草履蚧觸角嗅覺(jué)基因轉(zhuǎn)錄組及生物信息學(xué)分析
發(fā)布時(shí)間：2018-09-01 18:59
【摘要】：草履蚧Drosicha corpulenta(Kuwana)(半翅目:蚧總科:綿蚧科),是一種聚集在枝芽處刺吸汁液取食的植食性昆蟲(chóng),當(dāng)蟲(chóng)口密度過(guò)大時(shí)往往會(huì)造成枝芽枯死。由于草履蚧體表分泌蠟質(zhì)形成保護(hù)層,人工清除和化學(xué)防治難以對(duì)其進(jìn)行有效控制,而大量化學(xué)殺蟲(chóng)劑的使用不僅會(huì)滅殺天敵,還會(huì)對(duì)果實(shí)和環(huán)境造成污染。因此本文從昆蟲(chóng)化學(xué)通訊對(duì)害蟲(chóng)防治的角度出發(fā),對(duì)草履蚧觸角中參與嗅覺(jué)形成過(guò)程的相關(guān)蛋白進(jìn)行研究,發(fā)現(xiàn)嗅覺(jué)基因在蚧蟲(chóng)中的作用,為今后的生物防治提供新的視角及理論依據(jù)。本文的研究?jī)?nèi)容包括:(1)對(duì)草履蚧雌成蟲(chóng)觸角轉(zhuǎn)錄組進(jìn)行測(cè)序,并進(jìn)行組裝、注釋和同源性分析;從Unigenes中預(yù)測(cè)草履蚧嗅覺(jué)基因并歸納基因信息;對(duì)草履蚧的氣味結(jié)合蛋白(OBPs)及化學(xué)感受蛋白(CSPs)序列進(jìn)行比對(duì),并構(gòu)建系統(tǒng)發(fā)育樹(shù);(2)在轉(zhuǎn)錄組測(cè)序和分析的基礎(chǔ)上,得到氣味結(jié)合蛋白OBP(DcorOBP4,5,6,7,8,14)和化學(xué)感受蛋白DcorCSP2的編碼序列;利用生物信息學(xué)方法分析其序列信息,并構(gòu)建三維結(jié)構(gòu)。結(jié)果如下:1.本文對(duì)草履蚧雌成蟲(chóng)觸角轉(zhuǎn)錄組進(jìn)行高通量測(cè)序。在轉(zhuǎn)錄組中,共獲得33,741,845條序列標(biāo)簽reads,其中16,334條非重復(fù)序列unigenes被組裝。這些unigenes通過(guò)GO分析和eggNOG分析進(jìn)行基因功能的分類統(tǒng)計(jì)。草履蚧觸角中unigenes與其他物種相似性比較發(fā)現(xiàn),與豌豆蚜相似的數(shù)量最多,達(dá)到18.31%。2.本文從unigenes中預(yù)測(cè)到16條編碼氣味結(jié)合蛋白OBPs的基因、2條編碼化學(xué)感受蛋白CSPs的基因、1條編碼感覺(jué)神經(jīng)元膜蛋白SNMP的基因、21條氣味受體基因ORs和7條編碼味覺(jué)受體GRs的基因,并總結(jié)歸納了 47條嗅覺(jué)基因信息。此外,對(duì)草履蚧觸角中預(yù)測(cè)的氣味結(jié)合蛋白OBPs和化學(xué)感受蛋白CSPs序列與蚜蟲(chóng)、盲蝽和扶桑綿粉蚧相關(guān)序列構(gòu)建系統(tǒng)發(fā)育樹(shù)。結(jié)果顯示,8條氣味結(jié)合蛋白與蚜蟲(chóng)聚在一起,3條草履蚧氣味結(jié)合蛋白OBPs聚在一起。2條化學(xué)感受蛋白與扶桑綿粉蚧的2條化學(xué)感受蛋白分別聚類在一起。根據(jù)聚在一起且已知其功能的蚜蟲(chóng)和盲蝽的物種,可以對(duì)草履蚧的OBPs功能進(jìn)行預(yù)測(cè)。草履蚧嗅覺(jué)基因的發(fā)現(xiàn)對(duì)認(rèn)識(shí)其在蚧蟲(chóng)行為反應(yīng)中的作用提供新的視角,同時(shí)提高蚧蟲(chóng)的生物防治策略。3.本研究在草履蚧雌成蟲(chóng)觸角轉(zhuǎn)錄組測(cè)序和分析的基礎(chǔ)上,通過(guò)RT-PCR技術(shù)得到的氣味結(jié)合蛋白OBPs(DcorOBP4,5,6,7,8,14)和化學(xué)感受蛋白DcorCSP2的編碼序列,利用生物信息學(xué)的方法對(duì)草履蚧這些蛋白的理化性質(zhì)、信號(hào)肽、親疏水性等進(jìn)行預(yù)測(cè)分析。分析表明,這些蛋白均有完整的開(kāi)放閱讀框ORF,N端帶有19～29個(gè)氨基酸的信號(hào)肽,等電點(diǎn)在4.12～9.30之間,相對(duì)分子質(zhì)量為15.6～17.0KDa,這些蛋白均為親水性蛋白。OBPs(DcorOBP4,5,6,7,8,14)含有六個(gè)保守的半胱氨酸位點(diǎn),符合典型的氣味結(jié)合蛋白家族的特征�；瘜W(xué)感受蛋白DcorCSP2包含有四個(gè)保守的半胱氨酸位點(diǎn),符合化學(xué)感受蛋白家族的特征。草履蚧典型的氣味結(jié)合蛋白(DcorOBP4,5,6,7,8,14)和化學(xué)感受蛋白DcorCSP2通過(guò)同源建模后發(fā)現(xiàn),三維結(jié)構(gòu)都是由六個(gè)α螺旋組成。DcorOBP4,6,7,8這四個(gè)氣味結(jié)合蛋白的六個(gè)保守半胱氨酸形成了三對(duì)二硫鍵,與典型的氣味結(jié)合蛋白特征相符�；瘜W(xué)感受蛋白DcorCSP2也構(gòu)建出兩對(duì)二硫鍵,這些二硫鍵均起到穩(wěn)定蛋白質(zhì)結(jié)構(gòu)的作用。以上6條OBPs均由α1,α2,α4,α5,α6這5個(gè)α螺旋形成疏水口袋,α3在口袋的一端不參與疏水口袋的形成。此外,這些蛋白的C-末端均折合進(jìn)入蛋白質(zhì)的口袋內(nèi)部,使得口袋結(jié)構(gòu)變得更加穩(wěn)固。
[Abstract]:Drosicha corpulenta (Kuwana) is a kind of herbivorous insect that gathers in the shoots and sucks juice. When the population density is too high, it often causes the shoots to wither. Quantitative chemical insecticides not only kill natural enemies, but also cause pollution to fruits and environment. Therefore, from the point of view of insect chemical communication for pest control, this paper studied the proteins involved in olfactory formation in the antennae of paramecium, and found the role of olfactory genes in scale insects, providing a new way for biological control in the future. The main contents of this study include: (1) Sequencing, assembly, annotation and homology analysis of antennal transcriptome of female Paramecium paramecium; Predicting olfactory genes of Paramecium from Unigenes and inducing gene information; aligning and constructing odorant binding protein (OBPs) and chemosensory protein (CSPs) sequences of Paramecium paramecium. Establishing phylogenetic trees; (2) On the basis of transcriptome sequencing and analysis, the coding sequences of odorant binding protein OBP (DcorOBP 4, 5, 6, 7, 8, 14) and chemosensory protein DcorCSP 2 were obtained; the sequence information was analyzed by bioinformatics method, and the three-dimensional structure was constructed. The results were as follows: 1. Sequencing. A total of 33,741,845 reads were obtained in the transcriptome, of which 16,334 non-repetitive unigenes were assembled. These unigenes were classified and counted by GO analysis and eggNOG analysis. The similarity of unigenes in the antennae of Paramecium paramecis to other species was the highest, reaching 18.31%. 16 genes encoding odorant binding protein OBPs, 2 genes encoding chemoreceptor protein CSPs, 1 gene encoding sensory neuron membrane protein SNMP, 21 odorant receptor genes ORs and 7 genes encoding taste receptor GRs were predicted from unigenes, and 47 olfactory genes were summarized. The predicted odor-binding proteins OBPs and chemosensory proteins CSPs were sequenced to construct phylogenetic trees associated with aphids, bugs and Fusang mealworms. The results showed that eight odor-binding proteins were clustered with aphids, and three odor-binding proteins OBPs were clustered with paramecium. Two chemosensory proteins and two chemosensory eggs of Fusang mealworm were clustered with two chemosensory proteins. The OBPs function of Paramecium can be predicted according to the species of aphids and bugs that are clustered together and whose functions are known. The discovery of the olfactory genes of Paramecium provides a new perspective for understanding their role in behavioral responses of scale insects and improves the biological control strategies of scale insects. 3. This study was conducted in female Paramecium caterpillars. On the basis of antennal transcriptome sequencing and analysis, the coding sequences of odor-binding proteins OBPs (DcorOBP4, 5, 6, 7, 8, 14) and chemosensory proteins DcorCSP2 were obtained by RT-PCR. The physicochemical properties, signal peptides, hydrophilicity and hydrophobicity of these proteins were predicted by bioinformatics method. All of them have complete open reading frame ORF, N-terminal with 19-29 amino acid signal peptide, isoelectric point between 4.12-9.30, relative molecular weight of 15.6-17.0 KDa, these proteins are hydrophilic proteins. OBPs (DcorOBP4, 5, 6, 7, 8, 14) contain six conserved cysteine sites, which conform to the characteristics of typical odor binding protein family. The receptor protein DcorCSP2 contains four conserved cysteine sites, which conform to the characteristics of the chemoreceptive protein family. Typical odorant-binding proteins (DcorOBP4, 5, 6, 7, 8, 14) and chemoreceptive proteins (DcorCSP2) of Paramecium indicum were homologously modeled and found to be composed of six alpha-helices. The six conserved cysteines formed three pairs of disulfide bonds, which were consistent with the characteristics of typical odor-binding proteins. The chemosensory protein DcorCSP2 also constructed two pairs of disulfide bonds. These disulfide bonds stabilized the structure of proteins. All the six OBPs formed hydrophobic pockets from the five alpha helixes of alpha 1, alpha 2, alpha 4, alpha 5, and alpha 6. Alpha 3 was located at one end of the pocket. It is not involved in the formation of hydrophobic pockets. In addition, the C-terminals of these proteins are folded into the inner pockets of the proteins, making the structure of the pockets more stable.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S433;Q78
本文編號(hào)：2218065
[Abstract]:Drosicha corpulenta (Kuwana) is a kind of herbivorous insect that gathers in the shoots and sucks juice. When the population density is too high, it often causes the shoots to wither. Quantitative chemical insecticides not only kill natural enemies, but also cause pollution to fruits and environment. Therefore, from the point of view of insect chemical communication for pest control, this paper studied the proteins involved in olfactory formation in the antennae of paramecium, and found the role of olfactory genes in scale insects, providing a new way for biological control in the future. The main contents of this study include: (1) Sequencing, assembly, annotation and homology analysis of antennal transcriptome of female Paramecium paramecium; Predicting olfactory genes of Paramecium from Unigenes and inducing gene information; aligning and constructing odorant binding protein (OBPs) and chemosensory protein (CSPs) sequences of Paramecium paramecium. Establishing phylogenetic trees; (2) On the basis of transcriptome sequencing and analysis, the coding sequences of odorant binding protein OBP (DcorOBP 4, 5, 6, 7, 8, 14) and chemosensory protein DcorCSP 2 were obtained; the sequence information was analyzed by bioinformatics method, and the three-dimensional structure was constructed. The results were as follows: 1. Sequencing. A total of 33,741,845 reads were obtained in the transcriptome, of which 16,334 non-repetitive unigenes were assembled. These unigenes were classified and counted by GO analysis and eggNOG analysis. The similarity of unigenes in the antennae of Paramecium paramecis to other species was the highest, reaching 18.31%. 16 genes encoding odorant binding protein OBPs, 2 genes encoding chemoreceptor protein CSPs, 1 gene encoding sensory neuron membrane protein SNMP, 21 odorant receptor genes ORs and 7 genes encoding taste receptor GRs were predicted from unigenes, and 47 olfactory genes were summarized. The predicted odor-binding proteins OBPs and chemosensory proteins CSPs were sequenced to construct phylogenetic trees associated with aphids, bugs and Fusang mealworms. The results showed that eight odor-binding proteins were clustered with aphids, and three odor-binding proteins OBPs were clustered with paramecium. Two chemosensory proteins and two chemosensory eggs of Fusang mealworm were clustered with two chemosensory proteins. The OBPs function of Paramecium can be predicted according to the species of aphids and bugs that are clustered together and whose functions are known. The discovery of the olfactory genes of Paramecium provides a new perspective for understanding their role in behavioral responses of scale insects and improves the biological control strategies of scale insects. 3. This study was conducted in female Paramecium caterpillars. On the basis of antennal transcriptome sequencing and analysis, the coding sequences of odor-binding proteins OBPs (DcorOBP4, 5, 6, 7, 8, 14) and chemosensory proteins DcorCSP2 were obtained by RT-PCR. The physicochemical properties, signal peptides, hydrophilicity and hydrophobicity of these proteins were predicted by bioinformatics method. All of them have complete open reading frame ORF, N-terminal with 19-29 amino acid signal peptide, isoelectric point between 4.12-9.30, relative molecular weight of 15.6-17.0 KDa, these proteins are hydrophilic proteins. OBPs (DcorOBP4, 5, 6, 7, 8, 14) contain six conserved cysteine sites, which conform to the characteristics of typical odor binding protein family. The receptor protein DcorCSP2 contains four conserved cysteine sites, which conform to the characteristics of the chemoreceptive protein family. Typical odorant-binding proteins (DcorOBP4, 5, 6, 7, 8, 14) and chemoreceptive proteins (DcorCSP2) of Paramecium indicum were homologously modeled and found to be composed of six alpha-helices. The six conserved cysteines formed three pairs of disulfide bonds, which were consistent with the characteristics of typical odor-binding proteins. The chemosensory protein DcorCSP2 also constructed two pairs of disulfide bonds. These disulfide bonds stabilized the structure of proteins. All the six OBPs formed hydrophobic pockets from the five alpha helixes of alpha 1, alpha 2, alpha 4, alpha 5, and alpha 6. Alpha 3 was located at one end of the pocket. It is not involved in the formation of hydrophobic pockets. In addition, the C-terminals of these proteins are folded into the inner pockets of the proteins, making the structure of the pockets more stable.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S433;Q78
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