本文選題：斜紋夜蛾 + Dorsa　； 參考：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：斜紋夜蛾是一種重要的食葉昆蟲,對(duì)很多農(nóng)作物極其具有破壞性。在本研究中從斜紋夜蛾幼蟲中克隆到Rel/NF-kB的家庭成員Dorsal和Relish可變剪切體(Dorsal 14,Dorsal]B,Relish 1A和Relish 1B)。Dorsal和Relish全長(zhǎng)序列和他們的2個(gè)剪切體從同一個(gè)斜紋夜蛾幼蟲中克隆得到的。對(duì)保守結(jié)構(gòu)域分析得到Dorsal和Relish有兩個(gè)共同的結(jié)構(gòu)域,第一個(gè)是Rel同源結(jié)構(gòu)域(RHD),即真核細(xì)胞特征Rel/NF-kB轉(zhuǎn)錄影響因子,第二個(gè)是免疫球蛋白-叢蛋白-轉(zhuǎn)錄(IPT)結(jié)構(gòu)域,其是類免疫球蛋白折疊域主要負(fù)責(zé)DNA結(jié)合。Dorsal 1B具有RHD、IPT、NLS和LZ結(jié)構(gòu)域,而Dorsal1A具有和Dorsal1B相同的結(jié)構(gòu)域,并且Dorsal 14比Dorsal 1多一個(gè)叉頭N末端區(qū)域。Relish 1A具有RHD,IPT,ANK和死亡結(jié)構(gòu)域,而Relish7B有著和Relish 1A 一樣的結(jié)構(gòu)域。Dorsal的氨基酸序列與無脊椎動(dòng)物和脊椎動(dòng)物的數(shù)據(jù)庫(kù)中19個(gè)Dorsal和3個(gè)Relish蛋白序列對(duì)比,進(jìn)化樹顯示出Dorsal與棉鈴蟲(HaDorsal)和煙草天蛾(MsDorsal)同源基因聚集在一起。推導(dǎo)的Relish氨基酸序列與無脊椎動(dòng)物和脊椎動(dòng)物的數(shù)據(jù)庫(kù)中19個(gè)Relish和3個(gè)Dorsal蛋白序列對(duì)比。進(jìn)化樹顯示Relish與棉鈴蟲(HaRelish)和甜菜夜蛾(SeRelish)同源基因聚集在一起。Dorsal和Relish所有的氨基酸蛋白序列都包含Rel/NF-kB家族轉(zhuǎn)錄因子所具有的特異保守結(jié)構(gòu)域,故克隆的Dorsal和Relish基因均屬于Rel/NF-kB家族。通過獲得Dorsal和Relish在不同組織和發(fā)育階段mRNA表達(dá)量,從而分析出基因組織表達(dá)和齡期表達(dá)的規(guī)律。Dorsal的兩個(gè)剪切體在斜紋夜蛾不同的組織和發(fā)育階段均有表達(dá),并且在不同階段轉(zhuǎn)錄水平具有顯著差異。Dorsal 1A在卵和雌蛹階段有更高的表達(dá)水平。Dorsal 1A在雌蛹和雌成蟲階段轉(zhuǎn)錄水平顯著高于雄蛹和雄成蟲階段,但是Dorsal 1B在蛹和成蟲階段雌雄成蟲轉(zhuǎn)錄水平無顯著性差異。Dorsal 1A和Dorsal 1在不同組織中的轉(zhuǎn)錄水平有顯著性差異,Dorsal 1A在脂肪體和體壁具有較高的表達(dá)水平,而在中腸的表達(dá)水平最低。Dorsal 1B在體壁和脂肪體中的表達(dá)量較高。同樣的,Relish的兩個(gè)剪切體在斜紋夜蛾不同的組織和發(fā)育階段都有表達(dá)。他們?cè)诓煌陌l(fā)育階段的轉(zhuǎn)錄具有顯著差異。在蛹和成蟲階段雌蛹和雌成蟲個(gè)體Relish 1A轉(zhuǎn)錄水平比雄性顯著高。但是Relish 1B在蛹和成蟲階段雌雄個(gè)體并沒有表現(xiàn)出顯著差異。Relish在不同組織中的轉(zhuǎn)錄水平具有顯著差異�？臻g的表達(dá)譜表明,Dorsal在體壁中表達(dá)水平最高,而Relish在脂肪體的轉(zhuǎn)錄水平最高。RelishlA在脂肪體和血淋巴中具有較高表達(dá)量在中腸中表達(dá)量最低。Relish1B在脂肪體中的表達(dá)量最高,在中腸中的表達(dá)量最低。并且在脂肪體中表達(dá)水平均比其他階段高。Dorsal和Relish的兩種剪切體均在脂肪體內(nèi)具有高水平轉(zhuǎn)錄,這表明脂肪體在先天免疫中發(fā)揮了重要的作用。分別注射大腸桿菌、爪哇棒束孢和球孢白僵菌,分析斜紋夜蛾幼蟲Dorsal和Relish在微生物侵染后表達(dá)規(guī)律的特性和差異。Dorsal在被真菌侵染后表達(dá)量上調(diào),而細(xì)菌侵染后表達(dá)量并不上調(diào)。Relish在被大腸桿菌(革蘭氏陰性菌)侵染后表達(dá)量上調(diào),但是真菌侵染后表達(dá)量并無明顯變化。進(jìn)一步證明了在Toll通路下游的Dorsal與真菌之間的相關(guān)性,在IMD通路下游的Relish與革蘭氏陰性菌的相關(guān)性。Dorsal被球孢白僵菌上調(diào)的表達(dá)持續(xù)時(shí)間比爪哇棒束孢長(zhǎng)。Relish7B對(duì)大腸桿菌注入后反應(yīng)速度比Relish 1A慢。這四個(gè)基因應(yīng)對(duì)不同的微生注射后表現(xiàn)出不同的表達(dá)特性。斜紋夜蛾幼蟲同時(shí)注射先天免疫基因(Dorsal/Relis)的dsRNA和微生物懸浮液,幼蟲的死亡率明顯升高。dsRNA注射后下調(diào)了Dorsal/Relish的表達(dá)量,降低了幼蟲對(duì)于微生物侵染的抵抗作用,從而增加微生物侵染的成功率。研究了 Dorsal和Relish在所測(cè)斜紋夜蛾幼蟲的不同組織和不同發(fā)育階段表達(dá)特性。通過注射微生物懸浮液觀察不同微生物對(duì)于Dorsal和Relish誘導(dǎo)表達(dá)特點(diǎn)。同時(shí)注射微生物懸浮液和Dorsal和Relish基因的dsRNA,發(fā)現(xiàn)微生物對(duì)斜紋夜蛾幼蟲致病性明顯提高。并且構(gòu)建了可以在絲狀真菌中表達(dá)Dorsal dsRNA的載體P-F-R。目前的數(shù)據(jù)表明,Dorsal和Relish是斜紋夜蛾先天免疫重要組成部分。通過對(duì)斜紋夜蛾如何通過先天免疫應(yīng)對(duì)各種病原體侵染的研究,可以為生物防治該類害蟲提高理論基礎(chǔ)的研究。
[Abstract]:The Spodoptera Spodoptera is an important leaf eating insect that is extremely destructive to many crops. In this study, the family members of the Spodoptera Spodoptera cloned to Rel/NF-kB, Dorsal and Relish variable shear bodies (Dorsal 14, Dorsal]B, Relish 1A and Relish 1B).Dorsal and Relish full-length sequences and their 2 shear bodies from the same twill There are two common domains of Dorsal and Relish in the conservative domain analysis. The first is the Rel homologous domain (RHD), the Rel/NF-kB transcription influence factor of the eukaryotic cell, the second is the immunoglobulin - plexin - transcription (IPT) domain, and the immune globulin folding domain is mainly responsible for the DNA junction. The.Dorsal 1B has the RHD, IPT, NLS, and LZ domains, while Dorsal1A has the same domain as Dorsal1B, and Dorsal 14 is more than Dorsal 1. In the comparison of 19 Dorsal and 3 Relish protein sequences in the database, the evolutionary tree showed that Dorsal was clustered together with the homologous genes of the cotton bollworm (HaDorsal) and the tobacco moth (MsDorsal). The deduced Relish amino acid sequence was compared with the sequence of 19 Relish and 3 Dorsal proteins in the database of invertebrates and vertebrates. The evolutionary tree showed Relish All the amino acid protein sequences of the homologous genes of HaRelish and the beet armyworm (SeRelish) together with.Dorsal and Relish contain the specific conserved domain of the Rel/NF-kB family transcriptional factors, so the cloned Dorsal and Relish genes belong to the Rel/NF-kB family. Dorsal and Relish are obtained by obtaining Dorsal and Relish in different tissues and development. The expression of phase mRNA, thus analyzing the expression of gene tissue expression and age expression, two shear bodies expressed in different tissues and developmental stages of the Spodoptera Spodoptera, and there were significant differences in the transcriptional level of.Dorsal at different stages and.Dorsal 1A had higher expression level.Dorsal 1A in the female and female adults in the egg and the female pupae stage. The transcriptional level of the stage was significantly higher than the male and male adult stage, but there was no significant difference in the transcriptional level of Dorsal 1B in the pupae and adult stage..Dorsal 1A and Dorsal 1 were significantly different in the transcriptional level in different tissues. The Dorsal 1A had a high level of expression in the fat body and the body wall, while the lowest level of expression in the midgut was.D. The expression of orsal 1B in the body wall and fat body is higher. Similarly, the two shear bodies of the Relish are expressed in different tissues and developmental stages of the Spodoptera Spodoptera. They have significant differences at different developmental stages. The Relish 1A transcriptional level of female and female adults in the pupae and adult stage is significantly higher than that in the male. But Relish 1B in the pupa and adult stage did not show significant differences in the transcriptional level of.Relish in different tissues. Spatial expression profiles showed that the highest level of Dorsal was expressed in the body wall, while the highest level of Relish in the fat body was.RelishlA in the fat body and in the hemolymph. The lowest expression of.Relish1B in the fat body, the lowest expression in the midgut, and the high level of two kinds of shear bodies expressed in the fat body which are higher than the other stages of.Dorsal and Relish, show that the fat body plays an important role in the innate immunity. Bacteria, Java spore and Beauveria bassiana, analysis of the characteristics of the expression of Dorsal and Relish in the larvae of Spodoptera Spodoptera after microbe infection and the difference in expression of.Dorsal after being infected by fungi, and the expression of the bacteria after infection does not up increase the expression of.Relish after the infection of Escherichia coli (Gram-negative bacteria), but fungal infection There was no obvious change in post expression. The correlation between Dorsal and fungi downstream of the Toll pathway was further demonstrated. The correlation between Relish and Gram-negative bacteria downstream of the IMD pathway was higher than that of Bacillus bassiana. The reaction rate was slower than that of Relish 1A after the injection of Java bar bundle spores.Relish7B to bacillus Enterobacter. The four genes showed different expression characteristics after different microinjection. The larvae of the Spodoptera litura were injected with dsRNA and microorganism suspension of innate immune gene (Dorsal/Relis) at the same time. The mortality of the larvae was significantly higher than that of Dorsal/Relish after.DsRNA injection, and the resistance of larva to microbial infection was reduced. In order to increase the success rate of microbial infection, the expression characteristics of Dorsal and Relish in different tissues and different developmental stages of the larvae of the Spodoptera Spodoptera were studied. The induction of Dorsal and Relish by different microbes was observed by injecting microorganism suspension. Meanwhile, microorganism suspension and dsRNA of Dorsal and Relish genes were injected. The pathogenicity of the microbes to the larva of the Spodoptera litura was obviously improved and the vector P-F-R. that could express Dorsal dsRNA in filamentous fungi was constructed. The current data showed that Dorsal and Relish were important components of the innate immunity of the Spodoptera litura. To improve the theoretical basis for biological control of such pests.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.4

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本文編號(hào)：1852950