中国境内黑腹果蝇Drosophila melanogaster群体由对P族转座子敏感性形 …

中国境内从新疆,西藏到沿海,包括中国台湾,海南及崇明３大岛,共取样７０多个地区品系,遗传分析和单个果蝇的ＤＮＡ压印分子杂交分析后确定,在中国境内该果蝇主要是Ｍ型,中在遗传分析中,除沿海地区外,大多表现很高的对Ｐ敏感性（高达１００％左右）,但在分子检测中都带有Ｐ因子的Ｐ１和Ｐ２的片段,故尔确定为Ｍ型,而大连半岛,崇明岛和台中３地区的Ｐ活性较高,它们分别是１１％,１８％和２８％,为Ｑ型（台湾的其他地区     

中国东北地区黑腹果蝇（Drosophila melanogaster）P因子分布和来源

真核生物的转座因子(transposable elements)特别是果蝇P因子在研究生物进化上有重要的意义。以我国东北地区13个地方及毗邻的北京、烟台和呼和浩特三个地方共130个黑腹果蝇(D.melanogaster)单雌系为材料,对P因子序列的ORF2-ORF3区段进行PCR扩增,统计不同地方黑腹果蝇群体的P因子在此区段的缺失频率,再从整个地区来分析P因子缺失的分布规律,以推导东北地区黑腹果蝇中P因子的传递和扩散途径。结果显示P因子缺失频率由边境地区向内地逐渐递减,群体相对隔离的地方也较低,推断我国东北地区黑腹果蝇中P因子由朝鲜和俄罗斯向中国边境入侵后,逐步向中国内地扩散。     

黑腹果蝇的分离变相因子

众所周知,一对基因在杂合状态中保持相对的独立性,而在配子形成时,又按原样以相同比例分离到不同的配子中去,这是生物中最基本的遗传规律———孟德尔分离定律。但实际上并不是所有基因的分离都严格遵循孟德尔分离定律,存在于黑腹果蝇中的分离变相因子（ＳｅｇｒｅｇａｔｉｏｎＤｉｓｔｏｒｔｅｒ,以下简称ＳＤ）就是一种具有减数分裂驱动（ｍｅｉｏｔｉｃｄｒｉｖｅ）性质的、违反孟德尔分离定律的特殊遗传因子,从六十年代发现至今引起了人们的广泛关注,本文从其遗传背景,结构特征及进化规律等方面分别介绍一些近期的研究结果。１…     

piggyBac转座子及其在转基因昆虫中的应用

piggyBac是一种从粉纹夜蛾Trichoplusiani.中分离到的、具有TTAA插入位点特异性的DNA转座子。piggyBac可在昆虫基因组中准确切离,转化频率较高,并且不受宿主因子的限制,是目前转基因昆虫研究中应用最广的转座子载体。近年来的研究发现,piggyBac类转座子广泛分布于昆虫和其他生物基因组中。文章从piggyBac的结构、转座特性、在转基因昆虫中的应用以及piggyBac类转座子的分布等几个方面综述了piggyBac的研究进展。     

黑腹果蝇的性别控制

性别的形成包括两个过程,即性别决定和性别分化。果蝇的性别控制研究包括性别决定、性别分化、性别鉴定、性别诱导和性别控制5个方面。性别决定是在两种不同发育途径之间的选择,它提供了一个研究基因调控的模式系统。果蝇的性别决定问题已经研究得相当详细［1］。性别分化是使胚胎向着雌性或雄性发育的过程,决定了性别表型。果蝇的性别分化也取得了不少研究成果。近年来,许多重要的性别调控基因已被克隆和鉴定。随着果蝇基因组全序列测定的完成,果蝇的性别控制研究将会更为深入而完善。本文对与黑腹果蝇性别决定和性别分化相关的一些问题进行综述。     

樱桃新害虫黑腹果蝇的生物学特性

果蝇是近几年发现危害樱桃果实的一类重要害虫,在国内外樱桃产区均有发生。天水地区危害甜樱桃的果蝇有3个种,分别是黑腹果蝇Drosophila melanogaster Meigen、铃木氏果蝇Drosophila suzukii(Matsumura)和海德氏果蝇Drosophila hydei(Sturtevant),黑腹果蝇为优势种。作者记述黑腹果蝇对甜樱桃果实的危害情况、寄主范围及其生活史、生活习性、发育历期与温度的关系等,调查发现蚂蚁是樱桃果蝇的天敌之一。     

黑腹果蝇细胞谱系分析方法进展

对动物体内单个细胞的谱系进行分析有助于追踪其在发育过程中的作用,但是体内各种组织都是由很多形态、结构、功能各不相同的细胞构成的复杂系统,这种复杂性严重阻碍了对单个细胞的研究。嵌合克隆技术(Mosaic technique)和标记技术(Labeling technique)的出现为这一研究提供了强有力的手段。文章介绍了近几年来黑腹果蝇(Drosophila melanogaster)研究中常用的7种嵌合克隆标记方法,包括FRT介导的有丝分裂重组(FRT-mediated mitotic recombination)、MARCM(Mosaic analysis with a repressible cell marker)、TSG(Twin spotgenerator)、Twin-spot MARCM、Q-MARCM(Q system-based MARCM)、Coupled MARCM和G-TRACE(Gal4technique for real-time and clonal expression)技术,详述了这些技术的原理及应用,并对不同技术进行了对比。运用这些技术研究者可以从单细胞水平进行遗传学标记和操作,特别是在神经系统等复杂系统中追踪单个细胞的发育过程。果蝇中的这些技术也将为其他模式生物追踪细胞谱系提供参考。     

黑腹果蝇先天免疫研究进展

先天免疫是昆虫适应复杂环境的关键,也是新型害虫防治的重要研究方向。昆虫通过模式识别受体识别环境中不同的病原物,激活先天免疫系统以清除病原物。昆虫的先天免疫系统主要包括体液免疫与细胞免疫,体液免疫包括免疫信号通路诱导产生抗菌肽、活性氧以及黑化作用等,细胞免疫包括血细胞的吞噬、包囊和凝结。本文将重点总结黑腹果蝇Drosophila melanogaster在模式识别受体、免疫信号通路和细胞免疫相关方面的研究进展,为进一步研究其他经济昆虫与农业害虫的免疫机制,提高生产经济效益提供参考。     

黑腹果蝇黑条体突变体的求偶行为

黑腹果蝇Drosophila melanogaster黑条体果蝇(e^bsr)与黑檀体果蝇(e)为同一个基因(ebony)的不同突变体, 两者具有相似的形态表型, 但行为特征表现出明显的差异。本研究以黑条体、 黑檀体和野生型果蝇为研究对象, 首先检测果蝇的视力和活跃度, 再采用不同交配组合进行求偶成功率、交配时间和求偶模式的分析。结果表明: 黑条体果蝇视力与活跃度与野生型果蝇比较无显著差异; 黑条体果蝇的交配成功率和交配潜伏期与野生型果蝇不存在显著的差异; 黑檀体果蝇的交配成功率和交配潜伏期与野生型果蝇存在极显著的差异（P<0.000）。黑条体果蝇表现出异于黑檀体果蝇的活跃度和交配活力, 可能是由于黑条体果蝇ebony基因的新突变导致了果蝇体内多巴胺水平异常, 从而形成了黑条体果蝇独特的求偶模式。     

Comparison of transformation efficiency of piggyBac transposon among three different silkworm Bombyx mori Strains

The transformation rate of three different strains of silkworm Bombyx mori was comparedafter the introduction of enhanced green fluorescence protein (EGFP)-encoding genes into the silkwormeggs by microinjection of a mixture of piggyBac vector and helper plasmid containing a transposase-encodingsequence.Although there were no significant differences among the three strains in the percentages offertile moths in microinjected eggs (P=0.1258),the percentages of G_0 transformed moths in fertile mothsand injected eggs were both significantly different (P=0.01368 and P=0.02398, respectively).Thetransformation rate of the Nistari strain (Indian strain) was significantly higher than that of the other twostrains,Golden-yellow-cocoon (Vietnamese strain) and Jiaqiu (Chinese strain),which had similar rate. Theseresults indicate that the transformation efficiency of the piggyBac-based system might vary with silkwormstrains with different genetic backgrounds.The presence of endogenous piggyBac-like elements might bean important factor influencing the transformation efficiency of introduced piggyBac-derived vectors,andthe diverse amount and activation in different silkworm strains might account for the significant differences.     

Minos transposon causes germline transgenesis of the ascidian Ciona savignyi

An ascidian, Ciona savignyi, is regarded as a good experimental animal for genetics because of its small and compact genome for which a draft sequence is available, its short generation time and its interesting phylogenic position. ENU-based mutagenesis has been carried out using this animal. However, insertional mutagenesis using transposable elements (transposons) has not yet been introduced. Recently, one of the Tc1/mariner superfamily transposons, Minos, was demonstrated to cause germline transgenesis in the related species Ciona intestinalis. In this report, we show that Minos has the ability to transpose from DNA to DNA in Ciona savignyi in transposition assays. Although the activity was slightly weaker than in Ciona intestinalis, Minos still caused germline transgenesis in Ciona savignyi. In addition, one insertion seemed to have caused an enhancer trapping. These results indicate that Minos provides a potential tool for transgenic techniques such as insertional mutagenesis in Ciona savignyi.     

Evolution of the Xenopus piggyBac transposon family TxpB: domesticated and untamed strategies of transposon subfamilies

A new family, termed TxpB, of DNA transposons belonging to the piggyBac superfamily was found in 3 Xenopus species (Xenopus tropicalis, Xenopus laevis, and Xenopus borealis). Two TxpB subfamilies of Kobuta and Uribo1 were found in all the 3 species, and another subfamily termed Uribo2 was found in X. tropicalis. Molecular phylogenetic analyses of their open reading frames (ORFs) revealed that TxpB transposons have been maintained for over 100 Myr. Both the Uribo1 and the Uribo2 ORFs were present as multiple copies in each genome, and some of them were framed by terminal inverted repeat sequences. In contrast, all the Kobuta ORFs were present as a single copy in each genome and exhibited high evolutionary conservation, suggesting domestication of Kobuta genes by the host. Genomic insertion polymorphisms of the Uribo1 and Uribo2 transposons (nonautonomous type) were observed in a single species of X. tropicalis, indicating recent transposition events. Transfection experiments in cell culture revealed that an expression vector construct for the intact Uribo2 ORF caused precise excision of a nonautonomous Uribo2 element from the target vector construct but that for the Kobuta ORF did not. The present results support our viewpoint that some Uribo2 members are naturally active autonomous transposons, whereas Kobuta members may be domesticated by hosts.     

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.     

Excision and transposition of piggyBac transposable element in tobacco budworm embryos

The TTAA-specific lepidopteran transposon piggyBac has already proved useful as a gene-transfer vector for efficient transformation of a wide variety of insects. Transposable element excision and transposition assays are useful indicators of an element's ability to be mobilized in vivo and, thus, potentially serve as a transforming vector. Here, we report that this transposon is capable of excision and transposition in tobacco budworm embryos with relatively low frequency.     

Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools

Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc‐fusion proteins. Creating and characterizing the stable CHO clonally‐derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon‐mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2–10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in‐depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534–540, 2017     

A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary

Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example, nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.     

P element regulation and X-chromosome subtelomeric heterochromatin in Drosophila melanogaster

In Drosophila melanogaster, crossing males carrying autonomous P elements with females devoid of P copies results in hybrid dysgenesis in the germline of progeny. The reciprocal cross produces non-dysgenic progeny due to a maternally inherited state non-permissive for P transposition. The capacity of a P copy to repress transposition depends on both its structure and its chromosomal location. Naturally occuring regulatory P elements inserted at the telomere of the X chromosome have been genetically isolated in a genomic context devoid of other P elements. One or two copies of autonomous P elements at this site (1A) are sufficient to elicit a strong P repression in the germline. These elements are flanked by Telomeric Associated Sequences, previously identified and described by Karpen and Spradling (1992) as having heterochromatic properties. The regulatory properties of P elements at 1A are strongly impaired by mutations affecting Su(var)205, which encodes Heterochromatin Protein 1, a non-histone heterochromatin protein. The regulatory properties of classical P strains are not sensitive to Su(var)205. Models based on chromatin structure or on nuclear localisation of the telomeres are discussed in order to explain both the strong regulatory properties of P elements at the X chromosome telomere and their sensitivity to Su(var)205. This revised version was published online in August 2006 with corrections to the Cover Date.     

Tirant Stealthily Invaded Natural Drosophila melanogaster Populations during the Last Century

It was long thought that solely three different transposable elements (TEs)—the I-element, the P-element, and hobo—invaded natural Drosophila melanogaster populations within the last century. By sequencing the “living fossils” of Drosophila research, that is, D. melanogaster strains sampled from natural populations at different time points, we show that a fourth TE, Tirant, invaded D. melanogaster populations during the past century. Tirant likely spread in D. melanogaster populations around 1938, followed by the I-element, hobo, and, lastly, the P-element. In addition to the recent insertions of the canonical Tirant, D. melanogaster strains harbor degraded Tirant sequences in the heterochromatin which are likely due to an ancient invasion, likely predating the split of D. melanogaster and D. simulans. These degraded insertions produce distinct piRNAs that were unable to prevent the novel Tirant invasion. In contrast to the I-element, P-element, and hobo, we did not find that Tirant induces any hybrid dysgenesis symptoms. This absence of apparent phenotypic effects may explain the late discovery of the Tirant invasion. Recent Tirant insertions were found in all investigated natural populations. Populations from Tasmania carry distinct Tirant sequences, likely due to a founder effect. By investigating the TE composition of natural populations and strains sampled at different time points, insertion site polymorphisms, piRNAs, and phenotypic effects, we provide a comprehensive study of a natural TE invasion.