基于转录组分析的哈茨木霉Thga1基因功能研究
发布时间：2018-09-06 13:03
【摘要】：木霉菌(Trichoderma)是一类重要的生防真菌,具有适应性强、抗菌谱广、诱导植物抗性和多重拮抗作用机制等特点。木霉菌生防相关基因的表达由内源信号途径所调节,G蛋白介导的信号传递系统是真核生物中一类重要的细胞跨膜信号传递系统,在细胞外信号向胞内传递及调控细胞内反应中起到关键的分子开关的作用。本实验室从生防菌哈茨木霉Th-33中克隆到一种I型G蛋白α亚基基因Thga1,Thga1基因敲除后,突变株的生物学特性和理化特性均发生显著变化,包括菌丝生长速度下降,分生孢子梗分枝和产孢量均减少,突变株疏水性降低,胞内c AMP水平降低了50%左右,对立枯丝核菌的重寄生作用下降。为进一步阐明Thga1的功能,本研究开展了Thga1基因的过表达研究,并对Th-33进行了基因组测序,以及Thga1突变株的转录组测序,结果如下:1、通过原生质体转化方法获得了Thga1基因的过表达菌株,过表达菌株菌落形态未发生明显变化,但产孢量是野生型的1.63倍,生长速度快于野生型,对病原菌拮抗能力增加。2、采用Hiseq2500高通量测序平台完成了哈茨木霉Th-33的基因组测序,共产生21,579,163条高质量reads,组装成196个大片段,获得了10849个基因,平均长度为1776bp,CDS平均长度为1528bp,平均每个基因含有3个外显子,外显子平均长度为540bp,内含子平均长度为95bp,在KEGG数据库中共注释了6789个基因,在GO数据库共注释了6238个基因。3、采用Illumina Hiseq2000高通量测序平台完成了哈茨木霉Th-33及敲除突变株1-1的转录组测序,分别产生2,838,821,746和3,242,007,080条reads。突变株相对野生型Th-33,共有差异表达基因(DEG)888个,427个上调,461个下调。差异表达基因中,有318个基因被注释到184条KEGG代谢途径中,其中双酚降解和对氨基苯甲酸甲酯降解代谢途径涉及的差异基因最多,并且以细胞色素P450家族的编码基因最多。GO富集分析显示,507个差异表达基因分到707个功能亚类,涉及差异表达基因最多的为催化活性和代谢过程,其中发现大量编码碳水化合物活性酶、次生代谢物质、分泌蛋白及转录因子的差异表达基因。Kog功能分析显示,463个差异表达基因分到23个功能亚类中,其中次生代谢物的合成、运输及分解代谢过程中差异表达基因最多。为验证转录组测序的可靠性,我们随机选取16个基因进行实时荧光定量PCR验证,所有基因的表达趋势均与转录组测序结果一致。
[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 伪 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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.1
本文编号：2226432
[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 伪 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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.1
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