筛胸梳爪叩甲幼虫与绿僵菌互作机制研究

发布时间：2018-06-07 08:38

  本文选题：筛胸梳爪叩甲 + 平沙绿僵菌　； 参考：《中国林业科学研究院》2017年博士论文

【摘要】：筛胸梳爪叩甲(Melanotus cribricollis)幼虫,俗称“金针虫”,是中国南方竹林地最为重要的笋期害虫,监测及防控难度大。平沙绿僵菌(Metarhizum pingshaense)WP08菌株是筛胸梳爪叩甲僵虫上分离的一种高毒力的昆虫致病真菌,可能突破竹林金针虫的生物防治瓶颈。然而,关于地下植食性昆虫与和虫生真菌互作的分子机制研究罕见报道。本文以筛胸梳爪叩甲幼虫和平沙绿僵菌WP08菌株为研究对象,探索绿僵菌侵染筛胸梳爪叩甲幼虫后保护酶系统应答反应,同时利用新一代高通量测序技术,对筛胸梳爪叩甲幼虫和平沙绿僵菌WP08菌株转录组水平的遗传背景开展研究,并挖掘昆虫免疫基因和绿僵菌毒致病基因。研究主要取得以下成果:(1)测定了平沙绿僵菌WP08菌株、金龟子绿僵菌(M.anisopliae)WTKH菌株及蝗绿僵菌(M.acridum)3株绿僵菌在不同孢子浓度条件下对筛胸梳爪叩甲幼虫的致病力,同时测定了不同侵染时期金针虫超氧化物歧化酶(SOD)、过氧化物酶(POD)及过氧化氢酶(CAT)3种保护酶含量及活力变化情况。结果表明,3株绿僵菌对筛胸梳爪叩甲幼虫均具有致死性,平沙绿僵菌WP08菌株最优,金龟子绿僵菌WTKH菌株次之,蝗绿僵菌最低。不同绿僵菌菌株的致病力随着浓度的增加而提高,WP08在1.3×108孢子.g-1干土浓度条件下的致病效果最好,仅在16d时供试金针虫已经全部死亡,但与1.3×107孢子.g-1干土浓度条件下的致病效果无显著差异,与1.3×106孢子.g-1干土的浓度条件下的致病效果差异显著。筛胸梳爪叩甲幼虫感染平沙绿僵菌后3种保护酶活性的变化趋势不同,SOD在不同侵染时期其活性变化无显著差异,而POD和CAT酶活性变化均表现为先下降后上升再下降的趋势。由此可知,平沙绿僵菌WP08对筛胸梳爪叩甲幼虫的致病性显著优于金龟子绿僵菌WTKH菌株和蝗绿僵菌,可作为林间应用的首选菌株。绿僵菌侵染可能对筛胸梳爪叩甲幼虫体内POD和CAT的活性产生的影响较SOD明显。(2)运用RNA-Seq技术,对接染平沙绿僵菌WP08菌株不同时间(CK、1 d、3 d、4 d、5 d、7 d)的筛胸梳爪叩甲幼虫进行测序,共获得近800M的高质量的Reads,组装拼接获得约64000个Unigene,各组样品与Unigene库的比对效果也较理想(80%),同时与NCBI-Nr、Swiss-Prot、KEGG、COG、KOG、GO和Pfam数据库比对,获得40%Unigene功能注释率。筛胸梳爪叩甲与黑腹果蝇、赤拟谷盗和山松大小蠹的氨基酸序列比对,获得9512个同源基因cluster,4145个单拷贝基因Cluster。运用RNA-Seq技术对接染筛胸梳爪叩甲幼虫不同时间的平沙绿僵菌WP08菌株进行测序,共获得约134M的高质量的Reads,28107个Unigene和70%的注释率。平沙绿僵菌WP08菌株与烟曲霉、玉蜀黍黑粉菌、稻瘟病菌、尖孢镰刀菌和新型隐球菌氨基酸序列比对,获得9189个同源基因Cluster,1703个单拷贝基因的Cluster。同时,分别对两个物种的Unigene进行CDs预测,AS、SSR和SNP分析。(3)统计分析筛胸梳爪叩甲幼虫不同感染时期差异表达基因(DEGs)的数量、功能注释、GO富集、Pathway富集和免疫相关基因。筛胸梳爪叩甲幼虫的DEGs的数量随着侵染时间整体呈上升趋势,免疫相关通路和“生物学过程”GO term的种类和数量也逐渐增加。部分基因在不同的侵染时间具有特异性,参与不同的通路和GO terms,但大部分基因在不同阶段普遍存在。筛胸梳爪叩甲幼虫Unigene库中与昆虫免疫防御相关的部分基因亦被挖掘,包括GNBP、β-1,3-GRP、PGRP、lysozyme、GST、P450、SDR等。另一方面,将平沙绿僵菌WP08菌株氨基酸序列与PHI数据库比对,以发掘昆虫病原真菌的毒力基因。PHI比对共获得1529个已被验证的基因注释结果,其中35个基因注释的参考菌株为绿僵菌和白僵菌,包括BbbqrA、BbCHIT1、Chi2、CYP52X1、Kre2、Mnt1、Ktr1、Ktr4、MaSnf1、Pr1、OpS1、mrGAT、MrpacC、Ohmm、Fkh2、MBZ1、mas5、mhk1、Bck1、Fus3、Mkk1/2、Slt2、Ste11、Ste7、Hog1、Pbs2、Ssk2、ras3、vosA和wetA等基因,通过对昆虫体壁降解、氧化还原、碳源利用、磷酸化、转录因子调控、细胞周期、渗透压调节、生长发育等各方面调控致病菌株的多重胁迫抗性和致病力。本研究通过从生理和分子水平初步探究虫-菌的互作机理,不仅为昆虫免疫机制的研究提供关键信息和理论支持,同时也为高效生防菌株的基因改造奠定基础,有利于害虫生物防控技术的发展。
[Abstract]:The Melanotus cribricollis larva, commonly known as the "golden needle", is the most important bamboo pest in southern China, and is very difficult to monitor and control. The WP08 strain of Metarhizum pingshaense is a highly poisonous insect pathogenic fungus separated from the sieved thorax carbs, which may break through the golden needle of bamboo forest. A rare report on the molecular mechanism of interaction between subterranean herbivorous insects and entomophytic fungi, however, is rarely reported. In this paper, the response of Bacillus anisopliae to the response to the enzyme system after the infection of the sieved grizzle claw larva was explored and the new generation high pass was used in the study of the WP08 strain of the ethfic clawed larva and the Bacillus anisopliae. Quantitative sequencing technology was carried out to study the genetic background of the transcriptional level of the WP08 strain of the sifter carding claw larva and the Bacillus anisopliae, and to excavate the insect immune genes and the virulence genes. The main results were as follows: (1) the WP08 strain, the M.anisopliae WTKH strain and the M.a (M.a) were measured. Cridum) the pathogenicity of 3 strains of Bacillus anisopliae on the larvae of the sifting claw claw at different spores concentration, and the changes of the 3 protective enzymes and activities of the 3 kinds of protective enzymes of the superoxide dismutase (POD), peroxidase (POD) and catalase (CAT) at different infestation period. The results showed that 3 strains of Pseudomonas anisopliae on the larva of the simoid comb claw The WP08 strain of Pseudomonas sp. was the best, the Bacillus anisopliae WTKH was the next, and the Bacillus bassiana was the lowest. The pathogenicity of different strains of Bacillus anisopliae increased with the increase of concentration, and the pathogenicity of WP08 under the concentration of 1.3 x 108 spores.G-1 dry soil was the best. There was no significant difference in the pathogenicity of the sub.G-1 dry soil concentration. The difference of the pathogenic effect was significant with the concentration of 1.3 x 106 spores.G-1 dry soil. There were different changes in the activity of the 3 protective enzymes after the infection of the larva of the sifter carb claw larvae. There was no significant difference in the activity of SOD during the different infestation period, while the POD and CAT enzyme activities were not significantly different. As a result, the pathogenicity of WP08 on the simoid carb claw larvae was significantly better than that of Bacillus anisopliae WTKH and aristizium anisopliae, and it could be used as the first selection strain of the forest application. The infection of Bacillus anisopliae could produce the activity of POD and CAT in the larvae of the simoid comb claw. The effect of SOD was more obvious than that of SOD. (2) RNA-Seq technology was used to sequenced the larva of the sieved comb claw larva of the WP08 strains of Bacillus bassiana at different time (CK, 1 D, 3 D, 4 D, 5 d, 7 d). The high quality Reads of the 800M was obtained, and about 64000 Unigene were obtained by assembly and splicing. The results of the comparison between the samples and the library were also ideal (80%). I-Nr, Swiss-Prot, KEGG, COG, KOG, GO and Pfam are compared to obtain the 40%Unigene function annotation rate. The comparison of the amino acid sequence of the sieved chest comb claw to the black abdominal Drosophila, the red carpenter and the Pinus mountain beetle, obtained 9512 homologous gene cluster, and 4145 single copy gene Cluster. using RNA-Seq technique to dye the simoid comb claw larva at the same time. WP08 strains were sequenced, with a total of about 134M of high quality Reads, 28107 Unigene and 70% annotation rate. The comparison of the WP08 strain of Bacillus versii with Aspergillus fumigatus, sicomonas sp., rice blast fungus, Fusarium oxysporum and Cryptococcus neoformans, obtained 9189 homologous genes, Cluster, and 1703 single copy genes of C. Luster. also carried out CDs prediction, AS, SSR and SNP analysis on two species of Unigene. (3) statistical analysis of the number of differentially expressed genes (DEGs), functional annotation, GO enrichment, Pathway enrichment and immune related genes in the different infection period of the sieved comb claw larva. The number of DEGs of the sieved comb claw larva was on the whole infection time. The species and number of GO term in the immune related pathway and "biological process" are also increasing gradually. Some genes are specific at different infection times, participate in different pathways and GO terms, but most of the genes are ubiquitous in different stages. The part of the Unigene Library of the sieves comb claw larva is related to the insect immune defense. The gene was also excavated, including GNBP, beta -1,3-GRP, PGRP, lysozyme, GST, P450, SDR and so on. On the other hand, the amino acid sequence of Bacillus anisopliae strain WP08 strain was compared with the PHI database to obtain 1529 confirmed results of the nucleotide annotation of the virulence gene.PHI comparison of the entomopathogenic fungi, of which 35 reference strains of gene annotation were green stiffness. Bacillus and Beauveria bassiana, including BbbqrA, BbCHIT1, Chi2, CYP52X1, Kre2, Mnt1, Ktr1, Ktr4, MaSnf1, Pr1, OpS1, etc., through the degradation of the body wall, oxidation and reduction, carbon source, phosphorylation, transcription factor regulation, cell cycle, infiltration This study provides the key information and theoretical support for the study of insect immune mechanism by exploring the interaction mechanism of the insect bacteria from the physiological and molecular levels. It also lays the foundation for the genetic transformation of the high effective bacteria resistant strains. The development of pest biological control technology.
【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S763.306.4
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本文编号：1990557