hct in NH (21.0%±2.1%) and HHH (21.5%±1.8%) were significantly higher than NS (14.2%±1.5%) and SH (12.5%±1.4%).Tissue Na＋-K＋-ATPase and Ca2＋-Mg2+-ATPase activities were stimulated in all shock groups. Na＋-K＋-ATPase in skeletal muscle, cardiac muscle, liver, kidney was significantly lower in the NH (5.42±1.41, 4.54±2.01, 4.13±0.62, 3.42±0.84) and HHH (3.97±0.91, 2.94±0.66, 3.22±1.42, 3.03±0.53) than that in NS (7.34±1.41, 6.23±1.53, 6.11±0.97, 5.82±0.69) and SH (9.11±0.52, 8.40±1.08, 7.04±1.13, 6.55±1.45). CONCLUSION: Hypotensive resuscitation with normal saline or HHES reduces blood loss, decreses total infused volume, leads to higher hematocrit and finally alleviates metabolism derangement after uncontrolled hemorrhagic shock.     

Mesenteric lymph reperfusion exacerbates brain injury in superior mesenteric artery occlusion shock rats

AIM: To observe the effect of mesenteric lymph reperfusion (MLR) on the brain morphology and neurotransmitter in rats with superior mesenteric artery occlusion (SMAO) shock, and to explore the mechanism of oxygen free radicals, nitric oxide (NO), polymorphonuclear neutrophils (PMN), membrane ATPase function and energy metabolism. METHODS: Twenty-four male Wistar rats were randomly divided into 4 groups: in sham group, the rats were given only anesthetization and operation; in MLR group, the rats were performed 1 h occlusion of mesenteric lymphatics (ML) followed by 2 h of reperfusion; in SMAO group, the rats were performed 1 h occlusion of superior mesenteric artery (SMA) followed by 2 h of reperfusion; in MLR+SMAO group, the rats were performed 1 h occlusion of SMA and ML followed by 2 h of reperfusion. After 2 h of reperfusion, the brain tissue was taken for preparing microscopic sections to observe the morphological change. At the same time, the brain tissue was homogenized for determining the choline acetyltransferase (ChAT), acetylcholine esterase (AChE), dopamine (DA), norepinephrine (NE), lactic acid (LA), malondialdehyde (MDA), superoxide dismutase (SOD), NO, nitric oxide synthase (NOS), myeloperoxidase (MPO), cell membrane ATPase and ATP. RESULTS: Morphological observation showed that the architecture of the brain was close to normal in sham and MLR groups. Necrosis, degeneration and occasional swelling were found in neuronal cells in SMAO group, and in MLR + SMAO group the injury of the neurons was more serious than that in SMAO group. The contents of MDA, NO and LA, the activities of AChE, NOS and MPO in brain homogenate in SMAO and MLR+SMAO groups were increased, the ChAT activity and DA, NE contents were reduced significantly than those in MLR and sham groups, respectively. The contents of MDA and NO, the activities of AChE, NOS and MPO in MLR+SMAO group were higher than those in SMAO group. The activities of Na+-K+-ATPase and SOD in brain homogenate in SMAO group were lower than those in sham and MLR groups, the Mg2+-ATPase activity and ATP content were lower than those in MLR group. The activities of SOD, Na+-K+-ATPase, Ca2+-ATPase, Mg2+-ATPase and Ca2+-Mg2+-ATPase in brain homogenate in MLR+SMAO group were lower than those in sham and MLR groups, and the DA, Ca2+-ATPase, Mg2+-ATPase, Ca2+-Mg2+-ATPase and ATP were also lower than those in SMAO group. CONCLUSION: MLR exacerbates the brain injury, reduces the DA level and increases the AChE activity in SMAO shock rats, indicating that MLR enhances the brain tissue free radical injury, NO synthesis and releases, PMN detention, and decreases the activity of cell membrane ATPase, also induces the energy metabolism dysfunction.     

低温胁迫下北海道黄杨叶肉细胞Ca2+和Ca2+-ATPase的变化

 利用焦锑酸钙沉淀和硝酸铅沉淀的电镜细胞化学方法,以室温生长的北海道黄杨植株为对照,研究了人工4 ℃低温胁迫过程中北海道黄杨（Euonymus japonicus‘Cuzhi’）叶肉细胞Ca²⁺和Ca²⁺-ATPase的动态变化。在4 ℃低温胁迫的初期（3 ~ 12 h）,北海道黄杨叶肉细胞间隙和液泡内的Ca²⁺沉淀颗粒减少,而细胞质和细胞核内的Ca²⁺水平升高,但Ca²⁺-ATPase在细胞的分布几乎没有变化,主要分布在质膜和液泡膜上,有较高的活性;低温胁迫24 h,细胞质和细胞核内增加的Ca²⁺开始回到细胞间隙和液泡中,Ca²⁺-ATPase在质膜和液泡膜上活性增强;在低温胁迫48 ~ 96 h,细胞内的Ca²⁺又回到低温胁迫前的低水平,但Ca²⁺-ATPase在质膜和液泡膜上仍有很高的活性。叶肉细胞内Ca²⁺稳态平衡和Ca²⁺-ATPase的活性变化与植物的抗寒性存在一定的相关性。     

Effects of volatile anesthetics on rat heart ischemia-reperfusion injury in vitro

AIM: To investigate the effect of volatile anesthetics on function,metabolism,ATPase activity and free radicals in isolated ischemia /reperfusion (I/R) rat hearts.METHODS: 136 SD rats were anesthetized with pentobarbital and randomly divided into six groups and 17 sub-groups (n=8),according to the given drug.In a normal thermal isolated Langendorff rat heart model,four volatile anesthetics in 1.5 MAC concentration were given before global ischemia 25 min and during reperfusion 30 min.Coronary flow (CF),LVEDP,left ventricular developed pressure (LVDP),±dp/dt were monitored at 15 min of equilibrium,15 min of drug treatment,the end of reperfusion.Myocardial adenosine triphosphate (ATP),malodialdehyde (MDA),activity of Ca2＋-ATPase and Na＋-K＋-ATPase,and superoxide dismutase (SOD) were determined at 15 min of equilibrium,15 min of drug treatment or absence,10 min global ischemia and the end of reperfusion.RESULTS: CF and LVEDP were increased significantly after exposured to volatile anesthetics 15 min,and LVDP,+dp/dtmax were significantly decreased.However,LVDP and +dp/dtmax were increased at the end of reperfusion in the treated groups.HR in halothane and isoflurane groups was decreased before ischemia and after reperfusion.The myocardial ATP content was significantly increased before and after ischemia in the treated groups.At the end of reperfusion,the activity of SOD was significantly higher and myocardial MDA content was significantly lower in the treated groups than those in control group.The activity of Ca2＋-ATPase,compared with the control group,was markedly decreased before ischemia in halothane,enflurane and isoflurane group.Nonetheless,the activity of Ca2＋-ATPase was clearly increased in the treated groups during ischemia and at the end of reperfusion.The activity of Na＋-K＋-ATPase was only enhanced in halothane group at the end of reperfusion among groups.CONCLUSION: The volatile anesthetics depress myocardial systolic function.There are markedly protective effects against myocardial I/R injury.Meanwhile,the volatile anesthetics improve the recovery of function and metabolism,and increase CF and the activity of Ca2＋-ATPase and Na＋-K＋-ATPase in rats.