【摘要】：選擇適宜的產卵地對于鱗翅目昆蟲的種群延續(xù)尤為重要,因為它們的初孵幼蟲個體小、運動能力差很難再更換寄主植物。昆蟲的寄主選擇行為是一個連續(xù)的過程,包括搜索、降落、接觸評價和最終的決定。寄主植物的揮發(fā)物和植物組織內的次生代謝物對于昆蟲的寄主定位和產卵地的選擇至關重要。一般來說,昆蟲通過其靈敏的嗅覺系統在遠距離上感受寄主植物的揮發(fā)物進行定位,而接下來對寄主植物組織內的非揮發(fā)性次生代謝物的接觸評價則涉及到味覺識別過程。觸角是昆蟲主要的嗅覺器官,上面分布的各類感受器使昆蟲能夠靈敏的感知到外界的信息化合物。茶尺蠖屬于鱗翅目尺蠖蛾科,是我國茶樹的主要害蟲之一,以幼蟲食葉為害,嚴重影響茶葉產量和質量�；瘜W方法防治茶尺蠖簡單有效,但會威脅食品安全,破壞生態(tài)環(huán)境。其它的方法如“推-拉”策略、人工合成性信息素等基于昆蟲寄主選擇機制的行為調控方法已得到了研發(fā)。茶尺蠖單一的取食幾種植物,因此選擇適宜的寄主植物需要精確的感覺系統來完成。此外有研究證明,茶尺蠖取食會誘導茶樹釋放大量的揮發(fā)物,并吸引其雌蛾前來產卵。目前對茶尺蠖與其寄主茶樹之間化學通訊的研究尚不完善,由于缺乏對茶尺蠖寄主選擇機制的知識給防治造成了被動。深入研究茶尺蠖與其寄主茶樹的化學通訊機制,闡明氣味結合蛋白和化學受體的生理功能,有助于發(fā)掘更具有潛力的害蟲靶標基因,便于設計更合理的昆蟲行為調控劑服務于茶產業(yè)發(fā)展。本文通過分子生物學的相關技術,對茶尺蠖的氣味結合蛋白、化學感受蛋白以及化學受體進行初步研究。利用電子顯微鏡觀察了成蟲雌雄觸角和前足跗節(jié)與寄主定位相關的各類感器的超微結構。結果如下:1.茶尺蠖成蟲觸角和足跗節(jié)的感器掃描及超微結構觀察為了研究各類感器在茶尺蠖寄主定位中的功能,通過掃描電鏡觀察茶尺蠖雌雄蛾觸角和雌蛾前足跗節(jié)的化學感受器的外部形態(tài),利用透射電鏡觀察感器內部的超微結構。共鑒定出8種感器(包括15個亞型)。在成蟲觸角上,毛形感器分為兩個亞型,是數量最多的一類感器,表皮較厚,氣孔數目少,感器淋巴液存在神經元樹突。刺形感器分為三個亞型,表皮壁較厚,沒有氣孔結構,頂部有單孔,感器淋巴液內腔中有樹突神經元。錐形感器(包含三個亞型)和耳形感器表皮壁比較薄,分布著大量的氣孔,感器淋巴液分布大量的樹突神經元。栓錐形感器表皮壁較厚,無孔道結構,具有2~3個樹突神經元。腔錐形感器屬于雙壁型感受器,具有4-7個樹突神經元。同時在觸角上也觀察到鱗形感器和B?hm氏鬃毛的存在。在雌蛾前足第5跗節(jié)上觀察到兩類刺形感器存在,構成了主要化學感受器,推斷刺形感器具有味覺感知的功能。參考之前其它昆蟲上的有功能報道的同類型感器,比較推斷茶尺蠖中相關感器的功能,為以后系統研究感器的功能做準備。2.茶尺蠖成蟲足轉錄組測序分析與寄主定位相關基因鑒定感受寄主植物表面非揮發(fā)性次生代謝物涉及到味覺感受決定了寄主選擇的偏好,茶尺蠖前足跗節(jié)分布有的化學感受器。對茶尺蠖雌雄成蟲足的轉錄組進行Hi Seq 2500測序,通過生物信息學分析和同源比對的方法,鑒定出化學感受的相關基因包括24個OBPs、21個CSPs、2個SNMPs、3個GRs、4個ORs。RPKM值量化基因表達豐度發(fā)現,Eobl OBP6在雌雄轉錄組中的表達量均最高,9個Eobl OBPs在雌雄足表達存在顯著差異,8個Eobl OBPs在雌蛾足中上調,只有1個Eobl OBPs在雄蛾足發(fā)生上調,這些雌蛾足中高表達的基因暗示了雌蛾對于搜尋產卵地的生態(tài)適應性。系統發(fā)育分析表明,茶尺蠖的Eobl GR2與糖受體Harm GR4序列同源性很高(相似度76%),推斷Eobl GR2是果糖受體。通過半定量RT-PCR和熒光定量q RT-PCR分析這些基因的組織表達:(1)大部分的Eobl OBPs在觸角有表達。其中16個Eobl OBPs在成蟲觸角特異性表達,此外有3個Eobl OBPs在成蟲足部高表達,Eobl OBP7主要存在于腹部。其余5個Eobl OBPs各組織分布比較廣泛,Eobl OBP5、Eobl OBP6、Eobl OBP22主要在成蟲足和觸角有表達。在觸角中高表達的OBP可能參與性信息素識別、寄主植物定位和搜尋適宜產卵地的過程,而在足和腹部高表達的OBP功能還有待研究。(2)21個Eobl CSPs組織表達分布廣泛,其中6個Eobl CSPs主要在足中表達,其中Eobl CSP11和Eobl CSP15在雄蟲的足特異性表達。Eobl CSP2、Eobl CSP10和Eobl CSP16主要在成蟲腹部表達。Eobl CSP6在觸角特異表達,Eobl CSP6和Eobl CSP9主要在觸角表達,推斷在嗅覺識別過程中發(fā)揮作用。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在成蟲足部都有較高的表達量。在足部高表達或者特異表達的CSP可能參與味覺物質的識別。(3)Eobl SNMP1和Eobl SNMP2在觸角中表達量顯著高于其它組織。4個Eobl ORs也主要在觸角表達。在鑒定出的3個Eobl GRs中,Eobl GR1主要在腹部表達而Eobl GR2在觸角高表達,Eobl GR3主要在足表達。3.EoblOBP3和EoblOBP6的結合特性分析選擇在成蟲足轉錄組高豐度Eobl OBP3和Eobl OBP6作為研究對象,通過熒光競爭結合試驗測定了Eobl OBP3和Eobl OBP6與10種茶樹揮發(fā)物、8種害蟲誘導茶樹釋放揮發(fā)物、16種非生境植物揮發(fā)物、18種味覺物質的結合能力。結果顯示:(1)Eobl OBP3與順-馬鞭草烯醇結合能力最強,其對茶尺蠖成蟲有顯著的驅避作用。Eobl OBP3能夠結合(Z)-己酸-3-己烯酯,此種揮發(fā)物為茶樹受到害蟲取食后產生,顯著吸引雌蛾。Eobl OBP3還結合茶樹的揮發(fā)物癸醛。Eobl OBP3結合α-法尼烯,由害蟲為害后誘發(fā)的一種揮發(fā)物。Eobl OBP3還能夠結合味覺物質槲皮素,這類物質為具有苦味的生物堿,對昆蟲有拒食作用。(2)Eobl OBP6與橙花叔醇和α-法尼烯的結合能力最強,這兩類物質在是害蟲取食茶樹后誘導產生。Eobl OBP6還能夠結合苯甲醛,此類物質為茶梢的揮發(fā)物成分之一。Eobl OBP6可以結合萜烯類物質α-松油烯和α-石竹烯。此外,Eobl OBP6和硫酸黃連素有很強的的結合能力,這種物質為生物堿具有苦味,對昆蟲有強烈的拒食作用。Eobl OBP6和Eobl OBP3均不結合糖類物質和氨基酸類化合物。利用膠體金免疫組織定位發(fā)現Eobl OBP6在觸角和雌蛾前足跗節(jié)的刺形感器淋巴液外腔中表達。4.嗅覺受體基因的鑒定與組織表達分析對茶尺蠖雌雄成蟲觸角進行轉錄組測序,共鑒定出27個嗅覺受體基因。組織表達譜分析顯示,大部分的嗅覺受體基因在觸角中高表達或特異表達,推斷這些基因在嗅覺識別中起重要作用。Eobl OR33在雄蛾觸角特異性表達,結合系統發(fā)育分析,推斷其是性信息素受體。
[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.
【學位授予單位】：中國農業(yè)科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S435.711
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本文編號：2132948