【摘要】：木霉菌(Trichoderma)是一類重要的生防真菌,具有適應(yīng)性強(qiáng)、抗菌譜廣、誘導(dǎo)植物抗性和多重拮抗作用機(jī)制等特點(diǎn)。木霉菌生防相關(guān)基因的表達(dá)由內(nèi)源信號(hào)途徑所調(diào)節(jié),G蛋白介導(dǎo)的信號(hào)傳遞系統(tǒng)是真核生物中一類重要的細(xì)胞跨膜信號(hào)傳遞系統(tǒng),在細(xì)胞外信號(hào)向胞內(nèi)傳遞及調(diào)控細(xì)胞內(nèi)反應(yīng)中起到關(guān)鍵的分子開關(guān)的作用。本實(shí)驗(yàn)室從生防菌哈茨木霉Th-33中克隆到一種I型G蛋白α亞基基因Thga1,Thga1基因敲除后,突變株的生物學(xué)特性和理化特性均發(fā)生顯著變化,包括菌絲生長(zhǎng)速度下降,分生孢子梗分枝和產(chǎn)孢量均減少,突變株疏水性降低,胞內(nèi)c AMP水平降低了50%左右,對(duì)立枯絲核菌的重寄生作用下降。為進(jìn)一步闡明Thga1的功能,本研究開展了Thga1基因的過(guò)表達(dá)研究,并對(duì)Th-33進(jìn)行了基因組測(cè)序,以及Thga1突變株的轉(zhuǎn)錄組測(cè)序,結(jié)果如下:1、通過(guò)原生質(zhì)體轉(zhuǎn)化方法獲得了Thga1基因的過(guò)表達(dá)菌株,過(guò)表達(dá)菌株菌落形態(tài)未發(fā)生明顯變化,但產(chǎn)孢量是野生型的1.63倍,生長(zhǎng)速度快于野生型,對(duì)病原菌拮抗能力增加。2、采用Hiseq2500高通量測(cè)序平臺(tái)完成了哈茨木霉Th-33的基因組測(cè)序,共產(chǎn)生21,579,163條高質(zhì)量reads,組裝成196個(gè)大片段,獲得了10849個(gè)基因,平均長(zhǎng)度為1776bp,CDS平均長(zhǎng)度為1528bp,平均每個(gè)基因含有3個(gè)外顯子,外顯子平均長(zhǎng)度為540bp,內(nèi)含子平均長(zhǎng)度為95bp,在KEGG數(shù)據(jù)庫(kù)中共注釋了6789個(gè)基因,在GO數(shù)據(jù)庫(kù)共注釋了6238個(gè)基因。3、采用Illumina Hiseq2000高通量測(cè)序平臺(tái)完成了哈茨木霉Th-33及敲除突變株1-1的轉(zhuǎn)錄組測(cè)序,分別產(chǎn)生2,838,821,746和3,242,007,080條reads。突變株相對(duì)野生型Th-33,共有差異表達(dá)基因(DEG)888個(gè),427個(gè)上調(diào),461個(gè)下調(diào)。差異表達(dá)基因中,有318個(gè)基因被注釋到184條KEGG代謝途徑中,其中雙酚降解和對(duì)氨基苯甲酸甲酯降解代謝途徑涉及的差異基因最多,并且以細(xì)胞色素P450家族的編碼基因最多。GO富集分析顯示,507個(gè)差異表達(dá)基因分到707個(gè)功能亞類,涉及差異表達(dá)基因最多的為催化活性和代謝過(guò)程,其中發(fā)現(xiàn)大量編碼碳水化合物活性酶、次生代謝物質(zhì)、分泌蛋白及轉(zhuǎn)錄因子的差異表達(dá)基因。Kog功能分析顯示,463個(gè)差異表達(dá)基因分到23個(gè)功能亞類中,其中次生代謝物的合成、運(yùn)輸及分解代謝過(guò)程中差異表達(dá)基因最多。為驗(yàn)證轉(zhuǎn)錄組測(cè)序的可靠性,我們隨機(jī)選取16個(gè)基因進(jìn)行實(shí)時(shí)熒光定量PCR驗(yàn)證,所有基因的表達(dá)趨勢(shì)均與轉(zhuǎn)錄組測(cè)序結(jié)果一致。
[Abstract]:Trichoderma (Trichoderma) is an important class of biocontrol fungi, which has the characteristics of strong adaptability, wide antibacterial spectrum, induced plant resistance and multiple antagonistic mechanism. The expression of genes related to biocontrol of Trichoderma is regulated by endogenous signaling pathway. The signal transduction system mediated by G protein is an important transmembrane signal transduction system in eukaryotes. Play a key role in extracellular signal transduction and regulation of intracellular response. A type I G protein 偽 subunit gene Thga1,Thga1 gene knockout was cloned from Trichoderma harzii in our laboratory. The biological and physical and chemical characteristics of the mutant were significantly changed, including the decrease of mycelium growth rate. The number of branches and sporulation of conidia was decreased, the hydrophobicity of mutant was decreased, the level of c AMP decreased by about 50%, and the hyperparasitism of Rhizoctonia solani was decreased. In order to further elucidate the function of Thga1, the overexpression of Thga1 gene, genomic sequencing of Th-33 and transcriptome sequencing of Thga1 mutants were carried out in this study. The results were as follows: 1. The over-expressed strain of Thga1 gene was obtained by protoplast transformation. The colony morphology of the over-expressed strain did not change significantly, but the sporulation of the over-expressed strain was 1.63 times of that of the wild type, and the growth rate was faster than that of the wild type. The Hiseq2500 high-throughput sequencing platform was used to complete the genome sequencing of Trichoderma harzii Th-33. A total of 21579163 high quality reads, fragments were assembled into 196 large fragments, and 10849 genes were obtained. The average length of CDS is 1 528 BP, the average length of exon is 540 BP, the average length of intron is 95 BP, and the average length of CDS is 1 528 BP. The average length of exon is 540 BP, and the average length of intron is 95 BP. A total of 6789 genes have been annotated in KEGG database. A total of 6238 genes .3 were annotated in the GO database. The transcription sequence of Trichoderma harzii Th-33 and its knockout mutant 1-1 was completed by Illumina Hiseq2000 high-throughput sequencing platform. 2838821746 and 3242007080 reads. were produced, respectively. Compared with wild-type Th-33, the mutant had 461 down-regulated differentially expressed genes (DEG) 888). Among the differentially expressed genes, 318 genes were annotated into 184 KEGG metabolic pathways, among which bisphenol degradation and methyl p-aminobenzoate degradation pathway involved the most differentially expressed genes. Moreover, the enrichment analysis of the coding genes of cytochrome P450 family showed that 507 differentially expressed genes were classified into 707 functional subclasses, and the most involved genes were catalytic activity and metabolic process. Among them, a large number of differentially expressed genes encoding carbohydrate active enzymes, secondary metabolites, secretory proteins and transcription factors were found. Kog functional analysis showed that 463 differentially expressed genes were divided into 23 functional subclasses, among which secondary metabolites were synthesized. The most differentially expressed genes were found in transport and catabolism. In order to verify the reliability of transcriptome sequencing, 16 genes were randomly selected for real-time fluorescence quantitative PCR validation. The expression trends of all genes were consistent with the results of transcriptome sequencing.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S476.1

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本文編號(hào)：2226432