葱蝇热激蛋白DaHSP23基因的克隆及在冬滞育和夏滞育蛹中的表达分析

[目的]低分子量(12 ～43 kDa)热激蛋白(sHSPs)具有抗逆应答的功能,滞育是昆虫抵抗不良环境的特殊发育形式,但sHSPs在昆虫滞育发育过程中的作用仍不清楚.本研究克隆和特征化葱蝇Delia antiqua sHSP基因,并研究它在夏滞育和冬滞育发育过程中的表达模式,为阐明sHSPs在滞育发育上的功能奠定基础.[方法]通过RACE-PCR方法克隆了葱蝇HSP23基因,通过相似性比较分析了其特征、结构域及与双翅目代表性同源基因的系统发育关系;采用实时荧光定量PCR研究了该基因在葱蝇冬滞育蛹和夏滞育蛹发育过程中的表达情况,通过表达的差异比较揭示了该基因与滞育发育的关系.[结果]克隆出了葱蝇HSP23基因,命名为DaHSP23(GenBank登录号:HQ392521.1),其cDNA全长序列为904 bp,编码186个氨基酸,推测蛋白分子量为20.9 kDa,等电点为6.42.该基因的编码蛋白与其他双翅目昆虫的sHSPs有超过66％的氨基酸序列一致性,与已报道的其他双翅目昆虫的滞育相关HSP23基因同源.基因组测序显示该基因无内含子.DaHSP23基因在葱蝇非滞育蛹的发育过程中一直保持在较低的水平,各发育阶段间的表达量不存在显著差异.但在冬滞育和夏滞育蛹中,该基因从滞育起始期开始逐渐显著升高表达,到滞育维持期的中后期达到峰值,在滞育终止期逐渐降到较低的水平.[结论]DaHSP23基因在葱蝇冬滞育和夏滞育发育过程中明显上调表达,但存在差异,它在滞育期的调控可能是种专化的.DaHSP23可能在葱蝇两种类型的滞育上起重要作用.     

高低温催青温度敏感期家蚕卵的差异蛋白筛查

滞育（Diapause）是昆虫发育停滞或减缓的生理状态，家蚕Bombyx mori作为卵滞育的代表，其滞育过程已得到广泛研究，但诱导滞育发生的分子机制尚不清楚。二化性家蚕滞育性由遗传和母系在胚胎期所处的环境条件决定，25℃催青蚕卵孵化后的家蚕产滞育卵，15℃低温催青则诱导家蚕产下非滞育卵。本研究分别用25℃和15℃催青蚕卵，在发生滞育诱导的温度敏感期取样，抽提蛋白质通过非标（Label-free）蛋白质组定量技术进行质谱测序。筛选出具有明显表达差异的蛋白104个，其中56个蛋白上调，48个蛋白下调。通过生物信息学对差异蛋白进行GO分类和KEGG功能富集分析，结果显示差异蛋白主要参与生长发育、物质代谢和胁迫应答等生物过程；同时差异蛋白主要参与胰岛素信号通路、乙醛酸和二羧酸代谢等相关途径。分别选取上调基因和下调基因进行qRT-PCR验证，其趋势与蛋白组学结果一致。该研究将为进一步解析家蚕滞育诱导发生机制提供靶标蛋白和数据参考。     

葱蝇热激蛋白DaHSP23基因的克隆及在冬滞育和夏滞育蛹中的表达分析（英文）

【目的】低分子量（12~43 kDa）热激蛋白(sHSPs)具有抗逆应答的功能,滞育是昆虫抵抗不良环境的特殊发育形式,但sHSPs在昆虫滞育发育过程中的作用仍不清楚。本研究克隆和特征化葱蝇Delia antiqua sHSP基因,并研究它在夏滞育和冬滞育发育过程中的表达模式,为阐明sHSPs在滞育发育上的功能奠定基础。【方法】通过RACE-PCR方法克隆了葱蝇HSP23基因,通过相似性比较分析了其特征、结构域及与双翅目代表性同源基因的系统发育关系;采用实时荧光定量PCR研究了该基因在葱蝇冬滞育蛹和夏滞育蛹发育过程中的表达情况,通过表达的差异比较揭示了该基因与滞育发育的关系。【结果】克隆出了葱蝇HSP23基因,命名为DaHSP23（GenBank登录号：HQ392521.1）,其cDNA全长序列为904 bp,编码186个氨基酸,推测蛋白分子量为20.9 kDa,等电点为6.42。该基因的编码蛋白与其他双翅目昆虫的sHSPs有超过66%的氨基酸序列一致性,与已报道的其他双翅目昆虫的滞育相关HSP23基因同源。基因组测序显示该基因无内含子。DaHSP23基因在葱蝇非滞育蛹的发育过程中一直保持在较低的水平,各发育阶段间的表达量不存在显著差异。但在冬滞育和夏滞育蛹中,该基因从滞育起始期开始逐渐显著升高表达,到滞育维持期的中后期达到峰值,在滞育终止期逐渐降到较低的水平。【结论】DaHSP23基因在葱蝇冬滞育和夏滞育发育过程中明显上调表达,但存在差异,它在滞育期的调控可能是种专化的。DaHSP23可能在葱蝇两种类型的滞育上起重要作用。     

二化螟滞育生物钟蛋白TIME-EA4基因的克隆及时空和温度诱导表达分析

【目的】生物测时蛋白TIME-EA4是昆虫特有的一种滞育生物钟蛋白。本文旨在克隆二化螟Chilo suppressalis TIME-EA4基因,研究其在二化螟不同发育时期和不同组织中的时空表达模式及在不同田间种群和不同温度诱导下的滞育和非滞育种群中的表达水平。【方法】通过RACE技术克隆二化螟TIME-EA4基因全长c DNA序列,利用荧光定量PCR的方法检测TIME-EA4基因在二化螟不同发育时期、6龄幼虫不同组织中的表达模式,及在不同季节采集的田间种群和不同温度处理1h后的滞育和非滞育种群6龄幼虫中的表达量变化。【结果】从二化螟中克隆获得TIME-EA4的c DNA序列(Gen Bank登录号:KU356855),全长821 bp,开放阅读框516 bp,编码172个氨基酸;TIME-EA4基因在二化螟不同发育时期和不同组织中均有表达,且在早期蛹、雌雄成虫以及6龄幼虫的头部、脂肪体和中肠中表达水平较高,同时在滞育幼虫组织中的表达高于非滞育幼虫。此外,TIME-EA4基因在滞育种群中的表达量是非滞育种群的3倍左右,同时在非滞育种群中,该基因的表达明显受10℃及以下低温的诱导,且在10℃时表达量最高,而在滞育种群中该基因的表达受温度影响不明显。【结论】结果说明TIME-EA4基因与二化螟的抗低温胁迫以及低温滞育相关。本研究为TIME-EA4在农业害虫滞育的分子作用机制研究奠定了基础。     

伞裙追寄蝇滞育关联基因和滞育关联代谢物的联合分析

为探索伞裙追寄蝇蛹滞育调控的分子机制,本文对伞裙追寄蝇Exorista civilis滞育蛹和非滞育蛹进行转录组测序以及代谢组检测,鉴定其关键的滞育关联基因（diapause-associated genes, DEGs）和滞育关联代谢物（diapasuse-associated metabolites, DEMs）。本研究基于高通量测序以及液质联用技术,通过筛选,在转录组中获得了差异表达基因7 513个,在代谢组中,获得差异代谢物501个,其中氨基酸占比最多。将所有差异表达基因与差异表达代谢物同时向KEGG映射,获得两者共同的pathway信息,明确差异表达基因与差异代谢物共同参与的主要生化途径和信号转导途径。本研究通过转录组和代谢组的联合分析,在正、负离子模式下,差异表达基因与差异代谢物共同富集到70条通路。在负离子模式下,滞育关联因子主要参与氨基酸代谢和神经系统;在正离子模式下,滞育关联因子主要参与消化系统和信号转导途径。本研究重点分析了柠檬酸循环、cAMP信号通路、氨酰-tRNA的生物合成,将为进一步深入研究伞裙追寄蝇滞育调控的分子机制奠定理论基础。     

昆虫滞育诱导的分子调控机制

滞育是昆虫长期适应不利环境的重要对策之一,对昆虫的生存、繁衍和进化都具有重要意义。滞育一般分为滞育前期、滞育期和滞育后期3个阶段,其中,滞育前期包括滞育诱导期和滞育准备期,是确保昆虫顺利进入滞育的重要时期。本文首先简要概括了光周期、温度、湿度、食料等外界环境因素在不同昆虫滞育诱导中的作用;然后系统综述了滞育激素、保幼激素、蜕皮激素、促前胸腺激素和胰岛素等内分泌激素,生物钟通路的节律基因,以及DNA甲基化、小RNA和组蛋白修饰等表观遗传修饰在昆虫滞育过程,尤其是滞育诱导调控中的作用,并以黑腹果蝇Drosophila melanogaster的生殖滞育为例,总结了昆虫滞育诱导调控的分子通路;最后就目前昆虫滞育诱导调控研究中亟待解决的问题和后续重点研究方向进行了探讨,展望了将昆虫滞育研究应用于害虫防控实践的主要策略和前景。     

基于转录组的葱蝇表皮蛋白基因家族的鉴定及在非滞育和冬滞育条件下的表达分析

【目的】昆虫表皮蛋白在昆虫的生长发育、抑制水分蒸发和抵御其他外界不良环境等方面起到重要的作用。通过转录组水平上葱蝇Delia antiqua表皮蛋白基因家族的鉴定及部分基因在非滞育和冬滞育条件下的表达规律分析,为进一步研究葱蝇表皮蛋白在滞育期的抗逆功能奠定基础。【方法】基于转录组数据,通过生物信息学方法对葱蝇表皮蛋白基因进行鉴定分析,系统进化和保守结构域分析并采用实时荧光定量PCR法比较分析了12个基因在葱蝇非滞育和冬滞育不同发育时期的表达变化。【结果】在葱蝇中鉴定出81个表皮蛋白基因,分别属于RR(71个基因)、CPAP(5个基因)和CPLC(5个基因)亚家族,其编码的蛋白质含有92~495个氨基酸,分子量约为8.692~52.2 k D,等电点为3.90~9.93。亚细胞定位预测结果显示67个全长表皮蛋白均为外分泌型。12个葱蝇表皮基因在非滞育和冬滞育过程中有明显不同的表达规律。【结论】葱蝇表皮蛋白基因RR-2型的保守基序中发生了基因突变,引起关键氨基酸的替换,这可能暗示它们在非滞育和滞育不同条件下,不同发育阶段中蛹皮结构重建和某些器官的发生方面发挥不同作用。该研究结果为进一步阐明葱蝇滞育蛹皮结构及抗逆功能的研究提供了重要理论基础。     

抑制性消减杂交技术在昆虫滞育中的利用和展望

滞育是多数天敌昆虫固有的遗传属性,掌握天敌昆虫滞育诱导、维持和解除技术,能及时将扩繁的天敌昆虫以滞育状态进行储存,在释放前再打破滞育使其进入活跃状态以防控害虫。开展天敌昆虫滞育研究,还有助于掌握其发育特点与发生动态,提高害虫防治效率,加深对天敌昆虫发育机制的认识,探寻昆虫对环境适应机制及进化途径。因此,滞育调控及机理研究成为近年来昆虫学研究的一个重要领域。抑制性消减杂交技术（Suppression Subtractive Hybridization, SSH）是近年来发明的用于挖掘差异性调控或特异表达cDNA探针和构建文库的一种方法。本文在总结抑制性消减杂交技术的测试原理及其优越性的基础上,归纳了该技术在昆虫滞育研究中的应用,概述了通过构建抑制性消减文库,在鞘翅目、直翅目、鳞翅目、双翅目等昆虫中探明的滞育差异表达基因,进一步探讨了差异表达基因与滞育的关系。随着昆虫全基因组测序和转录组测序的信息增加,抑制性消减杂交技术将更广泛的应用于昆虫学各个领域的差异基因的筛选与挖掘,促进昆虫分子生物学科学体系的发展。     

西伯利亚蝗卵发育过程的比较转录组分析及滞育关联基因筛选

【目的】通过筛选西伯利亚蝗Gomphocerus sibiricus卵滞育基因及代谢通路,初步明确西伯利亚蝗卵滞育分子机理。【方法】利用Illumina NovaSeq 6000测序平台对西伯利亚蝗早期发育(ES)、滞育(DS)和滞育后发育阶段(PS)的卵进行转录组测序;采用GO和KEGG富集分析并结合文献,预测滞育相关通路并聚类热图分析,筛选蝗卵滞育关联基因,并选取其中6个重要基因进行qRT-PCR验证。【结果】DSvs ES和PS vs DS两比较组分别富集到12 419和4 789个差异表达基因,且多上调表达;两比较组共表达差异基因为2 206个,主要与糖代谢、环境胁迫和生长发育相关。DS vs ES组富集最显著的GO条目为蛋白结合,PS vs DS组GO条目主要包含酶活性、细胞骨架构建及蛋白结合等过程。滞育关联基因主要集中在Wnt信号通路、胰岛素信号通路、细胞周期通路以及昆虫激素生物合成等通路。6个重要的滞育关联基因的表达量变化趋势与转录组数据结果基本一致。【结论】本研究初步明确了西伯利亚蝗卵滞育调控的重要代谢途径,并筛选出20个滞育关联基因,为后续深入研究该物种的适应机理奠定了基础。     

昆虫滞育的分子机理——Ⅰ.家蚕胚子发育期的温度条件与滞育激素基因表达

运用分子生物学方法 ,解决了环境条件通过什么途径决定昆虫滞育的关键一步 :即环境信号能诱导家蚕胚期滞育激素基因表达 .胚期滞育激素cDNA被克隆 ,测序结果表明胚期的滞育激素基因表达完全属实 .测定了不同胚期的滞育激素基因的发育表达 ,推定滞育激素基因表达在头部的食道下神经节 .根据实验结果 ,提出环境条件诱导滞育的一种假说 .     

What makes a fly enter diapause?

Diapause is a dormant state that insects may undergo as a response to changing environmental conditions. In flies, like many insects inhabiting temperate zones, diapause occurs generally during the winter months when ambient temperatures are cool and food sources scarce. Whilst the environmental factors involved in determining diapause have been known for a long time, the genes and molecular events controling its initiation are poorly understood. Here I outline the factors that initiate diapause and highlight recent studies that implicate insulin signaling in its control.     

What Makes a Fly Enter Diapause?

Diapause is a dormant state that insects may undergo as a response to changing environmental conditions. In flies, like many insects inhabiting temperate zones, diapause occurs generally during the winter months when ambient temperatures are cool and food sources scarce. Whilst the environmental factors involved in determining diapause have been known for a long time, the genes and molecular events controlling its initiation are poorly understood. Here I outline the factors that initiate diapause and highlight recent studies that implicate insulin signaling in its control.     

PROTEOMICS ANALYSIS OF OVEREXPRESSED PLASMA PROTEINS IN RESPONSE TO COLD ACCLIMATION IN Ostrinia furnacalis

Many insects in temperate regions overwinter in diapause. In these insects, one of the metabolic adaptations to cold stress is the synthesis of responsive proteins. Using proteomic analysis, an investigation aimed to a better understanding of the molecular adaptation mechanisms to cold stress was carried out in Ostrinia furnacalis larva. Proteins were extracted from the larval hemolymph collected from both control and overwintering larva. By polyethylene glycol precipitation, approximately 560 protein spots were separated and visualized on two‐dimensional (2D) gels after silver staining. Eighteen protein spots were found to be upregulated in overwinter larval plasma in different patterns. As an initial work, 13 of these proteins were identified using MALDI TOF/TOF MS. The differentially overexpressed proteins include heat shock 70 kDa cognate protein, small heat shock protein (sHSP), putative aliphatic nitrilase, arginine kinase, phosphoglyceromutase, triosephosphateisomerase, and glutathione transferase. Alterations in the levels of these proteins were further confirmed by qPCR. This study is the first analysis of differentially expressed plasma proteins in O. furnacalis diapause larvae under extremely low temperature conditions and gives new insights into the acclimation mechanisms responsive to cold stress. Our results also support the idea that energy metabolism, alanine and proline metabolism, and antioxidative reaction act in the cold acclimation of O. furnacalis diapause larvae.     

ArHsp22, a developmentally regulated small heat shock protein produced in diapause-destined Artemia embryos, is stress inducible in adults

Diapause embryos of the crustacean Artemia franciscana exhibit extreme stress tolerance, a property thought to involve molecular chaperones known as small heat shock proteins. To further explore this idea, the structure, function and synthesis of ArHsp22, an Artemia small heat shock protein, were characterized. ArHsp22 contains amino-terminal WXDPF motifs, an alpha-crystallin domain with a highly conserved arginine, and a carboxy-terminal I/VXI/V motif, all typical of small heat shock proteins. ArHsp22 formed large oligomers and exhibited molecular chaperone activity in vitro, protecting citrate synthase and insulin from denaturation. Quantitative PCR and immunoprobing of western blots revealed that ArHsp22 synthesis is restricted to diapause-destined Artemia embryos and that the protein is degraded during post-diapause development. ArHsp22 was observed in cyst nuclei, a location shared by p26 but not ArHsp21, which are two other diapause-specific Artemia small heat shock proteins. ArHsp22 production was enhanced by thermal stress, but only in adults, thus representing the first crustacean small heat shock protein whose synthesis is known to be both developmentally regulated and stress inducible. The results demonstrate that expression of the gene for ArHsp22 is modulated by multiple cues that vary with life history stage. Such findings are of importance in understanding diapause maintenance in Artemia embryos and the survival of adult animals experiencing environmental insult.     

Multiple role of temperature during insect diapause: a review

The review emphasizes that there are multiple pathways to diapause completion. The programmed course of events is modified by environmental cues. Often chilling is not a prerequisite for the completion of hibernation diapause (examples tabulated). Diapause completion progresses well at intermediate or high temperatures, sometimes it is even stimulated by high or increasing temperature. Low temperatures are important, as they (1) conserve metabolic reserves, (2) prevent resumption of post-diapause morphogenesis and thus synchronize the life-cycle, (3) represent contrast to the later increase in temperature. Diapause consists of phases with different prerequisites. There is a principal difference between diapause development and photoperiodic activation as indicated by the subsequent physiological condition of insects.     

Activity enhancement of Zygogramma bicolorata,a biocontrol agent of Parthenium hysterophorus,by temperature regulated diapause aversion

Diapause is a unique strategy of dormancy in insects to avoid unfavourable conditions. The exotic beetle Zygogramma bicolorata, is an effective biological control agent of Parthenium hysterophorus in India, Adults diapause in soil during December to May. As a result, there is delay in its effectiveness on the plant that reaches to flowering and seed production by the time the beetle is able to build up its population after emerging from diapause. Therefore, a study was conducted to explore possibilities of diapause aversion by temperature regulation. Results indicated that exposure of newly emerged adults to heat treatment of 35°C and to low temperature of 10°C could reduce diapause in Z. bicolorata. The low temperature can also be used as a medium for the storage of the mass reared beetles for a long time without having negative effect on their longevity and fecundity.     

滞育昆虫营养物质的积累、转化与调控

滞育是昆虫度过不良环境的一种生存适应策略,营养物质的利用、积累和转化影响昆虫的滞育。滞育期间,昆虫体内发生一系列生理生化变化,包括脂类、碳水化合物和氨基酸等内源营养物质呈特异性地积累或转化,保障了滞育个体在逆境中存活及滞育解除后发育的能量需求。外源营养物质对昆虫滞育的影响较复杂,其种类、丰度、质量能通过"食料-寄主-天敌"营养关系进行传递,影响昆虫体内营养物质的积累和转化,改变昆虫的耐寒性、滞育率等,制约昆虫的滞育深度、滞育存活率。滞育昆虫营养物质积累、利用机制复杂多样,胰岛素信号通路-叉头转录因子以及激脂激素的调控效应在滞育诱导及营养物质转化中起到了关键的调控作用,但滞育的系统性调控仍需进一步深入研究。     

Diapause induction in aphid parasitoids

Diapause is one of the adaptations that insects have evolved for the synchronisation of their life cycle with seasonal climatic changes and resources. In aphid parasitoids, univoltine species have an obligatory, genetically determined diapause. Polyvoltine species, on the other hand, use a variety of abiotic (temperature, photoperiod) and biotic (host insect or/and host plant) signals for the induction of diapause. We present an overview of the role of these environmental cues in diapause induction in specialist and generalist aphid parasitoids, and discuss possible endocrine factors that may be involved in diapause induction.