斜纹夜蛾免疫基因Dorsal和Relish的克隆表达以及免疫通路研究

发布时间：2018-05-06 16:01

本文选题：斜纹夜蛾 + Dorsa　；参考：《南京农业大学》2015年硕士论文

【摘要】：斜纹夜蛾是一种重要的食叶昆虫,对很多农作物极其具有破坏性。在本研究中从斜纹夜蛾幼虫中克隆到Rel/NF-kB的家庭成员Dorsal和Relish可变剪切体(Dorsal 14,Dorsal]B,Relish 1A和Relish 1B)。Dorsal和Relish全长序列和他们的2个剪切体从同一个斜纹夜蛾幼虫中克隆得到的。对保守结构域分析得到Dorsal和Relish有两个共同的结构域,第一个是Rel同源结构域(RHD),即真核细胞特征Rel/NF-kB转录影响因子,第二个是免疫球蛋白-丛蛋白-转录(IPT)结构域,其是类免疫球蛋白折叠域主要负责DNA结合。Dorsal 1B具有RHD、IPT、NLS和LZ结构域,而Dorsal1A具有和Dorsal1B相同的结构域,并且Dorsal 14比Dorsal 1多一个叉头N末端区域。Relish 1A具有RHD,IPT,ANK和死亡结构域,而Relish7B有着和Relish 1A 一样的结构域。Dorsal的氨基酸序列与无脊椎动物和脊椎动物的数据库中19个Dorsal和3个Relish蛋白序列对比,进化树显示出Dorsal与棉铃虫(HaDorsal)和烟草天蛾(MsDorsal)同源基因聚集在一起。推导的Relish氨基酸序列与无脊椎动物和脊椎动物的数据库中19个Relish和3个Dorsal蛋白序列对比。进化树显示Relish与棉铃虫(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家族转录因子所具有的特异保守结构域,故克隆的Dorsal和Relish基因均属于Rel/NF-kB家族。通过获得Dorsal和Relish在不同组织和发育阶段mRNA表达量,从而分析出基因组织表达和龄期表达的规律。Dorsal的两个剪切体在斜纹夜蛾不同的组织和发育阶段均有表达,并且在不同阶段转录水平具有显著差异。Dorsal 1A在卵和雌蛹阶段有更高的表达水平。Dorsal 1A在雌蛹和雌成虫阶段转录水平显著高于雄蛹和雄成虫阶段,但是Dorsal 1B在蛹和成虫阶段雌雄成虫转录水平无显著性差异。Dorsal 1A和Dorsal 1在不同组织中的转录水平有显著性差异,Dorsal 1A在脂肪体和体壁具有较高的表达水平,而在中肠的表达水平最低。Dorsal 1B在体壁和脂肪体中的表达量较高。同样的,Relish的两个剪切体在斜纹夜蛾不同的组织和发育阶段都有表达。他们在不同的发育阶段的转录具有显著差异。在蛹和成虫阶段雌蛹和雌成虫个体Relish 1A转录水平比雄性显著高。但是Relish 1B在蛹和成虫阶段雌雄个体并没有表现出显著差异。Relish在不同组织中的转录水平具有显著差异。空间的表达谱表明,Dorsal在体壁中表达水平最高,而Relish在脂肪体的转录水平最高。RelishlA在脂肪体和血淋巴中具有较高表达量在中肠中表达量最低。Relish1B在脂肪体中的表达量最高,在中肠中的表达量最低。并且在脂肪体中表达水平均比其他阶段高。Dorsal和Relish的两种剪切体均在脂肪体内具有高水平转录,这表明脂肪体在先天免疫中发挥了重要的作用。分别注射大肠杆菌、爪哇棒束孢和球孢白僵菌,分析斜纹夜蛾幼虫Dorsal和Relish在微生物侵染后表达规律的特性和差异。Dorsal在被真菌侵染后表达量上调,而细菌侵染后表达量并不上调。Relish在被大肠杆菌(革兰氏阴性菌)侵染后表达量上调,但是真菌侵染后表达量并无明显变化。进一步证明了在Toll通路下游的Dorsal与真菌之间的相关性,在IMD通路下游的Relish与革兰氏阴性菌的相关性。Dorsal被球孢白僵菌上调的表达持续时间比爪哇棒束孢长。Relish7B对大肠杆菌注入后反应速度比Relish 1A慢。这四个基因应对不同的微生注射后表现出不同的表达特性。斜纹夜蛾幼虫同时注射先天免疫基因(Dorsal/Relis)的dsRNA和微生物悬浮液,幼虫的死亡率明显升高。dsRNA注射后下调了Dorsal/Relish的表达量,降低了幼虫对于微生物侵染的抵抗作用,从而增加微生物侵染的成功率。研究了 Dorsal和Relish在所测斜纹夜蛾幼虫的不同组织和不同发育阶段表达特性。通过注射微生物悬浮液观察不同微生物对于Dorsal和Relish诱导表达特点。同时注射微生物悬浮液和Dorsal和Relish基因的dsRNA,发现微生物对斜纹夜蛾幼虫致病性明显提高。并且构建了可以在丝状真菌中表达Dorsal dsRNA的载体P-F-R。目前的数据表明,Dorsal和Relish是斜纹夜蛾先天免疫重要组成部分。通过对斜纹夜蛾如何通过先天免疫应对各种病原体侵染的研究,可以为生物防治该类害虫提高理论基础的研究。
[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.

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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