诱抗菌激发子诱导黄瓜产生对炭疽病的抗性

用细胞壁提取法从诱抗菌中提取出激发子,经点滴法测定发现该激发子与活菌一样能诱导黄瓜产生对炭疽病的抗性反应。黄瓜经激发子处理后,体内与抗病反应有关的过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶活性显著增强;过氧化物酶同功酶酶带增多,着色加深;酚类物质和木质素含量也明显提高。     

诱抗菌激发子诱导黄瓜产生对炭疽病的抗性

用细胞壁提取法从诱抗菌中提取出激发子,经点滴法测定发现该激发子与活菌一样能诱导黄瓜产生对炭疽病的抗性反应。黄瓜经激发子处理后,体内与抗病反应有关的过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶活性显著增强;过氧化物酶同功酶酶带增多,着色加深;酚类物质和木质素含量也明显提高。     

丛枝菌根真菌对茄子黄萎病的防治效果和茄子植株生长的影响

研究了接种丛枝菌根真菌(AMF)对茄子生长和对黄萎病的影响,并且探讨了AMF诱导植物抗病性的生理生化变化。结果表明:接种AMF能促进茄子的生长,明显降低茄子黄萎病的发病率和发病指数;与只接黄萎菌处理比较,在先接种AMF然后接种黄萎病原菌的情况下,可以降低茄子叶内脯氨酸含量和相对电导率,提高根系活力,提高过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)的活性。试验显示,AMF对茄子黄萎病具有一定的生防效果。这种抗性可能来源于AMF提高了茄子营养水平,激活了植物抗病机制。     

丁香精油对冷藏冬枣果实腐烂及诱导抗病相关酶活性的影响

对不同浓度丁香精油处理冬枣果实在0℃贮藏期间(60d)及藏后25℃货架期(5d)的果实腐烂率、诱导抗病相关酶活性和总酚含量的变化特征进行分析,以探索丁香精油抑制冬枣果实腐烂与抗病性诱导的关系。结果表明:丁香精油处理能有效抑制冬枣贮藏期果实腐烂的发生,提高其苯丙氨酸解氨酶、多酚氧化酶和过氧化物酶活性,诱导总酚含量的增加。经丁香精油处理冬枣果实在贮藏60d后25℃货架期5d时的腐烂指数得到明显下降,同时保持了较高的苯丙氨酸解氨酶、多酚氧化酶、过氧化物酶活性和总酚含量,并以0.50%丁香精油处理的效果最显著,其贮藏后货架期的果实腐烂指数较对照下降了45.5%。可见,丁香精油抑制贮藏冬枣果实的腐烂与抗病相关酶活性的升高密切相关,抗病性诱导是丁香精油处理抑制冬枣采后果实腐烂的重要原因之一。     

蜜环菌激发子诱导猪苓细胞产生活性氧及其相关酶的变化*

研究了蜜环菌激发子处理猪苓细胞后活性氧产生及相关酶的变化。结果表明 :蜜环菌激发子分别处理猪苓菌丝和菌核后 ,都能引起活性氧迸发 ,且出现两个迸发高峰 ,高峰期分别在加入激发子后约 1 0min和 90min。与此同时 ,猪苓菌丝酶活性也发生相应变化 ,超氧化物岐化酶 (SOD)、过氧化物酶 (POD)活性下降 ,过氧化氢酶 (CAT)活性没有变化 ,而苯丙氨酸解氨酶 (PAL)活性出现先下降后上升的趋势。     

采前壳寡糖处理对杏果实黑斑病的抗性诱导

以新疆赛买提杏为试验材料,分别在果实坐果期、膨大期、转色期及采收前48h,采用分子量为5 000、浓度为0.05%的壳寡糖(GOS)溶液对杏果实进行喷施处理,以喷施清水为对照(CK);采收后的杏果实在机械损伤接种链格孢菌后置于4℃、相对湿度90%~95%的条件下贮藏,定期统计接种链格孢菌杏果实的病斑直径和发病率,测定抗病相关酶苯丙氨酸解氨酶(PAL)、β-1,3-葡聚糖酶(GLU)和几丁质酶(CHT)的活性及木质素、富含羟脯氨酸糖蛋白(HRGP)的含量,探讨采前壳寡糖处理对杏果实黑斑病的抗性诱导及其生理机制。结果显示,贮藏结束时,采前壳寡糖处理的果实发病率与病斑直径分别比对照显著降低了16.37%和17.57%。随着贮藏期间的延长,壳寡糖处理杏果实PAL、GLU、CHT的活性和木质素、HRGP的含量均表现出先上升后下降的变化趋势,且始终显著高于同期对照,并分别在接种后第21、28、21、28和14天达到峰值,峰值比同期对照分别显著提高12.17%、78.22%、31.41%、34.81%和77.44%。研究表明,采前壳寡糖处理能通过诱导提高杏果实病程相关蛋白及细胞壁HRGP和木质素的含量来增强杏果实对黑斑病的抗性。     

臭氧与海藻酸钠涂膜对葡萄的保鲜效果及其贮藏生理特性的影响

以采后"红地球"葡萄果实为试材,分别设置对照(CK)、250μL/L臭氧处理(O3)、0.3%海藻酸钠涂膜处理(M)、250μL/L臭氧+0.3%海藻酸钠涂膜处理(O3+M),在(0±0.5)℃条件下贮藏,通过测定贮藏过程中葡萄可溶性固形物含量、可滴定酸含量、呼吸强度、硬度、过氧化物酶(POD)、超氧化物歧化酶(SOD)、几丁质酶(CHI)、β-1,3-葡聚糖酶(GLU)的活性以及膜脂过氧化物质丙二醛(MDA)和总酚含量等的变化,统计果实失重率与腐烂率情况,观察各处理对葡萄保藏效果的影响。结果表明:与对照相比,250μL/L臭氧处理、0.3%海藻酸钠涂膜及250μL/L臭氧+0.3%海藻酸钠涂膜复合处理均能显著降低葡萄果实的失重率和腐烂率,抑制葡萄果实的呼吸上升,延缓硬度下降,提高果实抗性相关酶(POD、SOD、GLU、CHI)的活性,减少膜脂的过氧化程度,延缓果实总酚含量下降,有效改善葡萄的贮藏品质,并以250μL/L臭氧+0.3%海藻酸钠涂膜复合处理对葡萄果实保鲜效果最佳。     

米曲霉激发子促进紫草色素合成中细胞内Ca^2＋和cAMP的变化（简报）

米曲霉激发子促进滇紫草细胞合成紫草色素,这种作用与细胞内Ca(2 )相对浓度下降相关。离子载体A23187有抑制激发子的作用,Ca(2 )通道阻断剂Verapamil可部分模拟采曲霉激发子的作用,促进紫草色素合成。激发子处理后,胞内cAMP浓度快速上升。     

油菜素内酯与美极梅奇酵母复配对葡萄灰霉病的抑制作用

【背景】生防酵母具有繁殖速度快、抗逆性强和不产生抗生素等特点,但其对病害的防控效果易受环境影响,油菜素内酯(brassinosteroid,BR)能调控植物生长发育与逆境响应平衡,可有效抑制葡萄果实灰霉病的发生。【目的】本研究旨在明确BR与生防酵母复配对葡萄果实灰霉病的抑制效果及作用机制,为新型生防制剂的研制和应用提供理论依据。【方法】采用美极梅奇酵母P01C004 (Y)、酵母和BR (YBR)、酵母和BR抑制剂(YBZ)处理“红地球”葡萄果实,处理6 h后人工接种灰霉菌孢子悬液。灰霉菌接种7 d后评价BR与生防酵母复配对灰霉菌的防治效果,并检测其抗氧化酶活性和13种酚类物质含量,利用qRT-PCR检测不同处理对葡萄BR、白藜芦醇和抗病相关基因表达水平的影响。【结果】7 d时,与Y处理相比,YBR对葡萄果实灰霉病防治效率提高了23.64%,显著提高了“红地球”葡萄果实中PPO酶活。YBR显著提高了2 d时果实中绿原酸、原儿茶酸、咖啡酸、表儿茶素和芹菜素等酚类物质含量,激活了果实内多种酚类物质的快速合成。YBR在48 h时激活了果实BR信号转导途径,显著上调VvBZR1和VvPR1基因的表达水平,更好地维持植物免疫反应,提高果实对病原菌的防御力。【结论】BR与美极梅奇酵母复配能触发果实多种抗病防御机制,提高葡萄果实灰霉病的防治效率,在采后病害防控方面表现出良好的应用前景。     

24-表油菜素内酯预处理对干旱胁迫下葡萄幼苗抗氧化系统和渗透调节物质的影响

以酿酒葡萄‘雷司令’(Riesling)一年生营养袋扦插苗为材料,采用人工气候室水培试验,考察在聚乙二醇6000(PEG)模拟干旱条件下,不同浓度(0.05、0.10和0.20mg/L)24-表油菜素内酯(EBR)预处理对‘雷司令’幼苗活性氧、抗氧化物质、渗透调节物质含量和抗氧化酶活性的影响,以揭示EBR预处理对干旱胁迫下葡萄幼苗的抗旱机理。结果显示:(1)与正常生长(对照)相比,干旱胁迫显著提高葡萄幼苗叶片中超氧阴离子自由基(■)、过氧化氢(H_2O_2)和丙二醛(MDA)含量;与干旱胁迫处理(PEG)相比,不同浓度EBR预处理均可降低叶片中■、H_2O_2和MDA的含量。(2)与对照相比,PEG处理显著降低葡萄幼苗叶片的抗坏血酸(AsA)和还原型谷胱甘肽(GSH)含量;与PEG处理相比,各浓度EBR预处理均可显著提高葡萄叶片AsA与GSH的含量,且以0.10mg/LEBR处理效果最好。(3)随着干旱胁迫时间的延长,葡萄幼苗叶片中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)与抗坏血酸过氧化物酶(APX)活性均呈先上升后下降的变化趋势,而在正常生长条件下酶活性基本保持不变;EBR预处理的葡萄叶片SOD、CAT、POD和APX活性均始终高于同期PEG处理。(4)PEG处理条件下,渗透调节物质脯氨酸和可溶性蛋白的含量整体高于对照;与PEG处理相比,不同浓度EBR预处理在干旱胁迫中后期均能显著提高葡萄叶片中脯氨酸和可溶性蛋白含量。研究表明,在干旱胁迫下,外源EBR预处理能够提高葡萄叶片抗氧化系统酶活性和渗透调节物质含量,有效降低干旱胁迫诱导的活性氧过度积累及膜脂过氧化程度,提高葡萄幼苗的抗旱能力,且以0.10mg/L EBR处理效果最佳。     

我国棉花主产区棉蚜对吡虫啉的抗性监测及抗性机理

【目的】由于生长周期短、繁殖率高,棉蚜Aphis gossypii容易对杀虫剂产生抗药性。本研究旨在明确我国棉花主产区棉蚜对吡虫啉的抗性水平及抗性机理。【方法】采用浸叶法测定了北京海淀,河北廊坊和邯郸,山东德州,河南许昌,以及新疆奎屯和阿克苏地区棉蚜对吡虫啉的抗性水平;测定了不同种群棉蚜3种解毒酶（多功能氧化酶、羧酸酯酶、谷胱甘肽S-转移酶）及乙酰胆碱酯酶的活性;并对靶标基因烟碱型乙酰胆碱受体（nAChR）β1亚基基因进行了突变检测。【结果】北京海淀、河南许昌和河北邯郸的棉蚜对吡虫啉敏感;河北廊坊、新疆阿克苏、山东德州及新疆奎屯地区的棉蚜对吡虫啉的抗性倍数(resistance ratio, RR)分别为22.6, 26.3,53.5和61.1倍,为中等水平抗性。酶活力对比研究发现,阿克苏和奎屯地区的棉蚜多功能氧化酶的比活力分别是敏感种群(北京种群)的1.7和1.8倍,羧酸酯酶的比活力分别是敏感种群的1.6和1.7倍,谷胱甘肽S-转移酶的比活力均是敏感种群的1.5倍,但是乙酰胆碱酯酶比活力在棉蚜种群间差异不显著。靶标基因突变检测表明,河北廊坊、新疆阿克苏、山东德州及新疆奎屯棉蚜种群nAChR β1亚基均存在与吡虫啉抗性相关的精氨酸到苏氨酸（R81T）突变。【结论】结果提示,多功能氧化酶、羧酸酯酶和谷胱甘肽S-转移酶活力升高以及nAChR β1亚基R81T突变与棉蚜对吡虫啉的抗性形成相关。     

小麦品种（系）对麦红吸浆虫抗性指标筛选与抗性评价

【目的】筛选小麦对麦红吸浆虫Sitodiplosis mosellana抗性的准确鉴定方法, 明确生产上栽培小麦品种（系）对吸浆虫的抗性, 为抗虫小麦品种的筛选和利用提供科学依据。【方法】2012-2014年在陕西周至县建立麦红吸浆虫抗性鉴定圃, 调查并分析各参试小麦材料的估计损失率、粒被害率、穗被害率、单穗虫口和实际产量损失率及其相关性, 筛选出较准确的指标; 并以筛选到的指标为依据, 评估参试材料的抗性。【结果】估计损失率连续两年与其他3个抗性指标及实际产量损失率的相关性最强, 且均达到极显著水平。2012-2013年参试的85份和2013-2014年评估的80份材料中, 高抗、中抗和低抗材料合计分别为25份和40份; 重复种植的16份材料中, 14份两年均表现为抗性, 其中科农1006和晋麦47连续表现为高抗。【结论】估计损失率为具代表性且较准确的吸浆虫抗性鉴定指标。筛选出的抗性材料可作为抗吸浆虫的主推品种或后备品种, 也可作为亲本材料进行抗性育种研究。     

Transient transformation of Podosphaera xanthii by electroporation of conidia

Background

Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.

Results

To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.

Conclusion

This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.     

P-glycoprotein effect on the properties of its natural lipid environment probed by Raman spectroscopy and Langmuir-Blodgett technique

Behavior of P-glycoprotein (Pgp) natural lipid environment within the membrane of CEM cells expressing Pgp in the quantities varying from 0% to 32% of the total amount of all membrane proteins is described for the first time. Observed cooperative effect of Pgp-induced increase of membrane stability, decrease of the temperature of gel-to-crystal lipids transition and predominance of the lipid liquid crystalline phase at physiological temperatures should have an impact in development of multidrug resistance phenotype of tumor cells by favoring the Pgp intercellular transfer and Pgp ATPase activity.     

Hydraulic resistance of developing Actinidia fruit

Background and Aims

Xylem flows into most fruits decline as the fruit develop, with important effects on mineral and carbohydrate accumulation. It has been hypothesized that an increase in xylem hydraulic resistance (R_T) contributes to this process. This study examined changes in R_T that occur during development of the berry of kiwifruit (Actinidia deliciosa), identified the region within the fruit where changes were occurring, and tested whether a decrease in irradiance during fruit development caused an increase in R_T, potentially contributing to decreased mineral accumulation in shaded fruit.

Methods

R_T was measured using pressure chamber and flow meter methods, the two methods were compared, and the flow meter was also used to partition R_T between the pedicel, receptacle and proximal and distal portions of the berry. Dye was used as a tracer for xylem function. Artificial shading was used to test the effect of light on R_T, dye entry and mineral accumulation.

Key Results

R_T decreased during the early phase of rapid fruit growth, but increased again as the fruit transitioned to a final period of slower growth. The most significant changes in resistance occurred in the receptacle, which initially contributed 20 % to R_T, increasing to 90 % later in development. Dye also ceased moving beyond the receptacle from 70 d after anthesis. The two methods for measuring R_T agreed in terms of the direction and timing of developmental changes in R_T, but pressure chamber measurements were consistently higher than flow meter estimates of R_T, prompting questions regarding which method is most appropriate for measuring fruit R_T. Shading had no effect on berry growth but increased R_T and decreased dye movement and calcium concentration.

Conclusions

Increased R_T in the receptacle zone coincides with slowing fresh weight growth, reduced transpiration and rapid starch accumulation by the fruit. Developmental changes in R_T may be connected to changes in phloem functioning and the maintenance of water potential gradients between the stem and the fruit. The effect of shade on R_T extends earlier reports that shading can affect fruit vascular differentiation, xylem flows and mineral accumulation independently of effects on transpiration.     

烟粉虱MED隐种抑制蛋白基因Btarrestin的克隆及特性分析

【目的】明确烟粉虱Bemisia tabaci MED隐种抑制蛋白基因Btarrestin是否参与吡虫啉耐药性。【方法】根据已有烟粉虱转录组数据,利用RT-PCR克隆Btarrestin的全长序列,进行生物信息学分析。通过qPCR分析Btarrestin在烟粉虱MED隐种各个龄期(卵,1-2龄、3龄、4龄若虫,雌、雄成虫)以及吡虫啉处理(100 mg/L)后成虫中的表达量变化,明确其时空表达模式。利用RNAi技术沉默Btarrestin,观察沉默前后Btarrestin表达量和烟粉虱MED隐种成虫死亡率变化。【结果】成功克隆烟粉虱MED隐种Btarrestin的cDNA序列(GenBank登录号: MK204377),编码区全长1 227 bp,编码409个氨基酸,预测所编码的蛋白分子量约为45.33 kD,理论等电点(pI)为8.38。保守结构域分析表明,Btarrestin具有Arrestin_N和Arrestin_C两个超家族保守结构域,符合抑制蛋白家族特征。分子系统树分析表明,Btarrestin与褐飞虱Nilaparvata lugens的Arrestin亲缘关系最近。Btarrestin的表达量随烟粉虱MED隐种的生长发育逐渐升高,成虫期表达量最高。100 mg/L吡虫啉处理成虫24 h后Btarrestin的表达量较对照增加了2.39倍。RNAi干扰Btarrestin后进行生物测定,烟粉虱MED隐种成虫的死亡率上升了31.27％。【结论】Btarrestin可能与烟粉虱MED隐种对吡虫啉的耐药性有关。     

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.     

Photoaffinity labeling of the multidrug resistance protein 2 (ABCC2/cMOAT) with a photoreactive analog of LTC4

Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of chemotherapeutic drugs and normal cell metabolites, including Leukotriene C (LTC₄); however direct binding of the LTC₄ to MRP2 has not been demonstrated. In this study, a photoreactive analog of LTC₄ (IAALTC₄) was used to demonstrate its direct binding to MRP2. Our results show specific photoaffinity labeling of MRP2 with IAALTC₄ in plasma membranes from MDCKII^MRP2 cells. The photoaffinity labeling signal of MRP2 with IAALTC₄ was much lower than that of MRP1, consistent with previous studies whereby the measured K_m values of MRP1 and MRP2 for LTC₄ were 1 μM and 0.1 μM LTC₄, respectively. Competition of IAALTC₄ photoaffinity labeling to MRP2 with MK571, a well characterized inhibitor of MRP2 function, showed ~75% reduction in binding in the presence of 50 μM excess MK571. Interestingly, unmodified LTC₄ enhanced the photoaffinity labeling of IAALTC₄ to MRP2, whereas excess GSH and Quercetin had no significant effect. Mild tryptic digestion of photoaffinity labeled MRP2 revealed several photoaffinity labeled peptides that localized the IAALTC₄ binding to a 15 kDa amino acid sequence that contains transmembrane 16 and 17. Together these results provide the first demonstration of direct LTC₄ binding to MRP2.     

Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae

Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC(50) of 35μM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC(50)=19μM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray 'hits', peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking.