Effect of glyphosate on tryptamine production and Sekiguchi lesion formation in rice infected with Magnaporthe grisea

When exposed to light, the Sekiguchi lesion (sl) rice mutant has an enhanced resistance to Magnaporthe grisea infection responsible for Sekiguchi lesion formation and tryptamine accumulation. Glyphosate [N-(phosphonomethyl) glycine] pretreatment suppressed Sekiguchi lesion formation and tryptamine accumulation in the sl mutant after M. grisea infection even under light. This inhibition by glyphosate was blocked by the supply of exogenous tryptophan, but not by exogenous phenylalanine. In glyphosate-pretreated leaves, 5-enol-pyruvyl-shikimate-3-phosphate synthase gene expression and tryptophan biosynthesis were significantly suppressed. During tryptophan starvation, catalase activity was maintained at a high level even under light, leading to the suppression of H₂O₂ generation and DNA fragmentation. These results show a strong relationship between the tryptophan and tryptamine pathways in the induction of light-enhanced resistance to M. grisea infection in the sl mutant.     

Possible roles and functions of LPL1 gene encoding lysophospholipase during early infection by Magnaporthe grisea

The rice blast fungus Magnaporthe grisea differentiates appressoria, which are required to attack its rice plant host. Clone A26, tentatively named LPL1, was previously found to be homologous to the known lysophospholipase genes from our subtractive cDNA library. The LPL1 protein had a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence, and the protein expressed in Escherichia coli had lysophospholipase activity. To clarify the functions and possible roles of LPL1, the gene was disrupted by targeted gene replacement. The ΔLPL1 mutants formed fewer appressoria on the hydrophobic surface of GelBond film, and the appressoria had reduced turgor pressure and penetration into cells of the leaf sheath. The ΔLPL1 mutants and wild-type differentiated normal appressoria on other artificial substrata such as polycarbonate plate and on rice leaf sheath. Cytological analysis of the appressoria indicated that ΔLPL1 mutants had a delay in the disappearance of lipid droplets. These findings imply that LPL1, phospholipid metabolism, or both are involved in glycerol biosynthesis and accumulation to generate turgor pressure in the appressorium. LPL1 was, however, dispensable for full pathogenicity, suggesting that other complementary pathways or similar genes related to phospholipid metabolism probably function in M. grisea.     

黑龙江省部分地区稻瘟病菌致病性分析及鉴别体系优化

为明确哈尔滨市及鸡西市稻瘟病菌致病性变化情况并优化鉴别体系,以24个水稻单基因系为鉴别体系,对2006—2008年采自2市的稻瘟病菌菌株进行致病型划分,并应用聚类分析方法完成鉴别体系优化。结果表明,应用水稻单基因系鉴别体系可将哈尔滨市2006—2008年采集的稻瘟病菌菌株划分为34、12和27个致病型,致病性相似系数分别为0.32~1.00、0~1.00和0.20~1.00;水稻单基因系对其抗性频率分别为2.86%~97.14%、8.33%~100.00%和10.00%~96.67%;鸡西市2006—2008年采集的菌株可划分为26、19和20个致病型,致病性相似系数分别为0.47~0.92、0.15~0.86和0.39~1.00;水稻单基因系对其抗性频率分别为3.85%~96.15%、5.26%~73.68%和4.76%~95.24%。适用于哈尔滨市的优化鉴别体系包括Pi-9、Pi-11、Pi-a、Pi-ks、Pi-5、Pi-i、Pi-sh、Pi-3、Pi-km和Pi-ta共10个基因,2006—2008年累计方差贡献率分别为86.15%、98.77%和87.39%;适用于鸡西市的优化鉴别体系包括Pi-9、Pi-11、Pi-a、Pi-ks、Pi-5、Pi-i、Pi-sh、Pi-1、Pi-7和Pi-t共10个基因,2006—2008年累计方差贡献率分别为82.58%、94.55%和90.37%。应用优化后的鉴别体系可将哈尔滨市2006—2008年采集的菌株划分为22、12和23个致病型,将鸡西市2006—2008年采集的菌株划分为22、14和18个致病型。     

高温胁迫下绿豆象内参基因筛选及其Hsp70基因的表达特性

为明确Hsp70蛋白在绿豆象Callosobruchus chinensis高温耐受性中的作用,该研究筛选并克隆β-actin、α-tubulin、β-tubulin、EF1-α、GAPDH、Hsc70、AK和RPL40八个候选内参基因,采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术测定其在绿豆象雌雄成虫体内的表达量,通过geNorm、BestKeeperr和NormFinde软件对这8个候选内参基因的表达稳定性进行评价,选择最适宜的内参基因对不同高温胁迫下绿豆象体内Hsp70基因的表达特性进行测定。结果表明,8个候选基因引物均有良好的特异性和引物扩增效率,引物扩增效率介于91.6%～108.1%之间。高温胁迫下,β-tubulin基因较其他7个候选基因有较小的平均变异度、稳定值和标准差,为最适宜的内参基因。39、42和45℃高温处理1 h后绿豆象雌雄成虫体内Hsp70表达水平较对照显著上调,当处理时间延长至3 h后,Hsp70基因的相对表达量较对照也有不同程度上调,但差异不显著,表明绿豆象Hsp70基因可以响应高温胁迫,Hsp70蛋白在绿...     

苹果中新抗旱相关基因GR- RBPs的克隆与表达分析

在构建楸子叶片SSH cDNA文库及EST分析的基础上,通过电子克隆(in silico cloning)和RT-PCR方法分别从楸子和平邑甜茶叶片中各克隆得到了1个富含甘氨酸RNA结合蛋白(GR-RBP)基因,分别命名为MpGR-RBP1( HM042682)和MhGR-RBP1 (HQ380209).它们的cDNA...     

温度和药剂胁迫下茶刺盲蝽内参基因稳定性分析

为筛选特定条件下实时荧光定量PCR检测体系中能稳定表达的茶刺盲蝽Helopeltis theivora内参基因,从其转录组中筛选并克隆11个候选基因Actin、β-tubulin1、RPL13A、EF1α、RPS3A、GAPDH、18S RNA、G6PDH、EIF4A、TBP和UBQ,测定它们在不同温度及药剂胁迫下mRNA的表达水平,并通过geNorm、NormFinder、BestKeeper、Delta Ct和RefFinder软件(算法)分析各基因的稳定性。结果表明,11个候选基因引物均具有良好的特异性和扩增效率(90.10%～96.87%)。温度胁迫下,EIF4A有最小的平均变异度(mean variability,M)、稳定值(stability value,SV)和几何平均值,分别为0.200、0.096和2.060;RPS3A的平均标准偏差(average of standard deviation,STDEV)最小,为0.605;二者是表达最稳定的基因。药剂胁迫下,RPL13A的M值和STDEV最小,分别为0.123和0.660;RPS3A的SV、标准偏差(standard deviation,SD)和几何平均值均最小,分别为0.063、0.336和1.189;二者是表达最稳定的基因;Actin为最不稳定基因。全样品评价中,RPL13A(M值为0.282、几何平均值为1.565)和RPS3A(SV为0.099、STDEV为0.614)的评价值同比最小,是表达最稳定的基因;最不稳定的基因为Actin、β-tubulin1和TBP。geNorm分析结果还显示所有处理条件下最适内参基因数目均为2个。表明EIF4A和RPS3A可作为茶刺盲蝽与温度相关的抗性基因表达研究的内参基因,RPS3A和RPL13A可作为该虫抗药基因表达研究的内参基因。     

基于转录组测序解析干旱胁迫对祁连山黄参基因表达的调控

以栽培2个月的黄参为试材,设置对照（土壤相对含水量70%~80%）和适度干旱胁迫（土壤相对含水量55%~60%）处理,利用高通量转录组测序BGISEQ-500平台,对测序结果进行基因功能注释、差异表达基因(DEGs, differentially expressed genes)筛选。结果表明：(1)获得的68193条Unigene中,分别有34230(50.20%)、34170(50.11%）、31727(46.53%)、27701(40.62%)、27092(39.73%)和22793(33.42%)个Unigene分别被分配到NCBI非冗余蛋白(NR)、eggNOG(基因的进化谱系, Evolutionary genealogy of genes: Non\|supervised Orthologous Groups)、基因本体(Gene ontology,GO)、Pfam (Protein family)、SwissProt (Reviewed protein sequence database)和KEGG (Kyoto encyelopedia of genes and genomes)六大功能数据库。(2)DEGs分析显示,黄参块状根和叶中分别有10674个和13402个DEGs;GO富集结果表明,根和叶中的DEGs功能部位中的分布基本一致,主要富集在生物过程、DNA的复制和翻译调控、氧化还原过程、蛋白质磷酸化、防御响应等;KEGG富集分析表明,根中DEGs显著富集在苯丙烷类生物合成、半乳糖代谢、半胱氨酸和甲硫氨酸代谢、淀粉和蔗糖代谢、植物-病原菌相互作用、植物激素信号转导等途径,叶中DEGs则主要富集在半乳糖代谢、淀粉和蔗糖代谢、苯丙烷类生物合成、戊糖、葡萄糖醛酸转换、植物激素信号转导等途径,说明淀粉和蔗糖代谢、半乳糖代谢、苯丙烷类生物合成途径、植物激素信号转导途径在黄参应对干旱胁迫中起重要作用。干旱胁迫影响黄参不同器官中差异基因的表达,为解析黄参耐受干旱的生物学途径、黄参药效成分的生物合成和分子机制提供了理论依据。     

Expression of genes related to Parkinson’s disease after paraquat treatment in Drosophila melanogaster

This study was designed to determine the minimum effective concentration of paraquat that modulated the expression of PKD-related genes in Drosophila. We first studied the viability of Drosophila and then tested the expression of the PKD-related genes—Parkin, UCH, and tau—in various concentrations of paraquat in the water sucked by Drosophila. The lowest effective concentration of paraquat was approximately 20 μM and the gene expression was induced at paraquat doses between 20 mM and 20 μM. Parkin and tau expression was inhibited, while that of UCH was significantly increased.Next, we examined the expression of the Parkin and UCH genes in the neurons of SOD-reduced mutants under oxidative stress conditions and found that Parkin was up regulated, while UCH was down regulated. We also found that the expression of Parkin was regulated by JNK. This study revealed that paraquat affects the expression of PKD-related genes via oxidative stress.In conclusion, our results showed that paraquat in the water sucked by Drosophila altered the gene expression at a minimum concentration of 20 μM, and that it not only promoted but also inhibited PKD-related gene expression via signal transduction mediated by oxidative stress. In order to confirm whether paraquat is a causal factor of PKD, more balanced and in-depth tests seem to be done looking into multiple aspects.     

辣椒PP2C家族基因鉴定与响应低温胁迫的表达分析

对辣椒PP2C基因家族成员进行全基因组鉴定和特征分析,并研究CaPP2Cs基因在低温胁迫下的表达模式,结果表明：在辣椒参考基因组共系统鉴定出99个CaPP2Cs基因家族成员,并不均匀地分布在12条染色体上,CaPP2Cs基因编码的氨基酸数介于99~1 572 aa,分子量介于10.94~178.40 Da,等电点介于4.08~9.51。系统进化树分析可将辣椒和拟南芥的PP2C基因分为13个亚族,每个亚族均含有辣椒PP2C基因,基因结构分析表明CaPP2Cs基因的外显子数量介于1~18;在CaPP2Cs基因的启动子中发现许多与光、低温和激素响应有关的顺式作用元件。转录组数据分析表明,CaPP2C31基因在叶片中的表达量显著高于其他基因,CaPP2C16则特异性地表达于授粉后50 d的胎座中。低温处理24 h时,CaPP2C97和CaPP2C3在叶片中呈现显著的下调表达;低温处理1 h时,CaPP2C36、CaPP2C5、CaPP2C53、CaPP2C43、CaPP2C99等基因在根中明显上调,CaPP2C19、CaPP2C23、CaPP2C44、CaPP2C95则显著下调,表明CaPP2Cs可能在辣椒响应低温胁迫中发挥作用。     

Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquito fish, Gambusia affinis after lethal effect of an organophosphate pesticide, monocrotophos

Recovery study was performed at regular intervals to establish the time course of 50% and 100% recovery in neurotransmitter enzyme (acetylcholinesterase, AChE, EC 3.1.1.7) and locomotor behaviour response of mosquito fish, Gambusia affinis exposed to lethal concentration (20.49 mg L⁻¹) of an organophosphorous pesticide, monocrotophos (MCP) for 96 h. In vitro AChE activity studies indicated that MCP could cause 50% inhibition (I₅₀) at 10.2 × 10⁻⁵ M. A positive correlation was observed between brain AChE activity and swimming speed during the recovery study. Also, the recovery response of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) as well as lipid peroxidation (LPO) as biomarkers of oxidative stress were assessed in viscera of G. affinis. The results showed that the MCP besides its inhibitory effect on target enzyme AChE activity and induction in antioxidant enzyme activities as a characteristic of oxidative stress, which can be used as biomarkers in the pesticide contaminated aquatic streams.     

低温胁迫下白菜型冬油菜脂肪酸组分及FAD3的差异表达分析

为探究白菜型冬油菜(Brassica rapa L.)脂肪酸组分对低温胁迫的响应及脂肪酸去饱和酶FAD3(ω-3-fatty acid desaturase)基因与抗寒性的关系,采用气相色谱-质谱法测定了3个不同抗寒性白菜型冬油菜品种在低温胁迫下根和叶的脂肪酸组分变化;利用同源克隆法获得白菜型油菜陇油7号FAD3的CDS序列,利用生物信息学方法对其进行分析。结果显示:低温胁迫下,强抗寒品种陇油7号根部亚油酸和亚麻酸的含量较不抗寒品种Lenox分别高157%和89%,陇油7号根部脂肪酸不饱和指数(IUFA)较Lenox高91%,即低温胁迫下,强抗寒品种的亚油酸、亚麻酸和IUFA均显著高于不抗寒品种;陇油7号FAD3序列CDS区全长1 290 bp,编码429 aa,分子质量约49.16 kD,等电点为8.60,是一种稳定的亲水蛋白。多序列比对分析发现其编码蛋白与同一科属中大白菜相似性最高,为100%,进化树分析结果也表明FAD3蛋白与大白菜亲缘关系较近。荧光定量表达分析发现低温胁迫下,FAD3基因在陇油7号、天油2号和Lenox根中的表达量较对照(22℃)分别增加了690%、590%和710%,说明FAD3基因通过上调表达来适应低温,且其表达量与IUFA呈正相关关系。     

水稻苗期感染SRBSDV后赤霉素相关基因表达量及赤霉素含量的变化

为明确水稻在苗期被南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)浸染后植株体内赤霉素合成相关基因表达量和赤霉素含量的变化情况,利用q RT-PCR技术检测水稻在3叶期感染SRBSDV后的5个时期Os01g0883800、Os03g0856700、Os07g0169700三个赤霉素的生物合成基因表达量;采用高效液相色谱法检测GH998和Y11植株感染SRBSDV后的赤霉素含量;观察GH998病株施用外源赤霉素后病症的缓解效果。结果表明,感染SRBSDV后在GH998叶片中,以在第0天为对照组,Os01g0883800和Os07g0169700相对表达量最大分别下调25.6倍和21.1倍,Os03g0856700在第3天相对表达量下调20.5倍,在第6天上调2.3倍;在茎中,Os01g0883800、Os07g0169700、Os03g0856700的相对表达量在第3天分别下调8.5倍、6.4倍和1.4倍。在Y11叶片中Os01g0883800、Os03g0856700和Os07g0169700相对表达量最大下调分别为6.6倍,42.7倍和16.9倍;在茎中,Os01g0883800、Os07g0169700相对表达量最大下调分别为5.3倍和10.5倍;Os03g0856700在第3天时表达量上调1.5倍,在第12天下调2.8倍。GH998感染SRBSDV后植株体内赤霉素含量显著降低;施用外源赤霉素能缓解SRBSDV部分病症,表现为株高、剑叶长、剑叶宽和根长等均增加。     

外源ABA和钨酸钠对干旱胁迫下白菜型冬油菜ZEP表达的影响

为研究白菜型冬油菜玉米黄质环氧酶(ZEP)基因在干旱胁迫下响应外源ABA的分子机制,本研究选择白菜型冬油菜陇油7号和天油4号为试验材料,分别喷施蒸馏水、ABA溶液、钨酸钠溶液、ABA+钨酸钠溶液200 mL后置于植物培养箱干旱处理7 d。采用RT-PCR和qPCR等技术进行ZEP基因克隆和表达分析,结果表明:该基因包含一个长度为2 013 bp的ORF,编码含670个氨基酸残基的蛋白质,理论等电点为6.16,属于亲水性蛋白。该基因与拟南芥、甘蓝型油菜的同源性分别为89.25%和99.40%。实时荧光定量分析表明,干旱胁迫可诱导白菜型冬油菜ZEP基因上调表达,外源喷施ABA后陇油7号叶片ZEP基因相对表达量较CK升高66.64%,喷施外源ABA合成抑制剂钨酸钠后陇油7号叶片ZEP基因表达量较CK降低12.74%,而同时喷施外源ABA和钨酸钠后,干旱胁迫下陇油7号、天油4号叶片ZEP基因表达量较CK分别增加了3.3倍和2.28倍,陇油7号叶片中的ZEP基因表达量较根系高21.61%,表明ZEP基因在白菜型冬油菜抗旱中可能发挥一定作用。     

温度胁迫下沙葱萤叶甲小热激蛋白基因GdHsp20.6的克隆及表达分析

为明确小热激蛋白在沙葱萤叶甲Galeruca daurica应对温度胁迫中的作用,采用PCR方法克隆沙葱萤叶甲小热激蛋白基因Hsp20（GdHsp20.6）完整的开放阅读框（open reading frame,ORF）序列,应用在线软件对GdHsp20.6基因进行生物信息学分析,通过原核表达技术诱导表达及纯化其编码蛋白,并通过实时荧光定量PCR（real-time quantitative PCR,RT-qPCR）技术分析不同温度胁迫下GdHsp20.6基因的表达量。结果显示,GdHsp20.6基因ORF序列长度为543 bp,编码180个氨基酸,预测分子量为20.6 kD,无跨膜区和信号肽。GdHsp20.6氨基酸序列有高度保守的α-结构域。GdHsp20.6氨基酸序列与其它鞘翅目昆虫的Hsp20氨基酸序列有较高的一致性,其中与花绒寄甲Dastarcus helophoroides的Hsp20.99氨基酸序列的一致性最高,为63%。GdHsp20.6基因在大肠杆菌Escherichia coli BL21（DE3）细胞系中成功表达,经异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-β-d-thiogalactoside,IPTG）诱导后GdHsp20.6蛋白成功表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）和Western-blot分析表明融合蛋白大小与预测大小一致,并纯化获得了纯度较高的目的蛋白GdHsp20.6。低温（-10~5℃）和高温（35~40℃）处理1 h以及处理后25℃恢复30 min均能诱导GdHsp20.6基因表达上调,并且0℃处理30~120 min也能诱导GdHsp20.6基因表达上调。表明GdHsp20.6基因在沙葱萤叶甲应对低温和高温胁迫中可能起着重要作用。     

沙葱萤叶甲热激蛋白基因GdHsp10a的克隆、分子特征与表达分析

为明确热激蛋白HSP10在沙葱萤叶甲Galeruca daurica生长发育过程中的作用,采用RTPCR及RACE技术克隆沙葱萤叶甲Hsp10基因cDNA的全长序列,采用生物信息学方法分析其序列特征,并利用qPCR技术对该基因在沙葱萤叶甲不同发育阶段、成虫羽化后不同时期以及不同温度下的表达谱进行分析。结果表明,克隆获得1条新的沙葱萤叶甲Hsp10基因,命名为GdHsp10a,GenBank登录号为MG460308,cDNA序列全长为526 bp,开放阅读框为333 bp,编码蛋白含110个氨基酸,预测分子量为11.97 kD,等电点为9.74;无信号肽及跨膜结构。同源比对及系统进化分析表明,GdHSP10a与光肩星天牛Anoplophora glabripennis HSP10亲缘关系最近,氨基酸序列一致性为53.15%。qPCR结果表明,GdHsp10a在沙葱萤叶甲各发育阶段均有表达,其中在卵期和成虫期的表达量显著高于幼虫期、预蛹期及蛹期;成虫羽化后不同时期表达量差异显著,25 d时表达量最高,其次为100、10和7 d时的表达量;温度对GdHsp10a表达量有显著影响,30℃下表达量最高,35℃下表达量次之,15、20、25及40℃下表达量最低且无显著差异。表明GdHsp10a可能在沙葱萤叶甲生长发育及成虫越夏中起着多重作用。     

三叶草斑潜蝇hsp70的克隆及其表达量在高低温胁迫下的变化

为了明确高低温胁迫对三叶草斑潜蝇hsp70表达量的影响,采用RT-PCR和RACE技术获得了1条三叶草斑潜蝇诱导型热激蛋白基因hsp70,命名为Lthsp70-1,并利用实时荧光定量PCR技术检测其在温度胁迫后的表达量.该基因的开放阅读框为1923 bp,编码640个氨基酸.氨基酸序列中含有HSP70家族的签名序列IFDLGGGTFDVSIL和IVLVGGSTRIPK、DnaK特征基序IDLGTT(Y)S(C)V、非细胞器基序RARFEEL,以及C末端的保守序列EEVD.实时荧光定量PCR结果显示:成虫在31～33℃范围内,其hsp70表达量随温度升高而上升,33℃时达到最高峰;35～39℃时,hsp70表达量迅速下降;蛹经0℃胁迫0.5～2.0 h,其hsp70表达量随时间延长呈上升趋势.由此可见,高低温胁迫均能诱导三叶草斑潜蝇hsp70的表达.     

Co-stressors chilling and high light increase photooxidative stress in diuron-treated red alga Kappaphycus alvarezii but with lower involvement of H2O2

Diuron is one of the most commonly found N-phenylurea herbicides in marine/estuarine waters that promotes toxic effects by inhibiting photosynthesis and affecting the production of reactive oxygen species (ROS) in autotrophs. Since photo- and thermoacclimation are also ROS-mediated processes, this work evaluates a hypothetical additive effect of high light (HL) and chilling (12 °C) on 50 nM diuron toxicity to the highly-photosynthetically active apices of the red alga Kappaphycus alvarezii. Additive inhibition of photosynthesis was mainly evidenced by significant decreases of quantum yield of photosystem II and electron transfer rates upon co-stressors exposure to diuron-treated algae. Under extreme 12 °C/HL/diuron conditions, unexpected lower correlations between H₂O₂ concentrations in seawater and radical-sensitive protein thiols were concomitantly measured with the highest indexes of photoinhibition (parameter β). Altogether, these data support the hypothesis that co-stressors chilling/HL additively inhibit photosynthesis in diuron-exposed K. alvarezii but with less involvement of H₂O₂ in injury effects than with only chilling or HL.     

紫外线、高压静电和大麦黄矮病毒胁迫下荻草谷网蚜SOD基因表达

为探索环境胁迫影响昆虫适合度的内在机制,通过cDNA末端快速扩增（rapid amplification of c DNA end,RACE）技术克隆得到荻草谷网蚜Sitobion miscanthi体内超氧化物歧化酶（superoxide dismutase,SOD）基因MnSOD和CuZnSOD的cDNA序列全长,对该序列进行分析,检测紫外线、高压静电和大麦黄矮病毒（barley yellow dwarf virus,BYDV）胁迫下荻草谷网蚜体内MnSOD和CuZnSOD基因表达量的变化。结果显示,荻草谷网蚜体内MnSOD和CuZnSOD基因的cDNA序列全长分别为1 517 bp和954 bp(GenBank登录号分别为MT533627和MT533626),开放阅读框分别为669 bp和459 bp,分别编码222个和152个氨基酸,预测蛋白质相对分子量分别为24.99 kD和15.84 kD。荻草谷网蚜体内MnSOD和CuZnSOD蛋白均与半翅目蚜科昆虫同源蛋白氨基酸序列相似性高,亲缘关系最近。0.50 m W/cm2（低强度）和0.70 m W/cm2（高强度）紫外线胁迫下...