灰飞虱Wolbachia群体生物学的遗传特性研究

Ｗｏｌｂａｃｈｉａ是存在于多种昆虫生殖器官的一类共生菌,它可能在宿 主群体中引起细菌质不亲和性而给宿 主带来生殖优势。对灰飞虱体内的Ｗｏｌｂａｃｈｉａｆｊｔｆｄｇｐｗ,ｖ ｇｍ ｐｇｃｆｍｗ ｑｎｎｋｕｄｙｊｒ ｄｏ ｎｕｉ     

Wolbachia在灰飞虱体内的分布

灰飞虱（Laodelphax striatellus)能传播水稻条纹叶枯病,是我国主要的水稻害虫,有些灰飞虱体内含有细胞内共生菌－Wolbachia。Wolbachia是一种细胞质遗传的细胞内共生菌,这类细菌改变了宿主的生殖行为,引起细胞质不相容（CI）,雌性化及孤雌生殖等现象,导致带有Wolbachia的宿主具有生殖优势。采用PCR和Western杂交的方法分析Wolbachia的灰飞虱体内不同组织的分布状况,发现灰飞虱体内Wolbachia除存在于生殖组织外,还广泛分布在头、胸、腹、唾液腺、消化道等非生殖组织中。这种广泛的分布状态说明Wolbachia是在昆虫体内引入、表达与传播外源基因的良好媒介。     

灰飞虱共生菌Wolbachia引起的细胞质不亲和性

对我国６个地区和日本３个地区灰飞虱的Ｗｏｌｂａｈｉａ感染率，用ＰＣＲ技术进行了检测，结果表明中国的辽宁、北京、上海和云南的灰飞虱的Ｗｏｌｂａｃｈｉａ感染率均接近１００％；四川为５９．６％；而宁夏为０。各地区灰飞虱间的交配实验证明了在Ｗｏｌｂａｃｈｉａ引起的细胞质不亲和现象的存在，Ｗｏｌｂａｃｈｉａ１６ＳｒＤＮＡ的部分测序分析表明，上海、云南和日本出云的灰飞虱发的同为Ｗｏｌｂａｃｈｉａ ｐｉｐｉｅｎ     

灰飞虱体内共生菌Wolbachia的groEL基因克隆及序列分析

利用特异性引物,首次在国内克隆获得灰飞虱(Laodelphax striatellusFalln)体内共生菌Wolbachia的groEL基因全长序列,其开放阅读框为1659bp,可编码552个氨基酸,GenBank登录号为:EF468716,与已发布的韩国分离物核苷酸一致性达99.9%,为进一步研究其与灰飞虱传播水稻条纹病毒的关系奠定了基础.     

扑虱灵对水稻褐飞虱的抗药性筛选及其监测

用扑虱灵对水稻褐飞虱进行抗药性筛选及抗药性监测，在室内筛选８个世代后，褐飞虱没有产生抗药性；对田间褐飞虱种群连续３ａ的抗药性监测中，也未发现褐飞虱对扑虱灵的抗药性。可见室内抗药性筛选和田间抗药性监测的结果是致的。这些结果说明，褐飞虱对扑虱灵不容易产生抗药性，或者抗药性发展缓慢。     

水稻抗性品种对褐飞虱和白背飞虱 羧酸酯酶活力影响的比较研究

比较了ASD7、IR36、JX89、Mudgo四个抗褐飞虱水稻品种及感性品种TN1对褐飞虱和白背飞虱羧酸酯酶活力的影响.两种稻飞虱在各供试的抗性水稻品种上的羧酸酯酶活力均低于在感性品种TN1上的羧酸酯酶活力.在抗性水稻品种上,白背飞虱的羧酸酯酶活力只在Mudgo上低于褐飞虱,而在其余3个抗性品种上均较褐飞虱高.结果表明,抗褐飞虱水稻品种对褐飞虱和白背飞虱的羧酸酸酶活力均有不同程度的抑制作用,但对褐飞虱的抑制作用显然要大于对白背飞虱的抑制作用.供试水稻品种的这种低抑制作用,可能会导致白背飞虱持续取食抗褐飞虱的水稻品种,从而致使其在这类品种种区植内种群数量上升.     

转基因水稻纯系对褐飞虱的抗性研究

利用基因枪法将含潮霉素抗性基因、ＧＵＳ报告基因和雪花莲凝集基因的２个质粒ｐＷＲＧ１５１５和ｐＲＳＳＧＡ１共同转化粳稻品种鄂宜１０５的成熟胚诱导的愈伤组织,从轰击的１５２块愈伤组织中共再生出２６株独立转基因植株,ＰＣＲ／Ｓｏｕｔｈｅｒｎ印迹法分析发现,７３％的转基因植株含有所有３个外源基因,遗传分析证实外泊基因在转基因植株后代中以孟德尔方式遗传,从其Ｒ１代亲本为孟德尔３：１方式遗传的Ｒ２代中,鉴定出     

重庆市秀山县褐飞虱空间格局分析

根据重庆市秀山县褐飞虱Nilaparvata lugens(Stl)发生高峰期的田间调查资料,应用地理信息系统(GIS)结合传统空间分布分析方法,研究了褐飞虱于2007-2013年不同年份在全县范围的空间分布格局.结果表明:2007-2013年的褐飞虱种群在全县范围呈现明显的聚集分布;反向距离插值图显示,不同年份褐飞虱发生的聚集中心和聚集程度不断变化;秀山县中西部的溶溪、塘坳、官庄、中和等乡镇发生较重,南部的兰桥、梅江等地发生相对较轻.褐飞虱这种空间分布格局和动态变化是多种生态因素综合影响的结果,本研究为该地区褐飞虱的综合管理提供了一定的理论依据.     

Molecular Identification of a Wolbachia endosymbiont in Trichogramma dendrolimi

Wolbachia is a common and widespread group of bacteria found in arthropods. These bacteria have evolved various mechanisms for manipulating reproduction of their host. The presence of Wolbachia in a lab strain of the arrhenotokous species Trichogramma dendrolimi was observed by the amplification and sequencing of part of the wsp gene. Aligning the resulting sequences with already published ones, the phylogenetic relationships between Wolbachia found in Trichogramma dendrolimi and in other Trichogramma wasps was established, and the phylogenetic relationships of Wolbachia in Trichogramma were not congruent with their hosts Trichogramma. Some factors contributing to this uncongruence are discussed here.     

Infection of Wolbachia may improve the olfactory response of Drosophila

The endosymbiotic bacterium Wolbachia infects various insects and is primarily known for its ability to manipulate host reproduction.Recent investigations reveal that Wolbachia also affects the activity of somatic cells.We here demonstrated by trap method and T-maze that Wolbachia infection had signifi-cant impact on the olfactory response of Drosophila simulans.Wolbachia-infected flies took shorter time to enter the food trap and were more sensitive to odorant in T-maze than those uninfected controls.The t...     

Wolbachia在Drosophila auraria复合种和Drosophila simulans中的感染

用 Ｐ Ｃ Ｒ 方法检测了多种果蝇中共生菌 Ｗ ｏｌｂａｃｈｉａ 的感染,发现 Ｄｒｏｓｏｐｈｉｌａ ａｕｒａｒｉａ 复合种以及采集于北京地区的 Ｄｒｏｓｏｐｈｉｌａ ｓｉｍ ｕｌａｎｓ为 Ｗｏｌｂａｃｈｉａ 所感染, Ｒ Ｆ Ｌ Ｐ分析证实为单一感染．克隆了编码 Ｗ ｏｌｂａｃｈｉａ 外膜蛋白质的 ｗ ｓｐ 基因并进行了序列测定．同时比较了 Ｄｒｏｓｏｐｈｉｌａ ａｕｒａｒｉａ 复合种内 ４ 个种和采集于北京及美国加州的 Ｄｒｏｓｏｐｈｉｌａ ｓｉｍ ｕｌａｎｓ 的线粒体的细胞色素氧化酶 ２ 亚基基因的部分序列． 进而讨论了 Ｗｏｌｂａｃｈｉａ 对 Ｄｒｏｓｏｐｈｉｌａ ａｕｒａｒｉａ 复合种成员之间进化关系的影响．     

山东野生果蝇体内Wolbachia的检测及WSP基因序列分析

Wolbachia是当前分布最广的共生菌类,参与调控寄主的多种生殖活动.通过对山东8个地理种群野生果蝇Wolbachia的WSP基因进行检测,在5个地理种群的黑腹果蝇和3个地理种群拟果蝇中分别分离到长度为632bp和611bp的Wolbachia的WSP基因片段,命名为WSPSD632和WSPSD611,在GeneBank所获注册号分别为EU395833和EU395834.将两序列和CeneBank上公布的WSP基因序列构建系统发育树,表明山东野生黑腹果蝇和拟果蝇所感染的Wolbachia分属于WMel和WRi类群.序列比对结果显示,两核苷酸序列的同源性为86.39%,但碱基替换率和缺失率存在一定差异.     

MS-RDA的改进及在白背飞虱的应用研究*

甲基化敏感性代表性差异分析方法（MS-RDA）适用于特异、低丰度基因组DNA甲基化的筛查与鉴定,已大量应用于哺乳动物的疾病发生和植物发育机理方面的研究,但昆虫中尚未发现该方法的应用。从DNA提取、酶切、扩增子制备、消减杂交等方面对该方法进行了改进,并在水稻重要害虫白背飞虱上得到成功应用,获得了4条与雌、雄性别相关联的特异性甲基化差异条带,其中有3条序列已被GenBank收录,登陆号分别为JX847621、JX624161、JX472453,经BLAST比对,有3条未发现同源性序列,属于功能未知的新基因;同时还获得了2条与长短翅型相关联的特异性甲基化差异条带,也已被 GenBank 收录,登陆号分别为 JX514031、JX472454,同源性分析发现与多种动物的18S核糖体和28S核糖体RNA基因序列高度相似,因此有关核糖体基因的甲基化可能对白背飞虱的翅型分化起到一定的调控作用。     

Isolation and characterization of a potential transposable element from Wolbachia

Wolbachia are a group of Rickettsia-like bacteria which parasitize the cells of a wide range of anthropoid. These microorganisms are associated with the reproductive and developmental abnormalities io their hosts. To study the molecular mechanism underlying such phenomena, we analyzed the genomic difference between Wolbachia with different cytoplasmic incompatibility (CI) phenotype using representational difference analysis method. A potential transposable element, which exists in the strong CI-inducing strain wRi, was isolated. This element was designated as Wolbachia insertion sequence element (WISE).     

MSAP在水稻害虫白背飞虱中的应用研究

甲基化敏感的扩增多态性分析方法 (MSAP)是在扩增片段长度多态性技术基础上建立起来的主要用来检测样品基因组DNA甲基化情况的一种方法,特别适用于全基因组范围内检测CCGG位点的胞嘧啶甲基化情况,在高等动物和许多植物上已广泛应用,但尚未有在昆虫中应用该方法的报道。从DNA提取、酶切基因组DNA用量、酶切和接头连接时间、电泳图谱显影方法等方面对MSAP方法进行了改进,并在水稻重要害虫白背飞虱上得到成功应用。这为将来在其它昆虫中使用该方法研究基因组DNA甲基化情况提供了一定借鉴和指导价值。且通过比较白背飞虱雌雄性别间和长短翅型间的MSAP图谱,发现雌雄性别间和长短翅型间的基因组DNA甲基化式样都存在明显的差异,说明DNA甲基化可能参与了性别和翅型分化的调节。     

Cloning and characterization of a gene encoding phage-related tail protein (PrTP) of endosymbiont Wolbachia

Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.