茶尺蠖化学感受相关基因的克隆与功能研究
发布时间：2018-07-20 08:50
【摘要】：选择适宜的产卵地对于鳞翅目昆虫的种群延续尤为重要,因为它们的初孵幼虫个体小、运动能力差很难再更换寄主植物。昆虫的寄主选择行为是一个连续的过程,包括搜索、降落、接触评价和最终的决定。寄主植物的挥发物和植物组织内的次生代谢物对于昆虫的寄主定位和产卵地的选择至关重要。一般来说,昆虫通过其灵敏的嗅觉系统在远距离上感受寄主植物的挥发物进行定位,而接下来对寄主植物组织内的非挥发性次生代谢物的接触评价则涉及到味觉识别过程。触角是昆虫主要的嗅觉器官,上面分布的各类感受器使昆虫能够灵敏的感知到外界的信息化合物。茶尺蠖属于鳞翅目尺蠖蛾科,是我国茶树的主要害虫之一,以幼虫食叶为害,严重影响茶叶产量和质量。化学方法防治茶尺蠖简单有效,但会威胁食品安全,破坏生态环境。其它的方法如“推-拉”策略、人工合成性信息素等基于昆虫寄主选择机制的行为调控方法已得到了研发。茶尺蠖单一的取食几种植物,因此选择适宜的寄主植物需要精确的感觉系统来完成。此外有研究证明,茶尺蠖取食会诱导茶树释放大量的挥发物,并吸引其雌蛾前来产卵。目前对茶尺蠖与其寄主茶树之间化学通讯的研究尚不完善,由于缺乏对茶尺蠖寄主选择机制的知识给防治造成了被动。深入研究茶尺蠖与其寄主茶树的化学通讯机制,阐明气味结合蛋白和化学受体的生理功能,有助于发掘更具有潜力的害虫靶标基因,便于设计更合理的昆虫行为调控剂服务于茶产业发展。本文通过分子生物学的相关技术,对茶尺蠖的气味结合蛋白、化学感受蛋白以及化学受体进行初步研究。利用电子显微镜观察了成虫雌雄触角和前足跗节与寄主定位相关的各类感器的超微结构。结果如下:1.茶尺蠖成虫触角和足跗节的感器扫描及超微结构观察为了研究各类感器在茶尺蠖寄主定位中的功能,通过扫描电镜观察茶尺蠖雌雄蛾触角和雌蛾前足跗节的化学感受器的外部形态,利用透射电镜观察感器内部的超微结构。共鉴定出8种感器(包括15个亚型)。在成虫触角上,毛形感器分为两个亚型,是数量最多的一类感器,表皮较厚,气孔数目少,感器淋巴液存在神经元树突。刺形感器分为三个亚型,表皮壁较厚,没有气孔结构,顶部有单孔,感器淋巴液内腔中有树突神经元。锥形感器(包含三个亚型)和耳形感器表皮壁比较薄,分布着大量的气孔,感器淋巴液分布大量的树突神经元。栓锥形感器表皮壁较厚,无孔道结构,具有2~3个树突神经元。腔锥形感器属于双壁型感受器,具有4-7个树突神经元。同时在触角上也观察到鳞形感器和B?hm氏鬃毛的存在。在雌蛾前足第5跗节上观察到两类刺形感器存在,构成了主要化学感受器,推断刺形感器具有味觉感知的功能。参考之前其它昆虫上的有功能报道的同类型感器,比较推断茶尺蠖中相关感器的功能,为以后系统研究感器的功能做准备。2.茶尺蠖成虫足转录组测序分析与寄主定位相关基因鉴定感受寄主植物表面非挥发性次生代谢物涉及到味觉感受决定了寄主选择的偏好,茶尺蠖前足跗节分布有的化学感受器。对茶尺蠖雌雄成虫足的转录组进行Hi Seq 2500测序,通过生物信息学分析和同源比对的方法,鉴定出化学感受的相关基因包括24个OBPs、21个CSPs、2个SNMPs、3个GRs、4个ORs。RPKM值量化基因表达丰度发现,Eobl OBP6在雌雄转录组中的表达量均最高,9个Eobl OBPs在雌雄足表达存在显著差异,8个Eobl OBPs在雌蛾足中上调,只有1个Eobl OBPs在雄蛾足发生上调,这些雌蛾足中高表达的基因暗示了雌蛾对于搜寻产卵地的生态适应性。系统发育分析表明,茶尺蠖的Eobl GR2与糖受体Harm GR4序列同源性很高(相似度76%),推断Eobl GR2是果糖受体。通过半定量RT-PCR和荧光定量q RT-PCR分析这些基因的组织表达:(1)大部分的Eobl OBPs在触角有表达。其中16个Eobl OBPs在成虫触角特异性表达,此外有3个Eobl OBPs在成虫足部高表达,Eobl OBP7主要存在于腹部。其余5个Eobl OBPs各组织分布比较广泛,Eobl OBP5、Eobl OBP6、Eobl OBP22主要在成虫足和触角有表达。在触角中高表达的OBP可能参与性信息素识别、寄主植物定位和搜寻适宜产卵地的过程,而在足和腹部高表达的OBP功能还有待研究。(2)21个Eobl CSPs组织表达分布广泛,其中6个Eobl CSPs主要在足中表达,其中Eobl CSP11和Eobl CSP15在雄虫的足特异性表达。Eobl CSP2、Eobl CSP10和Eobl CSP16主要在成虫腹部表达。Eobl CSP6在触角特异表达,Eobl CSP6和Eobl CSP9主要在触角表达,推断在嗅觉识别过程中发挥作用。除此之外,Eobl CSP1、Eobl CSP3、Eobl CSP4、Eobl CSP9、Eobl CSP12、Eobl CSP13、Eobl CSP14、Eobl CSP17、Eobl CSP19和Eobl CSP20在成虫足部都有较高的表达量。在足部高表达或者特异表达的CSP可能参与味觉物质的识别。(3)Eobl SNMP1和Eobl SNMP2在触角中表达量显著高于其它组织。4个Eobl ORs也主要在触角表达。在鉴定出的3个Eobl GRs中,Eobl GR1主要在腹部表达而Eobl GR2在触角高表达,Eobl GR3主要在足表达。3.EoblOBP3和EoblOBP6的结合特性分析选择在成虫足转录组高丰度Eobl OBP3和Eobl OBP6作为研究对象,通过荧光竞争结合试验测定了Eobl OBP3和Eobl OBP6与10种茶树挥发物、8种害虫诱导茶树释放挥发物、16种非生境植物挥发物、18种味觉物质的结合能力。结果显示:(1)Eobl OBP3与顺-马鞭草烯醇结合能力最强,其对茶尺蠖成虫有显著的驱避作用。Eobl OBP3能够结合(Z)-己酸-3-己烯酯,此种挥发物为茶树受到害虫取食后产生,显著吸引雌蛾。Eobl OBP3还结合茶树的挥发物癸醛。Eobl OBP3结合α-法尼烯,由害虫为害后诱发的一种挥发物。Eobl OBP3还能够结合味觉物质槲皮素,这类物质为具有苦味的生物碱,对昆虫有拒食作用。(2)Eobl OBP6与橙花叔醇和α-法尼烯的结合能力最强,这两类物质在是害虫取食茶树后诱导产生。Eobl OBP6还能够结合苯甲醛,此类物质为茶梢的挥发物成分之一。Eobl OBP6可以结合萜烯类物质α-松油烯和α-石竹烯。此外,Eobl OBP6和硫酸黄连素有很强的的结合能力,这种物质为生物碱具有苦味,对昆虫有强烈的拒食作用。Eobl OBP6和Eobl OBP3均不结合糖类物质和氨基酸类化合物。利用胶体金免疫组织定位发现Eobl OBP6在触角和雌蛾前足跗节的刺形感器淋巴液外腔中表达。4.嗅觉受体基因的鉴定与组织表达分析对茶尺蠖雌雄成虫触角进行转录组测序,共鉴定出27个嗅觉受体基因。组织表达谱分析显示,大部分的嗅觉受体基因在触角中高表达或特异表达,推断这些基因在嗅觉识别中起重要作用。Eobl OR33在雄蛾触角特异性表达,结合系统发育分析,推断其是性信息素受体。
[Abstract]:The selection of suitable spawning sites is particularly important for the population of Lepidoptera, because their hatchable larvae are small, and their poor exercise ability is difficult to replace host plants. The host selection behavior of insects is a continuous process, including search, landing, contact evaluation and final decision. The volatiles and plant tissues of host plants. Secondary metabolites are important for host location and oviposition selection of insects. In general, insects locate the volatiles of host plants through their sensitive olfactory system at a long distance, and the subsequent contact evaluation of non volatile secondary metabolites in host plants involves the taste recognition process. Antennae is the main olfactory organ of insects, and the various receptors on it make insects sensitive to the information compounds of the outside world. The tea inchworm is a Lepidoptera inchworm moth. It is one of the main pests of tea trees in China, which seriously affects the yield and quality of tea. The chemical method is simple and effective for the control of the tea inchworm. It will threaten food safety and destroy the ecological environment. Other methods such as "push pull" strategy, synthetic pheromone based behavior control method based on insect host selection mechanism have been developed. The tea inchworm feeding on several plants, so the selection of suitable host plants needs accurate sensory system to complete. It is proved that the tea inchworm feeding can induce the tea tree to release a large number of volatiles and attract the female moth to lay eggs. The study of chemical communication between the tea inchworm and its host tea tree is not perfect. The lack of knowledge of the host selection mechanism of the camellia inchworm has caused the control of the chemical communication of the tea inchworm and its host tea tree. The mechanism, clarifying the physiological functions of odorin and chemoreceptor, helps to discover more potential pest target genes and facilitates the design of more reasonable insect behavior regulators to serve the development of tea industry. In this paper, the gas flavor binding protein, chemoreceptor and chemoreceptor of the tea inchworm are based on the related techniques of molecular biology. The ultrastructure of all kinds of sensilla related to the female and male antennae and the tarsus of the forefoot were observed with the electron microscope. The results are as follows: 1. the sensilla scanning and ultrastructural observation of the adult antennae and the foot tarsus of the inchworm in order to study the function of various sensors in the host location of the tea char worm, through scanning electron microscope The external morphology of the antennae of the female and male moth of the moth and the tarsus of the female moth was observed. The ultrastructure in the sensory organ was observed by transmission electron microscopy. 8 sensilla (including 15 subtypes) were identified. On the adult antennae, the hair sensilla was divided into two subtypes, which were the most abundant, the epidermis was thicker, the number of stomata was less and the sense organ was drenched. There are three subtypes of neurons in the liquid. The epidermis is thicker, there is no stomatal structure, there is a single hole in the top, and there are dendritic neurons in the inner cavity of the lymph. The cone sense organ (including three subtypes) and the epidermis of the ear shaped sensor are thinner, a large number of air holes are distributed, and the lymph nodes of the sensilla are distributed in a large number of dendritic neurons. Taper taper is conical. The epidermal wall of the sensilla is thick and has no pore structure. It has 2~3 dendritic neurons. The cavity conical sensilla belongs to the double wall receptor and has 4-7 dendritic neurons. At the same time, the squamous sensor and the B? HM mane are also observed on the antennae. The presence of two types of spiny receptors on the fifth tarsus of the female moth is the main chemoreceptor, and the main chemoreceptor is formed. The spines have the function of taste perception. Refer to the same type of sensilla on other insects that have been reported before, and compare the function of the related sensilla in the tea inchworm, and prepare the sequence analysis of the.2. tea inchworm adult foot transcriptional group and the host location related gene to identify the host plant surface for the later systematic research on the function of the sensilla. The non volatile secondary metabolites involve the preference of the taste sensation, and the chemoreceptor is distributed in the tarsus of the tea inchworm. The Hi Seq 2500 sequencing of the transcriptional group of the adult and male adult worms of the inchworm is carried out. By bioinformatics analysis and homologous comparison, the related genes of the chemoreceptor are identified as 24 OBPs, 21 CSPs, 2 SNMPs, 3 GRs, and 4 ORs.RPKM quantized gene expression abundances, the expression of Eobl OBP6 in both male and male transcripts is the highest, 9 Eobl OBPs in the male and female feet expression is significant difference, 8 Eobl OBPs in the female moth foot up, only 1 Eobl OBPs in the male moth's foot rise, these female moth's high expression gene hints that The analysis of phylogenetic analysis showed that the homology of the Eobl GR2 of the tea inchworm and the sugar receptor Harm GR4 sequence was high (similarity degree 76%), and that the Eobl GR2 was a fructose receptor. The analysis of these genes by semi quantitative RT-PCR and fluorescence quantitative Q RT-PCR: (1) the majority of Eobl OBPs were expressed in the antennae. 16 Eobl OBPs were expressed in the adult antennae, in addition, 3 Eobl OBPs were expressed in the adult foot, and Eobl OBP7 mainly existed in the abdomen. The other 5 Eobl OBPs tissues were distributed widely, Eobl OBP5, Eobl OBP6, and expressed mainly in the adult's feet and antennae. No, the host plants locate and search for the suitable spawning sites, and the high expression of OBP in the foot and abdomen remains to be studied. (2) the expression of 21 Eobl CSPs tissues is widely distributed, of which 6 Eobl CSPs are mainly expressed in the foot, of which Eobl CSP11 and Eobl CSP15 Express.Eobl CSP2 in the male's foot specificity. Eobl CSP6 and Eobl CSP9 are expressed in the antennae, and Eobl CSP6 and Eobl CSP9 are mainly expressed in the antennae. It is concluded that Eobl CSP1, Eobl CSP3, Eobl CSP4 have higher expression in the adult foot. The high expression or specific expression of CSP may be involved in the identification of taste substances. (3) the expression of Eobl SNMP1 and Eobl SNMP2 in the antennae is significantly higher than that of.4 Eobl ORs in other tissues and mainly in the antennae. In the identified 3 Eobl GRs, Eobl GR1 is mainly expressed in the abdomen. The combination characteristic analysis of oblOBP3 and EoblOBP6 was selected as the research object of high abundance Eobl OBP3 and Eobl OBP6 in the adult adult's foot transcriptional group. The volatiles of Eobl OBP3 and Eobl OBP6 and 10 kinds of tea trees were measured by the fluorescence competitive binding test. 8 kinds of pests induced the volatiles from the tea tree, 16 kinds of non habitat plant volatiles and 18 kinds of taste substances binding ability. The results were as follows: (1) Eobl OBP3 has the strongest binding ability to verbenalenol, and it has a significant repellent to the adult worm of the tea worm..Eobl OBP3 can combine (Z) - hexanoate hexenyl Hexanate -3- hexene. The volatiles of tea trees are produced by insect pests, and they attract female moth.Eobl OBP3 to combine the volatile decanal.Eobl OBP3 of tea tree with alpha - farinene. .Eobl OBP3, a volatile matter induced by insect pests, also combines the taste substance quercetin, which is a bitter alkaloid and is antifeedant to insects. (2) the combination of Eobl OBP6 and Nero TERT alcohol and alpha farenene is the strongest, and these two kinds of substances can also be combined to induce.Eobl OBP6 after the pest is fed to the tea tree. Benzaldehyde, one of the volatile components of tea shoots,.Eobl OBP6 can combine terpene with alpha pine and alpha carenene. In addition, Eobl OBP6 and berberine sulfate have strong binding ability. This substance has a bitter taste for alkaloids, and it has a strong antifeeding effect on insects,.Eobl OBP6 and Eobl OBP3 are not combined with carbohydrates. Eobl OBP6 expressed the.4. olfactory receptor gene in the antennae of the antennae and the tarsus of the female moth by colloidal gold immuno tissue localization. The identification and tissue expression analysis of the.4. olfactory receptor gene were sequenced to the female and male adult antennae of the tea inchworm, and a total of 27 olfactory receptor genes were identified and the tissue expression profiles were identified. The analysis shows that most of the olfactory receptor genes are highly expressed or expressed in the antennae. It is inferred that these genes play an important role in olfactory recognition..Eobl OR33 is expressed in the antennae of the male moth and is a sexual pheromone receptor combined with phylogenetic analysis.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.711
，
本文编号：2132948
[Abstract]:The selection of suitable spawning sites is particularly important for the population of Lepidoptera, because their hatchable larvae are small, and their poor exercise ability is difficult to replace host plants. The host selection behavior of insects is a continuous process, including search, landing, contact evaluation and final decision. The volatiles and plant tissues of host plants. Secondary metabolites are important for host location and oviposition selection of insects. In general, insects locate the volatiles of host plants through their sensitive olfactory system at a long distance, and the subsequent contact evaluation of non volatile secondary metabolites in host plants involves the taste recognition process. Antennae is the main olfactory organ of insects, and the various receptors on it make insects sensitive to the information compounds of the outside world. The tea inchworm is a Lepidoptera inchworm moth. It is one of the main pests of tea trees in China, which seriously affects the yield and quality of tea. The chemical method is simple and effective for the control of the tea inchworm. It will threaten food safety and destroy the ecological environment. Other methods such as "push pull" strategy, synthetic pheromone based behavior control method based on insect host selection mechanism have been developed. The tea inchworm feeding on several plants, so the selection of suitable host plants needs accurate sensory system to complete. It is proved that the tea inchworm feeding can induce the tea tree to release a large number of volatiles and attract the female moth to lay eggs. The study of chemical communication between the tea inchworm and its host tea tree is not perfect. The lack of knowledge of the host selection mechanism of the camellia inchworm has caused the control of the chemical communication of the tea inchworm and its host tea tree. The mechanism, clarifying the physiological functions of odorin and chemoreceptor, helps to discover more potential pest target genes and facilitates the design of more reasonable insect behavior regulators to serve the development of tea industry. In this paper, the gas flavor binding protein, chemoreceptor and chemoreceptor of the tea inchworm are based on the related techniques of molecular biology. The ultrastructure of all kinds of sensilla related to the female and male antennae and the tarsus of the forefoot were observed with the electron microscope. The results are as follows: 1. the sensilla scanning and ultrastructural observation of the adult antennae and the foot tarsus of the inchworm in order to study the function of various sensors in the host location of the tea char worm, through scanning electron microscope The external morphology of the antennae of the female and male moth of the moth and the tarsus of the female moth was observed. The ultrastructure in the sensory organ was observed by transmission electron microscopy. 8 sensilla (including 15 subtypes) were identified. On the adult antennae, the hair sensilla was divided into two subtypes, which were the most abundant, the epidermis was thicker, the number of stomata was less and the sense organ was drenched. There are three subtypes of neurons in the liquid. The epidermis is thicker, there is no stomatal structure, there is a single hole in the top, and there are dendritic neurons in the inner cavity of the lymph. The cone sense organ (including three subtypes) and the epidermis of the ear shaped sensor are thinner, a large number of air holes are distributed, and the lymph nodes of the sensilla are distributed in a large number of dendritic neurons. Taper taper is conical. The epidermal wall of the sensilla is thick and has no pore structure. It has 2~3 dendritic neurons. The cavity conical sensilla belongs to the double wall receptor and has 4-7 dendritic neurons. At the same time, the squamous sensor and the B? HM mane are also observed on the antennae. The presence of two types of spiny receptors on the fifth tarsus of the female moth is the main chemoreceptor, and the main chemoreceptor is formed. The spines have the function of taste perception. Refer to the same type of sensilla on other insects that have been reported before, and compare the function of the related sensilla in the tea inchworm, and prepare the sequence analysis of the.2. tea inchworm adult foot transcriptional group and the host location related gene to identify the host plant surface for the later systematic research on the function of the sensilla. The non volatile secondary metabolites involve the preference of the taste sensation, and the chemoreceptor is distributed in the tarsus of the tea inchworm. The Hi Seq 2500 sequencing of the transcriptional group of the adult and male adult worms of the inchworm is carried out. By bioinformatics analysis and homologous comparison, the related genes of the chemoreceptor are identified as 24 OBPs, 21 CSPs, 2 SNMPs, 3 GRs, and 4 ORs.RPKM quantized gene expression abundances, the expression of Eobl OBP6 in both male and male transcripts is the highest, 9 Eobl OBPs in the male and female feet expression is significant difference, 8 Eobl OBPs in the female moth foot up, only 1 Eobl OBPs in the male moth's foot rise, these female moth's high expression gene hints that The analysis of phylogenetic analysis showed that the homology of the Eobl GR2 of the tea inchworm and the sugar receptor Harm GR4 sequence was high (similarity degree 76%), and that the Eobl GR2 was a fructose receptor. The analysis of these genes by semi quantitative RT-PCR and fluorescence quantitative Q RT-PCR: (1) the majority of Eobl OBPs were expressed in the antennae. 16 Eobl OBPs were expressed in the adult antennae, in addition, 3 Eobl OBPs were expressed in the adult foot, and Eobl OBP7 mainly existed in the abdomen. The other 5 Eobl OBPs tissues were distributed widely, Eobl OBP5, Eobl OBP6, and expressed mainly in the adult's feet and antennae. No, the host plants locate and search for the suitable spawning sites, and the high expression of OBP in the foot and abdomen remains to be studied. (2) the expression of 21 Eobl CSPs tissues is widely distributed, of which 6 Eobl CSPs are mainly expressed in the foot, of which Eobl CSP11 and Eobl CSP15 Express.Eobl CSP2 in the male's foot specificity. Eobl CSP6 and Eobl CSP9 are expressed in the antennae, and Eobl CSP6 and Eobl CSP9 are mainly expressed in the antennae. It is concluded that Eobl CSP1, Eobl CSP3, Eobl CSP4 have higher expression in the adult foot. The high expression or specific expression of CSP may be involved in the identification of taste substances. (3) the expression of Eobl SNMP1 and Eobl SNMP2 in the antennae is significantly higher than that of.4 Eobl ORs in other tissues and mainly in the antennae. In the identified 3 Eobl GRs, Eobl GR1 is mainly expressed in the abdomen. The combination characteristic analysis of oblOBP3 and EoblOBP6 was selected as the research object of high abundance Eobl OBP3 and Eobl OBP6 in the adult adult's foot transcriptional group. The volatiles of Eobl OBP3 and Eobl OBP6 and 10 kinds of tea trees were measured by the fluorescence competitive binding test. 8 kinds of pests induced the volatiles from the tea tree, 16 kinds of non habitat plant volatiles and 18 kinds of taste substances binding ability. The results were as follows: (1) Eobl OBP3 has the strongest binding ability to verbenalenol, and it has a significant repellent to the adult worm of the tea worm..Eobl OBP3 can combine (Z) - hexanoate hexenyl Hexanate -3- hexene. The volatiles of tea trees are produced by insect pests, and they attract female moth.Eobl OBP3 to combine the volatile decanal.Eobl OBP3 of tea tree with alpha - farinene. .Eobl OBP3, a volatile matter induced by insect pests, also combines the taste substance quercetin, which is a bitter alkaloid and is antifeedant to insects. (2) the combination of Eobl OBP6 and Nero TERT alcohol and alpha farenene is the strongest, and these two kinds of substances can also be combined to induce.Eobl OBP6 after the pest is fed to the tea tree. Benzaldehyde, one of the volatile components of tea shoots,.Eobl OBP6 can combine terpene with alpha pine and alpha carenene. In addition, Eobl OBP6 and berberine sulfate have strong binding ability. This substance has a bitter taste for alkaloids, and it has a strong antifeeding effect on insects,.Eobl OBP6 and Eobl OBP3 are not combined with carbohydrates. Eobl OBP6 expressed the.4. olfactory receptor gene in the antennae of the antennae and the tarsus of the female moth by colloidal gold immuno tissue localization. The identification and tissue expression analysis of the.4. olfactory receptor gene were sequenced to the female and male adult antennae of the tea inchworm, and a total of 27 olfactory receptor genes were identified and the tissue expression profiles were identified. The analysis shows that most of the olfactory receptor genes are highly expressed or expressed in the antennae. It is inferred that these genes play an important role in olfactory recognition..Eobl OR33 is expressed in the antennae of the male moth and is a sexual pheromone receptor combined with phylogenetic analysis.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.711
，
本文编号：2132948
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2132948.html
最近更新
教材专著
