泡桐丛枝病传病昆虫研究(Ⅰ) 茶翅蝽传病试验及电镜观察

泡桐丛枝病传病昆虫试验,是深入开展病原、发病规律及防治措施研究的关键.我们自1978年开展了泡桐丛枝病病原等项研究时,对泡桐树上刺吸式口器害虫作了全面调查,并以茶翅蝽(Halyomorpha halys(Stal))等6种昆虫作了传病试验.本文报道了我们1980—1981年对茶翅蝽传病试验和电镜观察结果.其他虫种的传病试验,尚在进行中.     

美国白蛾核型多角体病毒ORF72原核表达载体的构建、表达及纯化

[目的]根据Ikdea对Hycu NPV全基因组序列的分析,筛选出功能还未被研究的、保守性高的ORF72基因。[方法]将ORF72基因构建到原核表达载体p GEX-4T-1上,对其进行诱导表达,优化表达的条件,并通过GST亲和层析色谱柱纯化融合蛋白。[结果]重组质粒p GEX-4T-1-ORF72的酶切检测以及测序结果均正确,证明重组质粒载体构建成功。通过SDS-PAGE凝胶电泳显示,ORF72蛋白能够与p GEX-4T-1载体上的GST标签蛋白进行融合表达,诱导表达后的融合蛋白大小约为38. 2 k Da。优化后的表达条件为:异丙基硫代β-D-半乳糖苷(IPTG)诱导浓度为1. 0 mmol·L-1,最佳诱导温度为25℃,最佳诱导时间为4 h,并且该蛋白在大肠杆菌BL21和Rosetta表达菌株中均能很好的表达,但在Rosetta菌株中表达量更高。[结论]成功构建了p GEX-4T-1-ORF72原核表达载体,确定出ORF72融合蛋白最佳诱导表达条件,诱导表达并纯化出ORF72融合蛋白,为进一步研究其生物功能奠定基础。     

泡桐丛核病病原及传毒途径的研究

本文研究了泡桐丛枝病病原和传毒途径。结果表明:类菌原体多为圆形或椭圆形,直径180～800mμ。多存在于病丛枝韧皮部筛管细胞内。繁殖方式分芽殖和两两分裂两种。种根和茶翅蝽、小绿叶蝉是泡桐丛枝病的传毒介体。种子不传毒,嫁接能够传毒。     

茶翅蝽对植原体的传播能力

经１９９６年至１９９８年室内接种试验,分别比较了人工饲养带毒和自然带毒的杀翅蝽成虫和若虫的传病特点。结果显示：室内人工饲养带毒的３龄、４龄若虫接种泡桐发病率分别达６１．７％和４６．５％,高于自然带毒的若虫１６％的传病率。室内饲带毒成虫的接种发病率为１４．１％,高于自然带毒成虫４．５％的传病率。寄主体内病原的最短潜育期（ＭＩＰ）室内饲毒３龄若虫为３４ｄ,４龄为４５ｄ,成虫处理为２１９ｄ;自然带毒若虫     

吸枣疯病病原的中国拟菱蚊叶蝉传染泡桐丛枝病

泡桐丛枝病和枣疯病病害症状相似,病原皆为MLO。传播媒介前者是茶翅蝽和虻蝽,后者为中拟菱蚊叶蝉(简称枣叶蝉)。1981年6月,将40多头饲枣疯病病毒7天的枣叶蝉成虫,接于锦纶纱笼内的5株泡桐实生苗上,8月中旬成虫全部死亡。1982年8月上旬,有一株苗木呈现典型的丛枝病症状,3个月后逐渐枯死。1983年初,又有一株苗木表现丛枝症状。1982年9月初、10底分别将枣叶蝉传毒发病的泡桐苗上的小叶柄、叶脉进行固定包埋,做超簿切片进行电镜观察。发现叶脉     

利用平腹小蜂防治茶翅蝽田间应用效果

试验借鉴广东省利用平腹小蜂Anastatus sp防治荔枝蝽Tessaratoma papillosa Drury的成功经验防治北京地区的茶翅蝽Halyomorpha halys(Stal).室内规模化繁殖平腹小蜂后,在野外进行大量释放,结果发现平腹小蜂对第1,2代荼翅蝽卵粒的平均寄生率分别为64.7%和52.6%,能较好地控制茶翅蝽.     

荔蝽、稻绿蝽和茶翅蝽生物防治研究概况

综述国内外研究较多的荔蝽Tessaratoma papillosa、稻绿蝽Nezara viridula、荼翅蝽Halyomorpha hatys生物防治研究进展,概括对这3种蝽起主要控制作用的天敌种类及其利用情况,并重点探讨了北方地区有机生态果园危害严重的茶翅蝽的生物防治研究及持续控制技术.     

毛白杨PtCDD基因5'片段的原核表达及功能分析

根据GenBank中毛白杨钙离子依赖型脱氧核糖核酸酶(PtCDD)基因序列,以毛白杨形成层区域的cDNA为模板,经PCR扩增出该基因上游637 bp的cDNA片段.将该片段与PET-30b( )载体连接,构建杨树PtCDD基因片段原核表达载体并进行表达研究,获得大量的PtCDD-(HIS)6 融合蛋白.并进一步对该蛋白进行纯化和功能检测.结果表明:PtCDD基因片段能够在大肠杆菌体内表达出具有DNase功能的蛋白,克服了因表达全酶可能强烈降解DNA所带来的不能最终获得表达产物的问题,为今后PtCDD蛋白的抗体制备以及进一步研究奠定了基础.     

注射传播泡桐丛枝病类菌原体的初步研究

以泡桐丛枝病类菌原体的敏感指示植物长春花苗为材料,用叶柄注射法注入感病泡桐叶汁液进行接种试验,结果表明,有1/4供试植株感病,其症状与泡桐丛枝病相一致,将感病植株叶脉切片镜检,发现有类菌原体,其形态、大小及结构,均与泡桐丛枝病类菌原体相同。为该病病原的传播及回接提供了一条简便的途径。     

BtCry1Ac、BADH双价基因对烟草的遗传转化及表达检测

利用农杆菌介导法,将植物转化载体p209-BtCry1Ac-BADH转化烟草,获得了Kan筛选的完整再生植株。经PCR检测证明,4个株系中检测到BtCry1Ac和BADH基因。荧光定量PCR检测表明,检测到目的基因的株系中有3个株系的2个目的基因均在转录水平得到表达,且存在表达差异;有1个株系未检测到BADH基因的表达。ELISA毒蛋白检测表明,经荧光定量PCR筛选的3个株系均检测到毒蛋白的表达,含量最高为414.63ng/g。室内饲虫试验表明,3个转基因株系中只有2个株系对斜纹夜蛾幼虫表现出抗虫性,校正死亡率最高为38.2%,其余均未表现出明显抗虫性。选取2个株系进行组培苗耐盐试验,表明在不同NaCl浓度下,转基因株系与对照之间没有明显差异。研究中,有些系号经PCR检测说明BtCry1Ac和BADH基因已整合到植物基因组中,但荧光定量PCR和ELISA检测表明虽然基因整合到植物基因组中却未表达,有可能发生了基因沉默。     

泡桐丛枝植原体胸苷酸激酶的原核表达、纯化及酶活性测定

胸苷酸激酶是d TTP从头合成和补救途径的关键酶,催化d TMP形成d TDP,在DNA复制和生物的生存中发挥着必不可少的作用。本文在前期研究的基础上,对从泡桐丛枝(Pa WB)植原体中获得的的3个同源蛋白TMK-a-1、TMK-a-2及TMK-b与已报道的小麦蓝矮(WBD)、洋葱黄化(OY-W)植原体的TMK-a、TMK-b的氨基酸序列进行了比对和相似性分析,结果显示Pa WBPS TMK-b与WBD TMK-2和OY-W TMK-b之间的相似性分别为95.65%和99.03%;Pa WBPS TMK-a-1与Pa WBPS TMK-a-2的相似性为90.57%,且二者与WBD TMK-1和OY-W TMK-a之间的相似性为87.32%-94.26%;而Pa WBPS、WBD、OY-W三种植原体的TMK-a与TMK-b之间的相似性仅为22.22%-25.95%。构建了Pa WBPS TMK-b、TMK-a-1、TMK-a-2的p ET28a原核表达载体,对Pa WBPS TMK-b、TMK-a-1、TMK-a-2 3种蛋白进行了原核表达,经Ni-NTA柱纯化后,利用双酶法进行了胸苷酸激酶催化活性测定,结果表明,Pa WBPS TMK-b具有较高的胸苷酸激酶活性,为85.96±0.74 U·mg-1,而Pa WBPS TMK-a-1和TMK-a-2几乎没有胸苷酸激酶活性。本文为进一步研究胸苷酸激酶在植原体繁殖过程中的作用机理奠定了基础。     

杨树生物钟节律基因PtCCA1的克隆及表达模式研究

生物钟节律基因CCA1在植物的光周期反应中起着重要的调控作用.利用RT-PCR方法克隆获得了一条包含1902 bp开放阅读框的杨树CCA1基因cDNA序列,其编码633个氨基酸残基,蛋白的分子量约为68.90 kDa,等电点为6.33.分析显示含有1个Myb-like DNA结合域及2个蛋白跨膜结合域.实时荧光定量PCR分析发现,PtCCA1基因主要在杨树叶组织中表达;随着光照时间的变化,该基因在杨树中的表达量呈现白昼不断降低而夜晚逐渐升高的昼夜变化趋势.研究结果为进一步探索PtCCA1基因在调控杨树光周期响应途径中的功能以及杨树光周期敏感机制提供了科学基础.     

刚毛柽柳富含甘氨酸RNA结合蛋白ThGRP1基因克隆与表达分析

在有机体的生长和发育过程中,基因表达在转录、转录后和翻译水平都受到严格地调控。转录调控是基因表达调控的第一步,曾被认为是基因表达的主要调控机制,但是随着对转录后调控机制越来     

日本落叶松谷胱苷肽还原酶的生化特性研究

本研究从日本落叶松中克隆到一个谷胱苷肽还原酶(GR)基因,命名为LaGR。该基因编码563个氨基酸组成的蛋白质,预测分子量为61.06 kDa。表达模式分析发现LaGR基因在芽、成熟针叶、茎韧皮部和根韧皮部均表达,是组成型表达基因。蛋白质亚细胞定位研究发现LaGR蛋白定位在叶绿体。在大肠杆菌中表达并纯化了LaGR重组蛋白。酶学性质分析发现,LaGR蛋白对底物GSSG和NADPH具有较高的催化活性和亲和力,是热稳定蛋白,而且最适pH值范围在7.0 9.0之间。Cd²⁺、Pb²⁺和Cu²⁺等重金属离子对LaGR蛋白的催化活性具有明显的抑制作用。将LaGR蛋白的第528位His突变为Gln后,其突变蛋白的催化活性显著降低,而且与野生型LaGR蛋白相比,突变蛋白的动力学常数、最适pH值范围和热稳定性均发生了显著变化,预示着第528位的His在LaGR的催化特性和蛋白结构稳定性方面发挥着重要作用。     

Behavioral responses of the egg parasitoid <Emphasis Type="Italic">Trissolcus japonicus</Emphasis> to volatiles from adults of its stink bug host, <Emphasis Type="Italic">Halyomorpha halys</Emphasis>

Halyomorpha halys, originating from East Asia, has become an important agricultural pest in northern temperate regions since its accidental introduction into North America in the mid-1990s and Europe in 2007. Trissolcus japonicus is the predominant egg parasitoid of this pest in its native range. Here, we tested the hypothesis that T. japonicus uses volatiles associated with H. halys adults during host location. The effects of these volatile compounds on H. halys adults were also assessed. Chemical analysis with GC-MS and electrophysiological analysis with GC-EAD revealed the presence of two bioactive volatile compounds from H. halys adults, n-tridecane and (E)-2-decenal. In the Y-tube assays, female T. japonicus were attracted by n-tridecane but strongly repelled by (E)-2-decenal. Furthermore, host searching time of female T. japonicus was significantly reduced when an H. halys egg mass was treated with n-tridecane and prolonged when treated with (E)-2-decenal or a mixture of (E)-2-decenal and n-tridecane. Male H. halys were significantly attracted by n-tridecane, whereas females had no response to this compound in the Y-tube assays. (E)-2-Decenal strongly repelled male and female H. halys in the Y-tube assays. We discuss the multifunctional kairomonal and other possible roles of these semiochemicals at different trophic levels in the context of intra- and inter-specific chemical communication, in the H. halys–T. japonicus system.     

白蜡虫蜡酯合酶基因cDNA全长克隆及原核表达

正白蜡虫(Ericerus pela)是我国特有的资源昆虫,泌蜡是白蜡虫二龄雄虫最为特化的性状[1]。白蜡虫分泌的白蜡在食品、医药、纺织等行业具有重要的经济价值[2]。白蜡的主要成分是26酸26酯[3],已有研究表明,蜡酯的生物合成在动植物中存在保守途径[4]。蜡酯生物合成的第一步由脂酰辅酶A还原酶催化脂酰辅酶A转化为脂肪醇,第二步由蜡酯合酶(WS)催化脂肪醇和脂肪酸的酯化反应[5]。WS     

蓝花楹JaCBF1转录因子片段的序列分析及耐寒性功能验证

CBF类转录因子是调控植物冷害应答反应的关键因子.从蓝花楹中克隆到一个457 bp CBF类基因片段,序列分析表明其编码142氨基酸的片段,并与多个物种CBF1基因序列高度同源,且具有CBF家族的AP2核心结构域,因此将其命名为JaCBF1.原核表达的结果显示此基因片段编码16 kDa的蛋白,表达分析结果显示JaCBF1于低温条件下在蓝花楹根、茎、叶中有差异性转录.Southern杂交结果揭示JaCBF1在蓝花楹基因组中存在至少2个拷贝.通过脓杆菌介导方法将JaCBF1异源转录到拟南芥中,荧光共聚焦显微照相结果显示其编码的蛋白质定位于细胞核,冷处理实验发现转基因拟南芥植株的耐冷性得到了显著提高,表明此新片段具有CBF类植物耐冷因子的典型功能.     

Natural biological control of <Emphasis Type="Italic">Halyomorpha halys</Emphasis> by native egg parasitoids: a multi-year survey in northern Delaware

Halyomorpha halys (Stål) is a highly polyphagous plant pest native to eastern Asia. Since its accidental introduction to the USA in the mid-1990s, it has spread across North America and caused serious economic damages. Chemical control has been a widely applied management strategy which is not sustainable on a long-term basis. A nine-year survey using sentinel and naturally laid H. halys was conducted to investigate the extent of natural biological control by parasitoids in managed and non-managed landscapes in northern Delaware, USA. Naturally laid egg masses of native pentatomids were collected to compare the resident parasitoid complex to the one attacking H. halys eggs. Mean parasitism rates of sentinel eggs were below 6% in any given year of the survey with a grand mean of 1.79%. Parasitism of naturally laid H. halys eggs was higher than that of sentinel eggs, but there was no significant difference in parasitism between years and the grand mean was 4.31%. Parasitoid species richness on sentinel and naturally laid H. halys eggs was similar but both were lower than the richness on native pentatomid eggs. H. halys eggs were parasitized by eight species in the genera Anastatus, Trissolcus, Telenomus, and Ooencyrtus. The level of parasitism by native egg parasitoids is insufficient to provide long-term control of H. halys. Trissolcus japonicus (Ashmead), an Asian egg parasitoid that has been recovered in the USA in several locations since 2014, may be the most promising candidate for long-term biological control of H. halys.     

Field surveys of egg mortality and indigenous egg parasitoids of the brown marmorated stink bug, <Emphasis Type="Italic">Halyomorpha halys</Emphasis>, in ornamental nurseries in the mid-Atlantic region of the USA

The brown marmorated stink bug, Halyomorpha halys (Stål), is an invasive species native to regions of China, Japan, Korea, and Taiwan. In its native and introduced range, H. halys is considered a pest of tree fruits, vegetables, legumes, and ornamental trees. The highly polyphagous nature of this insect as well as its vast dispersal capabilities, require an integrated approach to management. Here we focus on the potential impact of indigenous natural enemies on H. halys in woody ornamental nurseries in Maryland, USA. We sampled naturally field laid H. halys egg masses for mortality and parasitism rates in 2012 and 2013. Overall, egg mortality averaged 54% for both years, and increased within season and between years. The largest source of mortality was from egg parasitoids with mean parasitism rates of 30.73 and 39.63% in 2012 and 2013, respectively. Mortality from predation was much lower and averaged 4.61% by chewing and 2.53% by sucking predators. We found seven species of Hymenopteran egg parasitoids attacking H. halys eggs, with Anastatus reduvii being the dominant species comprising 61.17 and 79.12% of all parasitoids in 2012 and 2013, respectively. The observed increase in parasitism over time and female biased sex ratio of parasitoids suggests that native parasitoids may be responding to the novel host, H. halys. Consequently, the use of these native eupelmid egg parasitoids in augmentative or conservation biological control may be a viable H. halys management strategy in ornamental nurseries.     

Pheromone-based decision support tools for management of <Emphasis Type="Italic">Halyomorpha halys</Emphasis> in apple orchards: development of a trap-based treatment threshold

The invasive brown marmorated stink bug, Halyomorpha halys (Stål), has become a serious pest in mid-Atlantic apple orchards. Because no decision support tools exist for H. halys management, calendar-based insecticide applications have been the only successful technique for mitigating H. halys injury in orchards. Following the identification of the two-component aggregation pheromone of H. halys, we conducted a study to determine whether biological information generated by traps baited with this pheromone and pheromone synergist could be used to make management decisions. In this study, experimental apple orchards were treated with a H. halys-targeted insecticide only when a predetermined cumulative threshold of either 1, 10, or 20 adults per trap was reached. Once threshold was reached, two alternate row middle sprays 1 week apart were triggered and the threshold was reset. For comparison, some orchards were also subjected to a weekly alternate row middle spray or left untreated as controls. At harvest, significantly less fruit injury was observed when treated weekly or using a threshold of 1 or 10 H. halys adults per trap to trigger insecticide applications. Orchards treated using a cumulative threshold of 20 adults per trap or when left unsprayed had significantly higher fruit injury. In addition, insecticide applications were reduced by 40% using a threshold of 10 adults per trap. Our results suggest that baited pheromone traps can be used as decision support tools to trigger insecticide applications when needed to mitigate risk and effectively manage H. halys in apple orchards.