重组棉铃虫病毒的外源基因向其他生物的转移

本文报道了基因工程棉铃虫核多角体病毒(HaSNPV-AaIT)的外源蝎子毒素基因(AaIT)是否会向环境中的植物病原微生物或捕食性天敌转移的实验结果.首先,于实验室内将重组病毒HaSNPV-AaIT与棉花黄萎病病菌(Verticillium dahliae Lleb.)进行了长达90d的混合培养,在混合培养30d,60d和90d后分别提取棉花黄萎病病菌的基因组DNA,用AaIT基因作探针进行点杂交,结果显示无阳性信号.另外,从多次施用过重组病毒的棉花田中采集了120只龟纹瓢虫和七星瓢虫,利用健康蚜虫饲养3-4d,用碱解液处理瓢虫体表后,从处理液中可以检测到病毒DNA;但瓢虫体表经碱解液和Dnase处理后,从瓢虫体内提取的基因组DNA,用PCR和斑点杂交的方法,都没有检测到AaIT的序列存在.本研究的实验结果说明,基因工程病毒的外源基因向其它生物转移的可能性极低.     

表达昆虫特异性神经麻痹毒素AaIT的杀虫杆状病毒的构建

根据杆状病毒核多角体基因及植物基因的密码子选择偏向、G+C含量和密码子第三位碱基的G+C含量,同时综合在真核系统中影响基因转录、翻译及影响mRNA稳定性等因素,在不改变所编码的氨基酸序列的基础上,设计并人工合成了昆虫特异性神经蝎毒素AaIT基因。合成的AaIT基因与合成的gp67信号肽DNA序列正确融合后通过杆状病毒转移载体pSXIV VI⁺X3体内重组到粉纹夜蛾多角体病毒(Trichoplusia ni nuclear polyhedrosis virus,TnNPV)上,得到重组病毒TnNPV-AaIT。经Southern blotting验证了AaIT基因整合在TnNPV基因组中,SDS-PAGE证明AaIT基因在重组病毒中的表达。通过用重组病毒口服感染供试昆虫,证明了含AaIT基因的重组病毒能显著缩短杆状病毒的杀虫时间,提高杀虫效率。这是迄今为止我国构建的第一株具有实用价值和应用前景的基因工程病毒。     

表达蝎毒素的重组斜纹夜蛾核型多角体病毒(SpltMNPV)的构建及毒力

【目的】开发高毒力的重组斜纹夜蛾核型多角体病毒(Spodoptera litura multicapsid nucleopolyhedroviruse,SpltMNPV)杀虫剂。【方法】构建编码蜕皮激素UDP-葡萄糖基转移酶(ecdysterioid UDP-glucosyl transferase gene,egt)基因缺失并插入东亚钳蝎神经毒素(B.martensi Karsch,BmK ITa1)基因的重组转移载体,重组转移载体与SpltMNPVⅡ基因组DNA共转染斜纹夜蛾细胞,通过荧光斑法与有限稀释法相结合筛选重组病毒。【结果】成功筛选出缺失egt基因的、早期启动子(ie-1)启动的、表达BmK ITa1成熟肽的重组病毒SpltMNPV-Δegt-Pph-egfp-ie-1-BmK ITa1。生物测定结果显示,重组病毒的杀虫速度(LT50)较野生病毒提前0.7-0.8 d。【结论】通过在SpltMNPV病毒基因组中插入外源毒素基因可明显增强病毒的杀虫效果,结果说明开发高毒力SpltMNPV生物杀虫剂具有可行性。     

电脉冲作用将外源基因导入稀有ju鲫精子的研究

将稀有ju鲫（Gobiocypris rarus）精子与重组质粒pGAhLFc线性DNA混合温育,经电脉冲处理后与卵子受精,孵化出苗。从鱼苗中提取DNA,经PCR检测,25.5%～66.7%鱼苗带有外源基因。在显微镜下观察经电脉冲处理过的精子,发现其活力有不同程度下降,受精率也有不同程度下降,说明不同的电脉冲条件对精子有不同程度的损害作用。精子与外源DNA混合温育,经电脉冲处理后,用DNA外切酶消化后,提取精子DNA,经PCR检测,仍有阳性电泳带,证明电脉冲可以促使稀有ju鲫精子摄入外源基因。     

总DNA介导鱼类基因转移的初步研究

自七十年代我国开始将外源总DNA转移技术应用于棉花和水稻品种改良的研究中,但至今尚未见有关鱼类总DNA转移成功的报道。本实验采用显微注射和精于载体的方法将鲫鱼肝总DNA转移到红鲤受精卵,利用鯽鱼和红鲤在孵化前后色素表达的差异来筛选转移基因个体。此方法无需克隆目的基因及DNA重组操作,而且筛选简便,目的在于探讨总DNA转移方法应用于鱼类品种改良的可能性。     

表达昆虫特异性神经毒素AaIT基因的转基因烟草的抗虫性

经改造的昆虫特异性神经毒素AaIT基因插入植物高效表达载体得到重组质粒pNGY-2。重组质粒中AaIT基因5＇端与烟草花叶病毒(TMV)的Ω序列3＇端融合,受两个串联的35S启动子控制,通过土壤农杆菌LBA4404介导转化烟草NC89,经NPTⅡ选择后再经GUS染色挑选出阳性再生植株,Southern blotting进一步证实了AaIT基因已经整合到烟草基因组中,对棉铃虫(Heliothis armigera)的抗虫实验表明,转基因烟草有显著的抗虫活性。     

大珠母贝精子介导外源基因转移研究

将大珠母贝(PinctadamaximaJameson)精子与“全鱼”GH基因重组体pCAgcGH和pCAgcGHc的线性DNA混合,温育30min,经6次、2⁷、10kV脉冲电处理后,与卵子受精,得到若干贝苗。从贝苗中提取DNA,经PCR扩增和Southernblot分子杂交表明,部分受体带有外源基因,当与精子温育的外源基因浓度分别为2μg/mL,6μg/mL及18μg/mL时,相应贝苗携带外源基因比率分别为56%,20%和50%。即在此范围内,基因转移的阳性率与外源基因的浓度呈正相关。     

鸡痘病毒NX10株TK基因在病毒复制中不是完全非必需的

【目的】禽痘病毒(FPV)是痘病毒科禽痘病毒属的成员。FPV因其基因组庞大,含有大量复制非必需区,目前作为活病毒载体在禽类和哺乳动物中广泛使用。重组位点的选择是禽痘病毒载体构建的先决条件,外源基因的插入不影响病毒复制是筛选插入位点的前提。因此,鉴定可供外源基因插入的复制非必需位点将为重组病毒的构建提供更多选择。本研究拟鉴定FPV NX10株胸苷激酶(TK)基因在病毒复制中的必要性。【方法】以TK基因作为靶基因,增强型绿色荧光蛋白(EGFP)为筛选标记构建转移载体,FPV NX10株为亲本病毒,通过同源重组筛选重组病毒r FPV-ΔTK-EGFP。通过在CEF细胞培养物中添加5-溴脱氧尿苷(BUd R)验证TK基因在FPV复制中的作用。【结果】构建了转移载体p UC19-TK AB-EGFP。在转染后重组病毒克隆纯化过程中,蚀斑克隆中绿色荧光病变所占的比例逐渐增加,但在荧光蚀斑的边缘能观察到不带荧光病变的存在。对第9-15轮随机选取的蚀斑克隆的Western blot分析表明,重组病毒中插入的EGFP基因均能够正确表达,但PCR结果显示在重组病毒中始终存在野生型病毒。在细胞培养液中添加BUd R后,重组病毒不能继续生长。【结论】FPV NX10毒株TK基因在该病毒的复制中不是完全非必需的。     

双基因双重组AcNPV的构建及其杀虫活性

质粒pAcIEneo携带杆状病毒极早期基因IE1启动子驱动的新霉素抗性基因(neo),经酶切回收后插入到质粒pAc34DZ1的SacI位点上,构建成多角体外膜蛋白基因(pe)失活的转移载体pAc34DZ2。我们曾构建了一个多角体完整(ocu+)的表达苏云金杆菌(Bt)截短cryIab基因的重组病毒(1)vAcPhBtT。为了改进这一重组病毒的杀虫效率,将转移载体pAc34DZ2与重组病毒(1)vAcPhBtT DNA共转染Sf9细胞,进行第二次同源重组。由于neo基因的表达,用G418筛选得到重组病毒(2)vAcPhBtTPE^-;Southern blot证明vAcPhBtTPE-的构建是正确的,经SDS-PAGE分析,重组病毒(2)仍然能在昆虫细胞中表达80kD的Bt截短毒蛋白,但不表达34kD的多角体外膜蛋白。电镜观察重组病毒(2)无多角体外膜,碱解时病毒粒子释放的速度快于重组病毒(1)。以重组病毒(2)感染甜菜夜蛾三龄幼虫,LC₅₀比野生型病毒小了接近1倍,LT₅₀提前近2d。     

电脉冲作用将外源基因导入稀有(鱼句)鲫精子的研究

将稀有鲫 (Gobiocyprisrarus)精子与重组质粒pCAhLFc线性DNA混合温育 ,经电脉冲处理后与卵子受精 ,孵化出苗。从鱼苗中提取DNA ,经PCR检测 ,2 5 .5 %～ 6 6 .7%鱼苗带有外源基因。在显微镜下观察经电脉冲处理过的精子 ,发现其活力有不同程度下降 ,受精率也有不同程度下降 ,说明不同的电脉冲条件对精子有不同程度的损害作用。精子与外源DNA混合温育 ,经电脉冲处理后 ,用DNA外切酶消化后 ,提取精子DNA ,经PCR检测 ,仍有阳性电泳带 ,证明电脉冲可以促使稀有鲫精子摄入外源基因     

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: Intraspecific heterogeneity within the intergenic spacer region. Fungal Genetics and Biology 29, 19-27. The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.     

Electrophoretic karyotype and gene mapping of the vascular wilt fungus Verticillium dahliae

The karyotype profile of Verticillium dahliae was resolved by pulse-field gel electrophoresis. It revealed 6 chromosomal bands that corresponded to 7 chromosomes as shown by RFLP analysis using as probe the telomeric consensus sequence (AACCCT)(5). The number of chromosomes was further verified by the sensitivity of the hybridization signals to Bal31 digestion and the exclusion of interfering mitochondrial DNA signals. The corresponding sizes of the seven separated chromosomes were 6.7, 5.6, 4.1, 3.4, 3.1, 3.1 and 2.4Mb, raising the total genomic size of the fungus to approximately 28.4Mb. Twenty five homologous V. dahliae genes obtained either from randomly sequenced clones or PCR amplification were used as hybridization probes and were located onto the seven chromosomes.     

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region

The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.     

Direct DNA extraction for PCR-mediated assays of soil organisms.

By using the rDNA of a plant wilt pathogen (Verticillium dahliae) as the target sequence, a direct method for the extraction of DNA from soil samples which can be used for PCR-mediated diagnostics without a need for further DNA purification has been developed. The soil organisms are disrupted by grinding in liquid nitrogen with the natural abrasives in soil, and losses due to degradation and adsorption are largely eliminated by the addition of skim milk powder. The DNA from disrupted cells is extracted with sodium dodecyl sulfate-phenol and collected by ethanol precipitation. After suitable dilution, this DNA extract can be assayed directly by PCR amplification technologies. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of specific soil organisms or pathogens on a large-scale basis.     

Molecular analysis of Japanese isolates of Verticillium dahliae and V. albo-airum

Sixteen isolates of different pathogenicity groups of the plant pathogen Verticillium dahliae and four isolates of V. albo-atrum from Japan were analysed by means of an RAPD (random amplified polymorphic DNA) method using a PCR (polymerase chain reaction). Verticillium dahliae and V. albo-atrum could be distinguished by RAPD analysis. Four pathogenicity groups of V. dahliae could also be classified to a certain extent by this method. Similarities and differences in banding patterns obtained by RAPD may be a useful molecular tool in phylogenetic studies of the pathogenicity groups.     

DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae

The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin (Act), β-tubulin (β-tub), calmodulin (Cal) and histone 3 (H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium alboatrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.     

Field inactivation of wild-type and genetically modified Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus in cotton

Cotton bollworm (Helicoverpa armigera) is a serious pest on cotton in China. A specific baculovirus, H. armigera nucleopolyhedroviruses (HaSNPV) is used as a commercial biopesticide to control this pest. To improve the pesticidal properties, HaSNPV has been genetically engineered by both deleting the ecdysteroid UDP-glucosyltransferase (egt) gene from its genome (recombinant HaSNPV-EGTD) and incorporating an insect-selective toxin gene from the scorpion Androctonus australis (AaIT) (recombinant HaSNPV-AaIT). In the field, there was no significant difference among the inactivation rates of the two recombinant HaSNPVs and their parent wild-type, HaSNPV-WT. The inactivation rate of these viruses was significantly different in different years. The average half-life of HaSNPV was 0.57, 0.90 and 0.39 days in 2000, 2001 and 2002, respectively. Inactivation rates correlated well with solar radiation over these years.     

Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray

Detection, identification and quantification of plant pathogens are the cornerstones of preventive plant disease management. To detect multiple pathogens in a single assay, DNA array technology currently is the most suitable technique. However, for sensitive detection, polymerase chain reaction (PCR) amplification before array hybridization is required. To evaluate whether DNA array technology can be used to simultaneously detect and quantify multiple pathogens, a DNA macroarray was designed and optimized for accurate quantification over at least three orders of magnitude of the economically important vascular wilt pathogens Verticillium albo-atrum and Verticillium dahliae. A strong correlation was observed between hybridization signals and pathogen concentrations for standard DNA added to DNA from different origins and for infested samples. While accounting for specific criteria like amount of immobilized detector oligonucleotide and controls for PCR kinetics, accurate quantification of pathogens was achieved in concentration ranges typically encountered in horticultural practice. Subsequently, quantitative assessment of other tomato pathogens (Fusarium oxysporum, Fusarium solani, Pythium ultimum and Rhizoctonia solani) in environmental samples was performed using DNA array technology and correlated to measurements obtained using real-time PCR. As both methods of quantification showed a very high degree of correlation, the reliability and robustness of the DNA array technology is shown.     

Horizontal and vertical transmission of wild-type and recombinant Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus

Transmission plays a central role in the ecology of baculoviruses and the population dynamics of their hosts. Here, we report on the horizontal and vertical transmission dynamics of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV-WT) and a genetically modified variant (HaSNPV-AaIT) with enhanced speed of action through the expression of an insect-selective scorpion toxin (AaIT). In caged field plots, horizontal transmission of both HaSNPV variants was greatest when inoculated 3rd instar larvae were used as infectors, transmission was intermediate with 2nd instar infectors and lowest with 1st instar infectors. Transmission was greater at a higher density of infectors (1 per plant) than at a lower density (1 per 4 plants); however, the transmission coefficient (number of new infections per initial infector) was lower at the higher density of infectors than at the lower density. HaSNPV-AaIT exhibited a significantly lower rate of transmission than HaSNPV-WT in the field cages. This was also the case in open field experiments. In the laboratory, the vertical transmission of HaSNPV-AaIT from infected females to offspring of 16.7+/-2.1% was significantly lower than that of HaSNPV-WT (30.9+/-2.9%). Likewise, in the field, vertical transmission of HaSNPV-AaIT (8.4+/-1.1%) was significantly lower than that of HaSNPV-WT (12.6+/-2.0%). The results indicate that the recombinant virus will be transmitted at lower rates in H. armigera populations than the wild-type virus. This may potentially affect negatively its long-term efficacy as compared to wild-type virus, but contributing positively to its biosafety.     

Characterization of Moroccan Isolates of Verticillium dahliaeKleb Using RAPD Markers

Isolates of Verticillium dahliae were sampled from different olive tree orchards in Morocco. These olive trees were located in different commercial culture locations in southern, central and northern Morocco. The isolates were characterized using genetic markers obtained after their DNA PCR amplification with random amplified polymorphic DNA (RAPD) primers. Among the 40 primers tested, 10 generated a total of 66 polymorphic fragments. Among the 38 isolates of V. dahliae tested, RAPD markers were successful in the characterization of groups based on their geographic origin. With the exception of one specific isolate, no correlation could be established among the isolates, based on the morphological appearance of the colony in culture.