Stable germline transformation of the malaria mosquito Anopheles stephensi

Anopheline mosquito species are obligatory vectors for human malaria, an infectious disease that affects hundreds of millions of people living in tropical and subtropical countries. The lack of a suitable gene transfer technology for these mosquitoes has hampered the molecular genetic analysis of their physiology, including the molecular interactions between the vector and the malaria parasite. Here we show that a transposon, based on the Minos element and bearing exogenous DNA, can integrate efficiently and stably into the germ line of the human malaria vector Anopheles stephensi, through a transposase-mediated process.     

Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite

Malaria is estimated to cause 0.7 to 2.7 million deaths per year, but the actual figures could be substantially higher owing to under-reporting and difficulties in diagnosis. If no new control measures are developed, the malaria death toll is projected to double in the next 20 years. Efforts to control the disease are hampered by drug resistance in the Plasmodium parasites, insecticide resistance in mosquitoes, and the lack of an effective vaccine. Because mosquitoes are obligatory vectors for malaria transmission, the spread of malaria could be curtailed by rendering them incapable of transmitting parasites. Many of the tools required for the genetic manipulation of mosquito competence for malaria transmission have been developed. Foreign genes can now be introduced into the germ line of both culicine and anopheline mosquitoes, and these transgenes can be expressed in a tissue-specific manner. Here we report on the use of such tools to generate transgenic mosquitoes that express antiparasitic genes in their midgut epithelium, thus rendering them inefficient vectors for the disease. These findings have significant implications for the development of new strategies for malaria control.     

Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission

Genetic manipulations of insect populations for pest control have been advocated for some time, but there are few cases where manipulated individuals have been released in the field and no cases where they have successfully invaded target populations. Population transformation using the intracellular bacterium Wolbachia is particularly attractive because this maternally-inherited agent provides a powerful mechanism to invade natural populations through cytoplasmic incompatibility. When Wolbachia are introduced into mosquitoes, they interfere with pathogen transmission and influence key life history traits such as lifespan. Here we describe how the wMel Wolbachia infection, introduced into the dengue vector Aedes aegypti from Drosophila melanogaster, successfully invaded two natural A. aegypti populations in Australia, reaching near-fixation in a few months following releases of wMel-infected A. aegypti adults. Models with plausible parameter values indicate that Wolbachia-infected mosquitoes suffered relatively small fitness costs, leading to an unstable equilibrium frequency <30% that must be exceeded for invasion. These findings demonstrate that Wolbachia-based strategies can be deployed as a practical approach to dengue suppression with potential for area-wide implementation.     

具有发育阶段结构的野生与不育蚊子交互作用的数学模型（英文）

Using the sterile insect technique,in which sterile mosquitoes are released to reduce or eradicate the wild mosquito population,is an effective weapon to prevent transmission of mosquito-borne diseases. To study the impact of the sterile insect technique on the disease transmissions,we formulate stage-structured discrete-time mathematical models,based on difference equations,for the interactive dynamics of the wild and sterile mosquitoes. We incorporate different strategies for releasing sterile mosquitoes,investigate the model dynamics,and compare the impact of the different release strategies.Numerical examples are also provided to demonstrate dynamical features of the models.     

具有发育阶段结构的野生与不育蚊子交互作用的数学模型

Using the sterile insect technique,in which sterile mosquitoes are released to reduce or eradicate the wild mosquito population,is an effective weapon to prevent transmission of mosquito-borne diseases. To study the impact of the sterile insect technique on the disease transmissions,we formulate stage-structured discrete-time mathematical models,based on difference equations,for the interactive dynamics of the wild and sterile mosquitoes. We incorporate different strategies for releasing sterile mosquitoes,investigate the model dynamics,and compare the impact of the different release strategies.Numerical examples are also provided to demonstrate dynamical features of the models.     

Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles ste- phensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I di- gestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively con- served in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.     

The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations

Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.     

Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.     

汽车无级变速器底层驱动系统设计与开发

提出用于驱动步进电机和高速数字开关阀的无级变速器底层驱动系统.详细介绍此系统的底层驱动原理和系统中使用的电子元器件,设计了关键元器件的驱动原理图,以及变速箱接口单元与发动机管理系统之间的控制器局域网通信协议.进行硬件在环仿真,成功驱动步进电机和高速数字开关阀,精确快速地实现速比控制,夹紧力控制和离合器控制,验证了无级变速器底层驱动系统.     

基于磁势矢模型的电磁直线驱动装置解析建模

针对目前有限元法求解电磁直线驱动装置稳态电磁特性时运算周期长、计算效率较低的问题,提出研究此类驱动装置的解析建模方法。以直驱式自动变速器用动圈式电磁直线驱动装置为例,建立了驱动装置磁场泊松方程,引入磁势矢模型推导了磁场分布方程,并结合边界条件得到了径向气隙磁通密度及电磁力表达式,以此获悉驱动装置稳态电磁特性。结果表明,解析模型与有限元模型的求解误差最小为0.6%,最大为6.6%,与试验误差不超过2.5%,有限元及试验均验证了模型的正确性与精确性。所提方法在确保求解精度的前提下求解过程简单且通用性好,为实现电磁直线驱动装置的模块化设计和无位移传感器控制提供了理论参考,对丰富和发展全电集成动力系统控制方法有一定的理论意义和工程应用价值。     

多杀性巴氏杆菌幽灵的研究

采用基因工程方法,将PhiX174噬菌体裂解基因E和温敏控制表达系统与质粒pPBA1100基因杂交,构建了重组子pPBA1100-E.将重组子转化到多杀性巴氏杆菌中,通过温度诱导使裂解基因表达.用限制性内切酶检验重组子.扫描电镜观察多杀性巴氏杆菌细菌幽灵和菌落形成单位评价遗传灭活率.结果表明:重组质粒经限制性内切酶酶切鉴定,琼脂糖电泳呈现三条谱带,相对分子质量与理论值一致.扫描电镜观察显示,重组子在多杀性巴氏杆菌中成功表达,获得了细菌幽灵.菌落形成单位检验结果显示,多杀性巴氏杆菌遗传灭活率达到99%.     

Efficient and stable generation of transgenic silkworm, Bombyx mori with the lepidopteran derived transposon piggyBac

Transposable elements, such as P element, have be-come important tools in the study of gene function in Drosophila melanogaster. Their applications are manifold, serving as mutagens and molecular tags for identification and isolation of new genes, and as vehicles for introducing custom-made gene sequences into organism’s genome[1]. However, the use of P elements appears to be only re-stricted to Drosophila[2,3]. Recently, foreign genes have been successfully introduced into several other gr…     

弹性蛋白酶保护因子--Elafin结构基因腺病毒载体穿梭质粒的构建、鉴定及表达

构建能表达弹性蛋白酶保护因子———elafin基因腺病毒表达载体的穿梭质粒,为进一步包装能高效表达结构蛋白的腺病毒载体做准备.以含有elafin基因的真核质粒pEGFP-N1-Elafin为模板,PCR扩增elafin基因,PCR产物以SalⅠ、EcoRⅤ双酶切,定向插入到腺病毒穿梭质粒pAdTrack-CMV中CMV启动子下游SalⅠ与EcoRⅤ位点之间,获得重组腺病毒穿梭质粒pAdTrack-CMV-Elafin,通过SalⅠ及EcoRⅤ双酶切、PCR及插入片段序列测定对该质粒进行鉴定,将pAdTrack-CMV-Elafin转染293细胞,以RT-PCR及Western-Blot检测其在293细胞中的瞬时表达.结果表明:双酶切、PCR及测序鉴定证实,pAdTrack-CMV-Elafin穿梭质粒的插入片段为elafin基因,用pAdTrack-CMV-Elafin穿梭质粒转染293细胞后可见绿色荧光蛋白的表达,RT-PCR和Western-blotting表明其可在293细胞中瞬时表达.     

武汉地区2017年—2019年蚊类群落组成及其多样性分析

2017年－2019年,在武汉地区选取公园、居民区、医院、农户和猪圈5种生境为调查点,采用诱蚊灯法对武汉地区蚊类进行多样性调查.结果表明,共捕获蚊类95 072只,隶属1科5属8种.城市生境如公园、居民区、医院蚊虫密度消长呈双峰型,密度高峰出现在6月－7月,9月－10月,优势种为致倦库蚊;农村生境如猪圈、农户蚊虫密度消长呈单峰型,密度高峰为6月－7月,猪圈优势种为三带喙库蚊,农户优势种为三带喙库蚊、致倦库蚊和中华按蚊.农户和猪圈的丰富度(8)较高,农户多样性指数、均匀度指数最高,猪圈次之.公园、居民区、医院丰富度(4,4,3)较低,多样性指数、均匀度指数相似,年际间有波动.公园、医院与居民区,猪圈与农户相似性指数范围在0.75～1.00,均为极相似.这些结果表明武汉地区蚊类多样性受外界影响较大,提示蚊媒防控应根据不同季节不同生境下的不同蚊种特性,采取有针对性的综合防控措施.     

工程中瞬态温度场的有限元分析

本文考虑瞬态温度场问题，先后将空间域和时间域离散，从具有更广泛意义的加权剩余法导出瞬态温度场的有限元方程，应用等参数单元分析瞬态温度场．     

Analysis of the characteristic sequence of intein and revision of its motifs

Since the first intein (Sce VMA) was found inSaccharomydes cerevisiae ATPases gene in 1990, more and more inteins were identified. It is necessary to analyze the new inteins to understand the sequence charateristics of inteins. By searching protein and nucleic acid database systematically, 101 inteins were found, of which 69 inteins contain homing endonuclease motifs. We only analyze the 69 inteins since most inteins are the classic inteins with homing endonuclease motifs. We found that the distribution of these inteins is particular among species and protein. By multiple sequence alignment, some new sequence characteristics were found and the motifs described previously were revised.     

质量管理体系的三维空间结构模型

运用系统工程方法、ISO9000族标准和TQM的基本原理,建立了质量管理体系的三维空间结构模型,以产品实现过程维中的“生产与服务运作”为例,分析了产品实现过程维、过程策划维和质量改进维三维之间的网络关系。该空间结构模型可用于评价企业质量管理体系的有效性,并为进一步研究质量管理体系的经济性奠定了基础。     

动力定位船矢量推进器回转动力学响应分析

为研究全回转矢量推进器回转动力学系统的响应特性,对瓦锡兰某型矢量推进器的液压回转系统的组成进行深入分析,研究了泵控液压马达驱动齿轮减速机构带动推进器进行回转的工作原理,建立了矢量推进器回转动力学系统的数学模型,提出了一种全回转矢量推进器实现目标方位角快速跟踪的改进控制方法,构建了一种矢量推进器回转动力学异步控制频率仿真系统,分析了不同目标方位角时推进器的方位角、角速度、液压系统的动态响应特性,建立了矢量推进器回转响应的物理约束条件.仿真结果表明,建立的动力学模型可模拟真实矢量推进器的回转运动响应过程,同时可实现矢量推进器回转方位角的快速、准确控制,对全回转矢量推进器和动力定位船舶推力分配方法的研究具有较重要的指导意义和工程价值.      

基于四参数的工程车辆自动变速节能换挡策略

以保证工程车辆液力机械传动系统的传动效率为前提，在现有工程车辆三参数换挡规律基础上详细介绍了四参数换挡原理及其实现方法，即在制定自动换挡策略时，考虑工作油泵所消耗的发动机功率的波动问题．台架试验结果表明，四参数自动换挡策略能够根据工况的变化自动调整变速器换挡．更符合工程车辆的实际作业情况．     

基于矩阵式变换器的异步电动机矢量控制

为提高交流调速系统的运行性能并满足节能的要求,提出了一种适用于矩阵式变换器驱动异步电动机调速系统的组合控制策略,实现了矩阵式变换器的空间矢量调制和异步电动机的直接磁场定向矢量控制。将矩阵式变换器的优点和矢量控制的优点结合起来,实现了异步电动机的高性能控制。仿真结果表明:使用该控制策略的调速系统在加减速运行和负载转矩变化等场合均具有良好的动态性能;四象限运行结果证明了该系统可将能量回馈至电网,实现了节能的目的。稳态运行实验结果验证了上述控制方法的可行性和有效性。