本文選題：篩胸梳爪叩甲 + 平沙綠僵菌　； 參考：《中國林業(yè)科學研究院》2017年博士論文

【摘要】：篩胸梳爪叩甲(Melanotus cribricollis)幼蟲,俗稱“金針蟲”,是中國南方竹林地最為重要的筍期害蟲,監(jiān)測及防控難度大。平沙綠僵菌(Metarhizum pingshaense)WP08菌株是篩胸梳爪叩甲僵蟲上分離的一種高毒力的昆蟲致病真菌,可能突破竹林金針蟲的生物防治瓶頸。然而,關于地下植食性昆蟲與和蟲生真菌互作的分子機制研究罕見報道。本文以篩胸梳爪叩甲幼蟲和平沙綠僵菌WP08菌株為研究對象,探索綠僵菌侵染篩胸梳爪叩甲幼蟲后保護酶系統(tǒng)應答反應,同時利用新一代高通量測序技術,對篩胸梳爪叩甲幼蟲和平沙綠僵菌WP08菌株轉(zhuǎn)錄組水平的遺傳背景開展研究,并挖掘昆蟲免疫基因和綠僵菌毒致病基因。研究主要取得以下成果:(1)測定了平沙綠僵菌WP08菌株、金龜子綠僵菌(M.anisopliae)WTKH菌株及蝗綠僵菌(M.acridum)3株綠僵菌在不同孢子濃度條件下對篩胸梳爪叩甲幼蟲的致病力,同時測定了不同侵染時期金針蟲超氧化物歧化酶(SOD)、過氧化物酶(POD)及過氧化氫酶(CAT)3種保護酶含量及活力變化情況。結(jié)果表明,3株綠僵菌對篩胸梳爪叩甲幼蟲均具有致死性,平沙綠僵菌WP08菌株最優(yōu),金龜子綠僵菌WTKH菌株次之,蝗綠僵菌最低。不同綠僵菌菌株的致病力隨著濃度的增加而提高,WP08在1.3×108孢子.g-1干土濃度條件下的致病效果最好,僅在16d時供試金針蟲已經(jīng)全部死亡,但與1.3×107孢子.g-1干土濃度條件下的致病效果無顯著差異,與1.3×106孢子.g-1干土的濃度條件下的致病效果差異顯著。篩胸梳爪叩甲幼蟲感染平沙綠僵菌后3種保護酶活性的變化趨勢不同,SOD在不同侵染時期其活性變化無顯著差異,而POD和CAT酶活性變化均表現(xiàn)為先下降后上升再下降的趨勢。由此可知,平沙綠僵菌WP08對篩胸梳爪叩甲幼蟲的致病性顯著優(yōu)于金龜子綠僵菌WTKH菌株和蝗綠僵菌,可作為林間應用的首選菌株。綠僵菌侵染可能對篩胸梳爪叩甲幼蟲體內(nèi)POD和CAT的活性產(chǎn)生的影響較SOD明顯。(2)運用RNA-Seq技術,對接染平沙綠僵菌WP08菌株不同時間(CK、1 d、3 d、4 d、5 d、7 d)的篩胸梳爪叩甲幼蟲進行測序,共獲得近800M的高質(zhì)量的Reads,組裝拼接獲得約64000個Unigene,各組樣品與Unigene庫的比對效果也較理想(80%),同時與NCBI-Nr、Swiss-Prot、KEGG、COG、KOG、GO和Pfam數(shù)據(jù)庫比對,獲得40%Unigene功能注釋率。篩胸梳爪叩甲與黑腹果蠅、赤擬谷盜和山松大小蠹的氨基酸序列比對,獲得9512個同源基因cluster,4145個單拷貝基因Cluster。運用RNA-Seq技術對接染篩胸梳爪叩甲幼蟲不同時間的平沙綠僵菌WP08菌株進行測序,共獲得約134M的高質(zhì)量的Reads,28107個Unigene和70%的注釋率。平沙綠僵菌WP08菌株與煙曲霉、玉蜀黍黑粉菌、稻瘟病菌、尖孢鐮刀菌和新型隱球菌氨基酸序列比對,獲得9189個同源基因Cluster,1703個單拷貝基因的Cluster。同時,分別對兩個物種的Unigene進行CDs預測,AS、SSR和SNP分析。(3)統(tǒng)計分析篩胸梳爪叩甲幼蟲不同感染時期差異表達基因(DEGs)的數(shù)量、功能注釋、GO富集、Pathway富集和免疫相關基因。篩胸梳爪叩甲幼蟲的DEGs的數(shù)量隨著侵染時間整體呈上升趨勢,免疫相關通路和“生物學過程”GO term的種類和數(shù)量也逐漸增加。部分基因在不同的侵染時間具有特異性,參與不同的通路和GO terms,但大部分基因在不同階段普遍存在。篩胸梳爪叩甲幼蟲Unigene庫中與昆蟲免疫防御相關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,將平沙綠僵菌WP08菌株氨基酸序列與PHI數(shù)據(jù)庫比對,以發(fā)掘昆蟲病原真菌的毒力基因。PHI比對共獲得1529個已被驗證的基因注釋結(jié)果,其中35個基因注釋的參考菌株為綠僵菌和白僵菌,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,通過對昆蟲體壁降解、氧化還原、碳源利用、磷酸化、轉(zhuǎn)錄因子調(diào)控、細胞周期、滲透壓調(diào)節(jié)、生長發(fā)育等各方面調(diào)控致病菌株的多重脅迫抗性和致病力。本研究通過從生理和分子水平初步探究蟲-菌的互作機理,不僅為昆蟲免疫機制的研究提供關鍵信息和理論支持,同時也為高效生防菌株的基因改造奠定基礎,有利于害蟲生物防控技術的發(fā)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.
【學位授予單位】：中國林業(yè)科學研究院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S763.306.4
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本文編號：1990557