【作者】 杨文佳；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 几丁质和保幼激素在昆虫发育和变态过程中扮演着关键角色。几丁质是昆虫表皮、气管和中肠围食膜的主要成分,昆虫的生长发育依赖于体内几丁质生物合成与代谢的精确控制；昆虫体内保幼激素的含量由保幼激素合成与代谢共同维持平衡,保幼激素直接影响昆虫的变态和发育。几丁质和保幼激素的合成与代谢途径成为昆虫发育研究的热点,也是探索害虫控制新策略的重要途径。桔小实蝇是一种重要的果蔬害虫,主要分布于热带和亚热带地区,寄主范围广,可以危害多达250种的水果和蔬菜。桔小实蝇成虫产卵于果实内,主要以幼虫潜居果瓤内取食,造成果实腐烂和落果。目前对桔小实蝇的控制主要依赖于化学杀虫剂,但是各地区相继报道了桔小实蝇对多种杀虫剂产生了抗性,因此需要寻找新的途径进行持续防控。本学位论文以桔小实蝇为研究对象,基于转录组数据库,对几丁质和保幼激素合成与代谢中重要基因的分子特性及功能开展了较为系统和深入的研究,主要结果如下：1.桔小实蝇几丁质合成酶1基因克隆、mRNA表达特性及功能分析采用RT-PCR结合RACE技术,克隆获得了桔小实蝇几丁质合成酶1(Chitin synthase1, CHS1)基因的全长序列,并发现该基因存在选择性剪切现象,包括2个可变外显子,分别命名为BdCHSla和BdCHSlb。两个基因的cDNA序列全长均为5552bp,包含5’非编码区域685bp和3’非编码区88bp,开放阅读框4776bp,编码1592个氨基酸残基。两个CHS1基因仅在编码区(3784-3960bp)内的一个外显子的核苷酸序列存在差异,包含177bp,编码59个氨基酸残基,且核苷酸水平上的一致性为65%。在桔小实蝇生长发育过程中,BdCHSl和BdCHSla在幼虫-蛹和蛹-成虫的转化过程中表达量明显高于其他时期,而BdCHSlb在蛹-成虫转化过程中以及蛹中期表达量较高。BdCHSla主要在表皮中表达,BdCHSlb主要在气管中表达,而在脂肪体、中肠和马氏管的表达量较低。20-Hydroxyecdyson (20E)能够诱导BdCHSl及其两个可变外显子的表达上调。其中,BdCHSl和BdCHSla在处理8h后表达量显著升高,而BdCHSlb在处理1h后表达量明显升高,说明BdCHSlb能够更加迅速对20E作出响应。除虫脲处理桔小实蝇幼虫后,BdCHSl及其两个可变外显子的表达量明显高于对照,推测可能是由于几丁质合成受阻后基因表达水平的代偿性增加所造成的,几丁质合成酶可能是除虫脲作用的靶标之一。桔小实蝇3龄幼虫注射BdCHSl及其两个可变外显子的dsRNA后,目的基因的表达量明显降低,试虫在生长发育过程中出现了表型异常的现象,并导致部分虫体死亡。其中,干扰BdCHSl和BdCHSla后试虫表皮出现黑化、皱缩进而死亡等现象,而干扰BdCHSlb后却未出现表型变化。这些结果说明BdCHSl在桔小实蝇幼虫-蛹转化过程中起主要作用,BdCHSl基因的表达可以被20E调节。2.桔小实蝇几丁质酶基因克隆、mRNA表达特性及功能分析克隆获得了桔小实蝇2条几丁质酶基因的cDNA全长序列,分别命名为BdCht2和BdCht5。BdCht2开放阅读框1449bp,编码483个氨基酸,5’非编码区和3’非编码区的序列长度分别为126bp和296bp。 BdCht2基因组具有4个外显子和3个内含子。BdCht5开放阅读框1785bp,编码595个氨基酸。预测BdCht2和BdCht5蛋白的分子量分别为54.3kDa和67.5kDa,等电点分别为5.97和5.70。根据所获得基因的氨基酸序列信息与其他昆虫几丁质酶进行系统发育分析,发现这两个几丁质酶基因分别属于Group VII和Group I;通过对其推导的氨基酸进一步分析,发现功能区域的分布特征与上述的Group VII和Group I几丁质酶相同。另外,还克隆获得了BdCht2基因的5’端侧翼区域,并发现了转录调控元件。qPCR结果表明,BdCht2和BdCht5的表达量均在幼虫-蛹和蛹-成虫的转化期明显高于其他时期,可能与它们主要在桔小实蝇变态发育中发挥作用有关BdCht2和BdCht5分别在表皮和气管表达量较高,中肠和其他组织表达量相对较低。20E均能不同程度地诱导2个几丁质酶基因的表达上调,与对照相比分别在处理后8h和12h出现明显的上调,并具有一定的剂量效应。桔小实蝇3龄幼虫进行饥饿处理,可以提前化蛹,导致提前变态发育；同时饥饿也能诱导2个几丁质酶基因表达上调,重新饲喂后其表达量下降。向桔小实蝇3龄幼虫体内注射BdCht2和BdCht5基因的dsRNA进行RNAi,其中BdCht5的沉默效率较高,而BdCht2的RNAi敏感度较低且基因丰度下降不明显,但均没有观察到试虫表型有异常现象。3.桔小实蝇保幼激素合成通路中重要基因的克隆及mRNA表达特性分析克隆获得了6个桔小实蝇保幼激素合成酶基因,其中包括参与JH合成上游甲羟戊酸(Mevalonic acid, MVA)途径的BdHMGS、BdHMGR、BdIPPI、 BdFPS等4个基因,主要负责将乙酰辅酶A催化生成异戊烯醇焦磷酸；以及参与JH合成下游途径的BdSDR和BdJHAMT-like2个基因。通过序列分析明确了6个基因的开放阅读框,并推导了所编码的氨基酸序列,并发现其具有高度保守的结构域。利用qPCR技术分析了保幼激素合成通路中重要基因在桔小实蝇不同发育阶段和组织的mRNA表达特性,结果表明,BdFPS和BdSDR基因在3龄幼虫期高表达,其它基因表达较低或不表达；除BdSDR外,其他基因均在蛹期高表达：多数基因在雌虫的表达量明显高于雄虫,具有明显的性别差异。所有JH合成酶基因在桔小实蝇成虫的头部和脂肪体特异性表达,这可能与咽侧体主要位于头部,而脂肪体在成虫性成熟过程中发挥重要作用有关。解剖桔小实蝇成虫脂肪体进行体外组织培养,加入JH类似物后,大多数JH合成酶基因的表达受到明显的抑制,而20E在一定的程度上也对这些基因表现为抑制作用,说明保幼激素合成酶基因受外源激素的反馈调节。另外,与正常饲喂相比,BdHMGS、 BdIPPI和BdJHAMT-like基因在饥饿48h后其表达量明显降低,合成JH的速率明显变慢,导致其体内合成JH的量大幅度减少,桔小实蝇幼虫发生提前变态而形成蛹。另外,饥饿可以诱导BdHMGR基因的表达上调,而BdFPS和BdSDR基因的表达没有显著的变化。综上所述,推测桔小实蝇JH合成通路中这些基因应对激素和饥饿胁迫的调控模式存在差异。4.桔小实蝇3-羟甲基戊二酰辅酶A-还原酶基因(BdHMGR)的功能分析利用RNAi技术进一步研究了桔小实蝇BdHMGR基因的功能。结果表明,对5日龄雌成虫注射BdHMGR基因dsRNA后,与注射dsGFP后相比,目的基因的表达量显著降低,且卵巢中BdVg2表达量明显降低,卵巢发育不健全,以及直径减小而导致产卵量显著降低。研究结果说明BdHMGR可以作为一个潜在的靶标来开发新的害虫防控手段,以及基于RNAi技术的桔小实蝇及其它害虫的有效控制。5.保幼激素代谢酶基因的克隆及mRNA表达特性分析昆虫体内保幼激素的代谢由保幼激素酯酶、保幼激素环氧水解酶和保幼激素二醇激酶等3种酶共同催化完成。本研究克隆获得了桔小实蝇4个保幼激素代谢酶相关基因的cDNA全长序列,包括BdJHE、BdJHEH2、BdJHEH3和BdJHDK。通过序列分析明确了4个基因的开放阅读框,推导了其编码的氨基酸序列,并发现其均具有高度保守的结构域。系统发育分析表明3种保幼激素代谢酶在进化中均与双翅目昆虫相关基因的亲缘关系最近。BdJHE、BdJHEH2和BdJHDK均在幼虫-蛹的转化过程中高表达,可能与这3个基因在幼虫化蛹的过程中发挥作用相关。BdJHEH3在3龄幼虫早期有较高的表达,而在变态过程中表达较低,推测其可能不是专一性降解JH的环氧水解酶。3种JH代谢酶在成虫7、10日龄的表达量较高,且雌、雄虫之间存在明显的差异,雌虫BdJHE和BdJHEH2的表达量明显高于雄虫,而BdJHDK则为雄虫高于雌虫。桔小实蝇脂肪体的BdJHE和BdJHEH2mRNA表达量明显高于其他组织,而BdJHEH3和BdJHDK分别在中肠和马氏管的表达量高于其他组织。3龄幼虫体内注射20E后,除BdJHE外,其余3个JH代谢酶基因的表达量均出现明显的变化。与对照相比,BdJHEH2和BdJHEH3在处理1h后表达量明显上升,而BdJHDK基因表达量却明显下降。注射4h和8h后,BdJHEH2和BdJHEH3的表达量也明显上升,而BdJHDK在12h后表达量也明显上升。说明3种保幼激素代谢酶的作用方式不同,主要由JHEH和.JHDK作出回应并具有诱导效应。进一步分析了JH代谢酶应对饥饿胁迫的表达变化,结果表明BdJHE和BdJHDK的表达量明显上升,而恢复喂食后其基因表达量又下降；BdJHEH2和BdJHEH3的表达量却下降,重新饲喂后表达量与正常取食的一致。不同保幼激素代谢酶基因表达的变化表明其在幼虫生长发育过程中具有不同的作用。综上所述,本研究基于桔小实蝇转录组数据,通过克隆获得了13条桔小实蝇几丁质和保幼激素合成与代谢中重要基因的cDNA全长序列,并进行了分子特性分析。采用qPCR技术系统地分析了13个基因在桔小实蝇不同发育阶段、不同组织部位的表达特性,以及受激素、饥饿等因子的调节特性,并对重要基因进行了基于RNAi技术的功能验证。研究结果为进一步认识其生理生化特性和生物学功能提供了基础数据,为基于几丁质和保幼激素合成与代谢酶基因研究的桔小实蝇有效控制提供了科学依据。更多还原

【Abstract】 Chitin and juvenile hormone (JH) play key roles in insect development and metamorphosis. Chitin is the main component of integument, trachea, and peritrophic matrix of midgut. The growth and development of insect will reliance on rigorously controlling the synthesis and degradation of chitin. The physiological balance of JH in insects depends on its biosynthesis and degradation pathway. The regulation of JH titer is crucial to normal development in insects. Chitin and JH biosynthetic and metabolic pathway is not only a hot spot in the area of insect metamorphosis, but also an important research field in exploring the new strategies for controlling pests. The polyphagous tephritid fruit fly Bactrocera dorsalis is an important pest in the tropical and subtropical areas, damaging more than250plant species, including numerous fruits and vegetables. Females typically oviposit in fruit, and the developing larvae tunnel through the fleshy mesocarp on which they feed, causing fruit damage and drop. Current control techniques mainly rely on spraying chemical insecticides, but there are some successive reports about the insecticides resistance from B. dorsalis. Therefore, it is an urgent need to develop new pest management strategies. Based on the B. dorsalis transcriptomic database, the molecular characteristics and functions of the important genes in the biosynthetic and metabolic pathways of chitin and JH from B. dorsalis were analyzed comprehensively. The main contents are as follows:1. Molecular characterization and functional analysis of chitin synthase1gene and its two alternative splicing variants in B. dorsalisUsing RT-PCR and RACE techniques, the full-length cDNA sequence of chitin synthase1gene(BdCHSl) were cloned and characterized from B. dorsalis. Two alternative splicing variants of BdCHSl were identified, and termed as BdCHSl a and BdCHSlb, respectively. The cDNA of both variants consisted of5552bp, with an open reading frame (ORF) of4776bp, encoding a protein of1592amino acid residues, plus685bp and88bp of5’-and3’-noncoding regions, respectively. The alternative splicing site was located between positions3784-3960and formed a pair of mutually exclusive exons (a/b) that were same in size (177bp), but showed only65%identity at the nucleotide level. During B. dorsalis growth and development, BdCHSl and BdCHSla were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHSlb was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHSla was predominately expressed in the integument whereas BdCHSlb was mainly expressed in the trachea, and lowly expressed in fat body, midgut and Malpighian tubules. The20-hydroxyecdysone (20E) induced the expression of BdCHSl and its variants. Both BdCHSl and BdCHSla were up-regulated dramatically at8h post-injection compared with control insects, whereas the expression of BdCHSlb was up-regulated as early as at1h post-injection, indicating that BdCHSlb quickly responded to20E. The results showed the expressions of BdCHSl and its two variants in the larvae of B. dorsalis exprosed to diflubenzuron increased significantly compared to the control. Increased expression of these genes mRNA may be due to compensation response of the BdCHSl, BdCHSla, and BdCHSlb gene at the transcritional level that is caused by the retarded chitin synthesis, indicating that chitin synthase may be one of targets of diflubenzuron. Injection of dsRNA of BdCHSl, BdCHSla, and BdCHSlb into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHSl and BdCHSla had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHSlb has no effect on insect morphology. These results demonstrated that BdCHSl plays an important role in the larval-pupal transition and the expression of BdCHSl in B. dorsalis is regulated by20E.2. Molecular characterization and functional analysis of two chitinase genes in B. dorsalisIn this study, we identified and characterized two full-length cDNAs of chitinase genes in B. dorsalis. The chitinase genes were named BdCht2and BdCht5, respectively. The cDNA of BdCht2contains an open reading frame (ORF) of1449bp that encodes483amino acid residues, and126-and296-bp non-coding regions at the5’-and3’-ends, respectively. The BdCht2genome has four exons and three introns. The cDNA of BdCht5contains an ORF of1785bp that encodes595amino acid residues. Phylogenetic analysis with other insect chitinases suggested that the two chitinases were grouped into two classes, Group Ⅶ and Group Ⅰ, respectively. The predicted molecular mass of BdCht2and BdCht5proteins are approximately54.3kDa and67.5kDa and isoelectric point are5.97and5.70, respectively. The977bp5’flanking region of BdCht2was identified, and the transcription factor binding sites were predicted.The qPCR analysis showed that BdCht2and BdCht5were both mainly expressed during the larval-pupal and pupal-adult transitions, which suggested that two genes might play important roles in development and metamorphosis. BdCht2and BdCht5were all expressed in five tissues, with its higher expression occurring in the integument and trachea, followed by the fat body, and other tissues. BdCht2was predominately expressed in the integument whereas BdCht5was mainly expressed in the trachea. Moreover, the expressions of BdCht2and BdCht5were up-regulated significantly upon20E at different doses injection, compared to that of the control after8h and12h of20E treatment, respectively. In B. dorsalis, starvation can shorten the growth duration of third instar larvae, and lead to precocious metamorphosis. Starvation also increased the expression of BdCht2and BdCht5, and was suppressed again by re-feeding the insects. Although we performed RNAi for two chitinase genes by injection of dsRNA into the third-instar larvae, no phenotypic abnormalities were observed in the treated larvae. The knockdown efficiency of dsRNA for BdCht5was highly, whereas BdCht2was a limited RNAi response.3. Molecular characterization and expression analysis of JH biosynthetic pathway genes in B. dorsalisTo data, six JH biosynthesis pathway genes were identified, of which, four genes, including BdHMGS, BdHMGR, BdIPPI and BdFPS, involve in upstream mevalonic acid pathway (MVA) of JH biosynthesis and catalyze acetyl-CoA to generate isopentenyl diphosphate. BdSDR and BdJHAMT-like participate in downstream of JH biosyhthesis pathway. The ORF of BdHMGS was1383bp in length encoded460amino acids while the full-length cDNA of BdHMGR was a3157bp sequence with an ORF encoded928amino acids. Furthermore, BdIPPI had an ORF encoding245amino acids, while the1434bp cDNA of BdFPS had an ORF encoding408amino acids. Moreover, the920bp cDNA of BdSDR had an ORF encoding248amino acids, while BdJHAMT-like possessed an ORF of276amino acids. It is found that the domain regions of encoded proteins were highly conserved.The qPCR was used to analyze the mRNA expression of important genes involved in JH biosynthesis in different developmental stages and tissues of B. dorsalis. During the third-instar larval stage, the highest expressions of BdFPS and BdSDR were observed, but low or no expressions of other genes were observed. During the pupal stage, the expression of these genes was abundantly observed except BdSDR. The JH biosynthetic pathway genes have sex-specific expression patterns, and most genes were higher expressed in females. These genes were highly expressed in the adult fat body and head. Corpora allata is mainly localized in the head, and fat body is an important organ that plays important roles in sexual maturity. After adding JH analog to the incubated fat body in vitro, the expression levels of most JH biosynthetic pathway genes were inhibited, and20E also could inhibited the expressions of BdSDR and BdJHAMT-like. The results suggested that exogenous hormones might regulate the expression of these genes involved in JH biosynthesis. Furthermore, the expressions of BdHMGS, BdIPPI and BdJHAMT-like were significantly decreased after48h starvation compared to the fed larvae, and the rate of JH biosynthesis was effectively reduced. The JH titer was significantly decreased, leading to precocious metamorphosis in the larvae. However, the expression of BdHMGR was up-regulated, no significant change of BdSDR and BdFDR was detected after starvation. Based on these results, it is deduced that the regulation mechanism of important genes involved in JH biosynthesis may be different in response to hormone and starvation treatment.4. Functional analysis of BdHMGR gene in B. dorsalisRNAi was applied to further explore the functions of BdHMGR in B. dorsalis. Injection dsRNA of BdHMGR into day-5female adult significantly inhibited the expression level of the corresponding gene compared to control groups injected with dsGFP, significantly reduced BdVg2mRNA levels in ovaries, inhibited the development of ovaries. Moreover, the diameter of ovaries of the females was significantly lower than that in control groups, leading to effectively inhibit oviposition. The present study demonstrated the potential implications for developing novel pest management strategies using BdHMGR RNAi in the control of B. dorsalis and other insect pests.5. Molecular characterization and expression analysis of JH metabolic enzyme genes in B. dorsalisThree key enzymes, namely JH esterase (JHE), JH epoxide hydrolase (JHEH), and JH diol kinase (JHDK) are required for JH degradation in insects. In this study, we identified four JH metabolism-related genes, including BdJHE, BdJHEH2, BdJHEH3and BdJHDK. The cDNA of BdJHE contains an ORF of1788bp that encodes596amino acid residues, and52-and443-bp non-coding regions at the5’-and3’-ends, respectively. The ORF of BdJHEH2was1395bp in length encoded465amino acids while the full-length cDNA of BdJHEH3was a1651bp sequence with an ORF encoded459amino acids. The full-length cDNA of BdJHDK was a1065bp sequence with an ORF encoded184amino acids. It is found that the domain regions of encoded proteins were highly conserved. Phylogenetic analyses showed that separated clusters of each gene and the evolutional conservation in insects with a high similarity in Diptera.BdJHE, BdJHEH2and BdJHDK were mainly expressed during the larval-pupal transition, suggesting that these genes play important roles in the metamorphosis. However, BdJHEH3were predominately expressed in the early third-instar larvae, and lowly expressed during insect metamorphosis, indicating that this gene might be not JH metabolism enzyme. Furthermore, these genes were highly expressed on day-7and day-10adult, and have sex-specific expression patterns. The relative expression levels of BdJHE and BdJHEH2are substantially higher in females than males, while BdJHDK showed higher expression in males. Furthermore, BdJHE and BdJHEH2were mainly expressed in fat body, whereas BdJHEH3and BdJHDK were highly expressed in midgut and Malpighian tubules, respectively. The expressions levels of JH metabolism enzyme genes in third instar larvae were remarkably changed except BdJHE gene by20E injection. Specifically, At1h after20E injection, the expression levels of BdJHEH2and BdJHEH3were increased significantly. However, the expression level of BdJHDK was significantly decreased compared with control larvae. At4h and8h after20E application, the expression levels of BdJHEH2and BdJHEH3were increased remarkably, respectively. Expression level of BdJHDK was increased significantly at12h after20E injection. The results suggested that different juvenile hormone metabolism enzyme genes play various roles, and the induced expressions of JHEH and JHDK were mainly response to20E. Experiment was performed to analyze whether or not the JH metabolism enzymes expressions were regulated by starvation, the results showed that the expressions of BdJHE and BdJHDK were significantly increased after starvation treatment compared to the fed larvae, however, the expression of BdJHEH2and BdJHEH3were decreased, no significant change was observed after re-feeding. The changes in expression levels of these genes in response to food suggest that they may be have different roles in regulating growth and development of larvae during feeding.In summary, thirteen full-length cDNAs of genes involved in chitin and JH biosynthetic and metabolic pathway were cloned based on the B. dorsalis transcriptome database, and the sequences were analyzed using bioinformatics methods. The qPCR was applied to analyze the expression patterns of these genes in different developmental stages and tissues, but also evaluate the effects of hormone and starvation treatment in molecular responses under stress. Furthermore, RNAi was performed to investigate the functions of important genes. Results of our investigation provide foundational information for understanding the physiological and biological functions of these genes, and for facilitating the development of new strategies for pest control.更多还原