药用野生稻应答稻飞虱取食过程中基因表达的研究

褐飞虱和白背飞虱是水稻的刺吸式害虫。为了探讨水稻对虫害反应的分子机理,采用cDNA—AFLP(互补DNA扩增片段长度多态性)技术,研究了药用野生稻受这两种飞虱取食后基因表达的变化。通过调查约7％的虫．崮答后的药用野生稻转录本组,共分离了258个差异片段,测序112个;44个片段对应于已知或假定功能基因,其中35个基因的表达水平在白背飞虱为害后发生改变,24个基因在褐飞虱为害后改变。下调的基因比上调的基因多。有14个基因受这两种飞虱共同调节(2个上调,12个下调)。结果表明,药用野生稻对两种飞虱为害作出的反应有所不同。     

Characterization of Rice Black-Streaked Dwarf Virus- and Rice Stripe Virus-Derived siRNAs in Singly and Doubly Infected Insect Vector Laodelphax striatellus

Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi) which generates viral-derived small interfering RNAs (siRNAs). However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus) was infected by Rice black-streaked dwarf virus (RBSDV) (Reoviridae; Fijivirus), more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV), a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5′- and 3′-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.     

RNA干扰的研究进展及应用

RNA干扰(RNAi)是生物体的一种在进化上保持高度保守的,能抵御外源基因或外来病毒侵犯的重要防御机制,是一种序列特异性的转录后基因沉默现象。它由双链RNA引发,广泛存在于动、植物等各种生物体内。我们简要综述了RNAi发生的机制、特点、哺乳动物与RNAi现象,以及RNAi的应用等。     

稻飞虱迁飞种群的上灯行为节律研究

【目的】明确稻飞虱迁飞种群的上灯行为节律,以指导其大田迁入种群和迁出种群的发生预测与灾变预警。【方法】本研究运用逐时自动灯诱装置对2010和2011连续两年稻飞虱迁飞种群的上灯行为节律进行了系统研究。【结果】灰飞虱Laodelphax striatellus迁飞种群上灯始见期和灯诱虫量年际间差异不明显,白背飞虱Sogatella furcifera和褐飞虱Nilaparvata lugens迁飞过境种群上灯始见期和灯诱虫量年际间差异较大。此外,灰飞虱迁飞种群的特大高峰期和高峰期逐时灯诱虫量百分比与一般上灯期和零星上灯期相比突出了晨暮双峰型中的暮峰型上灯行为特点;白背飞虱迁飞种群特大高峰期逐时灯诱虫量百分比与高峰期和一般期相比突出了晨暮双峰型中的晨峰型生物学特性。【结论】稻飞虱迁飞种群的上灯行为节律存在种的特异性,这一行为节律除了受环境因素的影响外主要与其生物学特性有关。     

蜘蛛对稻纵卷叶螟和白背飞虱的选择捕食作用

讨论蜘蛛的主要种群拟水狼蛛、食虫瘤胸蛛、粽管巢蛛对稻纵卷叶螟、白背飞虱的选择捕食特性。由结果可见：猎物被捕食量与参试种群编码值（密度）的数量关系可由二次多项式模型技术。当取每科水稻上有稻纵卷叶螟５头,白背飞虱１０头,拟水狼蛛２头,粽管巢蛛２头,食虫瘤胸蛛２头时,蜘蛛种群对白背飞虱有较大的选择捕食比率。     

水稻白背飞虱发生程度的长期预测模型

气象条件影响白背飞虱Sogatellafurcifera发生发展 ,根据海气相互作用原理 ,海温的变化会引起大气环流系统的变化 ,同样制约各地气象条件。作者将西太平洋海温作为长期预报因子 ,根据场相关分析方法 ,找出与水稻白背飞虱发生程度各指标相关的强信号海区 ,应用最优化相关处理技术 ,大大增加了虫情发生指标和海温之间的相关度 ,建立了水稻白背飞虱始见期、迁入虫量及第五 ( 2 )代虫量可靠的长期预测模型 ,模型均通过了α=0 0 1显著性水平 ,拟合及试报结果理想 ,预报模型切实可行 ,可以投入业务使用 ,此研究对虫害防治和水稻生产具有重要指导意义     

RNA干扰技术在基因治疗中的应用进展

RNA干扰(RNA interference,RNAi)是一种双链RNA分子在mRNA水平上关闭相应序列基因的表达或使其沉默的过程,在基因治疗方面有着无可比拟的优势,已成功的应用于肿瘤、病毒感染、遗传性疾病及神经系统疾病等重大疾病的治疗.本文将主要介绍siRNA基因治疗的导入方法与途径,以及在不同疾病中,RNAi技术进行基因治疗的应用.     

大气环流特征量的水稻白背飞虱发生程度预报模型的研究

根据500hPa大气环流特征量能表征天气形势和控制天气条件的这一特性,利用线性及单调曲线相关与最优化因子相关两种技术对环流特征量因子进行普查、对比分析,发现白背飞虱虫情指标与环流因子之间关系不仅是线性及几种单调曲线的关系,而且还存在非线性、非单调的单峰(谷)型的相关关系,并从中挑选一批与水稻白背飞虱虫情指标相关极其显著、稳定性强、因子间相互独立、可靠的大气环流特征量作为预报因子,在此基础上,建立了江苏省水稻白背飞虱虫情指标预报的环流模型,且还证实了模型中的环流因子与影响白背飞虱迁入、发生发展的气象条件呈显著相关关系,为预报白背飞虱发生程度提供了新的长期因子和新途径。     

Spl基因RNA干扰载体的构建及鉴定

目的:构建干扰载体pSilencer 3.1-spl,并初步研究其对Spl基因的干扰作用.方法:根据Spl cDNA编码序列,设计并合成针对Spl基因的特异性RNA干扰片段,并将其克隆入pSilencer 3.1-Hl neo干扰载体中,构建Spl基因小干扰RNA(siRNA)真核表达载体pSilencer 3.1-Spl;分别将阴性对照载体pSilencer 3.1与重组载体pSilencer 3.1-Spl经脂质体LipofectAMINE2000介导转染HeLa细胞,采用RT-PCR、Western blot方法分别检测Spl基因的转录与表达水平.结果:构建了Spl基因siRNA真核表达载体pSilencer 3.1-Spl,经酶切、测序鉴定证实克隆正确,并在mRNA水平和蛋白水平证实了载体的干扰效果.结论:特异性siRNA能明显抑制Spl基因在HeLa细胞中的表达,为进一步研究Spl的生物学功能和作用机制奠定了实验基础.     

慢病毒介导RNA干扰SOCS3基因在猪前体脂肪细胞和成肌细胞中的表达

构建细胞信号抑制因子3(suppressor of cytokine signaling 3,SOCS3)慢病毒干扰载体,获得有感染性的病毒颗粒,感染猪前体脂肪细胞和成肌细胞,并检测其对前体脂肪细胞的干扰效率.首先设计并合成3对针对目的基因SOCS3的siRNA序列,退火后连接于LentiH1上,测序验证后,与包装质粒△8.9和vsv-g共转染到293T细胞中进行包装和浓缩,纯化后测定病毒滴度,然后感染猪前体脂肪细胞和成肌细胞.重组慢病毒载体LentiH1-siRNA经酶切和测序鉴定正确,病毒滴度为3×107tu/mL,感染猪成肌细胞和前体脂肪细胞后,可见报告基因GFP的表达;RT-PCR和Western印迹分析表明,前体脂肪细胞中SOCS3的表达被显著下调,其中LentiH1-siRNA3介导对SOCS3基因mRNA和蛋白的干扰效率分别达53%和71%.本研究成功构建了猪SOCS3慢病毒干扰载体,感染猪前体脂肪细胞能稳定沉默SOCS3基因的表达,为深入研究SOCS3的功能奠定了基础.     

Maximizing the Potential of Cropping Systems for Nematode Management

Quantitative techniques were used to analyze and determine optimal potential profitability of 3-year rotations of cotton, Gossypium hirsutum cv. Coker 315, and soybean, Glycine max cv. Centennial, with increasing population densities of Hoplolaimus columbus. Data collected from naturally infested on-farm research plots were combined with economic information to construct a microcomputer spreadsheet analysis of the cropping system. Nonlinear mathematical functions were fitted to field data to represent damage functions and population dynamic curves. Maximum yield losses due to H. columbus were estimated to be 20% on cotton and 42% on soybean. Maximum at-harvest population densities were calculated to be 182/100 cm³ soil for cotton and 149/100 cm³ soil for soybean. Projected net incomes ranged from a $17.74/ha net loss for the soybean-cotton-soybean sequence to a net profit of $46.80/ha for the cotton-soybean-cotton sequence. The relative profitability of various rotations changed as nematode densities increased, indicating economic thresholds for recommending alternative crop sequences. The utility and power of quantitative optimization was demonstrated for comparisons of rotations under different economic assumptions and with other management alternatives.     

结缔组织生长因子基因RNA干扰复制缺陷型腺病毒载体的构建及鉴定

目的：构建能介导结缔组织生长因子（CTGF）基因RNA干扰的复制缺陷型腺病毒表达载体。方法：以大鼠CTGF基因为靶序列,设计并合成含编码短发夹RNA序列的寡核苷酸,构建腺病毒穿梭质粒P-shuttle-CTGF,酶切及测序分析正确后,与腺病毒骨架质粒pAdEasy-1共转染AD-293细胞,进行病毒包装,得到腺病毒载体Ad．H1-CTGF,用该载体感染HSC-T6细胞,观察其对CTGF基因表达抑制的效果。结果：构建的腺病毒穿梭质粒p-shuttle-CTGF经酶切、测序分析证实正确;包装的病毒载体滴度为4×1010PFU／mL,感染HSC-T6细胞后,Western印迹证实CTGF表达显著减少。结论：构建的腺病毒载体Ad．H1-CTGF可有效抑制HSC-T6中CTGF的表达,为抗纤维化研究提供了有力的工具。     

白背飞虱体内金属元素的含量动态及模拟分析

摘要: 为明确金属元素在白背飞虱Sogatella furcifera (Horváth)体内的分布及其含量动态, 将白背飞虱长翅成虫在实验室中用超纯水分别饲养0, 24, 48, 96 h后测量其体重变化; 并用电感耦合等离子体质谱(inductively coupled plasma mass spectrometry, ICP-MS)检测各个处理虫体内12种金属元素(Cu, Zn, Cd, Mn, Ca, Fe, Na, As, U, Mg, K和Pb)的含量。以白背飞虱整个体重(W)、 有机体组织重量(O)、 体内可排泄内容物重量(E)及可排泄内容物排出体外的速率(r)为参数, 构建其体重变化的修正指数模型W_t=O+E(1-r)^t。基于白背飞虱体内金属元素总含量(WC_t)为有机体组织内含量(OC)和可排泄内容物中含量(EC)按两者百分率(OP_t和EP_t)加权之和, 即WC_t=OC·OP_t+EC·EP_t, 分别估计白背飞虱体内组织和可排泄内容物中金属元素含量。模拟分析结果表明： Cu, Zn, Cd, Mn和Ca这5种元素在白背飞虱组织内的含量显著高于其可排泄内容物中的含量(P<0.05), 表明这些金属元素可被昆虫有机体组织吸收、 积累, 并稳定地存在于昆虫的有机体组织之中。Fe, Na, As, U, Mg, K和Pb这7种元素在白背飞虱有机体组织内的含量与其可排泄内容物中的含量接近, 其在有机体组织中和可排泄内容物中的分布差异不显著(P>0.05), 以相对平衡状态存在于白背飞虱有机体组织和可排泄内容物中。该分析方法可能为其他小型和微型昆虫体内金属元素的分布、 积累、 排泄的研究提供借鉴。     

DJ-1基因siRNA对三阴性乳腺癌细胞侵袭和迁移影响的研究

目的:探讨DJ-1基因siRNA对三阴性乳腺癌细胞体外侵袭和迁移能力的影响。方法:设计DJ-1基因的小分子干扰RNA(siRNA)片段,脂质体介导转染入三阴性乳腺癌细胞株MAD-MB-23l,转染分3个组:A组(空白对照control组)、B组(转染非特异性对照Scramble组)、C组(转染si DJ-1组)。应用Western blotting免疫印迹法检测转染前后DJ-1表达水平;运用细胞迁移和侵袭实验检测细胞迁移和侵袭能力的变化。结果:C组DJ-1蛋白的表达强度弱于A组和B组(t=9.831,P0.05),而A组与B组比较,DJ-1蛋白表达水平则无明显差异(t=1.629,P0.05)。细胞迁移实验中,A组细胞为(218.37±12.75);B组的细胞为(214.46±11.38);C组的细胞为(129.65±8.59),C组细胞明显少于A组和B组(t=10.927,9.984,P0.05),而A组与B组之间,差异无统计学意义(t=0.512,P0.05)。细胞侵袭实验中,A组细胞为(127.28±12.65);B组的细胞为(123.06±13.08);C组的细胞为(52.85±9.58),C组穿过人工基底膜的细胞明显少于A组和B组(t=7.927,8.643,P0.05),而A组与B组之间,差异无统计学意义(t=0.627,P0.05)。结论:DJ-1基因siRNA可抑制三阴性乳腺癌细胞侵袭和迁移。     

Isolation and identification of the probing stimulants in the rice plant for the white-back planthopper, Sogatella furcifera (homoptera: delphacidae)

Adult females of the white-back planthopper, Sogatella furcifera, showed characteristic behavior of stylet sheath deposit on a parafilm membrane when fed on a 2% aqueous crude rice leaf and stem extract containing 15% sucrose. Subsequent bioassays revealed that the butanol-soluble fraction of the extract was highly active against the insects. When the butanol fraction was chromatographed on an ODS open column and eluted in sequence with a mixture of an increasing concentration of methanol in water, the 40 % methanol fraction was separated as the most active. A further bioassay of the HPLC components in the active fraction revealed that two major components (1 and 3) stimulated the high probing activity of the white-back planthopper only when they were combined. Of the active components, one component (3) was identified to be tricin 5-O-glucoside by spectroscopic analyses.     

Ovarian development status and population characteristics of Sogatella furcifera (Horváth) and Nilaparvata lugens (Stål): implications for pest forecasting

Migratory behaviour in insects correlates with reproductive development in females, and migration often occurs during the pre‐reproductive stage of adults. The relationship between ovarian development and population status of the white‐backed planthopper Sogatella furcifera (Horváth) and the brown planthopper Nilaparvata lugens (Stål) was evaluated. Females of both species were captured in rice fields and light traps and then dissected in double‐season rice‐farming regions of southern China. The ovarian development of S. furcifera and N. lugens was divided into five levels, following previous studies. The population statuses of both species were examined based on the ovarian development of female adults caught in rice paddies. The ovarian development in N. lugens females caught in light traps mostly ranged from level I to level II, whereas that in S. furcifera females caught in light traps mostly ranged from level I to level III. During peak immigration, ovarian development in N. lugens females was mainly at level II, whereas that in S. furcifera females was mainly at level II and sporadically at level III. During peak emigration, both S. furcifera and N. lugens showed level I ovarian development. The temporal dynamics of ovarian development in light trap catches revealed that (i) significant emigration and partial immigration periods occur in S. furcifera, with ovarian development mainly at level I and sporadically from level II to level III and (ii) numerous immigrants of N. lugens were detected during sedentary and local breeding periods. The temporal dynamics of ovarian development provides more information than does the paddy population. Thus, this study proposes another method for pest forecasting, which is more precise and efficient than conventional forecasting methods such as light trap catching and monitoring population dynamics in rice fields.     

甲壳动物RNAi技术研究与应用进展

RNA干扰(RNA interference,RNAi)是指由双链RNA(double-stranded RNA,ds RNA)诱发的一种使特定基因沉默的现象。作为一种研究基因功能、发现新基因和抗病毒的新型手段,近年来RNAi技术在昆虫、真菌、植物和哺乳动物等的研究中已被广泛应用,但是在甲壳动物研究中尚处于起步阶段。对甲壳动物RNAi技术实施方法做了简要介绍;着重综述了RNAi技术在甲壳动物CHH家族神经肽类基因功能、蜕皮和生长调控机制、配子及性腺发育调控和甲壳动物抗病毒机制研究上的应用进展。