桔小实蝇消化和解毒代谢相关基因鉴定及其对药剂胁迫的应激反应
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摘要
昆虫通过消化代谢从食物中吸收营养物质,为自身的生长、发育和生殖提供必要的能量,同时通过解毒代谢抵御在取食和活动中接触到的外源毒素对自身的不利影响,这是昆虫体内两条非常重要的代谢通路。而由于此前相关分子生物学信息的不足,关于消化代谢和解毒代谢的研究,往往仅局限于某几个基因或者酶的特性解析,对大多数昆虫的消化和解毒代谢系统的分子生物学信息还缺乏全面的了解。随着第二代高通量测序技术的出现和成熟,使得即使在缺乏全基因组信息的情况下,也可以快速、高效的在转录组水平上获得某一个个体、器官或者组织的基因信息。该技术在昆虫研究中的应用和推广,极大地推动了昆虫分子生物学研究的进程。
本学位论文以果树重要害虫桔小实蝇为研究对象,利用高通量测序技术,对其不同发育阶段的基因表达谱进行了检测,在发掘不同发育阶段差异表达基因的同时,明确了桔小实蝇体内主要消化酶和解毒代谢酶在不同虫态的表达分布情况。中肠是昆虫体内的第二大器官,是消化食物和吸收营养的主要场所,也是进行解毒代谢、阻隔外源毒素的一大屏障,因此进一步利用转录组测序技术在整体水平上解读了桔小实蝇幼虫中肠转录本信息,对功能基因进行了注释,为相关分子生物学研究的开展提供了充实的数据。并且从害虫防治的角度出发,围绕中肠的主要功能,筛选鉴定出了与食物消化、有毒物质代谢以及dsRNA作用通路上相关的基因,解析了它们的序列特征和系统进化关系。进而根据得到的丰富的基因信息,在内参基因筛选的基础上,利用qPCR技术系统开展了桔小实蝇解毒代谢酶基因(CCEs, P450s和GSTs)和消化酶基因在高效氯氰菊酯胁迫下的应激表达模式研究。研究发现桔小实蝇应对药剂胁迫时可能存在组织间的能量再分配,即通过组织间能量的转移和集中,高效表达解毒代谢酶基因的应对机制。本研究的主要结果如下：
1桔小实蝇不同发育阶段基因表达谱分析
1.1不同发育阶段基因表达谱测序概况
以采自海南并在实验室建立的桔小实蝇种群为试虫,分别从卵、幼虫、蛹以及成虫体内提取高质量RNA,在Illumina HiSeq2000上进行基因表达谱测序。获得的数据提交至NCBI的SRA数据库,登录号分别为SRA043792, SRA043786,SRA043785以及SRA043783。经过对数据的统计分析发现,4个样品的reads数均在5百万以上,且所含有的低质量reads、不确定碱基以及接头序列的比例都低于1%,以数据库中已公布的桔小实蝇不同发育阶段转录组数据为参考,对4个发育阶段的基因表达谱序列进行比对后,有超过65%的序列的覆盖度达到50%以上,说明表达谱测序的质量较高。
1.2不同发育阶段差异表达的基因
按卵期和幼虫期、幼虫期和蛹期、蛹期和成虫期的分组对4个基因表达谱进行两两对比,统计桔小实蝇不同发育阶段差异表达的基因数量发现,卵期和幼虫期之间差异表达的基因数量最多。与卵期相比,幼虫期分别有7352和8164个基因的表达存在上调和下调的现象。蛹期和幼虫期相比,共有7581个基因在表达丰度上存在显著差异,在蛹期表达上调的基因数量为3786个,表达下调的基因数量为3795个。蛹期与成虫期相比,在表达丰度上存在显著差异的基因有10272个,其中在成虫期表达上调的基因数(3077)远少于表达下调的基因数(7195)。通过对各个发育阶段差异表达显著的基因进行功能分析发现,在卵期表达丰度较高的基因主要涉及胚胎发育和细胞分裂,而幼虫期则主要参与能量代谢以及肌肉形成,在蛹期表达丰度高的是与表皮形成相关的基因,同时,在卵期高量表达的与细胞分裂相关的基因在蛹期的表达也明显活跃。成虫期与幼虫期相似,负责能量代谢和运动能力的基因表达活跃,此外成虫期与嗅觉和视觉有关的基因的表达丰度也较高。这些差异表达的基因反映了桔小实蝇各个虫态生命活动的特征,卵期和蛹期调控细胞分裂分化的基因表达活跃,是变态发育的关键时期,而幼虫期和成虫期与能量代谢、解毒代谢以及行动能力相关的基因表达活跃。
1.3消化、解毒代谢相关基因在不同发育阶段的表达分布
基因注释结果表明,多种编码消化酶和解毒代谢酶的基因在桔小实蝇不同发育阶段均有表达。消化酶中,基因数量最多的是胰蛋白酶,共发现15个编码胰蛋白酶的Unigene,这类基因的表达具有非常明显的发育阶段特异性,即主要集中在成虫期和幼虫期表达,并且大部分在幼虫期的表达量最高,但是几乎都不在卵期表达。另外氨基肽酶N和羧肽酶基因亦有类似的表达分布。解毒代谢酶中,基因数量最多的是P450s,共发现76个编码P450s的Unigene,成虫期和幼虫期是该类基因表达的高峰期,分别有62个Unigene在这两个时期表达。另外,还发现编码GSTs的Unigene23个,编码CCEs的Unigene17个,它们在幼虫期和成虫期的表达最为活跃,而在卵期的表达丰度都非常低。这些基因在不同虫态的表达分布说明消化和解毒代谢主要在幼虫期和成虫期进行。
2桔小实蝇中肠转录组分析
2.1桔小实蝇中肠转录组测序及Unigene注释概况
为明确中肠的基因转录本信息,以采自海南并在实验室建立的桔小实蝇种群为试虫,从幼虫中肠提取高质量RNA,在Illumina HiSeq2000上进行转录组测序。获得的数据提交至NCBI的SRA数据库,登录号为SRA056311。经测序共获得4755425400个核苷酸。使用Trinity软件将这些核苷酸组装成为53318个平均长度为379nt的contig,并运用paired-end joining和gap-filling技术将这些contig进一步拼接成为25236个Unigene。
将得到的Unigene提交NCBI蛋白质数据库(Nr)进行BLASTX比对,设阈值为E=10-5,25236个Unigene中,共有16531个返回有效结果。此外,还在其它数据库中进行了Unigene的注释工作,其中在Swiss-Prot数据库比对成功的Unigene为13026个,在KEGG数据库中比对成功的Unigene为11488个,在GO数据库中比对成功的Unigene为11575个,在Nt数据库中比对成功的Unigene为9825个,在COG数据库中比对成功的Unigene为5595个。综合以上所有数据库的比对结果,共有17308个Unigene得到成功注释。
2.2Unigene分布概况
在Nr数据库中成功注释的16531个Unigene中,E值小于10-45的占48.6%,另外51.4%的序列的E值在10-45到10-5之间。与数据库中的序列同源性在80%以上的Unigenen占24%。Unigene的同源性分布主要集中在果蝇属,在成功注释的序列中,75.74%的Unigene与果蝇属的相关基因同源性最高。其中,同源性最高的物种依次为Drosophila. virilis (11.03%),D. willistoni (10.32%)和D. mojavensis(9.86%)等。与其它双翅目昆虫相比较结果发现,桔小实蝇与刺舌蝇Glossina morsitans morsitans同源性较高(5.51%),其次分别为埃及伊蚊Aedes aegypti(1.03%)、致乏库蚊Culex quinquefasciatus (0.79%)和冈比亚按蚊Anopheles gambiae(0.62%)。仅有136个Unigene与果实蝇属的昆虫同源性最高,分别为橄榄实蝇B.oleae (68个)、木瓜实蝇B. papaya (7个)、昆士兰实蝇B. tryoni (6个)、瓜实蝇B.cucurbitae (5个)、以及菲律宾实蝇B. philippinensis (4个)。而与数据库中已登录的桔小实蝇B. dorsalis序列相吻合的Unigene仅有46个,这说明目前数据库中的桔小实蝇基因信息还有很大的不足。
2.3Unigene的GO以及COG分类
对Unigene进行GO功能分类发现,桔小实蝇中肠转录组数据库中共有11575个Unigene在GO分类中被划分到61个功能组中,这些组分别属于"biological process"、"cellular component"和"molecular function"3个大类。在"biological process"中,包含Unigene较多的是"cellular process","metabolic process","multicellular organismal process"以及‘'biological regulation",有多达4000余个Unigene参与了这些生命活动。"biological process"中最小的组为‘'carbon utilization",仅仅包含2个Unigene o在“cellular component"类群里,“cell"和“cell part"是最大的两个组,包含了超过6000个Unigene o在‘" molecular function "中,最大的功能组‘" binding "包含了6845个Unigene。
同时对Unigene进行了COG功能分类,结果表明该分类共注释了5595个Unigene o其中,2130个Unigene属于‘'General function prediction",另外,1173个Unigene属于"Transcription ",926个Unigene属于‘'Translation, ribosomal structure and biogenesis"以及886个Unigene属于"Replication, recombination and repair"等类群。GO和COG分类的结果说明桔小实蝇体内绝大多数的基因都承担着与基础生命活动相关的功能,如参与细胞的组成,新陈代谢以及生物调控等。
3功能基因分析
3.1消化代谢酶相关基因分析
将Unigene提交Nr数据库,与近源物种的基因序列进行比对,从桔小实蝇中肠转录组数据中筛选鉴定出106个与消化代谢酶相关的Unigene,包括蛋白酶、酯酶以及糖酶等。其中,共发现6种蛋白酶,分别是氨基肽酶(22个)、胰蛋白酶(22个)、天冬氨酸蛋白酶(3个)、羧肽酶(7个)、糜蛋白酶(2个)以及半胱氨酸蛋白酶(3个)。通过同源性比对分析,这些序列与双翅目昆虫如黑腹果蝇D.melanogaster、刺舌蝇G. morsitans morsitans、致乏库蚊C. quinquefasciatus以及埃及伊蚊A. aegypti等具有较高的同源性。说明蛋白酶在桔小实蝇中肠的消化过程中起着非常重要的作用,其中胰蛋白酶和氨基肽酶是两种最活跃的蛋白酶。
3.2RNAi作用通路相关基因分析
中肠作为dsRNA进入昆虫体内的一个重要器官,其中与dsRNA吸收、剪切等功能相关基因的表达对RNAi的效果起着至关重要的影响。通过在Nr数据库中的比对,从中肠转录组数据中筛选出了RNAi作用通路上的一系列基因。包括将dsRNA剪切成siRNA的Dicer-2,与siRNA形成复合蛋白的R2D2以及AGO2。但是在桔小实蝇的中肠没有发现对dsRNA的吸收起着关键作用的基因如SID-1,SR-CI等。只发现了1个可能与dsRNA在中肠里的跨膜运输有关的基因Eater。桔小实蝇中肠dsRNA作用通路上的基因具有双翅目昆虫的特征,但同时也缺失了一些在果蝇中较为典型的基因,说明桔小实蝇中可能存在其它的替代基因。
3.3解毒代谢酶相关基因分析
通过将Unigene与Nr数据库中多种昆虫的解毒代谢酶基因进行比对,从桔小实蝇中肠转录本数据中鉴定出编码GSTs的Unigene9个,平均长度为793nt,其中8个包括完整的开放阅读框,新发现GSTs基因1个。这8个GSTs基因的开放阅读框平均长度为639nt,编码177～234个氨基酸。根据同源性分析,这些基因属于Delta家族的有4个,属于Theta、Zeta家族以及微粒体型的GSTs基因各1个,此外,还有1个基因暂未有明确的分类。
在桔小实蝇中肠转录组中编码CCEs的Unigene10个,包含完整开放阅读框的基因有6个,其中5个基因为新发现的CCEs。在中肠表达的CCEs基因开放阅读框平均长度为1695nt,编码545～574个氨基酸。同源性分析发现,这些基因均属于α酯酶,具有典型的"FGESAG"和"EDCLYLN"胆碱酯酶和催化三联体的标签序列。并且这些CCEs基因在酯酶中属于"dietary"类群,该类群与高效氯氰菊酯以及氰戊菊酯的代谢相关。
此外经比对,桔小实蝇中肠转录组中鉴定出编码P450s的Unigene22个,平均长度为1461nt,包含完整开放阅读框的基因有16个,其中新发现的P450s基因有8个。P450s基因开放阅读框平均长度为1556nt,编码489～542个氨基酸,均具有典型的K螺旋结构"ExxR"以及血红素结合位点"PFxxGxRxCxG/A"。同源性比对分析发现,这些基因分属CYP4、CYP6、CYP12、CYP317、CYP309和CYP9六个家族。其中基因最多的是CYP4和CYP6家族,分别包含了6和5个基因。
4药剂胁迫下解毒代谢酶和消化酶基因的应激反应模式
4.1高效氯氰菊酯对桔小实蝇幼虫的胁迫及其对看家基因稳定性的影响
以混入不同剂量高效氯氰菊酯的人工饲料将桔小实蝇幼虫从一龄饲喂至三龄,统计相对存活率。发现当高效氯氰菊酯的剂量为1μg/g(药剂/饲料)和33μg/g时,桔小实蝇幼虫存活率显著降低,仅为14.8%和10.8%。而当高效氯氰菊酯的剂量为0.3μg/g时,桔小实蝇幼虫的存活受到的影响相对较小,存活率约为88.5%。因此,本研究选择0.3μg/g这一对幼虫的存活有一定压力但是又不会引起大量死亡的剂量作为测试解毒代谢酶应激反应的胁迫压力。
运用geNormplus和NormFinder两种软件,对桔小实蝇9个看家基因18S、Actin、Ef-la、GAPDH、RPL13、α-Tubulin、β-Tubulin、SDHA以及RPE经高效氯氰菊酯胁迫后,在幼虫、中肠和脂肪体中的表达稳定性进行了评估。结果表明,在整个幼虫和中肠里,经药剂处理前后,表达最为稳定的看家基因是EFla;而脂肪体经药剂处理前后,表达最为稳定的看家基因是RPL13。据此分别以这两个基因作为后续定量表达分析中的内参基因。
4.2高效氯氰菊酯胁迫下桔小实蝇解毒代谢酶以及消化酶基因的应激表达模式
在内参基因筛选的基础上,以桔小实蝇EFla和RPL13基因分别作为桔小实蝇幼虫、中肠和脂肪体定量表达分析的内参基因,采用qPCR方法对3种主要解毒代谢酶在高效氯氰菊酯胁迫下的应激表达模式进行了解析。
桔小实蝇8个GSTs基因的定量分析结果表明,参与高效氯氰菊酯代谢的主要是Delta家族的4个基因,它们在中肠和脂肪体均出现了5.6～32.5倍的过量表达,而其它家族的GSTs经药剂胁迫后没有出现明显的应激反应。同时发现,Delta家族的GSTs基因虽然在中肠和脂肪体对药剂胁迫有积极的响应,但是它们的表达在整虫却没有明显的变化。说明此时其它组织器官中,GSTs的表达量呈下调的趋势。可以推测,桔小实蝇在应对药剂压力时,将能量转移集中到了重要的组织器官进行相关基因的表达。
分析6个CCEs基因的定量结果发现,药剂胁迫后,桔小实蝇CCEs基因在整头幼虫中出现一定程度的上调,其中α-E3基因的表达量上调了4.1倍。但是这些基因在中肠和脂肪体均没有出现上调的情况。结合GSTs的表达模式分析,推测部分CCEs基因参与了高效氯氰菊酯的代谢,但是CCEs基因的主要作用组织器官不是脂肪体和中肠,而可能是其它使用解毒代谢功能的器官,比如马氏管。
桔小实蝇16个P450s基因的定量分析结果表明,有多个P450s基因参与了高效氯氰菊酯的代谢,其中大部分属于CYP4和CYP6两个家族。CYP4家族中出现明显上调反应的主要有CYP4D47(中肠)、CYP4E9(幼虫、脂肪体)和CYP4P5(幼虫、脂肪体)。CYP4AD1的表达量在中肠和脂肪体均有一定程度的上调,而在整头幼虫没有变化。另外,CYP4S18和CYP4AC4没有出现应激表达或者表达量的变化不明显。CYP6家族中,CYP6A48和CYP6A41在脂肪体的表达量显著增加,分别提高了18.0和9.6倍,CYP6G6和CYP6A50在脂肪体的表达量也有一定程度的上升,而CYP6EK1的表达量虽然在整头幼虫提高了4.8倍,但是在中肠和脂肪体并没有明显的变化,推测该基因主要在其它组织如马氏管起作用。其它家族的P450,如CYP12C2,在脂肪体中出现了12.6倍的高表达,并且在幼虫和中肠的表达量也均提高了两倍。CYP9F6在脂肪体的表达量提高了5.4倍,CYP12N1,CYP309B1表达量在脂肪体也有两倍以上的变化,但是CYP317B1的表达量在药剂压力下并没有明显的变化。
除了解毒代谢酶,本研究还检测了药剂胁迫对桔小实蝇中肠消化酶基因表达的影响。结果发现,所检测的6种消化酶基因的表达量均出现了3倍以上的提高,特别是胰蛋白酶,其表达量提高了10.7倍,说明经药剂处理后,桔小实蝇幼虫中肠的消化代谢活动增强。
综上所述,本研究应用高通量测序技术完成了桔小实蝇不同发育阶段的基因表达谱检测以及幼虫中肠的转录组测序工作,通过对数据进行详尽的解析和注释,在分析各虫态差异表达基因的同时,明确了消化酶和解毒代谢酶基因在各虫态的表达分布,并重点筛选出了在中肠特异表达的与消化、RNAi作用通路以及解毒代谢相关的基因,阐释了这些基因的分子生物学特性。在此基础上,利用qPCR技术检测了主要的解毒代谢酶以及消化酶基因在药剂胁迫下的应激反应,鉴定出了参与高效氯氰菊酯代谢的主要基因,同时发现这些基因的表达存在明显的组织特异性。研究结果提供了桔小实蝇大量详实的基因信息,为从分子生物学角度深入研究中肠等组织器官的功能奠定了坚实的基础。
Digestion and detoxification are two important metabolic pathways in insects. The digestion provides enough energy for growth, development and reproduction, and detoxification can prevent damage from xenobiotics. During a long period, researches have been just focused on a few genes or enzymes. Without sufficient molecular information, it was hard to get a broad view of this part. At present, high throughput sequencing technology has become the best choice to screen out the gene information of the organism of interest without reference genome. This technology provides a short cut that can effectively and rapidly characterize the unique transcripts from individuals or specific tissues, and make great promotion to molecular studies.
This study focused on an important agriculture pest, the oriental fruit fly, Bactrocera dorsalis. Firstly, we compiled four digital gene expression (DGE) libraries to investigate the expression profiles of genes at different developmental stages (egg, third-instar larva, pupa, and adult). The differently expressed genes were annotated and the expression distribution of digestion and detoxification genes were determined. Larva was found an important stage for the expression of these genes. As the second largest organ in insects, the insect midgut is the major tissue for digestion of food and detoxification of xenobiotics, and the first barrier and target against exogenous toxin. The functions and related mechanisms of midgut are always deemed as hot points by researchers. Secondly, we performed a midgut-specific transcriptome analysis of this insect. The annotation of gene transcripts provided sufficient molecular information for the fruit fly database and was useful for studying the biochemical and physiological mechanisms at molecular level. Meanwhile, for the pest control, we screened out the genes involved in digestion, RNAi mechanism and detoxification, which were important functions of the midgut, and conducted sequence characterization and phylogenic analysis of these genes. It is meaningful for finding the potential insecticide target, understanding the mechanism of detoxification, or developing new ways for pest control via RNAi. Using the transcriptome data, we futhur examined the expression reaction of detoxification and digestion genes under the stress. Based on the reference evaluation, our qPCR data showed the expression model of these genes in the midgut after stimulated by β-Cypermethrin. Compared with the data from the whole body of larvae and fat body, tissue specific genes related to detoxification were identified, and revealed some energy distribution strategies of this insect when stimulated by insecticides.
In conclusion, this study forms a solid basis for the function study of insect digestion and detoxification at molecular level, and provids a insight view for the future studies in this aspect. The analysis of gene sequences related to digestion, RNAi mechanism and detoxification will give new ideas and directions for pest control, especially for the identification of a number of specific genes that were considered to be significant to resistance.
1Comparison of gene expression among different developmental stages of Bactrocera dorsalis
1.1Summary of DGE libraries
RNA of different development stages was extracted from the lab population, which were initially collected from Hai Nan province, China. Based on the previous transcriptome data of developmental stages, four DGE libraries were constructed by Illumina HiSeq2000to identify the Unigene expression profiles of the different developmental stages (accession numbers:SRA043792for egg, SRA043786for third-instar larva, SRA043785for pupa, and SRA043783for adult). After excluding low-quality reads, each library generated above five million clean reads, and the distribution of genes with coverage above50%was75%in egg,60%in larva,77%in pupa, and63%in adult. These results indicated high quality of DGE library sequencing.
1.2Differently expressed genes among developmental stages
The four developmental stages were evaluated in three pairwise comparisons:egg vs. third-instar larva (E vs. L), third-instar larva vs. pupa (L vs. P), and pupa vs. adults(P vs. A). Genes found to have significant differences in expression were identified in each comparison. The results suggested that the expression of15516genes was significantly different between egg and third-instar larva. Of these genes7352were up-regulated and8164were down-regulated in the E vs. L comparison. In the comparison of third-instar larva and pupa, the expression profiles of7581genes were changed. There were3786genes up-regulated in pupa and3795genes down-regulated. When comparing pupa and adult,3077genes were up-regulated in adults and7195genes were down-regulated.
By the gene function annotation, we found in egg, the highly expressed genes were involved in embryo development and cell mitosis regulation. In larva, the expressions of genes involved in energy metabolism and muscle composition were the most active. In pupae, genes encoding chitinase were highly expressed, which indicated that in this period, cuticle was remodeling, and meanwhile, the the expressions of genes related to cell mitosis regulation were also very active. The genes highly expressed in adults were similar to larva, most of them were related to energy metabolism and muscle composition, and some genes related to visual and auditory sense were specifically identified in this stage. The differently expressed genes in different developmental stages reflected the most important actions in each stage. In egg and pupa, genes related to cell mitosis regulation were highly expressed, wich indicates these two period were crucial for metamorphosis. In larva and adult, the highly expressed genes were involved in energy, detoxification and movability.
1.3Expression distribution of digestion and detoxification genes
The expression distribution of genes involved in digestion and detoxification were determined in different developmental stages. The most abundant digestion enzyme was trypsin.15seqences were found highly expressed in adult and larva, but most of them were not found in egg. The expression distribution of some other digestion enzymes were similar to that of trypsin, such as aminopeptidase N and carboxypeptidase.
The most abundant detoxification enzyme was P450s.76sequences were found expressed in different developmental stages. P450s were also enriched in adult and larva, and62sequences were expressed in these two stages, respectively. Besides,23sequences encoding GSTs and17sequences encoding CCEs were identified. They were also most active in larva, but not enriched in egg.
2The midgut-specific transcriptome analysis of B. dorsalis
2.1Data assembly and annotation of midgut transcripts
The insect was initially collected from Hai Nan province, China, and RNA was extracted from the midgut of larva. A cDNA library (SRA submission number: SRA056311) was constructed by Illumina HiSeq2000, and this generated52838060total clean reads and4755425400nucleotides. These reads with certain length of overlap were assembled to53318contigs with an average length of379nt via the Trinity program. Using paired-end joining and gap-filling, the contigs were further assembled to25236Unigenes.
To annotate Unigenes, sequences were searched in the nonredundant (Nr) NCBI protein database using BLASTX with a cutoff E-value of10-5. A total of16531distinct sequences returned a blast result, which meant that these Unigenes were successfully mapped to known function genes. Besides the Nr database, there were13026Unigenes successfully annotated in Swiss-Prot,11488in KEGG,11575in GO,9825in Nt, and5595in COG. Totally,17308Unigenes were annotated across these databases.
2.2Unigene distribution
Of the16531annotated Unigenes in Nr,48.6%had an E-value of10-45, showing a significant homology matching in the NCBI database, while51.4%had an E-value ranging from10-45to10-5. The similarity distribution showed that24%of the sequences had a significant homology higher than80%. Among the annotated Unigenes,5273sequences were longer than1000nt.4386sequences between500nt to1000nt were successfully annotated, and6873sequences shorter than500nt returned a blast result. According to the best hit at Nr database, the majority of Unigenes (75.74%) had strong homology with Drosophila. Of these,11.03%Unigenes were best matched to sequences from D. virilis, followed by D. willistoni (10.32%), D. mojavensis (9.86%), and other species of Drosophila.24.26%of Unigenes matched to other Diptera species, such as Glossina morsitans morsitans (5.51%), Aedes aegypti (1.03%), Culex quinquefasciatus (0.79%) and Anopheles gambiae (0.62%). Only136Unigenes had a best hit to Bactrocera, such as B. oleae (68Unigenes), B. papaya (7), B. tryoni (6), B. cucurbitae (5), and B. philippinensis (4). There were only46sequences matched the genes of B. dorsalis, which indicated that in the data base, the sequence imformation of B. dorsalis was still far from sufficiency.
2.3Classification of gene ontology (GO) and Clusters of orthologous groups (COG)
The result of GO classification showed that11575Unigenes (45.87%of total) were mapped to61functional groups. These functional groups were classified into three categories:nameliy "biological process","cellular component" and "molecular function". In the "biological process","cellular process" was the most abundant group, followed by "metabolic process","multicellular organismal process", and "biological regulation". More than4000Unigenes were identified to be involved in these processes. In contrast, only2Unigenes were classified in "carbon utilization", which was the smallest group in this category,"cell" and "cell part" were the two largest groups in "cellular component" with>6000Unigenes in each group. In "molecular function", "binding" was the largest group containing6854Unigenes, while there was only1Unigene in "receptor regulator activity".
5595sequences had a COG annotation. Among the COG categories, most Unigenes (2130) were mapped to the "General function prediction". The largest three groups presenting specific functions were "Transcription"(1173),"Translation, ribosomal structure and biogenesis"(926),"Replication, recombination and repair"(886).
3Gene function classification
3.1Analysis of genes involved in digestion
In the midgut-specific transcriptome data of this study, we identified106Unigenes encoding various proteases, lipases, and carbohydrases related to digestion by BLAST in Nr. Among the proteases, Unigenes were divided into6groups:namely trypsins (22Unigenes), aminopeptidases (22), chymotrypsins (2), cysteine proteases (3), aspartic proteases (3), and carboxypeptidases (7). These sequences shared homology with aminopeptidases from Drosophila, C. quinquefasciatus, A. aegypti and G. morsitans morsitans.4sequences were characterized as aminopeptidases N, which were the target for Bt. The identification of these sequences will provide useful information for control and resistance management of B. dorsalis at molecular level.
3.2Transcripts encoding genes involved in RNAi mechanisms
Midgut is an important entrance for dsRNA. The uptake and cleavage of dsRNA are key factors for RNAi. To ensure the RNAi working in B. dorsalis, we characterized the genes involved in RNAi mechanisms. Four kinds of genes with great significance to RNAi were classified, such as Dicer-2i, that can cut dsRNA fragments into short nucleotides; R2D2and AGO2, that were important for RNA-initiated silencing complex (RISC). However, no SID-1orthologs were found in Diptera insects, as well as B. dorsalis, and we could not find a sequence encoding SR-CI. In addition, Eater and another scavenger receptor was classified. Further study can be focused on the RNAi mechanism and pest management via RNAi.
3.3Midgut specific sequences encoding detoxification genes
By BLAST in Nr,9sequences encoding GSTs were identified from the annotation results of the midgut transcriptome data of B. dorsalis, and8of them were found to represent full length of ORF. It was confirmed by ORF finding and protein BLAST at NCBI. The average length of ORF was639nt, ranging from531to702nt. Based on the BLAST results and phylogenetic analysis, these sequences were divided into different GSTs families. We found4delta genes and1gene in each of the theta, zeta family and a microsomal GST, and there was1unclassified gene (U).
In the transcriptome data,10sequences were found encoding CCEs, and6of these were fully sequenced, containing the complete ORF. ORF finder analysis and protein BLAST results at NCBI showed that the average length of CCEs ORF was1695nt, ranging from1635to1722nt. Further phylogenetic analysis with esterase genes from other insect species demonstrated that these6esterase genes were all a-esterase, and the highly conserved motif "FGESAG" and "EDCLYLN" of cholinesterases and the catalytic triad were found in all amino acid sequences. All of these genes were classified to the dietary class. Hence, it is known that the genes of this class are also involved in detoxification and xenobiotic resisatance.
22sequences encoding P450s were isolated, and16of these were fully sequenced with the complete ORF. The average length of fully sequenced ORFs was1556nt, ranging from1467to1626. In the deduced amino acid sequences of these P450genes, the typical conserved P450motifs were identified, including the absolutely conserved ExxR motif found in the Helix-K (hydrogen bonding), and the heme-binding domain (PFxxGxRxCxG/A). Phylogenetic analysis identified6families:6genes in the CYP4family,5genes in the CYP6family,2genes in the CYP12family, and1gene in the CYP317, CYP309and CYP9family each.
4The expression model of detoxification and digestion genes when stimulated by insecticide
4.2Effect of P-Cypermethrin exposure on larval survival and the stability of housekeeping genes
To determine the appropriate concentration of β-Cypermethrin against larvae in the exposure experiment, different doses of β-Cypermethrin were mixed into the diet that was fed to the larvae. The survive rate and the relative mortality were normalized by a control group.0.33μg/g (P-Cypermethrin/artificial diet) was determined as a suitable concentration to stimulate the larvae. Such a concentration would not cause apparent death as1μg/g and33μg/g, and most individuals could normally develop under the toxicity stress.
To ensure the accuracy of qPCR results, we firstly evaluated the expression stability of housekeeping genes under the experimental conditions. GeNormplus and Normfinder were used to estimate the stability of9housekeeping genes in the whole body of larvae, midgut and fat body under toxicity stress. EFla was found the best one for both larvae and midgut. In fat body, the most stable housekeeping gene was RPL13. Thus, these two genes were used as reference gene in qPCR study, respectively.
4.2The expression model of detoxification and digestion genes when stimulated by insecticide
Based on the reference evaluation, we analyzed the expression model of detoxification genes under toxicity stress. The qPCR results of8GSTs genes showed the genes from Delta family were involved in the detoxification, but other genes did not show very active reaction. The expressions of Delta genes were up-regulated5.6～32.5fold in the midgut and fat body. But in larvae, the expression changes of these genes were not so apparent, which indicated that their expression in other tissues were down-regulated. The results revealed some energy distribution strategy of this insect under survival stress. The energy was focused to important tissues when stimulated by insecticide to expression detocification genes.
In larvae, the expression of6CCEs genes was up-regulated in some degree, and a-E3was up-regulated4.1fold. But in the midgut and fat body, the expressions of most CCEs genes were not significantly changed. We assume that the up-regulation in larvae showed the relation between the CCEs and detoxcification of β-Cypermethrin. This kind of gene did not work in the midgut or fat body, but in other tissues, such as Malpighian tubes.
The qPCR results of16P450genes showed that most P450s genes were involved in the detoxification of β-cypermethrin, especially the CYP4and CYP6family. In CYP4family, the expression of CYP4D47(midgut), CYP4E9(larvae and fat body), CYP4P5(Larvae and fat body) was highly up-regulated. The expression of CYP4AD1increased in some degree in the midgut and fat body, but no significant changes were found in larvae. CYP4S18and CYP4AC4did not show any positive reaction when stimulated. In CYP6family, the expression of CYP6A48and CYP6A41has a significant increase in the fat body,18.0and9.6fold up-regulated, respectively. The expression of others like CYP6G6and CYP6A50also increased but not highly. The expression of CYP6EK1was up-regulated by4.8fold, but showed no apparent changes in midgut and fat body, which indicated that it might work in Malpighian tubes. It suggested that this gene play a role in metabolic of β-cypermethrin, and worked in fatbody. CYP12C2was12.6fold up-regulated in fat body, and2fold upregulated in larvae and midgut. Other genes, such as CYP12N1, CYP9F6, and CYP309B1also returned a positive reaction to stimulation in the fat body. But no significant expression of CYP317B1was detected under the stress.
We also detected the expression of6digestion genes. They all expressed highly after stimulated, more than3fold up-regulated. The increasing of trypsin was up to10.7fold. The results showed that under toxicity stress, the digestion of midgut was improved.
In conclusion, this study conducted DGE analysis of different developmental stages and transcriptome analysis of midgut from the larvae of B. dorsalis via high throughput sequencing technology. Through data analysis and annotation, we characterized the differently expressed genes, and showed the expression distribution of detoxification and digestion genes in developmental stages. From the transcriptome data, we screened out the genes related to digestion, RNAi mechanism, and detoxification, and characterized their molecular information. The expression models of detoxification and digestion genes under toxicity stress were further detected. The results revealed some energy distribution strategy of this insect when stimulated by insecticide. Our data provides sufficient gene information of B. dorsalis, and will promote the function study of midgut at molecular level.
本学位论文以果树重要害虫桔小实蝇为研究对象,利用高通量测序技术,对其不同发育阶段的基因表达谱进行了检测,在发掘不同发育阶段差异表达基因的同时,明确了桔小实蝇体内主要消化酶和解毒代谢酶在不同虫态的表达分布情况。中肠是昆虫体内的第二大器官,是消化食物和吸收营养的主要场所,也是进行解毒代谢、阻隔外源毒素的一大屏障,因此进一步利用转录组测序技术在整体水平上解读了桔小实蝇幼虫中肠转录本信息,对功能基因进行了注释,为相关分子生物学研究的开展提供了充实的数据。并且从害虫防治的角度出发,围绕中肠的主要功能,筛选鉴定出了与食物消化、有毒物质代谢以及dsRNA作用通路上相关的基因,解析了它们的序列特征和系统进化关系。进而根据得到的丰富的基因信息,在内参基因筛选的基础上,利用qPCR技术系统开展了桔小实蝇解毒代谢酶基因(CCEs, P450s和GSTs)和消化酶基因在高效氯氰菊酯胁迫下的应激表达模式研究。研究发现桔小实蝇应对药剂胁迫时可能存在组织间的能量再分配,即通过组织间能量的转移和集中,高效表达解毒代谢酶基因的应对机制。本研究的主要结果如下：
1桔小实蝇不同发育阶段基因表达谱分析
1.1不同发育阶段基因表达谱测序概况
以采自海南并在实验室建立的桔小实蝇种群为试虫,分别从卵、幼虫、蛹以及成虫体内提取高质量RNA,在Illumina HiSeq2000上进行基因表达谱测序。获得的数据提交至NCBI的SRA数据库,登录号分别为SRA043792, SRA043786,SRA043785以及SRA043783。经过对数据的统计分析发现,4个样品的reads数均在5百万以上,且所含有的低质量reads、不确定碱基以及接头序列的比例都低于1%,以数据库中已公布的桔小实蝇不同发育阶段转录组数据为参考,对4个发育阶段的基因表达谱序列进行比对后,有超过65%的序列的覆盖度达到50%以上,说明表达谱测序的质量较高。
1.2不同发育阶段差异表达的基因
按卵期和幼虫期、幼虫期和蛹期、蛹期和成虫期的分组对4个基因表达谱进行两两对比,统计桔小实蝇不同发育阶段差异表达的基因数量发现,卵期和幼虫期之间差异表达的基因数量最多。与卵期相比,幼虫期分别有7352和8164个基因的表达存在上调和下调的现象。蛹期和幼虫期相比,共有7581个基因在表达丰度上存在显著差异,在蛹期表达上调的基因数量为3786个,表达下调的基因数量为3795个。蛹期与成虫期相比,在表达丰度上存在显著差异的基因有10272个,其中在成虫期表达上调的基因数(3077)远少于表达下调的基因数(7195)。通过对各个发育阶段差异表达显著的基因进行功能分析发现,在卵期表达丰度较高的基因主要涉及胚胎发育和细胞分裂,而幼虫期则主要参与能量代谢以及肌肉形成,在蛹期表达丰度高的是与表皮形成相关的基因,同时,在卵期高量表达的与细胞分裂相关的基因在蛹期的表达也明显活跃。成虫期与幼虫期相似,负责能量代谢和运动能力的基因表达活跃,此外成虫期与嗅觉和视觉有关的基因的表达丰度也较高。这些差异表达的基因反映了桔小实蝇各个虫态生命活动的特征,卵期和蛹期调控细胞分裂分化的基因表达活跃,是变态发育的关键时期,而幼虫期和成虫期与能量代谢、解毒代谢以及行动能力相关的基因表达活跃。
1.3消化、解毒代谢相关基因在不同发育阶段的表达分布
基因注释结果表明,多种编码消化酶和解毒代谢酶的基因在桔小实蝇不同发育阶段均有表达。消化酶中,基因数量最多的是胰蛋白酶,共发现15个编码胰蛋白酶的Unigene,这类基因的表达具有非常明显的发育阶段特异性,即主要集中在成虫期和幼虫期表达,并且大部分在幼虫期的表达量最高,但是几乎都不在卵期表达。另外氨基肽酶N和羧肽酶基因亦有类似的表达分布。解毒代谢酶中,基因数量最多的是P450s,共发现76个编码P450s的Unigene,成虫期和幼虫期是该类基因表达的高峰期,分别有62个Unigene在这两个时期表达。另外,还发现编码GSTs的Unigene23个,编码CCEs的Unigene17个,它们在幼虫期和成虫期的表达最为活跃,而在卵期的表达丰度都非常低。这些基因在不同虫态的表达分布说明消化和解毒代谢主要在幼虫期和成虫期进行。
2桔小实蝇中肠转录组分析
2.1桔小实蝇中肠转录组测序及Unigene注释概况
为明确中肠的基因转录本信息,以采自海南并在实验室建立的桔小实蝇种群为试虫,从幼虫中肠提取高质量RNA,在Illumina HiSeq2000上进行转录组测序。获得的数据提交至NCBI的SRA数据库,登录号为SRA056311。经测序共获得4755425400个核苷酸。使用Trinity软件将这些核苷酸组装成为53318个平均长度为379nt的contig,并运用paired-end joining和gap-filling技术将这些contig进一步拼接成为25236个Unigene。
将得到的Unigene提交NCBI蛋白质数据库(Nr)进行BLASTX比对,设阈值为E=10-5,25236个Unigene中,共有16531个返回有效结果。此外,还在其它数据库中进行了Unigene的注释工作,其中在Swiss-Prot数据库比对成功的Unigene为13026个,在KEGG数据库中比对成功的Unigene为11488个,在GO数据库中比对成功的Unigene为11575个,在Nt数据库中比对成功的Unigene为9825个,在COG数据库中比对成功的Unigene为5595个。综合以上所有数据库的比对结果,共有17308个Unigene得到成功注释。
2.2Unigene分布概况
在Nr数据库中成功注释的16531个Unigene中,E值小于10-45的占48.6%,另外51.4%的序列的E值在10-45到10-5之间。与数据库中的序列同源性在80%以上的Unigenen占24%。Unigene的同源性分布主要集中在果蝇属,在成功注释的序列中,75.74%的Unigene与果蝇属的相关基因同源性最高。其中,同源性最高的物种依次为Drosophila. virilis (11.03%),D. willistoni (10.32%)和D. mojavensis(9.86%)等。与其它双翅目昆虫相比较结果发现,桔小实蝇与刺舌蝇Glossina morsitans morsitans同源性较高(5.51%),其次分别为埃及伊蚊Aedes aegypti(1.03%)、致乏库蚊Culex quinquefasciatus (0.79%)和冈比亚按蚊Anopheles gambiae(0.62%)。仅有136个Unigene与果实蝇属的昆虫同源性最高,分别为橄榄实蝇B.oleae (68个)、木瓜实蝇B. papaya (7个)、昆士兰实蝇B. tryoni (6个)、瓜实蝇B.cucurbitae (5个)、以及菲律宾实蝇B. philippinensis (4个)。而与数据库中已登录的桔小实蝇B. dorsalis序列相吻合的Unigene仅有46个,这说明目前数据库中的桔小实蝇基因信息还有很大的不足。
2.3Unigene的GO以及COG分类
对Unigene进行GO功能分类发现,桔小实蝇中肠转录组数据库中共有11575个Unigene在GO分类中被划分到61个功能组中,这些组分别属于"biological process"、"cellular component"和"molecular function"3个大类。在"biological process"中,包含Unigene较多的是"cellular process","metabolic process","multicellular organismal process"以及‘'biological regulation",有多达4000余个Unigene参与了这些生命活动。"biological process"中最小的组为‘'carbon utilization",仅仅包含2个Unigene o在“cellular component"类群里,“cell"和“cell part"是最大的两个组,包含了超过6000个Unigene o在‘" molecular function "中,最大的功能组‘" binding "包含了6845个Unigene。
同时对Unigene进行了COG功能分类,结果表明该分类共注释了5595个Unigene o其中,2130个Unigene属于‘'General function prediction",另外,1173个Unigene属于"Transcription ",926个Unigene属于‘'Translation, ribosomal structure and biogenesis"以及886个Unigene属于"Replication, recombination and repair"等类群。GO和COG分类的结果说明桔小实蝇体内绝大多数的基因都承担着与基础生命活动相关的功能,如参与细胞的组成,新陈代谢以及生物调控等。
3功能基因分析
3.1消化代谢酶相关基因分析
将Unigene提交Nr数据库,与近源物种的基因序列进行比对,从桔小实蝇中肠转录组数据中筛选鉴定出106个与消化代谢酶相关的Unigene,包括蛋白酶、酯酶以及糖酶等。其中,共发现6种蛋白酶,分别是氨基肽酶(22个)、胰蛋白酶(22个)、天冬氨酸蛋白酶(3个)、羧肽酶(7个)、糜蛋白酶(2个)以及半胱氨酸蛋白酶(3个)。通过同源性比对分析,这些序列与双翅目昆虫如黑腹果蝇D.melanogaster、刺舌蝇G. morsitans morsitans、致乏库蚊C. quinquefasciatus以及埃及伊蚊A. aegypti等具有较高的同源性。说明蛋白酶在桔小实蝇中肠的消化过程中起着非常重要的作用,其中胰蛋白酶和氨基肽酶是两种最活跃的蛋白酶。
3.2RNAi作用通路相关基因分析
中肠作为dsRNA进入昆虫体内的一个重要器官,其中与dsRNA吸收、剪切等功能相关基因的表达对RNAi的效果起着至关重要的影响。通过在Nr数据库中的比对,从中肠转录组数据中筛选出了RNAi作用通路上的一系列基因。包括将dsRNA剪切成siRNA的Dicer-2,与siRNA形成复合蛋白的R2D2以及AGO2。但是在桔小实蝇的中肠没有发现对dsRNA的吸收起着关键作用的基因如SID-1,SR-CI等。只发现了1个可能与dsRNA在中肠里的跨膜运输有关的基因Eater。桔小实蝇中肠dsRNA作用通路上的基因具有双翅目昆虫的特征,但同时也缺失了一些在果蝇中较为典型的基因,说明桔小实蝇中可能存在其它的替代基因。
3.3解毒代谢酶相关基因分析
通过将Unigene与Nr数据库中多种昆虫的解毒代谢酶基因进行比对,从桔小实蝇中肠转录本数据中鉴定出编码GSTs的Unigene9个,平均长度为793nt,其中8个包括完整的开放阅读框,新发现GSTs基因1个。这8个GSTs基因的开放阅读框平均长度为639nt,编码177～234个氨基酸。根据同源性分析,这些基因属于Delta家族的有4个,属于Theta、Zeta家族以及微粒体型的GSTs基因各1个,此外,还有1个基因暂未有明确的分类。
在桔小实蝇中肠转录组中编码CCEs的Unigene10个,包含完整开放阅读框的基因有6个,其中5个基因为新发现的CCEs。在中肠表达的CCEs基因开放阅读框平均长度为1695nt,编码545～574个氨基酸。同源性分析发现,这些基因均属于α酯酶,具有典型的"FGESAG"和"EDCLYLN"胆碱酯酶和催化三联体的标签序列。并且这些CCEs基因在酯酶中属于"dietary"类群,该类群与高效氯氰菊酯以及氰戊菊酯的代谢相关。
此外经比对,桔小实蝇中肠转录组中鉴定出编码P450s的Unigene22个,平均长度为1461nt,包含完整开放阅读框的基因有16个,其中新发现的P450s基因有8个。P450s基因开放阅读框平均长度为1556nt,编码489～542个氨基酸,均具有典型的K螺旋结构"ExxR"以及血红素结合位点"PFxxGxRxCxG/A"。同源性比对分析发现,这些基因分属CYP4、CYP6、CYP12、CYP317、CYP309和CYP9六个家族。其中基因最多的是CYP4和CYP6家族,分别包含了6和5个基因。
4药剂胁迫下解毒代谢酶和消化酶基因的应激反应模式
4.1高效氯氰菊酯对桔小实蝇幼虫的胁迫及其对看家基因稳定性的影响
以混入不同剂量高效氯氰菊酯的人工饲料将桔小实蝇幼虫从一龄饲喂至三龄,统计相对存活率。发现当高效氯氰菊酯的剂量为1μg/g(药剂/饲料)和33μg/g时,桔小实蝇幼虫存活率显著降低,仅为14.8%和10.8%。而当高效氯氰菊酯的剂量为0.3μg/g时,桔小实蝇幼虫的存活受到的影响相对较小,存活率约为88.5%。因此,本研究选择0.3μg/g这一对幼虫的存活有一定压力但是又不会引起大量死亡的剂量作为测试解毒代谢酶应激反应的胁迫压力。
运用geNormplus和NormFinder两种软件,对桔小实蝇9个看家基因18S、Actin、Ef-la、GAPDH、RPL13、α-Tubulin、β-Tubulin、SDHA以及RPE经高效氯氰菊酯胁迫后,在幼虫、中肠和脂肪体中的表达稳定性进行了评估。结果表明,在整个幼虫和中肠里,经药剂处理前后,表达最为稳定的看家基因是EFla;而脂肪体经药剂处理前后,表达最为稳定的看家基因是RPL13。据此分别以这两个基因作为后续定量表达分析中的内参基因。
4.2高效氯氰菊酯胁迫下桔小实蝇解毒代谢酶以及消化酶基因的应激表达模式
在内参基因筛选的基础上,以桔小实蝇EFla和RPL13基因分别作为桔小实蝇幼虫、中肠和脂肪体定量表达分析的内参基因,采用qPCR方法对3种主要解毒代谢酶在高效氯氰菊酯胁迫下的应激表达模式进行了解析。
桔小实蝇8个GSTs基因的定量分析结果表明,参与高效氯氰菊酯代谢的主要是Delta家族的4个基因,它们在中肠和脂肪体均出现了5.6～32.5倍的过量表达,而其它家族的GSTs经药剂胁迫后没有出现明显的应激反应。同时发现,Delta家族的GSTs基因虽然在中肠和脂肪体对药剂胁迫有积极的响应,但是它们的表达在整虫却没有明显的变化。说明此时其它组织器官中,GSTs的表达量呈下调的趋势。可以推测,桔小实蝇在应对药剂压力时,将能量转移集中到了重要的组织器官进行相关基因的表达。
分析6个CCEs基因的定量结果发现,药剂胁迫后,桔小实蝇CCEs基因在整头幼虫中出现一定程度的上调,其中α-E3基因的表达量上调了4.1倍。但是这些基因在中肠和脂肪体均没有出现上调的情况。结合GSTs的表达模式分析,推测部分CCEs基因参与了高效氯氰菊酯的代谢,但是CCEs基因的主要作用组织器官不是脂肪体和中肠,而可能是其它使用解毒代谢功能的器官,比如马氏管。
桔小实蝇16个P450s基因的定量分析结果表明,有多个P450s基因参与了高效氯氰菊酯的代谢,其中大部分属于CYP4和CYP6两个家族。CYP4家族中出现明显上调反应的主要有CYP4D47(中肠)、CYP4E9(幼虫、脂肪体)和CYP4P5(幼虫、脂肪体)。CYP4AD1的表达量在中肠和脂肪体均有一定程度的上调,而在整头幼虫没有变化。另外,CYP4S18和CYP4AC4没有出现应激表达或者表达量的变化不明显。CYP6家族中,CYP6A48和CYP6A41在脂肪体的表达量显著增加,分别提高了18.0和9.6倍,CYP6G6和CYP6A50在脂肪体的表达量也有一定程度的上升,而CYP6EK1的表达量虽然在整头幼虫提高了4.8倍,但是在中肠和脂肪体并没有明显的变化,推测该基因主要在其它组织如马氏管起作用。其它家族的P450,如CYP12C2,在脂肪体中出现了12.6倍的高表达,并且在幼虫和中肠的表达量也均提高了两倍。CYP9F6在脂肪体的表达量提高了5.4倍,CYP12N1,CYP309B1表达量在脂肪体也有两倍以上的变化,但是CYP317B1的表达量在药剂压力下并没有明显的变化。
除了解毒代谢酶,本研究还检测了药剂胁迫对桔小实蝇中肠消化酶基因表达的影响。结果发现,所检测的6种消化酶基因的表达量均出现了3倍以上的提高,特别是胰蛋白酶,其表达量提高了10.7倍,说明经药剂处理后,桔小实蝇幼虫中肠的消化代谢活动增强。
综上所述,本研究应用高通量测序技术完成了桔小实蝇不同发育阶段的基因表达谱检测以及幼虫中肠的转录组测序工作,通过对数据进行详尽的解析和注释,在分析各虫态差异表达基因的同时,明确了消化酶和解毒代谢酶基因在各虫态的表达分布,并重点筛选出了在中肠特异表达的与消化、RNAi作用通路以及解毒代谢相关的基因,阐释了这些基因的分子生物学特性。在此基础上,利用qPCR技术检测了主要的解毒代谢酶以及消化酶基因在药剂胁迫下的应激反应,鉴定出了参与高效氯氰菊酯代谢的主要基因,同时发现这些基因的表达存在明显的组织特异性。研究结果提供了桔小实蝇大量详实的基因信息,为从分子生物学角度深入研究中肠等组织器官的功能奠定了坚实的基础。
Digestion and detoxification are two important metabolic pathways in insects. The digestion provides enough energy for growth, development and reproduction, and detoxification can prevent damage from xenobiotics. During a long period, researches have been just focused on a few genes or enzymes. Without sufficient molecular information, it was hard to get a broad view of this part. At present, high throughput sequencing technology has become the best choice to screen out the gene information of the organism of interest without reference genome. This technology provides a short cut that can effectively and rapidly characterize the unique transcripts from individuals or specific tissues, and make great promotion to molecular studies.
This study focused on an important agriculture pest, the oriental fruit fly, Bactrocera dorsalis. Firstly, we compiled four digital gene expression (DGE) libraries to investigate the expression profiles of genes at different developmental stages (egg, third-instar larva, pupa, and adult). The differently expressed genes were annotated and the expression distribution of digestion and detoxification genes were determined. Larva was found an important stage for the expression of these genes. As the second largest organ in insects, the insect midgut is the major tissue for digestion of food and detoxification of xenobiotics, and the first barrier and target against exogenous toxin. The functions and related mechanisms of midgut are always deemed as hot points by researchers. Secondly, we performed a midgut-specific transcriptome analysis of this insect. The annotation of gene transcripts provided sufficient molecular information for the fruit fly database and was useful for studying the biochemical and physiological mechanisms at molecular level. Meanwhile, for the pest control, we screened out the genes involved in digestion, RNAi mechanism and detoxification, which were important functions of the midgut, and conducted sequence characterization and phylogenic analysis of these genes. It is meaningful for finding the potential insecticide target, understanding the mechanism of detoxification, or developing new ways for pest control via RNAi. Using the transcriptome data, we futhur examined the expression reaction of detoxification and digestion genes under the stress. Based on the reference evaluation, our qPCR data showed the expression model of these genes in the midgut after stimulated by β-Cypermethrin. Compared with the data from the whole body of larvae and fat body, tissue specific genes related to detoxification were identified, and revealed some energy distribution strategies of this insect when stimulated by insecticides.
In conclusion, this study forms a solid basis for the function study of insect digestion and detoxification at molecular level, and provids a insight view for the future studies in this aspect. The analysis of gene sequences related to digestion, RNAi mechanism and detoxification will give new ideas and directions for pest control, especially for the identification of a number of specific genes that were considered to be significant to resistance.
1Comparison of gene expression among different developmental stages of Bactrocera dorsalis
1.1Summary of DGE libraries
RNA of different development stages was extracted from the lab population, which were initially collected from Hai Nan province, China. Based on the previous transcriptome data of developmental stages, four DGE libraries were constructed by Illumina HiSeq2000to identify the Unigene expression profiles of the different developmental stages (accession numbers:SRA043792for egg, SRA043786for third-instar larva, SRA043785for pupa, and SRA043783for adult). After excluding low-quality reads, each library generated above five million clean reads, and the distribution of genes with coverage above50%was75%in egg,60%in larva,77%in pupa, and63%in adult. These results indicated high quality of DGE library sequencing.
1.2Differently expressed genes among developmental stages
The four developmental stages were evaluated in three pairwise comparisons:egg vs. third-instar larva (E vs. L), third-instar larva vs. pupa (L vs. P), and pupa vs. adults(P vs. A). Genes found to have significant differences in expression were identified in each comparison. The results suggested that the expression of15516genes was significantly different between egg and third-instar larva. Of these genes7352were up-regulated and8164were down-regulated in the E vs. L comparison. In the comparison of third-instar larva and pupa, the expression profiles of7581genes were changed. There were3786genes up-regulated in pupa and3795genes down-regulated. When comparing pupa and adult,3077genes were up-regulated in adults and7195genes were down-regulated.
By the gene function annotation, we found in egg, the highly expressed genes were involved in embryo development and cell mitosis regulation. In larva, the expressions of genes involved in energy metabolism and muscle composition were the most active. In pupae, genes encoding chitinase were highly expressed, which indicated that in this period, cuticle was remodeling, and meanwhile, the the expressions of genes related to cell mitosis regulation were also very active. The genes highly expressed in adults were similar to larva, most of them were related to energy metabolism and muscle composition, and some genes related to visual and auditory sense were specifically identified in this stage. The differently expressed genes in different developmental stages reflected the most important actions in each stage. In egg and pupa, genes related to cell mitosis regulation were highly expressed, wich indicates these two period were crucial for metamorphosis. In larva and adult, the highly expressed genes were involved in energy, detoxification and movability.
1.3Expression distribution of digestion and detoxification genes
The expression distribution of genes involved in digestion and detoxification were determined in different developmental stages. The most abundant digestion enzyme was trypsin.15seqences were found highly expressed in adult and larva, but most of them were not found in egg. The expression distribution of some other digestion enzymes were similar to that of trypsin, such as aminopeptidase N and carboxypeptidase.
The most abundant detoxification enzyme was P450s.76sequences were found expressed in different developmental stages. P450s were also enriched in adult and larva, and62sequences were expressed in these two stages, respectively. Besides,23sequences encoding GSTs and17sequences encoding CCEs were identified. They were also most active in larva, but not enriched in egg.
2The midgut-specific transcriptome analysis of B. dorsalis
2.1Data assembly and annotation of midgut transcripts
The insect was initially collected from Hai Nan province, China, and RNA was extracted from the midgut of larva. A cDNA library (SRA submission number: SRA056311) was constructed by Illumina HiSeq2000, and this generated52838060total clean reads and4755425400nucleotides. These reads with certain length of overlap were assembled to53318contigs with an average length of379nt via the Trinity program. Using paired-end joining and gap-filling, the contigs were further assembled to25236Unigenes.
To annotate Unigenes, sequences were searched in the nonredundant (Nr) NCBI protein database using BLASTX with a cutoff E-value of10-5. A total of16531distinct sequences returned a blast result, which meant that these Unigenes were successfully mapped to known function genes. Besides the Nr database, there were13026Unigenes successfully annotated in Swiss-Prot,11488in KEGG,11575in GO,9825in Nt, and5595in COG. Totally,17308Unigenes were annotated across these databases.
2.2Unigene distribution
Of the16531annotated Unigenes in Nr,48.6%had an E-value of10-45, showing a significant homology matching in the NCBI database, while51.4%had an E-value ranging from10-45to10-5. The similarity distribution showed that24%of the sequences had a significant homology higher than80%. Among the annotated Unigenes,5273sequences were longer than1000nt.4386sequences between500nt to1000nt were successfully annotated, and6873sequences shorter than500nt returned a blast result. According to the best hit at Nr database, the majority of Unigenes (75.74%) had strong homology with Drosophila. Of these,11.03%Unigenes were best matched to sequences from D. virilis, followed by D. willistoni (10.32%), D. mojavensis (9.86%), and other species of Drosophila.24.26%of Unigenes matched to other Diptera species, such as Glossina morsitans morsitans (5.51%), Aedes aegypti (1.03%), Culex quinquefasciatus (0.79%) and Anopheles gambiae (0.62%). Only136Unigenes had a best hit to Bactrocera, such as B. oleae (68Unigenes), B. papaya (7), B. tryoni (6), B. cucurbitae (5), and B. philippinensis (4). There were only46sequences matched the genes of B. dorsalis, which indicated that in the data base, the sequence imformation of B. dorsalis was still far from sufficiency.
2.3Classification of gene ontology (GO) and Clusters of orthologous groups (COG)
The result of GO classification showed that11575Unigenes (45.87%of total) were mapped to61functional groups. These functional groups were classified into three categories:nameliy "biological process","cellular component" and "molecular function". In the "biological process","cellular process" was the most abundant group, followed by "metabolic process","multicellular organismal process", and "biological regulation". More than4000Unigenes were identified to be involved in these processes. In contrast, only2Unigenes were classified in "carbon utilization", which was the smallest group in this category,"cell" and "cell part" were the two largest groups in "cellular component" with>6000Unigenes in each group. In "molecular function", "binding" was the largest group containing6854Unigenes, while there was only1Unigene in "receptor regulator activity".
5595sequences had a COG annotation. Among the COG categories, most Unigenes (2130) were mapped to the "General function prediction". The largest three groups presenting specific functions were "Transcription"(1173),"Translation, ribosomal structure and biogenesis"(926),"Replication, recombination and repair"(886).
3Gene function classification
3.1Analysis of genes involved in digestion
In the midgut-specific transcriptome data of this study, we identified106Unigenes encoding various proteases, lipases, and carbohydrases related to digestion by BLAST in Nr. Among the proteases, Unigenes were divided into6groups:namely trypsins (22Unigenes), aminopeptidases (22), chymotrypsins (2), cysteine proteases (3), aspartic proteases (3), and carboxypeptidases (7). These sequences shared homology with aminopeptidases from Drosophila, C. quinquefasciatus, A. aegypti and G. morsitans morsitans.4sequences were characterized as aminopeptidases N, which were the target for Bt. The identification of these sequences will provide useful information for control and resistance management of B. dorsalis at molecular level.
3.2Transcripts encoding genes involved in RNAi mechanisms
Midgut is an important entrance for dsRNA. The uptake and cleavage of dsRNA are key factors for RNAi. To ensure the RNAi working in B. dorsalis, we characterized the genes involved in RNAi mechanisms. Four kinds of genes with great significance to RNAi were classified, such as Dicer-2i, that can cut dsRNA fragments into short nucleotides; R2D2and AGO2, that were important for RNA-initiated silencing complex (RISC). However, no SID-1orthologs were found in Diptera insects, as well as B. dorsalis, and we could not find a sequence encoding SR-CI. In addition, Eater and another scavenger receptor was classified. Further study can be focused on the RNAi mechanism and pest management via RNAi.
3.3Midgut specific sequences encoding detoxification genes
By BLAST in Nr,9sequences encoding GSTs were identified from the annotation results of the midgut transcriptome data of B. dorsalis, and8of them were found to represent full length of ORF. It was confirmed by ORF finding and protein BLAST at NCBI. The average length of ORF was639nt, ranging from531to702nt. Based on the BLAST results and phylogenetic analysis, these sequences were divided into different GSTs families. We found4delta genes and1gene in each of the theta, zeta family and a microsomal GST, and there was1unclassified gene (U).
In the transcriptome data,10sequences were found encoding CCEs, and6of these were fully sequenced, containing the complete ORF. ORF finder analysis and protein BLAST results at NCBI showed that the average length of CCEs ORF was1695nt, ranging from1635to1722nt. Further phylogenetic analysis with esterase genes from other insect species demonstrated that these6esterase genes were all a-esterase, and the highly conserved motif "FGESAG" and "EDCLYLN" of cholinesterases and the catalytic triad were found in all amino acid sequences. All of these genes were classified to the dietary class. Hence, it is known that the genes of this class are also involved in detoxification and xenobiotic resisatance.
22sequences encoding P450s were isolated, and16of these were fully sequenced with the complete ORF. The average length of fully sequenced ORFs was1556nt, ranging from1467to1626. In the deduced amino acid sequences of these P450genes, the typical conserved P450motifs were identified, including the absolutely conserved ExxR motif found in the Helix-K (hydrogen bonding), and the heme-binding domain (PFxxGxRxCxG/A). Phylogenetic analysis identified6families:6genes in the CYP4family,5genes in the CYP6family,2genes in the CYP12family, and1gene in the CYP317, CYP309and CYP9family each.
4The expression model of detoxification and digestion genes when stimulated by insecticide
4.2Effect of P-Cypermethrin exposure on larval survival and the stability of housekeeping genes
To determine the appropriate concentration of β-Cypermethrin against larvae in the exposure experiment, different doses of β-Cypermethrin were mixed into the diet that was fed to the larvae. The survive rate and the relative mortality were normalized by a control group.0.33μg/g (P-Cypermethrin/artificial diet) was determined as a suitable concentration to stimulate the larvae. Such a concentration would not cause apparent death as1μg/g and33μg/g, and most individuals could normally develop under the toxicity stress.
To ensure the accuracy of qPCR results, we firstly evaluated the expression stability of housekeeping genes under the experimental conditions. GeNormplus and Normfinder were used to estimate the stability of9housekeeping genes in the whole body of larvae, midgut and fat body under toxicity stress. EFla was found the best one for both larvae and midgut. In fat body, the most stable housekeeping gene was RPL13. Thus, these two genes were used as reference gene in qPCR study, respectively.
4.2The expression model of detoxification and digestion genes when stimulated by insecticide
Based on the reference evaluation, we analyzed the expression model of detoxification genes under toxicity stress. The qPCR results of8GSTs genes showed the genes from Delta family were involved in the detoxification, but other genes did not show very active reaction. The expressions of Delta genes were up-regulated5.6～32.5fold in the midgut and fat body. But in larvae, the expression changes of these genes were not so apparent, which indicated that their expression in other tissues were down-regulated. The results revealed some energy distribution strategy of this insect under survival stress. The energy was focused to important tissues when stimulated by insecticide to expression detocification genes.
In larvae, the expression of6CCEs genes was up-regulated in some degree, and a-E3was up-regulated4.1fold. But in the midgut and fat body, the expressions of most CCEs genes were not significantly changed. We assume that the up-regulation in larvae showed the relation between the CCEs and detoxcification of β-Cypermethrin. This kind of gene did not work in the midgut or fat body, but in other tissues, such as Malpighian tubes.
The qPCR results of16P450genes showed that most P450s genes were involved in the detoxification of β-cypermethrin, especially the CYP4and CYP6family. In CYP4family, the expression of CYP4D47(midgut), CYP4E9(larvae and fat body), CYP4P5(Larvae and fat body) was highly up-regulated. The expression of CYP4AD1increased in some degree in the midgut and fat body, but no significant changes were found in larvae. CYP4S18and CYP4AC4did not show any positive reaction when stimulated. In CYP6family, the expression of CYP6A48and CYP6A41has a significant increase in the fat body,18.0and9.6fold up-regulated, respectively. The expression of others like CYP6G6and CYP6A50also increased but not highly. The expression of CYP6EK1was up-regulated by4.8fold, but showed no apparent changes in midgut and fat body, which indicated that it might work in Malpighian tubes. It suggested that this gene play a role in metabolic of β-cypermethrin, and worked in fatbody. CYP12C2was12.6fold up-regulated in fat body, and2fold upregulated in larvae and midgut. Other genes, such as CYP12N1, CYP9F6, and CYP309B1also returned a positive reaction to stimulation in the fat body. But no significant expression of CYP317B1was detected under the stress.
We also detected the expression of6digestion genes. They all expressed highly after stimulated, more than3fold up-regulated. The increasing of trypsin was up to10.7fold. The results showed that under toxicity stress, the digestion of midgut was improved.
In conclusion, this study conducted DGE analysis of different developmental stages and transcriptome analysis of midgut from the larvae of B. dorsalis via high throughput sequencing technology. Through data analysis and annotation, we characterized the differently expressed genes, and showed the expression distribution of detoxification and digestion genes in developmental stages. From the transcriptome data, we screened out the genes related to digestion, RNAi mechanism, and detoxification, and characterized their molecular information. The expression models of detoxification and digestion genes under toxicity stress were further detected. The results revealed some energy distribution strategy of this insect when stimulated by insecticide. Our data provides sufficient gene information of B. dorsalis, and will promote the function study of midgut at molecular level.
引文
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