昆虫差异蛋白质组: 进展和展望

黑腹果蝇Drosophila melanogaster、家蚕Bombyx mori、意大利蜜蜂Apis mellifera和冈比亚按蚊Anopheles gambiae等昆虫的基因组测序已经基本完成,蛋白质组学技术将是阐明这些基因组功能的重要工具。本文综述了应用差异蛋白质组学技术在昆虫诱导性免疫、抗性机制和分子病理研究方面取得的一些成果：（1） 细菌、真菌、脂多糖或机械损伤诱导的昆虫血淋巴或血细胞来源细胞系的蛋白质表达的改变; （2） Bt抗性昆虫中肠刷状缘膜和抗锥虫采采蝇Glossina morsitans唾液腺多种蛋白质表达的改变; （3）化学农药处理和姬蜂Chelonus inanitus携带的多分DNA病毒引起的昆虫蛋白质组的变化。最后讨论了昆虫差异蛋白质组学研究的瓶颈与对策。     

昆虫基因组大小及其进化

基因组是物种遗传信息的集合,其大小是研究基因组进化、结构及功能的重要参数之一。本文介绍了测定基因组大小的方法,简述了基因组大小的进化假说及分子机制,综述了近年来昆虫基因组大小的研究进展,尤其是昆虫基因组大小变化的相关影响因子。总体而言,昆虫基因组大小变化是一个复杂的过程,与转座子的活性有密切的关系,是基因组序列丢失和获得两种过程平衡的结果。基因组大小的变化仍在不断地进化中,其对生物造成的影响是剧烈的,因此对昆虫的表型特征产生了重要的影响,但影响的程度和关系在不同的类群有明显的差异,表现出一定的随机性,目前尚未总结出明显的规律。昆虫是生物多样性最为丰富的动物类群,是研究基因组大小进化的最佳材料,随着越来越多的昆虫基因组被测序公开,对昆虫基因组数据进行深入分析,有利于破解基因组大小进化的"C值之谜",可为生物基因组大小的研究提供重要参考。     

中华卵索线虫线粒体基因组多态性分析

为完善昆虫病原索科线虫线粒体基因组全序列数据库, 更系统地研究其基因组特征和系统演化规律, 进而为发挥该线虫生防潜力打下基础, 我们开展了中华卵索线虫Ovomermis sinensis线粒体全基因组的研究。该研究通过线粒体基因组滚环复制及酶切图谱, 揭示了中华卵索线虫线粒体基因组具有种内遗传多态性, 即群体中单体线虫具有独特的酶切条带, 且条带累加之和变化范围较大, 为16.5～24.5 kb。为进一步了解线粒体基因组多态性特征及产生的分子机制, 采用两步长PCR方法对2条代表性成虫线粒体基因组进行了测序及拼接, 得其全长分别为18 864和16 777 bp。对这2条序列的比对表明, 线粒体基因组中位于ND2和ND4之间的可变区域, 不仅基因排列顺序不同, 且存在ND3基因重复现象, 这是导致中华卵索线虫线粒体基因组呈现多态性的主要原因。通过对以上研究结果的分析及与GenBank中已有的6种索科线虫线粒体基因组序列进行比对, 概括出其线粒体基因组基本特点: ①线粒体基因排列顺序各不相同;②部分线虫线粒体基因存在重复现象, 且重复次数不同;③线粒体基因组大小存在很大差异。     

家蚕免疫相关基因和信号途径的鉴定和比较分析

家蚕Bombyx mori是一种重要的经济昆虫, 在中国约有5 000年的驯化历史。家蚕分子免疫学方面的最新研究已经初步勾勒出其先天免疫的轮廓。本研究基于更新的家蚕基因组数据, 通过与黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae、意大利蜜蜂Apis mellifera和赤拟谷盗Tribolium castaneum基因组的比较分析, 鉴定了家蚕21个免疫相关基因家族的218个基因, 其编码产物包括模式识别受体、信号传导因子、效应分子和氧化防御相关的酶类。尽管信号传导因子的序列分化较大, 但系统进化分析显示它们在不同昆虫间呈明显的直系同源关系。相反, 与识别、调制和效应因子相关的基因的序列保守性更高, 但是这些基因家族明显缺乏直系同源基因, 由此推测这些基因是由物种特异的基因复制机制产生的。结果提示家蚕拥有与其他昆虫相同的免疫应答调控的分子机制, 而且家蚕同样可以通过基因复制及其序列分化等方式调节防御策略。     

家蚕核型多角体病毒egt基因的分子进化分析

过PCR方法获得家蚕核型多角体病毒（Bombyx mori nuclearpolyhedrosis virus,BmNPV）的蜕皮甾体尿苷二磷酸葡萄糖基转移酶基因（egt）片段,序列分析表明该片段带有EGT的完整ORF,推测的多肽可形成EGT结构域的高级结构。为了研究egt的起源,利用家蚕基因组数据库,电子克隆了多个家蚕尿苷二磷酸葡萄糖醛酸转移酶（UGT）基因,在此基础上进行了进化分析,表明BmNPV的EGT为antennal-enriched型UGT;推测核型多角体病毒（nucleopolyhedrivirus,NPV）和颗粒体病毒（granulovirus,GV）的egt基因在进化上来源于昆虫的UGT基因,但GV的egt基因在进化上的起源可能要早于NPV的egt基因;可能在昆虫祖先种进化形成不同昆虫目的某一时期,杆状病毒的祖先种从昆虫中获得了antennal-enriched型UGT基因,并进化为egt基因。家蚕的部分UGT基因与转座子元件连锁的基因组结构特点反映了杆状病毒的egt基因可能通过转座子的传递而获得。     

直翅目昆虫线粒体基因组研究进展

本文总结了本实验室对40余种直翅目昆虫的线粒体基因组序列的研究方法和主要结果.直翅目线粒体基因组研究中最重要的发现包括：（1）在直翅目昆虫线粒体基因组中发现了3种基因排列次序.蝗亚目除蜢总科外都具有DK排列.蜢总科的变色乌蜢为KD 排列,与蝗亚目其他总科不同,而与螽亚目昆虫的排序方式相同.已测出的螽亚目大多数昆虫的KD 排列顺序与典型节肢动物的完全相同,但在黄脸油葫芦Teleogryllus emma发生了tRNA^Glu,tRNA^Ser和tRNA^Asn的倒置;（2）在疑钩额螽Ruspolia dubia中发现了一种到目前为止具有最短控制区（70 bp）的线粒体基因组;（3）采用多种方法分析了昆虫A+T富集区存在的调控序列和二级结构特征,获得了昆虫A+T富集区保守序列的一致结构.采用Z曲线分析蝗虫的A+T富集区,表明也存在与原核生物复制起点类似的信号;（4）构建了30种蝗虫12S rRNA和16S rRNA的二级结构.在昆虫线粒体基因组非编码链中发现了一些类tRNA结构和tRNA异构体;（5）构建了基于线粒体基因组数据的直翅目昆虫主要亚科以上分类单元之间的系统发育关系.     

昆虫基因组及数据库研究进展

基因组序列为昆虫分子生物学研究提供丰富的数据资源,推动系统生物学在古老的昆虫学中蓬勃发展。昆虫基因组学研究已经成为当前的研究热点,目前在NCBI登录注册的昆虫基因组测序计划有494项,其中已提交原始测序数据的昆虫有225种,完成基因组拼接的有215种,具有基因注释的有65种,公开发表的昆虫基因组有43篇。本文综述了测序技术发展的历史及其对昆虫基因组研究的推动作用、昆虫基因组的组装和注释及其存在的问题、昆虫基因组测序进展、昆虫基因组数据库的发展及基因数据挖掘利用的基本思路和对策,以及昆虫基因大数据在害虫防治和资源昆虫利用中的应用前景。     

丝兰属6种植物叶绿体基因组特征分析

为了理清丝兰属（Yucca）叶绿体基因组特征和序列变异情况,进行丝兰属植物叶绿体比较基因组学分析,并构建基于叶绿体基因组的系统发育树。利用高通量测序技术获得无刺龙舌兰（Y. treculeana）叶绿体基因组序列,结合丝兰属现已发表的叶绿体基因组,使用生物信息学方法对6种丝兰属植物叶绿体全基因组进行基本结构、重复序列、边界收缩与扩张以及序列变异分析等在内的比较基因组学研究,并进行系统发育分析。结果表明：6种丝兰属植物叶绿体基因组大小、基因的类型及数目相近,种间基因组结构比较保守;从丝兰属植物叶绿体基因组中检测到多条重复序列,其中SSR位点多是由单核苷酸、双核苷酸和四核苷酸组成,且偏好使用A、T碱基;根据核酸多态性指数π≥0.008,在6种丝兰属植物叶绿体基因组中筛选出了psbK-psbl-trnS-GCU、rpl20-rps12和ccsA-ndhD 3个高变异区域;基于叶绿体全基因组和LSC+SSC区序列构建的系统发育关系基本一致,确定了6种丝兰属植物间的系统发育关系,其中无刺龙舌兰与克雷塔罗丝兰（Y. queretaroensis）的亲缘关系最近。本研究测序获得了无刺龙舌兰叶绿体基因组,揭示了6种丝兰属植物叶绿体基因组特征和序列变异情况,明确了各物种间的亲缘关系,研究结果可为后续丝兰属植物分子标记开发及系统发育研究提供参考。     

几种化学因子对三种赤眼蜂离体酚氧化酶活性的影响

为比较不同赤眼蜂酚氧化酶（PO）活性的大小, 明确赤眼蜂种间免疫防御能力强弱, 本研究通过研磨、离心提取松毛虫赤眼蜂Trichogramma dendrolimi、螟黄赤眼蜂T. chilonis、玉米螟赤眼蜂T. ostriniae匀浆液, 首次测定了几种化学因子（Ca²⁺、昆布多糖、纤维素、右旋糖酐、脂多糖和丝氨酸蛋白酶抑制剂）对3种供试赤眼蜂匀浆液中酚氧化酶原激活系统（proPO-AS）的激活程度。结果表明: 玉米螟赤眼蜂匀浆液PO活性最高, 螟黄赤眼蜂次之, 松毛虫赤眼蜂最低。Ca²⁺是激活供试赤眼蜂proPO的必需因子, 且在0.03 mol/L浓度下激活作用最强; 昆布多糖和脂多糖也能有效激活proPO, 0.01及0.05 mg/mL昆布多糖能强烈激活PO活性; 纤维素和右旋糖酐对PO活性存在显著抑制作用。结果证明了赤眼蜂种间存在明显的免疫能力差异。本研究建立了小型昆虫proPO-AS研究体系, 为研究Wolbachia-小型昆虫的免疫互作体系奠定了基础。     

毛囊蠕形螨与皮脂蠕形螨基因组DNA的RAPD分析和序列比对

【目的】分析毛囊蠕形螨Demodex folliculorum（D.f.）和皮脂蠕形螨D. brevis（D.b.）基因组DNA的多态性, 对相关条带进行测序分析。【方法】采用改良小昆虫DNA提取法提取两种人体蠕形螨基因组DNA, 选择RAPD技术对其进行多态性分析, 将相关条带分别与pMD18-T载体连接, 克隆、测序后进行酶切鉴定和分析。【结果】毛囊蠕形螨共扩增15条带, 皮脂蠕形螨共扩增12条带;两种蠕形螨既有共有条带, 又有特异性条带;根据条带差异计算得到两种间的遗传距离为0.5556. 毛囊蠕形螨约800 bp处特异性条带测序结果显示, 序列片段长度为855 bp（GenBank登录号为FI277970）;特异性引物扩增和酶切鉴定均为毛囊蠕形螨所特有. 序列比对显示与阿糖胞苷DNA区域结合蛋白有46%的序列相似度。两种人体蠕形螨约300 bp处共有条带序列分析显示, 碱基序列均为341 bp（GenBank登录号分别为D.f. FI520176;D.b. FI520175）, 在第84和第165位点有2个碱基不同, 分别是A/G和C/T互换, 同源性高达99.4%. 但未发现有开放阅读框和相似度高的序列。 【结论】序列片段为855 bp的特异性条带为毛囊蠕形螨所特有;341 bp碱基序列为毛囊蠕形螨和皮脂蠕形螨所共有, 同源性高达99.4%. RAPD技术可用于两种人体蠕形螨基因组DNA的多态性分析和物种鉴定。     

Is genome size influenced by colonization of new environments in dipteran species?

Genome size differences are usually attributed to the amplification and deletion of various repeated DNA sequences, including transposable elements (TEs). Because environmental changes may promote modifications in the amount of these repeated sequences, it has been postulated that when a species colonizes new environments this could be followed by an increase in its genome size. We tested this hypothesis by estimating the genome size of geographically distinct populations of Drosophila ananassae, Drosophila malerkotliana, Drosophila melanogaster, Drosophila simulans, Drosophila subobscura, and Zaprionus indianus, all of which have known colonization capacities. There was no strong statistical differences between continents for most species. However, we found that populations of D. melanogaster from east Africa have smaller genomes than more recent populations. For species in which colonization is a recent event, the differences between genome sizes do not thus seem to be related to colonization history. These findings suggest either that genome size is seldom modified in a significant way during colonization or that it takes time for genome size of invading species to change significantly.     

Polyploidy does not control all: Lineage‐specific average chromosome length constrains genome size evolution in ferns

Recent studies investigating the evolution of genome size diversity in ferns have shown that they have a distinctive genome profile compared with other land plants. Ferns are typically characterized by possessing medium‐sized genomes, although a few lineages have evolved very large genomes. Ferns are different from other vascular plant lineages as they are the only group to show evidence for a correlation between genome size and chromosome number. In this study, we aim to explore whether the evolution of fern genome sizes is not only shaped by chromosome number changes arising from polyploidy but also by constraints on the average amount of DNA per chromosome. We selected the genus Asplenium L. as a model genus to study the question because of the unique combination of a highly conserved base chromosome number and a high frequency of polyploidy. New genome size data for Asplenium taxa were combined with existing data and analyzed within a phylogenetic framework. Genome size varied substantially between diploid species, resulting in overlapping genome sizes among diploid and tetraploid spleenworts. The observed additive pattern indicates the absence of genome downsizing following polyploidy. The genome size of diploids varied non‐randomly and we found evidence for clade‐specific trends towards larger or smaller genomes. The 578‐fold range of fern genome sizes have arisen not only from repeated cycles of polyploidy but also through clade‐specific constraints governing accumulation and/or elimination of DNA.     

The smallest avian genomes are found in hummingbirds

It has often been suggested that the genome sizes of birds are constrained relative to other tetrapods owing to the high metabolic demands of powered flight and the link between nuclear DNA content and red blood cell size. This hypothesis predicts that hummingbirds, which engage in energy-intensive hovering flight, will display especially constrained genomes even relative to other birds. We report genome size measurements for 37 species of hummingbirds that confirm this prediction. Our results suggest that genome size was reduced before the divergence of extant hummingbird lineages, and that only minimal additional reduction occurred during hummingbird diversification. Unlike in some other avian taxa, the small amount of variation observed within hummingbirds is not explained by variation in respiratory and flight-related parameters. Unexpectedly, genome size appears to have increased in four unrelated hummingbird species whose distributions are centred on humid forests of the upper-tropical elevational zone on the eastern slope of the Andes. This suggests that the secondary expansion of the genome may have been mediated by biogeographical and demographic effects.     

Genome size is a strong predictor of cell size and stomatal density in angiosperms

Across eukaryotes phenotypic correlations with genome size are thought to scale from genome size effects on cell size. However, for plants the genome/cell size link has only been thoroughly documented within ploidy series and small subsets of herbaceous species. Here, the first large-scale comparative analysis is made of the relationship between genome size and cell size across 101 species of angiosperms of varying growth forms. Guard cell length and epidermal cell area were used as two metrics of cell size and, in addition, stomatal density was measured. There was a significant positive relationship between genome size and both guard cell length and epidermal cell area and a negative relationship with stomatal density. Independent contrast analyses revealed that these traits are undergoing correlated evolution with genome size. However, the relationship was growth form dependent (nonsignificant results within trees/shrubs), although trees had the smallest genome/cell sizes and the highest stomatal density. These results confirm the generality of the genome size/cell size relationship. The results also suggest that changes in genome size, with concomitant influences on stomatal size and density, may influence physiology, and perhaps play an important genetic role in determining the ecological and life-history strategy of a species.     

Genome size estimation with quantitative real‐time PCR in two Tephritidae species: Ceratitis capitata and Bactrocera oleae

The medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae belong to the Tephritidae family of Diptera, a family whose members cause severe damages in agriculture worldwide. For such insect pests, the utmost concern is their population control. The sterile insect technique (SIT) has been used in the Tephritidae family with varying degrees of success. Its efficient use usually depends on the development of genetic sexing strains and the release of only male flies. However, such advances are based on modern genetic, molecular and genomic tools. The medfly is clearly the prototype of the family, since such tools have advanced considerably, which has resulted in effective SIT efforts around the world. A whole‐genome sequencing project of this insect is already underway. In contrast, similar tools in the olive fly lag behind, even though the insect is considered a promising candidate for a next SIT target. An accurate estimate of genome size provides a preliminary view of genome complexity and indicates possible difficulties in genome assembly in whole‐genome projects. Taking advantage of a quantitative real‐time PCR approach, we determined the genome size of these two species C. capitata and B. oleae as 591 Mb (CI range: 577–605 Mb) and 322 Mb (CI range: 310–334 Mb) respectively.     

Smallest angiosperm genomes found in lentibulariaceae, with chromosomes of bacterial size

Nuclear holoploid genome sizes (C-values) have been estimated to vary about 800-fold in angiosperms, with the smallest established 1C-value of 157 Mbp recorded in Arabidopsis thaliana. In the highly specialized carnivorous family Lentibulariaceae now three taxa have been found that exhibit significantly lower values: Genlisea margaretae with 63 Mbp, G. aurea with 64 Mbp, and Utricularia gibba with 88 Mbp. The smallest mitotic anaphase chromatids in G. aurea have 2.1 Mbp and are thus of bacterial size (NB: E. coli has ca. 4 Mbp). Several Utricularia species range somewhat lower than A. thaliana or are similar in genome size. The highest 1C-value known from species of Lentibulariaceae was found in Genlisea hispidula with 1510 Mbp, and results in about 24-fold variation for Genlisea and the Lentibulariaceae. Taking into account these new measurements, genome size variation in angiosperms is now almost 2000-fold. Genlisea and Utricularia are plants with terminal positions in the phylogeny of the eudicots, so that the findings are relevant for the understanding of genome miniaturization. Moreover, the Genlisea-Utricularia clade exhibits one of the highest mutational rates in several genomic regions in angiosperms, what may be linked to specialized patterns of genome evolution. Ultrasmall genomes have not been found in Pinguicula, which is the sister group of the Genlisea-Utricularia clade, and which does not show accelerated mutational rates. C-values in Pinguicula varied only 1.7-fold from 487 to 829 Mbp.     

内蒙古草原不同基因组大小植物对氮水添加的响应

被子植物基因组大小的种间差异巨大,约为2400倍.基因组大小与植物从细胞核到个体水平的一系列性状密切相关,进而影响植物对环境变化的响应.作为水分和养分共同限制的生态系统,内蒙古草原植物群落对氮素、水分有效性变化的响应具有明显的种间差异,这种差异可能与种间基因组大小不同有关.本研究利用流式细胞术测定了内蒙古典型草原水分、氮素添加实验平台植物的基因组大小,研究了不同基因组大小植物地上净初级生产力(ANPP)和物种丰富度对水分、氮素添加及其交互作用的响应.结果表明:基因组大小显著影响了不同植物ANPP对水分的响应,小基因组植物ANPP对氮水添加响应更敏感,加水和氮水共同添加显著增加了小基因组植物ANPP,而大基因组植物ANPP对所有处理响应均不显著.加氮对大小基因组植物ANPP都无显著影响.大小基因组植物的物种丰富度对氮水添加的响应也均不显著.基因组大小影响内蒙古草原不同植物ANPP对水分增加的响应.作为植物细胞核水平上十分稳定且种间差异巨大的物种性状,将基因组大小引入生态学研究将对全球变化背景下生态系统结构与功能变化研究起到重要作用.